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METALLURGICA

LAPPLICATION
S of
BACTERIA
LLEACHIN
G andRELATE
D
MICROBIOLOGICA
LPHENOMEN
A

Edited by

L A W R E N C E E. M U R R
A R P A D E. T O R M A
John D. Sullivan Center for In-Situ Mining Research,
Department of Metallurgical and Materials Engineering

J A M E S A. B R I E R L E Y
Department of Biology

New Mexico Institute of Mining and Technology


Socorro, New Mexico

ACADEMIC PRESS New York San Francisco London 1978


A Subsidiary of Harcourt Brace Jovanovich, Publishers
T h e material was prepared with the support o f National Science F o u n d a t i o n
Grant N o . A E R 7 7 - 1 2 2 2 1 . However, any opinions, findings, conclusions, or
recommendations expressed herein are those o f the author(s) and do not
necessarily reflect the views of N S F .

C O P Y R I G H T © 1 9 7 8 , New M e x i c o Institute o f Mining and T e c h n o l o g y


ALL RIGHTS RESERVED.
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United Kingdom Edition published by


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Library of Congress Cataloging in Publication Data

M a i n entry under title:

Metallurgical applications of bacterial leaching and


related microbiological phenomena.

Proceedings of an international symposium which took


place at the New Mexico Institute o f M i n i n g & Technology,
Socorro, Ν . M . , August 3-5, 1977.
Includes bibliographical references and index.
1. Leaching—Congresses. 2. Metallurgy—Congresses.
3. Industrial microbiology—Congresses. I. Murr,
Lawrence Eugene. II. Torma, Arpad Ε. III. Brierley,
James Α . IV. New M e x i c o . Institute o f M i n i n g and
Technology, Socorro. [ D N L M : 1. Metallurgy—Congresses.
2. Microbiology—Congresses. Q W 7 5 M587 1977]
TN673.M47 622'.75 78-693
I S B N 0-12-511150-9
PRINTED I N T H E UNITED STATES OF AMERICA
Dedicated to

Marguerite M. and John D. Sullivan

microbiologist and leaching pioneer respectively·,


whose professionalism and marriage
are the epitome of the objectives of this book
Contributor
s

Numbers in parentheses indicate the pages on which the authors' contributions begin.

WILLIAM A. APEL (45, 223), Department of Microbiology, The Ohio State


University, Columbus, Ohio
A. S. ATKINS (403), Virginia Polytechnic Institute and State University,
Blacksburg, Virginia
H. BALAKRISHNAN (427), National Chemical Laboratory, Poona, India
V. K. BERRY (103), Department of Metallurgical and Materials Engineering,
New Mexico Institute of Mining and Technology, Socorro, New Mexico
K. BOSECKER (389), Bundesanstalt fur Geowissenschaften und Rohstoffe, Han-
nover, Federal Republic of Germany
CORALE L. BRIERLEY (345, 477), New Mexico Bureau of Mines and Mineral
Resources, Socorro, New Mexico
JAMES A. BRIERLEY (477, 491), Department of Biology, New Mexico Insti-
tute of Mining and Technology, Socorro, New Mexico
A. BRUYNESTEYN (441), British Columbia Research, Vancouver, British Co-
lumbia, Canada
A. M. CHAKRABARTY (137), General Electric Company, Corporate Research
and Development, Schenectady, New York
DOUGLAS J. CORK (207), Department of Chemistry, University of Arizona,
Tucson, Arizona
MICHAEL A. CUSANOVICH (207), Department of Chemistry, University of
Arizona, Tucson, Arizona
CRAWFORD S. DOW (61), University of Warwick, Coventry, England
PATRICK R. DUGAN (45, 223), Department of Microbiology, The Ohio State
University, Columbus, Ohio
HANS GEORG EBNER (195), University of Dortmund, Dortmund, Federal
Republic of Germany
MARTIN ECCLESTON (61), University of Warwick, Coventry, England
STEFAN S. GAIDARJIEV (253), Higher Institute of Mining and Geology,
Sofia—Darvenitza, Bulgaria
FRATIO N. GENCHEV (253), Higher Institute of Mining and Geology,
Sofia—Darvenitza, Bulgaria
STOYAN N. GROUDEV (253), Higher Institute of Mining and Geology,
Sofia—Darvenitza, Bulgaria

XV
xvi Contributors

KAZUTAMI IMAI (275), Faculty of Agriculture, Okayama University,


Tsuchima, Okayama-shi, Japan
C. A. JONES (19), Glaxo Research Ltd., Stoke Poges, Bucks, England
DONOVAN P. KELLY (19, 61, 83), University of Warwick, Coventry, England
V. S. KRISHNAMACHAR (427), National Chemical Laboratory, Poona, India
NORMAN W. LE ROUX (167, 463), Warren Spring Laboratory, Stevenage,
Herts, England
DONALD LUNDGREN (151), Syracuse University, Syracuse, New York
R. O. McELROY (441), British Columbia Research, Vancouver, British Colum­
bia, Canada
Κ. Β. MEHTA (463), Warren Spring Laboratory, Stevenage, Herts, England
L. E. MURR (103, 491), Department of Metallurgical and Materials Engineering,
New Mexico Institute of Mining and Technology, Socorro, New Mexico
D. NEUSCHUTZ (389), Friedrich Krupp GmbH, Krupp Forschungsinstitut, Es­
sen, Federal Republic of Germany
P. R. NORRIS (83), University of Warwick, Coventry, England
S. G. PATIL (427), National Chemical Laboratory, Poona, India
VIVIAN F. PERRY (167), Warren Spring Laboratory, Stevenage, Herts, England
P. N. RANGACHARI (427), National Chemical Laboratory, Poona, India
GIOVANNI ROSSI (297), University of Cagliari, Sardinia, Italy
Μ. N. SAINANI (427), National Chemical Laboratory, Poona, India
U. SCHEFFLER (389), Friedrich Krupp GmbH, Krupp Forschungsinstitut, Es­
sen, Federal Republic of Germany
MARVIN SILVER (3), Universite Laval, Quebec, Canada
TATSUO TANO (151), Okayama University, Okayama, Japan
NOBORU TOMIZUKA (321), Fermentation Research Institute, Chiba, Japan
A. E. TORMA (375), Department of Metallurgical and Materials Engineering,
New Mexico Institute of Mining and Technology, Socorro, New Mexico
OLLI H. TUOVINEN (61), University of Helsinki, Helsinki, Finland
Η. M. TSUCHIYA (365), University of Minnesota, Minneapolis, Minnesota
DON S. WAKERLEY (167), Warren Spring Laboratory, Stevenage, Herts, Eng­
land
MITSUO YAGISAWA (321), Fermentation Research Institute, Chiba, Japan
Prefac
e

The synergistic aspects of science and engineering and o f their many disciplines
and subdisciplines stand out as perhaps the major influence on the bulk o f industrial
and technical advances at least in the latter part of the twentieth century. Indeed, the
complexity of the world society and its concomitant and interconnected social,
economic, and industrial problems have been syndetic to science and engineering,
with the outcome being the interdisciplinary and multidisciplinary approaches to the
development of and solutions to the technological innovations necessary to sustain
and advance the standard of living in many parts of the industrialized world.
In many respects, this book reflects the synergistic aspects of microbiology in
metallurgy—particularly hydrometallurgy—culminating in or converging to a
syndetic subdiscipline: biohydrometallurgy. The chapters comprising this volume
represent the invited and contributed papers that composed an international sym-
posium having the same title as the book. The theme of the symposium, which is so
obviously reflected in these chapters, addresses an attempt to provide some basic
understanding of the role o f bacteria in leaching processes and other metallurgical
applications, particularly hydrometallurgical. The book emphasizes the role played
by microorganisms in the kinetics o f leaching and similar metallurgical processes,
and this was achieved by stimulating a strong interaction—a priori—between mi-
crobiologists and metallurgists. In many respects, this book represents a very suc-
cessful attempt to bridge the gap between those involved in the basic study of
microorganisms, in the strictly microbiological aspects of metal extraction and
attendant conversion kinetics, and in the practical, engineering aspects of extrac-
tion. In this respect, it should be o f interest to a wide range of students, researchers,
and practitioners in microbiology, biophysics, biochemistry, mineral processing
and preparation, extractive and/or chemical metallurgy, mining engineering, and
many related disciplines including chemical engineering and bioengineering. A
particular aim of the book, like the symposium from which it originated, is to
discuss and ascertain the projected role of microbiological applications in areas of
mineral extraction, especially from low-grade, nonrenewable waste-ore deposits,
and the arrangement of the chapters addresses both the fundamental and practical
aspects of such applications. This is accomplished somewhat syndetically by the
arrangement of the chapters into three major sections:

I. Basic Microbial Studies Applied to Leaching, which also addresses fundamen-


tal microbial phenomena;
II. Waste Treatment and Environmental Considerations;
III. Bioextractive Applications and Optimization.

xvi
i
xviii Preface

The major objectives of the symposium, which are forcefully reflected in these
collected works, were
(1) to establish a strong interaction between the fundamentalists and practitioners
in the area of bacterial leaching in its broadest sense, to have industry input, and to
discuss industrial problems in bacterial leaching;
(2) to review the latest developments in optimizing bacterial activity, and in
understanding the role of microorganisms in large-scale metallurgical applications;
(3) to address and discuss new metallurgical applications of bacterial leaching and
the attendant economics;
(4) to establish the optimum conditions for conventional bacterial leaching of
metal values and recommend appropriate research efforts to address leach dump
optimization; and
(5) to establish a priority for research in the area of bacterial leaching with
particular emphasis on the determination of directions of future research in the
development of existing microorganisms or the search for new microorganisms.

It is not unlikely that this book would serve as a text/reference in advanced


courses in biometallurgy, extractive metallurgy, hydrometallurgy, and applied or
industrial microbiology. In fact, the symposium was offered for graduate credit and
assignments were structured to direct the student toward an assessment of various
concepts and processes after having been exposed to the topics presented herein.
The arrangement of this book into distinct but interconnected regimes involving both
the fundamental and practical (applied) aspects of bacterial leaching and related
phenomena is indeed conducive to such usage.
The editors wish to thank those who participated in the symposium and who
contributed to this volume. We are especially grateful for the support of this effort
through a grant (No. AER77-12221) from the National Science Foundation's Re-
search Applied to National Needs (RANN) Division. Finally, we are especially
grateful for the patient and competent typing of most of the chapters and the
discussions of the sections, as well as editorial help from Lorraine Valencia and
Elizabeth Fraissinet.
I BASIC MICROBIAL STUDIES APPLIED TO LEACHING

An understanding o f the underlying principles controlling the function o f


thiobacilli and their reaction to environment will ultimately provide the informa­
tion necessary for their successful use in solving leaching problems. Thus, this
symposium included papers dealing strictly with basic cellular phenomena.
Chapter 1 o f this section presents a brief review of sulfur, iron, and carbon
dioxide metabolism in thiobacilli. Three chapters follow presenting new data
concerning the oxidation of iron by Thiobacillus ferrooxidans. The kinetics o f
biological iron oxidation are greatly affected by the ferric to ferrous ratio, an
aspect of importance in considering use o f microbes for generating lixiviant for
metal sulfide oxidation. The requirement for hydrogen ion by Τ. ferrooxidans for
ferrous iron oxidation is also a factor affecting cell function. Chapter 8 reports
development of a procedure for study of the iron oxidizing system using isolated
cell membranes. Chapter 5 deals with the affect o f silver and other metals toxicity
on ferrobacilli. This chapter indicates that these microbes may also have a role in
concentration of various metals by cellular accumulation. Some basic characteris­
tics o f cell structure and the phenomenon o f apparent interspecies transition of
acidophilic thiobacilli was presented. Chapter 7 speculates on the potential for
alteration of thiobacilli characteristics by methods of mutation and plasmid trans­
fer. Chapters 6 and 9 consider the microbes involved in the leaching process; one
through direct observation o f the microbes; the other discussing the use of mi­
crobes other than thiobacilli, an aspect certainly to receive much attention in
the future.
Future symposia concerned with bacterial leaching will undoubtedly include
more chapters of basic cellular studies. This is a field in which basic research has
been greatly stimulated by a study of applications o f leaching procedures as dis­
cussed in the chapters of the following sections.
METABOLIC MECHANISMS

OF IRON-OXIDIZING THIOBACILLI

Marvin Silver

U n i v e r s i t y Laval
Quebec, Quebec, Canada

The iron-oxidizing thiobacilli are remarkable for the range


of inorganic compounds that are acted upon. The principal if not
sole source of carbon, C02* is assimilated by two methods simul-
taneously: the Calvin-Benson or reductive pentose phosphate cycle
and the carboxylation of PEP (phospho-enol-pyruvate) with the for-
mation of oxalo-acetate by the enzyme PEP-carboxylase.
Similar to other thiobacilli, elemental sulfur and reduced
inorganic sulfur compounds are used for the generation of energy;
several studies have demonstrated a number of pathways, which may
not be mutually exclusive, and some of the enzymes catalyzing in-
dividual reactions have been isolated and characterized. Unique
amongst the thiobacilli is the ability of these organisms to ox-
idize ferrous iron for the generation of chemical energy. Metal
sulfide mineral ores also serve this function, resulting in the
oxidation of the sulfide moiety to sulfate and the dramatic mo-
dification of the initial substrate, in many cases resulting in
the solubilization of the metallic entity.
The mechanisms, both known and postulated, of the above reac-
tions are described and discussed. In addition, evidence is pre-
sented indicating that the organism known as T h i o b a c i l l u s f e r r o o x i -
dans may not be one distinct bacterium, but rather a group of me-
tabolically similar microbes.

3
4 Marvin Silver

I. INTRODUCTION TO THE IRON OXIDIZING THIOBACILLI

Since the f i r s t of the t h i o b a c i l l i , Thiobaoillus thioparus,


was discovered by Nathansohn in 1902 ( 1 ) , many more s u l f u r - o x i d i -
zing autotrophs have been i s o l a t e d and i d e n t i f i e d . Among these
are the iron o x i d i z i n g bacteria. The f i r s t of these was named T.
ferrooxidans by Temple and Colmer ( 2 ) , and was followed s h o r t l y
afterwards by Ferrobacillus ferrooxidans (3) and F. sulfooxidans
(4). Nutritional and taxonomic studies (5-16) have a l l indicated
that there are no major differences between these three b a c t e r i a ,
and thus should a l l be regarded as T. ferrooxidans. More recent-
l y , however, i t was demonstrated that the iron o x i d i z i n g bacteria
adapted to and grown on d i f f e r e n t substrates showed both q u a n t i -
t a t i v e and q u a l i t a t i v e differences in t h e i r a b i l i t i e s to o x i d i z e
metal s u l f i d e m i n e r a l s , and to use some of these o x i d a t i o n s f o r
the a s s i m i l a t i o n of C 0 2 (17), Furthermore, these bacteria when
grown on d i f f e r e n t substrates were found to contain deoxyribonu-
c l e i c acid (DNA) of d i f f e r e n t base compositions (Table 1) ( 1 8 , 1 9 ) .
The DNA of Escherichia coli and Rhodospirillum rubrum were i n c l u -
ded in t h i s study as standards of known guanine plus cytosine
(G + C) content. Thus, the question i s r a i s e d : i s the organism
c u r r e n t l y known as T. ferrooxidans one d i s t i n c t s p e c i e s , or a
s e r i e s of metabolically s i m i l a r b a c t e r i a ?

Adaptation to growth of T. ferrooxidans on organic compounds


have been reported (20,21) accompanied by p h y s i o l o g i c a l and mor-
phological a l t e r a t i o n s (22,23). Guay and S i l v e r (24) repeated
these procedures, and obtained a microorganism s i m i l a r i f not
identical to those already described. As reported by Shafia and
Wilkinson ( 2 0 ) , the organism l o s t i t s a b i l i t y to o x i d i z e or grow
on ferrous iron after a number of t r a n s f e r s on ferrous iron-free
glucose medium. A n a l y s i s and comparison of the DNA of both the
bacteria of the o r i g i n a l i r o n - o x i d i z i n g c u l t u r e and the new i s o l -
ate, renamed T. acidophilus , showed great d i f f e r e n c e s in the gua-
TABLE I DNA Base Composition of Iron-Oxidizing Bacteria and T h i o b a c i l l u s
acidophilus Grown on Different Substrates

Guanine plus cytosine content of DNA^ Methods:


Growth
Bacteria
Substrate Τ CsCl absorbance other
m
density ratio methods

T. ferrooxidans FeSOj 56. 5 a


57.l a
56. 2 a
5S.0 '
b d

4
T. ferrooxidans PbS 54. 5 a
54. 4 a
54. 2 a

T. ferrooxidans CuFeS 59. 9 a


60. 2 a
60.l a

T. acidophilus S° 62. 8 b
64. 3 b
62. 7 h
60.0 >
b d
5

T. acidophilus glucose 62. 8 b


66. 3 h
60. 5 h
62.5 '
h d

E. c o l i glucose 51.1° 50.4° 51. 5 a


51.8 '
h d

R. rubrum malate 62.8° 65.0° 6Z.5 >


h d

62.4 '
G e

a
data of Guay Silver and Torma (19).
3 , data of Guay and Silver (24).
c data of Silver 3 et al. (71). calculated by the normal probability method (of 24).
3

e calculated by the O.D. 260/0. D. 280 ratio method (cf 71).


6 Marvin Silver

nine plus cytosine content (24) (Table 1 ) . T. acidophilus i s


s i m i l a r to both the glucose-adapted s t r a i n of Shafia and Wilkinson
( 2 0 ) , and the glucose-grown organism of Tabita and Lundgren (21)
with regard to metabolic properties and enzyme content (24, D.P.
K e l l y , personal communication). I t may also be s i m i l a r to T. or-
ganoparus ( 2 5 ) . As not a l l cultures of T. ferrooxidans can be
adapted to growth on glucose (20, B.J. Ralph, personal communica-
t i o n ) , and those that are adaptable to growth on substrates other
than ferrous iron manifest fundamentally d i f f e r e n t properties of
taxonomic importance ( 1 7 , 1 9 , 2 4 , 2 6 ) , the hypothesis formulated
e a r l i e r (12) that cultures of i r o n - o x i d i z i n g autotrophs may be
o r i g i n a l l y heterogeneous i s supported.

II. OXIDATION OF FERROUS IRON

The oxidation of ferrous iron by T. ferrooxidans can be des-


cribed by the equation:

Fe 2 +
+ H +
+ i0 2 + Fe 3 +
+ iH 0?

f o r c a l c u l a t i o n s of the free energy made a v a i l a b l e to the bacte-


r i a between pH 1.5 and 3.0 ( 2 7 ) . The free energy of the reaction
at physiological concentrations (AG) was found to be dependent
upon the pH of the environment of the r e a c t i o n ; Tuovinen and K e l l y
(23) have calculated these values between pH 1.5 and 3.0 to be
from 7.8 to 5.9 kcal/mole Fe +
oxidized. Therefore, i n s u f f i c i e n t
energy would seem to be a v a i l a b l e f o r the phosphorylation of ADP
to form ATP, which requires between 8.9 and 14 k c a l . The p o s s i -
b i l i t y e x i s t s that ferrous iron might be complexed with an organic
molecule in order that the potential of the Fe / Fe + +
couple be
lowered from 0.77 v o l t to around 0 ( 2 8 ) . This has been confirmed
59
by the polarographic and Fe uptake studies of Dugan and Lundgren
(29). Then, assuming that electrons are required to be t r a n s f e r -
red to oxygen v i a the cytochrome transport chain in p a i r s , about
14 kcal/2 Fe +
oxidized should be a v a i l a b l e for ATP formation.
Basic Microbial Studies Applied to Leaching 7

Cytochromes of the types a and Q have been detected in i r o n -


o x i d i z i n g bacteria ( 3 0 , 3 1 ) . Coenzyme Q has been detected by Du-
gan and Lundgren ( 3 2 ) . Results of these studies indicate that the
electron transport scheme for the o x i d a t i o n of ferrous iron in
these bacteria i s as f o l l o w s :

Fe -cytochrome c
2 +

oxidoreductase ADP + P.. + ATP

Fe cytochrome Q ( O X . ) ^ c y t o c h r o m e a ( r e d , ) ^ ^ + H +

Fe 3 + 1
^ cytochrome β (red. ^ c y t o c h r o m e a ( o x , ) ^ > i H 0 2

with coenzyme Q p o s s i b l y acting as an intermediary e l e c t r o n c a r ­


r i e r between a ferrous i r o n - s u l f a t e - o r g a n i c complex associated
with the c e l l u l a r envelope. That s u l f a t e i s required f o r the ox­
idation of ferrous iron i s supported by the f i n d i n g s of Lazaroff
(33) and Lees, Kwok and Suzuki ( 2 7 ) .

Iron-cytochrome <?-oxidoreductase has been p u r i f i e d and cha­


racterized by Yates and Nason (34) and D i n , Suzuki and Lees ( 3 1 ) .
The mechanism of the enzyme reaction has been shown by Din and
Suzuki (35) to be of the type known as Ping Pong Bi Bi - - that i s ,
each substrate i s bound and modified s e q u e n t i a l l y by the enzyme
with a corresponding change in the enzyme. This i s shown d i a -
gramatically below:

Fe 2 +
Fe 3 +

4- t
E ( F e ) -> E ( F e ) F e
3 + 3 + 2 +
- E(Fe )Fe 2 + 3 +
+ E(Fe ) 2 +
-

cyt. β ( F e ) 3 +
cyt. ο ( F e ) 2 +

+- +

ψ^) . ^ e 3 +
) . E ( F e 3 + )

cyt. ο ( F e ) 3 +
cyt. c ( F e ) 2 +

The enzyme containing one atom of f e r r i c iron binds one atom of


ferrous i r o n , which reduces the enzyme bound iron and i s then r e l ­
eased. Then one molecule of oxidized cytochrome β i s bound by the
enzyme, whose ferrous iron reduces the f e r r i c iron of the cyto-
8 Marvin Silver

chrome a. The iron of the enzyme i s thus o x i d i z e d , and the r e d ­


uced cytochrome β i s then released. Although t h i s mechanism i s
known to work on s o l u b l e ferrous s a l t s , there i s no reason to a s ­
sume that i t does not occur regardless of the form of i r o n , that
i s , iron in i n s o l u b l e s u l f i d e , oxide or carbonate m i n e r a l s , as
long as the iron i s in the 2 +
valency s t a t e .

III. OXIDATION OF ELEMENTAL SULFUR AND REDUCED SULFUR COMPOUNDS

By d e f i n i t i o n , for a bacterial species to be a member of the


genus Thiobaoillus , i t must be demonstrated to be able to use the
oxidation of inorganic s u l f u r compounds f o r i t s energy r e q u i r e ­
ments. A l l s t r a i n s of T. ferrooxidans do t h i s , although with
q u a n t i t a t i v e d i f f e r e n c e s noted with d i f f e r e n t s t r a i n s ( 8 , 1 6 ) , or
with the same s t r a i n grown on d i f f e r e n t substrates ( 1 2 ) .

Most of the research on the o x i d a t i o n of elemental s u l f u r and


reduced compounds of s u l f u r have been c a r r i e d out with t h i o b a c i l l i
other than T. fevvooxidans . However, i t i s not unreasonable to
assume that r e s u l t s obtained from the other t h i o b a c i l l i apply at
l e a s t i n part to the i r o n - o x i d i z i n g members of t h i s genus.

A generalized scheme f o r the o x i d a t i o n of elemental sulfur


and reduced s u l f u r compounds by the t h i o b a c i l l i i s shown in F i g .
1. The oxygenation of elemental s u l f u r with the formation of
s u l f i t e i s catalyzed by the s u l f u r - o x i d i z i n g system. Sulfide is
oxidized by a p a r t i c u l a t e system to the oxidation level of e l e ­
mental s u l f u r , which then might be oxygenated, p o s s i b l y by the
s u l f u r - o x i d i z i n g enzyme, to s u l f i t e . T h i o s u l f a t e can e i t h e r be
s p l i t with the formation of s u l f i t e and s u l f i d e , or i t may be
oxidized to t e t r a t h i o n a t e , which then y i e l d s s u l f i t e and t r i t h i o -
nate, which in turn i s converted to s u l f i t e and t h i o s u l f a t e . Sul­
f i t e can then be oxidized to s u l f a t e e i t h e r by a cytochrome-c?-
mediated o x i d a t i o n , or via adenosine phosphosulfate.
9

Fig. 1. Generalized scheme for the oxidation of elemental sulfur and reduced sulfur corn-pounds
by the thiobacilli.
10 Marvin Silver

The reactions involved in the oxidation of elemental sulfur


are:

S 8 + GSH + GSgSH
s u l f u r o x i d i z i n g enzyme
GSgSH + 0 2
GS S0 H
g 2

GS S0 H + H 0
8 2 2
GS SH + H S0
7 o

Elemental s u l f u r , which e x i s t s most commonly in the form of a c i r -


cular eight atom molecule i s attacked by a s u l f h y d r y l containing
agent r e s u l t i n g in an organic p o l y s u l f i d e . The s u l f u r o x i d i z i n g
enzyme catalyzes the oxidation of the terminal atom which i s then
removed h y d r o l y t i c a l l y as s u l f i t e . The organic p o l y s u l f i d e , minus
one molecule of s u l f u r i s then able to be acted upon again by the
s u l f u r o x i d i z i n g enzyme, in vitro, the s u l f i t e i s acted upon by
the elemental s u l f u r with the formation of t h i o s u l f a t e ; whether
t h i s reaction i s important in vivo i s not known. In the presence
of a s u i t a b l e s u l f i t e trapping reagent, such as formaldehyde ( 3 6 ,
3 7 ) , or an active s u l f i t e - o x i d i z i n g system ( 3 8 ) , s u l f i t e has been
shown to be the true end product of t h i s r e a c t i o n . Besides T.
ferrooxidans ( 3 7 ) , t h i s enzyme has been p a r t i a l l y p u r i f i e d and
characterized from T. thiooxidans ( 3 9 ) , and T. thioparus (36) and
i t s presence has been confirmed in T. novellus (40) and T. neapo-
litanus ( 4 1 ) . Takakuwa (42) has resolved the s u l f u r - o x i d i z i n g
enzyme of T. thiooxidans into two protein components, one of mo-
l e c u l a r weight of 120,000 and the other of molecular weight of
23,000 d a l t o n s .

The mechanism of the entry of elemental s u l f u r into the cell


has not as yet been s a t i s f a c t o r i l y explained. I t i s known that
when grown on elemental s u l f u r , the ends of the c e l l s of T. fer-
rooxidans are more pointed in form than when grown on ferrous
iron ( 2 2 ) , and small evaginations appear on the external membrane
which, when stained with s i l v e r i o n , show the presence of s u l f h y -
dryl groups ( 4 3 ) .
Basic Microbial Studies Applied to Leaching 11

The i n i t i a l reactions of the o x i d a t i o n of s u l f i d e have been


investigated by Moriarty and Nicholas (44,46) in T. ooncretivorus,
T. thiooxidans and T. thioporus and by Aminuddin and Nicholas 9

(47) in T. denitvificons. They demonstrated a cell-membrane a s ­


sociated enzymic process which oxidized s u l f i d e to the o x i d a t i o n
level of elemental s u l f u r . A c t u a l l y , a l i n e a r polymeric chain of
s u l f u r atoms bound to a l i p o p r o t e i n membrane f r a c t i o n was formed,
p o s s i b l y dependent on more than one enzyme; t h i s process was
mediated by a copper-containing p r o t e i n , cytochromes and u b i q u i ­
none and was dependent upon oxygen in T. concvetivorus, T. thio-
parus and T. thiooxidans and upon n i t r i t e in T. denitrificons.

^ s s u l f i d e oxidase <^ p o l y s u l f i d e oxidase ^ ^


^ ψ ^ ψ ^
(Cu) (flavin?)
ψ ψ
(flavin?) ubiquinone
ψ ψ
cyt, b cyt. b
ψ ψ
cyt. ο cyt. c

{ c^y t· . d
« τγ c y t . α·\

N0 2 + N 2 + NO + N0
2 0 2 + H0
2 ( N O 3 ? ) ( ? )

Two d i s t i n c l y d i f f e r e n t mechanisms, which may not be mutual­


l y e x c l u s i v e have been proposed for the oxidation of t h i o s u l f a t e
based p r i m a r i l y on the existance of two enzymes, rhodanese and
the t h i o s u l f a t e o x i d i z i n g enzyme. Rhodanese has been p a r t i a l l y
p u r i f i e d from T. denitrificons ( 4 8 ) , T. ferrooxidons (49) and
Thiobacillus A2 ( 5 0 ) , and has been shown to e x i s t in T. novellus
(40) and T. neopolitonus ( 5 1 ) . T h i s enzyme has been shown to
catalyze the s c i s s i o n of t h i o s u l f a t e to s u l f i t e and a s u l f i d e
moiety bound to e i t h e r cyanide, d i h y d r o l i p o a t e or dihydrolipoam-
ide ( 5 0 , 5 2 ) .
12 Marvin Silver

S - S 0 ~ + SCN"
3
2
-> S 0 ' + SCN" 3
2

ο 2
S-S0 + dihydrolipoate + S 0 " + dihydrolipoate
3 3 persulfide
2
S-S0 3 " + dihydrol ipoamide ->
2
S0 3 " + dihydrolipoamide p e r s u l f i d e
An enzyme with s i m i l a r f u n c t i o n , t h i o s u l f a t e reductase using
reduced g l u t a t h i o n e , has been isolated from T. thioparus ( 5 3 ) ,
and catalyzes the following r e a c t i o n :
S - S 0 " + 2GSH3
2
+ S 0 " + H S + GSSG
3
2
2

The t h i o s u l f a t e - o x i d i z i n g enzyme, which catalyzes the o x i d a ­


t i v e condensation of two molecules of t h i o s u l f a t e to form t e t r a -
thionate:

2 H
2 2°3
S H
2 4°6
S + 2 H +

has been p a r t i a l l y p u r i f i e d and characterized from T. neapolita-


nus ( 5 4 ) , T. novellus ( 5 5 ) , T. ferrooxidans (13) and T. thioparus
(56). Ferricyanide and cytochrome ο have been found to act as
electron acceptors in t h i s r e a c t i o n . Based on the detection of
polythionates in the medium of t h i o b a c i l l i growing on t h i o s u l f a t e
( 5 7 ) , tetrathionate has been postulated to be metabolized as f o l ­
lows ( 5 8 , 1 3 ) :

s
4°6 ' 2
^ 3°6 "
s 2 s
2° 3
2
"
S 0
3 S 0
3

S u l f i t e i s the central intermediate in s u l f u r o x i d a t i o n ; it


i s through t h i s compound that a l l pathways pass in the formation
of s u l f a t e and ATP. There are two p r i n c i p a l methods f o r the ox­
idation of s u l f i t e in the t h i o b a c i l l i ; v i a adenosine phosphosul-
fate (57) or by a cytochrome e-mediated oxidation ( 3 8 ) . The
f i r s t of these pathways:
Basic Microbial Studies Applied to Leaching 13

APS reductase
APS
ADP s u l f u r y l a s e
APS S 0 ^ + ADP
4
2

adenylate_kinase
ADP - iAMP + iATP

contains three r e a c t i o n s : these are catalyzed by APS reductase,


causing the formation of APS from AMP and s u l f i t e , ADP s u l f u r y ­
l a s e , which replaces the s u l f a t e moiety by a phosphoric acid
group r e s u l t i n g in the formation o f ADP and the l i b e r a t i o n of s u l ­
f a t e , and adenylate k i n a s e , which t r a n s f e r s a phosphoric acid
group from one ADP molecule to another, r e s u l t i n g in the forma­
t i o n of one ATP and one AMP. That t h i s pathway i s important in
at l e a s t some of the t h i o b a c i l l i i s emphasized by the f a c t that
in T. thioparus, APS reductase comprises 3 to 4% of the c e l l u l a r
protein ( 5 9 ) , and 4 to 5% in T. denitrifioans ( 6 0 ) . I t i s only
from these two t h i o b a c i l l i that t h i s enzyme has been p u r i f i e d ,
although i t has been reported to e x i s t in T. thiooxidans ( 6 1 ) , and
T. denitrifioans ( 4 7 ) .

A cytochrome-mediated AMP-independent s u l f i t e o x i d i z i n g s y s ­
tem,
2-
S0~ + cytochrome ο
oxidoreductase ADP + Ρ · -> ATP η

2_
S0^ ^/cytochrome β (ox.) {^cytochrome a ( r e d J ^ O ^
S O ^ " " cytochrome β (red. ^ c y t o c h r o m e a ( o x . J ^ ^ i ^ O
2

has been studied in T. novellus ( 3 8 ) , T. thioparus ( 6 2 ) , T. thio­


oxidans ( 6 3 ) , T. ferrooxidans ( 6 4 ) , and Thiobaoillus A2 ( S i l v e r ,
unpublished o b s e r v a t i o n s ) . A s u l f i t e o x i d i z i n g enzyme has been
i s o l a t e d from T. neapolitanus in which cytochromes are not i n v o l ­
ved in the oxidation ( 6 5 ) ; s u l f i t e i s oxidized d i r e c t l y by oxy­
gen. Although t h i s o x i d a t i o n i s not dependent on AMP, i t is
stimulated by t h i s compound, suggesting the formation of an e n -
zyme-sulfite intermediate which might react with AMP to form APS or
with water to form s u l f i t e .
14 Marvin Silver

IV. FIXATION OF CARBON DIOXIDE


As with other autotrophic bacteria ( 6 6 ) , the i r o n - o x i d i z i n g
t h i o b a c i l l i use the reductive pentose phosphate (Calvin-Benson)
cycle and the carboxylation of phospho-enol-pyruvate for the a s ­
s i m i l a t i o n of C 0 2 (67,68) with ferrous iron serving as the sole
energy source. Elemental s u l f u r , reduced inorganic s u l f u r com­
pounds and metal s u l f i d e minerals (12,14,17) could a l s o support
C0 2 fixation.

Each molecule of C 0 2 converted to organic carbon by the C a l ­


vin-Benson cycle requires three molecules of ATP and two mole­
cules of reduced pyridine nucleotide. The ATP i s derived d i r e c t l y
from the oxidation of the growth s u b s t r a t e s , whereas pyridine
nucleotides are reduced by the reversal of the electron transport
chain. That s u l f u r compounds and ferrous iron can be used for
t h i s process i s documented by Aleem (69) and Aleem, Lees and
Nicholas (70) r e s p e c t i v e l y .

V. REFERENCES

1. Nathansohn h.,Mitt. Zool. Sta. Neapel., 15, 655 (1902).


2. Temple, K.L., and Colmer, A . R . , J. Baoteriol., 62, 605 (1951).
3. Leathen, W.W., K i n s e l , N.A., and B r a l e y , S.A., J. Bacteriol.,
72, 700 (1956).

4. K i n s e l , N.A., J. Baoteriol., 80, 628 (1960).

5. Silverman, M.P., and Lundgren, D.G., J. Bacteriol., 77, 642


(1959).
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(1959).
7. Beck, J . V . , J. Baoteriol., 79, 502 (1960).
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9. Ivanov, V . I . , and L y a l i k o v a , N.N., Mikrobiologiya, 3 1 , 468
(1962).
10. Hutchinson, M., Johnstone, K . I . , and White, D., J. Gen.
Microbiol., 44, 373 (1966).
11. Hutchinson, Μ., Johnstone, K . I . , and White, D., J. Gen.
Microbiol., 57, 397 (1969).
Basic Microbial Studies Applied to Leaching 15

12. M a r g a l i t h , P., S i l v e r , M., and Lundgren, D.G., J. Bacteriol.,


92, 1706 (1966).
13. S i l v e r , Μ., and Lundgren, D.G., Can. J. Biochem., 46, 1215
(1968).
14. S i l v e r , Μ., Can. J. Microbiol., 16, 845 (1970).
15. K e l l y , D.P., and Tuovinen, O.H., Int. J. Syst. Bacterid.,
22, 170 (1972).
16. Bounds, H . C , and Colmer, A . R . , Can. J. Microbiol., 18, 735
(1972).
17. S i l v e r , Μ., and Torma, A . E . , Can. J. Microbiol., 20, 141
(1974).
18. Guay, R., S i l v e r , Μ., and Torma, A . E . , IRCS Med. Sci.:
Biochem. Microbiol. Parasitol. Inf. Dis., 3, 417 (1975).
19. Guay, R., S i l v e r , M., and Torma, A . E . , Rev. Can. Biol., 35,
61 (1976).
20. S h a f i a , F., and W i l k i n s o n , R.F., J. Bacterid., 97, 256
(1969).
21. T a b i t a , F.R., and Lundgren, D.G., J. Bacterid., 108, 328
(1970).
22. Lundgren, D.G., V e s t a l , J . R . , and T a b i t a , F.R., in "Water
P o l l u t i o n M i c r o b i o l o g y " , (R. M i t c h e l l , Ed.) p. 69. Wiley-
I n t e r s c i e n c e , New York, 1972.
23. Tuovinen, O.H., and K e l l y , D.P., Z. Allg. Mikrobiol., 12,
311 (1972).
24. Guay, R., and S i l v e r , M., Can. J. Microbiol., 2 1 , 281 (1975).
25. Markosyan, G.E., Dokl. Akad. Nayuk SSSR, 2 1 1 , 1205 (1973).
26. Tuovinen, O.H., K e l l e y , B.C., and N i c h o l a s , D.J.D., Can. J.
J. Microbiol., 22, 109 (1976).
27. Lees, H., Kwok, S . C . , and S u z u k i , I . , Can. J. Microbiol.,
15, 43 (1969).
28. B l a y l o c k , B.A., and Nason, Α . , J. Biol. Chem., 238 3453
(1963).
29. Dugan, P.R., and Lundgren, D.G., j. Bacteriol., 89, 825
(1965).
30. Vernon, L.P., Mangum, J . H . , Beck, J . V . , and S h a f i a , F.M.,
Arch. Biochem. Biophys., 88, 227 ( I 9 6 0 ) .
31. D i n , G.A., S u z u k i , I . , and Lees, H., Can. J. Biochem., 45
1523 (1967).
32. Dugan, P.R., and Lundgren, D.G., Anal. Biochem., 8, 312
(1964).
16 Marvin Silver

33. L a z a r o f f , N., J. Bacteriol., 8 5 , 78 (1963).


34. Yates, M.G., and Nason, Α . , J. Biol. Chem., 2 4 1 , 4872 (1966).
35. D i n , G.A., and S u z u k i , I . , Can. J. Biochem., 4 5 , 1547 (1967).
36. S u z u k i , I . , and S i l v e r , Μ., Biochem. Biophys. Acta, 122, 22
(1966).
37. S i l v e r , M., and Lundgren, D.G., Can. J. Biochem., 46, 457
(1968).
38. Charles, A.M., and S u z u k i , I . , Biochem. Biophys. Acta, 128,
522 (1966).
39. S u z u k i , I . , Biochim. Biophys. Acta, 104, 359 (1965).
40. Charles, A.M., and S u z u k i , I . , Biochim. Biophys. Acta, 128,
510 (1966).
41. T a y l o r , B.F., Biochim. Biophys. Acta, 170, 112 (1968).
42. Takakuwa, S . , Plant and Cell Physiol., 16, 1027 (1975).
43. Karavaiko, G . I . , L y a l i k o v a , N.N., and Pivovarova, in " E c o l ­
ogical and Geochemical A c t i v i t y of Microorganisms", (M.V.
Ivanov, Ed.) p. 25. Science Centre of the B i o l o g i c a l I n s t i ­
t u t e , USSR, 1976.
44. M o r i a r t y , D.J.W., and N i c h o l a s , D.J.D., Biochem. Biophys.
Acta, 184, 114(1969).
45. M o r i a r t y , D.J.W., and N i c h o l a s , D.J.D., Biochim. Biophys.
Acta, 197, 143 (1970).
46. M o r i a r t y , D.J.W., and N i c h o l a s , D.J.D., Biochim. Biophys.
Acta, 216, 130 (1970).
47. Aminuddin, Μ., and N i c h o l a s , D.J.D., Biochim. Biophys. Acta,
325, 81 (1973).
48. Bowen, T . J . , B u t l e r , P . J . , and Happold, F.C., Biochem. J.,
97, 651 (1965).
49. T a b i t a , R., S i l v e r , M., and Lundgren, D.G., Can. J. Biochem.,
47, 1141 (1969).
50. S i l v e r , Μ., and K e l l y , D.P., J. Gen. Microbiol., 97, 277
(1976).
51. K e l l y , D.P., and Tuovinen, O.H., Plant and Soil, 4 3 , 77
(1975).
52. S i l v e r , M., Howarth, O.W., and K e l l y , D.P., J. Gen. Micro­
biol., 97, 285 (1976).
53. Peck, H.D., and F i s h e r , E., J. Biol. Chem., 237, 190 (1962).
54. Trudinger, P.Α., Biochem. J., 78, 680 (1961).
55. Aleem, M . I . H . , J. Bacteriol,, 90, 95 (1965).
Basic Microbial Studies Applied to Leaching 17

56. L y r i c , R.M., and S u z u k i , I . , Can. J. Biochem., 48, 355


(1970).
57. Roy, A . B . , and Trudinger, P.Α., "The Biochemistry of I n o r ­
ganic Compounds of S u l f u r " , p. 207. Cambridge U n i v e r s i t y
P r e s s , London, 1970.
58. S i n h a , D.B., and Walden, C.C., Can. J. Microbil., 12, 1041
(1966).
59. L y r i c , R.M., and S u z u k i , I . , Can. J. Biochem., 48, 344
(1970).
60. Bowen, T . J . , Happold, F.C., and Taylor, B.F., Bioohim.
Biophys. Acta, 118, 566 (1966).
61. Peck, H.D., J. Bacteriol., 8 2 , 933 (1961).
62. L y r i c , R.M., and S u z u k i , I., Can. J. Biochem., 4 8 , 334 (1970).
63. Kodama, Α . , Kodama, Τ . , and M o r i , T . , Plant and Cell Physiol.
1 1 , 701 (1970).
64. V e s t a l , J . R . , and Lundgren, D.G., Can. J. Biochem., 49,
1125 (1971).
65. Hempfling, W.P., Trudinger, P.Α., and V i s h n i a c , W., Arch.
Mikrobiol., 59, 149 (1967).
66. K e l l y , D.P., Ann. Rev. Microbiol., 25, 177 (1971).
67. Maciag, W.J., and Lundgren, D.G., Biochem. Biophys. Res.
Commun., 17, 603 (1964).
68. D i n , G.A., S u z u k i , I . , and Lees, H., Can. J. Microbiol.,
13, 1413 (1967).
69. Aleem, M . I . H . , Symp. Soc. Gen. Microgiol., 27, 351 (1977).
70. Aleem, M . I . H . , Lees, H., and N i c h o l a s , D.J.D., Nature, 200
759 (1963).
71. S i l v e r , Μ., Friedman, S . , Guay, R., Couture, J . , and Tanguay,
R., J. Bacteriol., 107, 368 (1971).
FACTORS AFFECTING METABOLISM AND FERROUS IRON

OXIDATION IN SUSPENSIONS AND BATCH CULTURES OF

THIOBACILLUS FERROOXIDANS: RELEVANCE TO FERRIC IRON

LEACH SOLUTION REGENERATION

D. P. Kelly

U n i v e r s i t y of Warwick, Coventry, England

C. A. Jones

Glaxo Research L t d . , Stoke Poges, Buck, England

Ferric iron in acid solution is the essential reagent for


the bacterially-assisted solubilization of uranium from ores such
as uraninite, and is also significant in the dissolution of metal
sulphides. The regeneration of ferric iron by bacterial reoxid-
ation of ferrous sulphate is an essential step in the recycling
of leach liquors in heap or other percolation leach systems.
Consequently it is of use to know the factors likely to affect
bacterial development and activity. The principal factor limiting
growth in batch culture of T h i o b a c i l l u s ferrooxidans on ferrous
iron is ferric iron product inhibition of the rate of ferrous iron
oxidation. Ferric iron inhibits oxidation competitively, lowering
substrate saturation coefficient (K ) without affecting maximum
s

attainable specific growth rate (\i ). In normal batch culture,


max
W a s
Vmnr. °* l^ZhT , minimum observed K was 36mM ferrous sulphate,
1
ff

19
20 D. P. Kelly and C. A. Jones

and growth yield coefficient was 0. 35g dry wt/g atom iron oxidized.
Growth rates were exponential, measured as iron oxidation and
fixation of labelled carbon dioxide. Growth rate was not limited
by carbon dioxide concentration, but exhaustion of carbon dioxide
in sealed systems caused a switch from exponential to linear iron
oxidation, indicating growth-uncoupled oxidation of the residual
iron by the static population. High potassium concentrations
partially relieved ferric iron inhibition. With non-growing cell
suspensions, oxygen electrode methods indicated multiple K values m

for ferrous iron oxidation: 0.7mM FeSO^ between 0.4-4ZmM;


20-12ZmM between 100-400mU; and unmeasurably high between 400-
700mM. Above Ο. 7M ferrous sulphate became an inhibitory substrate.
Increased Η or uranyl sulphate inhibited non-competitively.
Efficiency of coupling of carbon dioxide fixation to Fe oxidation
was decreased at low (25mM) and high (SOOmM) ferrous sulphate
concentrations and was maximal in the 100-ZOOmM range. Ferric
sulphate did not inhibit carbon dioxide fixation and so affected
metabolism and growth only by its effect on ferrous iron oxidation.

I. INTRODUCTION

Thiobacillus ferrooxidans i s one of the most important


organisms i n the leaching of metal sulphides ( 1 , 2 ) , although
recent work indicates that i t i s not uniquely responsible for t h i s
phenomenon and i t s a c t i v i t y may be enhanced by the presence of
other organisms ( 3 - 5 ; N o r r i s and K e l l y , t h i s volume; Tuovinen,
e t a l . , t h i s volume). In many leaching systems, the oxidation of
p y r i t e or of ferrous i r o n plays a central r o l e , i n which f e r r i c
i r o n o x i d i z e s and consequently s o l u b i l i z e s other metals, such as
uranium from i t s oxides ( 2 , 6 - 1 0 ) . The regeneration of soluble
f e r r i c i r o n from ferrous sulphate i s a l s o important i n any
economic process with l i q u o r r e c y c l i n g ( 1 , 1 1 , 1 2 ) . Consequently
i t i s of some importance to understand the physiology of
T. ferrooxidans and in p a r t i c u l a r to be able to define the
factors l i m i t i n g the rate and extent of i t s development. Among
f a c t o r s l i m i t i n g development could be nutrient supply (especially
carbon d i o x i d e , which i s the organism's main carbon s o u r c e ) ,
a v a i l a b i l i t y of oxygen and oxidizable i r o n , p o s s i b l e t o x i c i t y of
d i s s o l v e d metals and the innate capacities of the bacteria
( i . e . maximum p o s s i b l e growth and oxidation rates and biomass
Basic Microbial Studies Applied to Leaching 21

yields). In t h i s paper we are concerned with the l i m i t a t i o n s


placed on growth and i r o n oxidation by the a v a i l a b i l i t y of carbon
dioxide and the concentrations of ferrous and f e r r i c ions to which
the bacteria are exposed i n batch culture or non-growing
suspensions.

P r e v i o u s l y (13) we have demonstrated that Thiobacillus


ferrooxidans i n continuous chemostat culture i s capable of
s p e c i f i c growth rates (μ) on ferrous i r o n at pH 1.6 of at l e a s t
μ = 1.33 h"^ with a p o s s i b l e y m = 1.78 h""'. In chemostat
culture i t s true substrate s a t u r a t i o n constant (K ) f o r FeSCL
s' x
4
was as low as 0.7mM, and i t s true growth y i e l d was 1.33g dry wt/g
2+
atom Fe oxidized ( 1 3 , 1 4 ) . Such f i g u r e s are i n marked contrast
to p r e v i o u s l y published batch culture values f o r μ of 0.145
and 0.2 at pH values above pH 1.9 or μ of 0.109 at pH 1.3, and
to K $ values between 7.2 and 35.9mM ( 1 5 - 1 7 ) . Apparent M i c h a e l i s
constants ( K ) f o r FeSO^ o x i d a t i o n by c e l l suspensions are
m

reported to l i e i n the range 0.568-9.4mM ( 1 8 - 2 2 ) ; the K m for


a p a r t i a l l y separated i r o n oxidase was 0.59mM ( 2 3 ) . Growth y i e l d s
(Y) i n batch culture are a l s o below those p o s s i b l e in the chemostat
2+
and f a l l i n the range 0.2-0.5g dry wt/g atom Fe oxidized. The
most r e l i a b l e f i g u r e s a v a i l a b l e to date f o r batch culture Y seem
to be 0.35 and 0.392 (17; A. C a g l a r , personal communications,
1974). L i m i t a t i o n of growth rate or e f f i c i e n c y have i n the past
v a r i o u s l y been attributed to l i m i t a t i o n of a v a i l a b l e oxygen or
carbon d i o x i d e : a view we contend i s not u n i v e r s a l l y tenable.
Our chemostat data i n d i c a t e the usual values for batch cultures to
be gross underestimates of the maximum potential of
T. ferrooxidans» but also showed growth-linked i r o n oxidation to
be subject i n d i f f e r e n t steady state experiments to competitive or
non-competitive i n h i b i t i o n by the f e r r i c i r o n produced by the
chemostat culture ( 1 3 ) . The former state was associated with a
low y i e l d of 0.36-0.38 ( 1 3 , 2 4 ) , p o s s i b l y i n d i c a t i n g a
p h y s i o l o g i c a l s i m i l a r i t y between batch cultures and chemostat
22 D. P. Kelly and C. A. Jones

steady states subject to predominantly competitive product


inhibition. A consequence of competitive i n h i b i t i o n of iron
oxidation by f e r r i c ions would be an apparently poor a f f i n i t y for
ferrous i r o n , reflected as a low growth rate and high e f f e c t i v e
Κ .
s

These experiments provide data for an i n t e r p r e t a t i o n of the


r e l a t i v e l y poor values commonly found for μ , K $ and Y; and s e t
out to e s t a b l i s h (a) whether competitive i n h i b i t i o n by f e r r i c
i r o n i s found during iron oxidation by batch cultures and s u s ­
pensions; (b) whether K § values deduced from the chemostat are
v a l i d for suspensions o x i d i z i n g i r o n i n the v i r t u a l absence of
f e r r i c ion product; (c) whether ferrous iron at high concen­
t r a t i o n s i s i n h i b i t o r y to i t s own o x i d a t i o n ; (d) whether f e r r i c
ions or high l e v e l s of ferrous ions can uncouple carbon dioxide
f i x a t i o n from i r o n o x i d a t i o n .

II. EXPERIMENTAL METHODS

A. Organism, Cultural Conditions and A n a l y t i c a l Procedures

The s t r a i n of Thiobaeillus ferrooxidans used and culture


methods are described in d e t a i l elsewhere (17, 2 4 ) . All cultures
were grown at 30°C i n media adjusted to pH 1.6 with H S 0 . 2 4 For
use i n manometry or oxygen electrode c e l l experiments, organisms
were harvested from chemostat cultures or from 1 or 2% of batch
cultures grown i n U amounts shaken i n 2£ Erlenmeyer f l a s k s until
near the end of the growth phase. Batch culture medium contained
(g/A): K H P 0 , 0.4;
2 4 ( N H ) S 0 , 0.4; M g S 0 . 7 H 0 , 0 . 4 ; F e S 0 ,
4 2 4 4 2 4

27.8 or as i n d i c a t e d . For chemostat c u l t u r e , and batch


experiments i n which the e f f e c t of potassium on i r o n oxidation was
t e s t e d , the medium contained (g/l): K H P 0 , 0.054; 2 4 (NH ) S0 ,
4 2 4

0.36; M g S 0 . 7 H 0 , 0.015; and FeS0zp7H 0 as r e q u i r e d .


4 2 2 Harvested
organisms were washed with water at pH 1.6 and suspended i n
pH 1.6 water or chemostat medium lacking i r o n . Bacterial protein
Basic Microbial Studies Applied to Leaching 23

i n suspensions or cultures was determined after collecting


organisms on membrane f i l t e r s and d i s s o l v i n g them i n 0.5N NaOH
(24). Ferrous i r o n oxidation in cultures was monitored by
t i t r a t i o n with eerie sulphate.

1. Fixation of ^C-Carbon Dioxide

a. Growing cultures. Culture medium (50 ml) i n 250 ml


14
f l a s k s sealed with Suba-seals received NaH CO^ or C 0 2 by
i n j e c t i o n and was shaken at 30°C for one hour before i n j e c t i n g
T. ferrooxidans. Duplicate samples (1 ml) were taken with s t e r i l e
syringes f o r i r o n e s t i m a t i o n ; f u r t h e r samples were f i l t e r e d
through 25 mm M i l l i p o r e membranes (0.45 ym pore s i z e ) , washed
with 10 ml 0.01 Ν H S 0 2 4 and 2 χ 10 ml d i s t i l l e d water, then dried
i n 20 ml s c i n t i l l a t i o n v i a l s over s i l i c a g e l .
b. Suspensions. Double-armed Warburg f l a s k s (25 ml) were
used with 2.0-3.5 ml l i q u i d volumes at pH 1.6. NaH C014
3 (30 mM,
10yc/ml) was tipped i n t o the main chamber, which contained FeSO^
(and f e r r i c sulphate i n some cases) i n pH 1.6 H S 0 , and C 0 2 4 2

release measured for 10-15 min before t i p p i n g a suspension of


T. ferrooxidans from the second side bulb. After measurement of
14
oxygen consumption, samples (1 ml) were removed to measure C
f i x e d i n t o f i l t e r e d bacteria as described above. To measure total
1 4
C0 2 f i x e d , samples (1 ml) were mixed with 1 ml 10% acetic acid
i n ethanol. Samples of t h i s were dried on Whatman GF/A f i b r e
g l a s s d i s c s i n s c i n t i l l a t i o n v i a l s or the whole samples were
14
d i l u t e d to 10 ml with water and 1 ml a l i q u o t s counted f o r C.
a. Measurement of C. 14
Membrane f i l t e r s and g l a s s f i b r e
d i s c s were immersed i n 5 ml 0.5% (v/v) butyl-PBD i n toluene.
Aqueous samples (1 ml) were mixed with 15 ml o f a mixture of 8 g
butyl-PBD, 500 ml T r i t o n X-100 and 1000 ml toluene. V i a l s were
counted i n a P h i l i p s Liquid S c i n t i l l a t i o n Analyzer programmed to
14
compute absolute dpm rates f o r C using predetermined channels
r a t i o equations.
24 D. P. Kelly and C. A. Jones

2. Removal of Carbon Dioxide from the Medium in CO^-free n fT

Growth Experiments

Mineral s a l t s medium (minus FeSO^) with a s u r p l u s of 10 ml


water was boiled i n the culture f l a s k s u n t i l the volume was
reduced by 10 ml. FeSO^ s o l u t i o n , s i m i l a r l y b o i l e d , was added
hot to the s a l t s s o l u t i o n and the f l a s k s sealed with rubber
stoppers having i n l e t and o u t l e t tubes. The medium was allowed
to cool i n a stream of a i r freed of CO^ by passage through
2N NaOH and saturated Ba(0H)£. Outgoing a i r passed through a
f u r t h e r NaOH trap. The tubes were then sealed with vaccine caps.
Some cultures were a l s o grown i n sealed "Katz f l a s k s " (300 ml)
with centre w e l l s containing a r o l l of Whatman No. 40 f i l t e r
paper and 1 mi 40% (w/v) Κ0Η.

3. Measurement of FeSO^ Oxidation by Suspensions of T h i o b a c i l l u s


ferrooxidans

a. Standard Warburg manometry. F l a s k s (20 ml) normally


contained 2 ml l i q u i d at pH 1.6, 0 . 5 - 0 . 8 mg T. ferrooxidans
p r o t e i n , and FeSO^ (tipped from a side bulb) between 10-800 mM.
Flasks were a i r - f i l l e d and shaken at 30°C at 150 ± 10 s t r o k e s /
min.

b. Oxygen electrodes. Water-jacketed perspex c e l l s (Rank


B r o s , Bottisham, Cambs) with Clark oxygen electrodes were used
at 30°C with remote magnetic s t i r r i n g of 2 ml or 3 ml reaction
solutions. Oxygen consumption i n a i r - s a t u r a t e d s o l u t i o n pH 1.6
was recorded on chart recorders (Kipp and Zonen, Switzerland) and
the systems calibrated to measure d i s s o l v e d oxygen i n nmoles/ml.
Generally, T. ferrooxidans suspensions (25-300 yg protein i n
0.1-0.2 ml) were injected into appropriate pH 1.6 FeSO^ s o l u t i o n s .
No endogenous oxygen consumption by the organisms was detectable.
Oxygen consumption due to non b i o l o g i c a l autooxidation of ferrous
i r o n was only s i g n i f i c a n t (10-15% of the total rate) with very
high concentrations, and, where necessary, corrections f o r
Basic Microbial Studies Applied to Leaching 25

autooxidation were always made. For i n h i b i t i o n experiments,


organisms were injected into f e r r o u s - f e r r i c mixtures. Appropriate
control t e s t s showed that only b i o l o g i c a l reactions were recorded
i n these experiments, and there was no interference from chemical
i n t e r a c t i o n of the iron species or reaction with the oxygen
electrode system.

III. RESULTS AND DISCUSSION

A. Effect of Carbon Dioxide A v a i l a b i l i t y on the Growth of


Batch Cultures

Batch cultures i n unsealed f l a s k s effected exponential


oxidation of FeSO^ ( F i g . 1) with a s p e c i f i c growth rate (Up ) of e

0.11-0.12 h'\ Using sealed f l a s k s containing normal a i r ,


i n i t i a l l y exponential FeSO^ oxidation became l i n e a r after 20-30%
of the iron was consumed ( F i g . 1 ) . Removal of 00^ i n t o KOH i n
sealed f l a s k s containing CO^-free media resulted i n e n t i r e l y
l i n e a r oxidation k i n e t i c s , i n d i c a t i n g that no growth of organisms
was occurring ( F i g . 1 ) . Autooxidation i n s t e r i l e controls was

0 20 40 60 80 100 120 140


TIME (HOURS)

Fig. 1. Effect of CO^limitation on kinetics of FeS0 4

oxidation by T h i o b a c i l l u s Terrooxidans cultures (50 ml).


Curve 1: unsealed flask; Curve 2: sealed flask with 260 ml air;
Curve 3: sealed Katz flask, C0 removed by KOH. Initial FeSO^
2

concentration, 82-88mM.
26 D. P. Kelly and C. A. Jones

These r e s u l t s are consistent with exponential FeSO^


oxidation being a measure of increase i n bacterial biomass and
numbers, while l i n e a r oxidation i s due to growth-uncoupled-
oxidation by a standing population of b a c t e r i a . Our sealed f l a s k s
contained a free a i r space of 260 ml and 50 ml of medium. From
14
isotope d i l u t i o n data i n our C0 2 experiments we calculated the
a i r space to contain 0.036% (v/v) C 0 . 2 Consequently, while the
f l a s k s contained ample oxygen (about 52 ml) to allow complete
FeSO^ oxidation i n our experiments, they contained only enough
C0 2 (about 0.1 ml) to allow growth of about 120 yg dry wt of new
organisms.
The absolute c o r r e l a t i o n of exponential growth and C 0 ~ 2

f i x a t i o n was proved i n cultures i n sealed f l a s k s , supplemented


with ^ C 0 . 9 Exponential i r o n oxidation became l i n e a r after 30%
14
of the a v a i l a b l e FeSO. was o x i d i z e d . C 0 - f i x a t i o n was exactly
9

14
proportional to exponential i r o n oxidation and C 0 was a l l
2

consumed at exactly the same time as the abrupt change to l i n e a r


oxidation r a t e . This indicated that rate of growth and
e f f i c i e n c y of coupling of C 0 - f i x a t i o n to i r o n oxidation did not
2

decline u n t i l carbon dioxide was e s s e n t i a l l y exhausted.


Comparison was made of the exponential rate of i r o n
14
oxidation and C 0 - f i x a t i o n with l i m i t i n g and n o n - l i m i t i n g
2

s u p p l i e s of C 0 , and of growth y i e l d with excess C 0


2 2 available.
S p e c i f i c growth rates (measured as y F e or y C Q ) were e s s e n t i a l l y
identical (Table 1 ) ; exponential growth rate^was e s s e n t i a l l y
14
unaffected by C 0 2 concentration and C 0 - f i x a t i o n was
2

proportional to i r o n oxidation at a l l concentrations. Viable


numbers of organisms produced were governed s o l e l y by the
quantity of iron a v a i l a b l e i n C 0 - u n l i m i t e d cultures and by the
2

amount of C 0 2 available in C0 -limited cultures.


2 The growth
y i e l d c o e f f i c i e n t was independent of absolute C 0 2 concentration
over the range 0.036% to 8% v/v (Table 2 ) .
Basic Microbial Studies Applied to Leaching 27

TABLE I Effect of Carbon Dioxide Supply on Growth Rates


of Batch Cultures on 84mM FeSO in Sealed and Unsealed Flasks. d

„ „ „ „ Increase in
a^TJl^
Λ Λ

U
™2 Viable count at 100%
a%r space ana ύ
_ , ., ..
nn J^IJ^J ι ι FeSO. oxidation,
nr

C0 2 supped (h-l } (h-l } ( $% b f o m t P o t )

Experiment 1
Unsealed (control) 0.110 - 100
260 ml + KOH Linear - 1
260 ml 0.107 - 33°
470 ml 0.107 - 72 d

Experiment 2
260 ml + 8 \il 00 14
£ 0.071 0.069 32
260 ml +0.5 ml C0 14
0 0.067 0.067 92
14
260 ml + 1.5 ml CO 2 0.069 0.069 100
Unsealed (control) - - 100

Fe and 00^ are specific growth rates calculated from


v μ

exponential rates of FeS0 oxidation and CO2 fixation as 0. 693/


4

doubling time (h).

^Control viable counts were 2.48 χ 10 and 2.39 χ 10 viable 8 8

organisms/ml determined by colony formation on membrane filters in


20 days (17). Initial count in all flasks, 2. 5 χ 10 /ml. 7

c d
* FeSO^ oxidation rate became linear after oxidation of 30% and
70% respectively.
Since s p e c i f i c growth rate was e s s e n t i a l l y constant at
0.11-0.12 h " 1
f o r FeSO^ concentrations of 77-165 mM, i t i s
reasonable to assume that t h i s represents the y m a v possible for
ill α λ
batch culture at pH 1.6 and 30°C, with a mean growth y i e l d
c o e f f i c i e n t (on 82 mM FeSO.) of 0.35 g dry wt/ g atom F e . ranging
12
from 0.33-0.39 and equal to about 3 χ 10 v i a b l e organisms.
28 D. P. Kelly and C. A. Jones

TABLE II Effect of carbon dioxide concentration on y ^ and e

growth yield of 50 ml cultures on 84-88mM FeSO^ in sealed flasks


with a 260 ml air space. Biomass was determined as protein and
dry weight calculated as protein X 1.67, after correcting for
initial 2.5 \xg dry wt/ml.

Carbon dioxide Final Biomass Yield


»Fe
supplied (ml) (\xg/ml) (mg dry wt/g
mole FeS0 ) 4

Unsealed control 0.113 29.4 350


1.5 0.105 29.0 333
5 0.112 31.0 354
10 0.115 31.0 352
20 0.124 33.0 386

B, E f f e c t of FeSO^ Supply on Batch Growth K i n e t i c s and


I n h i b i t i o n by F e r r i c I r o n

S p e c i f i c growth rates (y = y p ) increased i n response to


e

i n i t i a l FeSCL concentration at l e a s t over the range 10-60 mM and


ay of 0.143 h was indicated ( F i g . 2 ) . This r e s u l t suggests
max w v

a high K f o r FeSO^, but as accurate measurement of exponential


$

i r o n oxidation rate was p o s s i b l e only a f t e r about 15% of the FeSO


had been o x i d i z e d , s i g n i f i c a n t F e was always present. That the
3 +

apparently poor Κ f o r FeSO- was due to competitive i n h i b i t i o n by


3+
Fe was shown by adding f e r r i c sulphate to batch cultures with
d i f f e r e n t FeSO^ concentrations. Data a n a l y s i s by the method of
Lineweaver and Burk (25) p l o t t i n g 1/y χ 1/FeSO, for exponential
3+
rates with and without 5, 10 and 57 mM Fe showed competitive
i n h i b i t i o n of y p e ( F i g . 2) and an increase of K $ from 36 mM FeSO^
for the control (5-8 mM F e ) to 67 mM with 57 mM F e .
3 + 3 +
Added
f e r r i c ions did not a l t e r y m a x > i n d i c a t i n g a purely competitive
effect on i r o n oxidation ( F i g . 2)
Basic Microbial Studies Applied to Leaching 29

40 r

Fig. 2. Lineweaver-Burk analysis of exponential FeSO


oxidation in cultures with different initial FeSO levels Between
10-60 mM. \ip was measured over periods where the amount of
e

ferric iron produced was equivalent in all flasks. Flasks


contained the following supplements: Curve 1: control series;
Curve 2: 25mM K S0 ; Curve 3: 5mM Fe ;
2 4 Curve 4: lOmM Fe ; z+ 3+

Curve 5: lOmM Fe , 25mM K S0 ; Curve 6: 57mM Fe .


Z+
Cultures
2 4
3+

in curves 1, 3 and 5 contained 0.37 mM potassium ion.

C. E f f e c t of Potassium Ion on I r o n Oxidation K i n e t i c s in


Batch Culture

Growth on 30mM FeSO^ i n the low-potassium medium (0.37mM K ) +

was stimulated from y F e 0.114 to 0.138 and 0.15 h " 1


respectively
by r a i s i n g K +
(as K S 0 ) to 7.47 and 50.87mM.
2 4 High K +
partially
a l l e v i a t e d the competitive i n h i b i t i o n by f e r r i c i o n s , r e s u l t i n g
30 D. P. Kelly and C. A. Jones

i n a lessened e f f e c t by added f e r r i c sulphate and lowered the


control K $ to 19.6mM FeSO^ i n the absence of added f e r r i c i o n .
K $ with lOmM added F e 3 +
was 46.5mM with 0.37mM K +
but 22.7mM with
50.87mM K +
(Fig. 2). The observed y m a x was unaffected by K +

concentration ( F i g . 2 ) .

D. Effect of High Ferrous and F e r r i c Sulphate Levels on Iron


Oxidation in Batch Culture

Lag before growth was increased and rate of development


decreased as i n i t i a l FeSO^ concentration was increased from 62 to
249mM ( F i g . 3 ) .

TIME (HOURS)

Fig. 3. Effect of high ferrous and ferric sulphate levels on


FeSO4 oxidation in growing cultures. Initial concentrations of
ferrous + ferric ion (mM) were: Curve 1: 62 + 0; Curve 2:
59 + 120; Curve 3: 132; Curve 4: 126 + 120; Curve 5: 187;
Curve 6: 184 + 120; Curve 7: 249.

Addition of f e r r i c ions depressed growth f u r t h e r but


subsequently appeared stimulatory at high ferrous concentrations
(Fig. 3). These r e s u l t s indicate that FeSO^ could be an
i n h i b i t o r y substrate at high concentration.
Basic Microbial Studies Applied to Leaching 31

E. K i n e t i c s of Iron Oxidation by Suspensions of THobaoillus


ferrooxidans

1. Warburg Μanometry

The rate of FeSO^ oxidation increased up to about lOOmM FeS0 (

but then decreased at higher concentrations, i n d i c a t i n g substrate


i n h i b i t i o n (Table 3 ) .

TABLE III Kinetics of FeSO. oxidation in Warburg flasks.


Oxygen uptake rates were estimated 5-30 min after tipping
FeSOg to T. ferrooxidans at pH 1.7. Constants were calculated
from Lineweaver and Burk (25) plots.

FeS0 4 0 2 uptake £ £
concentration rate s ν
(mM) (\xl/h) (χ 10 )4
(x 10 ) b

8 391 1250 256


10 432 1000 232
16 536 625 187
24 611 417 164
25 588 400 170
32 657 313 152
40 688 250 145
50 708 200 141
60 735 167 136
80 761 125 131
100 800 100 125
250 680 40 147
500 590 20 170
800 280 12.5 357

max = 850 \il/hour


Κ = 9. 7mM FeSO
m 4 A

The apparent K m f o r FeSO^ was 9.7mM, but Warburg manometry


i s of questionable accuracy i n measuring rates at very low FeSO^
concentrations and i n a l l cases rates were i n e v i t a b l y determined
over a time period during which s i g n i f i c a n t f e r r i c i r o n had
accumulated. Consequently the K m value was probably r a i s e d by
ferric inhibition. In a l l cases the oxygen consumption supported
32 D. P. Kelly and C. A. Jones

the equation f o r normal iron o x i d a t i o n :


4FeS0 4 + 0 2 + 2H S0
2 4 = Fe (S0 )
2 4 3 + 2H 02

2. Oxygen Electrode Experiments

a. Normal kinetics. Using F e S 0 4 concentrations between


0.4-900mM, oxygen consumption always showed an acceleration phase
of 1-5 minutes before measurement of maximum oxidation rate was
possible. The presence of i n h i b i t o r y amounts of f e r r i c i r o n
produced longer acceleration phases before a constant rate was
established. I t was found that organisms harvested from late
exponential phase batch cultures increased 1.5-2.5 f o l d i n
s p e c i f i c rate of F e S 0 4 oxidation i f stored for 1-3 days at 20°C in
water at pH 1.6 before use. A c t i v i t y was r e t a i n e d , though
p r o g r e s s i v e l y d e c l i n e d , i n organisms k e p t a t 2 0 ° C f o r 1-3 weeks.
Storage at 4°C resulted i n considerable extension of the
acceleration phase i n o x i d a t i o n , although maximum rates were
r e l a t i v e l y unaltered.

Numerous experiments using 0.4-100mM F e S 0 4 indicated K^ f o r


FeS0 4 to l i e with high r e p r o d u c i b i l i t y between 0.43-0.9mM ( F i g . 4a,
b ) , although extreme values of 1.58, 2.0 and 3.26 were obtained
i n i n d i v i d u a l experiments. Recently a c r i t i c a l study has been
made of the various graphical c a l c u l a t i o n methods of d e r i v i n g K m

values from k i n e t i c data ( 2 6 ) . While we have presented data i n


F i g . 4 by the method of Lineweaver and Burk (25) we have used a
v a r i e t y of other procedures such as / s
v versus s or ν versus v
/ $

plots (27) and a computer programme on a CDC 6600 computer


( U n i v e r s i t y of London) designed to test the f i t of our data to
the Michaelis-Menten equation. Data from s i x separate experiments
with d i f f e r e n t cultures of bacteria using 0.4-43mM F e S 0 4

indicated a Κ of 0.70 ± 0.14mM FeSO-. I t was noted that at


m 4
higher F e S 0 concentrations the oxidation rate seemed further
4

accelerated, suggesting a second, higher value f o r K m (Fig. 4).


Basic Microbial Studies Applied to Leaching 33

1 1 2000 2500
- ο0 500 1000 1500

C 1.2

1 1
y

0.8
y'
^ 1 2 3
y
y
V x I
MA
3 7 9 y
0.4
yy
s
s

-8 -6 -4 -2 C) 2 H 6 8

l/ s (M FeS0 ) 4
_ 1

Fig. 4. Kinetics of FeSO^ oxidation measured in oxygen


electrode cells. A, 98 ]ig protein/ml; 0.43-11. 7mM; B , 29 \ig
protein/ml; 0.4-140mM, snowing biphasic response to concentration;
C 76.4 \ig protein/ml; 150-900mM FeS0 . ^ . is given as nmoles
y 4 TOa

O^/min/mg protein; as mM FeSO^.

Multiple values and substrate inhibition dependent on


FeSO. concentration. With F e S 0 between 100-700mM, oxidation
4 4

rates accelerated further with i n c r e a s i n g substrate concentration;


Lineweaver-Burk plots indicated ( F i g . 4c) a b i p h a s i c r e l a t i o n s h i p
of rate and concentration with apparent K m values of 123mM and
i n f i n i t y f o r the two quite d i s t i n c t phases obtained. The same
organisms i n t h i s experiment gave a K m of 0.43mM f o r l-9mM FeS04.
34 D. P. Kelly and C. A. Jones

In t h i s experiment, FeSO^ above 700mM produced substrate


inhibition (Fig. 4c). The absolute s i g n i f i c a n c e of these higher
Κ values i s questionable as we have obtained values of 20-40mM
m
l i s
i n some other experiments, calculated by the / χ / or / χ s v s

methods.

c. Effect of ferric ions on FeS0 4 oxidation. With organisms


f r e s h l y harvested from batch c u l t u r e , the addition of low
concentrations of f e r r i c sulphate stimulated oxygen uptake i n the
presence of 5-80mM F e S 0 4 ( F i g . 5 ) , but with i n c r e a s i n g F e 3 +

concentration, oxidation was p r o g r e s s i v e l y i n h i b i t e d ( F i g . 5 b ) .

A Β

I 1 1 Ι I Ι Ι Ι Ι Ι
0 10 20 30 0 100 200
FES0 4 (MH) FE 3 +
Ml)

Fig. 5. Stimulation of FeSO^ oxidation by low concentrations


of ferric iron using freshly harvested T. ferrooxidans A,
Oxidation by 104 \ig bacterial protein/ml; Curve 1: control series
Curve 2: with 20mM Fe +. B Oxidation of lOmM (Curve 1) or 20mM
s
3

FeSO$ (Curve 2) by 110 \ig bacterial protein/ml with various


amounts of Fe . 3+

Although low F e 3 +
concentrations stimulated the r a t e , they
did not a f f e c t the acceleration phase i n o x i d a t i o n . Storage of
the organisms at 20°C resulted i n increased oxidation rates over
1-2 days, but the s t i m u l a t i o n by F e 3 +
did not increase in
Basi
cMicrobia
lStudie
sApplie
dt oLeachin
g 35

proportion. Thus Q Q values (nmoles 0 2 consumed/min/mg


T. ferrooxidans protein) with 10mM F e S 0 4 (± 20mM F e ) were 785 3 +

(1009) a f t e r h a r v e s t i n g , 1258 (1240) after 1 d a y a t 2 0 ° C , and 1642


(1288) a f t e r 2 days. Thus as the a c t i v i t y of the organisms
increased, the stimulatory e f f e c t of F e 3 +
was completely l o s t .
For further study of inhibitory e f f e c t s of F e , organisms 3 +

subjected to p r i o r storage at 20°C were used, as K m values were


not apparently altered during t h i s period. Attempts to employ
f r e s h l y harvested organisms to estimate K. f o r i n h i b i t o r y
3+ι ι
concentrations of Fe by means of the graphical / versus
i n h i b i t o r concentration method (28) produced useless non-linear
plots (Fig. 5b).
z+

d. Competitive inhibition of FeSO^ oxidation by Fe . At


a l l i n h i b i t o r y concentrations t e s t e d , using 0.4-140mM FeSO^,
f e r r i c sulphate was demonstrated to act as a purely competitive
i n h i b i t o r of ferrous sulphate o x i d a t i o n . The competitive mode
f o r i n h i b i t i o n was c o n s i s t e n t l y indicated by f i v e graphical
procedures f o r a number of experiments ( F i g . 6 - 1 0 ) . Moreover,
computer f i t t i n g of experimental i n h i b i t i o n data to the
Michaelis-Menten equation was c o n s i s t e n t with no s i g n i f i c a n t
a l t e r a t i o n of maximum oxidation rate ( V 1 but increase of Κ
m

2 + max' m
( i . e . Kp) i n response to Fe concentration. While i n the
absence of F e was t y p i c a l l y about 1 mM FeSO., Κ values
3 +

3+ 4 p

observed with Fe ranged from 4.5-63 depending on concentration.


The i n h i b i t o r constant (K.) f o r F e was estimated by several
3 +

3+
procedures (27) and f i g u r e s ranging from 2.5mM Fe to 28mM using
high F e 3 +
l e v e l s were obtained.
Values of 12mM or 20mM were obtained by the graphical
methods of Dixon (28; F i g . 9) and Hunter and Davis (29; F i g . 10)
respectively. C a l c u l a t i o n s using the K m and Kp values (27)
suggest a value f o r K. of about 8-10mM F e . 3 +
36 D. P. Kelly and C. A. Jones

O 0.2 0,4 0.6 0.8 1.0

'[FESOJCMM)

Fig. 6. Lineweaver-Burk plot (1/v X 1/s) of oxidation rates


for 1-lOOmM FeSO* with (φ, C) and without (θ>Φ) lOOmM Fe by 3+

T. ferrooxidans (29 \xg protein/ml). The lines are computer fits,


giving identical V intercepts and Κ = 1.06 Κ = 9.6mM FeSO.. 3

0 20 40 60 80 100

FES0 4 CONCENTRATION (MM)

Fig. 7. Dixon and Webb plot (s/v X s) for oxidation of


1-lOOmM FeS0 with ferric iron at OmM (Q), lOmM ( V), or 25mM
4
Basic Microbial Studies Applied to Leaching 37

0 50 100 150 200

0 2 UPTAKE (NMOLES/MIN/MG PROTEIN)


FES0 4 CONCENTRATION (MM)

Fig. 8. Dixon and Webb plot (ν x v/s) for oxidation of


5-40mM FeS0 (54.7 \ig bacterial protein) with Fe
4 at OmM (1);
10 (2); 15 U); 20 (4); or 25mM (5).

e. Effect of pH and uranyl ion on FeSO^ oxidation. In


contrast to the purely competitive i n h i b i t o r y e f f e c t of f e r r i c
2+ +
ions on FeSO^ o x i d a t i o n , U 0 or increased Η (pH 1.2) were
2

c l e a r l y non-competitive i n h i b i t o r s of o x i d a t i o n , a l t e r i n g V m a x

(control 1183, others 824) without a f f e c t i n g K m (0.9 mM) f o r


FeS0 4 (Fig. 11).

14
F. E f f e c t of Ferrous and F e r r i c I r o n Concentration on C-Carbon
Dioxide F i x a t i o n
Using b a c t e r i a l suspensions i n Warburg f l a s k s supplied with
14
enough NaH CO^ to ensure that f i x a t i o n was independent of
carbon dioxide a v a i l a b i l i t y or rate of FeSO* o x i d a t i o n ( K e l l y ,
14
unpublished d a t a ) , the amounts of CO^ f i x e d were found to be
dependent on FeSO- concentration as well as on absolute quantity
14
oxidized. Thus i n one experiment the amount of C0£ (nmoles)
fixed per ymole FeSO. oxidized was 2.24 with 25mM FeSO.,
38 D. P. Kelly and C. A. Jones

IN
: ? 0,2-
1 1 1 I ι ' ι ι ι ι

0 m 80 120 160
FERRIC ION CONCENTRATION (mM)
Fig. 9. Estimation of K. for competitive ferric inhibition
of FeSO oxidation by the method of Dixon (28). FeSO^
4

concentrations were 5mM (O) and lOOmM (φ). is g^ven by the


intercept.

3,26 with 150mM and 3.36 with 300mM. F i x a t i o n e f f i c i e n c y was


more than doubled when 86mM FeSO^ was s u p p l i e d , compared with
8.6mM (Table 4 ) . Very high concentrations of FeSO^ depressed
fixation; thus 500mM depressed f i x a t i o n e f f i c i e n c y to about
70% of that with lOOmM (Table 4 ) .

Added f e r r i c i r o n did not i n h i b i t f i x a t i o n at the concen­


t r a t i o n s t e s t e d , even at 57mM which depressed FeSO^ oxidation to
45% of the control rate (Table 4 ) . In f a c t , the efficiency of
f i x a t i o n was apparently increased by f e r r i c i o n s .
Basic Microbial Studies Applied to Leaching 39

FES0 4 CONCENTRATION (MM)

Fig. 10. Estimation of for competitive ferric inhibition


of FeSOg oxidation by the method of Hunter and Downs (29) using 3

data from a number of experiments, i = inhibitor concentration


(mM Fe );
3+
α = ^/ν where ν = nmoles 0 consumed/min/mg
ν
Λ 2

bacterial protein in the absence of i, and v^ is ν in the


presence of i. The intercept on i ^ ^ is K^.

0 20 HQ 60 80 100

FES0 4 CONCENTRATION (MM)

Fig. 11. Non-competitive inhibition of FeSO^ oxidation by


increased E and by uranyl ion. s/v X s plots are given for
+

bacteria (29 \ig protein/ml) oxidizing FeSO. at pH 1.7 (O),


pH 1.2 ( V) and pH 1.7 with lOmM uranyl sulphate (φ). (Contrast
with purely competitive ferric effect in Fig. 7).
TABLE IV Effect of ferrous and ferric ion concentration
on CO^ fixation by T h i o b a c i l l u s ferrooxidans

(a) Total flask contents 3. 5 ml (940 \ig T. ferrooxidans protein).


Λ

3+
FeS0 uptake FeS0
4
Fe Incubation Oxygen 4
7 4
cone cone time Maximum Total oxidised C0 fixed?
£

(mM) (mM) (min) Rate (\imoles) nmoles/flask nmoles/\imole


(\il/hr)(\imoles) FeS0 oxidised
4

Total In filtered Total In filtered


fixed bacteria fixed bacteria

8.6 0 65 256 6.41 25.6 82.80 69.83 3.23 2.72


CD 8.6 28.6 75 256 7.05 28.2 102.29 83.25 3.59 2.95
8.6 57.0 85 114 5.26 21.4 89.88 65.40 4.20 3.06
86.0 0 45 720 17.32 69.3 537.53 414.21 7. 76 5.98
86.0 0 60 720 25. 71 102.8 777.84 667.10 7.57 6.49

(b) Total flask contents 2ml (744 \ig T. ferrooxidans protein).


Λ

FeS04 Fe z+
Incubation Oxygen FeS0 4 14
cone cone tvme Uptake Oxidised CO2 fixed nmoles/vmole FeSO^
(mM) (mM) (Min) (yjnoles) (\imoles) dpm/flask oxidised

100 0 45 20.4 81.6 182 300


Λ 3.003
500 0 45 18.1 72.5 111,752 2.072
100 50 55 20.8 83.2 213 900
3 3.456
a - 14
Specvfic activity, 744 dpm/nmole C0%
Basic Microbial Studies Applied to Leaching 41

IV. GENERAL DISCUSSION AND CONCLUSIONS

This study has revealed a number of fundamental properties of


Thiobacillus ferrooxidans that enable p r e d i c t i o n of how the
organism w i l l behave i n a v a r i e t y of environments of s i g n i f i c a n c e
to mineral leaching.

F i r s t , t h e e f f i c i e n c y with which growing cultures can couple


energy from i r o n oxidation i s e s s e n t i a l l y independent of the
concentration of carbon dioxide a v a i l a b l e over the range
< 0.001-8% and i n cultures i n which the FeSO^ concentration
decreases from at l e a s t 165mM to v i r t u a l l y zero, with a p a r a l l e l
increase i n f e r r i c i r o n . In some c o n t r a s t , non-growing
suspensions exhibited dependence both on C 0 2 concentration and
FeSO^ concentration f o r maximum e f f i c i e n c y of coupling of
oxidation to f i x a t i o n . Growing c u l t u r e s , whose growth ceases
because of C 0 2 exhaustion, are s t i l l capable of o x i d i z i n g FeSO^
at a high rate f o r long periods. Non-growing suspensions are
s i m i l a r l y capable of high rates of FeSO^ oxidation i n the absence
of carbon d i o x i d e . This i s biochemically i n t e r e s t i n g , since i t
i n d i c a t e s that oxidation can be "uncoupled" from a s s i m i l a t o r y
carbon metabolism and that the organisms have a mechanism by
which energy from i r o n oxidation can be d i s s i p a t e d . Moreover, i t
indicates that f o r the operation of f i x e d f i l m , tank reactors or
open oxidation ponds f o r the oxidation of i r o n ( 1 1 , 30, 31) an
abundant supply of C 0 2 i s not e s s e n t i a l , since non-growing
bacteria can be active f o r long p e r i o d s , and i r o n oxidation i s not
growth-dependent.

Secondly, the true K $ f o r FeSO^ i n batch cultures and


suspensions i s of the order of 1 mM, as was determined from
chemostat cultures (13, 2 4 ) , but i n batch cultures the e f f e c t i v e
K $ i s much higher (and growth rate consequently lowered) because
of competitive product i n h i b i t i o n by f e r r i c i r o n . Thus i n a l l
batch cultures and i r o n oxidation systems employing non-growing
organisms, the rate of i r o n oxidation i s depressed by f e r r i c ions
42 D. P. Kelly and C. A. Jones

and the observed rate w i l l be dependent on the f e r r o u s : f e r r i c


ratio. The f e r r i c i n h i b i t i o n was purely competitive, i n contrast
to our demonstration of non-competitive i n h i b i t i o n i n chemostat
culture ( 1 3 , 2 4 ) . We are thus i n disagreement with Wong, et a l .
( 2 2 ) , who claimed non-competitive i n h i b i t i o n i n a batch system,
but whose data are i n any case c o n s i s t e n t with at l e a s t an
element of competitive i n h i b i t i o n .

T h i r d l y , high concentrations of FeSO^ were a u t o i n h i b i t o r y to


oxidation by the bacteria i n growing cultures or non-growing
suspensions. Concentrations of 0.5 Μ and above considerably
depressed FeS0 -dependent C 0 - f i x a t i o n , p o s s i b l y
4 2 indicating
uncoupling as reported f o r d i n i t r o p h e n o l , uranium, copper and
nickel ( 2 , 1 8 ) . F e r r i c i r o n only depressed oxidation r a t e s ,
without uncoupling C 0 - f i x a t i o n .
2 Thus both s u b s t r a t e - f e r r o u s
and p r o d u c t - f e r r i c i r o n must be regarded as p o t e n t i a l l y t o x i c
metals to T. ferrooxidans when attempting to predict the
behaviour of the bacteria under a known s e t of c o n d i t i o n s . Even
s o , the organism has the a b i l i t y to o x i d i z e very high
concentrations of FeSO^, and apparently has oxidation mechanisms
with three d i s t i n c t sets of k i n e t i c parameters dependent on
FeSO^, concentration. We do not know i f these represent d i s t i n c t
oxidase systems or multiple binding or transport mechanisms.

In conclusion we can state that T. ferrooxidans e x h i b i t s iron


o x i d a t i o n k i n e t i c s i n chemostat and batch c u l t u r e , as well as in
non-growing s u s p e n s i o n s , that are f a r more complex than had
p r e v i o u s l y been presumed. The organism has a very high potential
f o r the turnover of ferrous i r o n i n both growing and non-growing
s t a t e s , and the capacity to develop i n media of low carbon dioxide
and oxygen supply. Many of the important systems f o r the
regeneration of f e r r i c leach l i q u o r s are e s s e n t i a l l y batch or
suspension systems and w i l l be governed by the phenomena described
i n t h i s paper. Chemostat k i n e t i c s ( 1 3 , 24) may apply to some
natural open culture systems such as heaps and drainage waters.
Basic Microbial Studies Applied to Leaching 43

V. REFERENCES

1. K e l l y , D.P., i n "Microbial Energy Conversion," (H.G. Schlegel


and J . Barnea, E d s ) , p. 329. E. Goltze KG, GBttingen, 1976.
2. Tuovinen, O.H., and K e l l y , D.P., Z. allg. Mikrobiol., 12,
311 (1972).
3. Balashova, V . V . , Vedenina, I.Y., Markosyan, G.E., and
Z a v a r z i n , G.A., Mikrobiologiya, 4 3 , 581 (1974).
4. Tsuchiya, H.M., T r i v e d i , N.C., and Schuler, M.L., Biotechnol.
Bioeng., 16, 991 (1974).
5. Bosecker, K., A b s t r a c t s , " F i f t h International Fermentation
Symposium," (H. Dellweg, E d ) , p. 4 5 1 . B e r l i n (1976).
6. Duncan, D.W., and Brynesteyn, Α . , "New Mexico State Bureau
of Mines and Mineral Resources," C i r c u l a r 118, 55 (1971).
7. Tomizuka, N., Yagisawa, M., Someya, J . , and Takahara, Y . ,
Agr. Biol. Chem., 40, 1019 (1976).
8. Guay, R., S i l v e r , Μ., and Torma, A . E . , European J. Appl.
Microbiol. , 3, 157 (1976).
9. Le Roux, N.W., North, Α . Α . , and W i l s o n , J . C . , "Tenth
International Mineral Processing Conference," Paper 45.
London (1973).
10. Gow, W.A., McCreedy, H.H., R i t c e y , G.M., McNamara, V.M.,
H a r r i s o n , V . F . , and Lucas, B.H., "The Recovery of Uranium,"
p. 195. IAEA, Vienna, 1970.
11. Derry, R., G a r r e t t , K.H., Le Roux, N.W., and Smith, S . E . ,
"Geology, Mining and E x t r a c t i v e Processing of Uranium,"
(M.L. Jones, E d . ) , p. 56. I n s t i t u t i o n of Mining and
M e t a l l u r g y , London, 1977.
12. McCreedy, H.H., CANMET Mineral Sciences Laboratories Report
No. MRP/MSL 76-342 OP.
13. Jones, C.A., and K e l l y , D.P., A b s t r a c t s , " F i f t h International
Fermentation Symposium," (H. Dellweg, E d . ) , p. 126. B e r l i n ,
1976.
14. K e l l y , D.P., E c c l e s t o n , M., and Jones, C.A., "GBF Monograph
S e r i e s No. 4 , " (W. Schwartz, E d . ) , Verlag Chemie, Weinheim,
1977.
15. Lacey, D.T., and Lawson, F., Biotechnol. Bioeng. , 12, 29
(1970).
16. MacDonald, D.G., and C l a r k , R.H., Can. J. Chem. Eng., 48,
669 (1970).
17. Tuovinen, O.H., and K e l l y , D.P., Arch. Mikrobiol., 88, 285
(1973).
44 D. P. Kelly and C. A. Jones

18. Tuovinen, O.H., and K e l l y , D.P., Arch. Microbiol.* 9 5 , 153,


165 (1974).
19. Schnaitman, C.A., K o r c z y n s k i , M . S . , and Lundgren, D.G.,
J. Bacterid., 9 9 , 552 (1969).
20. Bodo, C , and Lundgren, D.G., Can. J. Microbiol.. 20,
1647 (1974).
21. S t e i n e r , M., and L a z a r o f f , N., Appl. Microbiol. , 28, 872
(1974).
22. Wong, C.H., Sharer, J.M., and R i l e y , P.M., M.Sc. T h e s i s ,
U n i v e r i s t y of Waterloo (1973).
23. D i n , G.A., and S u z u k i , I . , Can. J. Biochem., 4 5 , 1547
(1967).
24. Jones, C.A., Ph.D. T h e s i s , U n i v e r s i t y of London (1974).
25. Lineweaver, H., and Burk, D., J. Amer. Chem. Soc. , 56, 658
(1934).
26. A t k i n s , G.L., and Nimmo, I.Α., Biochem. <7. , 149, 775 (1975).
27. Dixon, M., and Webb, E.C., "The Enzymes," Chapters 2 and 4.
Longmans, London, 1958.
28. D i x o n , M., Biochem. J . , 55, 170 (1953).
29. Hunter, Α . , and Downs, C . E . , J. Biol. Chem., 157, 427 (1945).
30. L i v e s e y - G o l d b l a t t , E . , Tunley, T . H . , and Nagy, I . F . , "GBF
Monograph S e r i e s No. 4 , " (W. Schwartz, Ed.) Verlag Chemie,
Weinheim, 1977.
31. Mehta, K.B., and Le Roux, N.W., Biotechnol. Bioeng. , 16,
559 (1974).
HYDROGEN ION UTILIZATION BY

IRON-GROWN THIOBACILLUS FERROOXIDANS

William A. Apel
Patrick R. Dugan

Department of Microbiology
The Ohio State U n i v e r s i t y
Columbus, Ohio, U.S.A.

The initial pH of aerobic suspensions of iron-grown T. f e r ­


rooxidans was shown to increase with the addition of FeSO . The
pH increase was proportional to the Fe 2 concentration in the
+

2 mM - 35 mM range and appears to be due to Ή+ uptake by the


cells. H removal also varied directly with initial pH of the
+

suspension in the range of 2.0 to 3.3. Initial pH increase was


followed in time by a net pH decrease; leading to the postula-
tion that the cells require R+ in proportion to the amount of
electrons produced from the oxidation of Fe % and that the net
+

acid production resulted from a slower abiotic hydrolysis of


Fe+3 (Fe + 3R%0
+3
>Fe(OH) + 3H+). Neither controls without
3

cells nor control suspensions of cells without substrate showed


any significant pH change. Suspensions of cells at pH 2.1 in
the presence of DNP (1 mM - 10 mM) or oxalacetate (80 μΜ -
120 uM) showed some uptake in the absence of added Fe ^ +

leading to the postulation that these compounds effected the


permeability of the cytoplasmic membrane.

45
46 William A. Apel and Patrick R. Dugan

I. INTRODUCTION

Thiobaeillus ferrooxidans i s an a c i d o p h i l i c , chemolithotro-


phic bacterium involved in both the leaching of metals from
s u l f i d e minerals ( 2 0 ) , and the formation of acid drainage i n
areas in which p y r i t i c minerals are exposed to a i r and water
(1,7,10). This autotrophic bacterium i s capable of d e r i v i n g a l l
of i t s energy from the oxidation of e i t h e r reduced inorganic
+2
s u l f u r or reduced iron compounds. Growth on ferrous iron (Fe )
+3
r e s u l t s in the formation of f e r r i c iron (Fe ) which undergoes
a b i o t i c h y d r o l y t i c reactions with consequent net production of
acid as shown in equations I and I I (6):

(I) 4 FeS0 4 + 2 H S0 2 4 + 0 2 2 Fe (S0 )


2 4 3 + 2 H0 2

(II) 2 Fe (S0 )
2 4 3 + 12 H 0 2 * - 4 Fe(0H) + 6H S0 3 2 4

In accordance with equation I , T. ferrooxidans growing on


+2
Fe consumes oxygen in the r a t i o of one mole 0 per four moles
+2 +3
9

of Fe oxidized to Fe ( 1 2 , 1 8 ) , energy being derived from the


+2
four electrons l i b e r a t e d from Fe . The electrons must be accom-
panied by protons f o r which there i s no b i o l o g i c a l source, since
+3
Fe i s not known to be deposited in the c e l l . Equation I I is
a non-biological reaction which f u r n i s h e s an environmental sup-
ply of H . +
When ( I ) and ( I I ) are summed (equation I I I ) , the
dilemma caused by the need to balance electrons taken up by the
c e l l i s not r e s o l v e d ; i . e . a source of protons i s required on
the l e f t side of the r e a c t i o n . F e r r i c hydroxide a l s o w i l l
react to form hydroxy s u l f a t e complexes (reaction IV) which have
buffering capacity and can a l t e r equations I I and I I I so that
l e s s H 0 enters the reactions and l e s s H S 0
2 2 4 i s produced.
Further, equation I does not consider the proton requirement for
the metabolic reduction of C 0 - 2

( I I I ) 4FeS0 + 0 + 10H 0
4 2 2 4 Fe(0H) + 4 H S0 3 2 4

(IV) Fe(0H) + 2H + S 0 3
+
4
=
* - F e ( 0 H ) ( S 0 ) + 2H 0
4 2
Basic Microbial Studies Applied to Leaching 47

A number of internal enzymes from T. ferrooxidans have been


i s o l a t e d and characterized. With the exception of crude prepar­
ations of a c e l l envelope associated iron oxidase which had a
pH optimum of 2.5 to 3.5 ( 5 ) , a l l of the above p u r i f i e d enzymes
had pH optima ranging from 5.0 to 9.0 ( 4 , 9 , 1 1 , 1 6 , 1 9 , 2 3 ) , which
i s s i g n i f i c a n t l y higher than the pH of the organism's e n v i r o n ­
ment. From t h i s difference in internal and external pH one may
conclude that the a c i d o p h i l i c Thiobacilli possess a c e l l enve­
lope which i s s e l e c t i v e l y impermeable to high concentrations of
H , a conclusion which has been d i r e c t l y supported by the
+

f i n d i n g s of Dewey and Beecher ( 8 ) . Due to the f a c t that r e s t i n g


T. ferrooxidans c e l l s do not r e s p i r e and are able to tolerate
long periods of storage at low p H ' s , i t was suggested by Beck
(2) that t h e i r Η b a r r i e r i s of a passive nature.

This report presents evidence to show that T. ferrooxidans


+ +2
takes Η from i t s environment in r e l a t i o n to the amount of Fe
u t i l i z e d and presents a model depicting H +
uptake. The e f f e c t s
of r e s p i r a t o r y uncouplers on H +
uptake by suspensions of T.
ferrooxidans a l s o are examined.

II. MATERIALS AND METHODS

A. Cell Culture

T. ferrooxidans were grown in 5 g a l . carboys under forced


aeration at 23± 1°C in the "9K" medium of Silverman and Lundgren
(17). After 4 days i n c u b a t i o n , the c e l l s were harvested by
c e n t r i f u g a t i o n at 10,000 χ g and the r e s u l t i n g c e l l p e l l e t was
resuspended in pH 3 F^SO^ s o l u t i o n and held overnight at 4°C
to allow p r e c i p i t a t i o n of residual i n s o l u b l e iron compounds.
The c e l l s then were c a r e f u l l y decanted, washed three times with
-4
pH 3 H S 0
2 4 s o l u t i o n , and resuspended to a density of 8.0 χ 10
g (dry wt.)/ml. C e l l s were stored at 4°C for a maximum of 48
hr. a f t e r harvesting before being e i t h e r u t i l i z e d or discarded.
48 William A. Apel and Patrick R. Dugan

B. H +
Uptake

H +
uptake was determined at 23t 1°C by placing 5 ml a l i q u o t s
of the cell suspension in a g l a s s polaragraphic electrolysis
vessel which contained a Corning 475060 pH electrode connected
to a Corning model 12 expanded scale pH meter. The pH meter
was coupled to a Heath EU-20B Servo Recorder. The gas i n l e t of
the vessel was connected to either a i r , Ν^ 5 or 0^» and the v e s ­
sel stopcock was adjusted to produce vigorous bubbling of the
+2 + 3 Μ + Μ 2 r +3 +2 +
.+ . ρ +2
suspension. Fe , Al , Na , Mg , Fe , Μη , Ni , and Cu
r Λ 1 M M

as s u l f a t e s o l u t i o n s , oxalacetate, and sodium t h i o s u l f a t e were


added i n d i v i d u a l l y to the c e l l suspension in pH 3 H^SO^ s o l u ­
t i o n s , while dinitrophenol (DNP) was d i s s o l v e d in 95% ethanol
and added to y i e l d 1 to 10 mM concentrations of DNP. Ethanol
controls showed no noticeable influence on the a c t i v i t y of the
c e l l s with the above concentration.

The pH of the suspension was adjusted with 5 Ν H^SO^ and


allowed to come to e q u i l i b r i u m a f t e r which the pH was c o n t i n u ­
ously monitored. Η uptake was calculated from the pH data.

C. X-ray M i c r o a n a l y s i s

Specimens for X-ray microanalysis were a i r dryed on carbon


planchets (E.F. Fullam, I n c . , Schnectady, N.Y. 12301) which were
attached with double sided tape to aluminum specimen stubs
which had been coated with Dag 154 graphite suspension (Achison
C o l l o i d s Co., Port Huron, Mi. 48060). The specimens were sput-
o
ter coated with 100 A of carbon and then viewed and analyzed
u t i l i z i n g an Hitachi model S-500 scanning electron microscope
equipped with an Ortec model 7844S-455 s i l i c o n - l i t h i u m (SiLi)
detector coupled to an Ortec model 6230 computer u n i t complete
with pulse height analyzer and d i g i t a l p r i n t o u t . All specimens
were analyzed for 400 sec at 30° t i l t , 15 mm working d i s t a n c e ,
condenser lens s e t t i n g 500, aperture s e t t i n g 2, and 30,000
V power.
Basic Microbial Studies Applied to Leaching 49

III. RESULTS

Hydrogen ion (proton) removal by c e l l s i s plotted as m i l l i -


moles (from the s t r i p chart pH curves) in order to f a c i l i t a t e
comparisons of data from d i f f e r e n t experiments which had d i f f e r ­
ent pH s t a r t i n g and terminating points due to differences in the
pH of s o l u t i o n s added to the reaction vessel (pH range 1.8 to
3.5). The data are only s e m i q u a n t i t a t i v e ^ accurate in terms
of millimoles H +
uptake, because f e r r i c and s u l f u r s a l t com­
plexes formed as reaction products in s o l u t i o n have some b u f f e r ­
ing capacity in the experimental pH range.

The addition of FeSO^ (5.0 mM) to an aerated T. ferrooxidans


suspension at an i n i t i a l pH of 2.4 was found to produce an
immediate increase in pH which was interpreted as an i n f l u x
of H +
into the c e l l s ( F i g . 1 ) . Approximately 16 min a f t e r the

I . , u
, , , , . I £
0 5 10 15 20 25 30 35 40
Time (min)

Fig. 1. Increases in initial pH and calculated Η uptake


in T. ferrooxidans suspensions in the presence of Fe at an +

initial pH of 2.4. (A) pH of cells with Fe ^ (B) calculated +2

uptake of cells with Fe+%, (C) calculated uptake of


cells without iron and (Ό) pH of cells without Fe ^.
3
+
50 William A. Apel and Patrick R. Dugan

5
J. 6
2
α
5
+
X
J>
-Q 4 V
I
φ 3
</>

Ο
σ
£
JO
-2
D
1
u
δ
2.0 2.5 ί(Γ~~ 3.5
Initial pH

F i ^ . 2. Calculated total observable changes in Ε uptake


relative to changes in initial pH of T. ferrooxidans suspen­
sions in the presence of Fe ^. +

addition of FeSO^ to the suspension, the H +


uptake leveled o f f
and approximately 20 min. a f t e r the addition of the FeSO^, the
pH began to drop. Under anaerobic c o n d i t i o n s , when the system
was purged with N , there was no observable H
?
+
uptake, although
when the system was oxygenated with 0^, Η uptake was comparable
to that attained with a i r .

Amounts of recordable H +
uptake were found to be pH
dependent in the range of 1.8 to 3.3 with the lower pH pro­
ducing the most noticeable uptake, while the addition of FeSO^
to a suspension with a pH greater than 3.3 resulted in an
immediate drop in pH ( i . e . no observable Η uptake ( F i g . 2 ) ) .

Magnitudes of H +
uptake by the c e l l suspension a l s o were
+2
found to d i r e c t l y correlate with the concentration of Fe
Basic Microbial Studies Applied to Leaching 51

0 10 20 _ 30 40
Fe (mM)
+ +

Fig. 3. Calculated total observable changes in E uptake +

relative to changes in Fe % concentrations in suspensions of


+

T. ferrooxidans at an initial pH of 2.3.


+2
present in the system i n the range of 2 mM to 35 mM. At Fe
concentrations greater than 35 mM, the system appeared to be
+2
saturated, and further increases in Fe concentrations resulted
in no net H +
uptake ( F i g . 3 ) .
+3 + +2
The addition of 50 mM concentrations of Al , Na , Mg ,
+2 + +2
Μη , Ni , and Cu as s u l f a t e s to the suspension had no obser­
vable e f f e c t s on H +
uptake under e i t h e r aerobic or anaerobic
c o n d i t i o n s , and under aerobic c o n d i t i o n s , the subsequent addition
of FeSO^ in the presence of the above ions resulted in normal
H +
uptake. When 50 mM concentrations of Fe^iSO^)^ were added to
the suspension there was an immediate net drop in pH, presumably
due to the a b i o t i c production of acid as p r e v i o u s l y described
in equations I and I I .
52 William A. Apel and Patrick R. Dugan

a*

Time(min)

Fig. 4. Effects of concentrations of DNP on calculated


Η uptake by suspensions of T. ferrooxidans at an initial pH
of 2.1. ( ) cells with 10 mM DNP, ( ) cells with 5 mM
DNP, ( ) cells with 1 mM DNP, and ( · ) cells with no
DNP.

When 1 to 1 0 mM concentrations of DNP were added to T.


ferrooxidans suspensions, a net increase in pH was observed i n ­
dicating H +
uptake by the c e l l s , with the rate of H +
uptake
being proportional to the DNP concentration ( F i g . 4 ) .

S i m i l a r data was obtained with T. ferrooxidans suspensions


which had been treated with oxalacetate in the range of 8 0 μΜ
to 1 2 0 μΜ ( F i g . 5 ) . Increases in pH a l s o appeared to be pro­
portional to concentrations of oxalacetate in the range examined.

X-ray microanalysis of the supernatants of c e l l suspensions


treated with 5 mM DNP and 8 0 JJM oxalacetate revealed no increase
Basic Microbial Studies Applied to Leaching 53

10 15 20
Time (min)

Fig. 5. Effects of concentrations of oxalacetate on calcu-


lated H+ uptake by suspensions of T. "ferrooxidans at an initial
pH of 2.1. ( ) cell with 120 uM oxalacetate, ( ) cells
with 80 uM oxalacetate, and ( . ) cells with no oxalacetate.

in elements which are detectable with t h i s technique and would


normally be associated with c e l l u l a r leakage ( i . e . Na, K,
P, e t c . ) ( F i g . 6 ) . Thus, i t was concluded that increases in
supernatant pH were p r i m a r i l y due to H +
uptake rather than
leakage of basic substances from the c e l l s which could r e s u l t in
a n e u t r a l i z a t i o n of H +
i n the suspending s o l u t i o n .

Any e f f e c t s of e i t h e r DNP or oxalacetate in the above con-


centrations on H uptake by T. ferrooxidans in the presence
+

+2
of Fe were below the detection l e v e l s of the monitoring
system.

IV. DISCUSSION

Iron grown T. ferrooxidans are unique since unlike many


other chemolithotrophic bacteria (e.g. Nitrosomonas,
54 William A. Apel and Patrick R. Dugan

Fig. 6. Photograph showing X-ray microanalysis spectrum of


supernatant containing cells treated with 80 μΜ oxalacetic acid.
Arrow denotes sulfur peak which was present due to acidifica­
tion of suspension to pH 2.1 with H^SO^. An identical spectrum
was obtained from the supernatant of cells treated with 5 mM DNP.

Hydrogenomonas, Methanobacterium, e t c . ) there are no protons


associated with i t s energy source. This may explain the o b i i -
gately a c i d o p h i l i c nature of the bacterium in that the oxidation
+2 +3
of Fe to Fe r e s u l t s in the removal by the c e l l of one
e l e c t r o n , and in order to maintain balance of e l e c t r i c a l charge,
a proton obtained from the c e l l ' s environment may ultimately be
u t i l i z e d for the reduction of CO^ and 0 · 2 Therefore, in t h i s
context, H seems to be an e s s e n t i a l n u t r i e n t f o r T. ferrooxidans.
+

The data presented in t h i s paper support t h i s viewpoint


+2
since i t appears that the addition of Fe to a culture of
T. ferrooxidans being maintained under conditions favorable f o r
iron oxidation by the organism ( i . e . low pH and aerobic c o n d i ­
t i o n s ) r e s u l t s in a s i g n i f i c a n t net increase in the pH of the
organism's environment for a period of several minutes a f t e r the
+2
addition of the Fe ( F i g . 1 ) . This pH increase i s a t t r i b u t a b l e
Basic Microbial Studies Applied to Leaching 55

to Η uptake by the c e l l s . Furthermore, i t seems p l a u s i b l e that


+ +2
Η uptake continues f o r as long as the c e l l s are o x i d i z i n g Fe .
The l e v e l i n g o f f and subsequent decrease in pH observed during
+3
these experiments i s thought to be due to the b u i l d up of Fe ,
r e s u l t i n g from the c e l l ' s metabolic a c t i v i t i e s , and the sub­
sequent a b i o t i c production of acid as p r e v i o u s l y d i s c u s s e d .
Thus, even though there i s a net production of acid in the
system, the c e l l s probably continue to u t i l i z e environmental Η .
This p o s t u l a t i o n i s c o n s i s t e n t with the chemiosmotic
coupling theory of Mitchell (13,14) which states that through
the a c t i v i t i e s of r e s p i r a t o r y c h a i n s , c e l l s pump H +
across a
H +
impermeable membrane r e s u l t i n g in both a H +
and e l e c t r o s t a t i c
gradient across that membrane. The c e l l s then u t i l i z e the
energy associated with t h i s gradient in the formation of ATP.
E s s e n t i a l l y , T. ferrooxidans possesses a pre-formed gradient
since the organism's external pH i s considerably lower than i t s
internal pH. Hence, T. ferrooxidans has a H +
impermeable
b a r r i e r and both pH and e l e c t r o s t a t i c gradients across that
b a r r i e r , a l l of which are primary c r i t e r i a f o r generation of
ATP according to M i t c h e l l ' s hypothesis.
Uncouplers, l i k e DNP, are believed to adversely e f f e c t the
i n t e g r i t y of H +
barriers (13). This i s evidently the case with
T. ferrooxidans since the a d d i t i o n of DNP to c e l l suspensions
resulted in an increase in the pH of the suspension, presumably
due to H +
uptake by the c e l l s . Similarly, H +
uptake was
observed by Noguchi, et al.(15) when they added DNP to low pH
suspensions of T. thiooxidans i n d i c a t i n g that a p a r a l l e l process
may e x i s t in both of these a c i d o p h i l i c s p e c i e s . Furthermore,
i t should be mentioned that i t has been shown by Beck and
Shafia (3) that l e v e l s of c e r t a i n phosphates, p a r t i c u l a r l y
orthophosphates, are c r i t i c a l r e l a t i v e to the e f f e c t s of DNP
on the p h y s i o l o g i c a l a c t i v i t i e s of T. ferrooxidans. Under low
phosphate c o n d i t i o n s , DNP was found to be much more e f f e c t i v e
56 William A. Apel and Patrick R. Dugan

in i n h i b i t i n g Fe 2 oxidation related metabolic a c t i v i t i e s of


+

the c e l l s than i t was under high phosphate c o n d i t i o n s . A l l DNP


related experiments included in t h i s report were performed
u t i l i z i n g suspensions of T. ferrooxidans containing l i t t l e if
any free phosphate.
-4
Oxalacetic acid in the 10 Μ range may a l s o e f f e c t T. ferro­
oxidans in a manner s i m i l a r to that of DNP. I t has been
previously shown that oxalacetic acid i n h i b i t e d iron oxidation
by T. ferrooxidans due to the e f f e c t s of the organic acid on the
i n t e g r i t y of the c e l l envelope of the organism ( 2 1 ) . When T.
-2
ferrooxidans was treated with 10 Μ concentrations of oxalacetic
a c i d , electron micrographs revealed that the c e l l wall and c y t o ­
plasmic membrane were g r o s s l y d i s r u p t e d , allowing leakage of
cytoplasmic components into the c e l l ' s environment (22). Thus,
the p o s s i b i l i t y e x i s t s that when T. ferrooxidans was treated
with oxalacetic acid in hundred-fold lower concentrations, a
more subtle d i s r u p t i o n of the organism's c e l l envelope occurred,
allowing leakage of environmental Η into the c e l l . This i s con­
s i s t e n t with X-ray microanalysis data which indicated that no
detectable c e l l u l a r elements leaked into the surrounding
solution.
When considering the concentrations of DNP and oxalacetic
acid necessary to r e s u l t in the above described e f f e c t s , one
should keep in mind that concentrations may vary g r e a t l y
r e l a t i v e to the density of the cell suspension u t i l i z e d , since
the reactions of the above chemical agents with the c e l l s pri­
marily appear to be surface r e l a t e d . The c e l l suspensions
used in the experiments contained a much greater c e l l density
and hence a greater surface area than one would normally expect
to f i n d under natural c o n d i t i o n s ; therefore, much lower con­
centrations of the chemical agents may accomplish e f f e c t s of
the same magnitude in natural c e l l populations. However, in
t h i s study high c e l l d e n s i t i e s were e s s e n t i a l in order to demon-
Basic Microbial Studies Applied to Leaching 57

s t r a t e measurable H +
uptake.

Figure 7 summarizes schematically the p o s s i b l e r o l e s of


+ +3
Η and iron in r e l a t i o n to c e l l envelope s t r u c t u r e . Fe
i s represented as bound to a receptor s i t e at the cytoplasmic
+2 +3
membrane surface. Fe exchanges with and replaces Fe at
the receptor s i t e and i s immediately o x i d i z e d , l i b e r a t i n g an
electron to an acceptor (cytochrome c) i n the membrane. The

Fig. 7. Schematic diagram representing the Fe and Fe


ions which are bound or complexed to the ceT^l envelope-membrane
area of T. ferrooxidans. The movement of Ε is also depicted.
58Willia mA .ApelandPatric
kR .Dugan

electron i s accompanied by a proton from the environment and


e l e c t r i c n e u t r a l i t y i s maintained. Higher concentrations of
+ +2
Η and Fe are depicted in the environment than in the envelope
+3
layers. A high concentration of bound Fe in the envelope-
membrane area contributes to the H +
b a r r i e r due to the high
e l e c t r o s t a t i c charge.
In c o n c l u s i o n , the observations incorporated in t h i s
report explain in part the o b l i g a t e a c i d o p h i l i c nature of T.
ferrooxidans. Data has been presented which indicates that
H +
i s taken up during iron oxidation by the c e l l s and i t has
been postulated that H +
i s an e s s e n t i a l nutrient for the
bacterium. However, more d e f i n i t i v e experiments w i l l be
required to further elucidate the role of hydrogen ions in
the metabolic processes of the a c i d o p h i l i c Thiobacilli.

V. REFERENCES

1. Apel, W.A., P.R. Dugan, J.A. F i l p p i , and M.S. Rheins,


Appl. Environ. Microbiol., 32, 159 (1976).
2. Beck, J . V . , J. Bacteriol., 79, 502 (1960).
3. Beck, J . V . , and F.M. S h a f i a , J. Bacteriol., 88, 850 (1964).
4. Blaylock, B.A., and A. Nason, J. Biol. Chem., 238, 3453
(1963).
5. Bodo, C.A., and D.G. Lundgren, Can. J. Microbiol., 20,
1647 (1974).
6. Carpenter, L.V.,and L.K. Henderson, Res. B u l l . #10.
Eng. Experim. S t a t i o n , U. of West V i r g i n i a (1933).
7. Colmer, A.R., K.L. Temple, and M.E. Hinkle, J. Bacteriol.,
59, 317 (1949).
8. Dewey, D.L. and J . Beecher, Radiation Research, 28, 289
(1966).
9. D i n , G.A., I . S u z u k i , H. Lees, Can. J. Biochem., 45,
1523 (1967).
10. Dugan, P.R., Ohio J. Sci., 75, 266 (1975).
11. Howard, Α., and D.G. Lundgren, Can. J. Biochem., 48, 1302
(1970).
Basic Microbial Studies Applied to Leaching 59

12. Lees, H., S.C. Kwok, and I . S u z u k i , Can. J. Microbiol.,


15, 43 (1969).
13. M i t c h e l l , P., Biol. Rev., 4 1 , 445 (1966).
14. M i t c h e l l , P., J. Bioenergetics, 3, 5 (1972).
15. Noguchi, Α . , Μ. Takama, Τ. S e k i g u c h i , and Y. Nosoh,
Agric. Biol. Chem., 4 1 , 451 (1977).
16. S i l v e r , Μ., and D.G. Lundgren, Can. J. Biochem., 46, 1215
(1968).
17. Silverman, M.P., and D.G. Lundgren, J. Bacterid., 77,
642 (1959).
18. Silverman, M.P., and D.G. Lundgren, J. Bacteriol., 78,
326 (1959).
19. Tabita, R., M. S i l v e r , and D.G. Lundgren, Can. J. Biochem.,
47, 1141 (1969).
20. Tuovinen, O.H., and D.P. K e l l y , Int. Met. Rev., 19, 21
(1974).
21. T u t t l e , J . H . , and P.R. Dugan, Can. J. Microbiol., 22, 719
(1976).
22. T u t t l e , J . H . , P.R. Dugan, and W.A. Apel, Appl. Environ.
Microbiol., 33, 459 (1977).
23. V e s t a l , R., and D.G. Lundgren, Can. J. Biochem., 49, 1125
(1971).
METABOLIC TRANSITIONS IN CULTURES OF

ACIDOPHILIC THIOBACILLI

Olli E. Tuovinen

U n i v e r s i t y of H e l s i n k i
H e l s i n k i , Finland

Donovan P. Kelly
Crawford S. Dow
Martin Eooleston

U n i v e r s i t y of Warwick
Coventry, England

Differences in the lipopolysaccharide composition and fatty


acid profiles have been reported for acidophilic thiobacilli* The
studies do not generally account for the growth-phase related
changes which have been seen to occur also in the phospholipid
composition. There is no evidence for specific (stimulation of)
phospholipid production during growth on solid substrates. In
Thiobacilitis ferrooxidans a cfiange of the inorganic source of
energy may affect its iron-oxidizing capability and an autotrophy-
heterotrophy transition may result in apparent loss of the iron-
oxidation activity. This may be due to metabolic repression and
current data also suggest that some cultures are heterogeneous on
the basis of their DNA base composition depending on the growth
substrate. The isolation of purely autotrophic and heterotrophic
counterparts from such cultures has not been achieved although the
growth conditions selectively determine the predominance of one
type or another. The close association of mixed populations in-
dicates tight metabolic coupling and interdependence.

61
62 Oilϊ Η. Tuovinen ef al.

I. INTRODUCTION

The t h i o b a c i l l i are a group of chemolithotrophic bacteria


that can obtain energy from the oxidation of inorganic sulphur
compounds. Most of the Thiobacillus species are a l s o autotrophic.
During growth on an inorganic s u b s t r a t e , carbon dioxide i s a s s i m i ­
lated p r i m a r i l y v i a the C a l v i n cycle and the intermediary carbon
metabolism i s predominantly biosynthetic in autotrophic b a c t e r i a .
In some t h i o b a c i l l i t r a n s i e n t mixotrophic conditions may repress
both autotrophic and heterotrophic key enzymes. The t r a n s i t i o n to
heterotrophy involves a change i n the intermediary carbon metabo­
lism so that energy and reducing power can be generated by such
mechanisms as the TCA c y c l e .

The t h i o b a c i l l i e x h i b i t a remarkable d i v e r s i t y of p h y s i o l o ­
gical and biochemical c h a r a c t e r i s t i c s , d i f f e r e n t species being
able to grow at pH values between pH 1 and 10 and being o b l i g a t e l y
chemolithotrophic, f a c u l t a t i v e l y heterotrophic or even chemolitho-
t r o p h i c a l l y heterotrophic. Taxonomically they have been divided
into f a i r l y d i s t i n c t s p e c i e s , but a n a l y s i s of t h e i r DNA base com­
p o s i t i o n has shown that the G + C content ranges over nearly 20 %
between extreme v a l u e s . Such a range i s c o n s i s t e n t with genetic
d i f f e r e n c e s between the various species of such magnitude as to
indicate the group to be made up of d i s t i n c t genera. Examined
c r i t i c a l l y , the only u n i f y i n g feature of the t h i o b a c i l l i is their
a b i l i t y to oxidize inorganic sulphur compounds. Some a c i d o p h i l i c
t h i o b a c i l l i can a l s o oxidize f e r r o u s - i r o n . The best known of
these i s Thiobaoillus ferrooxidans which grows at pH 1 - 4 and can
use i r o n , p y r i t e , soluble or mineral sulphides and other inorganic
S-compounds as energy s u b s t r a t e s . T h i s organism i s of prime im­
portance in many mineral leaching operations. Some work indicates
that T. ferrooxidans can be adapted to heterotrophic growth and
that the a b i l i t y to o x i d i z e iron can be permanently lost a f t e r
adaptation to growth on, for example, glucose. Such a phenomenon
Basic Microbial Studies Applied to Leaching 63

i s p o s s i b l y unique among bacterial adaptation mechanisms and i n -


c i d e n t a l l y deprives T. ferrooxidans of i t s key d i a g n o s t i c charac-
ter: the a b i l i t y to o x i d i z e f e r r o u s - i r o n .

In recent years a number of observations have suggested that


some Thiobacillus species are capable of remarkable p h y s i o l o g i c a l
v a r i a t i o n or that some supposed pure c u l t u r e s are in f a c t c l o s e l y
associated mixtures of organisms possessing d i v e r s e p r o p e r t i e s ,
d i f f e r e n t members of which develop best under d i f f e r e n t e n v i r o n -
mental c o n d i t i o n s . Apparent i n t e r - s p e c i e s changes led Johnstone
et al. (1) to the conclusion that " s i n g l e organisms of some
s t r a i n s of t h i o b a c i l l i can give r i s e to the range of organisms
described" i n t h e i r paper.

I n t h i s paper we s h a l l d i s c u s s v a r i o u s metabolic and s t r u c -


tural aspects r e l a t i n g to a change of substrate and environmental
conditions f o r t h i o b a c i l l i . We s h a l l a l s o review the current e v i -
dence and show additional data f o r the reputed heterogeneity of
c u l t u r e s of a c i d o p h i l i c thiobacilli.

II. MORPHOLOGICAL AND STRUCTURAL FEATURES

Morphological v a r i a t i o n s have been encountered in c u l t u r e s


of i r o n - o x i d i z i n g t h i o b a c i l l i . Under forced aeration the normally
rod-shaped c e l l s develop enlarged coccoid forms ( 2 ) . These mor-
phological a l t e r a t i o n s are r e v e r s i b l e and dependent on the rate
of a e r a t i o n . The enlarged c e l l s have a smaller surface-to-volume
r a t i o and e x h i b i t a decreased i r o n - o x i d i z i n g a c t i v i t y . These ob-
servations may suggest a r e g u l a t o r y mechanism, causing morpholo-
gical v a r i a t i o n s , f o r oxygen uptake coupled with iron oxidation
in response to the oxygen tension in the environment. Morpholo-
g i c a l a l t e r a t i o n s have a l s o been reported i n c u l t u r e s grown on
p y r i t e and ferrous sulphate (3) but these studies are not compa-
rable because of the e n t i r e l y d i f f e r e n t growth c o n d i t i o n s . Mor-
p h o l o g i c a l l y heterogeneous T. ferrooxidans c u l t u r e s have c e l l u l a r
64 Olli Η. Tuovinen e i al.

forms ranging from s i n g l e rods to chains of several c e l l s depend­


ing on the growth stage of the bacteria ( 4 ) .

Autotrophic i r o n - and s u l p h u r - o x i d i z e r s have c h a r a c t e r i s t i c ­


a l l y polyhedral i n c l u s i o n bodies ( 5 , 6 ) . These are electron dense
areas in electron micrographs and have been shown ( i n T. neapoK-
tanus) to contain high ribulose-1,5-biphosphate carboxylase a c t i ­
v i t y (7 - 9} which i s the key enzyme of carbon dioxide f i x a t i o n
via the Calvin c y c l e . The abundance of these i n c l u s i o n bodies,
a l s o c a l l e d carboxysomes, decreases when the bacteria grow mixo-
trophically (10). These i n c l u s i o n s are completely absent in hete­
rotrophic c e l l s . The ribulose-1,5-biphosphate carboxylase a c t i ­
v i t y i s a l s o detected in the soluble cytoplasm f r a c t i o n . Carbo­
xysomes are very f r a g i l e and rupture r e a d i l y during preparation
and p u r i f i c a t i o n which may p a r t i a l l y account f o r the presence of
the soluble ribulose-1,5-biphosphate carboxylase a c t i v i t y unasso-
ciated with carboxysomes. The t r a n s i t i o n of t h i o b a c i l l i from au-
totrophy to heterotrophy seems to r e s u l t v i r t u a l l y in complete
repression of t h i s enzyme ( 1 1 , 1 2 ) . The r e l a t i o n s h i p between c a r ­
boxysomes and the ribulose-1,5-biphosphate carboxylase a c t i v i t y is
a close a s s o c i a t i o n and t h e i r disappearance under heterotrophic
growth conditions may indicate that carboxysomes are the main
s i t e s of carbon dioxide f i x a t i o n i n autotrophic c e l l s .

Another r e a d i l y d i s t i n g u i s h a b l e change takes place when t h i o ­


b a c i l l i are grown h e t e r o t r o p h i c a l l y . The c e l l s develop a granular
appearance due to the formation of poly-3-hydroxybutyrate (PHB)
i n c l u s i o n s which are completely absent in c e l l s grown a u t o t r o p h i -
c a l l y on iron or sulphur compounds ( 5 ) . PHB s y n t h e s i s represents
a deviation in the f a t t y acid metabolism and i s normally triggered
o f f by excess carbon in the medium. The lack of the PHB granules
in autotrophic bacteria may be due to the repression of one or
more of the enzymes of f a t t y acid metabolism involved in the PHB
synthesis. Carbon dioxide f i x a t i o n u t i l i z e s most of the energy
derived from inorganic substrate oxidation and therefore excessive
Basic Microbial Studies Applied to Leaching 65

C0 2 f i x a t i o n leading eventually to PHB formation i s effectively


prevented because of the u n a v a i l a b i l i t y of energy.

III. LIPOPOLYSACCHARIDES, FATTY ACIDS, PHOSPHOLIPIDS

A lipopolysaccharide layer has been established as the main


component of the c e l l envelope c o n s t i t u t i n g about 65 % of i t s dry
weight i n i r o n - o x i d i z i n g t h i o b a c i l l i ( 1 3 ) . Comparative studies
indicate q u a n t i t a t i v e d i f f e r e n c e s in the chemical composition of
lipopolysaccharide f r a c t i o n s i s o l a t e d from autotrophic and hete-
rotrophic T. ferrooxidans (Table I ) . The phosphorus content i s
c h a r a c t e r i s t i c a l l y low i r r e s p e c t i v e of the growth conditions u n l i k e
many other gram-negative heterotrophic b a c t e r i a . Although f e r r i c
iron has been detected as the major trace element in the l i p o p o l y -
saccharide f r a c t i o n i s o l a t e d from iron-grown bacteria (6) the neg-
l i g i b l e phosphorus content established in l a t e r studies (14, 15)
may cast some doubt on the involvement of LPS-phosphate i n f e r r o u s -
iron oxidation at the c e l l envelope in c o n t r a s t to a previous s u g -
gestion ( 6 ) .

The sugar composition i s q u a n t i t a t i v e l y altered i n response


to d i f f e r e n t growth substrates (Table I ) . In sulphur-grown c e l l s
the hexose and heptose content of the lipopolysaccharide f r a c t i o n
i s much lower than in i r o n - or glucose-grown c e l l s whereas the
methyl pentose level i s elevated. 2-deoxy-sugars were p r a c t i c a l l y
absent i n a l l three types of l i p o p o l y s a c c h a r i d e s . However, one
should be cautious in concluding chemical comparisons since d i f -
ferent studies indicate wide v a r i a t i o n depending on the growth
c o n d i t i o n s and a n a l y t i c a l procedures employed. The h i g h protein
l e v e l s of the lipopolysaccharide f r a c t i o n s (Table 1} indicate r e -
l a t i v e l y unpurified preparations. Immunological d i f f e r e n c e s of
lipopolysaccharides have a l s o been reported f o r the various c u l -
tures of autotrophic and heterotrophic T. ferrooxidans but the
immunodiffusion a n a l y s i s indicated the presence of heterogeneous
components in the material ( 1 4 ) .
66 Olli Η. Tuovinen e£ al.

TABLE I Comparisons of available data on


lipopolysaccharide fractions from autotrophic and heterotrophic
T h i o b a c i l l u s ferrooxidans

LPS isolated from bacteria


2 + grown on
Analysis Fe E> glucose Reference

Sedimentation 105 6
coefficient (S w 2Q) > 280 61; 9.3 128 15

Density in sucrose 1.28


gradient 1.252 1.335; 1.176 1.319; 1.284 15

Protein (%) 2.5 18 25 15

Nitrogen (%) 1.00 1.81 1.55 14

Sugars (%) a
40.8 56.43 32.24 14

Fatty acids (%) 14.2 8.6 13.8 14

'Sum of hexosamine, WO, heptose and methyl pentose.

The f a t t y acid composition of lipopolysaccharides has been


shown to d i f f e r in T. ferrooxidans grown on i r o n , sulphur or g l u ­
cose. Iron-grown bacteria accumulate r e l a t i v e l y more C 14 β-hyd-
roxymyristic acid and C 18:1 f a t t y acid in lipopolysaccharides
when compared with s u l p h u r - or glucose-grown bacteria ( 1 5 ) . The
study a l s o indicates a n a l y t i c a l d i f f i c u l t i e s since the major f a t t y
acid in each lipopolysaccharide preparation remained u n i d e n t i f i e d .

Over 50 % of the f a t t y acid content of the chloroform-metha-


nol extractable l i p i d f r a c t i o n i s composed of C 16:1 and C 18:1
f a t t y acids in each c e l l type ( 1 5 ) . A n a l y t i c a l d i f f i c u l t i e s are
apparent again as there appears to be a poor, i f any, r e l a t i o n s h i p
between the f a t t y acid d i s t r i b u t i o n i n whole c e l l s and extractable
lipids. A prominent f a t t y acid i s C 19 cyclopropane acid found
in T. ferrooxidans ( 1 5 ) , T. thiooxidans (16) and T. novellus ( 1 7 ) .
Basic Microbial Studies Applied to Leaching 67

C 19 cyclopropane acid production i s enhanced i n the presence of


b i o t i n (16, 17^. I t has been suggested (17) that b i o t i n may be a
r a t e - l i m i t i n g compound i n the f a t t y acid s y n t h e s i s which proceeds
via the carboxylation of acetyl-CoA i n v o l v i n g carboxybiotinyl e n -
zyme p r o t e i n s .

In t h i s context one should point out that C 19 cyclopropane


acid l e v e l s are increased i n s t a t i o n a r y phase c e l l s while the pre-
cursor (unsaturated f a t t y a c i d s ) l e v e l s f a l l down ( 1 8 ) . Hitherto
f a t t y acid a n a l y s i s has been c a r r i e d out using s t a t i o n a r y phase
t h i o b a c i l l i and the data do not a c t u a l l y i n d i c a t e the r e l a t i v e
f a t t y acid p r o f i l e s prevalent during growth. V a r i a t i o n s i n tem-
perature, a g i t a t i o n and phosphate level had l i t t l e e f f e c t on the
c e l l u l a r f a t t y a c i d s i n T. novellus whereas major d i f f e r e n c e s o c -
curred with respect to the autotrophic and heterotrophic type of
energy and carbon metabolism ( 1 7 ) . Increased l e v e l s of saturated
f a t t y acids (C 16, C 18) could be found in autotrophic b a c t e r i a .
I t i s emphasized, however, that e n t i r e l y d i f f e r e n t f a t t y acid p r o -
f i l e s have been presented in two studies (16, 19) f o r the same
s t r a i n of T. thiooxidans which may i n d i c a t e that besides the d i f -
ferent a n a l y t i c a l techniques employed other f a c t o r s such as the
age of bacteria may s i g n i f i c a n t l y a l t e r the c e l l u l a r f a t t y acid
distribution. In f a c t , the f a t t y acid data presented f o r T. de-
nitrifioans grown a e r o b i c a l l y and a n a e r o b i c a l l y (19) indicate that
the f a t t y acid p r o f i l e s are not comparable under d i f f e r e n t c u l t u -
ral c o n d i t i o n s and thus may have l i t t l e value in c h a r a c t e r i z i n g
d i f f e r e n t species of t h i o b a c i l l i .

S i m i l a r l y , contradictory reports have been published f o r the


phospholipid metabolism i n d i f f e r e n t s t r a i n s of autotrophic iron-
grown T. ferrooxidans. Phosphatidyl serine was f i r s t reported to
occur i n the outer membrane of the c e l l envelope (20) but was not
detected at a l l i n a l a t e r study of a d i f f e r e n t s t r a i n ( 2 1 ) . The
r e l a t i v e proportions of i n d i v i d u a l phospholipids vary during d i f -
ferent stages of growth ( 2 2 , 23) suggesting a coupling between
68 Olli Η. Tuovinen ei al.

phospholipid s y n t h e s i s and growth-related r e a c t i o n s . Phospholipid


compositions have been characterized in various t h i o b a c i l l i but i t
s t i l l remains to be elucidated how phospholipid metabolism i s r e ­
gulated and altered under various growth conditions i n v o l v i n g i n ­
organic and organic compounds as an energy and carbon source. A
comparative study (24) of Sulfolobus and " F e r r o l o b u s " , both t h e r ­
mophilic and a c i d o p h i l i c autotrophs, indicates somewhat s i m i l a r
l i p i d composition in autotrophic and heterotrophic c e l l s and
points out the presence of g l y c o l i p i d s which have not been de­
scribed in t h i o b a c i l l i . In T. thiooxidans the phospholipid com­
p o s i t i o n changes over the growth cycle (22) which again imposes
l i m i t a t i o n s for comparative evaluations based on published data.
S i m i l a r growth-phase-related t r a n s i t i o n s in the phospholipid pro­
f i l e s have been reported f o r T. neapolitanus ( 2 3 ) . Since phospho­
l i p i d s may have more or l e s s s p e c i f i c i n t e r a c t i o n s with membrane
proteins in v a r i o u s metabolic functions including r e s p i r a t o r y
chain a c t i v i t i e s ( 2 5 ) , metabolic t r a n s i t i o n s are n a t u r a l l y r e f ­
lected in bacterial phospholipid p r o f i l e s . I t has been suggested
that phospholipids are involved as "wetting" agents in the o x i d a ­
tion of sulphur and i n s o l u b l e metal s u l p h i d e s , f a c i l i t a t i n g the
attachment of t h i o b a c i l l i to the s o l i d surface. Current data do
not indicate any s p e c i f i c phospholipid production stimulated by
a solid substrate. Phospholipids detected i n the medium may in
fact represent excretion products r e s u l t i n g from bacterial mem­
brane turn-over and related r e a c t i o n s .

IV. ASSIMILATORY METABOLISM OF NUTRIENTS

T. intermedins i s reportedly unable to u t i l i z e sulphate as


a sulphur source but requires reduced sulphur compounds for hete­
rotrophic growth ( 2 6 ) . In c o n t r a s t , T. ferrooxidans a s s i m i l a t e s
sulphate during growth on f e r r o u s - i r o n and glucose i n d i c a t i n g that
a s s i m i l a t o r y sulphur metabolism i s unaffected by the two d i f f e r e n t
modes of growth ( 2 7 ) . During thiosulphate-dependent growth, s u l -
Basic Microbial Studies Applied to Leaching 69

phate i s not a s s i m i l a t e d and reduced f o r c e l l u l a r biosynthesis


(28). Under these c o n d i t i o n s the sulphur requirement i s p r i m a r i l y
met by the outer S-atom of thiosulphate a f t e r i t s cleavage r e a c ­
tion although the major portion of thiosulphate i s oxidized for
energy. The enzymes mediating sulphate a c t i v a t i o n and reduction
are present i n Τ. ferrooxidans i r r e s p e c t i v e of the three growth
substrates ( 2 9 ) . T h i s suggests a regulatory mechanism for sulphate
a s s i m i l a t i o n by S-intermediates rather than the repression of the
enzymes of the pathway. A l t e r n a t i v e l y , the c e l l wall may have
s i m i l a r s i t e s f o r binding and uptake of sulphate and thiosulphate
which would account for the preferential u t i l i z a t i o n of S from
thiosulphate i n the presence of sulphate.

Nitrogen metabolism has not been studied in d e t a i l i n a c i d o ­


philic thiobacilli. Ammonium i s the preferred nitrogen source but
growth a l s o occurs on amino a c i d s and other organic N-compounds
(30). Use of inorganic n i t r a t e has not been proved c o n c l u s i v e l y
nor has an a s s i m i l a t o r y n i t r a t e reductase been shown. T. neapoli-
tanus can use n i t r a t e as a sole N-source. Dissimilatory nitrate
reduction i s known only in T. denitrificons and Thiobacillus A2.
In the absence of s u i t a b l e N-compounds some s t r a i n s are able to
reduce nitrogen gas to ammonia ( 3 1 , 32) which i s then incorporated
into amino a c i d s . The s h i f t to nitrogen f i x a t i o n i s s t r o n g l y d e ­
pendent on the level of NH^ in the medium. In s i t u studies have
not indicated any nitrogen f i x a t i o n a c t i v i t y in heap leaching
operations (33) p o s s i b l y owing to the presence of minute amounts
of NH^ or because of i r o n - o x i d i z e r s unable to f i x N - g a s .
2

V. OXIDATIVE ACTIVITIES, SUBSTRATE SPECIFICITY AND DNA BASE RATIOS

A common feature of a l l s t r a i n s of T. ferrooxidans i s t h e i r


a b i l i t y to derive energy from the oxidation of f e r r o u s - i r o n . The
original i s o l a t i o n s of autotrophic i r o n - o x i d i z e r s (34 - 36) indi­
cated d i f f e r e n c e s in the a b i l i t y to u t i l i z e inorganic sulphur com­
pounds for energy (T. ferrooxidans, Ferrobacillus ferrooxidans,
70Oll i Η.Tuovine
n ei al.

Ferrobacillus sulfooxidans) . Since their i s o l a t i o n i t has been


shown that these three bacteria do in f a c t o x i d i z e sulphur as well
as thiosulphate and many other inorganic S-compounds a l s o support
t h e i r growth (37 - 4 0 ) . Differences have been reported f o r growth
parameters and the k i n e t i c s of thiosulphate oxidation between the
subcultures of the o r i g i n a l i s o l a t e s of Τ. ferrooxidans, F. ferro­
oxidans and F. sulfooxidans (38, 39) but they do not warrant spe­
cies differentiation. This view i s further supported by the s i ­
m i l a r i t y of the DNA base r a t i o s (Table I I ) which f e l l within the
range of 52.5 - 53.6 for the three organisms ( 3 8 ) . As a r e s u l t
of the reevaluation of the inorganic sulphur compound o x i d i z i n g
a c t i v i t i e s of the three i s o l a t e s of i r o n - o x i d i z e r s they are now
a l l labelled as s t r a i n s of T. ferrooxidans ( 4 0 ) .

The t r a n s i t i o n of T. ferrooxidans to growth on thiosulphate


has been shown to take place a f t e r a d i s t i n c t adaptation period
(41). I t i s p o s s i b l e that adaptation from iron to thiosulphate
involves changes in membrane permeability for substrate t r a n s l o ­
cation and enzymic induction. With the exception of the t h i o s u l ­
phate oxidase the enzymes mediating the oxidation of thiosulphate
are present a l s o in iron-grown T. ferrooxidans ( 2 9 ) . Naturally,
other changes such as those associated with electron t r a n s f e r com­
ponents accompany t h i s kind of metabolic s h i f t but have not been

TABLE II Base composition of DNA of


T h i o b a c i l l u s ferrooxidans, F e r r o b a c i l l u s ferrooxidans
and F. s u l f o o x i d a n s
a

Organism G+C %

T. ferrooxidans 52.5 ± 0.57


F. ferrooxidans 53.6 ± 0.63
F. sulfooxidans 52.9 ± 0.71

a'Data from reference (38).


Basic Microbial Studies Applied to Leaching 71

f u l l y characterized h i t h e r t o . Iron-grown T. ferrooxidans u s u a l l y


adapts more r e a d i l y to growth on tetrathionate or elemental s u l -
phur; t h e i r o x i d a t i o n does not n e c e s s a r i l y involve thiosulphate
oxidase but does, by analogy, imply changes i n membrane permeabi-
l i t y and other phenomena related to substrate binding and uptake.

After continued growth on thiosulphate the a b i l i t y to grow


on iron gradually declines ( 4 2 ) . T h i s may indicate s l i g h t r e p r e s -
sion by t h i o s u l p h a t e , or i t s intermediates, of enzymes mediat-
ing f e r r o u s - i r o n o x i d a t i o n . A l t e r n a t i v e l y , while eliminating the
p o s s i b i l i t y of a contaminating organism, the decline may r e f l e c t
increased s e n s i t i v i t y of thiosulphate-grown c e l l s to f a c t o r s p r e -
v a i l i n g during growth on f e r r o u s - i r o n . T h i s phenomenon has not
been observed in tetrathionate-grown c e l l s of T. ferrooxidans
from batch (42) or chemostat c u l t u r e s .

D i f f e r e n t s t r a i n s of T. ferrooxidans vary in t h e i r a b i l i t y
to o x i d i z e metal sulphides ( 4 3 ) . Unfortunately, the r e s u l t s are
not comparable since d i f f e r e n t s t r a i n s , rather than only one grown
with v a r i o u s s u b s t r a t e s , were tested f o r oxygen uptake and carbon
dioxide f i x a t i o n a c t i v i t i e s coupled with metal sulphide o x i d a t i o n .
Adaptation to f a c t o r s l i k e l a t t i c e s t r u c t u r e and metal toxicity
v a r i e s in d i f f e r e n t s t r a i n s of T. ferrooxidans and may thus i n -
fluence the amenability of metal sulphides to b a c t e r i a l o x i d a t i o n .
There i s c u r r e n t l y some evidence that T. ferrooxidans may o x i d i z e
selenide (44) and cuprous compounds (45 - 48) f o r energy. Their
bacterial o x i d a t i o n , p a r t i c u l a r l y that of C u , i s l i k e l y to be
+

associated with p a r t i c u l a r enzyme reactions to introduce electrons


into the r e s p i r a t o r y chain and t r a n s f e r them to oxygen with
coupled energy generation. T h i s i s indicated by the d i f f e r e n t
redoxpotentials of F e / F e 2 + 3 +
(+770 mV) and C u / C u + 2 +
(-153 mV).
The bacteria may have a mechanism of a l t e r i n g substrate redoxpo-
t e n t i a l f o r metabolic coupling by way of c h e l a t i o n and a s s o c i a t i o n
of l i p i d material as suggested p r e v i o u s l y ( 4 9 ) .
72 Olli Η. Tuovinen ef al.

A c i d o p h i l i c t h i o b a c i l l i have been demonstrated to be capable


of growing h e t e r o t r o p h i c a l l y on organic compounds ( 1 2 , 50 - 5 4 ) .
I t i s well established that major changes occur i n the pattern of
carbon metabolism on t r a n s i t i o n to heterotrophy. These have been
summarized at length elsewhere ( 1 1 , 12, 5 1 , 5 2 ) . The t r a n s i t i o n
from an inorganic substrate to an organic compound i s r o u t i n e l y
brought about by increasing gradually or stepwise the concentra­
t i o n of the organic substrate at the expense of the inorganic
energy source. In T. ferrooxidans the t r a n s i t i o n to heterotrophy
was believed to be an i r r e v e r s i b l e process leading to complete
l o s s of i r o n - o x i d i z i n g a c t i v i t y ( 5 3 , 5 4 ) .

Various arguments have been put forward to explain t h i s phe­


nomenon. I t may be that the derepression of i r o n - o x i d i z i n g a c t i ­
v i t y i s d i f f i c u l t to demonstrate a f t e r prolonged growth on glucose
(the bacterium may a l s o lack a coupling factor under these c o n d i ­
tions). A l t e r n a t i v e l y , the p o s s i b i l i t y has never been tested r i ­
gorously that some s t r a i n s of T. ferrooxidans may be mixed c u l ­
tures. Heterotrophic v a r i a n t s can s u r v i v e on organic excreta pro­
duced by the autotrophic s t r a i n in a medium containing f e r r o u s -
iron (or inorganic sulphur compound) as an energy source. In such
t i g h t l y coupled mixed populations, the autotrophic bacteria may
be able to survive at low concentrations i n heterotrophic c u l t u r e s .
Their level may be below the detection of standard microbiological
methods such as manometric techniques. The l o s s of iron oxidation
may thus represent a s i t u a t i o n where autotrophic iron-oxidizers
are gradually d i l u t e d to a very low level in the course of sub­
culture on glucose. Two organotrophic s t r a i n s of a c i d o p h i l i c thio­
b a c i l l i have been studied in t h i s respect.

I t has been shown with the heterotrophic s t r a i n KG-4 of T.


ferrooxidans that t r a n s f e r s of t h i s c u l t u r e give r i s e to a u t o t r o ­
phic i r o n - o x i d i z e r s ( 5 5 ) . Colony counting a f t e r growth on g l u c o s e -
and f e r r o u s - i r o n - a g a r indicated that glucose-grown c u l t u r e s con­
tained at most 0.1 % bacteria which could develop c o l o n i e s on
Basic Microbial Studies Applied to Leaching 73

iron-agar. T h i s estimate does not take into account the p o s s i b i -


l i t y of the greater s e n s i t i v i t y of glucose-grown bacteria to the
components of the autotrophic i r o n - c o n t a i n i n g medium as already
suggested f o r the thiosulphate ferrous-iron transition. Autotro-
phic iron oxidation was redeveloped by the bacteria a l s o in l i q u i d
cultures. These observations contradict the o r i g i n a l v e r d i c t i n -
d i c a t i n g i r r e v e r s i b l e l o s s of iron oxidation in the glucose-grown
culture of the KG-4 s t r a i n ( 5 3 ) .

The iron o x i d i z i n g s t r a i n TM of T. ferrooxidans was converted


to heterotrophy and the s t r a i n obtained renamed as T. acidophilus
in view of the reputedly i r r e v e r s i b l e l o s s of i r o n oxidation ( 5 4 ) .
The species d i f f e r e n t i a t i o n was further supported by the d i f f e r e n t
G + C % r a t i o s determined f o r the heterotrophic and o r i g i n a l iron-
grown c u l t u r e . The heterotrophic s t r a i n of T. acidophilus has
been restudied and t h i s presentation gives an account of the work
in progress on the growth and G + C % r a t i o s of T. acidophilus.

The o r i g i n a l glucose-grown culture of T. acidophilus was


kindly provided by Dr. M. S i l v e r . I t was r o u t i n e l y maintained in
shake f l a s k s i n the glucose medium (53) f o r about one and a h a l f
years before the studies described herein were c a r r i e d out. The
c u l t u r e s were adapted to the change of the energy source i n two
ways. F i r s t , c e l l s from 0.1 - 5.0 ml a l i q u o t s of c u l t u r e s were
collected onto membrane f i l t e r s and incubated on agar media u n t i l
v i s i b l e growth was apparent. The membrane f i l t e r s were then t r a n s -
ferred to the respective l i q u i d media. The media have been de-
scribed p r e v i o u s l y ( 4 1 , 53, 5 6 ) . A l t e r n a t i v e l y , 1 ml i n o c u l a t i o n s
were d i r e c t l y transferred to l i q u i d media. The f e r r o u s - i r o n c u l -
ture was further adapted to growth at pH 1.5 to avoid f e r r i c - i r o n
precipitate. Bulk c u l t u r e s on glucose were grown in the medium of
Guay and S i l v e r (54) or on FeSO* at pH 1.4 ( 5 6 ) .
74 Olli Η. Tuovinen et al.

The following t r a n s i t i o n s were achieved:


A Β C D Ε
&LUCMI - ΤΗ I O S U L P H A T E 4FERR0US~-TRON1 -> ΤΗ I O S U L P H A T E - T E T R A T H I ON A T E

F G Η
(GLUCOSE 1
IGLUCOSEI SULPHUR

The DNA base composition was determined f o r the organisms indicated


by the boxes and i s given i n Table I I I .

TABLE III Base composition of DNA of


T h i o b a c i l l u s acidophilus grown on different
substrates (see Textf*

Substrate Buoyant density G+C %

Glucose (A) 1.7227 64.0


Ferrous-iron (C) 1.7168 58.0
Glucose (G) 1.7227 64.0
Glucose (F) 1.7226 63.9

^Bacterial cultures were harvested by centrifugation and washed in


TES buffer, pH 7.1 (0.05 Μ Tris pH 7.2 + 0.001 Μ EDTA + 0.1 MNaCl).
Freshly prepared lysozyme was added to a final concentration of
2 mg/ml and the culture incubated at 30°C for 1 hour. The addition
of sodium lauryl sarcosinate to a final concentration of 2 % (w/v)
brought about lysis. Separation of DNA from cellular debris, RNA
and protein was achieved by centrifuging the lysate at 120,000 g^
for 36 hours in a linear CsCl gradient of mean density 1.71 g/cc .
Fractions were collected and dialyzed overnight at 4 C against
saline citrate (0.15 Μ NaCl + 0.015 Μ sodium citrate adjusted to
pH 7.0). Sample purity was determined by reading A280/A260 and
the DNA concentration calculated from the extinction coefficient.
If required, further purification was achieved by the addition of
1 ml redistilled, water saturated phenol per 2 ml sample, rotating
slowly for 10 minutes, centrifuging and retaining the aqueous phase
which was dialysed against saline citrate. Samples were stored in
saline citrate at 4 C over chloroform. Equilibrium sedimentation
was performed in a Beckman Model Ε analytical ultracentrifuge for
at least 22 hours at 44,000 rev/min and 25°C in a cell with a 12
mm 4° Kel F centre piece. Photographs were taken using ultravio­
let absorption optics and the films examined either by photographic
Basic Microbial Studies Applied to Leaching 75

enlargement or with a Joyce-Loeble recording microdensitometer.


DNA from Escherichia coli £p = 1.7100 g/cc ) and Micrococcus l y s o -
d e i k l i c u s (p = 1.7310 g/cc ) were used as markers and buoyant den­
sities were calculated by the method of Mandel et al. (67). The
buoyant density of each sample in neutral CsCl was determined at
least twice.

The growth studies indicated that T. acidophilus i s able to


grow a u t o t r o p h i c a l l y on t h i o s u l p h a t e , f e r r o u s - i r o n , tetrathionate
2+
and elemental sulphur. After repeated s u b c u l t u r i n g on Fe or
S 0
2 3 , the bacterium i s s t i l l able to r e v e r t back to heterotrophic
growth on glucose.
The DNA base r a t i o s f o r T. acidophilus and T. ferrooxidans
grown on iron are very s i m i l a r , i n d i c a t i n g p o s s i b l e genetic s i m i ­
l a r i t y of the organisms in c o n t r a s t to τ. acidophilus grown on
glucose (Table I I I and I V ) . This view i s a l s o supported by the
resemblance of the radiorespirometric pattern f o r glucose o x i d a ­
t i o n in T. acidophilus (61) and "T. ferrooxidans" ( 5 1 ) .

The change of the DNA base composition of T. acidophilus on


the t r a n s i t i o n from heterotrophy to autotrophy (Table I I I ) indi­
cates c u l t u r e heterogeneity. The d i f f e r e n t G + C r a t i o s may a l s o
indicate the involvement of plasmid DNA in the metabolic t r a n s i ­
t i o n between heterotrophy and autotrophy but t h i s p o s s i b i l i t y is
c u r r e n t l y not supportable as the changes are so large and DNA he­
terogeneity was not observed. The p r o b a b i l i t y of such a big
change in plasmid DNA i s extremely u n l i k e l y although extrachromo-
somal DNA has not been reportedly studied i n t h i o b a c i l l i .

Mixed c u l t u r e s may provide explanation f o r the heterotrophy -


autotrophy interconversions accompanied by G + C % changes. The
heterotrophic counterpart may s u r v i v e on the organic excreta i n
the autotrophic medium as already pointed out. A number of meta­
b o l i c products have been i d e n t i f i e d in the spent autotrophic me­
dium of T. ferrooxidans ( 6 2 , 6 3 ) . I f there are two types of bac­
t e r i a present i n the heterotrophic c u l t u r e of Τ. acidophilus* then
the i r o n - o x i d i z i n g autotrophic types must a l s o be able to maintain
76 Olli Η. Tuovinen e£ al.

TABLE IV Base composition of DNA of


T h i o b a c i l l u s ferrooxidans and T. acidophilus

Substrate G+C % Reference

A. T. FERROOXIDANS
Fe 2+
56.2 58 a

56.6 59 a

57 60 b

58 M. Mandel
58.3 This study

58.7 This study


PbS 53.6 58 a

54.4 S9 a

CuFeS 2 59.9 58 a

60.1 59 a

Glucose 59 This study^

Β. T. ACIDOPHILUS
Glucose 63.0 54a

Sulphur 63.2 54a

^Mean of various methods.


Personal communication.
^Experimental details described in Footnote to Table III.
Strain KG-4; DNA was extracted by J.F. Jackson (60) and the base
composition determined by M. Mandel.

a reasonable growth rate in the glucose medium as otherwise they


would be d i l u t e d out in the heterotrophic medium in the course of
subculturing. The l o s s of the i r o n - o x i d i z i n g a b i l i t y in the hete­
rotrophic T. acidophilus - p r e v i o u s l y a l s o shown in T. ferrooxidans
- may not s o l e l y be due to repression of the enzymes mediating the
oxidation of inorganic substrates but may rather suggest the pre­
dominant s e l e c t i o n of the heterotrophic counterpart since the t r a n ­
s i t i o n involves a d i s t i n c t change in the DNA base r a t i o . On the
other hand, i t i s evident that the autotrophic population i s ex­
tremely low in the heterotrophic culture of T. acidophilus since
Basic Microbial Studies Applied to Leaching 77

s a t e l l i t e bands of DNA were not observed and since the i r o n - o x i d i z ­


ing a c t i v i t y i s below the l i m i t of detection by manometric t e c h n i ­
ques in the glucose-grown c u l t u r e s . I t i s not known what a c t u a l l y
accounts for the maintenance of the autotrophic population in the
glucose-grown culture and further studies are c l e a r l y required to
e s t a b l i s h the cause of the t i g h t l y coupled T. ferrooxidans/T. aci­
dophilus mixture.

T r a n s i t i o n s of an autotrophic s t r a i n of T. ferrooxidans be­


tween tetrathionate and f e r r o u s - i r o n did not a f f e c t the DNA base
composition (Table V) suggesting that these growth conditions do
not r e s u l t i n s e l e c t i v e changes of the predominant population.

TABLE V Base composition of DNA of


T h i o b a c i l l u s ferrooxidans grown on
tetrathionate and ferrous-iron 2

Substrate Buoyant density G+C %

A. Tetrathionate 1.7176 58.8


B. Ferrous-iron (from A) 1.7171 58.3
C. Tetrathionate (from Β) 1.7166 57.8

DNA extracted and analyzed as described in Footnote to Table III.

I t i s noteworthy that the KG-4 s t r a i n of T. ferrooxidans (53)


has a G + C % of 59 (Table IV) when grown on glucose, i n d i c a t i n g
that at l e a s t some s t r a i n s of T. ferrooxidans are indeed capable
of true adaptation to heterotrophy. The Τ. ferrooxidans/T. acido­
philus mixture would always give r i s e to an i r o n - o x i d i z i n g T. fer­
rooxidans population (G + C 56 - 58 %) when grown on i r o n , but
would equally always give r i s e to a predominantly T. acidophilus
population (G + C 63 - 64 %) on glucose even if the T. ferrooxidans
consortant can grow on glucose, since the T. acidophilus organisms
must have a s e l e c t i v e advantage due to higher s p e c i f i c growth rates
on the concentration of glucose s u p p l i e d . Thus in batch culture
78 Olli Η. Tuovinen ef al.

both organisms would be expected to p e r s i s t during prolonged sub­


culture. In a g l u c o s e - l i m i t e d chemostat, however, T. ferrooxidans
would q u i c k l y be eliminated i f i t was s o l e l y competing with T. aci­
dophilus f o r glucose.

We must conclude that a c i d o p h i l i c t h i o b a c i l l i are e i t h e r ca­


pable of unusual p h y s i o l o g i c a l adaptation with changes in the ge­
nome DNA, or more l i k e l y , that there e x i s t mixed populations at
l e a s t in some cultures so c l o s e l y interdependent that i t i s not
easy to separate them. Not only may there be i n t e r - s p e c i e s changes
with respect to the substrate but a l s o other f a c t o r s may contribute
to the s e l e c t i v e development of mixed populations. An example of
t h i s kind i s the pH-limit for growth often quoted f o r various spe­
c i e s of t h i o b a c i l l i . Our studies show that the heterotrophic c u l ­
ture of T. acidophilus i s capable of adapting to pH-values ranging
from pH 1.5 to 6.5 while growing on glucose. The wide range of
substrates and pH-values s u i t a b l e for growth indicates unusual me­
t a b o l i c and p h y s i o l o g i c a l f l e x i b i l i t y which stretches the s u r v i v a l
of a c i d o p h i l i c t h i o b a c i l l i i n natural environments. I t i s evident
that further work i s required to e s t a b l i s h the nature of the o r g a ­
nisms present in acid leaching environments and the taxonomic r e ­
l a t i o n s h i p s of f a c u l t a t i v e l y heterotrophic a c i d o p h i l i c t h i o b a c i l l i .

VI. REFERENCES

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biol., 24, 201 (1961),
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Basic Microbial Studies Applied to Leaching 79

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23. Agate, A . D . , and V i s h n i a c , W., Arch. Mikrobiol., 89, 247 (1973).
24. Langworthy, T.A., J. Bacterid., 130, 1326 (1977).
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26. Smith, D.W., and Rittenberg, S . C . , Arch. Microbiol., 100, 65
(1974).
27. Tuovinen, O.H., in "Conference Bacterial Leaching", (W. Schwartz,
E d . ) , p. 9. Verlag Chemie, Weinheim, 1977.
28. K e l l e y , B.C., Tuovinen, O.H., and N i c h o l a s , D.J.D., Arch. Mic­
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29. Tuovinen, O.H., K e l l e y , B.C., and N i c h o l a s , D.J.D., Can. J.
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3 1 . Macintosh, M.E., J. Gen. Microbiol., 66, i (1971).


32. Macintosh, M . E . , Proc. Soc. Gen. Microbiol., 4, 23 (1976).
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Micro-environments in Aquatic and T e r r e s t r i a l Systems", (W.E.
Krumbein, E d . ) , in p r e s s . Ann Arbor, Michigan, 1977.
34. Temple, K.L., and Colmer, A . R . , J. Bacterid., 6 2 , 605 (1951).
35. Leathen, W.W., K i n s e l , N.A., and B r a l e y , S.A., j. Bacterid.,
72, 700 (1956).
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37. Colmer, A . R . , J. Bacteriol., 8 3 , 761 (1962).
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39. Bounds, H.C., and Colmer, A.R., Can. J. Microbiol., 18, 735
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43. S i l v e r , M., and Torma, A . E . , Can. J. Microbiol., 20, 141 (1974).
44. Torma, A . E . , and Habashi, F., Can. J. Microbiol., 18, 1780
(1972).
45. N i e l s e n , A.M., and Beck, J . V . , Science, 175, 1124 (1972).
46. Imai, K., Sakaguchi, H., S u g i o , T . , and Tano, T . , J. Perm.
Technol., 5 1 , 865 (1973).
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Chem., 13, 2499 (1974).
48. Lewis, A . J . , and M i l l e r , J.D.A., Can. J. Microbiol., 23, 319
(1977).
49. Tuovinen, O.H., and K e l l y , D.P., Z. Allg. Mikrobiol., 12, 311
(1972).
50. S h a f i a , F., and W i l k i n s o n , R.F., J. Bacterid., 97, 256 (1969).
5 1 . T a b i t a , R., and Lundgren, D.G., J. Bacteriol., 108, 334 (1971).
52. T a b i t a , R., and Lundgren, D.G., J. Bacterid., 108, 343 (1971).
53. S h a f i a , F., B r i n s o n , K.R., Heinzman, M.W., and Brady, J.M., j.
Bacterid., I l l , 56 (1972).
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Basic Microbial Studies Applied to Leaching 81

55. Tuovinen, O.H., and N i c h o l a s , D.J.D., Arch. Microbiol., in


press (1977}.
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(1973).
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Enzymology", ( S . P . Colowick, and N.O. Kaplan, E d s . ) , V o l . 12B,
p. 184. Academic P r e s s , New York, 1968.
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Microbiol. Parasitol. Inf. Dis., 3 , 417 (1975).
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(1976}.
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Microbiol., 53, 53 (1968).
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113, 265 (1977).
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robiol., 3 3 , 459 (1977).
TOXIC METALS IN LEACHING SYSTEMS a

P. R. Norris
D. P. Kelly

U n i v e r s i t y of Warwick, Coventry, England

For most organisms, the most toxic metals released into mine
waters are cadmium, silver, mercury and uranium. However, their
toxicity to acidophilic bacteria involved in leaching of metals
from ores ranges from the relatively non-toxic cadmium to the
very toxic silver. Slight inhibition of growth of Thiobaci11 us
ferrooxidans in ferrous sulphate medium at pH 1.5 was observed
at 1CT M silver nitrate (0.1 ppb silver) with more severe
9

inhibition as the silver concentration increased. Silver


toxicity was alleviated at higher pH but not by addition of
potassium, sodium or other cations to the medium. The toxicity
was related to specific accumulation of silver by the cells.
Serial subculturing of T. ferrooxidans in the presence of silver
enabled selection of a silver-resistant strain with increased
sensitivity to mercury, compared with organisms grown in the
absence of silver. Although T. ferrooxidans andl. thiooxidans
could not grow with silver sulphide as a sulphur source, the
toxicity of silver nitrate or silver sulphide to the organisms in
artificial leaching systems including pyrite or cadmium sulphide
was greatly reduced or insignificant compared to the effect of
silver on iron oxidation in a ferrous sulphate medium.

a
This work was supported in part by Imperial
Chemical Industries (UK) Ltd.

83
84 P. R. Norris and D. P. Kelly

I. INTRODUCTION

Among the factors that could a f f e c t the a c t i v i t y of the


bacteria involved i n mineral leaching i s the p o s s i b l e t o x i c i t y
of some of the metals released from o r e s . Metal t o x i c i t y could
lead to reduced rates of leaching which would be undesirable f o r
an i n d u s t r i a l leach process but b e n e f i c i a l i n reducing metal
contamination of mine drainage waters.

The tolerance of a c i d o p h i l i c b a c t e r i a , notably Thiobaoillus


ferrooxidans, to high concentrations (greater than 10 g/£) of
most metal cations i s well documented (1) and i s e s s e n t i a l for
d i r e c t microbial leaching of o r e s . However, soluble uranium (2)
and thallium (3) can be moderately t o x i c to T. ferrooxidans, and
s i l v e r (4) can be very t o x i c to the organism. Prevention of
growth of T. ferrooxidans i n ferrous i r o n medium by 1 ppm s i l v e r
has led to the suggestion that trace amounts of soluble silver
would adversely a f f e c t the leaching performance of the bacteria
(4).

Of metal cations that may be released in mine drainage or


process waters, mercury, s i l v e r and cadmium are the most toxic
to many organisms, i n c l u d i n g zooplankton ( 5 ) , s h e l l f i s h (6) and
fish (7). Furthermore, contamination of r i v e r s with s i l v e r and
other metals from mining sources can lead to abnormal concentrat-
ions of the metals i n organisms, p a r t i c u l a r l y i n f i l t e r - f e e d i n g
and deposit-feeding s h e l l f i s h (8).

Consequently, the a c t i v i t y of a c i d o p h i l i c bacteria in


s o l u b i l i z a t i o n of these t o x i c metals from ores i s of environmental
and biochemical interest.

The i n i t i a l studies described in t h i s report c h i e f l y


involved T. ferrooxidans, the prevalent organism i n leaching
s t u d i e s , and s i l v e r , the most t o x i c c a t i o n . Comparative s i l v e r
t o x i c i t y i s a l s o described for s u l p h u r - o x i d i z i n g Thiobaoillus
thiooxidans', f o r Leptospirillum ferrooxidans, an i r o n o x i d i z i n g
Basic Microbial Studies Applied to Leaching 85

acidophile which, i n mixed culture with the s u l p h u r - o x i d i z e r s


T. thiooxidans, T. acidophilus or T. organoparus, can release
i r o n from p y r i t e in laboratory cultures more e f f i c i e n t l y than
T. ferrooxidans ( N o r r i s and K e l l y , unpublished); and for a
thermophilic acidophile that o x i d i z e s s u l p h u r , i r o n and p y r i t e .

II. MATERIALS AND METHODS

A. Organisms and Culture Conditions

The following organisms were used: (a) Thiobacillus


thiooxidans ATCC 8085. (b) Thiobacillus ferrooxidans, the Yates
and Nason s t r a i n as described e a r l i e r ( 9 , 1 0 ) . A silver-resist­
ant s t r a i n , designated T. ferrooxidans Ag-R, was obtained by
s e r i a l subculture of T. ferrooxidans i n ferrous i r o n medium with
increasing concentrations of s i l v e r up to 2·10~^Μ AgNOg.
(c) Leptospirillum ferrooxidans (11) kindly provided by
G. A. Z a v a r z i n . (d) The thermophilic i r o n - and s u l p h u r - o x i d i z i n g
Thiobacillus-Mke bacterium i s o l a t e d from I c e l a n d i c thermal
springs (12).

For growth on ferrous i r o n , the medium contained (g/&):


K HP0 , 0.4;
2 4 ( N H ) S 0 , 0.4;
4 2 4 M g S 0 - 7 H 0 , 0.4;
4 2 FeS0 4 7H 0, 2

27.8 ( 1 0 " Μ ) .
Ί
K HP0 was replaced by ( N H ) H P 0 ( 0 . 4 g / £ ) to give
2 4 4 2 4

a K-deficient medium. The pH was adjusted to 1.5 with H S 0 2 4 for


growth of T. ferrooxidans, unless stated otherwise i n the t e x t .
The standard medium was d i l u t e d 1:1 with d i s t i l l e d water and
readjusted to pH 1.5 f o r growth of L. ferrooxidans. Yeast extract
(0.02% w/v) was added to the medium for growth of the thermophilic
bacterium. For growth of T. ferrooxidans on p y r i t e , the F e S 0 4

was replaced by 10 g T h a r s i s pyrite/£ and the pH was adjusted


to 2.0. Sulphur medium contained ( g / £ ) : K H P 0 , 3.0;
2 4 (NH ) S0 , 4 2 4

3.0; MgS0 -7H 0, 0.5;


4 2 C a C l . 2 H 0 , 0.25;
2 2 FeS0 -7H 0, 0.01;
4 2

S ( f l o w e r s ) , 5.0. The pH was adjusted to 3.0 with H S 0 . 2 4 Silver


sulphide ( A g S ) and cadmium sulphide (CdS) were added (5g/£) i n
?
86 P. R. Norris and D. P. Kelly

place of or with elemental S as r e q u i r e d . FeSO. and p y r i t e media


2+
were inoculated ( 1 % v/v) from FeSO^ grown (100% Fe oxidized)
cultures. S medium was inoculated ( 1 % v/v) from S grown cultures
i n which the pH had f a l l e n from 3.0 to 1.5. T. ferrooxidans,
L. ferrooxidans and T. thiooxidans were incubated at 30°C and the
thermophile at 50°C i n a l l growth and t o x i c i t y experiments u s i n g
100 ml of medium i n 250 ml Erlenmeyer f l a s k s on an o r b i t a l
shaker at 200 rpm. Larger volumes of T. ferrooxidans cultures
to provide c e l l s f o r metal accumulation experiments were grown
at 30°C in 600 ml of medium i n 2 £ f l a s k s on an o r b i t a l shaker.
2+
Growth on FeSO. medium was followed as Fe oxidation after
2+
determination of residual Fe with eerie sulphate and 1 , 10-
phenanthroline-ferrous sulphate complex s o l u t i o n as i n d i c a t o r .
Growth on S medium was estimated as acid production by measuring
culture pH. Growth on p y r i t e was followed as culture pH and
release of s o l u b l e Fe.
B. S i l v e r Accumulation Experiments
2+
T. ferrooxidans was harvested from FeSO^ medium when Fe
oxidation was complete, washed by resuspension and f i n a l l y
resuspended i n d i s t i l l e d water adjusted to pH 1.5 with H^SO^,
using an optical d e n s i t y - c e l l dry weight c a l i b r a t i o n curve to
give a cell suspension of 0.05 mg dry weight/ml. Suspensions
(25 ml) were mixed with F e S 0 4 (25 ml, 1 0 Μ at pH 1.5) or H S 0
-1
2 4

(pH 1.5) i n 250 ml Erlenmeyer f l a s k s . The f l a s k s were placed i n


a shaking water bath at 30°C for 30 mi η before adding AgNO^.
Other additions and AgNOg concentrations are indicated i n the
text. Samples (4 ml) were taken at i n t e r v a l s and f i l t e r e d through
0.6 ym pore s i z e membrane f i l t e r s (Sartorius). C e l l s were
washed immediately on f i l t e r s with 8 ml H^SO^ (pH 1.5). Filters,
i n c l u d i n g control f i l t e r s washed with H^SO^, were retained for
metal analyses and, where appropriate, f i l t r a t e s were assayed
for F e , 2 +
Basic Microbial Studies Applied to Leaching 87

C. Metal Analyses

F i l t e r s and c e l l s were boiled i n 10 Μ HNO^ to release c e l l -


associated metals. Samples were d i l u t e d with d i s t i l l e d water,
centrifuged to remove any undigested m a t e r i a l , and supernatants
assayed for Ag, Cu, Mg and Κ by atomic absorption
spectrophotometry. Quantities of cell-accumulated Ag and Cu and
residual cell c a t i o n s , Mg and K, are expressed as nmol/ml cell
suspension. Ag, Cd and Fe released from sulphides i n growth
experiments were a l s o determined by atomic absorption
spectrophotometry.

III. RESULTS
2+
A. E f f e c t of S i l v e r and Other Metals on Fe Oxidation
AgN0 at 1 0 " M , 5·10" Μ and 10" M caused 1 , 5 and 11 h
3
9 9 8

extensions r e s p e c t i v e l y to the lag phase of τ. ferrooxidans


cultures before subsequent growth proceeded almost at the control
2+
growth rate i n absence of Ag with a doubling time f o r Fe
oxidation of 8.5 h. No growth occurred after a d d i t i o n of 10~^M
AgN0~ (0.01 ppm A g ) . The period of i n h i b i t i o n was not affected
-2
by the presence of 5·10 Μ KHSO^ or NaHSO^ i n the medium but was
reduced at higher pH. A lag phase of 15 h after addition of
5·10~ Μ AgN0 at pH 1.7 was reduced to 1 h at pH 2.0.
8
3 Ag
t o x i c i t y to T. ferrooxidans and T. ferrooxidans Ag-R, an Ag-
resistant strain, is illustrated in F i g . l .
I n h i b i t i o n of growing T. ferrooxidans cultures was
investigated with a d d i t i o n of AgNO^ (arrowed) when 8-10% of medium
Fe 2 +
had been oxidized i n K - d e f i c i e n t and K - or N a - excess
+ + +

media ( F i g . 2 ) .
Growth was severely i n h i b i t e d by 10~^M AgNO^,
-2 +
but resumed after 100 h i n the presence of 5·10 Μ Κ and after
120 hours in K - d e f i c i e n t media. A s i m i l a r s l i g h t a l l e v i a t i o n of
+

-2 +
growth i n h i b i t i o n occurred i n the presence of 5·10 Μ Na (data
not shown).
88 P. R. Norris and D. P. Kelly

100 h

50 100 150 200


Time ( h )
Fig. 1. Effect of Ag on growth of T. ferrooxidans (open
symbols) and T. ferrooxidans Ag-R (closed symbols) in FeS0 4

medium. Control growth (Ο · ) and growth after addition of


Λ

4-10~ M
8
(V)> 6-10~ M 8
(<>), 8*10~ M 8
(Δ,Α^ 10 'Μ (m) and
2-10~ M
7
(1) AgN0 .3

Addition of 5 g Ag S/£ FeSO^ medium at pH 1.5 prevented


2

growth of T. ferrooxidans. A soluble Ag concentration of 1.3 x


-fi
10" Μ (0.14 ppm) had been reached i n the medium after incubation
at 30°C f o r 200 h.
AgNOg caused an extension of the lag phase preceding growth
of the thermophile at 50°C. Onset of growth was delayed f o r 6 h
and 15 h by 10~^Μ and 10~Sl AgNO^ r e s p e c t i v e l y ; subsequent
growth occurred as i n the absence of Ag with a doubling time
2+
for Fe oxidation of 4 h.
Basic Microbial Studies Applied to Leaching 89

1 1 1 ι 5*
75 100 125 150
Time ( h )
Fig. 2. Inhibition by AgNO? (10~ Μ Δ; 10~ Μ,Ώ;
8
Λ 10~ M,V )
7 6

ofl. ferrooxidans (control growth Ο) growing in K-deficient


Λ

FeS0 medium (A) and in FeSO medium plus 5*10 Μ KHS0 (B).
4 d 4
90 P. R. Norris and D. P. Kelly

Leptospirillum ferrooxidans grew slowly i n FeSCL medium


2+
(doubling time for Fe oxidation of 24 h) and only after several
days l a g . Growth of the organism has not yet been f u l l y
characterized but i t appeared l e s s s e n s i t i v e to Ag than
T. ferrooxidans or the thermophile with 10" M AgNOg causing a 32 h
6

extention of the lag phase ( i . e . l e s s than 1.5 χ the generation


time of the organism) before growth at the control r a t e .
Only mercury (Hg) and gold (Au) of other metals tested
showed a level of t o x i c i t y approaching that of Ag to
T. ferrooxidans. Au (added as sodium chloroaurate,
Na[AuCl 1.2H20) i n FeSO^ medium caused extended lag phases before
4

growth approximately 20% slower than growth i n i t s absence.


Maximum i n h i b i t i o n occurred with 10" M Au;7
less inhibition
occurred with 10"^M and 10~^M and higher concentrations at
-4
which Au p r e c i p i t a t e d . Addition of 10 Μ Au resulted i n a lag
phase shorter than that of control cultures but equivalent to
-4 +
that i n the presence of 10 Μ NaCl, i n d i c a t i n g an e f f e c t of Na
or C I " ions rather than of Au. Hg added as H g C l prevented 9

-5
growth at 10 Μ (2 ppm Hg) i n FeSO^ medium. I n h i b i t i o n of
T. ferrooxidans and T. ferrooxidans Ag-R by Hg ( F i g . 3) showed
increased s e n s i t i v i t y of the A g - r e s i s t a n t s t r a i n to the metal.
B. E f f e c t of S i l v e r on Growth i n Sulphur Medium

AgNOg was added to S medium before i n o c u l a t i o n with


T. thiooxidans or Τ. ferrooxidans. Growth, estimated as acid
production, was i n i t i a l l y i n h i b i t e d at the higher Ag
concentrations ( F i g . 4) but eventually proceeded at a s i m i l a r
rate and to a s i m i l a r extent as control growth i n the absence
of Ag.

Samples of c e l l - and S- free medium were prepared by


c e n t r i f u g a t i o n after growth had occurred at a l l Ag concentrations
tested. Only traces of Ag were detected i n supernatant l i q u i d s
whereas residual S i n the culture medium was coloured more
Basic Microbial Studies Applied to Leaching 91

i n t e n s e l y brown as the total Ag concentration increased,


i n d i c a t i n g removal of s o l u b l e Ag by S.

Φ
Ν
TD
Χ
Ο

ο
LO

100
Time ( h )
Fig. 3. Effect of Eg on growth of T. ferrooxidans (open
symbols) and T. ferrooxidans Ag-R (closed symbols) in FeSO^
medium. Control growth (Ο · ) and growth after addition of
3

S*10~ M (Δ,Α^
7
and 10' M (u,M) 6
EgCl . £

C. Release of Metals from Sulphides i n the Presence of S i l v e r


Sulphide

Growth of T. ferrooxidans and T. thiooxidans did not occur i n


mineral s a l t s medium with A g S as the only source of reduced S and
2

there was consequently no release of Ag i n the medium (pH 3 . 0 ) .


Growth of both organisms i n medium to which A g S and elemental S
2

were added was the same as on elemental S alone; pH of


s t a t i o n a r y phase cultures a f t e r 45 days incubation at 30°C
were 1.03 (T. thiooxidans) and 1.22 (T. ferrooxidans). During the
92 P. R. Norris and D. P. Kelly

Ω­

Χ
CL

100 200 300


Time ( h )
Fig. 4. Effect of Ag on growth (culture pH) of
T. thiooxidans (A) andT. ferrooxidans (B) in S medium. Control
growth (O) and growth after addition of 10~ M (£), 5·10~ Μ (Φ)
6 6

and 10" M (Δ) AgN0~.


5
Basic Microbial Studies Applied to Leaching 93

growth of both organisms, but not i n s t e r i l e medium a c i d i f i e d to


the same extent as the growing c u l t u r e s , some A g S became 2

p r o g r e s s i v e l y more f i n e l y d i v i d e d . After 45 days incubation and


24 h s t a n d i n g , up to 1.5 ppm Ag remained i n suspension.
F i l t r a t i o n through 0.45 ym membrane f i l t e r s gave clear f i l t r a t e s
which contained 0.25 μΜ (0.027 ppm Ag) " s o l u b l e 1
Ag from a
T. thiooxidans culture and 0.95 yM (0.1 ppm Ag) ' s o l u b l e ' Ag from
a T. ferrooxidans c u l t u r e . The bulk of the suspended Ag therefore
probably included f i n e l y divided s u l p h i d e , complexes and
p r e c i p i t a t e s with medium constituents such as phosphate, and
p o s s i b l y some c e l l - a s s o c i a t e d Ag.

The e f f e c t of the presence of Ag^S on leaching of another


metal sulphide was tested with CdS. CdS was s u i t a b l e to compare
the reaction of T. thiooxidans and T. ferrooxidans to A g S 2

because release of Cd by both organisms follows the same pattern


and requires the same c o n d i t i o n s , although Cd release i s slightly
f a s t e r during growth of T. thiooxidans ( N o r r i s and K e l l y ,
unpublished). Growth and Cd release required added S , as
described p r e v i o u s l y f o r T. thiooxidans ( 1 3 ) , and release of up
to 90% of Cd from 5 g CdS/£ through the action of both organisms
was not i n h i b i t e d by 5 g Ag S/&. 2

Release of s o l u b l e Fe from p y r i t e ( F e S ) was unaffected by


2

1 0 " ^ M A g N 0 ( 1 ppmAg) when AgNO^ was added to growing cultures


q

of T. ferrooxidans after 10% of a v a i l a b l e Fe was r e l e a s e d , but


-4
was t r a n s i e n t l y i n h i b i t e d by 10 Μ AgNO^ before Fe release
occurred at the control rate i n absence of Ag, and was completely
i n h i b i t e d by 10" M AgNO^.
3

D. Accumulation of S i l v e r by T. ferrooxidans

Addition of AgN0 to washed c e l l suspensions of


Q

2+
T. ferrooxidans o x i d i z i n g Fe resulted i n accumulation of Ag by
2+
the c e l l s and i n h i b i t i o n of o x i d a t i o n . Fe oxidation was
i n h i b i t e d to a l e s s e r extent and l e s s Ag was accumulated by
94 P. R. Norris and D. P. Kelly

T. ferrooxidans Ag-R than by T. ferrooxidans ( F i g . 5 ) .

Time after AgNO^ addition ( min )

2+ —2
Fig. 5. Oxidation of Fe (5.10 Μ FeSO A and accumulation
of Ag at 30°C and pH 1.5 by T. ferrooxidans (and
T. ferrooxidans Ag-R (±) after addition of 10~ M AgW^^P cell
5

suspensions (0.05 mg dry weight/ml) and oxidation of Fe by both


cell suspensions in the absence of Ag ( O).
Basic Microbial Studies Applied to Leaching 95

Further experiments (data not shown) have shown that the


2+
extent of Ag accumulation and rate of i n h i b i t i o n of Fe
oxidation was proportional to the Ag concentration with complete
i n h i b i t i o n occurring w i t h i n 3 h of addition of 10"*M AgNO^.
-4 -5
Addition of cysteine (10 M) before AgNO^ (10 M) prevented
2+
i n h i b i t i o n of Fe o x i d a t i o n but i t s e f f e c t on Ag accumulation
was not clear with retention of more Ag with c e l l s by f i l t e r s
p o s s i b l y r e s u l t i n g from c o l l e c t i o n of an Ag-cysteine p r e c i p i t a t e .
10"^M CuSO^ (6.35 ppm Cu) did not i n h i b i t F e oxidation and did2 +

not a f f e c t the accumulation of Ag from 10" M AgNO^ (1 PpmAg). Cu


5

-4
accumulation from 10 Μ CuSO^ was not measurable ( i . e . l e s s than
0.1 nmol Cu/ml suspension of 0.05 mg cell dry weight/ml).
The e f f e c t of Ag accumulation by T. ferrooxidans on the
l e v e l s of the major c e l l c a t i o n s , Mg and K, i s shown i n F i g . 6.
Mg l o s s from the c e l l s was gradual and almost complete i n 3 h
while approximatley 50% of the cell Κ was l o s t r a p i d l y , the
cell Κ concentration then remaining almost constant.
Further experiments (data not shown) with rapid sampling
have shown that the Κ l o s s occurred w i t h i n 1 mi η of AgN0 3

addition. The Κ l o s s was reduced to 25% of the cell Κ i n the


presence of 5 · 1 0 " Μ KHSO^ while the Ag accumulation and Mg l o s s
2

were unaffected. Ag accumulation was the same in the presence


or absence of FeSO^ but Κ l o s s (75% of cell K) was greater and
the Mg l o s s was more rapid i n the absence of FeSO^.

IV. DISCUSSION

The reported (4) high t o x i c i t y of Ag to T. ferrooxidans has


been confirmed with i n h i b i t i o n of the organism at Ag
concentrations several orders of magnitude l e s s than the
i n h i b i t o r y concentrations of most metal c a t i o n s . The thermophilic
bacterium appeared l e s s s e n s i t i v e than T. ferrooxidans to Ag but
components of yeast extract i n the growth medium probably
complexed some Ag and reduced i t s s o l u b l e concentration, i n a
96 P. R. Norris and D. P. Kelly

3F

Time ( min )
Fig. 6. Cell-associated Ag, Mg and Κ (open symbols) of
T. ferrooxidans (0.05 mg dry weight/ml) at 3(Pc and pH 1.5 after
addition of 10" M AgNO^ in the presence of 5 ΊΟ~ Μ FeS0 ; and
5 2
4

cell-associated Mg and Κ (closed symbols) in the absence of Ag.


Basic Microbial Studies Applied to Leaching 97

s i m i l a r but l e s s e f f e c t i v e manner than the cysteine which


2+
prevented Ag i n h i b i t i o n of Fe oxidation by non-growing
suspensions of T. ferrooxidans. L. ferrooxidans appeared l e s s
s e n s i t i v e than the other organisms but some i n h i b i t i o n of a l l
three organisms at an Ag concentration of 10'^M indicated the
2+
general t o x i c i t y of the metal to Fe oxidizing acidophiles.
Metal t o x i c i t y to microorganisms i s influenced by the
p h y s i o l o g i c a l state of the organisms, the chemical state of the
metal and by the extent of t h e i r i n t e r a c t i o n as governed by
t h e i r environment. The tolerance of acidophiles to most metals
i n low pH media probably r e s u l t s from e f f e c t i v e competition by
H ions for negatively-charged s i t e s at the c e l l surface. Thus,
+

2+ 2+
Cu was not accumulated and did not a f f e c t Fe oxidation by
T. ferrooxidans i n the present experiments, and even at the high
2+ 2+
concentrations of UC^ and Ni required to i n h i b i t
T. ferrooxidans, only small amounts of the metals were
associated with the c e l l s ( 1 4 ) . I n c o n t r a s t , the high t o x i c i t y of
2+
Ag to T. ferrooxidans o x i d i z i n g Fe appeared related to ready
accumulation of the metal by the c e l l s .
After accumulation of Ag by c e l l s , there are several
p o s s i b i l i t i e s f o r d i s r u p t i o n of normal metabolism, i n c l u d i n g the
i n h i b i t i o n of many enzymes and reactions dependent on a c t i v i t y
of t h i o l groups, as Ag forms a strong complex with cysteine ( 1 5 ) .
2+
Exposure of Escherichia coli to Cd r e s u l t s i n s i n g l e strand
breaks i n DNA which are repaired during long lag phases before
c e l l s p r o l i f e r a t e at a normal rate ( 1 6 ) . The s i m i l a r pattern of
Ag t o x i c i t y i n the growth experiments might have resulted from
i n t e r a c t i o n of accumulated Ag and the DNA of T. ferrooxidans. Ag
i s known to bind to DNA in vitro, p a r t i c u l a r l y to a nitrogen of
2+
guanosine bases ( 1 7 ) . I n h i b i t i o n of Fe oxidation of c e l l
suspensions of Τ. ferrooxidans by higher Ag concentrations than
those i n h i b i t i n g growth does not concern such p o s s i b l e d i r e c t
interference with growth and c e l l d i v i s i o n . A metal-
98 P. R. Norris and D. P. Kelly

concentration-dependent m u l t i p l i c i t y of t o x i c actions has


2+
previously been suggested with U0 i n h i b i t i o n of T. ferrooxidans
9
?+ 2+
i n which Fe oxidation was severely i n h i b i t e d by U 0 2

concentrations greater than those required to i n h i b i t C 0 9

2+
fixation (14). Ag i n h i b i t i o n of Fe oxidation and the rapid l o s s
of cell Κ after Ag accumulation might have resulted from i n t e r ­
ference with membrane cation and electron t r a n s p o r t . A
transmembrane pH gradient of T. ferrooxidans i n acid medium, with
an internal pH near n e u t r a l i t y to allow normal metabolism to
f u n c t i o n , could be used to conserve energy with the coupling of
the gradient to ATP s y n t h e s i s v i a a chemiosmotic ATPase reaction
(18). Net entry of protons would be prevented and the pH
gradient maintained by the h a l f - r e a c t i o n of H ions with oxygen +

i n s i d e the c e l l matrix (2e~ + J 0 + 2 H 92H 0) with the other


+
9

2 + 3 +
h a l f - r e a c t i o n (2Fe + 2Fe + 2e") occurring outside the c e l l
matrix. I n h i b i t i o n of the cytochrome-mediated 0^ h a l f - r e a c t i o n
by Ag could i n h i b i t a r e s p i r a t o r y mechanism of eliminating the
protons from the c e l l . Ag +
i s known to i n h i b i t the r e s p i r a t o r y
chain of E. coli with the most s e n s i t i v e s i t e s to i n h i b i t i o n
between the b^-cytochromes and cytochrome a^ ( 1 9 ) . F a i l u r e of
T. ferrooxidans to remove protons entering c e l l s would lead to a
collapse of the pH gradient and the proton motive force would f a l l
to zero. Rapid l o s s of Κ from c e l l s could have followed the
d i s r u p t i o n of an energized membrane s t a t e . Rapid i n h i b i t i o n of
r e s p i r a t i o n of y e a s t by Hg i s followed by a rapid depletion of
the endogenous ATP level and a l o s s of c e l l Κ ( 2 0 ) .
The observed c h a r a c t e r i s t i c s of Ag t o x i c i t y appear s p e c i f i c
compared with the other cations which i n h i b i t T. ferrooxidans.
The t o x i c i t y of Au was p a r t i c u l a r l y concentration-dependent as a
r e s u l t of the unstable nature of the Au complex i n the FeSO^
medium. A s i m i l a r action of Au and Ag has been shown with t h e i r
2+
i n h i b i t i o n of Hg reduction to elemental Hg by E. coli ( 2 1 ) , an
i n h i b i t i o n of s i m i l a r s p e c i f i c i t y to that seen with
Basic Microbial Studies Applied to Leaching 99

T. ferrooxidans i n that other metals were f a r l e s s i n h i b i t o r y than


Au or Ag. S i m i l a r actions of both metals in T. ferrooxidans are
p o s s i b l e , but each probably reached s e n s i t i v e s i t e s by d i f f e r e n t
routes. Au in s o l u t i o n was probably i n the form of (AuCl^OH)"
anions. Oxyanions of arsenate, s e l e n a t e , t e l l u r a t e and molybdate
are more t o x i c than most metal cations to T. ferrooxidans ( 1 ) ,
perhaps through r e l a t i v e ease of a c c e s s , without H +
ion
competition, to c e l l s .
2+
U0 9 t o x i c i t y i s of several orders of magnitude l e s s than
2+
that of Ag and i n the absence of clear UO^ accumulation, i t has
been suggested that the t o x i c i t y , which can be a l l e v i a t e d by l e s s
toxic cations such as Zn, probably involves loose binding of
υθ 2+
at membrane s i t e s (14).
The increased s e n s i t i v i t y of the A g - r e s i s t a n t s t r a i n to Hg
suggests d i f f e r e n t i n t e r a c t i o n s of the metals in i n h i b i t i o n of
T. ferrooxidans.
Thallium (Tl) t o x i c i t y to T. ferrooxidans i s a l l e v i a t e d i n
growth media with high K +
concentrations ( 3 ) , in keeping with T l +

accumulation by microorganisms in competition with K +


uptake v i a
a K +
transport pathway ( 2 2 ) . Although some Ag and Tl compounds
share s i m i l a r p r o p e r t i e s , and Ag +
i s of s i m i l a r i o n i c radius
(1.26 8 ) to K +
(1.33 8 ) and T l +
(1.40 8 ) and of the same charge -
both l i k e l y c r i t e r i a for e f f e c t i v e competition with K +
uptake in
y e a s t and E. coli (22) - there was no evidence to l i n k A g +

t o x i c i t y with K +
metabolism of T. ferrooxidans. In contrast to
i t s e f f e c t on T l +
toxicity, K +
(or N a ) did not a l l e v i a t e
+

extended lag phases before growth i n the presence of Ag, or


a l l e v i a t e t o x i c i t y of Ag added to growing c e l l s and did not a f f e c t
2+
Ag accumulation by non-growing suspensions of Fe - oxidizing
cells.
The mechanism of Ag accumulation by T. ferrooxidans i s not
known but comparison can be made with the accumulation of t o x i c
100 P. R. Norris and D. P. Kelly

non-essential Cd by another Gram-negative organism, E. coli ( 2 3 ) .


The cell Mg and c e l l Κ concentrations of T. ferrooxidans were
s i m i l a r to those of washed, non-growing c e l l suspensions of
E. coli ( 2 2 ) . Unlike uptake of C o 2 +
and N i 2 +
which are
accumulated by an energy-dependent pathway normally associated
with M g 2 +
accumulation, Cd accumulation by E. coli was independent
of an external energy source (glucose) j u s t as Ag accumulation by
T. ferrooxidans was independent of FeSO^, but accumulation of both
cations probably depended on an energized membrane state of the
cells. Cd accumulation experiments used d i f f e r e n t conditions
(pH 6 . 5 , 10 χ metal s a l t concentrations and 10 χ the biomass)
than Ag accumulation experiments with T. ferrooxidans but the
extent of metal uptake by c e l l s (excluding cell surface binding
of C d 2 +
by E. coli) was s i m i l a r i n both cases with up to about
+ 2+
50 ymol Ag or Cd accumulated/g c e l l dry weight. Further
experiments are i n progress with s p e c i f i c i n h i b i t o r s of cation
transport and membrane functions in attempts to define the
mechanism of accumulation of such t o x i c metal c a t i o n s .
I n t e r a c t i o n of Ag and T. ferrooxidans under conditions
where most cations do not a f f e c t the c e l l s could have been
f a c i l i t a t e d by the high a f f i n i t y of Ag f o r t h i o l groups. High
a f f i n i t y f o r sulphur (log Κ of Ag^S ~ - 5 0 ) a l s o explains the
r e l a t i v e n o n - t o x i c i t y of Ag i n S medium. The presence of S , C l "
ions and the higher pH a l l probably contributed to allow growth
and associated leaching a c t i v i t i e s of T. ferrooxidans and
T. thiooxidans i n the presence of greater total Ag concentrations
than would be found i n ore bodies or ore concentrates and
p r o v i d e s , i n comparison with Ag t o x i c i t y in FeSO^ medium, an
example of the influence of the environment on metal toxicity.
The development of an A g - r e s i s t a n t s t r a i n of T. ferrooxidans
suggests that i f bacteria active i n leaching were threatened by
i n c r e a s i n g Ag concentrations, an increase i n tolerance to the
metal could occur. Development of T. ferrooxidans populations
Basic Microbial Studies Applied to Leaching 101

with increased tolerance to UO^ has been attributed to s e l e c t i o n


of mutant organisms which retained r e s i s t a n c e i n the absence of
2+
(2). T. ferrooxidans Ag-R has not been i n v e s t i g a t e d i n the
same d e t a i l . Whether the acidophiles can contribute indirectly
to Ag release from A g S i s being f u r t h e r studied with leaching
9

3+
columns. Chemical oxidation of cinnabar by Fe in acid mine
waters occurs at much greater rates than release of Hg i n t o
s o l u t i o n as a r e s u l t of Hg binding to remaining cinnabar, but Hg
release i s increased by typical mine water l e v e l s of C l " ions
that complex Hg ( 2 4 ) . F e , produced by microbial a c t i o n , could
3 +

contribute to Ag release under s i m i l a r conditions while Ag


accumulation by c e l l s could contribute i n a small way to
m o b i l i z a t i o n of the metal i n t o mine drainage waters.
V. REFERENCES

1. Tuovinen, O.H., Niemeia, S . I . , and Gyllenberg, H.G., Antonie


v. Leeuwenhoek, 37, 489 (1971).
2. Tuovinen, O.H., and K e l l y , D.P., Arch. Microbiol., 9 5 , 153
(1974).
3. Tuovinen, O.H., and K e l l y , D.P., Arch. Microbiol., 9 8 , 167
(1974).
4. Hoffman, L . E . , and Hendrix, J . L . , Biotech. Bioeng. , 18, 1161
(1976).
5. B i e s i n g e r , K.E., and C h r i s t e n s e n , G.M., J. Fish Res. Bd.
Canada, 27, 1227 (1972).
6. Calabrese, Α . , C o l l i e r , R . S . , Nelson, D.A., and Macinnes,
J . R . , Mar. Biol. , 18, 162 (1973).
7. Hale, J . G . , Bull. Environ. Contam. Toxicol., 17, 66 (1977).
8. Bryan, G.W., and Hummerstone, L.G., J. Mar. Biol. Ass. U.K.,
57, 75 (1977).
9. Tuovinen, O.H., and K e l l y , D.P., Arch. Mikrobiol., 88, 285
(1973).
10. Tuovinen, O.H., and K e l l y , D.P., Arch. Microbiol., 9 8 , 351
(1974).
11. Balashova, V . V . , Vedenina, I . Y a . , Markosyan, G.E., and
Z a v a r z i n , G.A., Microbiology, 4 3 , 491 (1973).
102 P. R. Norris and D. P. Kelly

12. Le Roux, N.W., Wakerley, D . S . , and Hunt, S . D . , J. Gen.


Microbiol., 100, 197 (1977).
13. B r i s s e t t e , C , Champagne, J . , and J u t r a s , J . R . , Can. Mining.
Met. Bull. , 64, 85 (1971).
14. Tuovinen, O.H., and K e l l y , D.P., Arch. Microbiol., 9 5 , 165
(1974).
15. Gruen, L.C. Biochim. Biophys. Acta., 386, 270 (1975).
16. M i t r a , R.S., and B e r n s t e i n , I.Α., Biochem. Biophys. Res.
Commun. , 74, 1450 (1977).
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18. Ingledew, W . J . , Cox, J . C . , and H e l l i n g , P . J . , Proc. Soc. Gen.
Microbiol., 4, 74 (1977).
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(1974).
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(1972).
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Microbiol., 110, 279 (1976).
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24. B u r k s t a l l e r , J . E . , Mcarty, P.L., and P a r k s , G.A., Env. Sci.
Technol. , 9 , 676 (1975).
DIRECT OBSERVATIONS OF BACTERIA AND QUANTITATIVE

STUDIES OF THEIR CATALYTIC ROLE IN THE LEACHING

OF LOW-GRADE, COPPER-BEARING WASTE

V. K. Berry
and
L. E. Murr

New Mexico I n s t i t u t e of Mining and Technology


Socorro, New Mexico 87801 USA

Observations have been made of T h i o b a c i l l u s f e r r o o x i d a n s ,


T h i o b a c i l l u s t h i o o x i d a n s , and a high-temperature Sulfolobus-Z-z^e
microorganism attached to mineral-phase surfaces following
various stages of leaching low-grade copper waste ore. Attach-
ment in all cases is observed to be selective, that is bacteria
attach only to sulfide-phase surfaces (principally CuFeS2 and
FeS2)3 but attachment has been observed not to be a requirement
for leaching to occur. The regularity of attachment is much less
for T. ferrooxidans and T. thiooxidans than for the higher-temper-
ature Sulfolobus-Hfce microbe and this seems to be because of the
absence of a flagellum in the case of the latter. Attempts to
relate bacterial attachment on chalcopyrite surfaces to emergence
sites of dislocations and related crystal defects which can cause
local changes in surface stoichiometry (using the transmission
electron microscope) are also described. Direct, systematic ob-
servations and comparison of surface degradation (corrosion) of
FeS2 and CuFeS2 in sterile acid environments and inoculated acid
environments clearly indicate a significant enhancement in leach-
ing rate by bacteria. This bacterial catalysis is shown to be
important in enhanced conversion rates by galvanic processes when

103
104 V. Κ. Berry and L Ε. Murr

FeS2 and CuFeS2 phase regions are in intimate (electrical) con­


tact; and the implications of bacterial catalyzed galvanic con­
version as it relates to sulfide dissemination in large ore
bodies is briefly described.

I. INTRODUCTION

The subject of bacterial leaching has received a great deal


of attention only in recent y e a r s , although bacterial leaching
of s u l f i d e minerals has been occuring for centuries ( 1 ) . Micro­
b i o l o g i c a l leaching i s a process of metal e x t r a c t i o n i n which
certain microorganisms catalyze oxidation r e a c t i o n s . Due to the
great increase in demand f o r metals during t h i s century and the
l i k e l i h o o d of t h i s demand further increasing in the f u t u r e , i t is
necessary to look to the lower-grade deposits as ore resources
f o r the future. Known and a c c e s s i b l e high-grade deposits of many
minerals are now almost depleted. I t i s thus appropriate to
search for t e c h n o l o g i c a l l y and economically f e a s i b l e processes to
recover greater amounts of metals from the lower grade o r e s . It
i s in t h i s context that bacterial leaching and the role of micro­
organisms in a l t e r i n g minerals and metals in s o l u t i o n has assumed
a role of considerable importance. The bacterial leaching pro­
cess i s not only economical as compared to the conventional
methods but i t i s a l s o e s s e n t i a l l y p o l l u t i o n f r e e .

The e a r l i e s t recorded mining a c t i v i t y which can be a t t r i b u t e d


to bacteria involved the recovery of copper from mine waters at
Rio T i n t o , Spain (1) in 1670. The presence of bacteria in the
leaching f l u i d s was not confirmed in that operation u n t i l 1962 (2).
However, in 1922 Rudolf et a l . (3,4) reported the microbiological
leaching of metal s u l f i d e s by an u n i d e n t i f i e d autotrophic bac­
terium. The economic importance of the extraction of metals from
low-grade s u l f i d e - b e a r i n g ores was thus suggested. In 1947,
Colmer and Hinkle (5) f i r s t i s o l a t e d the microorganism, Thioba­
oillus ferrooxidans, from an acid drainage source which was
responsible for the oxidation of s u l f i d e minerals. Bryner et a l .
Basic Microbial Studies Applied to Leaching 105

(6,7) reported on the role of t h i s microrganism in the leaching


of s u l f i d e minerals. However, i t was Malouf and Prater (8) who
s y s t e m a t i c a l l y studied the r o l e played by bacteria i n the a l t e r a ­
t i o n of s u l f i d e minerals.

The chemolithotrophic 1
microorganism T. ferrooxidans can be
found wherever s u l f i d e minerals occur i n an a c i d i c environment.
In the e a r l i e s t reported s t u d i e s of autotrophic b a c t e r i a ,
Winogradsky (9) in 1888 proposed chemolithotrophy. Bacteria use
the energy released from the oxidation of ferrous iron (10)
or other inorganic substrates such as reduced inorganic s u l f u r
compounds ( 1 1 , 1 2 ) . The chemical energy i s converted p r i m a r i l y
by oxidative p h o s p h o r y l a t i o n to adeosine triphosphate (ATP)
(13). ATP plays an important r o l e in the energy metabolism
of these microorganisms as d i s c u s s e d for example in Chap. 1 .

The bacterial leaching of metal s u l f i d e s can be considered


as a biochemical oxidation process which i s catalyzed by micro­
organisms. This process i s represented by the following simple
equation

MS+20 2 Microorqanism S> ^ ( 1 )

where Μ i s i d e a l l y a b i v a l e n t metal.

Microbiological leaching processes involve complex i n s t r u c ­


t i o n s between b i o l o g i c a l (microorganisms), aqueous ( n u t r i e n t s i n
the medium) and s o l i d mineral phases. In order to obtain a
better understanding of the o v e r a l l leaching process and to make
better economical use of t h i s process the i n t e r a c t i o n s between
each of three phases need study.

A process in which microorganisms synthesize their protoplasmic


constituents from inorganic sources and carbon from CO^ and
derive the energy from the oxidation of inorganic compounds.
106 V. Κ. Berry and L Ε. Murr

In the past ten to f i f t e e n years s i g n i f i c a n t research has


been reported on several aspects of bacterial leaching (14-19),
Many l a b o r a t o r y , p i l o t , and f i e l d studies of microbial leaching
of copper minerals have been concerned p r i m a r i l y with determining
the conditions under which bacterial oxidation occurs and then
optimizing the process ( 2 0 - 2 4 ) . There have been studies of the
mechanism of bacterial oxidation of iron o x i d i z i n g bacteria T.
ferrooxidans ( 2 , 25,26) and s u l f u r o x i d i z i n g bacteria Thiobaoil­
lus thiooxidans ( 2 7 , 2 8 ) . Some recent i n v e s t i g a t i o n s have been
directed towards the i s o l a t i o n and development of bacteria able
to metabolize at high temperatures (29-31) ( i n excess of those
temperatures which optimize the growth of T. ferrooxidans and
T. thiooxidans).

One aspect of the bacterial leaching process which has not


been extensively studied i s the attachment c h a r a c t e r i s t i c s of the
bacteria to the mineral surfaces a f t e r microbe/mineral interaction.
The d i r e c t contact mechanism proposed by Silverman (32) has never
been v e r i f i e d by d i r e c t observations even though bacterial a t t a c h ­
ment to mineral surfaces has been described by various authors
(33-36) and observed for a number of systems ( 3 7 - 3 9 ) . Silverman
(32) has shown that the d i r e c t contact mechanism, which requires
physical contact between the bacteria and the s u l f i d e mineral
under aerobic c o n d i t i o n s , i s d i f f i c u l t to demonstrate with iron
containing s u l f i d e minerals such as p y r i t e (FeS2)» bornite
( C ^ F e S ^ and chalcopyrite (CuFeS2). Thus, in the bacterial
leaching of high-grade s u l f i d e minerals and low-grade o r e s , i t is
important to understand the contact or attachment mechanism. In
the case of low-grade ores i t i s of i n t e r e s t to know whether bac­
t e r i a attach s e l e c t i v e l y to s p e c i f i c mineral i n c l u s i o n s , and what
the mechanism and p r i n c i p a l features of attachment include.

Another s i g n i f i c a n t feature of bacterial/mineral interaction


involves the mode and character of bacterial attachment. Bac­
t e r i a l attachment to surfaces has been described as occurring
Basic Microbial Studies Applied to Leaching 107

through a v a r i e t y of h o l d f a s t s , i n c l u d i n g adhesive pi 1i (38,40,41)


as well as surface adhesion ( 4 2 , 4 3 ) . One feature of attachment
which has not been i n v e s t i g a t e d with regard to mineral leaching
with T. ferrooxidans involves the attachment l o c a t i o n . There
have been no attempts to determine i f the attachment s i t e i s a
r e s u l t of random s e l e c t i o n , or whether i t occurs at regions of
s p e c i f i c ion concentration, changed stoichiometry, or strained
regions containing c r y s t a l defects such as d i s l o c a t i o n s . I t may
be of i n t e r e s t to determine whether c r y s t a l defects such as emer-
gence s i t e s of d i s l o c a t i o n s , g r a i n boundaries, c r a c k s , etc.
influence the c h a r a c t e r i s t i c s and degree of attachment in bac-
terial leaching.

Another i n t e r e s t i n g feature in the oxidation of minerals is


the e f f e c t of c r y s t a l structure on the oxidation of these min-
erals. Silverman and E h r l i c h (19) reported that the c r y s t a l
structure of a given mineral i s an important factor in i n f l u e n c -
ing the oxidation of these minerals. They suggested that well
ordered c r y s t a l l a t t i c e s are l e s s s u s c e p t i b l e to oxidation than
imperfect c r y s t a l s t r u c t u r e s . They did not elaborate on t h i s .
There has been no d i r e c t attempt to s y s t e m a t i c a l l y study the
influence of d i f f e r e n t c r y s t a l l o g r a p h i c o r i e n t a t i o n s on the rate
of leaching.

The purpose of t h i s i n v e s t i g a t i o n was to study the mode and


c h a r a c t e r i s t i c s of attachment of bacteria to mineral surfaces
and to e s t a b l i s h a c o r r e l a t i o n , i f any, between the attachment
and c r y s t a l defects ( d i s l o c a t i o n s in p a r t i c u l a r ) in the minerals.
In the case of low-grade o r e s , p r e f e r e n t i a l attachment of bac-
t e r i a to s p e c i f i c mineral surfaces was s t u d i e d . The e f f e c t of
c r y s t a l l o g r a p h i c o r i e n t a t i o n s on the surface c o r r o s i o n (corrosion
features) of s u l f i d e phases in bacterial leaching was a l s o s t u -
died. These studies were performed using transmission and
scanning electron microscopy. Specimen preparation procedures
involved a combination of b i o l o g i c a l and physical science
108 V. Κ. Berry and L Ε. Murr

techniques. The degree of surface c o r r o s i o n with time was s t u ­


died with the scanning electron microscope (SEM) and correlated
with the metals s o l u b i l i z e d (Fe and Cu).

II. MATERIALS AND METHODS

A. Ores and Minerals

The following copper ores were obtained and used in the pre­
sent experiments, i) Phelps Dodge Corporation Ajo waste ore
having chalcopyrite as the p r i n c i p a l copper mineral, i i ) Kenne-
cott Corp., Chino Mines D i v i s i o n , waste copper ore having c h a l -
cocite as the p r i n c i p a l copper mineral being c h a l c o p y r i t e . Be­
sides these o r e s , museum grade specimens of chalcopyrite were a l s o
used. Waste ore from each of the three sources was crushed and
screened to give a -4 to +6 mesh s i z e (3 to 5mm) f r a c t i o n f o r the
leaching experiments.

B. Microorganisms: C u l t i v a t i o n and Harvesting

A Sulfolobus-Mke microorganism described by B r i e r l e y et a l .


(30) and T. ferrooxidans were used in the various s t u d i e s . The
Sulfolobue-Mke c e l l s were cultured in 125 ml Erlenmeyer f l a s k s
containing 75 ml of n u t r i e n t medium reported by Bryner and
Anderson (7) and 0.02% y e a s t - e x t r a c t (see r e f . 30 ). The i n i t i a l
pH of the medium was adjusted to 2 . 5 , and elemental s u l f u r was
used as an energy source. The f l a s k s were incubated at 60°C in a
water bath. An a l i q u o t of the culture was transferred into a new
medium every 20 days to maintain the stock c u l t u r e . The bacteria
free of s u l f u r were harvested from the remaining part whenever
needed, otherwise these were discarded.

Bacterial c e l l s of the Sulfolobus-Mke microorganism free of


s u l f u r were obtained by c a r e f u l l y decanting the medium containing
c e l l s into another f l a s k a f t e r 20 days of growth. Most of the
s u l f u r used as an energy source s e t t l e d to the bottom of the
flask. The decanted medium containing the c e l l s was centrifuged
Basic Microbial Studies Applied to Leaching 109

at 5000 rpm in a S o r v a l l superspeed Model RC2-B c e n t r i f u g e . The


heavier s u l f u r sedimented and the supernatant f l u i d containing
the bacteria were c a r e f u l l y removed so as not to d i s t u r b the
sediment. The decanted f l u i d contained the c e l l s essentially
free of elemental s u l f u r . This f l u i d was stored in a r e f r i g e r a -
tor at « 4°C. The c e l l s remained v i a b l e for s i x days.

Thiobacillus ferrooxidans c e l l s were grown in 125 ml. E r l e n -


meyer f l a s k s containing 75 ml of the n u t r i e n t medium reported by
Silverman and Lundgren using ferrous s u l f a t e as the energy source.
The i n i t i a l pH of the medium was adjusted to 2 . 5 , and the f l a s k s
containing the medium were incubated at room temperature (28°C).
An a l i q u o t of the culture was t r a n s f e r r e d into a f r e s h medium
every 14 days to maintain the stock c u l t u r e . To obtain c e l l s free
of i r o n , c u l t u r e s were grown in 2 £. Fernbach f l a s k s containing
1 £. of the n u t r i e n t medium (44) and ferrous s u l f a t e as the
energy source. The c e l l s were harvested a f t e r 14 days of growth.
To secure c e l l suspensions containing a minimum of p r e c i p i t a t e d
iron the harvesting procedure described by Silverman and Lundgren
(44) was followed. The contents of the Fernbach f l a s k s were cen-
t r i f u g e d ( S o r v a l l superspeed Model RC2-B) at 15000 rpm. The r e -
s u l t i n g paste of c e l l s and p r e c i p i t a t e d iron was suspended in
* 100 ml of cold d i s t i l l e d water ( a c i d i f i e d to pH 2,6 with H 2 S O 4 )

in a 500 ml Erlenmeyer f l a s k and shaken v i g o r o u s l y . The f l a s k


was then allowed to stand in a r e f r i g e r a t o r f o r at l e a s t 6 hours.
The t u r b i d supernatant f l u i d was c a r e f u l l y decanted from the
underlying layer of p r e c i p i t a t e d i r o n . This procedure was repeat-
ed at l e a s t two time. The c e l l s contained in the combined super-
natant were removed by c e n t r i f u g a t i o n at 15,000 rpm. The p e l l e t
was washed with d i s t i l l e d water (pH 2.6) two or three times and
f i n a l l y the p e l l e t containing the c e l l s was brought to a volume of
20 ~ 30 ml with d i s t i l l e d water ( a c i d i f i e d to pH 3.5 with H 2 S O 4 ) .

The clean cell suspensions were stored in a r e f r i g e r a t o r and used


as an inoculam of c e l l s free of i r o n .
110 V. Κ. Berry and L Ε. Murr

C. Experimental Procedures

1. Preparation of Chaloopyrite Thin Sections for Specific


Attachment Studies

Unlike molybdenite which was o r i g i n a l l y used as a t e s t ma­


t e r i a l to study s p e c i f i c bacterial attachment ( 4 5 ) , chalcopyrite
does not have any preferential cleavage p l a n s , although some pre­
ference f o r cleavage along {011} might be expected. Thus in
order to obtain electron transparent thin s e c t i o n s , natural c h a l ­
copyrite (Quebec Canada) was crushed. An optical microscope was
used to i s o l a t e small sections considered to be electron t r a n s ­
parent. These c a r e f u l l y picked thin sections were then examined
in a Hitachi Perkin-Elmer H.U. 200F transmission electron micro­
scope operated at 200 kV, and f i t t e d with a g o n i o m e t e r - t i l t s t a g a
The sections which were electron transparent were then used for
the study. Also small sections (3 to 4 mm on one s i d e ) of the
natural chalcopyrite samples were taken and polished by hand to
thin f l a k e s on a 600 g r i t ( f i n a l p o l i s h ) p o l i s h i n g paper. The
polished sections were washed in d i s t i l l e d water in an u l t r a s o n i c
cleaner (Bransonic 12) to remove debris and a i r d r i e d . The
polished thin sections were used to see bacterial attachment
c h a r a c t e r i s t i c s in the scanning electron microscope. A f t e r pre­
paring a s u f f i c i e n t number of thin chalcopyrite chips having
electron transparent edges, a special immersion apparatus was
devised. P l e x i g l a s s d i s c s 25 mm χ 25 mm were prepared from a 5
mm thick sheet. Small shallow wells about 2 mm in diameter were
d r i l l e d in the p l e x i g l a s s d i s c s . Each d i s c was then attached to
another s i m i l a r d i s c s l i g h t l y bigger but without holes with the
help of two p l e x i g l a s s rods ~ 3 mm in diameter and ~ 12 cm long
at the two diagonal ends. This arrangement was required to keep
the thin sections submerged during incubation and growth of the
microorganisms.

Thin sections of chalcopyrite prepared as described above


were placed in the shallow wells of the immersion apparatus and
Basic Microbial Studies Applied to Leaching 111

the d i s c s were covered with p l a s t i c mesh to prevent them from


f l o a t i n g when immersed in the medium. Two 250 ml Phillips flasks,
each containing 100 ml of n u t r i e n t medium ( 7 ) , were taken and
covered with aluminum f o i l . Two other s i m i l a r f l a s k s with 20 to
30 ml d i s t i l l e d water were kept ready. The immersion assembly
with specimens in place was suspended in these f l a s k s . The water
level was kept at l e a s t 10 mm below the lower surface of the
plexiglass disc. These two f l a s k s were then a l s o covered with
aluminum f o i l . The four P h i l l i p s f l a s k s (the two containing the
medium and the other two containing the immersion assemblies with
chalcopyrite specimens) were s t e r i l i z e d in autoclave at 120°C f o r
15 minutes and then cooled. On c o o l i n g 2 ml of 1% yeast extract
was added to one of the two f l a s k s containing the medium under
asceptic c o n d i t i o n s . This f l a s k was then inoculated with an a l i ­
quot of stock culture of Sulfolobus-λ ike microorganisms. The
immersion device containing the specimens was then transferred to
the inoculated f l a s k . The f l a s k was incubated at 60°C in a water
bath f o r two weeks.

At the conclusion of the incubation period the sample d i s c


arrangement was withdrawn from the medium. The samples were r e ­
moved with a f i n e tweezer (the end of the tweezer s t e r i l ized with
alcohol) and placed in a 1% w/v s o l u t i o n of uranyl acetate f o r at
l e a s t 5 minutes to achieve negative s t a i n i n g of the microbes
which were presumed to have attached to the mineral s u r f a c e s . The
s t a i n i n g of the microbes has to be done to obtain mass-thickness
contrast ( 4 6 ) . The samples a f t e r s t a i n i n g were l i g h t l y r i n s e d in
d i s t i l l e d water on a drop plate two times to wash o f f uranyl
acetate and then d r i e d . The samples with attached, stained
microbes were then examined in the transmission electron micro­
scope operated at 200 kV as described e a r l i e r .

Some of the leached chalcopyrite specimens without s t a i n i n g


with uranyl acetate were mounted on standard c y l i n d r i c a l aluminum
stubs (14 mm χ 10 mm) with s i l v e r conducting p a i n t . These were
112 V. Κ. Berry and L Ε. Murr

then coated with ~ 100 A 60/40 Au/Pd sputtered onto the s p e c i ­


mens mounted on aluminum stubs in a residual vacuum of z
500 mm
Hg (Torr) in a sputtering unit (Commonwealth S c i e n t i f i c Minicoater
Model CSC-100). The specimens were then examined in a H i t a c h i -
Perkin Elmer HHS-2R scanning electron microscope operated at 25
kV a c c e r l e r a t i n g voltage in the secondary electron emission mode.
The SEM i s f i t t e d with an ORTEC e n e r g y - d i s p e r s i v e X-ray micro-
analyser (Model 6200) u t i l i z i n g S i ( L i ) detector for nondispersive
X-ray a n a l y s i s of d i f f e r e n t phases in the specimen. The SEM exa­
mination was done to study the bacterial d i s t r i b u t i o n on the
surface of chalcopyrite and a l s o to study the bacterial morpholo­
gies.

2. Bacterial Attachment in Low-Grade Ore

Waste-ore sections having a mesh s i z e -4 to +6 (3 to 5 mm)


were somewhat randomly selected from Phelps Dodge Corp., Ajo waste
ore containing chalcopyrite as the main copper ore. The waste
ore sections were polished f l a t on one s i d e , the f i n a l p o l i s h was
done on 6 0 0 - g r i t p o l i s h i n g paper. The polished samples were
washed in d i s t i l l e d water in an u l t r a s o n i c cleaner to remove
debris and then a i r d r i e d .

100 ml of nutrient medium (7) was taken in each of f i v e


250 ml. Erlenmeyer f l a s k s . The pH of the medium was adjusted to
2.3 and the f l a s k s were covered with aluminum f o i l . The E r l e n ­
meyer f l a s k s containing the medium were s t e r i l i z e d in steam under
pressure (15 ~ 20 minutes, temp. ~ 120°C). The f l a s k s were c o o l ­
ed and 2 ml of 1% s t e r i l i z e d yeast extract s o l u t i o n was added to
each of the f l a s k s ( f i n a l concentration 0.02%). S i x to eight
polished sections were then transferred to each of the f l a s k s
under aseptic c o n d i t i o n s . The ore sections were not s t e r i l i z e d
to avoid chemical and related a l t e r a t i o n s . The f l a s k s were then
inoculated with an a l i q u o t from Sulfolobus-Mke stock culture and
incubated f o r periods ranging from 2 to 6 weeks at 60°C in a
water bath.
Basic Microbial Studies Applied to Leaching 113

The ore sections were removed from the culture f l a s k s a f t e r


varying periods (2 to 6 weeks) of incubation and were r i n s e d
l i g h t l y in d i s t i l l e d water. This was done to ensure that bacteria
not a c t u a l l y adhering to the mineral surface would not remain at
random l o c a t i o n s as a r e s u l t of surface tension e f f e c t s upon w i t h ­
drawal from the culture medium and a l s o to r i n s e o f f the d i s s o l v e d
s a l t s of the culture medium. The specimens were then dried in
a i r in a covered container for 24 hours.

The dried specimens were mounted on standard aluminum stubs


(14 mm χ 10 mm) with s i l v e r paste. These were then coated with
ο

approximately 300 A 60/40 Au/Pd in a u n i t operated at a residual


a i r pressure of ~ 500 mm Hg.
The specimens were examined in a scanning electron microscope
operated at 25 kV in secondary electron emission mode. Pyrite
and chalcopyrite phase areas in the specimens were i d e n t i f i e d by
s e l e c t i n g Fe-S or Cu-Fe-S regions simultaneously as regions of
i n t e r e s t s in the X-ray energy spectrum of the X-ray analyser
display. The sample was then scanned at low magnifications (100 X
to 500 X) while observing both the secondary electron image and
the associated Fe-S or Cu-Fe-S c h a r a c t e r i s t i c s X-ray map of the
imaged r e g i o n . Phase regions i d e n t i f i e d as Fe-S (Fe$2) or Cu-Fe-S
(CuFeS^) were then s y s t e m a t i c a l l y studied over a ran^e of much
higher magnifications to i d e n t i f y the extent of bacterial a t t a c h ­
ment and to characterize the s e l e c t i v i t y of the attachment.

The experiments described above for the Sulfolobus-Mke micro­


organisms were repeated using a T. thiooxidans in one case and
T. ferrooxidans inoculum in the second case. The two experiments
with T. thiooxidans and T. ferrooxidans were s i m i l a r to that
described above except that no yeast extract was added to the
nutrient medium and the f l a s k s were incubated at room temperature
(~ 28°C).
114 V. Κ. Berry and L. Ε. Murr

3. Surface Reactions at Sulfide Phases

Waste ore sections having a mesh s i z e -4 to +6 (3 to 5 mm)


were randomly selected from crushed and screened Duval Corpora-
t i o n - S i e r r i t a mine waste (chalcopyrite being the main copper
mineral). These sections were polished f l a t on one side on 220
g r i t p o l i s h i n g paper, then s u c c e s s i v e l y to 600 g r i t polishing
paper ( f i n a l p o l i s h ) . The polished sections were washed in d i s ­
t i l l e d water in an u l t r a s o n i c cleaner to remove d e b r i s , a i r d r i e d
and kept in a dessicator for l a t e r use. A l s o -4 to +6 mesh s i z e
ore (~ 200 g.) was given several washes in water to remove loose
dust adhering onto the surface and a l s o a i r dried and stored in a
d i s s i c a t o r for l a t e r use.

A complete ore a n a l y s i s of the S i e r r i t a ore was done. Cop­


per and iron a n a l y s i s of -4 to +6 mesh s i z e S i e r r i t a ore was a l s o
done. The sized ore was taken, washed and dried and crushed to
powder. A known weight of the powdered ore was digested by s t a n ­
dard procedure and brought to a known volume.

Total iron in the s o l u t i o n (digested ore) was analyzed by


t i t r a t i o n a g a i n s t standardized potassium dichromate s o l u t i o n as
t i t r a n t using diphenylamine sulfonate as i n d i c a t o r ( 4 7 ) . Copper
a n a l y s i s was done by atomic absorption spectroscopy using a Per-
kin Elmer Model 303 atomic absorption spectrophotometer.

a. Flask Leaching. 250 ml. Erlenmeyer f l a s k s containing


100 ml. nutrient medium (47) with pH adjusted to 2.0 were taken.
The f l a s k s were covered with aluminum f o i l and s t e r i l i z e d in an
autoclave (120°C, 15 minutes). 10 grams of e a r l i e r washed and
dried -4 to +6 mesh s i z e (3 to 5 mm) ore including 6 to 8 p o l i s h ­
ed s l i c e s were accurately weighed and t r a n s f e r r e d to each of the
f l a s k s on c o o l i n g . Two f l a s k s were prepared f o r each sample,
one used as a s t e r i l e control and the second was inoculated with
1 ml. of Sulfolohus-λ ike microorganisms. C e l l s free of s u l f u r
were used in the inoculum. The f l a s k s were incubated at 55+2°C
Basic Microbial Studies Applied to Leaching 115

in a water saturated a i r incubator ( P r e c i s i o n Thelco Model 6,


temperature range 65°C) with no a g i t a t i o n . Eight s e t s , each set
containing two f l a s k s ; one s t e r i l e and one inoculated with
microbes were used in the experiment. Any l o s s in water due to
evaporation during the course of t h i s experiment was made up by
adding s t e r i l e d i s t i l l e d water. Samples were taken out at weekly
i n t e r v a l s , up to eight weeks. At the end of the weekly p e r i o d s ,
the polished s l i c e s were removed from the medium, r i n s e d l i g h t l y
in d i s t i l l e d water and a i r dried f o r at l e a s t 24 hours. The long
drying period was u t i l i z e d to reduce morphological d i s t o r t i o n s by
surface tension e f f e c t s during drying because a c r i t i c a l point
drying apparatus was not a v a i l a b l e f o r t h i s study. The medium
3+ 2+
was removed for the chemical a n a l y s i s of Fe , Fe and Cu con-
2
tent in the s o l u t i o n and the bacterial count (MPN) .
The f l a t specimen, a f t e r drying were mounted on aluminum
ο

stubs with s i l v e r paste and coated with approximately 300 A layer


of Au/Pd in a sputtering u n i t . The specimens were then examined
in the scanning electron microscope at 25 kV f i t t e d with an ORTEC
energy d i s p e r s i v e X-ray analyzer. The l i q u i d medium was analyzed
3+ 2+
for Fe and Fe by t i t r a t i o n method using standarized potassium
dichromate s o l u t i o n as t i t r a n t and diphenylamine sulfonate as i n ­
dicator ( 4 7 ) . Cu was analyzed by atomic absorption spectroscopy.
The bacterial count in the medium was done using MPN (most pro-
2
bable number) estimates procedure ( 4 8 ) . This experiment was
repeated with the f l a s k s incubated at 60+2°C.
The experimental procedure as outlined above was followed
using Thiobaoillus ferrooxidans. The n u t r i e n t medium used was the
one reported by Bryner and Anderson ( 7 ) . The i n i t i a l pH of the
medium was adjusted to 2 . 1 . As before, two f l a s k s were prepared
for each sample. The f l a s k s were inoculated with 5 ml of

2
This is a statistical method using various dilutions of the
medium containing bacteria.
116 V. Κ. Berry and L Ε. Murr

inoculum (2.5 χ 10& c e l l s ) of iron free c e l l s , 5 ml of panacide


s o l u t i o n (0.8 g panacide/1 I ethyl a l c o h o l ) , which i s a bacteri­
c i d e , and a fungicide was added to each of the s t e r i l e flasks
used as c o n t r o l s . The f l a s k s were placed on a rotary shaker
(Lab-Line Junior Orbit Shaker) run at 150 rpm continuously. The
chemical a n a l y s i s of the medium at weekly i n t e r v a l s (1 to 8 weeks),
bacterial count (MPN) and the SEM observation of the f l a t ore
sections were performed as described in detail above and the
experiment was duplicated under the same c o n d i t i o n s . Another
experiment was performed with T, ferrooxidans using a s t a t i o n a r y
f l a s k technique f o r comparison with the s t a t i o n a r y f l a s k leaching
experiments u t i l i z i n g the Sulfolobus-Mke microorganism.

III. RESULTS AND DISCUSSION

A. Morphology of Microorganisms Attached to Sulfide-Phase


Surfaces

Figure 1 shows f o r comparison typical views of Thiobacillus


thiooxidans, Thiobacillus ferrooxidans, and the Sulfolobus-Mke,
t h e r m o p h i l i c microorganisms as they appear in the scanning e l e c ­
tron microscope when attached to e i t h e r pyrite or chalcopyrite
surfaces. While there i s a d i s t i n c t morphological difference
between the Thiobacilli and the Sulfolobus-Mke microorganism,
there i s a l s o a rather marked difference in the density of attach­
ment, with the Sulfolobus-Mke thermophile attaching more r e a d i l y
than the Thiobacilli. This may be due in part to the increased
mobility of the Thiobacilli as a r e s u l t of t h e i r flagellum [ F i g .
1 (b) i n s e r t ] , and the fact that the cell wall of the Sulfolobus-
Mke microorganism i s much l e s s r i g i d , and perhaps more conducive
to adhesive attachment. The contact area associated with the
attachment of the Sulfolobus-Mke microorganisms appears to be
generally l a r g e r than that associated with the Thiobacilli
although there i s some evidence, as apparent in the i n s e r t of
F i g . 1 ( b ) , that the Thiobacilli secrete a substance of some
Basic Microbial Studies Applied to Leaching 117

Fig. 1. Bacteria attached to pyrite surfaces. (a) T h i o -


b a c i l l u s thiooxidans on pyrite after incubation for 7 weeks,
(arrows), (b) T h i o b a o i l l u s ferrooxidans after 7 weeks. The
insert shows a magnified view of a single microbe on chalcopyrite.
Flagellum disturbed by drying is visible (arrow) and the micro-
organism appears to be resting upon a biomat of some kind, (c)
Su^fo^obus-like microorganisms. Insert shows magnified view. The
distortion results by drying in air· vacuum collapse of the cell wall.
118 V. Κ. Berry and L Ε. Murr

kind which might aid in t h e i r attachment. This observation i s ,


however, not well documented at present, and the nature of the
substance i s also unknown. I t does not, however, appear to be
a r t i f a c t u a l l y produced, but t h i s w i l l be apparent only after pre­
paration i n v o l v i n g c r i t i c a l - p o i n t d r y i n g .

B. Bacterial Attachment: S i t e S p e c i f i c i t y and the Possible


Role of Crystal Defects

In previous studies i n v o l v i n g bacterial attachment to molyb­


denite u t i l i z i n g t h i n electron transparent sections in which d i s ­
locations could be observed, i t was d i f f i c u l t to associate the
attachment s i t e of Sulflobus-Mke microorganisms with s p e c i f i c
d i s l o c a t i o n s or dense arrays of d i s l o c a t i o n s p a r t l y because in
molybdenite ( M 0 S 2 ) the thin sections cleave p r e f e r e n t i a l l y along
( 0 0 0 1 ) , and the d i s l o c a t i o n s l i e mainly p a r a l l e l to the surfaces
(45). Only d i s l o c a t i o n s associated with surface ledges can t e r ­
minate on the free surface. In an e f f o r t to circumvent t h i s
shortcoming, and to apply the technique to c h a l c o p y r i t e , of
i n t e r e s t in the present s t u d i e s , crushed and c a r e f u l l y selected
thin sections were examined and treated experimentally as des­
cribed p r e v i o u s l y . While i t was p o s s i b l e to prepare several
excellent thin s e c t i o n s , i t was not possible to induce attachment
of the Sulfolobus-Mke microorganisms near the thin edges, and i t
was therefore not p o s s i b l e to associate the s p e c i f i c attachment
s i t e with any c r y s t a l d e f e c t s , p a r t i c u l a r l y d i s l o c a t i o n s . How­
ever, bacteria were observed away from the chalcopyrite specimen
edges which were arranged i n patterns resembling grain or subgrain
boundaries, i . e . the emergence of such defects on the c r y s t a l
surface. An unambiguous demonstration of t h i s contention was,
however, not p o s s i b l e .

Figure 2 i l l u s t r a t e s some of the important features of the


experiments outlined above, and some of the r e s u l t s alluded to
in the preceeding paragraph. Figure 2 (a) and (b) illustrate
Basic Microbial Studies Applied to Leaching 119

Ο Ο Ο Ο XS--CT 0 0 0 0
O W o W o W o '
Ο

Fe:oS)

(e) (f)

Fig. 2. (a) and (b) show dislocation substructure in elec­


tron transparent thin sections of chalcopyrite which are imaged by
diffraction contrast (46). (a) shows numerous dislocations and
stacking faults while (b) shows a single dislocation, (c) and (d)
show cell-like attachment characteristics for Sulfolobus-like mi­
croorganisms attached to the thin section surfaces, (e) and (f)
illustrate schematic views of edge dislocations intersecting sur­
faces having a (001) or (Oil) orientation respectively. The arrows
in (b) show the dislocation ends which terminate on the surface.
120 V. Κ. Berry and L Ε. Murr

some examples of individual d i s l o c a t i o n s and other defects in the


i n i t i a l chalcopyrite samples p r i o r to immersion in the medium.
Figure 2(c) and (d) show the arrangement of Sulfolobus-Mke micro­
organisms on the surface of a chalcopyrite thin s e c t i o n . In F i g .
2(e) and ( f ) are shown two i d e a l i z e d views of d i s l o c a t i o n s inter­
secting the surface having d i f f e r e n t o r i e n t a t i o n s , and the local
(surface) stoichiometry changes with respect to both Fe and S.

C. Selective Bacterial Attachment to S u l f i d e Phase Surfaces

I t has already been demonstrated in previous work that when


the Sulfolobus-Mke microorganisms attach to the surface of a po­
lished section of a low-grade waste rock, the attachment i s
s p e c i f i c to s u l f i d e phase r e g i o n s , v i z . F e S 2 and CuFeS2 surface
regions ( 4 9 ) . These observations have been made repeatedly, and
were a l s o supported by observations made in the present i n v e s t i ­
gations. In a d d i t i o n , and as depicted somewhat generally in F i g .
1 , the attachment of T. ferrooxidans and T. thiooxidans to low-
grade waste-rock surfaces was a l s o s p e c i f i c to exposed FeS2 and
CuFeS 2 r e g i o n s , but the attachment was not as frequent, and the
density of attached microbes (number/unit area of exposed s u l f i d e
phase surface) was considerably l e s s than for the Sulfolobus-Mke
microorganism ( F i g . 1 ) .

The s p e c i f i t y of attachment of the Sulfolobus-Mke micro­


organisms i s i l l u s t r a t e d t y p i c a l l y in F i g . 3 which shows a sharp
boundary separating a CuFeS2 region on the l e f t where microbes
are attached from quartz matrix on the r i g h t which i s free of
attached microbes. Figure 4 a l s o i l l u s t r a t e s the s p e c i f i c a t t a c h ­
ment features and the i d e n t i f i c a t i o n of sulfide-phase regions
u t i l i z i n g energy d i s p e r s i v e X-ray spectrometry and X-ray mapping.
In a d d i t i o n , F i g . 4 i l l u s t r a t e s the complete surface coverage of
bacteria which has been observed to occur in some cases.
Basic Microbial Studies Applied to Leaching 121

Fig. 3. Sulfolobus-like microorganisms selectively attached


to a chalcopyrite region in a quartz host rock. Note the sharp
boundary between the CuFeS^ on the left and the quartz on the
right. Bacteria appear distorted as a result of cell wall col-
lapse during drying. This could be avoided by drying at the
critical point, but this process might then alter the mineral
substrate.

While i t has been observed that profuse attachment u s u a l l y


requires 3 weeks or more of i n c u b a t i o n , attachment i s not always
observed a f t e r t h i s time. In many experiments, e s s e n t i a l l y no
attachment was observed even a f t e r 8 weeks incubation, even though
122 V. Κ. Berry and L Ε. Murr

(c) (d)

Fig. 4. Selective attachment of Sulfolobus-Hfce microorga­


nisms on pyrite. (a) Pyrite grain with bacteria attached. The
insert shows a magnified view of the bacteria, (b) Energy-disper­
sive X-ray spectrum of the entire area in (a), including the
matrix region which consists mainly of K-Al silicates; (c) X-ray
map using Fe and region on which the bacteria are selectively
attached, (d) Dense (continuous) coverage of a pyrite surface
by Sulfolobus-Zi/ce microorganisms.
Basic Microbial Studies Applied to Leaching 123

there was a marked difference between the copper s o l u b i l i z e d in


the inoculated case when compared to the uninoculated (sterile)
control situation.

I t i s of course not unexpected that bacteria would attach to


the s u l f i d e regions because these regions ( F e S 2 and CuFeS^) a r e ,
a f t e r a l l , sources of energy in the metabolic processes. However
attachment does not seem necessary f o r enhanced s o l u b i l i z a t i o n of
copper, and as a consequence metabolism does not seem to be pre-
dicated upon d i r e c t attachment. Attachment c o u l d , nonetheless,
be important and could enhance the leaching k i n e t i c s , but measure-
ments of leaching k i n e t i c s related to bacterial attachment have
been neither c o n s i s t e n t nor systematic. Such measurements do not
e x i s t at present.

D. Surface Reactions at F e S 2 and CuFeS2 and Their Relation to


Bacterial Leaching

While bacterial attachment may not be necessary f o r leaching


to be stimulated as a consequence of the c a t a l y t i c role played by
the b a c t e r i a , leaching must involve the degradation, i . e . reaction
or corrosion of the surface of the exposed p y r i t e and chalcopyrite
phase i n c l u s i o n s . In the case of p y r i t e i n c l u s i o n s , the following
prominent reactions are taking place:

FeS 2 + 3.50 2 + H0 2 > FeS0 4 + H S0


2 4 [1]

2FeS0 + 0.5 0 4 2 + H S0 2 4
b a c t e r i a
> Fe^SO^ + H0 2 [2]

FeS 2 + Fe (S0 )
2 4 3 > 3FeS0 + 2S 4 [3]

2S + 3 0 2 + 2H 0 2
b a c t e r i a
> 2H S0 2 4 [4]

In the case of chalcopyrite r e g i o n s , the following reactions


are taking place:

2CuFeS + 8 . 5 0 + H S 0
2 2 2 4
b a c t e n a
> 2CuS0 + F e ( S 0 ) + H 0
4 2 4 3 2 [5]

CuFeS + 2 F e ( S 0 )
2 2 4 3 > CuS0 + 5FeS0 4 4 + 2S [6]
124 V. Κ. Berry and L Ε. Murr

where the s u l f u r formed in Eq. [6] i s converted to s u l f u r i c acid


according to Eq. [ 4 ] . While bacterial attachment to F e S 2 and
CuFeS^ might accelerate or optimize the c a t a l y t i c activity
indicated in Eqs. [ 2 ] , [ 4 ] and [ 5 ] , above, such a c t i v i t y would
a l s o be expected simply by the presence of s u f f i c i e n t numbers of
microorganisms in the s o l u t i o n proximate to the reacting surface.

Figures 5 and 6 c l e a r l y i l l u s t r a t e the c a t a l y t i c role played


by bacteria in the leaching of low-grade, copper-bearing waste
rock. The c a t a l y t i c a c t i v i t y i s e s p e c i a l l y graphic on comparing
3+ 2+
the Fe /Fe r a t i o in F i g . 6. I f t h i s i s taken as a q u a n t i t a t i v e
measure of bacterial c a t a l y s i s , then i t i s r e a d i l y apparent that
3
the enhancement i s as much as a factor 10 . I t should be noted
in F i g . 5 that the s o l u b i l i z a t i o n of copper i s very small in
s t a t i o n a r y leaching with T. ferrooxidans. This i s due in large
part to the formation of hydronium j a r o s i t e s which prevent d i r e c t
exposure of the s u l f i d e surfaces to the leach s o l u t i o n . The
3+ 2+
decline and v a l l e y in the Fe /Fe response noted in F i g . 6 i s
a l s o due to t h i s e f f e c t . In a d d i t i o n , differences in the leach­
ing rates f o r the T. ferrooxidans runs ( s t a t i o n a r y and agitated
f l a s k leaching experiments) and the Sulfolobus-Mke microorganism
studies (which were a l s o s t a t i o n a r y f l a s k runs) are due to the
temperature difference and not because of a difference in the
c a t a l y t i c a c t i v i t y being better f o r the Sulfolobus-Mke micro­
organisms. This feature i s treated in detail in Chap. 25.
Figures 7 and 8 show the corresponding surface corrosion
on F e S 2 and CuFeS which r e s u l t s from the reactions
2 indicated
in Eqs. [ 1 ] - [ 6 ] , and the q u a l i t a t i v e surface features c o r r e s ­
ponding to the q u a n t i t a t i v e leaching responses noted in F i g s . 5
and 6. There are several i n t e r e s t i n g features to note in F i g s .
7 and 8. F i r s t i t should be apparent that there i s in general
a dearth of attached microorganisms. While one might expect that
in the heavily corroded surface regions bacteria might remain
tenaciously attached or entrapped within the surface p i t s and
Basic Microbial Studies Applied to Leaching 125

7 5 R

AS(a)

./ 4

60

4 5
Ε
Θ­
α
" Ο
(D
'η »Tf(e)
3 3 0 1

19-

i ^ 8 — § — 5 — 9 — φ — φ — jaUib)
9d l
3 6 9
Elapsed time (weeks)
Fig. 5. Comparison of copper leaching with T, ferrooxidans
and Sulfolobus-Hfce microorganisms. S(a) and Sib) denote the
inoculated (a) and control or sterile (b) condition for the S u l f Ο ­
Ι obus-like stationary flask run at 60°C. The initial pH was 2.1
while the final inoculated (a) and control (b) pH was 2.5 and 2.8
respectively. Sic) and Sid) denote the inoculated ic) and control
id) condition for the Sulfolobus-like stationary flask run at55°C.
The intial pH was 2 and the final pH was 2.8 and 3 for ic) and id)
3

respectively. Tf ie) and Tf if) denote the inoculated (e) and con­
trol if) condition for T. ferrooxidans in a shake flask run at 28°C.
The initial pH was 2.1 while the final pH was 2. 6 and 2. 8 for ie) and if)
respectively. Tf (g) and Tf ih) denote the inoculated (g) and control
ih) condition for T. ferrooxidans in a stationary flask run at 28°C .
The initial pH was 2.3 while the final Ph was 2.8 and 3.0 for
(g) and ih) respectively.
126 V. Κ. Berry and L. Ε. Murr

.12
10
• agitated /inoculated ) , o \ +

ο agitated /sterile 1 / Fe
• stationary/inoculated Γ I "j=2+
• stationary/sterile
10*
• ?^
Α
Impn
ο stationary j
10'
3+ s

Fe'
7

Fe'
10'
ι \

V ο

,0 Ο ν,ο
10

10" .0-0'" V - a ^

10"
0

Elapsed timeiweete)
3+ 2+
Fig. 6. Comparison of Fe /Fe ratios for stationary and
agitated flask leaching of Duval-Sierrita waste using T. f e r r o o x i ­
dans. The most probable number (MPN) of microorganisms is also
shown corresponding to the leaching times. All experiments were
performed at 28°C and correspond to those recorded in Fig. 5.
3
Basic Microbial Studies Applied to Leaching 127

(c) (d)

Fig. 7. Corrosion of pyrite and chalcopyrite phase regions


in low-grade waste after 8 weeks in agitated flasks, (a)sterile
(control) pyrite surface, (b) pyrite surface exposed to medium
inoculated with T. f e r r o o x i d a n s , (c) Sterile (control) chalcopy-
rite surface, (d) chalcopyrite surface exposed to medium inocu-
lated with T. f e r r o o x i d a n s . All runs at 28°C. All magnifications
are given in (a).

and related corrosion morphology, there was no evidence of t h i s .


There simply was no prominent attachment of bacteria even though,
as noted in F i g s . 5 and 6, leaching in the presence of bacteria
was markedly enhanced. There were however, instances where
128 V. Κ. Berry and L Ε. Murr

Fig. 8. Corrosion of pyrite and chalcopyrite phase regions


low-grade waste after 8 weeks in stationary flasks. (a) sterile
(control) pyrite surface, (b) pyrite surface exposed to medium
inoculated with Sulfolobus-like microorganisms (c) sterile (con­
trol) chalcopyrite surface, (d) chalcopyrite surface exposed to
medium inoculated with the Sulfolobus-like microorganisms. All
runs at 60°C. All magnifications are given in (a).

bacteria were observed to attach to the corroded s u r f a c e , and t h i s


i s i l l u s t r a t e d in F i g . 9. In a d d i t i o n , i t was apparent that the
corrosion texture in FeS^ or CuFeS^ was not the same over the waste
p a r t i c l e surface treated in an identical way, and t h i s particular
feature was attributed to differences in the c r y s t a l l o g r a p h i c
Basic Microbial Studies Applied to Leaching 129

Fig. 9. Sulfolobus-like microorganisms attached to the


corroded surface of a pyrite inclusion after incubation at 60°C
for 4 weeks.

surface o r i e n t a t i o n exposed to the leach s o l u t i o n . This feature


i s shown in F i g . 10 (a) - ( c ) , and unambiguously characterized as
differences in grain surface o r i e n t a t i o n in F i g . 10 (d) and (e)
which show d i f f e r e n t g r a i n s separated by grain boundaries, with
the degree and texture of surface c o r r o s i o n changing across the
boundaries. This occurs simply because the rates of reaction are
d i f f e r e n t for d i f f e r e n t c r y s t a l l o g r a p h i c surface o r i e n t a t i o n s
j u s t as rates of c r y s t a l growth are d i f f e r e n t along d i f f e r e n t
crystallographic directions.

E. Galvanic I n t e r a c t i o n Between Chalcopyrite and Pyrite in


Intimate ( E l e c t r i c a l ) Contact

I t can be observed in F i g s . 7 and 8 that when the F e S 2 and


CuFeS 2 phase i n c l u s i o n s leach separately, there i s a greater rate
of reacation on the FeS2- The c h a l c o p y r i t e , in f a c t , leaches
130 V. Κ. Berry and L Ε. Murr

Fig. 10. Variations in the degree (texture) and rate of


corrosion for different phase (grain) surfaces exposed to identi­
cal leaching conditions; illustrating the effects of grain sur­
face (crystallographic) orientation. (a) - (c) show three dif­
ferent pyrite surface areas in the same waste particle leached in
the presence of S u l f o l o b u s - Z t k e microorganisms for 6 weeks at
60°C. (d) shows the junction of three grains of pyrite leached for
4 weeks and showing different corrosion on each grain surface,
(e) junction of three chalcopyrite grains showing differences in
corrosion after 6 weeks. (d) and (e) in medium inoculated with
Sulfolobus-like microorganisms.
Basic Microbial Studies Applied to Leaching 131

r e l a t i v e l y slowly by comparison with the p y r i t e . However, i t


was frequently observed that when the p y r i t e and chalcopyrite were
in contact, the trend normally observed was reversed, i . e . the
CuFeS£ reacted much f a s t e r than the FeSr,, which was e s s e n t i a l l y
passivated as a r e s u l t of i t s higher r e s t potential as compared
with chalcopyrite in forming a galvanic couple. This i s identical
to the galvanic conversion described recently by Hiskey and
Wadsworth (50) except that the conversion i s accelerated, a d d i -
t i o n a l l y , by the presence o f bacteria which l i m i t the formation
of elemental s u l f u r through the reaction of Eq. [ 4 ] . Figure 11
i l l u s t r a t e s t h i s e f f e c t , and shows f o r comparison the differences
in the surface reactions at a non-contacting F e S 2 phase. The
chalcopyrite surface c o r r o s i o n in F i g . 11 can a l s o be compared
with F i g . 8 ( d ) .

The implications of t h i s phenomena (as i l l u s t r a t e d in Fig.11)


in a low-grade waste heap or dump leaching operation would seem
to have great s i g n i f i c a n c e because i f a waste rock regime c o n t a i n -
ed a large proportion of contacting s u l f i d e s , then the leaching
rate would presumably be very large f o r the s u l f i d e member having
the lower rest potential. In the presence of b a c t e r i a , which i s
probably a c h a r a c t e r i s t i c of a l l leach dumps, the process would be
enchanced as described above, and i l l u s t r a t e d in F i g . 1 1 . The
galvanic i n t e r a c t i o n between chalcopyrite and p y r i t e during bac-
t e r i a l leaching i s treated in more detail elsewhere ( 5 1 ) .

IV. SUMMARY AND CONCLUSIONS

The morphologies of T. thiooxidans, T. ferrooxidans, and a


Sulfolobus-Mke, thermophilic microorganisms attached to s u l f i d e
surfaces have been examined by scanning electron microscopy. The
attachment of Sulfolobus-Mke microorganisms was more p r o l i f i c
than the Thiobacilli, and t h i s was a t t r i b u t e d to the difference
in c e l l wall r i g i d i t y and the absence of a f l a g e l l u m on the
Sulfolobus-Mke microorganisms.
132 V. Κ. Berry and L Ε. Murr

Fig. 11. (a) Contacting pyrite and chalcopyrite phases


showing selective galvanic corrosion of the anodically reacting
chalcopyrite and the passivation of the pyrite after 8 weeks in
medium inoculated with the Sulfolobus-Zifce microorganism. The
insert shows a separate (non-contacting) pyrite surface similarly
leached for only 6 weeks, (b) schematic representation of (a) depict­
ing the galvanic conversion model for the CuFeS2/FeS2~acid sustain
Basic Microbial Studies Applied to Leaching 133

In attempting to determine whether s i n g l e microbes attach


s e l e c t i v e l y to c r y s t a l defects which i n t e r s e c t the s u r f a c e ,
chalcopyrite thin sections were prepared and observed in the
transmission electron microscope. While the technique has been
demonstrated to be a viable one, and d i s l o c a t i o n s have been ob-
served, bacterial attachment to i n d i v i d u a l d i s l o c a t i o n s has not
been observed because for some reason i t was not p o s s i b l e to get
the bacteria to attach to the electron transparent edge regions
of the chalcopyrite s e c t i o n s , p o s s i b l y because of some edge
e f f e c t s or surface tension e f f e c t s . However, observations of
attached bacteria to the t h i c k e r regions of the specimen showed
them to be arranged in c e l l - l i k e arrays resembling g r a i n boundaries
or sub-grain boundaries. I t may be that because of local varia-
t i o n s in stoichiometry of the emergence s i t e s of d i s l o c a t i o n s in
c h a l c o p y r i t e , these may be favorable f o r attachment, but t h i s
question remains open.

The attachment of bacteria was, however, observed to be


s p e c i f i c to s u l f i d e phase regions on a polished waste rock surface
because these are regions o f f e r i n g an energy source. However,
while attachment was observed, and while i t was unquestionably
s p e c i f i c to only the s u l f i d e phase regions ( F e S 2 and C u F e S ) , i t
2

did not always occur. Nonetheless, bacterial c a t a l y s i s was shown


to have been important in the leaching of the waste rock in f l a s k
experiments, and correspondingly the surfaces of F e S 2 and CuFeS 2

phase regions were observed to have been reacted (corroded). The


corrosion observed was a l s o v a r i e d , and the degree of c o r r o s i o n
was observed to vary with c r y s t a l l o g r a p h i c o r i e n t a t i o n .

When F e S 2 and CuFeS 2 phases were in intimate contact, the


normally r a p i d l y corroding F e S 2 was observed to be passivated
while the CuFeS was very v i g o r o u s l y corroded.
2 This galvanic
conversion was enhanced in the presence of bacteria not only
because of the c a t a l y t i c oxidation of the c h a l c o p y r i t e , but a l s o
because of the bacterial conversion of elemental s u l f u r formed
134 V. Κ. Berry and L Ε. Murr

on the chalcopyrite to s u l f u r i c a c i d . This observation can have


s i g n i f i c a n t a p p l i c a t i o n s in a v a r i e t y of s u l f i d e leaching
processes.

There i s no question that bacteria attach to mineral surfaces


and that they attach to s p e c i f i c areas in a waste rock which can
function as an energy source, namely s u l f i d e phases in the present
study. However attachment does not always occur, and bacterial
c a t a l y s i s can be very s i g n i f i c a n t without d i r e c t attachment.

V. ACKNOWLEDGMENTS

This research was supported by the National Science Founda­


t i o n (RANN) under grants AER-76-03758 and AER-76-03758-A01. The
authors thank James A. and Corale L. B r i e r l e y f o r laboratory
p r o v i s i o n s and the p r o v i s i o n of the high-temperature microorganism
u t i l i z e d in t h i s i n v e s t i g a t i o n , as well as many f r u i t f u l discus­
sions.

VI. REFERENCES

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52, 36(1943).
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(1965).
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12. Peck, H.D., Bacteriol. Rev., 26, 67 (1962).


13. Brock, T . D . , " B i o l o g y of Microorganisms", P r e n t i c e - H a l l ,
I n c . , Englewood C l i f f s , New Jersey, 1970.
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16. Corrans, I . J . , H a r r i s , B., and Ralph, B . J . , J. South African
Inst. Min. Met., March, 221 (1972).
17. Tuovinen, O.H., and K e l l y , D.P., Zeit. fur Allg. Mikrobiol.
12 ( 4 ) , 311 (1972).
18. Trudinger, P.Α., Minerals Sci. Engr., 3 ( 4 ) , 13 (1971).
19. Silverman, M.P. and E h r l i c h , H.L., Adv. Appl. Microbiol.,
6, 153 (1964).
20. Bruynesteyn, Α . , and Duncan, D.W., Can. Met. Quart., 1 0 ( 1 ) ,
57 (1970).
21. Pinches, Α . , A l - J a i d , F.O., and W i l l i a m s , D.J.A., Hydrometal-
lurgy, 2, 87 (1976).
22. Torma, A . E . , in Proc. 3rd. I n t . Biodegradation Symposium,
(J.M. Sharpley and A.M. Kaplan, Eds.) p. 937 Applied Science
P u b l i s h e r s , L t d . , London, 1976.
23. Sakaguchi, H., S i l v e r , M., and Torma, A . E . , Biotechnol. Bio-
engr., X V I I I ( 8 ) , 1091 (1976).
24. Torma, A . E . , and I t z k o v i t c h , I . J . , Appl. and Environ. Micro-
Did., 32 ( 1 ) , 102 (1976).
25. Bryner, L . C , and Jameson, A . K . , Appl. Microbiol., 6, 281
(1958).
26. Duncan, D.W., Walden, C.C., T r u s s e l l , P.C., and Lowe, E.A.,
Trans. Soc. Min. Engrs., 238, 1 (1967).
27. Sutton, J . A . , and C o r r i c k , J . D . , Min. Engr. 15, 37 (1963).
28. Sutton, J . A . , and C o r r i c k , J . P . , U.S. Bureau of Mines Report
I n s . 6423, p. 1 , 1964.
29. Brock, T . D . , Brock, K.M., B e l l y , R.T., and Weiss, R.C.,
Arch Microbiol., 84, 54 (1972).
30. B r i e r l e y , C.L., and B r i e r l e y , J . A . , Can J. Microbiol., 19,
183 (1973).
31. de Rosa, M. Gambacorta, Α . , and Bulock, J . D . , J. Gen. Micro­
biol., 86, 156 (1975).
32. Silverman, M.P., J. Bacterid., 94, 1046 (1967).
33. McGoran, C.J.M., Duncan, D.W., and Walden, C.C., Can. J.
Microbiol., 15, 135 (1967).
136 V. Κ. Berry and L Ε. Murr

34. E h r l i c h , H.C., and Fox, S . I . , Biotechnol. Bioengr., 9, 471


(1967).
35. Duncan, D.W., and Drummond, A . D . , Can. J. Earth Sci., 10,
476 (1973).
36. Gormley, L.C., and Duncan, D.W., Can. J. Microbiol., 20,
1453 (1974).
37. B r i e r l e y , C.L., B r i e r l e y , J . Α . , and Murr, L.E., Res. Develop.
Mag.; 24 ( 8 ) , 24 (1973).
38. Weiss, R.C., J. Gen. Microbiol., 77, 501 (1973).
39. Baldensperger, J . , Guarraia, L . J . , and Humphreys, W . J . ,
Arch Microbiol., 99, 323 (1974).
40. Duguid, J . P . , J. Gen. Microbiol., 2 1 , 271 (1959).
41. H i r s c h , P., and Pankrantz, S r . H., Zeit. fur Allg. Mikrobiol.,
10, 589 (1970).
42. Schaeffer, W . I . , Holbert, P.E., and Umbreit, W.W., J. Bac­
teriol. , 85 137 (1963).
43. Meadows, P . S . , Arch fur Mikrobiol. 75, 374 (1971).
44. Torma, A . E . , Walden, C.C., and Branion, R.M.R., Biotechnol.
Bioengr., 12, 501 (1970).
45. Berry, V . K . , and Murr, L . E . , Met. Trans., 6B, 488 (1975).
46. Murr, L.E., "Electron Optical A p p l i c a t i o n s in M a t e r i a l s
S c i e n c e " , McGraw-Hill Book Co., I n c . , New York, 1970.
47. "Chemical A n a l y s i s of M e t a l s " , : Sampling and A n a l y s i s of
Metal Bearing O r e s " , American Soc. f o r Testing and M a t e r i a l s ,
P h i l a d e l p h i a , Pennsylvania, 1969.
48. C o l l i n s , C.H., " M i c r o b i o l g i c a l Methods", Plenum P r e s s ,
New York, 1967.
49. Murr, L . E . , and Berry, V.K., Hydrometallurgy, 2, 11 (1976).
50. Hiskey, J . B . , and Wadsworth, M . E . , Met. Trans., 6B, 183
(1975).
51. Berry, V.K., Murr, L.E., and Hiskey, J . B . , Met. trans., 8B,
in p r e s s .
GENETIC MECHANISMS IN

METAL-MICROBE INTERACTIONS

A. Μ. Chakrabarty

General E l e c t r i c Company
Corporate Research & Development
Schenectady, New York, 12301 USA

The genetic bases of the interaction of microorganisms with


metals have been reviewed. Many microorganisms develop resistance
to toxic concentrations of inorganic or organic forms of heavy
metals by reducing them to elemental metal. Genes specifying such
reduction steps have been shown to be plasmid-borne in several
instances. Plasmid-specified resistance to cadmium, on the other
hand, is due to reduced uptake of this metal by the resistant
cells. Microorganisms are also known to accumulate certain metal
ions, which are rendered innocuous because of their binding with
the intracellular protein. This ability has also been shown to be
due to plasmid-borne genes in some instances. Finally, plasmids
have also been implicated in enhancing methylation of metals such
as mercury. Although nothing is known about the genetic basis of
metal leaching by the T h i o b a c i l l i group of bacteria, some areas
where potential genetic improvements can be made with T h i o b a c i l l u s
ferrooxidans are suggested.

I. INTRODUCTION
Microorganisms are ubiquitous in nature; so are various metal
i o n s , which e i t h e r occur in ores or natural rocks and m i n e r a l s , or
else are released to the environment v i a i n d u s t r i a l and household

137
138 Α. Μ. Chakrabarty

uses of t h e i r compounds. Because metal i o n s , p a r t i c u l a r l y the


heavy metals, are t o x i c at high concentrations, microorganisms
have evolved various b i o l o g i c a l systems to cope with t h e i r i n t e r ­
actions with l i v i n g c e l l s . One such system has led to the devel­
opment of r e s i s t a n t c e l l s that can convert the reactive inorganic
form of the metal to the l e s s reactive elemental form. The metal
can then be v o l a t i l i z e d o f f (mercury, for example) or stored within
the c e l l s . A l t e r n a t i v e l y , r e s i s t a n c e may a r i s e from a change in
the permeability properties of the c e l l membrane so that c e l l u l a r
uptake of the s p e c i f i c metal ion i s g r e a t l y diminished. In a d d i ­
t i o n , several fungi and bacteria are known to methylate metal i o n s ,
which can then be e a s i l y v o l a t i l i z e d o f f from the c e l l s , or
elaborate proteins which keep the metal in an innocuous form.

Microorganisms, p a r t i c u l a r l y the chemolithotrophic Thio­


baoillus group that can release soluble form of metals in an
a c i d i c medium from i n s o l u b l e o r e s , which i s of great i n d u s t r i a l
s i g n i f i c a n c e , are known. These microorganisms develop r e s i s t a n c e
to very high concentration of leached metals by mechanisms as yet
unknown. An understanding of the genetic basis of leaching of
metals by bacteria such as Thiobaoillus ferrooxidans, as well as
the development of r e s i s t a n c e a g a i n s t the leached metals, could be
of great value in the enhanced leaching and recovery of the metals
for i n d u s t r i a l use.

In t h i s a r t i c l e , I would l i k e to review some of the genetic


and biochemical bases of the development of bacterial resistance
and i n t r a c e l l u l a r accumulation of metal i o n s . Additionally, I
would l i k e to point out some areas where genetic s t u d i e s , p a r t i c u ­
l a r l y with Thiobaoillus ferrooxidans, may contribute significantly
towards enhanced leaching of metals such as copper or uranium.

II. METAL ION RESISTANCE, ACCUMULATION, AND METHYLATION

In general, microorganisms are s e n s i t i v e to high concentra­


tions of heavy metal s a l t s . I t has a l s o been recognized, however,
Basic Microbial Studies Applied to Leaching 139

that microorganisms can develop r e s i s t a n c e a g a i n s t h i g h , toxic


concentrations of many metal ions when adapted in presence of
i n c r e a s i n g l y higher concentrations. In a d d i t i o n , organisms can
a l s o be i s o l a t e d that e x h i b i t high l e v e l s of r e s i s t a n c e a g a i n s t
some metal ions in t h e i r n a t u r a l l y - o c c u r r i n g s t a t e . Such r e s i s -
tant bacteria often harbor plasmids that s p e c i f y r e s i s t a n c e
against the t o x i c metal (Table 1 ) . Four a l t e r n a t i v e mechanisms
are used by the bacteria for achieving r e s i s t a n c e :

(1) Reduction to free metal. This mode of r e s i s t a n c e has


been widely studied both from genetic as well as enzymatic points
of view, p a r t i c u l a r l y in regard to r e s i s t a n c e a g a i n s t mercury.
Because of widespread i n d u s t r i a l use of inorganic mercury s a l t s
and the organomercurials, t h e i r consequent release into r i v e r s
and l a k e s , and t h e i r ultimate capture by f i s h used as human food,
the environmental transformation of mercury has received much
attention i n recent y e a r s . Various types of bacteria have been
demonstrated to play s i g n i f i c a n t r o l e s in the cycles of t r a n s f o r -
mations from m e t a l l i c to i o n i c to organomercurial states that
proceed in aquatic ecosystems and are responsible f o r the amounts
and nature of mercury compounds found i n various parts of the
food chain ( 1 , 2 ) .

I t appears that most bacteria develop r e s i s t a n c e against


mercury s a l t s and organomercurials by reducing such species to
m e t a l l i c mercury, which can then be v o l a t i l i z e d o f f from the
cells. The genes s p e c i f y i n g such enzymes have been shown to be
plasmid-borne and more than 200 of such plasmids in enteric
b a c t e r i a , Staphylococcus aureus and Pseudomonas have been charac-
terized (3). Some plasmids determine r e s i s t a n c e to mercuric ion
alone while others determine r e s i s t a n c e to a range of mercurials
2+ 2+
in addition to Hg . Plasmid-bearing bacteria thus convert Hg
to Hg°, phenylmercuric acetate to Hg° + benzene and methylmercuric
chloride to Hg° plus methane. The enzyme systems s p e c i f i e d by
such plasmids are i n v a r i a b l y i n d u c i b l e , i r r e s p e c t i v e of whether
140 Α. Μ. Chakrabarty

TABLE I Plasmids Specifying


Resistance to Toxic Elements

Plasmid Element Resisted! 2


References

PI 258 Hg, Cd, Bi, Pb, As, Sb 7


R factors Ni, Co, Hg 33
pMG 101 Ag, Hg, Te 34
R 733 As 40
R 477 As, Hg, Te 18
R 3108 ^9 3
MER Hg 4
pMG 6 Cr, Hg, Te 6
RMS 159 B, Hg, Te 6

Only resistance to toxic elements has been shown. Most of the


plasmids also carry antibiotic resistance genes.

they attack inorganic mercury or the organomercurials, and


generally c o n s i s t of a reducing enzyme, a C-Hg s p l i t t i n g enzyme,
and hydrolases s p e c i f i c for alkyl and aryl m e r c u r i a l s . The p e r t i ­
nent properties of such enzymes have recently been reviewed ( 4 ) .

Microbial reductions of oxyanions of heavy metals, such as


selenium and t e l l u r i u m , have a l s o been reported ( 5 , 6 ) . In many
c a s e s , the reducing enzymes are known to be s p e c i f i e d by plasmid-
borne genes. T e l l u r i t e (or t e l l u r a t e ) reduction to metallic
t e l l u r i u m i s s p e c i f i c f o r Pseudomonas plasmids belonging to P-2
i n c o m p a t i b i l i t y group. Plasmids belonging to other i n c o m p a t i b i l ­
i t y groups can specify r e s i s t a n c e to borates and chromates ( 6 ) ,
but the s p e c i f i c chemical mechanisms invoked to produce the
r e s i s t a n c e are unknown.

(2) Reduced uptake of metal. In some c a s e s , the r e s i s t a n c e


against metal ions i s manifested by a reduced uptake of the i o n .
2+
A typical example i s r e s i s t a n c e a g a i n s t Cd , which in Staphylo­
coccus aureus i s known to be s p e c i f i e d by a plasmid ( 7 ) . The
mechanism of Cd r e s i s t a n c e r e s u l t s from a permeability change in
the c e l l s , so that r e s i s t a n t c e l l s harboring the plasmid do not
accumulate the toxic l e v e l s of cadmium normally accumulated by the
Basic Microbial Studies Applied to Leaching 141

sensitive cells (8,9). Normally cadmium uptake by s e n s i t i v e c e l l s


i s temperature-dependent, and i n h i b i t e d by i n h i b i t i o n of energy
metabolism. There appears to be a h i g h l y s p e c i f i c relationship
between cadmium uptake and the manganese transport system of the
2+
cell. With the cadmium s e n s i t i v e c e l l s , high Cd rapidly
i n h i b i t s manganese t r a n s p o r t , whereas in r e s i s t a n t c e l l s , mangan­
ese transport system remains unaffected in the presence of high
2+
concentrations of cadmium. The Cd - r e s i s t a n c e plasmid thus
appears to block an energy-dependent transport system in the
resistant cells (10).
(δ) Intracellular concentration of metal. Resistance
against metal ions can a l s o be manifested by an e f f i c i e n t i n t r a ­
c e l l u l a r accumulation of the i o n s , which are stored in apparently
innocuous forms. In some c a s e s , t h i s type of r e s i s t a n c e has been
shown to be p l a s m i d - s p e c i f i e d . Thus, Kondo, et a l . (8) have
2+
demonstrated that more than 90% of Hg ions present in a t e s t
medium can be taken up by S. aureus c e l l s harboring the plasmid
Pc-ase. The Pc-ase plasmid i s known to a f f o r d r e s i s t a n c e to
mercury, cadmium, a r s e n i c , l e a d , and zinc i o n s , in addition to
penicillin. The mercury-accumulation process appears to be inde­
pendent of temperature and presumably i n d u c i b l e . S i m i l a r accumu-
2+
l a t i o n of Hg has been demonstrated f o r a r e s i s t a n t s t r a i n of
Enterobacter aerogenes, which was shown to concentrate 9 1 % of the
20^
total Hg in the i n t r a c e l l u l a r protein f r a c t i o n in three hours
(11). S i m i l a r mercury accumulating and m e r c u r y - v o l a t i l i z i n g
Pseudomonas s t r a i n s have been described ( 1 2 ) . The use of a
Pseudomonas c u l t u r e , harboring aggregates of mercury r e s i s t a n c e
and other plasmids, has been recommended f o r removal and recovery
of mercury from i n d u s t r i a l wastes ( 1 3 ) . The nature of the binding
2+
of Hg to the i n t r a c e l l u l a r protein i s not known, although
highly s p e c i f i c metal-binding metallothionen-1ike p r o t e i n s , such
as cadmium-binding p r o t e i n , have been described f o r mammalian
systems ( 1 4 ) .
142 Α. Μ. Chakrabarty

(4) Methylation of metals. The methylation of certain heavy


metals by microorganisms i s now well documented ( 2 , 1 5 ) . Such
methylated d e r i v a t i v e s can be quite t o x i c ; the i n i t i a l studies of
methylation of mercury were conducted because of the f i n d i n g that
e s s e n t i a l l y a l l the mercury in f i s h was the highly toxic methyl
mercury. Although the anaerobic bacteria in lake or r i v e r s e d i ­
ments were i n i t i a l l y believed to be the sole agents f o r conversion
of inorganic mercury to CH^H^ and ( C H ^ H g , i t i s now well docu­
mented that many aerobic mercury-resistant bacteria can methylate
2+
Hg . Thus Pseudomonas s t r a i n s harboring the plasmids pMGl and
2+
pMG2 can produce methyl mercury from Hg (10). S i m i l a r methyla-
tions of other metals such as selenium ( 1 6 ) , lead ( 1 7 ) , a r s e n i c ,
t e l l u r i u m ( 1 8 ) , t i n and cadmium (19) by microorganisms present in
the aquatic ecosystem are now known. Since most methylated metals
are v o l a t i l e , t h i s represents a simple bacterial mechanism f o r
developing r e s i s t a n c e against inorganic metal i o n s . Transmethyla­
t i o n r e a c t i o n s , where methyl groups are transferred to mercury
ions from b i o l o g i c a l l y methylated t i n compounds, are a l s o known
2+
(20). While the genes s p e c i f y i n g methylation of Hg are known to
be s p e c i f i e d by plasmids pMGl and pMG2 in some Pseudomonas strains
( 1 0 ) , the d i s p o s i t i o n of methylation genes in other bacteria has
not been i n v e s t i g a t e d .
III. PROSPECTS FOR GENETIC IMPROVEMENT OF THIOBACILLUS FERRO­
OXIDANS
Among microorganisms that are involved in the i n d u s t r i a l
production and recovery of various metals, Thiobacillus ferrooxi­
dans i s presumably the most widely used. I t has been estimated
that as much as 5% of the total world copper production i s based
on the s o l u b i l i z a t i o n and leaching of the metal from low-grade
ores by T. ferrooxidans (21). This bacterium i s capable of r e ­
leasing more than 90% of copper from common s u l f i d e ores such as
chalcopyrite or chalcocite (22,23,24). In a d d i t i o n , Τ. ferrooxi­
dans has a l s o been shown to e f f e c t s o l u b i l i z a t i o n and release of
Basic Microbial Studies Applied to Leaching 143

such important metals as uranium, c o b a l t , n i c k e l , z i n c , and lead


(25,26). Initial leaching of a low grade uranium ore containing
0.11% uranium with T. ferrooxidans f o r a period of 9 days has
resulted in the extraction of 68% of the uranium, and the extent
of extraction could be increased to 87% i f the residue were r e -
ground and leached further ( 2 7 ) . Optimization of the leaching
process by adjusting the ore pulp density to 5% i s reported to
r e s u l t in almost 100% extraction of uranium from a low grade ore
(28).

Considerable work r e l a t i n g to the optimization of temperature,


pH, aeration r a t e , ferrous iron concentration and pulp density
suspension in the leaching of various metals from t h e i r ores by
T. ferrooxidans i s now in the l i t e r a t u r e ( 2 7 , 2 9 ) . Scale-up of the
laboratory and p i l o t plant studies to i n d u s t r i a l scale operation
p a r t i c u l a r l y in the production of uranium, i s now p o s s i b l e (28).
However, the economic b a r r i e r s to such s c a l e - u p , which would
entail s i z a b l e expenditures f o r equipment and energy costs for
g r i n d i n g large q u a n t i t i e s of low-grade ores to permit e f f i c i e n t
microbial growth, are considerable. The i n e f f i c i e n t but simple
leaching of unprocessed t a i l i n g dumps i s therefore favored on
economic grounds.

In addition to optimizing the process f o r maximal leaching of


metals, s i g n i f i c a n t improvements can a l s o be made in the perform-
ance of the bacteria. A study of the p h y s i o l o g i c a l parameters
a f f e c t i n g leaching, as well as optimum n u t r i t i o n a l and growth
conditions (24) i s e s s e n t i a l f o r maximizing the recovery of the
metal. In addition genetic improvements, leading to an enhance-
ment of the rate and extent of metal s o l u b i l i z a t i o n , can and
should be made. In t h i s s e c t i o n , I would l i k e to point out some
areas where genetic improvements could be made to increase the
effectiveness of T. ferrooxidans f o r metal s o l u b i l i z a t i o n .
144 Α. Μ. Chakrabarty

Α. Oxidation of Fe to Fe

The chemolithotropic bacterium, T. ferrooxidans, i s known to


derive i t s energy from the oxidation of ferrous i r o n , metal s u l ­
f i d e s and soluble s u l f u r compounds in an a c i d i c medium. The o x i ­
dation processes can be described as f o l l o w s :

2FeS0 + H S 0
4 2 4 + ^0 2 = Fe (S0 ) 2 4 3 + H0 2

4FeS 2 + 150 2 + 2H 0 = 2 F e ( S 0 ) 2 2 4 3 + 2H S0 2 4

CuFeS + 4 02 2 = CuS0 + FeS0 4 4

MS + 2 0 2 = MS0 4 (where Μ = Cu, N i , Co, Zn, e t c . )

The oxidation of ferrous iron to f e r r i c iron i s an important


reaction in the overall process, since f e r r i c s u l f a t e can react
chemically with several ore minerals to oxidize them. Thus copper
and uranium can react as f o l l o w s :

CuFeS + 2 F e ( S 0 )
2 2 4 3 + 2H 0 + 3 02 2 = CuS0 4

+ 5FeS0 + 2 H S 0 4 2 4

Cu S + 2 F e ( S 0 )
2 2 4 3 = 2CuS0 + 4FeS0 + S 4 4

U0 + F e ( S 0 )
2 2 4 3 = U0 S0 2 4 + 2FeS0 4

This reaction i s p a r t i c u l a r l y important for the extraction of


uranium, since the tetravalent form of uranium present in the ore
i s i n s o l u b l e , while the oxidized hexavalent form in the leach
solution is soluble. The f e r r i c i o n , which serves as the o x i d i z ­
ing agent, i s regenerated by the microorganisms via the reoxida-
tion of the ferrous ion produced.

One of the primary r a t e - l i m i t i n g steps in the leaching of


metals from ores by T. ferrooxidans i s t h i s f e r r o u s - f e r r i c r e o x i -
dation. This i s due to product i n h i b i t i o n of the oxidation s t e p ,
i . e . , f e r r i c ion competitively i n h i b i t s the rate of ferrous ion
oxidation. Kelly and Jones ( t h i s volume) have shown that
f e r r i c s u l f a t e has no effect on the rate of C O 2 f i x a t i o n and
Basic Microbial Studies Applied to Leaching 145

a f f e c t s the growth of T. ferrooxidans s o l e l y due to i t s inhibitory


e f f e c t on the ferrous ion oxidation step. Since production of
large q u a n t i t i e s of f e r r i c iron i s necessary not only f o r a high
degree of uranium s o l u b i l i z a t i o n , but a l s o to effect underground
leach mining of other valuable metals, i t would be most desirable
to obtain a mutant of T. ferrooxidans where the oxidation step
would not be repressed by f e r r i c s u l f a t e . This would be reminis-
cent of the n i t r o g e n - f i x a t i o n derepressed mutants of Klebsiella
pneumoniae, where the f i x a t i o n of atmospheric nitrogen i s no long-
er subject to repression by the product NH^ (30). Since T. ferro-
oxidans can grow r a p i d l y i n synthetic medium with ferrous s u l f a t e
as an energy source, i s o l a t i o n of such mutants in presence of
high concentration of f e r r i c s u l f a t e but l i m i t i n g concentrations
of ferrous s u l f a t e in a chemostat should be f e a s i b l e . Successful
i s o l a t i o n of such a mutant could not only lead to appreciable en-
hancement of the s o l u b i l i z a t i o n of uranium from ores such as
u r a n i n i t e , but such mutants must a l s o be used f o r d i r e c t dump
leaching of other metals such as copper, lead or z i n c .

B. Toxic E f f e c t s of Metal Ions and U V - S e n s i t i v i t y

Thiobacillus ferrooxidans i s known to develop r e s i s t a n c e


against very high concentrations of the metals being leached.
Thus, r e s i s t a n c e of T. ferrooxidans to copper ion concentrations
as high as 55 gm/liter (24) or to uranium concentrations of
12 gm U^Og/liter (31) i s not unusual. However, there are other
contaminating metal ions that appear to i n h i b i t growth even at
very low concentrations. Notable among those are s i l v e r , mercury,
and cadmium. S i l v e r , even at concentrations as low as 1.0 ppm,
can appreciably i n h i b i t the growth of T. ferrooxidans ( 3 2 ) . Since
T. ferrooxidans might release s o l u b l e s i l v e r from i t s sulfide
(argentite) which i s o c c a s i o n a l l y present with complex s u l f i d e
o r e s , t o x i c i t y to s i l v e r i s a major problem in the microbial
leaching. This problem, however, ought to be solvable by
146 Α. Μ. Chakrabarty

introducing plasmids specifying r e s i s t a n c e to metal ions such as


s i l v e r , mercury, cadmium, etc. (3,33,34) to T. ferrooxidans de-
repressed mutants. This should markedly increase the r e s i s t a n c e
of T. ferrooxidans to the i n h i b i t o r y effects of toxic metals.
T. ferrooxidans i s a l s o known to be s e n s i t i v e to UV, and plasmids
are known that can g r e a t l y increase the r e s i s t a n c e of the host
c e l l s towards U V - i r r a d i a t i o n ( 3 5 , 3 6 ) . For at l e a s t one plasmid,
the resistance to UV has been shown to be due to a p l a s m i d - s p e c i -
f i e d DNA polymerase ( 3 6 ) . Introduction of such plasmids should
therefore protect T. ferrooxidans c e l l s from the k i l l i n g action of
UV or γ - r a y s .

C. Nitrogen N u t r i t i o n

Another area in which genetic improvements f o r enhanced


growth of T. ferrooxidans f o r dump leaching of metals might be
made i s in the s e l f - g e n e r a t i o n of nitrogenous n u t r i e n t s . Although
the benefits of addition of nitrogen and phosphorus sources, as
well as organic carbon compounds, to enhance metal dump leaching
by T. ferrooxidans have not been thoroughly evaluated, i t is
l i k e l y that the exogenous supply of ammonia would help to enhance
the growth of T. ferrooxidans and hence the consequent release of
soluble metals. Enhanced growth and release of soluble metals
during s y n e r g i s t i c growth of T. ferrooxidans with the aerobic N^-
f i x i n g organism Beijerinokia laoticogenes have been interpreted as
due to the f i x a t i o n and a v a i l a b i l i t y of a s s i m i l a b l e nitrogenous
compounds (37,38). Since i t might be d i f f i c u l t f o r the N^-fixing
Beijerinokia species to grow s y n e r g i s t i c a l l y with T. ferrooxidans
at high a c i d i t y and metal ion concentrations during dump l e a c h i n g ,
one convenient way to supply additional nitrogenous n u t r i e n t s to
T. ferrooxidans might be to g e n e t i c a l l y enable i t to f i x i t s own
nitrogen. Since T. ferrooxidans i s a gram-negative short r o d -
shaped bacterium j u s t l i k e Pseudomonas, i t might be p o s s i b l e to
introduce plasmids of P I - i n c o m p a t i b i l i t y groups such as RP4 into
Basic Microbial Studies Applied to Leaching 147

it. Plasmids l i k e RP4 have a very wide host range and can be i n ­
troduced into a large number of gram-negative bacteria ( 4 ) . An
RP4 plasmid having the his nif genes of Klebsiella pneumoniae
which can also be t r a n s f e r r e d to a large number of bacterial
genera has been constructed ( 3 9 ) . One of the problems in nitrogen
f i x a t i o n by s t r i c t l y aerobic bacteria i s the l a b i l i t y of n i t r o -
genase towards oxygen, and protective mechanisms, s i m i l a r to those
in Azotobacter, must be operative w i t h i n such h o s t s . It is likely
that RP4 plasmids containing the nitorgen f i x a t i o n genes from
Klebsiella, w i l l be t r a n s m i s s i b l e and stably maintained in T.
ferrooxidans. Whether presence of such plasmids can lead to f i x a ­
tion of nitrogen by T. ferrooxidans and can r e s u l t in the enhance­
ment of growth and metal release w i l l be very i n t e r e s t i n g areas of
future research on T. ferrooxidans b i o l o g y .

IV. REFERENCES

1. Jernelov, Α . , and M a r t i n , A . L . , Ann. Rev. Microbiol., 29, 61


(1975).
2. Wood, J.M., Science, 183, 1049 (1974).
3. S c h o t t e l , J . , Mandal, Α . , C l a r k , D., S i l v e r , S . , and Hedges,
R.W., Nature, 2 5 1 , 335 (1974).
4. Chakrabarty, A.M., Ann. Rev. Genet., 10, 7 (1976).
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biol., 107, 1 (1976).
6. Jacoby, G.A., in "Pseudomonas aeruginosa: C l i n i c a l Manifesta­
tions of I n f e c t i o n and Current Therapy", (R.G. Doggett, E d . ) ,
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7. Novick, R.P., and Bouanchaud, D., Ann. N.Y. Acad. Sci., 182,
279 (1971).
8. Kondo, I . , Ishikawa, T . , and Nakahara, H., J. Bacteriol.,
117, 1 (1974).
9. Chopra, I . , Antimicrob. Agents Chemother., 7, 8 (1975).
10. S i l v e r , S . , S c h o t t e l , J . , and Weiss, Α . , in "Proceedings of
the Third International Biodegradation Symposium", (J.M.
Sharpley and A.M. Kaplan, E d s . ) , p. 899. Appl. Science
P u b l i s h e r s , London, 1976.
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148 Α. Μ. Chakrabarty

12. S a y l e r , G.S., Nelson, J . D . , and C o l w e l l , R.R., Appl. Micro­


biol., 30, 91 (1975).
13. Chakrabarty, A.M., F r i e l l o , D.A., and M y l r o i e , J . R . , U.S.
Patent 3,923,597 (1975).
14. Cherian, M.G., Biochem. Biophys. Res. Commun., 6 1 , 920 (1974).
15. R i d l e y , W.P., D i z i k e s , L . J . , and Wood, J.M., Science, 197,
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16. Chau, Y.K., Wong, P.T.S., S i l v e r b e r g , B.A., Luxon, P.L., and
Bengert, G.A., Science, 192, 1130 (1976).
17. Wong, P.T.S., Chau, Y.K., and Luxon, P.L., Nature, 253, 263
(1975).
18. Summers, A.O., and Jacoby, G.A., J. Bacterid., 129, 276
(1977).
19. Huey, C.W., Brinckman, F . E . , Grim, S.O., and I v e r s o n , W.P.,
in "Proceedings of International Conference on Transport of
P e r s i s t e n t Chemicals in Aquatic Ecosystems I I " , p. 73.
Natl. Res. C o u n c i l , Ottawa, 1974.
20. Brinckman, F.E., I v e r s o n , W.P., and B l a i r , W., in "Proceed­
ings of the Third International Biodegration Symposium",
(J.M. Sharpley and A.M. Kaplan, E d s . ) , p. 919. Appl. Science
P u b l i s h e r s , London, 1976.
21. Malouf, E.E., Mining Eng., 2 3 , 43 (1971).
22. Bruynesteyn, Α., and Duncan, D.W., Can. Met. Quart., 10, 57
(1971).
23. S i l v e r , M., and Torma, A . E . , Can. J. Microbiol., 20, 141
(1974).
24. Sakaguchi, H., Torma, A . E . , and S i l v e r , M., Appl. Environ.
Microbiol., 3 1 , 7 (1976).
25. Torma, A . E . , Rev. Can. Biol., 30, 209 (1971).
26. Tuovinen, O.H., and K e l l y , D.P., Int. Metall. Rev., 19, 21
(1974).
27. Guay, R., S i l v e r , M., and Torma, A . E . , Biotechnol. Bioeng.,
19, 727 (1977).
28. Guay, R., S i l v e r , M., and Torma, A . E . , Eur. J. Appl. Micro­
biol., 3, 157 (1976).
29. Torma, A . E . , Walden, C.C., Duncan, D.W., and Branion, R.M.R.,
Biotechnol. Bioeng., 14, 777 (1972).
30. Andersen, K., Shanmugam, K.T., and Valentine, R.C., Develop.
Indust. Microbiol., 19 ( i n p r e s s ) .
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74, 116 (1971).
Basic Microbial Studies Applied to Leaching 149

32. Hoffman, L.E., and Hendrix, J . L . , Biotechnol. Bioeng., 18,


1161 (1976).
33. Smith, D.H., Science, 156, 1114 (1967).
34. McHugh, G.L., M o e l l e r i n g , R.C., Hopkins, C.C., and Swartz,
M.N., Lancet, i , 235 (1975).
35. K r i s h n a p i l l a i , V., Mutation Res., 29, 363 (1975).
36. Lehrbach, P., Kung, A . H . C , Lee, B.T.O., and Jacoby, G.A.,
J. Gen. Microbiol., 98, 167 (1977).
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Bioeng., 16, 991 (1974).
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1 (1975).
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(1973).
STRUCTURE-FUNCTION RELATIONSHIPS OF THIOBACILLUS

RELATIVE TO FERROUS IRON AND SULFIDE OXIDATIONS

Donald Lundgren

Syracuse U n i v e r s i t y
Syracuse, New York, USA

and

Tatsuo Tano

Okayama U n i v e r s i t y
Okayama, Japan

The acidophilic bacterium T h i o b a c i l l u s ferrooxidans used to


develop a conceptual framework for explaining how the organism may
oxidize reduced iron and sulfide. Structure-function relationships
are shown using cell envelope and spheroplast-like preparations.
The cell envelope is apprently involved in both iron and sulfide
oxidations and the envelope somehow establishes micro environments
which enable the appropriate oxidases to function in an acid
milieu.

I. INTRODUCTION
Autotrophic bacteria r e s t r i c t e d to inorganic oxidations
f o r t h e i r energy are generally gram-negative b a c t e r i a . I t has
been the b e l i e f of the present i n v e s t i g a t o r s that the envelope
of these bacteria i s in some way involved in those r e a c t i o n s .

lbr
152Donal dLundgre
nandTatsu
oTano

M O D E L O F

G R A M - NEGATIVE BACTERIAL ENVELOPE

Fig. 1. Modified drawing from Costerton et al (1) showing


the layering of the gram-negative cell envelope. The drawing
is used to underscore the complexity of structure and chemical
components consisting of lipopolysaccharide, phospholipids,
protein (enzyme) structures, cations, etc. The reader should
consult the original article (1) for identification of specific
labels.
Basic Microbial Studies Applied to Leaching 153

that allow microorganisms to use inorganic substrates such as


ferrous iron and reduced s u l f u r compounds f o r energy; the
energy in turn i s used to support the c e l l ' s biosynthetic pro-
cesses necessary for growth. The present conceptual framework
for the involvement of the envelope i n chemoautotrophy reported
here comes from knowledge gleaned in published studies of
heterotrophic bacteria including Escherichia, Salmonella, Pro-
teus and Pseudomonas and from work with Thiobacillus, a chemo-
autotroph.

The complex cell envelope i s schematically shown in Figure


1. An excellent overview of the importance of the cell enve-
lope to general b i o l o g i c a l functions in heterotrophic bacteria
has been given ( 1 ) . The presence of lipopolysaccharide in the
outer membrane layer e s t a b l i s h e s a zone outside the cytoplasmic
membrane and contains the movement of molecules into and out of
the space; t h i s zone i s referred to as the "periplasmic space".
The i n d i v i d u a l macromolecular and ion components of the c e l l
envelope l a y e r s influences the p e n e t r a b i l i t y of both substrates
and end products of metabolism by binding or trapping molecules
and ions and thereby screens those molecules and ions that
reach the cytoplasmic membrane. Further, the composite of the
many chemical groups making up the s t r u c t u r a l a r c h i t e c t u r e of
the c e l l envelope creates at times a v a r i e t y of m i n i e n v i r o n -
ments which undoubtedly are important f o r the proper function
of those enzyme c a t a l y s t s promoting surface physiology and meta-
bolism of the bacterium. An example of such an a f f e c t in a
heterotrophic bacterium i s the lipopolysaccharide (LPS) i n f l u -
ence on a l k a l i n e phosphatase, an enzyme found in a s s o c i a t i o n
with the c e l l envelope ( 1 ) ; here LPS provides a favorable
physicochemical environment f o r dimerization of subunits of
enzyme.
154 Donald Lundgren and Tatsuo Tano

Relative to the subject of inorganic o x i d a t i o n s , envelope


associated enzymes protected from a harmful environment such as
low pH or high metal concentrations, could s t i l l have d i r e c t
access to a non-soluble energy substrate such as F e S . 2 Further,
the sequestation of autotrophic processes i n the c e l l envelope
would serve to i s o l a t e t o x i c products of metabolism from the
cytoplasm of the c e l l while allowing non-toxic products a v a i l -
able for transport into the c e l l . As a consequence of t h i s
basic design of c e l l envelope architecture and the r e s u l t i n g
function of the envelope in t h i o b a c i l l i , a practical industrial
a p p l i c a t i o n has occurred which couples the oxidation of an
inorganic substrate to the leaching of a metal from a poor
grade ore.

I t i s the purpose of t h i s paper to review the s t r u c t u r e -


function r e l a t i o n s h i p s of the cell envelope of Thiobaoillus
ferrooxidans, a chemoautotrophic bacterium capable of o x i d i z i n g
e i t h e r reduced s u l f u r or i r o n . Some structure and function
r e l a t i o n s h i p s f o r ferrous i r o n and s u l f i d e oxidation are con-
sidered. I t i s the presence of both iron and s u l f u r in the
form of p y r i t e ( F e S ) that makes b i o l o g i c a l leaching of metals
2

economically f e a s i b l e .

The subject w i l l be introduced in the following manner:


(a) The nature of the c e l l envelope of the gram-negative
bacterium Thiobaoillus ferrooxidans. (b) The involvement of
the c e l l envelope i n the oxidation of F e + +
and S~.

II. EXPERIMENTAL METHODS

The organism used was Thiobaoillus ferrooxidans. Cultures


were grown in the standard 9K medium (2) and when grown on
s u l f u r , i r o n was replaced with c o l l o i d a l s u l f u r at 5 gm/liter.
Culture v e s s e l s used were 5-gallon carboys equipped with
spargers and aeration was accomplished by passing humidified
Basic Microbial Studies Applied to Leaching 155

compressed a i r through the culture medium. The incubation


temperature was 28 C and c u l t u r e s were aerated for 96 hours
with iron-grown c e l l s and f o r 5-6 days with c o l l o i d a l sulfur.
A l l i n o c u l a t i o n s f o r iron-grown c e l l s were made with f r e s h
washed cell suspensions (15% w/v) and f o r sulfur-grown c e l l s
residual s u l f u r from a p r e v i o u s l y grown c u l t u r e served as an
inoculum.

Cell f r a c t i o n s , envelope preparations and spheroplasts


were prepared using the French Pressure Cell (3) or a modified
spheroplast procedure designed around that commonly used f o r
Escherichia coli (4) and that procedure published f o r T. ferro-
oxidans ( 5 ) . In the l a t t e r procedure the Tris-HCl buffer was
replaced by phosphate buffer (pH 7.9) and the C a C l was ?

++
replaced by Mg . I t was immediately apparent that T. ferro-
oxidans grown on e i t h e r iron or s u l f u r did not respond to con-
ventional spheroplasting or c e l l envelope separation procedures
employed f o r heterotrophic bacteria and that many procedural
adjustments had to be made.
Iron oxidation was assayed by measuring 0 2 uptake with a
Clark 0 2 electrode.
S u l f i d e oxidation was followed by measuring 0 2 utilization
with the oxygen electrode using 0.12 ymoles of NaS 2 as the
substrate i n 3-alanine s u l f a t e b u f f e r , pH 3.0. Electron
microscopy was done following e s s e n t i a l l y the e a r l i e r proce-
dures c i t e d f o r T. ferrooxidans ( 3 , 6 ) .

Protein concentration was estimated c o l o r i m e t r i c a l l y ( 7 ) .


P r i o r to the a s s a y , c e l l s and c e l l envelopes were hydrolyzed
f o r 30 minutes in 0.5N NaOH in a b o i l i n g water bath.

III. RESULTS AND DISCUSSION

A review of the s t r u c t u r e and function r e l a t i o n s h i p s of


the c e l l envelope o f gram-negative bacteria has recently
156 Donald Lundgren and Tatsuo Tano

appeared. Figure 1 i s a drawing of the envelope of Gram-nega-


t i v e bacteria. I t s s t r u c t u r a l complexity i s apparent, but i t
i s t h i s complexity that accounts f o r the wide range of growth
habitats of Gram-negative bacteria including the acid e n v i r o n -
ment required by t h i o b a c i l l i used in metal leaching. The
envelope c o n s i s t s of three major zones: (1) Cytoplasmic
membrane which c o n s t i t u t e s the inner layer of the envelope
bordering on the cytoplasm. (2) The central zone comprising
both the r i g i d layer of peptidoglycan and the periplasmic
space. (3) The outer layer which contains the l i p o p o l y -
saccharide and l i p o p r o t e i n . Excellent d i s c u s s i o n s of each of
these zones in addition to that c i t e d e a r l i e r have appeared
(8, 9, 10, 1 1 ) .

Much of the f i n e structure d e t a i l s of T. ferrooxidans has


been reviewed ( 1 2 ) . For general o r i e n t a t i o n , c e l l s that have
been treated in three d i f f e r e n t ways to h i g h l i g h t t h e i r surface
features are shown in Figure 2. Figure 2a shows negatively
stained f e r r o b a c i l l i ; Figure 2b i s a t h i n section of a part
of the cell envelope revealing the outer membrane (0M), pep-
tidoglycan (PG) and cytoplasmic membrane (CM), the periplasmic
space resides between the outer and inner b a r r i e r s of the c e l l .
Figure 2c i s a frozen etched preparation of the same cell
revealing the aforementioned l a y e r i n g of the c e l l envelope.

Figure 3 i s a sketch of the envelope showing l a y e r i n g of


a bacterium in j u x t a p o s i t i o n to a c r y s t a l of p y r i t e (FeS^)
modeled a f t e r an e a r l i e r published idea ( 1 3 ) . How the outer
layer of the bacterium influences the s o l u b i l i z a t i o n and sub-
sequent oxidation of the two substrates ( F e + +
and 2S~") i s
the important question r e l a t i n g to how these organisms function
in metal freeing from poor grade o r e s . Both F e + +
and S "
substrates are capable of attack by the bacterium and both
the " i r o n oxidase" and " s u l f i d e o x i d a s e " , two complex enzyme
Basic Microbial Studies Applied to Leaching 157

Fig. 2. Electron micrographs of T h i o b a c i l l u s f e r r o o x i d a n s .


negative stained preparation, b. thin section of a chemi-
lly fixed cell. c. frozen etched preparation.
158 Donald Lundgren and Tatsuo Tano

Fig. 3. Sketch of the layering of the cell envelope in _


juxtaposition to an insoluble pyrite crystal. Both Fe* and S +

are energy substrates for the bacterium.

systems responsible for the respective o x i d a t i o n s , must co-


e x i s t and function depending upon what external environmental
conditions are needed by the organism to support the appropriate
biological reactions.

In the case of F e + + oxidation the overall reaction i s :

2Fe + + + h0 2 + 2H + > 2Fe + + + + H0 2

The two components of the overall reaction of iron oxidation


are apparently s p a t i a l l y separated within the c e l l envelope
(14). The energy associated reaction (2e~ + % 0 2 + 2H + >
H 0) i s probably located within the inner membrane whereas the
2

(2Fe + + ) 2Fe + + + + 2e~) reaction i s involved at the outer


Basic Microbial Studies Applied to Leaching 159

membrane or perplasmic space level ( 1 4 ) . How the two reactions


are coupled in situ i s s t i l l unknown but the separation from a
practical standpoint i s important f o r the product F e + + +
of t h i s
reaction never has to enter the bacterium and be handled by a
special transport system.

I t i s apparent that within the c e l l envelope the o r g a n i z a ­


t i o n of the necessary enzymes required to support the a f o r e ­
mentioned oxidation reactions are protected from both the
external acid m i l i e u and the high concentrations of metals.
F e r r i c iron i s extremely i n s o l u b l e at pH values above 4.0 and
large amounts of t h i s metal accumulates following F e + +
ion
oxidation. These substrate " o x i d a s e s " are so s t r u c t u r a l l y
organized i n t h e i r respective l a y e r s that microenvironments
favorable to the functioning of enzymes are formed even though
the natural acid environment of the bacterium i s harmful when
the enzymes are separated from the c e l l . This i s shown with
c e l l envelopes, i s o l a t e d from iron-grown t h i o b a c i l l i , which
possess approximately 40% of the iron o x i d i z i n g a b i l i t y of
intact c e l l s . Figure 4 shows a t h i n s e c t i o n of enzymatically
a c t i v e c e l l envelopes of T. ferrooxidans with an optimum pH
or iron o x i d a t i o n of 2.5 ( 3 ) . I t i s known that the optimum pH
of at l e a s t two p a r t i a l l y p u r i f i e d enzyme components o f the
" i r o n oxidase" complex (Fe -Cytochrome c reductase and
++

Cytochrome oxidase) are approximately pH 6.5 and 4.5 respec­


t i v e l y (15, 1 6 ) .

At t h i s point in time r e l a t i v e l y l i t t l e i s known about


the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s of iron o x i d a t i o n in τ.
ferrooxidans. S u f f i c e to say, an examination of Table 1
q u i c k l y reveals that compared to the cell envelopes of Sal­
monella and other heterotrophic bacteria l i t t l e i s a l s o known
about the chemical composition of c e l l envelopes of T. ferro­
oxidans. Results of some physical and chemical studies of
160Donal dLundgre
nan dTatsu
oTano

Fig. 4. Thin section of chemically fixed isolated cell


envelopes from iron-grown T h i o b a c i l l u s ferrooxidans.

T. ferrooxidans lipopolysaccharide have recently been published


(17). Lipopolysaccharide i s o l a t e d from iron-grown c e l l s was
stable to acid and a l k a l i , possessed a r i b b o n - l i k e morphology,
contained no phosphorous and very l i t t l e protein and possessed
an unknown f a t t y acid component.

The oxidation of the s u l f u r component of p y r i t e r a i s e s


comparable questions of s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s in
the bacterium. The oxidation of S~ i s reported to follow those
reactions given below (18, 19, 2 0 ) ;
Basic Microbial Studies Applied to Leaching 161

TABLE I Composition of Inner and Outer Membrane Fractions of


Salmonella typhimurium and T h i o b a o i l l u s ferrooxidans

Salmonella typhimurium T h i o b a o i l l u s ferroxidans


Inner (Cytoplasmic) Membrane
Phospholipids Phospho lipids
Proteins, including: Proteins, including:
Electron transfer Electron transfer
activities activities
Permease functions
Phospholipids biosynthetic
enzymes
Lipopolysaccharide
biosynthetic enzymes
Outer Membrane
Lipopolysaccharide Lipopolysaccharide
Phospholipids Phospholipids
Proteins, including: Proteins, including:
Phospholipase activity
Murein lipoprotein Murein lipoprotein
Major polypeptides of
known function
A. Iron binding and
transport
B. Colicin binding
C. Vitamin Β22 binding
D. Receptor F-pilus

SH" su^lde [ s ] + H + + 2 -
oxidase

2[S] * [S-S]

[S-S] + SH" > "S-S-SH

"S-S-SH + X > X-S-S-SH

zix-s-s-sH] p;^!!ir > de x


-v x
162 Donald Lundgren and Tatsuo Tano

S~~ oxidation proceeds in two s t a g e s . In the f i r s t s t a g e ,


s u l f i d e l o s e s two electrons mediated by s u l f i d e oxidase, and
polymerization of the r e s u l t i n g s u l f u r atoms f o l l o w s . The
oxidation of a s h o r t - c h a i n p o l y s u l f i d e to polymeric s u l f u r
compounds occurs. These p o l y s u l f i d e s are thought to be mem-
brane bound. X in the scheme represents a hypothetical group
l i n k i n g the p o l y s u l f i d e chain to a membrane f r a c t i o n of the
c e l l envelope. Formation of t h i s membrane bound p o l y s u l f i d e
when studied using membrane f r a c t i o n s o f T. thiooxidans
required a cytoplasmic component ( 2 1 ) . P o l y s u l f i d e oxidase
catalyzes the oxidation of the p o l y s u l f i d e .

S u l f i d e o x i d a t i o n was studied using T. ferrooxidans grown


on c o l l o i d a l s u l f u r at pH 3 . 5 ; membrane preparations of these
c e l l s were prepared according to the procedures reported f o r
iron-cfrown c e l l s ( 3 ) . However, with these membranes s u l f i d e
oxidation occurred only at pH 7.0, and not at pH 3.0. These
preliminary r e s u l t s suggested that the s t r u c t u r a l organiza-
t i o n of the membrane components involved in the oxidation was
v i t a l i f c e l l s were to mediate the f i r s t stage of s u l f i d e
oxidation in an acid m i l i e u .

A much milder treatment of breaking intact c e l l s than


that of d i s r u p t i o n by the French Press was used in an attempt
to prepare c e l l - f r e e membranes capable of supporting S~
oxidation. The spheroplasting procedure e a r l i e r used for
thiobacilli (5) was employed as a s t a r t i n g point in t h i s
study.

The spheroplasting treatment to sulfur-grown c e l l s and


the subsequent d i f f e r e n t i a l c e n t r i f u g a t i o n of the treated
c e l l s on sucrose gradients led to the i s o l a t i o n of three types
of membrane and c e l l envelope or leaky spheroplast prepara-
tions; Figure 5 shows thin sections of these preparations
Basic Microbial Studies Applied to Leaching 163

i d e n t i f i e d as F - l , F-2, F-3 and i n t a c t bacterium. Sulfide


oxidation at a low pH was observed in the F-3 preparation and

Fig. 5. Electron micrographs showing thin sections of


sulfur-grown T h i o b a c i l l u s ferroxidans treated for spheroplast
formation:
(a) Intact cell - Thin section of an intact sulfur-grown cell.
(b) F-l fraction - Membrane collected at a 40 ^ 46% sucrose
gradient in 0.01M phosphate buffer (pH 7.0) containing
10 mM MgS0 . 4

(c) F-2 fraction - Envelope membranes collected at a 46 ^ 51%


sucrose gradient as above.
(d) F-3 fraction - Envelope membranes and leaky spheroplasts
collected at a 52 ^ 54% sucrose gradient.
164 Donald Lundgren and Tatsuo Tano

t h i s f r a c t i o n a l s o possessed both NADH oxidase and succinate


dehydrogenase a c t i v i t i e s . The f r a c t i o n contained s p h e r o p l a s t -
l i k e s t r u c t u r e s which had l o s t t h e i r r i g i d i t y but were s t i l l
s t r u c t u r a l l y organized so that the respective c e l l envelope
l a y e r s appeared to be i n t a c t . The authors conclude that
enough s t r u c t u r e i s i n t a c t to e s t a b l i s h the necessary micro-
environments for the enzymes to permit the o x i d a t i v e reactions
within the natural acid environment of the bacterium.

Figure 6 shows the pH a c t i v i t y curve for s u l f i d e o x i d a t i o n


of the F-3 preparation; neither the F-l nor F-2 preparations

Ο' 1 1 -i r
1 2 3 4 5
pH

Fig. 6. pH activity curves for sulfide oxidation by


intact T h i o b a c i l l u s ferrooxidans and the F-3 fraction. Reaction
mixture contained for intact cells (0 0) $-alanine-H2SΟ 4

buffer; cells, 60 mg/dry wt.; Na S, 0.12 \imoles; total


volume, 2 ml. For the F-3 fraction the reaction mixture con­
tained spheroplasts and membranes, 2.74 mg protein; $-alanine-
#2^04 containing 1 mM MgS0 4and 10% sucrose; Na2S, 0.12 \wiole.
Temperature was 28 C.
Basic Microbial Studies Applied to Leaching 165

possessed s u l f i d e oxidase a c t i v i t y . Further, F-l and F-2 pre­


parations did not possess NADH oxidase nor succinate dehydro­
genase a c t i v i t i e s .

From these preliminary r e s u l t s , i t i s the a u t h o r s ' feeling


that the two membrane systems, that i s the 1ipopolysaccharide-
containing outer membrane and the inner cytoplasmic membrane
c o n s t i t u t e the necessary s t r u c t u r a l organization needed to
support s u l f i d e oxidation in an acid m i l i e u . Hopefully, a
successful approach in i s o l a t i n g active membrane f r a c t i o n s w i l l
enable us to answer t h i s question.

IV. REFERENCES

1. Costerton, J.W., Ingram, J.M., and Cheng, K . J . , Bacteriol.


Rev., V o l . 38, No. 1 , 87 (1974).
2. Silverman, Μ., and Lundgren, D., J. Bacteriol., 11, 642
(1959).
3. Bodo, C , and Lungren, D.G., Can. J. Microbiol., 20, 1647
(1974).
4. Osborn, M. J . , Gander, J . E . , P a r i s i , E., and Carson J . ,
J. Biol. Chem., 247, 3962 (1972).
5. Howard, Α . , and Lundgren, D.G., Can. J. Biochem., 48, 12,
1302 (1970).
6. Remsen, C , and Lundgren, D.G., J. Bacterid., 92, 6, 1765
(1966).
7. Lowry, 0 . , Rosebrough, M., F a r r , Α . , and Randall, R., J. Biol.
Chem., 193, 265(1951).
8. Bayer, M . E . , in "Mode of Action of A n t i b i o t i c s on Microbial
Walls and Membranes", (M.R.J. Sal ton and A. Tomasz, E d s . ) ,
Vol. 235, p. 6. N.Y. Acad. S c i . , 1974.
9. Osborn, M . J . , R i c k , P.D., Lehmann, V . , Rupprecht, E., and
S i n g h , Μ., in "Mode of Action of A n t i b i o t i c s on Microbial
Walls and Membranes", (M.R.J. Sal ton and A. Tomasz, E d s . ) ,
Vol. 235, p. 52. N.Y. Acad. S c i . , 1974.
10. L e i v e , L., in "Mode of Action of A n t i b i o t i c s on Microbial
Walls and Membranes", (M.R.J. Salton and A. Tomasz, E d s . ) ,
V o l . 235, p. 109, N.Y. Acad. S c i . , 1974.
11. S a l t o n , M . R . J . , and Owen, P., in "Annual Review of M i c r o ­
b i o l o g y " , (M.P. S t a r r , J . L . Ingraham, and S. R a f f e l l , E d s . ) ,
Vol. 30, p. 4 5 1 . Annu. Rev. I n c . , C a l i f o r n i a , 1976.
166 Donald Lundgren and Tatsuo Tano

12. Lundgren, D . J . , V e s t a l , J . R . , and T a b i t a , F.R., in "Water


P o l l u t i o n M i c r o b i o l o g y " , (R. M i t c h e l l , E d . ) , p. 69. John Wiley
& Sons, I n c . , 1972.

13. Dugan, P.R., "Biochemical Ecology of Water P o l l u t i o n " , p. 124.


Plenum P r e s s , New York, 1972.
14. Ingledew, W.J., Cox, J . C . , and H e l l i n g , P . J . , Proc. Soc. Gen.
Microbiol., V o l . 4, Part 2 , 74 (1977).
15. D i n , G.A., S u z u k i , I . , and Lees, H., Can. J. Biochem., 4 5 ,
1523 (1967).
16. B l a y l o c k , B.A., and Nason, Α . , J. Biol. Chem., 20, 3453
(1963).
17. H i r t , W.E., and V e s t a l , J . R . , J. Bacterid., 123, 642 (1975).
18. M o r i a r t y , D.J.W., and N i c h o l a s , D.J.D., Biochem. Biophys.
Acta, 184, 114 (1969).
19. Aminuddin, M., and N i c h o l a s , D.J.D., Biochim. Biophys. Acta,
325, 81 (1973).
20. Aleem, M . I . H . , Plant and Soil, 587 (1975).
21. Tano, T . , Oka, M., and Lundgren, D.G., Abstr. Annu. Meeting
Amer. Soc. Microbiol., 15 (1977).
LEACHING OF MINERALS USING BACTERIA OTHER THAN THIOBACILLI

Norman W. Le Roux
Don S. Wakerley
and
Vivian F. Perry

Warren Spring Laboratory


Stevenage
Herts SGI 2BX
England

The aim was to investigate the technical feasibility of


leaching metals using bacteria other than thiobacilli. Bacteria
were isolated from soil and grown on organic media so that
metabolic products were produced which were capable of leaching
nickel and copper from a copper/nickel concentrate. The bacteria
were not examined in detail but most of those used in the tests
were rod-shaped cells of various sizes. On solid media, they
grew on malt plus peptone agar plates at pH 5.0 as smooth, cream-
coloured colonies. Some indication of metal selectivity and
adaptation to metal inhibition was observed. The bacteria pro-
duced acids from the organic substrates and in one case 99% nickel
and 42% copper were leached. Comparative tests using sulphuric
acid for leaching were less efficient than the microbial tests.
Nickel solubilization, which was greater than copper, was favoured
by low pH and was directly related to the development of acidity.
Metal leaching was also favoured by elevated temperature and
agitation. The amount of nickel solubilized increased but the
percentage amounts of ore solubilized decreased with increase in
ore pulp density. At 25% pulp density the concentration of nickel
in solution was about lOg per litre.

167
168 Norman W. Le Roux et al.

I. INTRODUCTION
The a b i l i t y of t h i o b a c i l l i to a s s i s t in the recovery of
metals from ores i s now well known and t h e i r contribution to the
s o l i b i l i z a t i o n of metal s u l p h i d e s , while the mechanism i s not
completely c l e a r , i s accepted and well documented e.g. ( 1 ) . They
are the only microbes to be used for metal recovery on a commer-
cial scale.

In general, microbes are ubiquitous on t h i s planet and are


capable of producing many varied metabolities under d i f f e r e n t
environmental c o n d i t i o n s . I t has been shown that the m o b i l i z a t i o n
of metals can r e s u l t from microbial a c t i v i t y ( 2 ) . The extent and
magnitude of these ' n a t u r a l ' phenomena are not well documented
for at l e a s t two reasons. Various rate l i m i t i n g f a c t o r s lead to
very slow reaction k i n e t i c s , the amounts of metal involved are
very small and the time span long when considered against a man's
working l i f e . However, the amounts and effects can be large when
measured in geological time. What are the reactions taking place
in the natural environment? What are the rate l i m i t i n g factors?
Can these factors be optimized so that the a b i l i t y of microbes to
mobilize metals can be harnessed f o r the benefit of man? These
are far-reaching questions and cannot yet be answered.

In the preliminary studies reported here, f u r t h e r evidence


was sought to reinforce these ideas. The technical feasibility
f o r metal s o l u b i l i z a t i o n by microbes other than t h i o b a c i l l i was
studied on a laboratory s c a l e . I t i s emphasized that conditions
were not optimized and economic v i a b i l i t y was not sought. The
authors are well aware of the many problems in bridging the gap
between technical f e a s i b i l i t y and economic/commercial viability.

II. EXPERIMENTAL
A. Metal Source

Two copper/nickel sulphide concentrates were used as a source


of metals. Sample A contained 3.8% copper and 11.5% n i c k e l , and
Basic Microbial Studies Applied to Leaching 169

Sample B, 4.5% copper and 7 . 1 % n i c k e l . The copper and nickel


were present as chalcopyrite and pentlandite. Sample Β was only
used for the higher pulp density t e s t s .

B. Analyses

Copper, n i c k e l , and iron in the f i l t e r e d supernatant l i q u o r s


and in the r e s i d u e s , which were dried at room temperature, were
measured by atomic absorption spectrophotometry.

C. Inocula

Two s o l i d s w e r e used as sources for microbes. S o i l Ρ was a


Black Norfolk Peat and S o i l C a Hertfordshire Clay adjacent to an
organic compost heap. No p a r t i c u l a r s i g n i f i c a n c e should be
attached to these s o i l samples. They were chosen f o r t h e i r con­
venience and the potential presence of a large v a r i e t y of microbes.

D. Media

Two common bacterial media were used, Malt Extract (Oxoid


L t d ) , r i c h in carbohydrates and B a c t e r i o l o g i c a l Peptone (BDH L t d ) ,
r i c h in p r o t e i n s . These were prepared i n d i v i d u a l l y as 1.66g Malt
Extract/1 and 0.34g Peptone/1 and were a l s o used mixed.

For t e s t s with Thiobacillus ferrooxidans the inorganic s a l t s


medium of Silverman and Lundgren was used ( 3 ) .

E. I s o l a t i o n of Microbes

Bacteria were i s o l a t e d on agar plates prepared by adding 1%


Ionagar (BDH Ltd) and an acid i n d i c a t o r (Bromecresol Green or
BDH 4.5) to mixed Malt Extract/Peptone medium. After s t e r i l i z a ­
tion by autoclaving (121°C for 20 minutes) the medium was poured
into petri d i s h e s , cooled, streaked with samples from culture
f l a s k s and incubated at 30°C.
170 Norman W. Le Roux et al.

F. Microbial Testwork

A s e r i e s of t e s t s were made with the media and inocula des-


cribed and the v a r i a b l e s pH, temperature, a g i t a t i o n , and pulp
density studied.

Known weights (about 0.4g) of copper/nickel concentrate were


added to 100 ml of medium in 250 ml conical f l a s k s . The f l a s k s
were shaken in an o r b i t a l incubator (A.E. Gallenkamp Ltd) at 200
rpm and 30°C. S o l u t i o n analyses f o r copper and nickel and pH
measurements were done on the supernatant l i q u o r s every 3 to 5
days. The shaken t e s t s were allowed to stand f o r 4h before
samples were withdrawn f o r a n a l y s i s . The i n i t i a l t e s t s which
were done at c o n t r o l l e d pH v a l u e s , were readjusted when necessary
by the addition of NaOH or H^SO^ when the s o l u t i o n s were analysed.
Tests which showed the highest rates of leaching were selected for
making subcultures which were made with a 1% inoculum. Sterile
control t e s t s were c a r r i e d out by adding a biocide (5 ml of 1%
"Panacide", BDH L t d ) .

III. RESULTS AND DISCUSSION

A. Initial Tests

An i n i t i a l s e r i e s of t e s t s were done to determine the rates


of leaching of nickel and copper from Sample A in a mixed malt +
peptone medium adjusted to pH 5.0 or 7.0. P a i r s of f l a s k s were
either continuously shaken to g i v e good aeration or kept s t a t i o n -
ary to give poor a e r a t i o n . E i t h e r peat or clay s o i l ( l g ) was
used as an inoculum. I t was intended that a l l t e s t s would be
done at the c o n t r o l l e d temperature of 30°C. However, a f t e r the
f i r s t day the production of f o u l - s m e l l i n g fermentation products
from the s t a t i o n a r y t e s t s was so obvious that these f l a s k s had to
be removed to a fume hood at room temperature (about 20°C) f o r the
remainder of the t e s t period.
Basic Microbial Studies Applied to Leaching 171

In the f i r s t 50 days three t e s t s showed good s o l u b i l i z a t i o n


of nickel (Fig. 1). The highest rate of nickel solubilization
occurred in the aerated f l a s k containing the more a c i d , clay s o i l
culture. About 220 ppm Ni (50%) was s o l u b i l i z e d in 50 days.
Lower rates of nickel e x t r a c t i o n were obtained using 'non-aerated'
( s t a t i o n a r y ) cultures but t h i s might have been due to the fact
that the t e s t s were c a r r i e d out at room temperature (20°C) rather
than 30°C.

In the t e s t with clay s o i l at pH 7.0, nickel which had been


s o l u b i l i z e d was p r e c i p i t a t e d . In a l l these i n i t i a l t e s t s the
amount of copper in s o l u t i o n was l e s s than 3 ppm.

3 00 1 1 1 1 1 1 1 1 1
1 P E ATS O ILp H 5 0] ο2 P E A TS O I Lp H5 Ο
3 P E ATS O ILp H7 Ο I A E R A T E D "4 P E A TS O I Lp H7 Ο STATIONA YR
•7 C L AYS O I Lp H 5 0[ C U L T U R ES • βC L A YS O I Lp H5 Ο C U L T U RSE
.9 C L AYS O ILp H7 Ο J 1 0C L A YS O I Lρ Η 7 0
C3 S T E R IELC O N T R O
Lp H7 Ο
2 50

2 00

1 50

υ
ζ
1 00

50

Fig. 1. Leaching of nickel using matt + peptone medium at


pH 5.0 and 7.0 with peat or clay soil as inoculum.
172 Norman W. Le Roux ef al.

In the t e s t s at pH 5.0 and to a l e s s e r extent in those at


pH 7.0, acid was produced in the f l a s k s . The s o l u t i o n s originally
at pH 5.0 became a c i d , pH 3.5 to 4.0 every 3 to 5 days despite the
fact that the s o l u t i o n pH was restored to 5.0 each time pH mea­
surements were made. This acid production was associated with
nickel s o l u b i l i z a t i o n and maximum nickel extraction occurred when
acid production was most r a p i d .

B. Effect of Subculturing

In an attempt to increase the beneficial microbial activity


sucessive subcultures were made from the clay s o i l culture at pH
5.0. Some increases in the rate of nickel s o l u b i l i z a t i o n and

3 00

Ο2 04 06 08 01 0 01 2 0
TIME
,d

Fig. 2. Leaching of nickel with successive clay soil sub­


cultures on malt +-peptonemedium at pH 6.0.
Basic Microbial Studies Applied to Leaching 173

y i e l d were achieved ( F i g . 2 ) . Residue analyses showed that with


the second and t h i r d subcultures over 80% nickel and about 20%
copper had been extracted (Table 1 ) .

C. Tests Without pH Control

The i n i t i a l t e s t s had suggested that nickel solubilization


was favoured by low pH. Moreover, c u l t u r e s o r i g i n a l l y at pH 5.0
and to a l e s s e r extent at pH 7.0 produced a considerable amount
of acid and needed repeated pH adjustment. To examine the effect
of dispensing with pH adjustment a subculture from the aerated
clay s o i l culture at pH 5.0 was made into malt + peptone medium
i n i t i a l l y adjusted to pH 5.0 but thereafter no further pH a d j u s t -
ments were made. The rate of nickel d i s s o l u t i o n obtained ( F i g .
3a) was s i m i l a r to those obtained in the i n i t i a l aerobic t e s t s at
pH 5.0. A l s o , l i k e the i n i t i a l t e s t s copper in s o l u t i o n was very
low. This was u n l i k e l y to be due to a pH e f f e c t as the pH started
at 5.0 and then decreased during the course of the experiments.
Residue a n a l y s i s , which a l s o included the measurement of nickel
and copper which had been s o l u b i l i z e d and then precipitated
(Table 1) showed that 99% nickel and 42% copper had been extracted
in 66 days, which was considerably more than had been obtained
previously. The pH of the c u l t u r e was a l s o determined and good
c o r r e l a t i o n was shown between the rate of nickel solubilization
and the development of a c i d i t y ( F i g . 3 a ) .

Repeated subcultures of t h i s t e s t were made and increases in


the rates of nickel leaching and acid production were obtained
(Figs. 3b-3f). A f t e r 5 subcultures the time f o r maximum nickel
extraction was reduced from 73 to about 30 days ( F i g . 3 f ) . The
amounts of nickel and copper extracted, as determined by residue
analyses remained between 81-98% and 25-42%, r e s p e c t i v e l y
(Table 1 ) . In a l l these s u b c u l t u r e s , s o l u t i o n analyses showed
that a f t e r some time nickel was being p r e c i p i t a t e d . I t was
noticed t h a t , simultaneously with the onset of nickel p r e c i p i t a t i o n ,
TABLE I Residue and Solution Analyses of Bacterial
Leaching Tests on Malt and Peptone Media

Wt of wt of NickeI Coppier
ore residue
Test In In Solubi- Solubi- In In In Solubi- Solubi- In
Medium Ore Resi­ lized lized Solution Ore Resi­ lized lized Solution
due * due λ *

mg mg ppm ppm ppm % ppm ppm ppm ppm % ppm

Initial Tests at pH 5.0


M+P 2nd subculture 461 382 530 101 429 81 283 176 130 46 26 0.6
M+P 3rd subculture 393 373 450 70 380 84 270 150 123 27 18 0.3
174

Tests without pH Control


M+P 351 236 403 6 397 99 250 134 78 56 42
M+P 1st subculture 439 393 505 15 490 97 335 168 125 43 26 16
M+P 2nd subculture 467 426 536 22 514 96 308 155 115 40 26 8
M+P 3rd subculture 425 393 488 86 402 82 290 162 118 44 27 11
M+P 4th subculture 419 416 482 87 395 82 315 161 121 41 25 10
M+P 5th subculture 452 353 518 17 501 97 473 172 127 45 26 21
M+P 30°C then 40°C 428 366 493 35 458 93 320 164 117 47 29 9
M+P 40°C 399 403 460 14 446 97 433 153 131 22 14 14

* Calculated from residue analyses.


Basic Microbial Studies Applied to Leaching 175

450r—
r

Ο2 04 06 0Θ Ο1 0 0Ο 2 04 06 0Θ ΟΙ Ο ΟΟ 2 04 06 0Θ Ο1 0 0

Fig. δ. (3a-δf) Bacterial leaching of nickel and copper


using malt + peptone medium without pH control.

iron p r e c i p i t a t i o n could be v i s u a l l y observed. The time at which


iron p r e c i p i t a t i o n was f i r s t observed i s indicated in F i g . 3f and
a l s o in some l a t e r f i g u r e s . Because of t h i s observation the
behaviour of iron in s o l u t i o n was determined in the f i f t h c u l t u r e .
The maximum i r o n in s o l u t i o n was 247 ppm (17%) and the leaching
curve f o r iron had a s i m i l a r pattern to that f o r nickel (Fig. 3f).
176 Norman W. Le Roux ef al.

A t e s t was also made to determine i f an inoculum taken from


a culture which was a c t i v e l y leaching nickel a f t e r 7 days was
more e f f e c t i v e than inocula taken when nickel s o l u b i l i z a t i o n and
acid production were d i m i n i s h i n g . Contrary to expectation the
l a t t e r were found to be the most active inocula although the
numbers of bacteria per unit volume were not counted.

D. Effect of Temperature and A g i t a t i o n

Tests were done in shaken and s t a t i o n a r y f l a s k s at both 30°C


and room temperature ( c i r c a 20°C) using malt + peptone medium at
pH 5.0. The clay s o i l culture at pH 5.0 from the i n i t i a l tests
was used as inoculum f o r each f l a s k .

The rates of leaching of nickel were i n i t i a l l y more rapid in


both the shaken t e s t s . A f t e r 15 days the rate of leaching in the
s t a t i o n a r y t e s t at 30°C increased to that obtained in the shaken
t e s t at 30°C and a s i m i l a r quantity of nickel was f i n a l l y e x t r a c t -
ed in both t e s t s ( F i g . 4 ) .

The rate of leaching in the s t a t i o n a r y t e s t at room tempera-


ture was very slow although the rate did increase a f t e r day 36.
In the shaken t e s t at room temperature leaching was i n i t i a l l y
reasonably rapid but a f t e r about day 15 the rate decreased sharply.
This was probably the r e s u l t of rough shaking with l o s s of s o l u t i o n
on to the wool plug in the neck of the f l a s k . For t h i s reason the
t e s t must be regarded as unrepresentative. Although the r e s u l t s
indicate that the leaching of nickel i s favoured at 30°C and by
shaking i t i s not p o s s i b l e to a s s e s s which factor i s most impor-
tant without further t e s t i n g .

E. Tests at 40°C Without pH Control

The e a r l i e r leaching t e s t s had shown that better leaching


took place at 30°C than at room temperature. Thus, t e s t s were
carried out to determine the rate of leaching at 40°C in malt +
peptone medium at an i n i t i a l pH of 5.0 but with no subsequent
Basic Microbial Studies Applied to Leaching 177

300

°t 150 h

Fig. 4. Effect of aeration and temperature on the bacterial


leaching of nickel using malt + peptone medium at pH 5.0.

pH c o n t r o l . In one of the t e s t s the culture was grown at 30°C for


10 days and then transferred to 40°C while in a p a r a l l e l t e s t , the
culture was maintained at 40°C throughout the t e s t period.

There was an increase in the rate of leaching of nickel when


the culture was transferred from 30 to 40°C a f t e r 10 days but in
the t e s t at 40°C, nickel extraction was e r r a t i c and at no time did
the rate exceed that obtained at 30°C ( F i g . 5 ) . A pH curve i s
178 Norman W. Le Roux e£ al.

1 ι ι 1 1 1 1 1—
• 1 ΝI - CULTURE AT 30°C FOR ΙΟ DAYS, THEN AT40°C
A 2 Ni - CULTURE AT 40°C
• 3 Ni -CULTURE AT 30°C
Ο 4 Cu - CULTURE AT 30°C FOR 10 DAYS, THEN AT40°C
• 5 pH - CULTURE AT 30°C FOR 10 DAYS. THEN AT40°C

Ο ΙΟ 20 30 40 50 60 70 ΘΟ 90 ΙΟΟ
TIME.d

Fig. 5. Variation of extraction rates with temperature for


nickel and copper on malt + peptone medium.

a l s o plotted and shows a c o r r e l a t i o n between the rate of nickel


extraction and rate of acid production.

Residue a n a l y s i s (Table 1) showed that copper s o l u b i l i z a t i o n


was only about h a l f that obtained in comparable t e s t s at 30°C,
whereas nickel s o l u b i l i z a t i o n was unaffected.

In the t e s t at 40°C, microscopic examination showed that the


bacteria became very elongated a f t e r only a day at 40°C suggesting
that microbial growth was being adversely affected.

F. Effect of Pulp Density

Tests were made with copper/nickel concentrate at pulp


d e n s i t i e s of 1.2, 4 . 0 , 10.0, 15.0, and 25.0% using cultures grown
Basic Microbial Studies Applied to Leaching 179

on malt + peptone medium. The t e s t at 1.2% pulp density was done


with Concentrate A but because of a v a i l a b i l i t y Concentrate Β was
used f o r t e s t s at higher pulp d e n s i t y . A s t e r i l e control t e s t was
set up using 4% pulp d e n s i t y . The rates of n i c k e l , copper, and
iron s o l u b i l i z a t i o n and pH changes are shown in F i g s . 6a to 6 f .

A comparison of the rates of nickel s o l u b i l i z a t i o n obtained


in these t e s t s and a l s o i n a previous t e s t using 0.4% pulp density
( F i g . 3 f ) shows a steady increase with increase in pulp density
(Fig. 6g).

The calculated average nickel e x t r a c t i o n rates at d i f f e r e n t


pulp d e n s i t i e s f o r a 26 day period of maximum s o l u b i l i z a t i o n ,
showed the e f f i c i e n c y of nickel extraction decreased with i n c r e a s ­
ing pulp density (Table 2 ) . I t should be noted that Table 2 shows
r e s u l t s averaged only f o r a good 26 day period and not f o r the
completed t e s t .

At the end of the t e s t s nickel i n s o l u t i o n reached quite


high concentrations. For example, at 25% pulp density the con­
centration of nickel was almost 10g/l. The decrease in a c i d i t y
and the rate of nickel s o l u b i l i z a t i o n a f t e r 60 days i n t h i s t e s t
suggested that the malt + peptone medium was being exhausted and
that with f r e s h medium high rates of nickel s o l u b i l i z a t i o n would

TABLE II Effect of Pulp Density on the


Efficiency of Nickel Solubilization

Pulp Density Ni Solubilized Ni Solubilized/26 days


/100 ml Slurry/ %
26gdays

0.4 0.052 100.0


1.2 0.065 48.1
4.0 0.174 61.0
10.0 0.179 25.2
15.0 0.200 18. 7
25.0 0.322 17. 7
180 Norman W. Le Roux ef al.

2 5 00 » r τ
1
1 •-r - ι - τ r— — ιτ 1 1
α. 1 2%P U LPD E N S I TY b. 4%P U L P Ji
N
c. 4 %P U L PD E N S I TY
D E N S IYT m/p^z S T E R IELC O N T R OL

2 0 00
i
t P
f /
1 5 00

11 / . ^Ν S pH X

1000 - 30

5 00

tl 20

y \
P H

o
1000
0
d.10°/oPU
PDL E N S I TY
'C

β. 1 5%P U LPD E N S I TY
u i!

Y
f. 2 5%P U LP
D E N S IYT/
N i
f

8 0 00 A / W - 5Ο

6 0 00

/ \ / N
''

4 0 00

/ ΑΛ* 11

2 0 00 20
λ Λ * —

—1 ι ι ι
Ο2 0 4 0 6 0Θ ΟΟ 2 04 06 0Θ ΟΟ 2 0 4 06 0 8 01 0 0
Ρ - V i s ula
I r o nP r e c i p i t aetT I M E , d

Fig. 6. (6a-6f) Effect of pulp density on the bacterial


leaching of nickel, copper and iron (malt + peptone medium).
Basic Microbial Studies Applied to Leaching 181

TIME.d

Fig. 6g. Effect of pulp density on the bacterial leaching


of nickel (malt + peptone medium).

be resumed. Residue analyses showed that about 80% of the nickel


had been s o l u b i l i z e d at 10 and 15% pulp density and about 32% at
25% pulp d e n s i t y . These f i g u r e s are only approximate as d i f f i -
c u l t y was experienced in drying the residues at room temperature
due to the gelatinous nature of the residue.

The rates of copper e x t r a c t i o n remained low at a l l pulp den-


s i t i e s except f o r the unexplained high r e s u l t at 25%. In t h i s
t e s t appreciable q u a n t i t i e s of copper were s o l u b i l i z e d a f t e r 40
days ( F i g . 6 f ) .
182 Norman W. Le Roux et al.

In the s t e r i l e t e s t using 4% pulp density much l e s s acid was


produced and the rate of nickel s o l u b i l i z a t i o n was l e s s than a
quarter of that in the comparable inoculated t e s t s ( F i g . 6 c ) .

A check on the residue from the t e s t with concentrate at 25%


pulp density showed that of the 68% nickel l e f t in the leached
residue, 10% was soluble in 20% HC1 ( a f t e r correcting f o r nickel
s o l u b i l i z e d by 20% HC1 from unleached o r e ) . This nickel had been
s o l u b i l i z e d and reprecipitated in an acid soluble form. Thus, the
total nickel leached in t h i s t e s t was about 42%. I t is possible
that in some of the other t e s t s , nickel which was reported in the
residue had in fact been s o l u b i l i z e d and then r e p r e c i p i t a t e d .
This could a l s o have happened to copper. This should be kept in
mind when considering the s o l u b i l i z e d nickel and copper values in
Tables 1 , 2, and 3 which were calculated from residue a n a l y s e s .
They are probably minimal v a l u e s .

G. Leaching Tests With Sulphuric Acid

In the microbial t e s t s the leaching of nickel was apparently


associated with the production of a c i d . Therefore, t e s t s were
carried out to determine the amount of nickel which could be
s o l u b i l i z e d from t h i s ore by an acid l e a c h , using s i m i l a r c o n d i -
tions at 30°C. The t e s t s were set up using copper/nickel concen-
trate and s u l p h u r i c acid at pH 2.0 and 3.0 with subsequent pH
c o n t r o l , and at 3.0 and 5.0 without pH c o n t r o l . The highest rate
of nickel s o l u b i l i z a t i o n was obtained in the t e s t at pH 2.0 but
there was l i t t l e difference in the rates in the other t e s t s (Fig.
7a). Even at pH 2.0 the rates were lower than those attained in
the best microbial t e s t s . The pH curves f o r these t e s t s are
shown in F i g . 7b. The amounts of copper s o l u b i l i z e d in the
sulphuric acid leaching t e s t s were a l s o l e s s than those t y p i c a l l y
obtained in the microbial t e s t s (Table 3 ) .

In order to simulate the microbial t e s t s more c l o s e l y two


additional t e s t s were set up. One used a subculture from a
TABLE III Residue and Solution Analyses ο»f Sulphuric Acid
and T. ferrooxidans Leaohing Tests

Wt of Wt of Nickel Copper
ore residue
Test In In Solubi- Solubi- In In In Solubi- Solubi- In
Medium Ore Resi­ lized lised Solution Ore Resi­ lized lized Solution
due λ * due λ λ
mg mg ppm ppm ppm % ppm ppm ppm ppm % ppm
183

pH 2.0 controlled 440 305 505 18 487 97 330 168 147 22 13 16


pH 3.0 controlled 424 282 488 44 444 91 - 158 107 51 32 -
pH 3.0 no pH control 409 288 470 87 383 82 220 156 153 3 2 5.3
pH 5.0 no pH control 396 342 455 12 343 75 373 151 113 39 26 21
T. F E R R O O X I D A N S 403 833 462 3 460 99 330 153 77 76 50 25

* Calculated from residue analyses.


184 Norman W. Le Roux et al.

T I M Ed.

Fig. 7a. Leaching of nickel with sulphuric acid.

ι·5ι 1 1 1 1 . 1 1 1 1 1 1
O2 04 06 0Θ Ο1 0 01 2 0
T I M Ed.

Fig. 7b. pH of sulphuric acid leaching tests (the pH curves


of the controlled tests were obtained from measurements taken
immediately before pH adjustments were made).
Basic Microbial Studies Applied to Leaching 185

previous microbial t e s t i n malt + peptone medium and the other


had s u l p h u r i c acid alone with the pH adjusted every 3-4 days to
the value attained by the microbial c u l t u r e . The nickel leaching
curves show that leaching was more rapid in the microbial test
(Fig. 8).

Although the pH values i n the s u l p h u r i c acid t e s t lagged


behind those in the microbial c u l t u r e the difference seemen i n -
s u f f i c i e n t to account f o r the difference in leaching r a t e s . The
pH curve of the s u l p h u r i c acid t e s t was plotted from measurements
taken immediately before an adjustment was made to bring the pH
value to that of the microbial t e s t . Thus, the pH values were
measured when there was a maximum difference between them. In

TIME.d

Fig. 8. Nickel leaching in a micvobial test (malt + -peptone


medium) v. in a sulphuric acid leaching test, at similar pH values.
186 Norman W. Le Roux e i al.

with the pH curves obtained in a previous s i m i l a r microbial test


with concentrate present and in the s u l p h u r i c acid leaching t e s t
at pH 5.0 i s shown i n F i g . 9. This indicates that production of
acid i s due to both the microbes and concentrate.

H. Leaching with Thiobacillus ferrooxidans

The previous t e s t s had shown that the s o l u b i l i z a t i o n of


nickel and copper was, at l e a s t i n p a r t , dependent on the produc-
t i o n of acid from an organic medium by the microbes. The iron and
sulphur o x i d i z i n g bacterium T. ferrooxidans i s the organism norm-
a l l y considered f o r the acid leaching of sulphide minerals and a
comparative t e s t was therefore made to assess the rates of s o l u -
b i l i z a t i o n that could be achieved with a s t r a i n of T. ferrooxidans
growing on Concentrate A.

The concentrate (0.4g) in 100 ml of an inorganic s a l t s medium


containing 2g Fe /l at pH 2.0 was inoculated with an active
culture of T. ferrooxidans. Some 65% of the nickel was leached in
10 days. Thereafter there were f l u c t u a t i o n s in the nickel concen-
t r a t i o n u n t i l about 95% was s o l u b i l i z e d in 50 days ( F i g . 1 0 ) .

The f l u c t u a t i o n s in the nickel leaching curve might have


been associated with the changes in the form of iron p r e c i p i t a t e s .
The i n i t i a l rate of leaching of nickel was f a s t e r than in the most
active of the t e s t s using malt + peptone media and s o i l cultures.
The total amounts of nickel and copper s o l u b i l i z e d , based on
residue a n a l y s i s were 99 and 50%, r e s p e c t i v e l y (Table 3 ) .

I. Microbial Studies

During the t e s t s with malt and peptone media the supernatant


s o l u t i o n s were frequently examined for microbial growth. Apart
from a few protozoa and fungi seen i n the i n i t i a l t e s t s , only
bacteria were observed and the majority of these were rod-shaped
c e l l s of various s i z e s . The l a r g e s t number of bacteria were seen
Basic Microbial Studies Applied to Leaching 187

practice the average pH of the s u l p h u r i c acid t e s t would have


been c l o s e r to that of the microbial t e s t than i s indicated in
F i g . 8.

The i n d i c a t i o n s from t h i s were that the acid produced in the


microbial leaching t e s t s was more e f f i c i e n t than s u l p h u r i c acid
or that there were additional f a c t o r s produced by the microbes
which influenced leaching.

In the s u l p h u r i c acid leaching t e s t s at pH 3.0 and 5.0 w i t h ­


out pH control the pH curves showed a marked f a l l (Fig. 7b). This
suggested that acid could be released or produced from the copper/
nickel concentrate and may have contributed to the production of
acid in the microbial c u l t u r e s . To determine the r e l a t i v e c o n t r i ­
butions of the concentrate and the microbes to the production of
acid a microbial t e s t was a l s o set up to follow the pH changes in
malt + peptone medium, i n i t i a l l y at pH 5.0, in the absence of
concentrate. A comparison of the pH curve obtained in t h i s t e s t

SO| . 1 . . . . . • . . . •—,

2 Ο l

i-oi . 1 1 1 1 " • · « · • > — 1

Ο ΙΟ 20 30 40 50 60 70 βΟ 90 ΙΟΟ 110 120


TIME.d

Fig. 9. pH variation in teaching tests initiatty at pH 5.0;


1) microbiat test in matt + peptone medium with Cu/Ni concentrate;
2) microbial test in malt + peptone medium without Cu/Ni concen­
trate; 3) Η SO leaching test with Cu/Ni concentrate.
188 Norman W. Le Roux ef al.

500 I 1 1 ρ

°/o OF METAL
SOLUBILIZED
(CALCULATED FROM
RESIDUE ANALYSIS)

Fig. 10. Leaching of nickel and copper using T. ferrooxidans


at pH 2.0.

within 24h of inoculating a t e s t . Thereafter the numbers observed


in s o l u t i o n decreased.

Samples from some of the e a r l y t e s t s were inoculated on to


agar plates at pH 5.0. Numerous, very s m a l l , smooth, cream-
coloured colonies c o n s i s t i n g of motile rods (1.0-2.0 χ 0.5 micro­
metres) grew on a l l plates a f t e r 48h. Tests for acid production
by these bacteria were negative. In an attempt to i s o l a t e a c i d -
producing organisms, samples from the t e s t s on malt + peptone
without pH control were plated on agar at pH 5.0, 3.0, and 2.6.
The concentration of malt + peptone was a l s o increased in an
e f f o r t to increase the s i z e of the c o l o n i e s . Bacteria s i m i l a r in
s i z e and c o l o n i a l appearance to those i s o l a t e d from the i n i t i a l
t e s t s were obtained but only at pH 5.0. At pH 3.0 and 2.6 only a
few yeast colonies grew a f t e r 30 days.
Basic Microbial Studies Applied to Leaching 189

The i s o l a t e d bacteria were s i m i l a r in appearance to T. ferro-


oxidans but l e s s motile. As T. ferrooxidans i s known to grow on
organic substrates (4) i t was p o s s i b l e that the bacteria were
s t r a i n s of t h i s organism. Limited attempts were made to grow the
bacteria on ferrous iron and mixtures of ferrous iron and malt +
peptone media but no growth was obtained. Although the t e s t s were
of short duration i t seemed u n l i k e l y that the bacteria were T.
ferrooxidans. Oxidation of reduced sulphur compounds was not
tested.

In retrospect i t i s p o s s i b l e that concentrates were not wholly


s u i t a b l e f o r t e s t s such as these. They are not ' n a t u r a l ' metal
sources having been concentrated by man and would give high con-
centrations of s o l u b l e metal ions in a comparatively short time.
I t might have been more s a t i s f a c t o r y to use rocks containing low
concentrations of metal f o r these e a r l y s t u d i e s .

Although these studies were directed at a preliminary examin-


ation of the technical f e a s i b i l i t y f o r metal s o l u b i l i z a t i o n by
microbes i t i s i n t e r e s t i n g to r e f l e c t on these other forms of
microbial leaching. In some cases there could be i n t e r e s t in d i -
lute s o l u t i o n s of metals. To i l l u s t r a t e t h i s statement a compari-
son can be made between, f o r example, recovery of gold or uranium
and zinc or iron from s o l u t i o n . In the case of the two former
metals i t i s economic in some cases to recover them from s o l u t i o n s
containing 1-3 ppm of gold and 10-40 ppm of uranium. I t i s not
d i f f i c u l t to f i n d 'waste' s o l u t i o n s having zinc or iron concentra-
t i o n s much higher than these i . e . 1-2 g l _ 1
. These s o l u t i o n s do
not at present arouse much i n t e r e s t except perhaps as potential
p o l l u t i o n problems. An important f a c t o r i s obviously the volume
of s o l u t i o n involved and hence the total amount of metal present.
Uranium occurs in copper dump leach l i q u o r s in low concentrations,
3
2-15 ppm. The volume of l i q u i d in c i r c u i t can be h i g h , 250 m
min" 1
(50,000 g a l l s m i n " ) .
1
The amount of uranium i s large enough
to tempt an economic recovery. As metals such as zinc and i r o n
190 Norman W. Le Roux et al.

are not recovered at present from s o l u t i o n s containing a few grams


per l i t r e , t h i s should not preclude the p o s s i b i l i t y of recovery in
the future of metals from ' d i l u t e ' s o l u t i o n s . Perhaps microbial
leaching leading to d i l u t e s o l u t i o n s of metals could be the f i r s t
stage in an economically v i a b l e process. Therefore innovation
and the technology to concentrate and recover these metals must
advance to complement microbial leaching research. The concentra-
t i o n step we are seeking f o r the future might a l s o be m i c r o b i a l ,
but that i s another s t o r y .

IV. CONCLUSIONS

The r e s u l t s have indicated that bacteria other than t h i o


b a c i l l i can be used f o r leaching metals. Although the mechanism
appeared to be acid production there was evidence that the acid or
acids produced were more e f f e c t i v e than equivalent amounts of
sulphuric acid.

The rates of leaching obtained with the s o i l i s o l a t e d were


much slower than when a s t r a i n of T. ferrooxidans was used. This
l a t t e r s t r a i n had been adapted over long periods of time to t o l e r -
ate high concentrations of metals. The s o i l i s o l a t e s a f t e r 5
subcultures reduced the leaching time by a factor of 3 . 5 and i t is
p o s s i b l e that further adaptation would have taken place thus i n -
creasing t h e i r a c t i v i t y and tolerance to metals.

V. ACKNOWLEDGMENT

Warren Spring Laboratory are pleased to acknowledge the


f i n a n c i a l support and i n t e r e s t of Consolidated Gold F i e l d s L t d ,
London, in t h i s work and for t h e i r permission to publish i t .
Basic Microbial Studies Applied to Leaching 191

VI. REFERENCES
1. Tuovenen, O.H., and K e l l y , D.P., Z. Allg. Mikrobiol., 12,
311-346 (1972).
2. PareS, Y . , Rev. Ind. Miner., 50, 408-414 (1968).
3. Silverman, M . P . , and Lundgren, D.G., J. Bacteriol, 77, 642
(1959).
4. T a b i t a , R., and Lundgren, D.G., J. Bacteriol, 108, 322
(1971).
II WASTE TREATMENT AND ENVIRONMENTAL
CONSIDERATIONS

In many countries where enormous quantities of waste products are accumulat-


ing, a need has been created to develop effective methods to deal with metal
extraction problems associated with these various wastes, and with related en-
vironmental considerations.
The microbiological activity involved in the recovery of metal values from
mining and processing waste materials is very complex. The reaction mechanisms
involved in the associated metal dissolution processes are also not completely
known. In this regard, the topics presented in this section contribute toward a
solution to these problems and to a better understanding of the leaching
phenomena involved.
The first chapter of this section deals with the microbiological leaching of waste
materials such as fly ash, slags, and jarosite-type leach residues. The aim of this
research is to extract toxic metal ions (such as Cd, As, etc.) from the waste
materials and as a consequence to reduce the possibility of contaminating the
environment by these industrial wastes. The second chapter is an attempt to
provide a microbiological process for the recovery of sulfur in its elemental form
from sulfate and sulfuric acid-containing aqueous wastes which are to be released
in the environment. The final chapter of this section describes a microbiological
leaching process for the removal of inorganic sulfur contained in coal. This pro-
cess may be considered as a practical alternative to the chemical coal desulfuriza-
tion process. The effect of the parameters influencing the attendant oxidation
process is also discussed.
METAL RECOVERY AND ENVIRONMENTAL PROTECTION
BY BACTERIAL LEACHING OF INORGANIC WASTE
MATERIALS

Hans Georg Ebner

U n i v e r s i t y of Dortmund
Dortmund, Federal Republic of Germany

Industrial waste materials contain valuable metals in high


total amounts but in low concentrations. Sulfidic dust concentra-
tes and oxidic fly ash from a copper process, slag from a lead
smelting process and "Jarosite" from a zinc electrolysis were
leached with different thiobacillus species. The leaching effi-
ciency was specific to materials, treatment and inoculated bacteria
species. The maximum output for zinc was around 95%, for copper
around 70%. We were concerned about the high concentrations of
32 mg Cd/l, 5 mg Pb/l and 30 mg As/I in the leaching liquors be-
cause these values exceed the allowed toxic levels in drinking
water.

I. INTRODUCTION

The foreseeable shortage of raw materials i s one of the major


problems faced by the world today. But s o l e l y declaiming the
depletion of o i l as an energy source means f o r g e t t i n g about the
exhaustion of mineral ore deposits which are necessary f o r the
production of metals. While f o s s i l e energy sources are exchange-
able a g a i n s t each other t h i s w i l l - in most cases - not become

195
196 Hans Georg Ebner

TABLE I Reserves of the World's Energy and


Mineral Resources

Nickel 46 yrs. Lignite 270 yrs.


Aluminum 44 yrs. Coal 190 yrs.
Zinc 43 yrs. Gas 54 yrs.
Lead 29 yrs. Oil 34 yrs.
Copper 25 yrs.
Mercury 20 yrs.
Si Iver 12 yrs.
Gold 10 yrs.

f e a s i b l e for metals. Recent studies ( 1 , 2 , 3) about the estimated


l i f e t i m e of resources show that - even under the aspects of i n -
creasing haulage - the resources of f o s s i l e materials w i l l last
many decades longer than those of m e t a l l i c ores (Table I ) .

The depletion of high-grade ore deposits corresponds with an


exponential growth rate in the demand for metals. Between 1975
and the year 2000 the requirement for zinc i s estimated to r i s e
from 6 to 12 m i l l i o n t o n s , for copper from 8 to 24 m i l l i o n tons
and for aluminum from 13 to 53 m i l l i o n tons ( 2 , 3 ) .

Another problem of our century i s in s o l v i n g industrial


pollution. Forced by s t r i c t l y growing governmental regulations
industry has to pretreat a l o t more waste materials before de-
p o s i t i n g them as in former y e a r s . These data correspond pretty
well with the predictive model of Massachusetts I n s t i t u t e of
Technology which concludes that without the development of new
and more economical technologies the i n d u s t r i a l production of our
world w i l l break down within the f i r s t decades of the coming cen-
tury owing to depleted resources, a growing population and an
increasing p o l l u t i o n ( 4 ) .

Many inorganic i n d u s t r i a l wastes s t i l l contain valuable


metals. Although t h e i r metal concentrations are mostly very low
t h e i r total amount i t s e l f i s often high because of the large
masses which a r i s e during i n d u s t r i a l processes. A development
Waste Treatment and Environmental Considerations 197

of a cheap and simple process to pretreat these residues before


t h e i r storage on deponies can be regarded at l e a s t under two
beneficial aspects: toxic substances w i l l be removed from the
wastes which lowers the cost for t h e i r disposal and at the same
time valuable metals can be gained from the waste m a t e r i a l s .

To meet the needs of raw-material supply and to diminish


environmental p o l l u t i o n we i n v e s t i g a t e d the p o s s i b i l i t i e s of
r e c y c l i n g inorganic i n d u s t r i a l waste by bacterial leaching. In-
cluded in the i n v e s t i g a t i o n s were e s p e c i a l l y those residues which
contain remarkable concentrations of valuable metals or which have
to be stored on special deposits because of t h e i r high contents
of t o x i c elements or which accumulate in large q u a n t i t i e s during
industrial processes.

II. MATERIALS AND METHODS

The waste materials were supplied by d i f f e r e n t German i n d u s -


t r i a l companies. A l l materials possessed a very small grain s i z e
below 0.1 mm except the s l a g from a lead smelting process which
had to be crushed before the bacterial leaching treatment.

J a r o s i t e , J - l , was a residue which amasses during the p r o -


duction of z i n c . After r o a s t i n g of the zinc ore the o r i g i n a t e d
ore concentrate i s leached chemically with s u l f u r i c a c i d . The
zinc pregnant l i q u o r i s pumped to the recovery plant whereas the
iron r i c h residue c a l l e d " J a r o s i t e " has to be stored on special
deposits. KS-1 and KS-2 were s u l f i d i c dust concentrates from
copper processes in which the ores are chemically leached with
hydrochloric a c i d . The o x i d i c f l y a s h , K0-3, i s f i l t e r e d from
the fumes of a p y r i t e r o a s t i n g process. BS-1 was a basic s l a g
derived from a lead smelting process.

D i f f e r e n t Thiobacillus thiooxidans and Thiobacillus ferrooxi-


dans s t r a i n s had been i s o l a t e d during former experiments from
several l o c a t i o n s of European and American countries (mines, r i v e r s ,
198 Hans Georg Ebner

vulcanoes, sea water). They were grown e i t h e r on normal or d i l u -


ted LEATHEN's r e s p e c t i v e l y STARKEY's medium at incubation tempera-
tures of 30° C or higher.

Leaching experiments were performed in 300 and 500 ml E r l e n -


meyer-flasks as s t a t i c and/or s t i r r e d c u l t u r e s . To create
applied conditions for further i n - s i t u processes the pH values of
the culture media were neither lowered at the beginning nor main-
tained during the experiments with additional acid supply. The
pH was measured e l e c t r o m e t r i c a l l y with INGOLD-pH-electrodes and
a KNICK-pH-meter (Type 700).

The elements in s o l u t i o n were analysed by atomic absorption


spectroscopy with a PERKIN-ELMER AAS 400. Before t h e i r determina-
t i o n the leaching l i q u o r s had to pass a f i l t r a t i o n u n i t and the
remaining s o l i d s on the f i l t e r were washed twice with d i s t i l l e d
water. The measured element concentrations of the l i q u i d s m u l t i -
p l i e d by t h e i r volumes gave the total amount of the leached metals
in s o l u t i o n . C o r r e l a t i n g these r e s u l t s with the o r i g i n a l metal
contents the s o l i d s had before t h e i r treatment expresses the
leaching e f f i c i e n c y in per cent.

For the performance of the a r s e n i c analyses we g r a t e f u l l y


acknowledge the cooperation of the I n s t i t u t fur Spektrochemie,
Dortmund.

III. RESULTS AND DISCUSSION

The chemical analyses of the waste materials proved i n t e r e s t -


ing amounts of several valuable metals (Table I I ) . The dust
concentrates KS-1 and KS-2 from the copper processes contained
18.3% and 4.4% copper as well as 1 0 . 1 % and even 29.5% z i n c . The
f l y a s h , K0-3, from the p y r i t e r o a s t i n g however possessed only
0.9% copper and 2.5% z i n c . J a r o s i t e , J - l , held 0.38% copper and
s t i l l 9.6% z i n c . S i m i l a r concentrations showed the basic lead
s l a g with 0.19% copper and 9.2% z i n c . The total iron amount in
Wast
eTreatmen
tan dEnvironmenta
lConsideration
s19 9

TABLE II Chemical Analyses of Industrial Waste


Materials (%)

KS-1 KS-2 KO-3 J-l BS-1


dust oonc. dust cone, fly ash jarosite lead slag
sulfidio sulfidio oxidic

Cu 18.3 4.A 0.9 0.38 0.19


Zn 10.1 29.,5 2.5 9.6 0.2
Fe, , 22.9 25 37.4 27.2 29.2
S 37.2 33.,5 trace 9 1.23
6.1 1.,6 19.4 8.6 1.56
As 0.4 2.,2 4.1 0.21 0.015
Cd 0.15 0.005

in a l l materials varied between "a t r a c e " up to 37.2%. Remark-


able were a l s o the r e l a t i v e high concentrations of hazardous
components in the residues l i k e l e a d , arsenic and cadmium which
create the problems for the deposition of these waste m a t e r i a l s .

Our leaching experiments in Erlenmeyer-flasks with several


s t r a i n s of Thiobacillus thiooxidans and ferrooxidans demonstrated
that the complexity of the materials in mineral and ion content
requires special treatments for each residue. About 10 out of 30
d i f f e r e n t t h i o b a c i l l u s s t r a i n s could not even s u r v i v e concentra-
t i o n s of 1% w/v of the waste in s o l u t i o n . From the remaining 20
s t r a i n s meanwhile 3 of them are adapted to higher tolerances
against the complex m a t e r i a l s . Without any d i f f i c u l t y we were
able to suspend 15% of KS-1 or K0-3, 10% of the lead s l a g , but
only 5% of the s u l f i d i c dust concentrate KS-2 or the J a r o s i t e
J-l in the media. In special experiments the bacteria could
survive concentrations up to 20% of the waste in s o l u t i o n .

To t e s t the dependency of d i f f e r e n t s t r a i n s in d i f f e r e n t
waste suspensions we examined the growth of Thiobacillus thiooxi-
dans s t r a i n s T-E 1 and T-E 2 and of Thiobacillus ferrooxidans
F-E 2 in several concentrations of K S - 1 , KS-2 and K0-3 (Table
III). During these experiments Thiobacillus ferrooxidans, s t r a i n
200 Hans Georg Ebner

TABLE III Maximal Endured Waste Concentrations by Different


T. thiooxidans and T. ferrooxidans Strains (% w/v).

KS-1 KS-2 KO-3


Concentration 10% 0.75% 15%
T. thiooxidans
strain T-E 2 T-E 1 T-E 1
T-E 2
Concentration 15% 5% 15%
T. ferrooxidans F-E 2 F-E 2 F-E 2
strain

F-E 2, withstood the highest concentrations in the t e s t . Never­


t h e l e s s we selected Thiobaoillus thiooxidans s t r a i n T-E 2, from
our c o l l e c t i o n for the leaching experiments with the f l y ash KO-3.
Although t h i s s t r a i n could not endure the same high waste concen­
t r a t i o n s l i k e the s t r a i n F-E 2 i t had a better c a p a b i l i t y to leach
elements from t h i s f l y ash.

During the experiments our p a r t i c u l a r i n t e r e s t became mainly


concentrated on the f e a s i b i l i t y of leaching the lead s l a g BS-1 and
the J a r o s i t e J - l . Leaching the J a r o s i t e with Thiobaoillus ferro­
oxidans was not quite s u c c e s s f u l . Although the bacteria survived
more than 30 days in the s o l u t i o n s with λ% and 5% J a r o s i t e the
pH values did not decrease, but remained close to those of the
s t e r i l e control (5). In the experiments with the basic lead BS-1
the pH values of the inoculated s o l u t i o n s and the s t e r i l e control
even rose to pH 7 with 1% of lead s l a g and to pH 8.3 with 5% of
lead s l a g in s o l u t i o n ( 5 ) . An i n o c u l a t i o n of Thiobaoillus thio­
oxidans gave far better r e s u l t s . With 5% of J a r o s i t e in s o l u t i o n
the pH dropped to 0.9 a f t e r 20 days ( 5 ) .

In f u r t h e r i n v e s t i g a t i o n s we t r i e d to explore at which day(s)


during the progress of the leaching processes the metal concentra­
t i o n s became t h e i r maxima. At the beginning of the t e s t s a s t a t i c
culture time of seven days was given to l e t the bacteria attach
to the p a r t i c l e s ( F i g . 1 ) . At the end of t h i s time the pH began
Waste Treatment and Environmental Considerations 201

Acid formation
PH Jarosite 5 % [w/v]
Th. thiooxidans TE-2
3 . 5-
*s t e r i l e
c o n t rlo

3 . 0-

2 . 5-

2.Ο ­

Ι.5 -

13 7 9 1 1U 1 61 82 12 32 5 days -

Fig. 1. pH-curves of Jarosite suspensions inoculated with


T. thiooxidans and sterile control.

already to f a l l s l i g h t l y , but a f t e r s t a r t i n g to shake the culture


f l a s k s the pH almost immediately went up a g a i n . A f t e r two more
days of shaking the pH anewed to decrease and f i n a l l y f e l l sharply
between the 16th and 18th day. We w i l l have to prove now whether
the long time of seven days for an attachment of the bacteria to
the p a r t i c l e s can be reduced or may even not be necessary.

Following the leaching rates over 23 days of shaking - r e s ­


pectively 30 days in total - around 36% of the cadmium content
from the J a r o s i t e had been leached ( F i g . 2 ) . In the s t e r i l e
c o n t r o l , plotted as black bars in the f i g u r e , considerable
amount of cadmium was leached as w e l l . The leachinq rate of
zinc rose within the f i r s t seven days remained almost constant
u n t i l the 18th day a f t e r which i t climbed again ( F i g . 3 ) . Corre­
lated with the decrease of the pH-values in the s o l u t i o n there had
a l s o been a sharp decline of the pH-curve on the same 18th day of
202 Hans Georg Ebner

Cd-Leaching rate
LR Jarosite 5 %Iw /v)
[%1
Th. thiooxidans T E - 2
3S\ . staticishake^

30 ilil

2 ^

Γ Τ 1
' 'Γ Τ ΊΤ 1
Ί " Ί ? Ί ? ' 'J '2? 'g • • •
days

K E Y . 2 . Leaching rates of cadmium from Jarosite with and


without bacteria (black bars) in per cent from the total original
content.

LR Zn- Leaching rate


(%) I Jarosite 5 % Iw/vl
35- I Th. thiooxidans TE- 2
static I shake
culture
I
I

I 7 9 11 Η 16 18 21 23 25 30
days —

ί£<7· 2 . Leaching rates of zinc from Jarosite with and without


bacteria (blackbars) in per cent from the total original content.
Waste Treatment and Environmental Considerations 203

these experiments. I t i s worth noting that the d i s s o l v e d zinc


concentrations in the s t e r i l e control declimbed a f t e r the 18th
day. We suppose that a process of p r e c i p i t a t i o n or rearrangement
of the zinc ions takes place. Regarding the copper output we
observed s i m i l a r tendencies as during the zinc leaching ( F i g . 4 ) .
However in these experiments the bacteria did not d i s s o l v e more
than 7% of the total copper content which i s u n s u f f i c i e n t for
bacterial leaching processes i n s i t u . More experimental work to
c l a r i f y these problems w i l l be necessary.

During further approaches i t became obvious that the leaching


e f f i c i e n c y was s p e c i f i c to m a t e r i a l s , treatment and inoculated
bacteria. Leaching the concentrates KS-1 and KS-2 with Thiobacil­
lus ferrooxidans for example was much more e f f e c t i v e than with
Thiobacillus thiooxidans. Leaching KS-1 with a mixed culture of
Thiobacillus ferrooxidans s t r a i n s F-E 2 and F-E 21 gave better
r e s u l t s than leaching t h i s concentrate with one of these s t r a i n s

Cu-Leaching rate [%)


ι
ι Jarosite 5%(w
/v)
, static I shake ,
culture Th. thiooxidans TE-2
I
ι

[L

23 25 30
days-

Fig. 4. Leaching rates of copper from Jarosite with and without


bacteria (black bars) in per cent from the total original content.
204 Hans Georg Ebner

separately. On the other hand Thiobacillus thiooxidans created


the maximal e f f i c i e n c y when leaching the f l y ash KO-3, J a r o s i t e
and the lead s l a g .

The r e s u l t s (that were obtained in t h i s studv) with ootimized


growth conditions are encouraging enough for f u r t h e r experiments
(Table I V ) . The average r e l a t i v e y i e l d s of copper from a l l resi-
dues l i e in the region of 35 to 40% and f o r zinc above 50%. The
maximal y i e l d of zinc which was 94% was leached from the concen-
trate KS-2. The maximal output of copper came from the J a r o s i t e
with 69%. From the f l y ash KO-3 the bacteria had been able to
bring 8 1 % of the arsenic content into s o l u t i o n .

Not a l l of our examined residues are already stored on special


deposits. As we could i s o l a t e t h i o b a c i l l u s from a number of
deposits we are concerned about the high concentrations of toxic
elements which can be leached by the bacteria from the waste
materials (Table V ) . The concentrations of l e a d , cadmium and
arsenic leached from these wastes are s e v e r a l f o l d higher than the
permitted values in potable water in Germany. Uncontrolled de-
p o s i t s contaminated by bacteria therefore can cause severe ground
water i n t o x i c a t i o n .

From the r e s u l t s i t can be seen that inorganic industrial


residue materials contain valuable amounts of metals which are too
good f o r depositing or wasting e s p e c i a l l y at a time when natural
resources are getting scarce and the demand of developing i n d u s -
t r i e s i s growing e x p o n e n t i a l l y . With bacterial leaching i t should
be p o s s i b l e to create economically f e a s i b l e processes with which
metals from inorganic wastes can be brought into s o l u t i o n and then
gained by conventional recovery. Our experiments a l s o proved that
on i n d u s t r i a l deposits - in many cases - bacteria are already
growing and thus are leaching hazardous elements from the wastes.
We know that we j u s t scratched the envelope of the parcel with
i n d u s t r i a l waste materials but we are sure that i t contains a l o t
more gainable elements and many other i n t e r e s t i n g residues or
Maximal Leachin<3 Efficiency froiΗ different Was1:es (%)

KS-1 KS-2 K0-3 J-1 BS-1


Cu Zn As Cu Zn As Cu Zn As Cu Zn Cu Zn

Bact. Leaching 12 38 3 35 94 14 42 85 81 69 52 33 50
Ster. Control 0.1 16 0.4 10 0.2 10 16 5 10 0 0
Th. Strain F-E2 F-E2 F-E2 F-E2 F-E2 F-E2 T-E2 T-E2 T-E2 T-E2 T-E2 T-E2 T-E2
ο F-E21
c_n

F = Thiobacillus ferrooxidans
Τ = Thiobacillus thiooxidans

5-»

^4^

4^v
<o CO

CO4

<tt CO

CO

CO
4^

co

_
206 Hans Georg Ebner

TABLE V Concentrations of Hazardous Elements in Microbiolo-


gically Leached Industrial Wastes and Maximal Allowed Concentra-
tions in Drinking Water in Germany (mg/l).

Cd Pb As
KS-1 8.7 2.0 8.4
KS-2 5.1 2.5 18.0
KO-3 0.7 2.3 30.1
J-l 31.8 5.0 -
BS-1
allowed in
0.7 3.1 -
drinking water 0.006 0.04 0.04

tailings. I t i s to hope that our work w i l l be encouraging enough


to lead more s c i e n t i s t s to the f i e l d of microbiological recycling.

IV. ACKNOWLEDGMENT

Thanks are due to Mrs. B r i g i t t e S t e i n f e l d f o r her excellent


and s k i l l f u l technical assistance.

V. REFERENCES

1. Bundesanstalt fur Geowissenschaften und Rohstoffe: "Die


kunftige Entwicklung der Energienachfrage und deren Deckung",
Abschnitt I I I , Das Angebot von E n e r g i e r o h s t o f f e n , Hannover,
1976.
2. Deutsche Forschungsgemeinschaft: " D e n k s c h r i f t zur Lage der
Lagerstattenforschung", Teil I , Mineralische R o s h s t o f f e , H.
Boldt Verlag KG, Boppard, 1975.
3. von Engelhardt, "Raubbau an den E r z v o r r a t e n " , Bild der Wissens-
chaft, 11 , 78 (1976).
4. Meadows, D., "The Limits of Growth", Universe Books, New York,
1972.
5. Ebner, H.G., in "Conference Bacterial Leaching 1977", (W.
Schwartz, E d . ) , GBF Monograph S e r i e s , No. 4 , Verlag Chemie,
Veinheim - New York, 1977.
SULFATE DECOMPOSITION: A MICROBIOLOGICAL PROCESS

Douglas J. Cork
and
Michael A. Cusanovich

U n i v e r s i t y of Arizona
Tucson, A r i z o n a , U.S.A.

The disposal and treatment of sulfur, a major process stream


constituent of industry and mining, is rapidly expanding into one
of its most formidable challenges. Significant potential exists
for the treatment of industry s major effluents including excess
f

sulfuric acid, gypsum, coal de-sulfurization by-products, acid


mine waters, and general metallurgical effluents.
A variety of microorganisms are capable of metabolizing sul-
fate to elemental sulfur. In nature, these processes are gener-
ally slow and yield products toxic to the environment (e.g. hydro-
gen sulfide). We are reporting here on studies designed to quan-
titatively convert sulfate to elemental sulfur utilizing D e s u l f o v i -
brio d e s u l f u r i c a n s for sulfate conversion to sulfide and photosyn-
thetic bacteria for the sulfide s subsequent conversion to sulfur.
r

We have developed a purge system, using an inert carrier gas,


to continuously remove sulfide from actively growing cultures of
D e s u l f o v i b r i o and supply this substance to either Chlorobium t h i o -
sul fa tophi 1 urn or Chromatium vinosum. This technique has allowed
us to achieve quantitative conversion of sulfate (initial concen-
trations of 30 to 40 mm) to elemental sulfur. Conditions for
optimizing the process have been studied and the approach has
been successfully applied to solvent extraction raffinates.
Previous studies on static cultures at various sulfate and
sulfate concentrations or on mixed cultures of D e s u l f o v i b r i o

207
208 Douglas J. Cork and Michael A. Cusanovich

and photo synthetic bacteria yielded discouraging results. These


approaches did not quantitatively convert sulfate to sulfur and
bacterial growth was poor. The studies described here provides
elemental sulfur.

I. INTRODUCTION

A v a r i e t y of metallurgical e f f l u e n t s i n c l u d i n g solvent e x t r a c -
t i o n r a f f i n a t e s contain high concentrations of s u l f a t e i o n .
These e f f l u e n t s present s e r i o u s disposal problems in that the
s u l f a t e must be converted to a s t a b l e , non-leachable form. We
herein propose a s o l u t i o n to t h i s problem which u t i l i z e s the
natural b i o l o g i c a l a c t i v i t i e s of Desulfovibrio desulfuricans,
Chlorobium thiosulfatophilum, and/or Chromatium vinosum, o b l i g a t e
anaerobes of the aquatic s u l f u r cycle ( 1 , 2 ) . Desulfovibrio r e -
duces s u l f a t e to s u l f i d e , and the green and purple s u l f u r bacteria
Chlorobium and Chromatium p h o t o s y n t h e t i c a l l y o x i d i z e s u l f i d e to
elemental s u l f u r . Chlorobium thiosulfatophilum excretes the
c o l l o i d a l s u l f u r , while Chromatium vinosum s t o r e s s u l f u r i n t r a -
cellular^. In p r i n c i p l e , a solvent e x t r a c t i o n r a f f i n a t e would
provide s u l f a t e to Desulfovibrio, thereby i n i t i a t i n g the s u l f u r
cycle. The s u l f i d e thus produced can be made a v a i l a b l e to the
green and purple s u l f u r bacteria being q u a n t i t a t i v e l y oxidized to
elemental s u l f u r . These photosynthetic bacteria grow on auto-
trophic media and f i x ZQ^-

I t i s the purpose of the studies described herein to demon-


s t r a t e that a mutualism may be e s t a b l i s h e d between Desulfovibrio
and e i t h e r Chromatium or Chlorobium, so as to q u a n t i t a t i v e l y con-
vert s u l f a t e to elemental s u l f u r . Further, we have found that the
addition of a purge system u t i l i z i n g an i n e r t c a r r i e r gas leads to a
more e f f i c i e n t conversion of s u l f a t e to s u l f i d e , and then s u l f i d e to
s u l f u r , than can be obtained from s t a t i c cultures of each of the bac-
teria. F i n a l l y data w i l l be presented which demonstrate that t h i s
purge system can be adapted to solvent extraction r a f f i n a t e .
Waste Treatment and Environmental Considerations 209

II. MATERIALS AND METHODS

A. Organisms

Experiments were c a r r i e d out with Desulfovibrio desulfur icons


ATCC7757 Chromatium vinosum ( s t r a i n D ) , and Chlorobium thiosul-
fatophilum ( s t r a i n PM).

B. Media and S t a t i c Growth Conditions

Desulfovibrio was grown in a modified media of Starkey ( 3 ) :

Compound gm/1iter
NaS0 4 3.0
MgS0 4 1.5
NH C1
4 1.0
K0HPO4 · 3H 0 2 0.65
CaCl 2 0.08
F e ( N H ) o ( S 0 4 ) · 6H 0
4 2 2 0.01
L a c t i c Acid (85%) 5.0 ml
Yeast Extract 0.2

The pH was adjusted to 7.0 with NaOH and growth was c a r r i e d


out in completely f i l l e d 1 1 i t e r p r e s c r i p t i o n b o t t l e s which were
immersed in a 30°C water bath.

S t r i c t l y autotrophic media was used for the photosynthetic


bacteria. The media of Bartsch (4) was modified to contain:

Compound gm/1iter
NaCI 10.0
NH4C1 1.2
K2HPO4 · 3HoO 1.3
MgClo 0.55
CaCl 2 0.12
L a r s o n ' s Trace Elements (5) 2.0 ml
NaHC0 3 2.0
210 Douglas J. Cork and Michael A. Cusanovich

Solutions containing the appropriate amount of s u l f i d e were


autoclaved separately and added to the bulk media. The pH was
adjusted to 7.0 with HC1. Growth was r o u t i n e l y c a r r i e d out in
completely f i l l e d one l i t e r p r e s c r i p t i o n bottles which were
immersed in a 3 0 C w a t e r bath.
Q
The center of each bottle c o n t a i n -
ing photosynthetic bacteria was placed f i v e centimeters from two
f o r t y watt General E l e c t r i c showcase lamps. These photosynthetic
c u l t u r e s were illuminated continuously.

C. Gas Purged Bacterial Growth Conditions

A gas purged system of bacterial growth i s particularly


amenable to the following s u l f u r metabolism by Desulfovibrio and
e i t h e r Chlorobium or Chromatium:

<.Q = Desulfovibrio ^ ^ Chlorobium or <*0

^ > 2 chromatium >

This s u l f u r pathway reveals that the common intermediate i s


v o l a t i l e hydrogen s u l f i d e , which may be t r a n s f e r r e d from the
Desulfovibrio culture to the photosynthetic culture by an i n e r t
c a r r i e r gas (75% A r , 25% CO^), which was a l s o used to maintain
a constant pH (^7).

The purge system i s shown schematically in Figure 1 .


Desulfovibrio and the photosynthetic organisms are grown in
separate one l i t e r p r e s c r i p t i o n b o t t l e s , each culture occupying
80% of the bottle volume. The bottles containing separate
cultures are connected by latex tubing. Gas d i s p e r s i o n tubes
d e l i v e r the i n e r t c a r r i e r gas to Desulfovibrio, and the i n e r t
c a r r i e r gas plus the v o l a t i l e s u l f i d e to the photosynthetic
culture b o t t l e . L i g h t i n g and temperature conditions are i d e n t i -
cal with those described for s t a t i c c u l t u r e s . Constant gas
pressure was maintained while media was withdrawn for assay.
Waste Treatment and Environmental Considerations 211

ZINC
ACETATE
purging
GAS

PESULFCMBRIQ CHLORORIUM ΠΡ
CHROMATIUM

Fig. 1. The gas purged mutualistic system utilizing


D e s u l f o v i b r i o and either Chlorobium or Chromatium.

D. Chemical A n a l y s i s of Bacteria

S u l f i d e was determined according to the method of Truper and


Schlegel (6). S u l f a t e was determined by a B a C ^ - g e l a t i n t u r b i -
dimetric method ( 7 ) . T h i o s u l f a t e was analyzed by the method of
Sorbo,et a l . ( 8 ) . Elemental s u l f u r , Chromatium bacteriochlorophyll
and protein were determined as described by Schmidt and Kamen ( 9 ) .
Chlorobium chlorophyll was extracted in absolute methanol and
q u a n t i t a t i v e l y determined by use of the absorption c o e f f i c i e n t s
reported by S t a n i e r and Smith ( 1 0 ) .

After assaying f o r s u l f i d e , elemental s u l f u r , and b a c t e r i o ­


c h l o r o p h y l l , the c e l l s were centrifuged at 15,000 rpm. The super­
natant was analyzed f o r s u l f a t e and the p e l l e t was washed in 4%
t r i c h l o r o a c e t i c acid and then resuspended three times in 7:2
acetone-methanol and recentrifuged in a c l i n i c a l centrifuge before
proceeding with the Biuret protein assay as described by Schmidt
and Kamen ( 9 ) . For a l l a s s a y s , absorbances were recorded on a
Cary 118 Spectrophotometer.
212 Douglas J. Cork and Michael A. Cusanovich

III. RESULTS

As has been determined f o r a v a r i e t y of phototrophic bacteria


( 1 0 ) , s t a t i c cultures of both Chlorobium thiosulfatophilum and
Chromatium vinoswn are i n h i b i t e d by high concentrations of s u l f i d e
(greater than 4 and 9 mm Na S · 91^0, r e s p e c t i v e l y ) .
2 Figure 2
presents data on b a c t e r i o c h l o r o p h y l l , p r o t e i n , elemental sulfur,
s u l f a t e , t h i o s u l f a t e , and s u l f i d e production or u t i l i z a t i o n f o r
s t a t i c cultures of Chlorobium and Chromatium over a twenty-four
hour period. As seen in Figure 2, s u l f i d e i s r a p i d l y oxidized in
both cases: 60% and 31% of the s u l f i d e i s oxidized to elemental
s u l f u r by Chlorobium and Chvomatium, r e s p e c t i v e l y . However, at
l e a s t 40% of the s u l f i d e i s oxidized to t h i o s u l f a t e and s u l f a t e
in each case.

Cthiosulfatophilu m Cyinosu m

51 01 52 05 1 01 52 0
tim e(hours )

Fig. 2. Growth parameters for static cultures of Chlorobium


and Chromatium. α and b_: Protein (o) and bacteriochlorophyll
(0) versus time for Chlorobium and Chromatium, respectively.
c_and dj sulfide (M, sulfur, (o), sulfate (u) and thiosulfate
(0) versus time for Chlorobium and Chromatium, respectively. The
ordinate for bacteriochlorophyll concentration is χ 10"^.
Waste Treatment and Environmental Considerations 213

time (hours)

Fig. δ. The effect of gas purge rates on growth parameters


<rf D e s u l f o v i b r i o . a, b_, and c_: Protein (o) versus time, d, e,
and f_: Sulfate(Ώ) and sulfide (Δ) versus time.

Typical r e s u l t s with a s t a t i c culture of Desulfovibrio


inoculated in the presence of 35 mM s u l f a t e are presented in
Figure 3a and 3d. Although bacterial growth i s slow, s u b s t a n t i a l
reduction of s u l f a t e to s u l f i d e takes place in twenty-four hours.

A. S u l f a t e Reduction by Purged Cultures of Desulfovibrio

The purging of cultures of Desulfovibrio with an i n e r t


c a r r i e r gas (see M a t e r i a l s and Methods f o r the experimental
procedure) r e s u l t s in a profound e f f e c t on the reduction of
sulfate. Figures 3b and 3e present r e s u l t s at a gas purge rate
of 5 ml/min and Figures 3c and 3f at 30 ml/min. For a twenty-
four hour p e r i o d , a 5 ml/min purge y i e l d s r e s u l t s almost i d e n t i ­
cal to those found in s t a t i c c u l t u r e s (Figures 3a and 3d).
214 Douglas J. Cork and Michael A. Cusanovich

However, at a purge rate of 30 ml/min, a s u b s t a n t i a l increase in


the amount of s u l f a t e reduced and protein synthesized i s noted
at twenty-four hours. Further, s u l f i d e production does not exceed
8 mM and appears to drop o f f to a low steady-state rate of
production (2-3 mM) a f t e r 10-12 hours. These r e s u l t s suggest
that high concentrations of s u l f i d e were responsible at l e a s t
in part for the limited reduction of s u l f a t e observed at a purge
rate of 5 ml/min and in s t a t i c c u l t u r e s . Thus high purge rates
keep the amount of s u l f i d e in the desulfovibrio culture at a low
level and stimulate both cell growth and s u l f a t e reduction.

B. Effect of Purging on M u t u a l i s t i c Cultures

Figure 4 presents data on the e f f e c t s of providing the


e f f l u e n t gas of a purged culture (30 ml/min) of Desulfovibrio to
a culture of Chlorobium thiosulfatophilum. Over a twenty-four
hour period, approximately 90% of the s u l f a t e reduced by
Desulfovibrio i s converted to elemental s u l f u r by Chlorobium
(Figures 4a and 4 b ) . Further, unlike s t a t i c c u l t u r e s (Figure 2 ) ,
no t h i o s u l f a t e i s produced and l e s s than 4mM s u l f a t e i s formed.
Compared to the s t a t i c culture of Chlorobium, approximately four
times as much protein and nine times as much bacteriochlorophyll
i s synthesized in the purged system in twenty four hours (compare
Figure 2a to Figure 4 a ) . F i n a l l y , about 4.5 times as much
elemental s u l f u r i s formed in 24 hours in the purged system
as compared to a s t a t i c c u l t u r e .

Studies on elemental s u l f u r production by Chromatium in


the same purging system are presented in Figure 5. For these
experiments a purge rate of 5 ml/min was used as we observed an
i n h i b i t i o n of the growth and extent of s u l f i d e oxidation by
Chromatium at higher purge r a t e s . As observed with Chlorobium,
no t h i o s u l f a t e was formed when s u l f i d e was provided by the
c a r r i e r gas. However, in the f i r s t twenty four hours, l i t t l e
or no elemental s u l f u r was produced by Chromatium, and the total
Waste Treatment and Environmental Considerations 215

C. thiosulfatophitum

10 15 20

Fig. 4. Growth parameters for purged cultures of Chlorobium.


a: Protein (o) and bacteriochlorophyll (Q) versus time. b_:
Sulfur (o) and sulfate (Ώ) versus time. The ordinate for
bacteriochlorophyll is χ 10~1. The gas purge rate is 30
ml/min.

amount of elemental s u l f u r was s u b s t a n t i a l l y l e s s than formed by


Chlorobium. Further at l a t e r times ( > 40 hours) large amounts
of s u l f a t e are formed. Thus, in terms of net production of
elemental s u l f u r from s u l f a t e , Chlorobium i s far superior to
Chromatium.

C. Raffinate Studies

Twenty-five percent (162 mM s u l f a t e ) and 50% (324 mM s u l f a t e )


hydrometallurgical solvent e x t r a c t i o n r a f f i n a t e s (composition
given in Table 1) were supplemented with l a c t i c acid (47 or 94 mM),
yeast extract (1 gm/liter) K HP0 2 4 · 3H 0 (0.65 gm/liter and
2

MgCl 2 (1 g m / l i t e r ) . Undiluted r a f f i n a t e was unable to support


Desulfovibrio growth.
216 Douglas J. Cork and Michael A. Cusanovich

P. desulfuricans C. vinosum

15-

10-

4- "20 4 0 " 60 Ί0 3fe 5 070 "


time (hours)

Fig. 5. Growth parameters for purged cultures of Chromatium.


a: Protein (o) versus time for D e s u l f o v i b r i o . b_: Protein (o)
and bacteriochlorophyll (Q) versus time for Chromatium.
c_: sulfate (U) and sulfide (Δ) versus time for Chromatium. The
ordinate for bacteriochlorophyll concentration is χ 10~^ . The
gas purge rate is 5 ml/min.

Figure 6 presents typical r e s u l t s for s u l f i d e production by


Desulfovibrio obtained with 25% r a f f i n a t e (47 and 94 mm l a c t i c
a c i d , Figure 6a and 6b, r e s p e c t i v e l y ) , and 50% r a f f i n a t e (94 mM
l a c t i c a c i d , Figure 6 c ) . For these experiments, the Desulfovibrio
culture was purged at a rate of 30 ml/min into a zinc acetate
trap for q u a n t i t a t i v e determination of s u l f i d e ( 6 ) .

The cultures in 25% r a f f i n a t e y i e l d e d a net amount of


s u l f i d e of 18-22 moles/24 hours i r r e s p e c t i v e of the i n i t i a l
l a c t i c acid concentration. These l e v e l s of s u l f i d e production
compare favorably with that obtained with Desulfovibrio grown on a
defined media (Figure 3f) and indicate that d i l u t e d r a f f i n a t e
contains no compounds detrimental to Desulfovibrio growth.
Waste Treatment and Environmental Considerations 217

TABLE I Chemical Analysis of Solvent Extraction Raffinate

Constituent Concentration (mM) a

646
S0=
4
M+ 1170
P0= <.001
Mg** 1.42
Fe++ .54
Ca 6.50
Na* 17
+
Κ .80
NO; .03
As .12
.004
Co ++
.05
Zn .009
Cu <.001

'Chemical analysis was done by the University Analytical Center,


Department of Chemistry, University of Arizona.

The experiments done in the presence of 50% r a f f i n a t e (Figure 6c)


indicate a s u b s t a n t i a l decrease in the rate of production of
s u l f i d e in twenty-four hours and suggest that the concentration
of some substance deleterious to the growth of Desulfovibrio i s
s u f f i c i e n t l y high to i n h i b i t growth.

IV. DISCUSSION

In Table 2 we have summarized the r e s u l t s of our studies in


order to provide a comparison of s t a t i c and purged c u l t u r e s of
Chromatium and Chlorobium in terms of the various parameters
218 Douglas J. Cork and Michael A. Cusanovich

time (hours)

Fig. 6. Sulfide-productionfor purged cultures of


D e s u l f o v i b r i o grown on diluted raffinate. a: 25% raffinate,
47 mM lactic acid, bj 25% raffinate, 94 mM lactic acid,
cj 50% raffinate, 94 mM lactic acid. The gas purge rate is
30 ml/min. See text for concentrations of other constituents
added to the raffinate.

measured. Because s u l f a t e reduction was becoming l i m i t i n g at


24 hours in s t a t i c c u l t u r e s of Desulfovibrio, we used the data
from t h i s time point f o r a l l cultures in these comparisons. Pre
sented in Table 2 are the amount of elemental s u l f u r formed
(A[S°]) in m i l l i m o l e s / l i t e r , the r a t i o of elemental s u l f u r formed
to s u l f a t e and t h i o s u l f a t e formed (A[S°]/(A[S0=] + [ S ^ ] ) ) , the
r a t i o of elemental s u l f u r formed by the photosynthetic bacteria
to s u l f a t e reduced by Desulfovibrio in m u t u a l i s t i c cultures
( A [ S ° ] / A [ S 0 ^ ] ) , the amount of protein synthesized
R (A[protein])
in mg/liter, and the amount of bacteriochlorophyll synthesized
( A [ B s c h l ] ) in mg/liter.

For optimum conversion of s u l f a t e to elemental s u l f u r , the


ratio A[S°]/A[S0^] R should approach 1.0 and the r a t i o
A[S°]/(A[S0^] + A [ S 0 ^ ] ) should approach i n f i n i t y .
2 Although
we have not achieved the ideal state to date, the data presented
Waste Treatment and Environmental Considerations 219

TABLE II Comparison of static and purged cultures after 24


hours of growth

„ , Purged Static Purged Static


V
s e m
Chlorobium Chlorobium Chromatium Chromatium

Δ[5°] α
21.5 4.6 2.3 0.9
klS°]/AlS0 +S 0 f =
4 2
=
3 6.1 1.4 1.6 0.4
Δ[5°]/Δ[5^ ] * 0.9 — <0.0 —
k[protein~\d 450 135 80 50
t\Bchl\ e
27 3.2 3.1 3.2

u
the -increase in elemental sulfur concentration (mM) in 24 hours.
2J
the ratio of the increase in elemental sulfur concentration to
the combined increase of sulfate and thiosulfate concentration
in 24 hours.
c
the ratio of the increase in elemental sulfur concentration to
the total concentration of sulfate reduced by D e s u l f o v i b r i o in
24 hours.
d
the increase in protein concentration (mg/l) in 24 hours.
e
the increase in bacteriochlorophyll concentration (mg/l) in 24
hours.
in Table 2 c l e a r l y demonstrates that the purge system u t i l i z i n g
Desulfovibrio and Chlorobium approaches t h i s c o n d i t i o n . Chlorobium
i s able to convert approximately 90% of the s u l f a t e reduced by
Desulfovibrio to elemental s u l f u r in twenty-four hours with the
net y i e l d of approximately 20 mmoles of elemental s u l f u r per l i t e r .
An overall process conversion for a two step s i n g l e stage reduc­
t i o n , s i n g l e stage oxidation system i s 55%. Further, the e l e ­
mental s u l f u r to oxidized s u l f u r r a t i o i s s u b s t a n t i a l l y improved
in the purged system. Chlorobium o f f e r s the added advantage that
the elemental s u l f u r produced i s excreted e x t r a c e l l u l a r l y and can
be e a s i l y i s o l a t e d by d i f f e r e n t i a l centrifugation.
220 Douglas J. Cork and Michael A. Cusanovich

In terms of conversion of s u l f i d e to elemental sulfur


Chromatium i s marginal at best. In s t a t i c c u l t u r e s , Chromatium
can only tolerate low l e v e l s of s u l f i d e and large amounts of
s u l f a t e are produced. In the purged system, Chromatium produces
elemental s u l f u r at reasonably high levels; however, this
process i s much slower than observed with Chlorobium (50-70 hours
are required for optimum l e v e l s ) , and the elemental s u l f u r to
s u l f a t e r a t i o never exceeds 2.0.

The studies reported here demonstrate that Desulfovibrio


can survive on d i l u t e d solvent extraction r a f f i n a t e and can
e f f i c i e n t l y convert s u l f a t e to s u l f i d e in the concentration
range 25-50% r a f f i n a t e . These observations r e a f f i r m the technical
f e a s i b i l i t y of using Desulfovibrio for i n d u s t r i a l applications.

Overall the studies reported here e s t a b l i s h that a g a s -


purged mutualiStic system of Desulfovibrio and Chlorobium can
be used for the e f f i c i e n t conversion of s u l f a t e to elemental
sulfur. P r e s e n t l y , s t u d i e s are underway u t i l i z i n g continuous
culture methods to determine optimum conditions f o r elemental
s u l f u r production in terms of pH, temperature, and s u b s t r a t e s .
I t i s anticipated that these studies w i l l y i e l d an even more
e f f i c i e n t process applicable to an i n d u s t r i a l scale.

Having e s t a b l i s h e d the technical f e a s i b i l i t y of using


microorganisms for the conversion of s u l f a t e to elemental sulfur,
i t i s of some i n t e r e s t to address the economics. The primary
"energy currency" of the system i s l a c t i c a c i d , the electron and
carbon source u t i l i z e d by Desulfovibrio f o r s u l f a t e reduction and
cell growth. In part the energy requirements can be minimized by
r e c y c l i n g Chlorobium i . e . , feeding i t to Desulfovibrio a f t e r
separating i t from the elemental s u l f u r . However, a s u b s t a n t i a l
amount of f i x e d carbon i s s t i l l required to drive s u l f a t e reduc-
tion. At the present time, studies are underway t e s t i n g a l t e r -
nate carbon sources including raw sewage. Considering the many
options a v a i l a b l e f o r further optimization of the microbiological
Waste Treatment and Environmental Considerations 221

conversion of s u l f a t e to elemental s u l f u r , i t i s our view that


a viable i n d u s t r i a l process can be developed. We feel the
approach described here p o t e n t i a l l y o f f e r s an economical and
environmentally sound i n d u s t r i a l procedure f o r the disposal of
s u l f a t e containing e f f l u e n t s in a non-leachable form.

V. ACKNOWLEDGEMENTS

Support f o r t h i s research was kindly provided by Anaconda


Copper Company. We are p a r t i c u l a r l y indebted to Dr. T. McNulty
and J . Spisak of Anaconda Copper f o r much encouragement and many
fruitful discussions.

VI. REFERENCES

1. Roy, A . B . , and Trudinger, P.Α., in "The Biochemistry of


Inorganic Compounds of S u l f u r " , p. 3, Cambridge U n i v e r s i t y
P r e s s , New York, 1970.
2. Postgate, J . R . , in " I n o r g a n i c S u l f u r Chemistry", (G. N i c k l e s s ,
Ed.) p. 259. E l s e v i e r P u b l i s h i n g Company, New York, 1968.
3. Starkey, R.L. Arch Mikrobiol. , 9,268 (1938).
4. B a r t s c h , R.G., in " B a c t e r i a l Photosynthesis" (H. Gest, A.
San P i e t r o , L.P. Vernon, E d s . ) , p. 5 0 1 . Antioch P r e s s ,
Yellow S p r i n g s , 1963.
5. Larsen, H., J. Bacterid., 64, 187 (1952).

6. Truper, H.G., and S c h l e g e l , H.G., Antonie van Leeuwenhoek


J. Microbiol. Serol. , 30, 225 (1964).
7. Tabatabai, M.A., Sulfur Inst. J., 10, 11 (1974).

8. Sorbo, B. Biochim. Biophys. Acta., 27, 324 (1958).


9. Schmidt, G.L., and Kamen, M.D., Arch. Mikrobiol., 73 (1970).
10. S t a n i e r , R.Y., and Smith, J.H. C , Biochem. Biophys. Acta, 4 1 ,
478 (1960).
MICROBIOLOGICAL DESULFURIZATION

OF COAL

Patrick R. Dugan
William A. Apel

The Ohio State U n i v e r s i t y


Columbus, Ohio 43210

Mixed enrichment cultures of acidophilic microorganisms are


effective in the removal of pyritic sulfur from 20% slurries of
a commercial grade of pulverized coal in the laboratory. Neither
T. ferrooxidans nor T. thiooxidans alone were effective in sulfur
removal from the coal. Microbial pyrite oxidation was most ef-
fective when the initial pH of the coal slurries was in the range
2.5 to 2.0 and when the slurries were supplemented with
(NR^)gSO.. Under these conditions approximately 97% of the py-
ritvc sulfur was removed within 5 days from coal which had an
initial pyritic sulfur content of 3.1%. This resulted in a bene-
ficiation from an average 4.6% total sulfur to about 1.5% total
sulfur.

I. INTRODUCTION

A. The High S u l f u r Coal Problem

I t i s anticipated that coal production in the U.S. w i l l more


than double by the year 1990 and the l a r g e s t source of t h i s coal
w i l l continue to be the Appalachian f i e l d s (33,43,45). A l l coal

223
224 Patrick R. Dugan and William A. Apel

contains s u l f u r in varying amounts and much of the coal found in


the Appalachian f i e l d s contains a r e l a t i v e l y high total sulfur
content of 3.0 to 5.5% ( 8 4 ) , much of which i s in the form of py-
r i t i c minerals deposited during the period of coal formation.

There are three forms of s u l f u r in c o a l : ( i ) organic s u l f u r


in which the s u l f u r i s either covalently bonded to carbon in the
general forms - R - S - S - R - , and - R - S - R - or bound as a s u l f a t e in the
general form R-0-SOg ( 1 5 , 3 4 ) , ( i i ) p y r i t i c s u l f u r in the form of
iron p y r i t e and marcasite, both of which have the same chemical
composition, FeS^, hut d i f f e r in physical s t r u c t u r e , and ( i i i )
sulfate. The total s u l f u r content of U.S. coal types ranges from
about 0.25% to nearly 7.0% ( 8 1 ) . The percentage of s u l f a t e in
non-weathered (non-oxidized) coal i s nearly always l e s s than 0 . 1 %
and i s therefore a minor consideration in the present d i s c u s s i o n .
Appalachian coal u s u a l l y contains from 0.5% to 2.0% organic s u l -
fur plus 0.5% to 4.0% p y r i t i c s u l f u r , although there are deposits
i n Kentucky, V i r g i n i a , West V i r g i n i a and elsewhere that contain
considerably l e s s than 1% total s u l f u r . As a gross g e n e r a l i t y ,
the content of p y r i t i c s u l f u r u s u a l l y exceeds the content of o r -
ganic s u l f u r in coals which contain more than 1% total sulfur.

The Clean A i r Act Amendments of 1970 provided for the e s t a -


blishment of national ambient a i r q u a l i t y standards which set a
l i m i t on SO^ of 0.03 ppm (80 pg/m ) as an annual arithmetic mean
concentration with an allowable 24 hour maximum of 0.14 ppm (365
3
jug/m ), not to be exceeded more than once each year. When t r a n s -
lated to source of performance standards, power plant emissions
are r e s t r i c t e d in some l o c a t i o n s to 1.2 l b s per m i l l i o n BTU.
Depending upon how c a l c u l a t i o n s are made, t h i s could r e s t r i c t the
combustion of coal at large power plants without scrubbers to
that coal which contains somewhere between 0.5% and 1.5% total
sulfur.
Combustion of coal r e s u l t s in conversion of most of the s u l -
fur present in the coal to S O 2 and ultimately to S O 4 . In
Waste Treatment and Environmental Considerations 225

general, the greater the s u l f u r content of the coal the greater


i s the stack emission of S 0 2 and SO^ into the atmosphere. In
order to maintain s a t i s f a c t o r y s u l f u r oxide l e v e l s in the atmos-
phere in regions where large scale coal combustion o c c u r s , either
coal with a total s u l f u r content of l e s s than 1.5% (some r e g u l a -
tory a u t h o r i t i e s argue l e s s than 1.0%) must be used or p r o v i -
s i o n s must be made to scrub s u l f u r oxides out of the stack gas
before entry into the atmosphere. Coal of l e s s than 1 % total
s u l f u r i s generally considered to be "low s u l f u r c o a l " and coal
with greater than about 1.5% to 2.0% total s u l f u r i s considered
by many regulatory a u t h o r i t i e s to be " h i g h " s u l f u r c o a l . It is
estimated that approximately 33% of our a v a i l a b l e coal cannot
presently be used without stack scrubbers because the s u l f u r con-
tent i s too high to meet a i r q u a l i t y standards.

Sixty-two percent of the l o w - s u l f u r reserves in the U.S. are


found West of the M i s s i s s i p p i whereas 90% of the present coal use
for e l e c t r i c power generation i s East of the M i s s i s s i p p i (26).
Increased costs associated both with production and shipping (ap-
proximately $.006 per ton mile) of Western coal to Eastern mar-
kets provides an incentive to remove s u l f u r from high s u l f u r
Eastern c o a l .

In t h i s report the phrases: pyrite oxidation, sulfur oxida-


t i o n , s o l u b i l i z a t i o n of p y r i t i c minerals and s u l f a t e release are
a l l tantamount to removal of p y r i t i c s u l f u r from coal v i a a s o l u -
b i l i z a t i o n and leaching process. S u l f a t e production can be due
to oxidation and s o l u b i l i z a t i o n of both p y r i t i c and o r g a n i c a l l y
bound s u l f u r .

B. The Oxidation of P y r i t e

Exposure of the chemically reduced compound F e S 2 to oxygen


and water r e s u l t s i n oxidation of the F e S 2 to f e r r i c s u l f a t e by
a complex s e r i e s of chemical reactions which are summarized in
the following r e a c t i o n s :
226 Patrick R. Dugan and William A. Apel

(I) Fe - — * ~ F e 0
+ electron

(II) 2 S" 2
+ 30 2 + 2H 0 - * - 2 ( S 0 " ) + 16 electrons + 4 H
2 4
2 +

(III) Sum: FeS 2 + 30 2 + 2H 0 — » - 2 H S 0


2 2 4 + Fe + 3

+3
The oxidized iron (Fe ) formed, subsequently reacts with
water to produce f e r r i c hydroxide and more acid according to the
following equation:
(IV) F e + 3H 0 - * - F e ( 0 H )
+ 3
2 3 + 3H (14,16,17,23,25,35,36,64)
+

The i n i t i a l report by Colmer and Hinkle in 1947 (16) on the


i s o l a t i o n of T. ferrooxidans from a c i d i c coal mine drainage gen-
erated considerable i n t e r e s t , both in the basic biology and e c o l -
ogy of the organism and in the potential f o r e x p l o i t a t i o n of the
organism to o x i d i z e p y r i t i c minerals.

Examination of the a c i d o p h i l i c iron and s u l f u r o x i d i z i n g


bacteria r e l a t i v e to the oxidation of p y r i t i c minerals found in
coal appears to have been o r i g i n a l l y reported both by Leathen, et
a l . ( 3 5 ) and by Temple and Del champs ( 6 8 ) . In these e a r l y r e -
ports the motivating i n t e r e s t f o r the research was in understand-
ing the formation of s u l f u r i c acid from p y r i t e ( i . e . the produc-
t i o n of a c i d i c coal mine d r a i n a g e ) . A p p l i c a t i o n of the a c i d o -
p h i l i c iron and s u l f u r o x i d i z i n g bacteria f o r the removal of py-
r i t e s in coal was studied by Zarubina,et a l . ( 8 3 ) and by Ashmead
(3) who demonstrated that the natural microbial f l o r a of a c i d i c
mine waters increased the rate of oxidation of p y r i t e in a 4%
p y r i t i c s u l f u r coal sample compared to oxidation i n the absence
of added bacteria. Silverman,et a l . ( 5 9 ) subsequently reported
that Ferrobacillus (presently T. ferrooxidans) accelerated the
oxidation of samples of p y r i t e and coarsely c r y s t a l l i n e marca-
s i t e extracted from coal (greater than 60% p y r i t e content) but
that the c e l l s were i n a c t i v e on coarsely c r y s t a l l i n e iron p y r i t e .
Oxidation rates in the presence of T. ferrooxidans were i n -
creased by reducing the p y r i t e p a r t i c l e s i z e . T. thiooxidans
Waste Treatment and Environmental Considerations 227

c e l l s were i n a c t i v e on a l l the p y r i t i c samples they examined.


These same i n v e s t i g a t o r s subsequently reported (50,60) that T.
ferrooxidans catalysed removal of appreciable amounts of p y r i t e
from coal w i t h i n 3 to 4 days and that p a r t i c l e s i z e reduction as
well as pretreatment with 2 Ν HC1 increased the s u s c e p t i b i l i t y of
most coal samples to bacterial d e s u l f u r i z a t i o n . They a l s o repor­
ted that a d d i t i o n of Fe^iSO^)^ increased p y r i t e removal from acid
treated c o a l . There was and s t i l l i s disagreement as to the
a b i l i t y of T. thiooxidans to o x i d i z e p y r i t i c m a t e r i a l s ; (37,38,
68,69). However, there appears to be universal agreement that T.
ferrooxidans o x i d i z e s p y r i t i c minerals by c a t a l y z i n g reaction I I I .
During the period between 1947 and 1965 attention was a l s o
directed toward bacterial leaching of other s u l f i d e minerals (57,
65) and i n t e r e s t in the bacterial leaching of s u l f u r from coal
subsequently diminished u n t i l stimulated by the energy p u b l i c i t y
of the e a r l y 1 9 7 0 ' s .

The only commercial scale data on bacterial removal of py­


r i t e from coal appears to be that of Capes e t a l . ( 1 3 ) who blended
3

various percentages of weathered coal as t h e i r inoculum of bac­


t e r i a with run of mine (r.o.m.) c o a l . They were able to show
that a d d i t i o n of 10% weathered coal resulted in a reduction of
the s u l f u r content of r.o.m. coal from 6 . 1 % to 2.7% during sub­
sequent agglomeration of p y r i t e from pulverized c o a l , using a
f l o t a t i o n technique. These r e s u l t s were reported to be much s u ­
p e r i o r to those obtained using chemical depressants. I t i s im­
portant to note that r e l a t i v e l y l i t t l e s u l f a t e was formed during
the bacterial p r o c e s s i n g , i n d i c a t i n g that p y r i t e r e j e c t i o n during
agglomeration in t h e i r system cannot be ascribed to simple
leaching but involves p r e f e r e n t i a l wetting of coal due to the
rendering of p y r i t e hydrophobic by bacterial action that a i d s in
p y r i t e r e j e c t i o n during agglomeration. I t must be pointed out
however, that use of weathered coal as inoculum may have been
f o r t u i t o u s as w i l l be discussed subsequently. At any rate i t
228 Patrick R. Dugan and William A. Apel

seems unreasonable to ascribe the total benefit obtained e x c l u ­


s i v e l y to T. ferrooxidans but rather to the mixed microbial
population.

Reports that iron o x i d i z i n g bacteria are more active than


the e x c l u s i v e l y s u l f u r o x i d i z i n g bacteria r e l a t i v e to rates of
p y r i t e oxidation (35,60,80) in view of p r i o r observations that
f e r r i c s u l f a t e could o x i d i z e p y r i t e (69) led to speculation that
the primary r o l e of bacteria in p y r i t e oxidation was the produc­
t i o n of f e r r i c ions and that the f e r r i c ions thus produced, ox­
idized more p y r i t e with concomitant regeneration of ferrous ions
according to Equation V. The recycled iron could again be
oxidized by the bacteria and the cycle would continue (35,59,62).

(V) 14 F e + FeS + 8 H 0 —
+ 3
2 2 15 F e + 2
+ 2(S) ^iobaoilU
i 2 2 ui

ferrooxidans ' 2S0^" + 16 Η


1
*—T. ferrooxidans Λ Ο Λ 2
'l r
- L,+

This microbiological process i s analogous to a chemically


catalysed reaction process. That i s , l i v i n g bacteria are o x i d a ­
tion c a t a l y s t s promoting the oxidation o f i n s o l u b l e m e t a l l i c s u l ­
f i d e to soluble s u l f a t e , which i s then removed by leaching. The
bacteria u t i l i z e the p y r i t e n u t r i t i o n a l l y and grow in the system.

Singer and Stumm (63) have concluded that under a c i d i c con­


d i t i o n s below pH 4 . 0 , the rate of p y r i t e oxidation by f e r r i c ion
i s considerably greater than the rate of ferrous ion oxidation
in the absence of bacteria. Hence, the bacteria must catalyze
+2 +3 +3
the oxidation of Fe to Fe ion in order to supply the Fe
to o x i d i z e the p y r i t e . They, therefore, concluded that the bac­
t e r i a l l y catalyzed reaction c o n t r o l s the rate of p y r i t e o x i d a ­
tion under a c i d i c c o n d i t i o n s . Lau, et a l . ( 3 4 ) a l s o came to e s s e n ­
t i a l l y the same conclusion in that the bacteria were e s s e n t i a l
+3 +2
to maintenance of the high Fe to Fe ion r a t i o in s o l u t i o n
which i s necessary to chemically o x i d i z e p y r i t e . The mechanism
of s u l f u r oxidation by T. thiooxidans would be somewhat d i f f e r e n t
in that s u l f u r i s e s s e n t i a l l y i n s o l u b l e , therefore r e q u i r i n g
Waste Treatment and Environmental Considerations 229

d i r e c t contact of bacterium to substrate (71,80).

C. The Microorganisms of Highly A c i d i c Environments

Thiobaoillus ferrooxidans i s c l a s s i f i e d on the b a s i s of i t s


a b i l i t y to u t i l i z e the oxidation of ferrous iron as i t s s o l e e n -
ergy source and C0 2 as i t s s o l e carbon source. I t i s placed in
the genus Thiobaoillus because i t can a l s o o x i d i z e reduced s u l f u r
as a sole energy source. T. ferrooxidans has been reported to
+2
adapt from autotrophic growth on Fe to heterotrophic growth on
glucose as a carbon and energy source a f t e r at l e a s t two t r a n s -
+2
f e r s in media containing both Fe and glucose ( 4 6 , 5 4 , 5 5 , 6 6 ) .
Adapted c e l l s contain a complete t r i c a r b o x y l i c acid cycle when
grown h e t e r o t r o p h i c a l l y , but unadapted c e l l s lack alpha-keto
glutarate dehydrogenase and reduced nicotinamide adenine d i n u c l e -
+2 +2
otide oxidase when grown on Fe , and the presence of Fe is
reported to repress glucose d i s s i m i l a t i o n (66). With reference
to alternate substrates for chemolithotrophic growth, the iron
o x i d i z i n g enzymes are reported to be inducible ( 4 0 ) . I t has
been proposed that the f a c u l t a t i v e heterotrophic bacteria derived
from T. ferrooxidans c u l t u r e s , which grow on e i t h e r organic or
+2
reduced s u l f u r , but not on Fe , be designated as a new s p e c i e s :
T. acidophilus (27). Differences in ( i ) enzymes ( 7 5 ) , (ii)
mole % guanine plus c y t o s i n e , 5 5 - 5 7 . 1 % f o r i r o n grown and
62.9-63.2% for glucose grown ( 2 2 , 2 7 , 4 9 ) , and ( i i i ) lack of the
iron o x i d i z i n g enzyme in glucose grown c e l l s , indicate that the
heterotrophic i s o l a t e s are d i f f e r e n t from the autotrophic i s o -
l a t e s and that they are selected from T. ferrooxidans c u l t u r e s
(54,55,67). Furthermore, the glucose o x i d i z i n g a c i d o p h i l e s exam-
ined do not react with f l u o r e s c e i n isothiocyanate labeled r a b b i t
anti T. ferrooxidans IgG, which i s s p e c i f i c f o r T. ferrooxidans
(2).
I t appears that T. ferrooxidans s t r a i n s are chemolithotro-
phic whereas the organic o x i d i z e r s which r e t a i n thp a b i l i t y to
230 Patrick R. Dugan and William A. Apel

oxidize reduced s u l f u r compounds are e i t h e r mixotrophic or f a c u l -


t a t i v e l y heterotrophic. Also there are many d i f f e r e n t s t r a i n s
of T. ferrooxidans with d i f f e r e n t metabolic c a p a b i l i t i e s (8,31)
present in the environment. There i s a l s o divergence of opinion
r e l a t i v e to the use of organic substrates by chemolithotrophic
bacteria (30,42,44,47,48,79).

Walsh et a l . (82)questioned how f r e s h l y mined s p o i l s i n i t i a l l y


achieve s u f f i c i e n t a c i d i t y to be optimal for the a c i d o p h i l i c
Thiobacilli. These i n v e s t i g a t o r s i s o l a t e d a d i f f e r e n t microor-
ganism; Metallogenium, which produces some acid via the oxidation
+2 +3
of Fe to Fe when accompanied by subsequent h y d r o l y s i s . The
acid then creates an environment s u i t a b l e for the succession of
the a c i d o p h i l i c Thiobacilli. Although t h i s could be an e c o l o g i -
cal explanation, i t need not be a p r e r e q u i s i t e to e s t a b l i s h i n g
T. ferrooxidans growth since chemical oxidation of p y r i t e would
accomplish a lowering of pH to about 4.0 over a longer time span.
Also many non-iron o x i d i z i n g a c i d o p h i l i c Thiobacillus i s o l a t e s
a c t u a l l y grow better when the pH i s above 3.5 to 4.0 thereby
lowering t h e i r own environmental pH s u f f i c i e n t l y . Leaching of
sandstone w i l l a l s o produce acid s u f f i c i e n t to lower the pH
down to 4.0.
A microorganism capable of o x i d i z i n g iron and s u l f u r as well
as growth at thermophilic temperatures (45°-70°C) and low pH was
i s o l a t e d by B r i e r l e y from a thermal region in Yellowstone Park
(9). The organism designated " f e r r o l o b u s " was l a t e r character-
ized more thoroughly (10). A s i m i l a r but d i f f e r e n t organism,
Sulfolobus acidocalderius was i s o l a t e d and characterized by
Brock, et a l . ( 1 2 ) and was reported to have an optimum temperature
of 70°-75°C with a range of 55-80°C and a pH optimum of 2 to 3.
Both organisms o x i d i z e reduced iron and s u l f u r and grow on simple
organic compounds and yeast extract (10,11,12) and both have been
shown to o x i d i z e metal s u l f i d e ores with potential for commercial
metal extraction processes (11) where high temperatures
Waste Treatment and Environmental Considerations 231

&5°-80°C) can be maintained. The organisms do not appear to grow


much below 45°C.

D. F i e l d Studies

F i e l d studies of the a c t i v i t y of bacteria i n coal refuse


14
showed a strong c o r r e l a t i o n between uptake of C0 2 and most pro-
bable numbers (MPN) of i r o n o x i d i z i n g bacteria but not with the
acid t o l e r a n t heterotrophic microorganisms which were a l s o
14
present in the refuse. Maximal CO^ uptake occurred in 2 to 3
year old coal refuse with only s l i g h t incorporation in f r e s h
refuse or material 40 years o l d . Maximal uptake was always found
in samples taken from the surface above 8 to 10 cm depth, at tem-
peratures between 20° and 30° C and at moisture content of be-
tween 23 and 35%, (5) a l l of which agree with data p r e v i o u s l y
published on the b a s i s of laboratory i n v e s t i g a t i o n s (20,34,56,
61,70,76).
Apel and Dugan developed a f l u o r e s c e n t antibody technique
which allowed them to follow i n c r e a s i n g numbers of Thiobaoillus

Fig. I. Scanning electron micrograph (EM) of a coal refuse


sample showing surface porosity and what appear to be microorgan-
isms on the surface.
232 Patrick R. Dugan and William A. Apel

ferrooxidans in coal refuse ( 2 ) . T. ferrooxidans c e l l s were de-


tected in the surface washings of the r e f u s e , but none were detec-
ted in refuse which had been prewashed and then p u l v e r i z e d , i n -
d i c a t i n g that T. ferrooxidans c e l l s were on the coal refuse
surface but not in the internal pores of the refuse m a t e r i a l s .
F i g . 1 i s a scanning electron micrograph showing the p o r o s i t y
of coal refuse. Typical bacterial growth curves with time were
observed and the exponential phase of these curves corresponded
to increases in t i t r a t a b l e a c i d i t y in the refuse samples, while
over the same period acid production in s t e r i l i z e d refuse con-
t r o l s was considerably l e s s ( 2 ) . The c o r r e l a t i o n between the
acid production rate and the exponential growth phase of T.
ferrooxidans in coal refuse suggests that bacteria are a dominant
stimulus leading to acid formation from p y r i t e in coal refuse (a
non-marketable coal with a high s u l f u r and mineral content) and
previous work on refuse i s d i r e c t l y applicable to higher grades
of c o a l .

The a c i d o p h i l i c Thiobacilli are known to produce autotoxic


metabolic by-products which i n h i b i t iron and s u l f u r oxidation by
the c e l l s when present in s u f f i c i e n t concentration (7,24,52,78,
79). Lower molecular weight organic a c i d s , p a r t i c u l a r l y alpha-
keto a c i d s , some of which are intermediates of the c e l l s meta-
b o l i c pathways i n h i b i t metabolism of iron and s u l f u r by Thio-
bacilli by causing the c e l l membrane to become leaky and u l -
timately d i s r u p t (79) which may a l s o allow i n t o l e r a b l e amounts of
H +
to enter the c e l l (1). Acid tolerant heterotrophs are a l s o
present in the environments of the a c i d o p h i l i c Thiobacilli ( 5 ,
20,21,22,41,76,77) and may therefore be an i n d i r e c t aid in
s u l f u r oxidation by v i r t u e of t h e i r removal of the autotoxic o r -
ganic by-products ( 7 8 , 7 9 ) . I t i s known that c e r t a i n organic sub-
stances retard iron and s u l f u r oxidation by Thiobacilli (19,24)
and a l s o to retard the rate of metallurgical leaching from
s u l f i d e s (28,51,72,73,74). The biochemical reactions which
Waste Treatment and Environmental Considerations 233

s o l u b i l i z e o r g a n i c a l l y bound s u l f u r are d i f f e r e n t from those of


-2
pyrite oxidation. The s p l i t t i n g of SO^ from R - 0 - S 0 3 i s due to
s u l f h y d r o l a s e enzymes, whereas the oxidation of - R - S - S - R - and
- R - S - R - would be caused by desulfhydrase enzymes. This topic
has not been adequately studied r e l a t i v e to coal b e n e f i c i a t i o n .
An indigenous population of acid t o l e r a n t heterotrophic bac-
t e r i a i s responsible f o r production of slime streamers in h i g h l y
acid mine water (pH 2 . 8 ) . This group of bacteria has not been
well characterized but at l e a s t one i s o l a t e has a pH optimum
near n e u t r a l i t y ( 2 1 , 2 2 ) . This suggests that the organisms c o l o -
nize in the stream creating a l o c a l i z e d microcosm that i s d i f f e r -
ent from the surrounding, h i g h l y acid environment.
Bohlool and Brock have reported the growth of a thermophilic
mycoplasma, Thermoplasma acidophilum, in refuse p i l e s (6).
Yeasts and filamentous fungi have frequently been i s o l a t e d from
a c i d i c mine water ( 5 , 1 8 , 2 0 , 2 2 , 2 9 , 3 2 ) . The source of organic
n u t r i e n t required to support the rather extensive amount of het-
erotrophic growth in mine drainage i s not evident. One p o s s i b l e
source would be from metabolic by-products produced by the a c i d o -
p h i l i c autotrophs. Schnaitman and Lundgren (52) reported that
organic acids accumulate from autotrophic growth of T. ferrooxi-
dans. Several species of a l g a e , e s p e c i a l l y Euglena and Ulothrix,
have a l s o been observed in rather large numbers in acid drainage
at pH 3.0 in the presence of s u n l i g h t ( 1 8 , 2 0 , 2 9 , 3 2 ) , which a l s o
may either enrich the stream with organic substances or remove
by-products that are t o x i c to the iron and s u l f u r o x i d i z e r s .
Other sources of heterotrophic n u t r i e n t would be from coal com-
ponents such as phenolics and the organic content of other s e d i -
mentary d e p o s i t s .

Microbiological leaching of s u l f u r from coal has many f e a -


tures that are s i m i l a r to m e t a l l u r g i c a l bacterial leaching
processes and some that are quite d i f f e r e n t . Both processes
involve the oxidation of s u l f i d e minerals by a v a r i e t y of
234 Patrick R. Dugan and William A. Apel

a c i d o p h i l i c microorganisms, including Thiobaoillus s p e c i e s ; u l -


_2
timately to soluble SO, . In the case of iron p y r i t e , bacteria
+2 +3
a l s o oxidize Fe to Fe . In contrast to metallurgical leaching
where oxidation of s u l f i d e mineral releases a commercially useful
metal ion from the ore p i l e that i s recovered from the ensuing
a c i d i c leachate, i t i s the coal leach substrate which i s the
desired end product; whereas, the a c i d i c s o l u t i o n of s o l u b i l i z e d
mineral ions presently represents an undesirable waste for which
a commercial use i s being sought. Again, by comparison, although
a coal s u l f u r content in excess of about 1.5% i s considered to be
h i g h , t h i s represents a small concentration of n u t r i t i o n a l iron
and/or s u l f u r for the bacteria which depend upon i t for growth.
F i n a l l y , i t must be emphasized that the s u l f u r content of coal
i s u s u a l l y reported on the b a s i s of a n a l y t i c averages from large
samples and there i s tremendous v a r i a b i l i t y within short d i s -
tances in coal seams or within short distances in coal piles
unless precautions are taken to obtain representative homogeneous
samples. P y r i t e c r y s t a l s may be many centimeters in s i z e or in
some coal samples lenses often l e s s than 10 micrometers in s i z e
remain i n t a c t , embedded within the coal (53) and exposure of
these microscopic p y r i t e lenses to bacterial action requires ex-
tensive p u l v e r i z a t i o n of the c o a l . The Thiobacilli which c a t a -
lyse the oxidation have an approximate diameter of 1 micrometer.
The purpose of t h i s paper i s to describe some of the parameters
which influence removal of s u l f u r from coal s l u r r i e s by microbial
metabolism in an e f f o r t to demonstrate that the process has
potential for commercial e x p l o i t a t i o n .

II. MATERIALS AND METHODS

A. Inocula
T. ferrooxidans was grown under forced aeration at ambient
temperature (23+ 1°C) in "9K" medium ( 5 8 ) . After 5 days incuba-
t i o n , the c e l l s were harvested, washed, suspended in pH 3 H S 0 , 9
Waste Treatment and Environmental Considerations 235

s o l u t i o n , and stored as p r e v i o u s l y described ( 2 ) . Cell suspen­


s i o n s were used as inoculum in 1 week or l e s s a f t e r harvesting.

T. thiooxidans was grown on ATCC medium #450, harvested, and


stored using the same method described for T. ferrooxidans.

Natural coal enrichment c u l t u r e s were obtained by e i t h e r i n ­


oculating a 10% s l u r r y (pH 3) of pulverized n o n - s t e r i l e , high
s u l f u r coal with acid mine drainage and incubating at ambient
temperature f o r 4 weeks on a gyratory shaker (200 rpm), or simply
incubating a 10% s l u r r y (pH 3) of n o n - s t e r i l e , uninoculated, p u l ­
verized high s u l f u r coal on a gyratory shaker (200 rpm) for 4
weeks at ambient temperature. The former coal enrichment was
designated coal enrichment #3, while the l a t t e r was designated
coal enrichment #4.

P y r i t e enrichment c u l t u r e s were obtained by i n o c u l a t i n g


s l u r r i e s of p y r i t e adjusted to pH 3 with H^SO^ with acid mine
drainage and incubating under the c o n d i t i o n s described for the
coal enrichments. A l l acid coal mine drainage samples were from
an acid stream (pH 3.5) located approximately 10 miles east of
McArthur, Ohio. After the i n i t i a l 4 week incubation p e r i o d , a l l
enrichment c u l t u r e s were maintained by t r a n s f e r r i n g to f r e s h
s l u r r i e s every 2 weeks.

B. E f f e c t s of I n i t i a l pH

Coal s l u r r i e s (20% coal in H^O) were prepared with a com­


m e r c i a l l y pulverized coal (Table 1) designated as coal sample #1
which was courteously provided by the Columbus and Southern Ohio
E l e c t r i c Co. The s l u r r i e s were adjusted to a v a r i e t y of pH
with 10 Ν H^SO^ and 12.5 ml a l i q u o t s were dispensed in 250 ml
Erlenmeyer f l a s k s and s t e r i l i z e d by autoclaving at 20 p . s . i . for
25 min. After c o o l i n g to ambient temperature, duplicate f l a s k s
containing s l u r r i e s at each of the above pH were inoculated
with 5% inoculum from the coal enrichment culture #3, while 2
236 Patrick R. Dugan and William A. Apel

additional f l a s k s from each of the above pH s l u r r i e s remained un-


inoculated and served as s t e r i l e c o n t r o l s . Both inoculated, and
s t e r i l e f l a s k s were placed on a gyratory shaker (200 rpm) and
incubated at ambient temperature. At 2-3 day i n t e r v a l s during the
incubation p e r i o d , 1 ml a l i q u o t s were a s e p t i c a l l y removed from
each of the f l a s k s and used to determine pH and s u l f a t e concen-
tration. The pH of the samples was determined u t i l i z i n g a
Corning model 12 expanded scale pH meter equipped with a Corning
475060 pH electrode. Sulfate concentrations were determined as
outlined below.

C. Quantitative Determination of S u l f a t e

The procedure used for determination of s u l f a t e was a modi-


f i c a t i o n of a barium s u l f a t e t u r b i d i m e t r i c procedure in which
samples were c o l l e c t e d , d i l u t e d in H^O ( i f n e c e s s a r y ) , and
treated with excess BaCl^. The t u r b i d i t y of each sample was
determined in a Shimadsu model 14PS-50L spectrophotometer set at
a wave length of 450 nm. Percent transmission of each sample
was recorded and compared to a standard s u l f a t e curve prepared
in the range of 0 to 600 ppm of s u l f a t e .

D. Effects of Bacterial Inoculum on D e s u l f u r i z a t i o n

Twenty percent coal-water s l u r r i e s at pH 2.5 were prepared


as described above and were inoculated with either 5% by volume
coal enrichment culture #3, 5% p y r i t e enrichment c u l t u r e , 5%
T. ferrooxidans suspension (10^ c e l l s / m l ) , 5% T. thiooxidans s u s -
pension (10^ c e l l s / m l ) , or 5% suspension containing h a l f T. fer-
rooxidans and h a l f T. thiooxidans (10^ c e l l s / m l ) . All flasks
were prepared in d u p l i c a t e , including a b i o t i c c o n t r o l s , with pH
and s u l f a t e concentrations in each f l a s k determined at 2-3 day
i n t e r v a l s as p r e v i o u s l y described.
Waste Treatment and Environmental Considerations 237

E. Supplemental Nutrients

In i n i t i a l studies on the e f f e c t s of supplemental nutrients


on the d e s u l f u r i z a t i o n of c o a l , sub 200 mesh coal (Table 1) was
employed in 20% s l u r r i e s ( i n i t i a l pH approximately 3.5) set up as
shaker c u l t u r e s as described above. "9K" basal s a l t s (58) were
added to one set of s l u r r i e s while another set of s l u r r i e s
remained unsupplemented. Two f l a s k s of supplemented and 2 f l a s k s
of unsupplemented coal s l u r r y were inoculated i n d i v i d u a l l y with
one of the f o l l o w i n g : 5% p y r i t e enrichment c u l t u r e , 5% coal en­
richment culture #3, 5% T. ferrooxidans (10^ c e l l s / m l ) , and 5%
acid mine drainage obtained from the p r e v i o u s l y described source.
Again, pH and s u l f a t e concentrations were determined at 2-3 day
intervals.

The e f f e c t s of additional supplemental n u t r i e n t s on commer­


c i a l l y blended coal (coal sample 1) a l s o was examined using 20%
s l u r r i e s in 250 ml shaker c u l t u r e s as p r e v i o u s l y described. Rep­
l i c a t e s of s l u r r i e s c o n t a i n i n g : no supplement, ATCC medium #450
trace m i n e r a l s , 9K basal s a l t s , 0.05% K H P 0 , and 0.3% ( N H ) S 0
2 4 4 2 4

were inoculated with coal enrichment #3 and incubated on a gy­


ratory shaker at ambient temperature. In a d d i t i o n , inoculated,
unsupplemented s l u r r i e s were incubated at ambient temperature on
a gyratory shaker under a 5% C 0 2 - 95% a i r atmosphere. In a l l
cases comparisons between inoculated s l u r r i e s were made to com­
parable uninoculated c o n t r o l s . A l l values reported are averages
of duplicate samples.

F. Scanning Electron Microscopy

Coal specimens f o r scanning electron microscopy (SEM) were


attached to Dag 154 (Achison C o l l o i d s Co., Port Huron, M i . 48060)
coated aluminum specimen stubs with double sided tape, a f t e r
ο
which they were sputter coated with 100 A of g o l d . Specimens
were examined at various magnifications (20° angle) with an
238 Patrick R. Dugan and William A. Apel

Hitachi model S-500 scanning electron microscope. Photomicro­


graphs were taken u t i l i z i n g Polaroid 4" χ 5" Land Film (type 5 5 ) .

III. RESULTS AND DISCUSSION

Coal sample # 1 , a power generating blend of pulverized coal


assumed to be a representative sample of commercial c o a l , was
separated into 3 mesh s i z e f r a c t i o n s and analysed for t o t a l ,
organic and p y r i t i c s u l f u r as shown in Table 1 .

Table I Values Showing % Sulfur Content


of Various Fractions of the Pulverized Coal

Sulfur Content as % of Fraction


Mesh size composition Total Pyritic SO ~% Organic

10% 60 to 100 (.297 to .149 mm) _ _ _ _


46% 100 to 200 (.149 to .074 mm) 4.2 2.9 0.1 1.2
46% sub 200 (sub .074 mm) 6.4 4.2 0.2 1.0
Combined Coal Mixture 4.6 3.1 0.1 1.4

Scanning electron micrographs of the coal sample and T. fer-


ooxidans are shown in F i g s . 2 and 3.

Fig. 2. A scanning EM of the pulverized coal sample showing


particles and what appear to be microbes on the surface. — = δ um

Fig. 3. Scanning EM of Τ. ferrooxidans for size comparison


to Fig. 2.— » 0 5 urn
S
Waste Treatment and Environmental Considerations 239

Fractions of the coal shaken in Erlenmeyer f l a s k s as 20%


s l u r r i e s in d i s t i l l e d Η,>0 (wt. to v o l . ) in the presence of v a r ­
ious types of inocula were examined f o r production of soluble
SO^ and changes in pH. Figure 4 presents data from the 100 to
200 mesh f r a c t i o n versus time in days. I t can be seen that d i f ­
ferent enrichment culture inocula varied with respect to rates of
_2
SO- released from the coal sample. The culture of T. fevroox-
-2
idans used in the experiment had v i r t u a l l y no e f f e c t on S0^
release when compared to release from the s t e r i l e (uninoculated)
c o n t r o l . I t i s a l s o noted that the c u l t u r e s with the most active
-2
SO^ release a l s o had the most dramatic drop in pH over the same
time span. The coal sample inoculated with coal enrichment #3
( F i g . 4) contained 1.8% total s u l f u r and 0 . 1 % p y r i t i c s u l f u r at
the end of the experiment.
Data s i m i l a r to that in F i g . 4 f o r smaller p a r t i c l e s i z e
coal (sub 200 mesh) i s presented in F i g . 5. In every case there
-2
was more S0^ released and at higher rates in the sub 200 mesh
coal than from the 100 to 200 mesh coal ( F i g . 4 ) , which i s con­
s i s t e n t with previous reports ( 3 5 , 3 9 ) . A g a i n , the T. ferroox-
_2
idans s t r a i n used had l i t t l e e f f e c t on e i t h e r S0^ release or
acid production and the pH drop in other samples had a p o s i t i v e
_2
c o r r e l a t i o n with SO- r e l e a s e . In a l l cases ( F i g . 4 and F i g . 5)
-2
a 6 to 7 day lag in production of S0^ was observed. The sample
inoculated with coal enrichment #3 contained 2% total s u l f u r and
0.5% p y r i t i c s u l f u r at the end of the experiment, whereas the
control s t i l l contained 5.2% s u l f u r .
_2
When considering the greater total amount of S0^ released
from sub 200 mesh coal ( F i g s . 5 and 6) compared to 100-200 mesh
( F i g . 4) i t must be emphasized that the total s u l f u r content i s
higher in the smaller p a r t i c l e f r a c t i o n (see Table 1) and d i f ­
ferences in r e s u l t s are a t t r i b u t a b l e both to p a r t i c l e s i z e
and o r i g i n a l p y r i t e content.
240 Patrick R. Dugan and William A. Apel

Fig. 4. Curves showing SO^~ release from 100-200 mesh


ooal and pH change vs time when in the presence of various types
of inoculum.

In an attempt to reduce the lag p e r i o d , a s e r i e s of 20%


s l u r r i e s of sub 200 mesh coal were supplemented with the "9K"
basal s a l t s ( 5 8 ) , inoculated with coal enrichment #3 and mon-
-2
itored f o r pH and SO. . Results are presented in F i g . 6 and
-2
c l e a r l y show l a r g e r amounts of S0^ release than non-nutrient
supplemented f l a s k s ( F i g s . 4 and 5 ) . However, the lag period
was not s i g n i f i c a n t l y reduced.
Waste Treatment and Environmental Considerations 241

[
χ Uninoc. control
Q. T. ferrooxidans
Acid mine enrich.
Coal enrich. #4
Pyrite enrich
Coal enrich. #3

Fig. 5. Curves showing SO^ release from sub 200 mesh


coal and pH change vs time when in the presence of various types
of inoculum.

When pH data in F i g s . 4, 5 and 6 are examined c o l l e c t i v e l y ,


-2
rapid release of SO^ i s observed a f t e r the pH drops below 2.5
and the highest rates of release are seen in the pH range 2.5
to 2.0, suggesting that the p y r i t e o x i d i z e r s are most a c t i v e
in t h i s pH range and that s l u r r i e s should be i n i t i a l l y adjusted
to about 2.5. Previous unpublished experiments showed an
242 Patrick R. Dugan and William A. Apel

Fig. 6. Curves showing SO release and pH change from


sub 200 mesh coal when supplemented with "9K" salts solution vs
time when in the presence of various types of inoculum.

optimum pH for iron oxidation by our s t r a i n of T. ferrooxidans in


the range 2.3 to 2.8 with d r a s t i c reduction of iron oxidation
below 2.0 and above 3.5, a l l of which i s c o n s i s t e n t with our
present data. Sulfate release versus time from a s e r i e s of pH
adjusted 20% s l u r r i e s of coal Sample #1 are shown in F i g . 7. The
lag period was s i g n i f i c a n t l y reduced in the pH range 2.5 to 2.0.
Waste Treatment and Environmental Considerations 243

2 4 6 8 ΪΟ 12 Ϊ4
Time in D a y s

Fig. 7. Curves showing decrease in lag period for SO^


release vs time as influenced by initial pH of a 20% slurry of
the commercial coal blend.

Addition of supplemental n u t r i e n t s was then examined using


a 20% s l u r r y of the commercial coal #1 when inoculated with coal
enrichment #3 at an i n i t i a l pH of 2.5. These data are shown in
F i g . 8. In these experiments there was no observable increase
244 Patrick R. Dugan and William A. Apel

in rate or amount of SO. release by addition of either trace


2

^ -2
minerals or by 0.05% K HP0 . The s l i g h t l y decreased S 0
2 4 re­ 4

lease in the presence of "9K" s a l t s i s unexplained. Addition


of 5% C 0 as a gas in the head space of a c o n t r o l l e d growth
2

chamber resulted i n i t i a l l y in an increased lag p o s s i b l y as a


r e s u l t of buffering the pH, followed by a s l i g h t p o s i t i v e e f f e c t

2.5|

χ +0.3%(ΝΗ ) 2 4 S0 4

1.5|

1
Ο Uninoc. control

0.5
Ε
Ο

>
Ο

1.5 + 5 \ C 0 2

6 N o Supplement
ιι + P450 Tr. minerals
+0.05% K HP0
Ο
2 4

Χ + 9 K salts
CO

Uninoc. control
0.5

2 4 6 8 10 12
Time in Days

Fig. 8. Curves showing SO^ release vs time from 20%


slurry of coal blend, initially adjusted to pH 2.5 when various
nutrient supplements were added.
Waste Treatment and Environmental Considerations 245

on S 0 4 release. Addition of 0 . 3 % (NH ) S0


4 2 4 had a beneficial
influence on both the rate and amount of SO^ released. Exper­
iments are continuing in our e f f o r t s to optimize the rate and
amount of SO^ release p r i o r to expanded volume (scale up)
experiments. F i n a l l y , we have a l s o used T. thiooxidans as an
inoculum without being able to show s i g n i f i c a n t SO^ release.
However, when T. thiooxidans and T. ferrooxidans are inoculated
together there was a s i g n i f i c a n t increase in SO^ release over
the s t e r i l e c o n t r o l s .

The above experiments on a s i n g l e pulverized coal sample (or


ο
f r a c t i o n s thereof) show that SO^ ions were released at greater
rates in the presence of mixed enrichments of a c i d o p h i l e s than
by e i t h e r of the species of Thiobacillus used in the experiments.
Since the T. ferrooxidans was a very a c t i v e iron o x i d i z i n g
s t r a i n i t i s p o s s i b l e e i t h e r that o x i d a t i o n of p y r i t e involves
+ 3

more than production of Fe as an oxidant, or that another organ­


ism i s responsible f o r i n i t i a t i n g the attack on p y r i t e surfaces
+ 3
to provide the i n i t i a l release of Fe necessary to carry out
the oxidation, or that neither bacterium alone could o x i d i z e py-
_2
r i t e a l l the way to S 0 ^ because we did not assay for i n t e r ­
mediate s u l f u r compounds. There i s no α priori reason to
a t t r i b u t e p y r i t e oxidation e x c l u s i v e l y to e i t h e r a d i r e c t or i n ­
d i r e c t mechanism. Undoubtedly both occur in the c o n t i n u a l l y
changing coal s l u r r y environment and several d i f f e r e n t species
and s t r a i n s contribute to p y r i t e o x i d a t i o n . I t i s our view that
some organisms aid the iron and s u l f u r o x i d i z i n g autotrophs by
removing otherwise autotoxic by-products of metabolism. Although
.2
there was l i t t l e SO. released by T. ferrooxidans, the c e l l s
remained a c t i v e F e + 2
o x i d i z e r s and SO^" a n a l y s i s may not account
f o r a c t i v i t y of t h i s organism in t h i s system - a point which
would be c o n s i s t e n t with the observation of Capes,et a l . ( 1 3 )
although they were not using pure c u l t u r e s of T. ferrooxidans.
_2
I t can a l s o be concluded that greater rates of S O 4 release
246 Patrick R. Dugan and William A. Apel

as well as reduction of lag periods are seen when i n i t i a l pH i s


adjusted to the range 2.5 to 2.0. Addition of ( N H ^ S O ^ as a
supplemental nutrient resulted in greater oxidation of p y r i t e
from the coal samples.

Although the data confirms previous reports of more rapid


pyrite oxidation as p a r t i c l e s i z e i s reduced; the smaller par-
t i c u l a t e f r a c t i o n s a l s o contain a higher percentage of s u l f u r
-2
and would be expected to release more total S 0 4 , necessitating
a d i s t i n c t i o n between rate and amount released.
I t can be concluded that microorganisms e f f e c t i v e l y remove
p y r i t i c s u l f u r from coal via oxidation and s o l u b i l i z a t i o n in l a b -
oratory scale experiments thereby demonstrating the potential for
commercial scale operations. The organic s u l f u r content of coal
was not s o l u b i l i z e d in these experiments and e f f o r t s are now
being directed toward the p o s s i b i l i t y of microbial removal of
the organic s u l f u r f r a c t i o n s of coal and to examine the e f f e c t of
other v a r i a b l e s on coal d e s u l f u r i z a t i o n .

IV. ACKNOWLEDGMENT

This work was supported in part by The Ohio State U n i v e r s i t y


Development Fund.

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Ill BIOEXTRACTIVE APPLICATIONS AND OPTIMIZATION

The fact that the majority of the papers composing this volume are contained in
this section is somewhat consistent with the relationship between the theoretical or
basic research areas and the applications. In a practical sense, this is of course a
healthy feature.
This section begins with a treatment of microfloral observations in an industrial
dump leaching operation and is clearly a bridge between the basic research areas in
Section I and the more applied areas which follow. The mechanism of bacterial
leaching applied to industrial dumps and conditions for optimizing the conditions
for leaching are presented. Many of the chapters of this section deal with specific
applications of bacterial leaching, the optimization of the leaching conditions, and
process optimization based upon economic considerations. Many of the test results
reported relate to laboratory and small pilot-plant experiments. However, large-
scale data is presented beginning with the very first chapter of this section and
especially in the concluding chapter. The final chapter also makes an attempt at
pointing out the advantages of large-scale testing compared with laboratory test-
ing.
The objectives of this book and the symposium upon which it was based, are
strongly amplified in the chapters composing this section. The applications of
fundamental concepts and basic research as outlined in the chapters composing
Section I are very clearly exemplified in this section, and the synergistic aspects of
metallurgy and microbiology in the extraction of metal values by leaching are
certainly apparent throughout.
OBSERVATIONS ON THE MICROFLORA IN AN INDUSTRIAL COPPER

DUMP LEACHING OPERATION

Stoyan N. Groudev,
Fratio N. Genchev
and
Stefan S. Gaidarjiev

Higher I n s t i t u t e of Mining and Geology


S o f i a - Darvenitza, Bulgaria

The paper presents many years of observations on the micro-


flora in a large dump in Vlaikov vrah, Bulgaria, containing about
twenty million tons of low-grade copper sulfide and oxide ores.
The observations cover two periods: from 1968 to 1972, when the
dump was an area of natural leaching processes, and since 1972,
when industry began using it for recovering copper by means of
bacterial leaching. The microflora of the dump is quite varied
and the paper gives the specific part played in the leaching pro-
cesses by some of its main representatives. Cases of microbial
mutualism in leaching, as well as cases of inhibition of the
ferrobacill'i by some other bacteria, moulds and protozoa are des-
cribed. The ferrobacilli in the dump are a very mixed population
and some rather special wild strains have been isolated: thermo-
philic, facultative autotrophic and strains, possessing systems
for substrate phosphorylation during the oxidation of sulfides and
sulfur, and capable of growth at low rHg. The microflora of each
separate component of the complex production process is described:
in the dump itself, the circulating solutions, the regeneration
pond and the cementation unit. The paper presents the results of
the attempts to stimulate the useful microflora by a suitable
change of some environmental factors and by adding surface active

253
254 Stoyan Ν. Groudev et al.

agents. In 1974 and in 1975, a laboratory selected mutant strain


having a high level of oxidizing activity and a well-distinguish­
able marker, was introduced into part of the dump after being
grown in fermentors, built next to the dump. The marker is a
mutation, caused by a post-replication repair mechanism following
UV-irradiation, and consists in the possession by the strain of a
specific direct metabolic pathway from S%~ and S° to sulphate via
polythionates. The fate of this selected strain and its influence
on the leaching are shown. The possibility for and the usefulness
of introducing laboratory selected strains into leaching opera­
tions are discussed.

I- INTRODUCTION
The ore p i l e s in the proximity of the Vlaikov vrah mine,
B u l g a r i a , contain about twenty m i l l i o n tons of waste low-grade
copper s u l f i d e and oxide o r e s , with an average copper content of
about 0.05%. Some parts of the dumps have a higher copper content-
about 0.15%. The p r i n c i p a l copper-containing mineral i s chalco­
p y r i t e , followed by c o v e l l i t e and c h a l c o c i t e . Pyrite is well-
represented, too and quartz and f e l d s p a r s are the basic minerals
of the host rock.

The observations conducted in that area during 1968 showed


that a f t e r r a i n f a l l , acid drainage waters with a high content of
copper and iron ions (over 0.5 and 2 g/£, r e s p e c t i v e l y ) and of
bacteria from the species Thiobacillus ferrooxidans ^ηά T. thiooxi­
dans flow out of the dumps. That f i n d i n g served as an argument
f o r the construction of an i n d u s t r i a l plant f o r bacterial leaching,
which was commissioned in 1972.

Since 1968 there have been regular checks of the microflora


in the dumps. After 1972 these checks a l s o covered the separate
stages of the i n d u s t r i a l plant and many attempts were made to
stimulate the bacterial leaching processes. Some of the more
i n t e r e s t i n g r e s u l t s and conclusions from these experiments are
shown in the present paper.
Bioextractive Applications and Optimization 255

II. PROCEDURES, RESULTS, AND DISCUSSION

The i s o l a t e d microorganisms were i d e n t i f i e d by means of


the guides o f Bergey, Gilman and Lodder ( 1 , 2 , 3 ) . The f e r r o b a c i l l i
in the s o l u t i o n were estimated by d i r e c t counting under a phase-
contrast microscope, using a bacterial counting chamber, and by
the most probable number method (MPN); the c e l l s , attached to the
s o l i d surface were estimated by means of determining bacterial
nitrogen. The number of T. thiooxidans was found by using s o l u -
t i o n p l a t i n g on the Waksman s o l i d thiosulphate medium.

Regardless of the s e l e c t i v e ecological c o n d i t i o n s , the


microflora of the i n d u s t r i a l plant i s characterized by considerable
v a r i e t y of i t s species of the [T. ferrooxidans, T. thiooxidans,
T. thioparus and T. denitrifioans), various sulphur bacteria
( c o l o u r l e s s , green and p u r p l e ) , iron bacteria [Gallionella and
Leptothrix), sulphate-reducing and other heterotrophic bacteria
[Desulfovibrio, Bacillus, Enterobacter (Aerobactor) Pseudomonas, 9

Caulobacter e t c . ) , moulds and y e a s t s [Cladosporium, Penicillium,


Trichosporon, Rhodotorula e t c . ) , microscopic algae [Ulothrix) and
protozoa [Amoeba, Euglena, Eutrepia) were found. The green algae
Hormidium fluitans, whose role in bacterial leaching was recently
shown by Madgwick and Ralph ( 4 ) , grow l u x u r i a n t l y in the acid
dump e f f l u e n t s .

A f t e r the beginning of the i n d u s t r i a l recovering of copper


the composition of the dump microflora suffered only n e g l i g i b l e
changes, the most important of which was the drop in the amount
of heterotrophic microorganisms.

The f e r r o b a c i l l i in the dumps are a very mixed population,


but some more i n t e r e s t i n g i s o l a t e s deserve special mention.

Thermophilic f e r r o b a c i l l i , capable of growth and oxidation of


ferrous iron and sulphur at 60°C, were i s o l a t e d from some r i c h - i n -
pyrite parts of the dump. The temperature in these parts exceeds
sometimes 80°C, a f i n d i n g noted a l s o by Beck (5) for dumps in the
256 Stoyan Ν. Groudev ef al.

United S t a t e s . Thermophils from the genus Sulfolobus (6) and the


Sulfolobus-Mke microbe (7) have not been i s o l a t e d . The thermo­
philic f e r r o b a c i l l i leached p y r i t e f a s t e r than the mesophilic
under laboratory c o n d i t i o n s . Unfortunately, t h e i r a c t i v i t y on
c h a l c o p y r i t e , even a f t e r a preliminary adaption, was not higher
(leaching r a t e s , comparable with these, noted by B r i e r l e y and
Murr (8) with the Sulfolobus-Mke microbe, were observed).

A considerable part (about 40%) of the i s o l a t e d ferrobacilli


were f a c u l t a t i v e autotrophs and were capable of using various
organic compounds as a source of energy. The f a c u l t a t i v e auto­
trophic nature of these i s o l a t e s was e s t a b l i s h e d beyond doubt
by means of the appropriate genetic t e s t s . On the other hand,
the heterotrophic companion of the T. ferrooxidans (9) was found
in the dump, too. The f a c u l t a t i v e autotrophic f e r r o b a c i l l i dis-
s i m i l a t e d the glucose by the Entner-Doudoroff pathway, and, in
p a r t , by the pentose-phosphaste pathway, and t h i s corroborates
the f i n d i n g s of Tabita and Lundgren (10) concerning the hetero­
trophic metabolism of the T. ferrooxidans. An i n t e r e s t i n g f a c t
must be mentioned: no c o r r e l a t i o n was found to e x i s t between the
number of the f e r r o b a c i l l i and the quantity of organic matter in
the acid dump e f f l u e n t s . The f a c u l t a t i v e autotrophic ferrobacilli
were i s o l a t e d mostly from the dump sections with a lower rH^. It
seems that under normal conditions the organic matter in the acid
drainage waters i s used e x c l u s i v e l y by the heterotrophic micro­
organisms, belonging to other taxonomic genera.

F e r r o b a c i l l i incapable of s u l p h u r - o x i d i z i n g a c t i v i t y were
isolated. They are very s i m i l a r to the species Ferrobacillus
ferrooxidans, described by Leathen»et a l . (11). Under laboratory
conditions we have so f a r f a i l e d to induce s u l p h u r - o x i d i z i n g
a c t i v i t y in these i s o l a t e s . The DNA base composition of these
i s o l a t e s was 57.3 + 0.09 % GC, which i s very close to that found
by Guay,et a l . (12) f o r T. ferrooxidans, grown on i r o n . It is
i n t e r e s t i n g to note that the i s o l a t e s , which possess a sulphur-
Bioextractive Applications and Optimization 257

o x i d i z i n g a c t i v i t y and which o x i d i z e most a c t i v e l y the chalcopy-


rite, had a % GC always over 58 - a f i n d i n g , which coincides
a l s o with the data obtained by the above-mentioned authors. These
r e s u l t s indicate that the a b i l i t y of the f e r r o b a c i l l i to adapt to
various substrates can be observed a l s o under natural conditions.
I t i s d i f f i c u l t to say what i s the genetic nature of that adapta-
tion.

Some f e r r o b a c i l l i , possessing systems for substrate phosphory-


l a t i o n during the oxidation of sulphides and sulphur have been
i s o l a t e d from the depth of the dump (about 30 m below the s u r f a c e ) .
These s t r a i n s grow well at rW^ < 20 and r e t a i n t h e i r viability
even at rW^ = 14-16. Their molar economic c o e f f i c i e n t (Y ) was
always greater than that of the s t r a i n s possessing systems only
for o x i d a t i v e phosphorylation. There are reasons to accept that
f e r r o b a c i l l i with substrate phosphorylation use f e r r i c iron as
e l e c t r o n i c acceptor during the oxidation of sulphur compounds,
i . e . act in accordance with the mechanism, suggested by Brock and
Gustafson ( 1 3 ) .

Among the various i s o l a t e s , the " t y p i c a l " s t r a i n from Vlaikov


vrah dump i s characterized by the data, shown in Table I .

Two terminal types of i s o l a t e s can be d i f f e r e n t i a l : one,


which includes i s o l a t e s , p o s s e s s i n g a pronounced ferrous iron-
o x i d i z i n g a c t i v i t y and a n e g l i g i b l e ( a r i s i n g only a f t e r induction)
s u l p h u r - o x i d i z i n g a c t i v i t y , and the other, i n c l u d i n g isolates
with pronounced s u l p h u r - o x i d i z i n g and moderate ferrous i r o n - o x i d i -
zing a c t i v i t y . The i s o l a t e s from the f i r s t type were found mainly
in the dump s o l u t i o n s , and those from the second type mainly on
the ore surface. The second type i s o l a t e s oxidized the chalcopy-
r i t e much more q u i c k l y .

The d i f f e r e n t points of the i n d u s t r i a l plant had d i f f e r e n t


f e r r o b a c i l l i counts (Table I I ) .
258 Stoyan Ν. Groudev ef al.

TABLE I. Physiological Characterization of T h i o b a c i l l u s


f e r r o o x i d a n s , Isolated from Vlaikov vrah Dump

Substrate
Parameter
Fe +
2

Maximum Qo^W value, \il/mg.h 20 840 680


Minimum doubling time h ^7.5 16
Maximum specific growth rate, h 0.092 0.043
Economic coefficient (Y),
g dry wt/g iron oxidized 0.006 0.112
Molar economic coefficient (Y ) m 3

g dry wt/g atom iron oxidized 0.315 3.650


Power coefficient (Υ&ψρ)»
g dry wt/mole ATP 0.630 ?
Effectiveness of CO2 fixation,
moles C0 /100 moles 0%
2 2.5 22.5
Effectiveness of free energy
utilization, % 22 15
Optimum pH 2.3 2.8
Optimum temperature, C 35 37
Tolerance to heavy metal ions,
inhibitory level, g/i
Cu > 12 > 5
Zn > 15 >10
Ni > 10 > 5
Co > 10 > 3

TABLE II. The Number of Ferrobacilli in Various Points


in the Industrial Plant during the Different Months

Point May July September October

I 9.10 -4.10
3 5
2.10 -6.10
4 6
1.10 -3.10
4 5
2.10 -2.10
4 5

II 7.10^-9.10 9.10^-9.10% 7.10\-1.10 9.10*-1.10*


III Z.10*-5.10* 2.10^-4.10^ 8.101-1.10* 7.10-8.10
IV 6.10'-4.10° 7. W-2.10° 8.10'-2.10° 5.10'-9.IO'
V 8.10-4.10 5.10-5.10 5.10-5.10 6.10-4.10°
I - pregnant solution (dump effluent)
II - barren solution (precipitation plant effluent)
III - solution taken from the collection pond II
IV- solution taken from the regeneration pond
V - ore taken from a dump/a depth of 50 cm/
Number of ferrobacilli: in the liquid samples are indicated
cells/ml; in the solid samples - cells/g.
Bioextractive Applications and Optimization 259

The basic " r e s e r v o i r " of f e r r o b a c i l l i in the plant i s the


leaching material i t s e l f , i . e . the ore in the dumps. Both the
absolute and r e l a t i v e numbers of f e r r o b a c i l l i in the d i f f e r e n t
l e v e l s of the dump are not exactly known, but i t seems that the
greatest number of them are located in the upper part - down to
50 cm from the surface. F e r r o b a c i l l i have been found a l s o farther
down the dump (down to as f a r as 30 m below the s u r f a c e ) , but only
in and around the gaps in the ore mass. Many of the f e r r o b a c i l l i ,
adsorbed on the ore surface were without f l a g e l l a , a f i n d i n g ,
which was noted by Le Roux,et a l . (14) during the i n v e s t i g a t i o n s
on the bacterial oxidation of p y r i t e .

The following f i n d i n g w i l l give an idea about the total


quantity of the f e r r o b a c i l l i , adsorbed on the ore surface and
swimming in the s o l u t i o n s . The leaching s o l u t i o n passes through
the dump in about eleven hours and during that time a l l the f e r -
3+
rous i r o n , combined in i t (3-4 g/l) turns into Fe . This i s a
rate which cannot be reached even under laboratory conditions and
that by using s p e c i a l l y selected s t r a i n s . Regardless of the
f e r r o b a c i l l i count, t h i s high rate i s p o s s i b l e only when the
oxdiation process i s c a r r i e d out in a t h i n f i l m . In f a c t , the
difference between the oxidation rates under laboratory conditions
3+
and in the dump i s much more marked, because the reduction of Fe
takes place during the oxidation of suphides in the dump. On the
other hand, the c a t a l y t i c e f f e c t of cupric ions during the o x i d a -
t i o n of ferrous i r o n , based on the reaction (1)

Fe 2 +
+ Cu 2 +
> Fe 3 +
+ Cu +
[1]

i s not s i g n i f i c a n t .

T. thiooxidans was i s o l a t e d much more r a r e l y and always in a


smaller number than the f e r r o b a c i l l i . I t s maximal quantity in the
leaching s o l u t i o n was 2.10 cells/m£; more frequently i t s number
1 2
varied between 5.10 - 4.10 cells/m&. These bacteria were found
mainly in the dump e f f l u e n t s . I t i s p o s s i b l e that t h e i r growth
260 Stoyan Ν. Groudev e i al.

depends on the elemental sulphur, produced by the chemical and


electrochemical processes. The t h i o b a c i l l i did not oxidize the
sulphide minerals.

Both T. thioparus and T. denitrifioans have been i s o l a t e d


s p o r a d i c a l l y from d i f f e r e n t points in the dump (mainly from s e c ­
t i o n s , where c o v e ! l i t e and chalcocite are the dominating copper
minerals). The s y n e r g i s t i c action of the d i f f e r e n t thiobacilli
during the leaching of sulphide minerals was s u c c e s s f u l l y demon­
strated under laboratory c o n d i t i o n s . That was the c l a s s i c a l case
of such kind of a c t i o n . T. thioparus (or T. denitrifioans) o x i ­
dizes the elemental sulphur (but not the sulphides) in an a l k a l i n e
medium of a pH range of 6 to 9. As the oxidation proceeds and
water-soluble metal sulphates are formed, both the h y d r o l y s i s and
the formation of s u l p h u r i c acid lower the pH. As the pH drops to
4.0 T. ferrooxidans continues the oxidation of minerals. The
i n a b i l i t y of our s t r a i n s of T. thioparus and T. denitrifioans to
o x i d i z e the copper sulphide minerals as well as t h e i r small c e l l
number suggest that they do not play an important r o l e in the
leaching of copper in Vlaikov vrah.

The presence of the heterotrophs and even of T. thiooxidans


i t s e l f had an unfavorable e f f e c t on the p h y s i o l o g i c a l a c t i v i t y of
the f e r r o b a c i l l i . This i s shown in Table I I I , which presents the
r e s u l t s from the leaching of sulphide minerals by means of a pure
culture of autochthonic f e r r o b a c i l l i as well as by means of a
mixed microbial c u l t u r e . In both cases the shake-flask technique
was used.

The harmful action of the other microbial species was due to


t h e i r competitive p a r t i c i p a t i o n in the consumption of oxygen and
some useful i o n s , and to t h e i r p r e c i p i t a t i o n on the mineral sur­
face. In some cases the influence was s p e c i f i c - thus some
protozoa (Amoeba) used the f e r r o b a c i l l i as food and Caulobacter
i n h i b i t e d them by means of organic compounds secreted in the
medium.
Bioextractive Applications and Optimization 261

TABLE III Leaching of Sulphide Minerals by means of a


Pure Culture of Ferrobacilli and a Mixed Microbial Culture

Mineral Pure Culture Mixed culture

copper, leached in 30 days, %


Chalcopyrite 5.9 3.5
Covellite 67.1 52.1
Chalcocite 93.6 77.0

Both cultures contained 10 ferrobacilli per ml each in the


beginning of the experiment; the mixed culture contained also six
other species of microorganisms in quantities and ratios, charac­
teristic of the leaching solution.

In some sections of the dump the number of Amoebae was


p a r t i c u l a r l y high (up to 500 per mi). Like E h r l i c h (15) we have
not been able to subculture the Amoebae in 9K medium or in other
a r t i f i c i a l media f o r f e r r o b a c i l l i , but these protozoa grew very
well in acid drainage waters from the dump. In t h e i r presence
the rate as well as the f i n a l degree of leaching decreased. After
90 days of l e a c h i n g , the number of v i a b l e f e r r o b a c i l l i i n the
experiment in the presence of Amoebae was only 5.10 cells/m£ in
ο
comparison with 10 cells/mil i n the absence of Amoebae.
The sulphate - reducing bacteria were most numerous in the
deeper l e v e l s of the dump, where the rH^ was low. I t is possible
that the H^S, formed by these b a c t e r i a , i s used by T. thioparus
as a source of energy. I t i s not known with c e r t a i n t y whether
the sulphate-reducing bacteria are capable of reducing sulphates
at a low pH ( 1 6 ) .

The quantity of the microbial s p e c i e s , concomitant to T.


ferrooxidans (expressed as dry weight of t h e i r biomass), depends
on the ecological parameters - mainly on the pH and on the con­
centration of copper ions in the s o l u t i o n . Under c e r t a i n special
262 Stoyan Ν. Groudev ef al.

conditions (pH over 3.5-4.0 and copper concentration below 1


g/£) the percentage of these microorganisms was close to 30% of
the total amount of the biomass (Table I V ) .

The experiments to i n h i b i t the harmful microflora by lower­


ing the pH were conducted under laboratory c o n d i t i o n s . I t was
found that the change in the pH from a previous 3.2 to 2.4
resulted in increasing the percentage of the f e r r o b a c i l l i from
76% to 93%, reaching 97% at pH 2 . 1 .

S i m i l a r experiments have been c a r r i e d out and in the i n d u s ­


t r i a l plant i t s e l f . The lowering of pH in an experimental section
of the dump by adding sulphuric acid in the leaching s o l u t i o n
resulted in increasing the f e r r o b a c i l l i number as well as the
rate of copper leaching (Table V ) . On the other hand, the a d d i ­
tion of ammonium and phosphate ions as well as of surface active
agents, including Tween 20, had no p o s i t i v e e f f e c t . The greater
part of these ions were adsorbed f i r m l y on the ore surface and
t h e i r concentration in the c i r c u l a t i n g s o l u t i o n was much smaller
than expected. When the leaching was c a r r i e d out mainly by
s t r a i n s , possessing strong d i r e c t mechanism, i . e . adsorbing

TABLE IV Percentage of the Ferrobacilli in the Total


Dvy Weight of Biomass

Analyzed point ferrobacilli, % of


the total biomass

Pregnant solution, pH 2.4 and 1.4 g/i Cu 94


Pregnant solution, pH 2.4 and 1.2 g/% Cu 91
Pregnant solution, pH 2.8 and 1.4 g/i Cu 83
Pregnant solution, pH 3.1 and 1.4 g/ϋ Cu 76
Solution taken from the cementators, pH 3.6 80
Solution taken from the cementators, pH 3.9 74
Solution taken from the cementators, pH 4.3 71
Collection pond II effluent, pH 4.0 74
Regeneration pond effluent, pH 2.5 85
feed to dump leaching solution, pH 3.6 87
Bioextractive Applications and Optimization 263

TABLE V The Effect of pH of Leaching Solution on the Copper


and Ferrobacilli Content in the Pregnant Solution

Period and
Sampling No. Leachinq Solution Preqnant Solution
pH T.f. pH Cu T.f.

1 4.45 2.10 5
2.68 1.05 4.10 9

I 2 4.37 5.10 5
2. 77 1.12 6.10 s

3 4.09 8.105 2.59 1.14 7.105


1 3.27 2.106 2.44 1.21 4.10 6

II 2 3.09 4.10 6
2.37 1.39 7.10 6

3 3.22 4.106 2.33 1.32 6.10 6

1 2.51 5.10$ 2,30 1.49 9.10 6

III 2 2.40 3.10 6


2.26 1.40 1.10 7

3 2.49 6.10 6
2.31 1.52 2.10 7

1 4.55 6.10 6
2.49 1.35 8.10 6

2 4.40 1.10 6
2.57 1.18 2.10 6

3 4.43 8.105
2.60 1.01 9.10 s

Duration of each period was 30 days. The samplings were


carried out at 10, 20 and 30th day, respectively. The copper
concentration is shown in g/%, and T. ferrooxidans count - in
cells/ml.

themselves in great number on the mineral s u r f a c e , the a d d i t i o n of


phosphate (over 10 mg/β P0^ " in the leaching s o l u t i o n ) was found
to be p a r t i c u l a r l y harmful to the speed of the process. These
data are v a l i d only f o r the copper sulphide ore from Vlaikov vrah.
The phosphate requirement f o r maximum rate and extraction during
the bacterial leaching of other substrates i s quite d i f f e r e n t ( 1 7 ) .
2+ 3+
The form (Fe or Fe ) under which the iron was present
in the leaching s o l u t i o n caused l i t t l e e f f e c t on the f e r r o b a ­
c i l l i number and rate of leaching. I t i s known that the r a t i o of
these two ions determines the Eh of s o l u t i o n [but see a l s o Bhappu,
et a l . (18)] and, in p r i n c i p l e , has a s i g n i f i c a n t influence on
the leaching. I t seems, however, that the a c t i v i t y of microflora
i s a more important f a c t o r in a leaching p r o c e s s , which takes place
in a big dump, containing mainly CuFeS2-
264 Stoyan Ν. Groudev ef al.

The c h a r a c t e r i s t i c peculiarities of the microflora of each


separate stage of the complex production process must be noted.
The dump e f f l u e n t s were characterized by r e l a t i v e l y uniform com­
p o s i t i o n of t h e i r m i c r o f l o r a . I t was due to t h e i r low pH ( u s u a l l y
below 2.5) and a high content of heavy metal i o n s . The hetero­
t o p i c microorganisms were represented only in small numbers. On
the other hand, the f e r r o b a c i l l i count i s not high in comparison
with that observed in other smaller dumps in Bulgaria and abroad.
There are various reasons f o r that,but mainly i t may be the dump
size i t s e l f . The s i z e determines to a great degree, the a v a i l a ­
b i l i t y of oxygen and carbon dioxide i n the d i f f e r e n t parts of the
dump.

A f t e r many years of e f f o r t , i t seems doubtful that the


f e r r o b a c i l l i number in a l l dump e f f l u e n t s can be increased. The
addition of s u l p h u r i c acid was e f f i c a c i o u s , but only in small
dump r e g i o n s , in which the secondary sulphides are well represented
and where the acid-consuming processes have been accomplished. It
i s a l s o doubtful that the increasing of the swimming f e r r o b a c i l l i
w i l l indicate an increasing of total f e r r o b a c i l l i number and the
rate of leaching. There are i n d i c a t i o n s that an e q u i l i b r i u m
e x i s t s between the adsorbed and swimming bacteria in the leached
systems. In various systems that e q u i l i b r i u m i s achieved at
d i f f e r e n t r a t i o s of these two groups. The i n c r e a s i n g of swimming
bacteria (which are ferrous iron o x i d i z e r s ) may cause a decreasing
of the a c t i v i t y of adsorbed b a c t e r i a , many of which d i r e c t l y
3+
o x i d i z e the s u l p h i d e s . In such cases the Fe ( f o r instance
CuFeS^) would be diminished. On the other hand, i t i s not c e r t a i n
that an increase of the number of adsorbed bacteria would r e s u l t
in an increase in the number of swimming f e r r o b a c i l l i . Unfortuna­
t e l y , only the numbers of swimming bacteria are e a s i l y c o n t r o l l e d .
In the p r e c i p i t a t i o n plant the f e r r o b a c i l l i count suffered
i n t e r e s t i n g changes. In the f r o n t cementation chambers i t de­
creased (by up to twenty f o l d in separate c a s e s ) , which was due
Bioextractive Applications and Optimization 265

to the increasing of pH and formation of hydrogen i n the


system:

H S0
2 4 + Fe > FeS0 4 + H 2 [2]

In the back chambers, however, the f e r r o b a c i l l i count i n -


creased (up to 9 . 1 0 4
cells/mil), which was connected with the r e l a -
t i v e l y long holding time of the sheet iron i n the cementation
chambers and with smaller formation of H^. The p r e c i p i t a t i o n
plant e f f l u e n t s were r i c h in heterotrophic microorganisms.
Among the l a t t e r , the bacteria and moulds dominated in numbers,
and y e a s t s and protozoa were found only s p o r a d i c a l l y . Sometimes
many microscopic algae were i s o l a t e d . The heterotrophic micro-
f l o r a , concomitant to T. ferrooxidans, exhibited a c e r t a i n
s t a b i l i t y in the time.

The microflora of the c o l l e c t i o n pond I I (the l a t t e r c o l l e c t s


the p r e c i p i t a t i o n plant e f f l u e n t s ; on the other hand, the c o l l e c -
t i o n pond I c o l l e c t s the dump e f f l u e n t s ) was very varied,up to
14 d i f f e r e n t species of microorganisms were i s o l a t e d from some
samples. The main reason f o r that was the r e l a t i v e l y high pH
(about 3.5-4.0) of the s o l u t i o n s .

The composition of m i c r o f l o r a became again much more uniform


and the f e r r o b a c i l l i count-higher i n the regeneration pond, where
favorable conditions f o r ferrous i r o n oxidation e x i s t e d . It is
necessary to note that only a part of the p r e c i p i t a t i o n plant
e f f l u e n t s pass through the regeneration pond. In the Vlaikov
vrah, the s i g n i f i c a n c e of the regeneration stage c o n s i s t s not i n
3+
the production of Fe , but in the p r e c i p i t a t i o n of iron s a l t s out
of dumps. In p a r t , such role i s played a l s o by the c o l l e c t i o n
ponds because of r e l a t i v e l y long holding time of the s o l u t i o n s
here. That holding time i s much shorter during the winter months
to avoid the cooling of s o l u t i o n s .
266 Stoyan Ν. Groudev et al.

I t i s obvious that the problem of maintenance of a s u i t a b l e


microflora in the leached material becomes of increasing impor­
tance f o r i n d u s t r i a l leaching operations. For that purpose, a
laboratory selected s t r a i n , possessing a high leaching a c t i v i t y
versus Vlaikov vrah ore has been introduced in a small part of the
dump. That part contained about 100,000 tons of ore and was p a r t i a l l y
separated from the main ore mass by elevations of l a y . The s t r a i n
(marked here as Pmv) has been obtained by U V - i r r a d i a t i o n of
another selected strain-Pm. The l a t t e r has been obtained as a
r e s u l t of gradual s e l e c t i o n of s t r a i n P, i s o l a t e d in 1968 from the
same place, where the introduction of s t r a i n Pmv has been made
subsequently. The gradual s e l e c t i o n was c a r r i e d out by consecutive
p l a t i n g s of f e r r o b a c i l l i on the 9K s i l i c a gel medium or on the
Manning's s o l i d medium (19) (each of the media has been pre-
2+
pared with 3 g/i Fe ) and by t e s t i n g of the a c t i v i t y of s i n g l e
c o l o n i e s , bred on the s o l i d surface. Everytime, the most active
colonies were subcultured.
Both w i l d s t r a i n Ρ and s t r a i n Pm oxidized tetrathionate by
the metabolic pathway, suggested by Tuovinen and K e l l y ( 2 0 ) . The
oxidation via that pathway i s described by the equation:
2K S 0
2 4 6 + 70 2 + 6H 0 = 2 K S 0
2 2 4 + 6H S0
2 4 [3]

i . e . each of the four sulphur atoms of tetrathionate i s oxidized to


sulphate.

The s t r a i n Pmv oxidized tetrathionate with formation of t r i -


thionate [ l i k e the s t r a i n of Sinha and Walden ( 2 1 ) ] , but had a
unique a b i l i t y to o x i d i z e the internal sulphur atom of t r i t h i o n a t e
to sulphate. Differences between the s t r a i n s have been demonstra­
ted c l e a r l y by using of sulphur compounds, containing l a b e l l e d
or
sulphur atoms ( S ) .
Bioextractive Applications and Optimization 267

pathway, suggested by Tuovinen and K e l l y (20) ( t r i t h i o n a t e has not


been formed)

2 S
OD
- S0 3 > 0 S -
3 S - s - S - so
x x x
3
2

I • so "
x
4
2

( p o s s i b l e mechanism: +2e, as i t is
proposed by Imai*et a l . (22) f o r
T. thiooxidans)

s - so " — > so ~ + s - so "


x
3
2
3
2 x
3
2

^ 2-
so/
pathway, used by s t r a i n Pmv pathway, suggested by Sinha
and Walden ( 2 1 ) .

The s p e c i f i c pathway, possessed by s t r a i n Pmv, i s a " b i g "


mutation, caused by a p o s t - r e p l i c a t i o n r e p a i r mechanism f o l l o w i n g
UV-irradiation. That metabolic change has been a stable charac-
t e r i s i t i c of the s t r a i n and the p r o b a b i l i t y f o r back mutation
has been i n s i g n i f i c a n t .

This p a r t i c u l a r feature of the Pmv s t r a i n was used as a


marker f o r i t s d i s t i n g u i s h i n g from the wild s t r a i n s of the Vlaikov
vrah. These w i l d s t r a i n s o x i d i z e tetrathionate via the pathway,
suggested by Tuovinen and K e l l y . A simple t e s t f o r estimation of
the c e l l number of both selected and autochthonic wild s t r a i n s in
mixed population was worked out on the b a s i s of the above-mentioned
268 Stoyan Ν. Groudev e i al.

marker. The test was the f o l l o w i n g . Aliquote samples from a


mixed population ( i . e . from the leaching or pregnant or barren
s o l u t i o n s ) were inoculated in Erlenmeyer f l a s k s , containing i r o n -
χ 2-
free 9K medium and a l a b e l l e d t r i t h i o n a t e (O^S - S - SO^ ) as a
sole source of energy. A f t e r a short c u l t i v a t i o n the culture was
analyzed by ion-exchange chromatography method (23-26) f o r detect­
ing various sulphur compounds. The immediate advent of l a b e l l e d
sulphate showed the presence of s t r a i n Pmv. The c e l l number of
the l a t t e r was calculated from the quantity of sulphate by
means of standard curve.
For introduction of the s t r a i n s i x concrete fermentors were
constructed near to the dump. Every one of them had a system f o r
3
aeration. The volume of a s i n g l e fermentor was 1.5 m . Barren
s o l u t i o n has been used as a n u t r i e n t medium. Ammonium and phos­
phate ions have been added to the s o l u t i o n . Sometimes, ferrous
sulphate was added to increase the substrate concentration. Pure
ο

culture of the selected s t r a i n Pmv, containing about 4.10 cells/


mil has been used as an inoculum. A f t e r the ferrous iron exhaus­
tion, the l a t t e r was pumped to a d e f i n i t e part of the experimen­
tal dump r e g i o n . Another part of the dump (possessing the same
volume and s i z e ) served as a c o n t r o l .
In 1974 and i n 1975 the introduction was repeated 18 times.
Content of copper and iron ions as well as the f e r r o b a c i l l i number
in the experimental and control dump e f f l u e n t s were checked r e g u ­
larly. Mineralogical c h a r a c t e r i z a t i o n of leaching material was
a l s o c a r r i e d out p e r i o d i c a l l y .
We are able to present here only the most p r i n c i p a l results
and conclusions of our experiments.
The introduction i t s e l f of the selected s t r a i n Pmv was s u c ­
cessful. In March 1976 about 55% of the f e r r o b a c i l l i , i s o l a t e d
from the experimental dump, had the s p e c i f i c p e c u l a r i t y of s t r a i n
Pmv. I t does not mean that a l l of them were decended from that
Bioextractiv
eApplication
san dOptimizatio
n26 9

selected s t r a i n . D i f f e r e n t ways of t r a n s f e r of genetic material


between the selected and wild s t r a i n s are p o s s i b l e .

Pmv-like f e r r o b a c i l l i were only about 4 - 5 % of the total


amount of these bacteria in the control dump (there was some con-
nection between the experimental and control dumps by soaking up
of s o l u t i o n s ) . In the main dump the selected f e r r o b a c i l l i were
found s p o r a d i c a l l y and only in regions near to the experimental
dump. I t i s important to note that only a part of the Pmv-like
i s o l a t e s had been retained the s t r i k i n g high a c t i v i t y of t h e i r
laboratory-bred r e l a t i v e s . That f i n d i n g i s an i n d i c a t i o n that
the high oxidative a c t i v i t y of s t r a i n Pmv i s not connected with
i t s s p e c i f i c pathway for oxidation of sulphur compounds.

Leaching k i n e t i c s in the experimental dump did not increase


in comparison with those of the control dump. Total ferrobacilli
count (selected and wild) a l s o did not increase. The l a s t f i n d i n g
was v a l i d f o r c e l l s swimming in c i r c u l a t i n g s o l u t i o n s as well as
for c e l l s adsorbed o n t h e o r e (rock) surface.

III. CONCLUSIONS

The above-mentioned experiment was preceded by analogous


experiments, conducted under laboratory and s e m i - i n d u s t r i a l con-
ditions. In some experiments other laboratory selected s t r a i n s
were used besides the s t r a i n Pmv. These s t r a i n s had s p e c i f i c
markers, which were not very stable (for example, d i f f e r e n t t o l e -
rance to heavy metal i o n s , organic compounds, a n t i b i o t i c s , tem-
perature, etc.) . Introductions were c a r r i e d out into o r e s ,
leached by d i f f e r e n t methods: by s h a k e - f l a s k technique, in
reactors with mechanical s t i r r i n g , and in percolation columns. The
more important conclusions from these experiments e s p e c i a l l y for
leaching of copper sulphide and mixed o r e s , are the f o l l o w i n g :
1. The r e s u l t s from i n t r o d u c t i o n are impossible to be
predicted in advance.
270 Stoyan Ν. Groudev e i al.

2. Introduction i s useful only for leaching of some o r e s ,


containing mainly chalcopyrite ( i . e . s u l f i d e , which i s very
stable versus o x i d a t i v e a g e n t s ) . No p o s i t i v e r e s u l t s were ob­
tained by s t r a i n introduction in o r e s , whose copper was repre­
sented mostly as secondary sulphides (CUoS, CuS).

3. Introduction i s successful only when selected s t r a i n s ,


possessing a pronounced d i r e c t o x i d a t i v e mechanism for sulphide
m i n e r a l s , are used. These s t r a i n s are capable of s e l e c t i v e
adsorption on the mineral surface and possess a high s u l p h u r - o x i ­
dizing activity. On the other hand, the selected s t r a i n s must
2+
oxidize Fe f a s t e r than the autochthonic wild s t r a i n s . That
a b i l i t y presents the p o s s i b i l i t y f o r barren s o l u t i o n s to be used
as a n u t r i e n t medium during the c u l t i v a t i o n in fermentors. When
the introduction i s s u c c e s s f u l l y advanced in some dump r e g i o n , the
l a t t e r can be used e f f e c t i v e l y as a fermentor.
I t has been found that the use of s t r a i n s i s very e f f i c a c i o u s
and they act probably by the mechanism suggested by Dugan and
Randies (27). That mechanism i s p o s s i b l e for sulphide minerals
where a c r y s t a l l a t t i c e having a s p e c i f i c state e x i s t s . This state
can be associated with a "reverse e x c i t o n " · I t i s known that the
exciton i s an excited state of a system of e l e c t r o n s , and that the
exciton i s not connected with the t r a n s f e r of e l e c t r i c charge. A
t r a n s f e r of an electron from the cation to the next anion i s c a r r i e d
out in the c r y s t a l l a t t i c e during the process of formation of an
exciton. At the "reverse e x c i t o n " the t r a n s f e r of an electron i s
c a r r i e d out from an anion to the next c a t i o n . That phenomena
gives the p o s s i b i l i t y for the f e r r o b a c i l l i , adsorbed on the
mineral s u r f a c e , to oxidize mainly the ferrous atoms of the
mineral. In the structure of c h a l c o p y r i t e , these atoms are more
available. The r e s u l t i n g f e r r i c atoms accept electrons from the
next cations (S ) and serve as intermediates in the e l e c t r o n i c
transport between the mineral and the bacterial c e l l . As the
ferrous i r o n - o x i d i z i n g enzyme of T. ferrooxidans acts by the s o -
Bioextractive Applications and Optimization 271

c a l l e d Ping Pong Bi Bi mechanism ( 2 8 ) , the oxidation w i l l be most


2- 3+
rapid when the t r a n s f e r s of electrons from S to Fe in the
c r y s t a l l a t t i c e , on the one hand, and from the bound iron in
2+
protein moiety of Fe -cytochrome c^ reductase to cytochrome £
in the bacterial c e l l , on the other hand, are in resonance. It
i s very d i f f i c u l t to e s t a b l i s h by experimental means that such a
state e x i s t s . In p r a c t i c e , only the actual (observed) rate of
leaching of a sulphide mineral can be accepted as a c r i t e r i o n f o r
e f f i c a c y of a c e r t a i n s t r a i n . The high ferrous iron oxidation rate
i t s e l f does not indicate that the s t r a i n w i l l a c t i v e l y oxidize
the c h a l c o p y r i t e .
Estimation of rhodanese a c t i v i t y can be used as a simple,
but quite sure t e s t i f s t r a i n s must be picked out to serve as an
i n i t i a l material of gradual s e l e c t i o n , whose aim i s the obtaining
of selected s t r a i n s , p o s s e s s i n g a high o x i d a t i v e a c t i v i t y versus
CuFeS . 2 A high rhodanese a c t i v i t y i s a very important f a c t o r , espe-
c i a l l y in dump l e a c h i n g , where the lower sulphur and s u l p h i t e -
o x i d i z i n g a c t i v i t i e s of f e r r o b a c i l l i permit a non-enzymatic forma-
t i o n of thiosulphate in the c e l l envelope. I t i s known, on the
one hand, that thiosulphate i n h i b i t e s the b i o l o g i c a l oxidation of
ferrous iron and sulphide minerals ( 2 9 ) , and, on the other hand,
that the s e n s i s i t i v y of f e r r o b a c i l l i versus heavy metal ions
increases during the oxidation of thiosulphate ( 3 0 ) .

4. The most successful introduction of selected biomass i s


c a r r i e d out by i t s preliminary d i l u t i o n in the leaching s o l u t i o n
5 6
to about 10 - 10 cells/mil (the most s u i t a b l e leaching s o l u t i o n
f o r i s a leaching s o l u t i o n with low i r o n content - below 1 g/&).
Introduction of that s o l u t i o n into the ore mass must be conducted
between two long pauses in dump i r r i g a t i o n .
5. Use of laboratory selected s t r a i n s i s very e f f i c a c i o u s
f o r leaching of f l o t a t i o n concentrations, which do not contain an
autochthonic f e r r o b a c i l l i population. Leaching of such concentra-
tes i s well i n v e s t i g a t e d (31-37) and the use of selected s t r a i n s
272 Stoyan Ν. Groudev ef al.

here i s only a question of time. The introduction can be rather


perspective and in some ore s e c t i o n s , intended f o r in-situ l e a c h ­
ing. These are s e c t i o n s , in which the autochthonic ferrobacilli
population i s not numerous or i s even absent, because of unfavor­
able natural conditions (low r ^ , absence of free oxygen e t c ) .

The use of laboratory selected s t r a i n s of f e r r o b a c i l l i for


industrial leaching operations poses some new problems, already
e x i s t i n g in the other microbiological operations (for i n s t a n c e ,
production of a n t i b i o t i c s ) . Some of those problems are: 1 . Devel­
oping of methods of experimental s e l e c t i o n of f e r r o b a c i l l i as well
as methods of express microbiological control in the i n d u s t r i a l
leaching operations; 2. Maintenance of the selected s t r a i n s under
laboratory and i n d u s t r i a l c o n d i t i o n s ; 3. Finding the optimal ways
and conditions for introduction i t s e l f to be c a r r i e d out; 4. Tech­
nological r e a l i z a t i o n of introduction process as well as i t s
i n s e r t i n g in the complex production scheme. These problems can
be s u c c e s s f u l l y solved only by close i n t e r a c t i o n between the
fundamentalists and the p r a c t i t i o n e r s in the area of bacterial
leaching.

IV. REFERENCES

1. Buchanan, R., and Gibbons, N., ( e d s . ) , "Bergey* Manual of


Determinative B a c t e r i o l o g y " , 8th E d i t i o n , The Williams and
Wilkins Co., Baltimore, 1974.
2. Gilman, G . , "A Manual of S o i l F u n g i " , Iowa, 1957.
3. Lodder, J . and Kreger, N., - Van R i j , "The Yeasts - a Taxono-
mic S t u d y " , Interscience P u b l i s h e r s , New York, 1952.
4. Madgwick, J . , and Ralph, B., "Round Table Conference of Leach­
i n g " , Braunschweig, Germany, 1977.
5. Beck, J . , Biotechnol. Bioeng. 9, 487 (1967).
6. Brock, T . , Brock, Κ., B e l l y R., and Weiss, R., Arch. Micro­
biol., 84, 54 (1972).
7. B r i e r l e y , C . L . , and B r i e r l e y , J . , Can. J. Microbiol. 19. 183
(1973).
8. B r i e r l e y , C . L . , and Murr, L.E. Science, 179, 488 (1973).
Bioextractive Applications and Optimization 273

9. Z a v a r z i n , G., Microbiology (Moscow), 4 1 , 369 (1972).


10. T a b i t a , R., and Lundgren, D., J. Bacteriol. 108, 334 (1971).
11. Leathen, W., K i n s e l , Ν., and B r a l e y , S . , J. Bacteriol. 72, 700
(1956).
12. Guay, R., S i l v e r , Μ., and Torma, Α . , Rev. Can. Biol.. 35
61 (1976).
13. Brock, T . , and Gustafson, J . , Appl. Environ. Microbiol. 32,
567 (1976).
14. Le Roux, Ν., North, Α . , and W i l s o n , J . , "Tenth International
Mineral Processing C o n g r e s s " , London 1973.
15. E h r l i c h , H., Bacteriol. J., 8 6 , 350 (1963).
16. T u t t l e , I . , Dugan P., Macmillan, C , and Randies, C , J. Bac­
teriol. 97, 594 (1969).
17. Bruynesteyn, Α . , Annual Meeting of the AIME, Denver, Colorado
1970.
18. Bhappu R., Johnson, P., B r i e r l e y , J . and Reynolds, D., Trans.
ΑΜΕ, 244, 307 (1969).
19. Manning, H., Appl. Microbiol., 30, 1010 (1975).
20. Tuovinen, 0 . , and K e l l y , D., Arch. Microbiol., 98, 351 (1974).
21. S i n h a , D., and Walden, C , Can.J. Microbiol. 12, 1041 (1966).
22. Imai, Κ., Okuxumi, M., and K a t a g i r i , H., Koso Kagaku Shimposi-
umu, 17, 132 (1962).
23. I g u c h i , Α . , Bull. Chem. Soc, Japan, 31 , 597 (1958).
24. I g u c h i , Α . , Bull. Chem. Soc, Japan, 31 , 600 (1958).
25. Trudinger, P., Biochem. J., 78 680 (1961).
26. Trudinger, P., Austral. J. Biol. Sciences, 17 446 (1964).
27. Dugan, P., and Randies, C , i n : "Acid Mine Drainage Forma­
t i o n and Abatement", The Ohio State U n i v e r s i t y Research
Foundation 1971.
28. D i n , G. and S u z u k i , I., Can. J. Biochem., 4 5 , 1547 (1967).
29. R a z z e l l , W., and T r u s s e l l , P., J. Bacterid., 8 5 , 595 (1963).
30. Tuovinen, 0 . , Niemela, S . , and Gyllenberg, H., Antonie van
Leeuwenhoek, 37, 489 (1971).
31. Torma, Α . , Walden, C , and Branion, R., Biotechnol. Bioeng.,
12, 501 (1970).
32. Bruynesteyn, Α . , and Duncan, D., Can. Metallurg. Quart., 10,
57 (1970).
33. ^" c a
^3 D e
- f 2 ^ | f ^ 6 ^ ) * ' T r u s s e l l , P., and Lowe, E., Trans.
a C
274 Stoyan Ν. Groudev ef al.

34. Torma, A . E . , Walden, C , Duncan, D., and Branion, R., Bio­


technol. Bioeng. 14, 777 (1972).
35. Torma A . E . , and Subramanian, Κ., Intr. J. Miner. Proc. 1 ,
125 (1974).
36. Sakaguchi, Η., S i l v e r , Μ., and Torma, A . E . , Biotechnol. Bio­
eng., 18, 1091 (1976).
37. R o s s i , G., Morra, Α . , and S a v o i n i , G . , Personal communication
(1977).
ON THE MECHANISM OF BACTERIAL LEACHING

Kazutami Imai

Faculty of A g r i c u l t u r e
Okayama U n i v e r s i t y
Tsuchima, Okayama-shi, Japan

Manganese dioxide is not soluble in sulfuric acid, but it is


solubilized in the growing culture of T h i o b a c i l l u s thiooxidans.
The leaching mechanism is postulated that manganese dioxide is
firstly reduced with hydrogen sulfide or sulfite which is formed as
an intermediate during the oxidation of sulfur to sulfuric acid
by the bacterium, and then solubilized with sulfuric acid. Intact
cells or cell-free extracts of T. ferrooxidans are able to oxidize
chalcocite in the absence of iron. It suggests the existence of
direct oxidizing mechanism, and the mechanism has been investi-
gated. The rate of covellite-leaching in the growing culture of
T. ferrooxidans is enhanced by a pre-treatment (shaking in water
for 10 days at 30°C) of the covellite. The phenomenon is also
observed in the leaching experiments using cells of the bacterium
suspended in iron-free 9K medium.

I. INTRODUCTION

I n t e r e s t in the action of autotrophic bacteria on inorganic


metal compounds has been increased since the e a r l y 1 9 5 0 ' s . How-
ever, b i o l o g i c a l reactions on inorganic metal compounds have not
been studied i n t e n s e l y in the f i e l d of biochemistry, and the

275
276 Kazutami Imai

mechanisms of the reactions are generally complicated with i n c i ­


dental chemical r e a c t i o n s .

This paper describes the mechanism of b i o l o g i c a l contri­


bution of Thiobaoillus thiooxidans and T. ferrooxidans in the
leaching of manganese dioxide and copper s u l f i d e s , r e s p e c t i v e l y .

II. MATERIALS AND METHODS

A. Microorganisms and Growth Conditions

For the leaching of manganese d i o x i d e , a s u l f u r - o x i d i z i n g


chemoautotrophic bacterium was used. The s t r a i n was i s o l a t e d
from a sewage and i d e n t i f i e d as Thiobaoillus thiooxidans ( 1 ) .
The bacterium was cultured at 30°C under shaking in a shaking
f l a s k containing 100 ml of a basal medium which had the following
composition: K H P 0 , 0.4 g; M g S 0 , 0.03 g; C a C l , 0.025 g;
o 4 4 n

F e S 0 - 7 H 0 , 0.001 g; ( N H ) S 0 , 0.2 g; S u l f u r , 1 g, Water, 100ml;


4 2 4 n 4

pH was adjusted to 2.0.

For the leaching of chalcocite (CuoS) and c o v e l l i t e (CuS),an


i r o n - o x i d i z i n g chemoautotrophic bacterium, Thiobaoillus ferro­
oxidans was used. The s t r a i n (2,3) was i s o l a t e d from acid water
of a mine at Yanahara (Japan) and cultured as in the case of
T. thiooxidans, except the culture medium. The bacterium was
cultured in 9K medium reported by Silverman and Lundgren ( 4 ) .
The composition of 9K medium i s as f o l l o w s : ( N H ^ S O ^ 3.0 g;
KC1, 0.1 g; K H P 0 , 0.5 g; M g S 0 - 7 H 0 , 0.5 g; C a ( N 0 ) , 0.01 g;
2 4 4 2 3 o

d i s t i l l e d water, to 700 ml; 10 Ν H S 0 , 1.0 ml; F e S 0 - 7 H 0 , 300 ml


o 4 4 o

of a 14.74% (w/v) s o l u t i o n .

B. Materials

In the experiments of manganese-leaching, a manganese dioxide


ore (ground to pass through a 100 mesh s i e v e ) or pure manganese
dioxide powder was used. The chemical composition of the ore was
Bioextractive Applications and Optimization 277

as f o l l o w s : (%) M n 0 , 10.6; S i 0 , 55.0; F e ^ , 25.0; MgO, 5.32;


2 o

and trace of Ca, A l , and S.

In the leaching experiments on c h a l c o c i t e , a chemical reagent


o f Cu^S was used. The sample was ground to pass through a 200
mesh s i e v e , treated with 1 Ν HC1 f o r 30 minutes at room tempera­
t u r e , washed throughly with water, d r i e d , and treated with
acetone.

In the leaching of c o v e l l i t e , f i n e powder of a n a l y t i c a l


grade of CuS (99.99% pure, obtained from Mitsuwa Chemicals Co.,
Ltd.) was used.

C. Assay Methods

S u l f a t e formed in culture media was determined by the method


of F r i t z and Fruland ( 5 ) . Ferrous iron was determined colorime-
t r i c a l l y by a modification of the o-phenanthroline method. The
i r o n - or c h a l c o c i t e - o x i d i z i n g a c t i v i t y of c e l l s or c e l l - f r e e
extracts of T. ferrooxidans was determined by oxygen-uptake in a
Warburg manometer.

The s o l u b i l i z e d manganese and copper were determined by


chelate t i t r a t i o n and Atomic Absorption Spectrometry (Model AA-1
of Jarrel A s h ) , r e s p e c t i v e l y . The growth of both bacteria in
culture media was determined counting the number of c e l l s with
Toma s Haematometer.

III. RESULTS AND DISCUSSIONS

A. Leaching of Manganese Dioxide with T. thiooxidans

I t i s well known that four valent manganese, f o r example


manganese d i o x i d e , i s not s o l u b l e in s u l f u r i c a c i d . However, i t
was found in our laboratory (6,7) that manganese i s solubilized
as manganese s u l f a t e from a manganese dioxide ore (see MATERIALS
AND METHODS) by the action of T. thiooxidans.
278 Kazutami Imai

As shown in F i g . 1 , when the culture medium was added with


3% of the ground ore, inoculated with the bacterium, and cultured
under shaking, about 1,300 ppm of manganese was s o l u b i l i z e d as
manganese s u l f a t e in nine days. In t h i s case, a manganese-acclima-
tized bacterium was used as inoculum. The a c c l i m a t i z a t i o n was
proceeded as f o l l o w s : the o r i g i n a l s t r a i n was subcultured three
to four times in 1% ore containing medium, and then s u c c e s s i v e l y
transferred to 1.5 and 3% ore containing media. On the other
hand, manganese was not s o l u b i l i z e d in the medium not inoculated
(Fig. 1).

Then, in order to explore the leaching mechanism of manganese


d i o x i d e , the effect of several additions on the extraction rate of
manganese was examined. In t h i s case, pure Mn0 powder (0.5 g/
2

100 ml medium) was used as the substrate. As shown in Table I,


the addition of metal s u l f i d e s enhanced the leaching rate of

Fig. 1. Extraction of manganese from manganese dioxide ore


in the growing culture of T h i o b a c i l l u s thiooxidans.
ΜηΟ^ content of the ore, 10.6%; Pulp density of the ore, 3.0%.
• · inoculated; Ο Ο uninoculated.
Bioextractive Applications and Optimization 279

TABLE I Effect of Metal Sulfides on the Extraction


of Manganese from MnO in the Growing Culture of T. thiooxidans

Addition Sulfuric acid, Mr? , extracted


+

(1.6 mmoles/100 ml) formed (g/100 ml) (ppm)

FeS 0. 7 1110
CuS 0.3 214
ZnS 0.2 1850
- 0.3 170

ΜηΟ^ (0.5 g/100 ml) and metal sulfides were added on the second
day after inoculation, and sulfuric acid and Mn%+ were determined
in 72 hours after the addition.

manganese d i o x i d e , and FeS gave good e f f e c t on both of the leach­


ing of manganese and bacterial growth. ZnS was e f f e c t i v e f o r the
former, but i n e f f e c t i v e f o r the l a t t e r . On the other hand, CuS
was not so e f f e c t i v e f o r both of them.

The leaching process of manganese in the culture with added


FeS i s shown in F i g . 2. Addition of ferrous s u l f a t e a l s o gave
good e f f e c t on the leaching of manganese, but the e f f e c t of f e r r i c
s u l f a t e was l i t t l e ( F i g . 3 . ) . From such o b s e r v a t i o n s , i t is
suggested that the leaching mechanism of manganese dioxide with
T. thiooxidans i s as f o l l o w s : At the o u t s e t , four valent manga­
nese i s reduced to two valency with some reducing substance (or
substances) which i s formed from s u l f u r as an intermediate during
the oxidation of s u l f u r to s u l f u r i c a c i d . Then, the two valent
manganese i s extracted with s u l f u r i c acid formed from s u l f u r by
the action of the bacterium. The reducing intermediate may be
thought to be hydrogen s u l f i d e or s u l f i t e , because the formation
of hydrogen s u l f i d e in the c u l t u r e s of T. thiooxidans has been
observed (8,9) and the addition of FeS enhances the leaching rate
of manganese (Table I and F i g . 2 ) . And i t has a l s o been e s t a b l i s h e d
by many i n v e s t i g a t o r s that s u l f i t e i s the important intermediate
in the oxidation of s u l f u r by T. thiooxidans ( 1 0 ) .
280 Kazutami Imai

Fig. 2. Effect of FeS on the leaching of manganese dioxide


in the growing culture of T. t h i o o x i d a n s .
Additions (%)
MnO FeS H^SO^
ΟΟ 4
A (inoculated) 0.5 0.1
Β (uninoculated) 0.5 0.1 3.0
C (inoculated) 0.5
Ό (uninoculated) 0.5 - 3.0

Such p o s t u l a t i o n on the leaching mechanism of manganese


dioxide w i l l be supported by the following chemical r e a c t i o n s :
(1) On introducing hydrogen s u l f i d e g a s , manganese dioxide
suspended in d i l u t e s u l f u r i c acid i s s o l u b i l i z e d immediately.
(2) Manganese dioxide suspended in water i s a l s o e a s i l y s o l u b i l i ­
zed upon the introduction of s u l f u r dioxide gas.

Although the chemical equations to reactions (1) and (2)


have not been e s t a b l i s h e d y e t , the following equations may be
inferred.

Mn0 + H S + H S0
2 2 2 4 > MnS0 +S+2H 0
4 2 [1-a]

4Mn0 + H S + 3 H S 0
2 2 2 4 > 4MnS0 + 4H 0
4 2 [1 - b ]
Bioextractive Applications and Optimization 281

1500

•g 1000

500

oi
0 1.0 2.0
CONCENTRATION (MMDES/IOOML)

Fig. 3. Effect of the concentration of additions on the


extraction of manganese in the growing culture of T. thiooxidans.
A) FeS; B) FeSO^; C) Fe2(S0^)Extracted manganese was deter­
mined on the seventh day after inoculation. Pulp density of Mn0 9

was 0.5%. 0

2Mn0 + 4 S 0 + H 0
2 2 2 > Mn (S0 ) + H S0
2 3 3 2 4

Mn (S0 )
2 3 3 > M n S 0 + MnS0
2 6 3 [2-a]

Mn0 + H S 0
o o o > MnSO. + H 0 o [2-b]

Among these equations, [ 1 - b ] and [ 2 - b ] w i l l be preferable to


reactions (1) and ( 2 ) , r e s p e c t i v e l y . The formation of
s u l f u r i s not detected during reaction ( 1 ) , and the product of
reaction (2) i s mainly manganese s u l f a t e .

The r e s u l t s indicated i n F i g . 3 suggest that ferrous iron i s


a l s o e f f e c t i v e in reducing four valent manganese.

B. Oxidation of Chalcocite (Cu S) by Thiobacillus ferrooxidans.


2

I t has been reported (11) that there are two mechanisms in


bacterial leaching of metal s u l f i d e s . One i s i n d i r e c t , that i s ,
282 Kazutami Imai

metal s u l f i d e s are oxidized and d i s s o l v e d chemically with f e r r i c


iron and s u l f u r i c a c i d , and ferrous iron formed in t h i s reaction
i s re-oxidized by the action of b a c t e r i a . The other i s d i r e c t ,
that i s , metal s u l f i d e s are oxidized d i r e c t l y by c e l l s of the
bacteria in the absence of a c i d - s o l u b l e i r o n . However, d e t a i l s
of the l a t t e r mechanism have not been c l a r i f i e d y e t . Landesman,
et a l . (12) as well as Beck and Brown (13) reported that when
p y r i t e or chalcopyrite i s used as a s u b s t r a c t , both iron and
s u l f u r moieties are oxidized simultaneously by T. ferrooxidans.

Torma (14) reported that the bacterium i s able to o x i d i z e


i r o n - f r e e a n a l y t i c a l l y pure c o b a l t , n i c k e l , and zinc s u l f i d e s in
the absence of a c i d - s o l u b l e i r o n . Nielsen and Beck (15) reported
that when chalcocite i s used as a s u b s t r a t e , the bacterium
o x i d i z e s the Cu moiety, but not the s u l f u r moiety. In order to
detect the d i r e c t mechanism and to explore the d e t a i l s of the
mechanism, we c a r r i e d out the following experiments:

1. Oxidation of Chalcocite with Intact Cells in the Absence


of Acid-Soluble Iron

As shown in F i g . 4, chalcocite was oxidized and the copper


was extracted by the action of c e l l s of T. ferrooxidans under the
condition without i r o n . From every value of oxygen-uptake and
copper-extraction indicated in the f i g u r e , t h e value of respective
tontrol using boiled c e l l s has been subtracted. The optimum pH
for the oxidation of chalcocite i s about 2.3 and i t coincides
with that f o r the extraction of copper. The molar r a t i o of
extracted copper to consumed oxygen i s about 2.0 at every pH
( F i g . 4 ) , and s u l f u r i s not detected during the o x i d a t i o n . These
facts suggest that the reaction proceeds according to the f o l l o w -
ing equation:

2Cu S + 2 H S 0
2 2 4 + 0 2 > 2CuS + 2CuS0 + 2H 0 4 2

When f e r r i c iron i s added to the reaction mixture, the rates


of oxygen uptake and copper extraction were both accelerated as
Bioextractive Applications and Optimization 283

1.0 2.0 3.0 4.0


PH

Fig. 4. Extraction of copper from chalcocite by intact


cells of T h i o b a c i l l u s f e r r o o x i d a n s . Reaction conditions: CU2S,
100 \imoles; glycine-Η^SO^ buffer, 500 \wioles; cells, 2 mg protein;
total volume, δ ml; gas phase, air; temp., δ0°Ο.
Cu * extraction;
1
0^ uptake

Cone, of Fe (x10" M)3

δ+
Fig. 5. Effect of Fe on the leaching of chalcocite with
cells of T. ferrooxidans. Reactions were carried out at pH 2.5.
Ο Cu^ extraction;
+
• · 0£ uptake
284 Kazutami Imai

indicated in F i g . 5. A l s o in t h i s case, the molar r a t i o of


extracted copper to consumed oxygen was 2.0 at every concentra­
tion of f e r r i c iron.

From the r e s u l t s shown in F i g s . 4 and 5, i t may be thought


that T. ferrooxidans has a d i r e c t leaching mechanism to chalco­
c i t e , because r e s t i n g c e l l s of the bacterium are able to o x i d i z e
chalcocite in the reaction mixture without i r o n . However,
i f the c e l l s were contaminated with a trace of i n s o l u b l e iron
coming from the c u l t u r e medium, i t leaves room f o r the operation
of i n d i r e c t mechanism. Therefore, we attempted to demonstrate
the existence of a directmechanism using c e l l - f r e e extracts of the
bacterium. As the c e l l s are disrupted by sonic o s c i l l a t i o n in
Tris-H S02 4 buffer (0.1 M) of pH 7.0 and then c e n t r i f u g e d , the
c e l l - f r e e extracts cannot contain any free i r o n .

2. Oxidation of Chalcocite with Cell-Free Extracts

As indicated in Table I I , c e l l - f r e e extracts of T. ferro­


oxidans possessed high a c t i v i t y of iron oxidase and a l s o showed
c h a l c o c i t e - o x i d i z i n g a c t i v i t y without the addition of i r o n , though
i t was weak. When f e r r i c iron was added to c h a l c o c i t e , the
o x i d i z i n g a c t i v i t y of the extracts was enhanced remarkably. On
the other hand, addition of quinacrine i n h i b i t e d the a c t i v i t y of
iron oxidase s t r o n g l y , but i t did not a f f e c t the oxidation of
chalcocite (Table I I ) .

These r e s u l t s suggest that oxidation of chalcocite by the


extracts without the addition of iron proceeds through d i r e c t
mechanism, since the rate of chalcocite oxidation must depend on
the rate of ferrous iron oxidation in i n d i r e c t mechanism.

In f a c t , the rate of chalcocite oxidation of the crude


extracts increased with the addition of f e r r i c i r o n , but decreased
to the level of chalcocite alone with further addition of
quinacrine (Table I I ) . On the other hand, when the crude c e l l -
free extracts were dialyzed a g a i n s t 0.01 Μ T r i s - H S 0 9 zl buffer
Bioextractive Applications and Optimization 285

TABLE II Oxidation of Chaloooite with


Cell-Free Extract of T. ferrooxidans

Activity (\ilΟ2/6Ο min/


40.7 mg protein) on
Condition Cu S Cu S 0 FeSO
+
Fe (S0 )
2 4 s

1. Crude cell-free extract 26 115 337


2. Crude cell-free extract
+ quinacrine (5 χ 10~ M) 3
28 35 50

Composition of the reaction mixture: each substrate, 100 \xmoles;


$-alanine-H2S0 buffer (pH 2.5), 150 \imoles; cell-free extract,
4

40.7 mg protein; total volume, 3.0 ml; 30°C. From every value
indicated in the Table, the value of respective control using
boiled cell-free extracts has been subtracted.

TABLE III Effect of Dialysis on Chalcocite-Oxidizing


Activity of Crude Cell-Free Extracts of T. ferrooxidans

Activity (\ilΟ2/6Ο min/


35.5 mg protein)
Addition Before dialysis After dialysis

None 25 4
Fe (S0 )
2 4 (6 χ 10~ Μ) +
3
δ

quinacrine (5 χ 10"^U) 32 32

Reactive conditions were the same as given in Table II.

(pH 7.0) f o r 41 hours in the c o l d , the a c t i v i t y of chalcocite


oxidation decreased markedly.

However, i t was recovered by the addition of f e r r i c iron


even in the presence of quinacrine (Table I I I ) . T h e s e results
suggest that a d i r e c t mechanism on chalcocite oxidation e x i s t s in
c e l l - f r e e extracts of T. ferrooxidans, and the enzyme system
needs trace amounts of iron as a c o - f a c t o r ( 1 6 ) .
286Kazutam iImai

Corrans, et a l . (17) suggested that both of the i n d i r e c t and


d i r e c t mechanisms function in the oxidation of chalcocite by
T. ferrooxidans, but i n d i r e c t mechanism i s prominent under usual
c o n d i t i o n s , that i s , in the presence of ferrous or f e r r i c iron.
From the r e s u l t s indicated in F i g . 5 and Table I I , we a l s o approve
of t h i s suggestion.

C. Leaching of C o v e l l i t e (CuS) with T. ferrooxidans

Regarding to bacterial leaching of c o v e l l i t e many papers have


been published ( 1 7 - 2 4 ) . However, in order to c l a r i f y the detailed
aspect of bacterial c o n t r i b u t i o n to the leaching of c o v e l l i t e , we
c a r r i e d out some basical experiments. In t h i s study, chemical
reactions which are thought to p a r t i c i p a t e in bacterial leaching
of c o v e l l i t e were considered.

3. Auto-Oxidation of Ferrous Iron in 9K Medium

100 ml of s t e r i l i z e d 9K medium in cotton pluged shaking f l a s k


was shaken a s e p t i c a l l y f o r 12 days at 30°C, and the rate of auto-
oxidation of ferrous iron was determined. In t h i s experiment the
concentration of ferrous s u l f a t e in 9K medium was varied as 1 , 2 ,
3, and 4% (as FeSO^^H^O). At the same time, four other media
containing cupric s u l f a t e i n a d d i t i o n to ferrous s u l f a t e were
2+
prepared and shaken under the same c o n d i t i o n . The content of Cu
in each of the media was the same (1000 ppm), but the concentra-
t i o n of ferrous s u l f a t e was varied as indicated above.
Auto-oxidation of ferrous iron in a l l of the media was
small even a f t e r 12 days, and the e f f e c t of cupric s u l f a t e
on the auto-oxidation of ferrous iron was not observed
(Fig. 6).
4. Chemical Leaching of Covellite with Free Sulfuric Acid

I r o n - f r e e 9K medium (excluded merely ferrous s u l f a t e from 9K


medium) was prepared and i t s pH was r e s p e c t i v e l y adjusted to
Bioextractive Applications and Optimization 287

Fig. 7. Effect of initial pH of iron-free 9K medium on the


chemical leaching of covellite.
288 Kazutami Imai

4.0 - 1.0 with s u l f u r i c a c i d . Each medium was s t e r i l i z e d , added


with c o v e l l i t e ( 1 % ) , and shaked a s e p t i c a l l y f o r 12 days at 30°C.

As shown in F i g . 7, the amount of copper extracted was


smaller t pH 4.0 compared with that at other lower pH v a l u e s . In
the range of pH 1.0 to 3.0, 300 to 500 ppm of copper was e x t r a c t e d ,
and a c o r r e l a t i o n was observed between the rate of copper e x t r a c -
tion and the pH of the medium.

5. Effect of Ferric Concentration on the Chemical Leaching of


Covellite in 9K Medium.

Ferrous s u l f a t e and f e r r i c s u l f a t e were added to i r o n - f r e e


9K medium in the following r a t i o ( 1 % of FeS0 4 · 7H 0:
2 % of
Fe (S0 )
2 4 3 in the medium): A. 5:0; B. 4 : 1 ; C. 3:2; D. 2 : 3 ; E . l : 4 ;
and F. 0 : 5 .

A f t e r s t e r i l i z a t i o n , 1% of c o v e l l i t e was added to each of


them, shaken a s e p t i c a l l y f o r 12 days at 30°C, and the amount of
extracted copper was determined. As shown in F i g . 8, copper
extraction occurred r a p i d l y in the i n i t i a l stage of the reaction
(within one h o u r ) , and the rate of copper extraction was nearly
proportional to the i n i t i a l concentration of f e r r i c s u l f a t e in
the medium. However, in the f i n a l stage of the r e a c t i o n , the
amount of extracted copper became l a r g e r in the media with the
mixture of ferrous and f e r r i c s u l f a t e (B and C. in F i g . 8 ) .

6. Leaching of Covellite in Growing Culture of T. ferrooxidans

9K medium with 1% of c o v e l l i t e added was inoculated with


T. ferrooxidans and cultured for 8 days at 30°C under shaking.
During the c u l t u r e , c e l l - g r o w t h , iron o x i d a t i o n , and copper
extraction were determined. As shown in F i g . 9, the growth of
the bacterium and the rate of iron oxidation in the medium were
almost s i m i l a r to those in normal 9K medium ( without c o v e l l i t e ) .
However, the rate of copper extraction in inoculated
medium was higher than in uninoculated medium. I t implies
Bioextractive Applications and Optimization 289

1500

α
ε
a.
Τ 1000
Ϊ

/ Β
500k.

10
Day
2+
2 Fig. 8. Effect of the initial concentration of Fe ^ and
+

Fe in 9K medium on the chemical leaching of covellite.


% FeS0 *7H 0 : % Fe (S0 )
4 2 in 9K medium: A. 5:0; B. 4:1; C. 3:2;
2 4 3

D. 2:3; E. 1:4; F. 0:5.

that the leaching of c o v e l l i t e i s accelerated by the action of


the bacterium, e s p e c i a l l y in the e a r l y phase of i t s growth.

7. Effect of Ferrous Iron Concentration on the Leaching of


Covellite with the Bacterium
The concentration of ferrous s u l f a t e in 9K medium was varied
r e s p e c t i v e l y as 2 , 3, and 4% (as F e S 0 * 7 H o 0 ) , and to each medium
4

1% of c o v e l l i t e was used. These media were inoculated with


T. ferrooxidans and cultured at 30°C under shaking. At the same
time, control media which had the compositions corresponding to
the respective t e s t media, but not inoculated were shaken under
the same c o n d i t i o n s .

As shown in F i g . 10, the rate of copper extraction in inocu­


lated media was not affected by a change in the concentration of
ferrous s u l f a t e in the range of 2 to 4%, and the amount of
290 Kazutami Imai

extracted copper was much l a r g e r in inoculated media than in un-


inoculated, except one case. The exceptional medium (A in F i g . 1 0 )
was one of the uninoculated c o n t r o l s , and the copper extraction was
comparable to that in inoculated media. E x c l u s i v e l y in t h i s
medium, a sample of c o v e l l i t e powder which had been exposed to
the a i r f o r a long time was used a c c i d e n t a l l y . The color of
fresh powder of pure c o v e l l i t e i s u s u a l l y deep indigo blue, but
that of the sample was rather black.

Thereupon, fresh powder of pure c o v e l l i t e was suspended in


water (7 g/100 ml) and shaked a s e p t i c a l l y f o r 10 days at 30°C
using a cotton pluged shaking f l a s k . After the treatment, the
Bioextractive Applications and Optimization 291

1500r

10

Fig. 10. Effect of Fe concentration on the leaching of


covellite in the growing culture of T. f e r r o o x i d a n s . Concentra­
tion of FeS0*7KJD:
J
4 2
Ο 2%, 0 3%, # 4%
inoculated> uninoculated

c o v e l l i t e was f i l t e r e d , washed with water, and d r i e d . The treated


c o v e l l i t e powder was used as a substrate f o r leaching experiments,
and the rate of copper extraction was compared with that of un­
treated f r e s h c o v e l l i t e .

8. Comparison of Copper Extraction from Treated - and Untreated -


Covellite

9K medium added with λ% of t r e a t e d - or u n t r e a t e d - c o v e l l i t e


was inoculated with T. ferrooxidans, and shaken f o r 12 days at
30°C.

As shown in F i g . 1 1 , the rate of copper extraction was higher


in the culture using t r e a t e d - c o v e l l i t e than in that using untreat­
ed c o v e l l i t e , and the phenomenon was a l s o observed in uninoculated
292 Kazutami Imai

900,

(ppm)

600h

300

Day

Fig. 11. Extraction of copper from treated- and untreated-


CuS in the growing culture of T. f e r r o o x i d a n s .
• Treated CuS 3 Ο Untreated CuS
inoculated Λ uninoculated

control media. In the next place, the leaching of both kinds of


the c o v e l l i t e with r e s t i n g c e l l - s u s p e n s i o n of T. ferrooxidans was
then examined.

C e l l s of the bacterium which were obtained from the culture


ο

in 9K medium and washed thoroughly were suspended (2 χ 10 cells/


ml) in i r o n - f r e e 9K medium containing 1% of the t r e a t e d - or
untreated-covellite. The suspensions were shaken for 15 days at
28°C. As the r e s u l t s , copper was extracted from both kinds of
the c o v e l l i t e , but the rate of the extraction was a l s o higher in
the t r e a t e d - c o v e l l i t e ( F i g . 1 2 ) .
The fundamental change which occurs in c o v e l l i t e during the
pretreatment has not been c l a r i f i e d y e t , but i t i s indicated
that the treatment g i v e s good e f f e c t on the leaching of c o v e l l i t e .
Bioextractive Applications and Optimization 293

Cu' +

(ppm)

1000

500

°0 5 10 15
Day

Fig. 12. Extraction of copper from treated- and untreated-


CuS with cells of T. f e r r o o x i d a n s .
• Treated CuS, Q Untreated CuS
inoculated uninoculated

As widely known, the mechanicsm of bacterial leaching i s


complicated with many reasons. However, I think that the most
important reason i s that many s e r i e s of chemical and b i o l o g i c a l
reactions occur and they a f f e c t one another in complex pattern
during the leaching process.

Furthermore, as I described above, the e f f e c t i v e reactions


are o x i d a t i v e in most of the leaching processes, e s p e c i a l l y in the
leaching of metal s u l f i d e s , but in c e r t a i n c a s e s , f o r example the l e a c h -
ing of manganese d i o x i d e , some reducing reactions play an important
role.

From such observations and o t h e r s , i t i s evident that the


mechanism of bacterial leaching i s not s i m i l a r , but diverse depending
upon the kind of metal compounds, leaching c o n d i t i o n , s o r t of
b a c t e r i a , and so on.
294 Kazutami Imai

IV. REFERENCES

Ί. Imai, K., Okuzumi, M., and K a t a g i r i , H., J. Ferm. Technol.,


42, 755 (1964).
2. Imai, K., S u g i o , T . , and Tano, T . , in "Abstracts of Papers,
F i f t h International Fermentation Symposium, B e r l i n , 1976",
p. 453.
3. Imai, K., in "World Mining and Metals Technology", (A. Weiss,
E d . ) , Vol. 1 , p. 3 2 1 . American I n s t i t u t e of M i n i n g , Metal­
l u r g i c a l , and Petroleum Engineers, I n c . , 1976.
4. Silverman, M.P., and Lundgren, D.G., J. Bacteriol., 78, 326
(1959).
5. F r i t z , I . , and Fruland, M., Anal. Chem., 26, 1593 (1954).
6. Tano, Τ . , and Imai, K., J. Agr. Chem. Soc. Japan, 37, 576
(1963).
7. Imai, Κ., Tano, T . , and Noro, H., U.S. Patent, 3433629,
March 1969.
8. Starkey, R., J. Bacteriol., 3 3 , 545 (1937).
9. Imai, K., Okuzumi, M., and K a t a g i r i , H., in "Symposium on
Enzyme Chemistry, Japan", V o l . 17, p. 132 (1962).
10. Peck, J r . H.D., Ann Rev. Microbiol., 22, 489 (1968).
11. Silverman, M.P., J. Bacterid., 94, 1046 (1967).
12. Landesman, J . , Duncan, D.W., and Walden, C.C., Can. J.
Microbiol., 12, 959 (1966).
13. Beck, J . V . , and Brown, D.G., J. Bacterid., 96, 1433 (1968).
14. Torma, A . E . , Rev. Can. Biol., 30, 209 (1971 ).
15. N i e l s e n , M.M., and Beck, J . V . , Science, 175, 1124 (1972).
16. Imai, Κ., Sakuguchi, H., S u g i o , T . , and Tano, T . , J. Ferm.
Technol., 5 1 , 865 (1973).
17. Corrans, I . J . , H a r r i s , B., and Ralph, B . J . , J. South African
Inst, of Mining and Metallurgy, 72, 221 (1972).
18. Bryner, L.C., Beck, J . V . , D a v i s , D.B., and W i l s o n , D.C.,
Ind. Eng. Chem., 46, 2587 (1954).
19. I t o , I . , J. Ferm. Assoc., Japan, 25, 1 (1967).
20. Watanabe, Α . , Uchida, T . , and F u r u t a n i , S . , J. Ferm. Assoc.,
Japan, 25, 21 (1967).
21. Sakaguchi, H., Torma, A . E . , and S i l v e r , M., Appl. Environ,
Microbiol., 3 1 , 7 (1976).
Bioextractive Applications and Optimization 295

22. S i l v e r , Μ., and Torma, A . E . , Can. J. Microbiol., 20, 141


(1974).
23. Torma, A . E . , Gabra, G.G., Guay, R., and S i l v e r , M., Hydro-
metallurgy , 1 , 301 (1976).
24. Torma, A . E . , in "Abstracts of Papers, F i f t h International
Fermentation Symposium, B e r l i n , 1976", p. 455.
POTASSIUM RECOVERY THROUGH LEUCITE BIOLEACHING:

P O S S I B I L I T I E S AND LIMITATIONS

Giovanni Rossi

U n i v e r s i t y of C a g l i a r i
Cagliari, Sardinia, Italy

Leuoite is a framework silicate (2SiO^AlO^)~ •K with a 1 tl

specific gravity of 2.5, Mohs hardness about 6, and vitreous


luster. It is tetragonal (pseudocubic) and can be differentiated
from orthoclase because of its lower silica content (four mole-
cules in leucite vs. six in orthoclase). It is more basic and
more susceptible to acid attack and weathering than orthoclase.
In the volcanic belt of Central Italy, comprised between Upper
Latium and the Alban Hills and in nearby areas, there exist large
occurrences of leucitic rocks containing estimated reserves of
nine billion tons of potassium oxide (K 0) equivalent and the same
2

amount of aluminum oxide (Al 0$). Numerous attempts aimed at


2

developing industrial processes of potassium extraction from leu-


cite ores date as far back as 1856, but so far they have not been
successful. Microbial solubilization of potassium and aluminum
from leucite using heterotrophic microorganisms from collections
or isolated from soils of leucitic areas and thiobacilli was
studied in static conditions (batch cultures). Leaching with
thiobacilli, even in the presence of pyrite, and with some strains
of soil microorganisms did not give good results. In 150 days,
strains of A s p e r g i 1 l u s niger (van Tieghen), S c o p u l a r i o p s i s b r e v i -
caule (Sacc. Bain) andPenici 11 ium expansum (Link) leached between
21 and 27% of the potassium.

297
298Giovann iRossi

I. INTRODUCTION

Leucite i s a potassium aluminum s i l i c a t e (a framework sili­


cate) with the formula ( 2 S i 0 * A 1 0 ) · Κ , or K A l ( S i 0 ) , s p e c i f i c
2 2
_ 1 + 1
3 2

g r a v i t y 2 . 5 , Mohs hardness 5.5 to 6 . 6 , which generally occurs as


white or gray trapezohedral c r y s t a l s (icositetrahedrons or " l e u -
citohedrons") or rounded g r a i n s disseminated in eruptive igneous
rock, with s i z e s comprised between a few millimeters and a few
centimeters ( 1 ) . I t i s tetragonal (pseudocubic) and can be
d i f f e r e n t i a t e d from orthoclase because of i t s lower s i l i c a content
(four molecules in l e u c i t e v s . s i x in orthoclase) (2) ( F i g . 1 ) .

Ο Κ · Si,Al

Fig. 1. Lower half of the unit cell of leucite (2).


Bioextractiv
eApplication
san dOptimizatio
n29 9

Leucite has v i t r e o u s l u s t e r , i s more basic and more s u s c e p t i -


ble to acid attack and weathering than orthoclase. I t contains
21.58% K 0 and 23.4% A l ^ .
2

Leucite occurs abundantly in the rocks of the volcanic belt


of Central I t a l y , between Acquapendente in Upper Latium and the
Alban H i l l s and, further South, in the province of Caserta as well
as in the Sessa Aurunca area and in the v i c i n i t y of the Vesuvius
volcano ( F i g . 2 ) .

Fig. 2. Map of Italian leucite deposits.


300 Giovanni Rossi

On the basis of estimates made by Washington, who extensively


investigated the I t a l i a n l e u c i t e occurrences ( 3 ) , potash equiva-
lent (K^O) and alumina (Al^O^) reserves contained i n the above
mentioned deposits can be calculated in more than nine b i l l i o n
tons each ( 4 ) .

The potassium mineral s a l t deposits discovered in the f i f t i e s


in S i c i l y and exploited since then are i n s u f f i c i e n t to s a t i s f y the
potash demands of the I t a l i a n economy, and the s i t u a t i o n i s even
worse as f a r as aluminum metal i s concerned, since the major
I t a l i a n bauxite deposits are depleted, and in the O.E.C.D. area
the only bauxite producer i s France.

The development of an i n d u s t r i a l process of potash and


aluminum extraction from l e u c i t e rocks could therefore have a
s i g n i f i c a n t effect on the I t a l i a n economy, and t h i s i s the reason
why several i n v e s t i g a t o r s and s p e c i a l i s t s in t h i s f i e l d have
tackled the problem.

Furthermore, one cannot ignore the presence of considerable


l e u c i t e deposits in other areas of the world, f o r instance in the
United S t a t e s .

The f i r s t attempts [made by B i c k e l l ( 5 ) ] to recover potash


from l e u c i t e date as f a r back as 1856. Since then, several pro-
cesses have been developed and tested a l l of them based on attack
by a strong inorganic acid or on a l k a l i n e treatment. In every
case the major d i f f i c u l t y c o n s i s t e d in the elimination of the
s i l i c a and the i r o n .

Among the processes which have been developed up to the


p i l o t plant scale the following should be mentioned ( 6 , 7 ) :
- those based on hydrochloric acid a t t a c k , with formation of
potassium c h l o r i d e and aluminum c h l o r i d e , subsequent separate
p r e c i p i t a t i o n of the l a t t e r and f i n a l decomposition of the hydra-
ted aluminum chloride by heating at about 350°C; among these, the
most important was that developed by Prof. Blanc, which was
Bioextractive Applications and Optimization 301

realized in a plant located at A u r e l i a , 70 km North of Rome, in


the t h i r t i e s (8,9);
- those based on n i t r i c acid attack, s i m i l a r , in p r i n c i p l e ,
to the previous process;
- those based on s u l f u r i c a c i d attack; most noteworthy i s
that patented by Prof. G a l l o , of Pisa U n i v e r s i t y , according to
which l e u c i t e i s attacked with s u l f u r i c acid a t 5 2 ° C , the s i l i c a
i s f i l t e r e d and the pure alum thus formed i s p u r i f i e d , c r y s t a l l i z -
ed, dehydrated and calcined at about 800°C with recovery of s u l f u r
t r i o x i d e , which i s r e c y c l e d ; the calcined residue i s treated with
water: the soluble alumina i s then separated and the potassium
s u l f a t e s o l u t i o n i s f i n a l l y evaporated. This process was tested
in a p i l o t plant b u i l t at C a s t e l l i n a in C h i a n t i , in Tuscany, by
the I t a l i a n Company S.A.L.P.A. in the f o r t i e s ;
- the processes based on a l k a l i n e attack, they consisted in
p r i n c i p l e in the reaction of l e u c i t e with limestone with formation
of calcium s i l i c a t e which was then separated, and of the water-
soluble potassium aluminate. Among these processes should be
mentioned the Messer-Schmidt p r o c e s s , tested by S.A. V u l c a n i a , the
Jourdan process, tested by S . I . P . (Societa I t a l i a n a P o t a s s a ) , and
the process developed and tested on a p i l o t plant scale by S . I . P .
in the late f o r t i e s i n a plant located at Bagnoli (Naples).

A l l of the above mentioned p r o c e s s e s , though t e c h n o l o g i c a l l y


sound, were too expensive due to the f a c t that the formation on
the l e u c i t e p a r t i c l e s of an i n s o l u b l e i n h i b i t i n g layer r e q u i r e d ,
for the process to be rendered commercially p r a c t i c a b l e , expensive
a u x i l i a r y o p e r a t i o n s , and the products, though excellent as f a r as
commercial requirements are concerned, were not competitive.

For these reasons, a l l of the i n d u s t r i a l attempts to e x p l o i t


l e u c i t e f o r aluminum and potash e x t r a c t i o n have been abandoned up
to now.
302 Giovanni Rossi

II. THE BIODEGRADATION OF ALUMINOSILICATES

The success of the bioleaching techniques developed in the


past decade in the f i e l d of s u l f i d e minerals has prompted the i n -
v e s t i g a t i o n of the p o s s i b i l i t i e s of b i o l o g i c a l s o l u b i l i z a t i o n of
aluminum and potassium from l e u c i t e .

The biodegradatidn of a l u m i n o s i l i c a t e s and carbonates has up


to now constituted a f i e l d of research mainly f o r a g r i c u l t u r a l
microbiologists. From l i t e r a t u r e , i t would seem that the i n v e s t i -
gations c a r r i e d out up to the present time have followed two l i n e s
of approach: the f i r s t u t i l i z e d mainly in the Western hemisphere
and in Europe, adopted the conventional procedures of a g r i c u l t u r a l
microbiology, and aimed at a s c e r t a i n i n g the action of the known
s o i l microorganisms on a l u m i n o s i l i c a t e s and carbonates; the second,
mainly followed by the Slav-speaking c o u n t r i e s , under the leader-
ship of the Russian School, aimed at the research of microbial
s t r a i n s s p e c i f i c a l l y s u i t a b l e f o r a l u m i n o s i l i c a t e biodegradation.

Among the f i r s t - one may very l i k e l y say "the f i r s t " -


i n v e s t i g a t o r s who tackled the problem can be considered the
I t a l i a n s De Grazia and Camiola ( 1 0 ) . The object of t h e i r i n v e s t i -
gation was to understand the process by which plants obtain the
soluble potassium supply needed f o r t h e i r n u t r i t i o n from the i n -
soluble volcanic rock. They worked on batches of very f i n e l y
ground museum-grade l e u c i t e c r y s t a l s mixed with 1,000 ml of culture
medium each in 2,000 ml s t a t i c f l a s k s which were inoculated with
various s t r a i n s of moulds. A culture y i e l d e d , in 70 days, more
than 70% d i s s o l u t i o n of K^0 equivalent, though the s t e r i l e control
y i e l d e d r e s u l t s quite high too (30%).

S h o r t l y a f t e r , B a s s a l i k (11) reported on an i n v e s t i g a t i o n of
orthoclase biodegradation by means of several s t r a i n s ; the best
r e s u l t s were obtained with s t r a i n s of Bacillus extorquens, n . s p . ,
with y i e l d s , expressed in terms of percent weight of the i n i t i a l
feldspar sample d i s s o l v e d , lower than 4% in 68 days in the most
Bioextractive Applications and Optimization 303

favourable case, but more than 40 times higher than in the s t e r i l e


controls.

I n v e s t i g a t i o n s aimed at throwing l i g h t on the problem have


recently been undertaken by the research workers of the "Bureau de
Recherches Geologiques et M i n i e r e s " (B.R.G.M.) in France, in the
framework of a wide program concerning the understanding of the
processes of geologic rock a l t e r a t i o n ( 1 2 , 1 3 ) . They experimented
with several rocks ( o l i v i n e from b a s a l t nodules of the Srmci
region in Northern Bohemia, dunite from New Caledonia, "green
rocks" (serpentine) from the Alps and mica muscovite) with various
s t r a i n s of moulds and b a c t e r i a , both of c o l l e c t i o n and i s o l a t e d
from s o i l s . The best r e s u l t s were obtained with Aspergillusniger,
which, in 18 days, produced a 26.8% s o l u b i l i z a t i o n of the potash
contained in the muscovite samples. Evidence of transformations
induced i n b i o t i t e c r y s t a l s by the same s t r a i n was a l s o produced
by Boyle, Voigt and Sawhney ( 1 4 ) . As has already been pointed out
in the i n t r o d u c t i o n , the philosophy underlying the research con-
ducted in t h i s f i e l d by the Russian School has been somewhat
d i f f e r e n t from the one o u t l i n e d so f a r , i t s objective being the
i s o l a t i o n of microorganisms s p e c i f i c a l l y capable of a l u m i n o s i l i -
cate d e s t r u c t i o n . The Russian Academician V . I . Vernadskii had
already d i s c u s s e d , as f a r back as 1934, the p o s s i b i l i t y of s i l i -
cate d e s t r u c t i o n by the s o i l microorganisms which l i b e r a t e potas-
sium together with other s i l i c a t e - f o r m i n g elements. In h i s study
on alumina and s i l i c a he expressed the view that the decomposition
of aluminum s i l i c a t e (AloSi^O^) could only take place through b i o -
chemical action ( 1 5 ) .

These i n v e s t i g a t i o n s were taken over, in the f o r t i e s , by


Alecsandrof, whose objective was to f i n d and i s o l a t e s o i l bacteria
capable of decomposing s i l i c a t e s and t h e i r u t i l i z a t i o n f o r improv-
ing the a g r i c u l t u r a l output as a s u b s t i t u t e f o r potash f e r t i l i z -
ers. According to the r e s u l t s reported by Alecsandrof in three
papers published in 1947 and in 1949, the bacteria i s o l a t e d from
304 Giovanni Rossi

Russian black s o i l ("cernoziom") were able to transform the


potassium contained in the l a t t e r into a form not susceptible to
a s s i m i l a t i o n by upper plants into an a s s i m i l a b l e form (16,17,18).

These f i n d i n g s were confirmed by a subsequent work published


in 1950 Alecsandrof and Zac, who, c u l t u r i n g the s i l i c o b a c t e r i a on
" s i l i c a t e d a g a r " , obtained the s o l u b i l i z a t i o n of 75.9% and 67.0%
of the a l u m i n o s i l i c a t e present i n the culture medium ( 1 9 ) . Un-
f o r t u n a t e l y , they did not indicate the chemical composition nor
the c r y s t a l l i n e structure of the a l u m i n o s i l i c a t e subjected to b i o -
degradation in t h e i r t e s t s .

Even though the existence of s i l i c o b a c t e r i a was taken f o r


granted by some researchers, who devoted a remarkable amount of
work to t h e i r c h a r a c t e r i z a t i o n ( 2 0 - 2 6 ) , Tesic and Todorovic (27-29)
have come to the conclusion that they r e a l l y belong to a genus
known f o r some time under the name of Bacillus circulans Jordan
(1890), and therefore, the bacteria discovered by Russian
s c i e n t i s t s can probably be considered as ecotypes of an already
known species of s i l i c o b a c t e r i a ; furthermore according to them, i t
does not seem to have been e n t i r e l y proved that these are bacteria
specific for s i l i c a . Alecsandrof himself, in l a t e r papers, (30-
35) seems to share t h e i r o p i n i o n .

Bacteria having c h a r a c t e r i s t i c s s i m i l a r to those described by


Alecsandrof have been reported a l s o , in the paper already mention-
ed, by Goni, Gugalski and Sima ( 1 3 ) , whereas Heinen assessed that
t h i s type of microorganism a s s i m i l a t e s s i l i c a in the place of
phosphorus (36,37).

III. MATERIALS AND METHODS

A. The Mineral Substrates

The l e u c i t e sample used in the experiments i s a concentrate


obtained by magnetic separation from a volcanic rock ("leucito-
f i r o " ) of the C i v i t a c a s t e l l a n a area, described in detail by
Bioextractive Applications and Optimization 305

V e n t r i g l i a ( 3 8 ) , with procedures, described elsewhere ( 3 9 , 4 0 ) . It


was dry-ground in a "Vibratom" v i b r a t i n g m i l l (Siebtechnik,
G.m.b.H., Muhlheim, German Federal Republic) with a g r i n d i n g
charge of ceramic pebbles. I t s composition i s reported in Table
I and i t s s i z e d i s t r i b u t i o n a n a l y s i s in Table I I .

The p y r i t e was the same as that described in a previous work


(41).

TABLE I Chemical Data on Leucite Concentrate


Utilized as Solid Substrate

Component Weight % Component Weight %

sio 2 55.44 MgO 0.14


22.58 0.08
Al
2°3 °3
S

0.50 Na 0 0.82
Fe
2°3 2

CaO 0.40 κο 2 19.00

TABLE II Size Distribution Analysis


of Leucite Concentrate

Size, \xn

Screen fractions Weight %

+208 11.83
-208 +147 8.58
-147 +104 10.53
-104 + 74 9. 75
-74 +61 21.96
-61 +53 26.90
-53 +43 4.03
-43 +38 0.19
- 38 6.24
306 Giovanni Rossi

Β. The Microorganisms

The Thiobaoillus ferrooxidans microorganisms used f o r the


bioleaching experiments of mixtures of l e u c i t e and p y r i t e , were
o r i g i n a l l y i s o l a t e d from acid mine waters in Southern S a r d i n i a
(42) and maintained by weekly t r a n s f e r s into Silverman and
Lundgren 9K medium ( 4 3 ) .

For leaching t e s t s on l e u c i t e alone, microorganisms both from


c o l l e c t i o n s and i s o l a t e d from I t a l i a n s o i l s were used. The
former were purchased from Central bureau voor Schimmelcultures
(CBS), Barn, The Netherlands, and are:

- Scopulariopsis brevioaule ( S a c c ) , CBS 467.48;


- Penicillium expansum L i n k , CBS 146.45;
- Aspergillus niger van Tieghen, CBS 131.52

The bacteria were i s o l a t e d from a sample of s o i l collected in


the v i c i n i t y of a volcanic rock quarry close to C i v i t a c a s t e l l a n a .
The samples were kept sealed in polythene bags f o r 24 months; at
the end of t h i s time i n t e r v a l , the bacteria were " r e v i v e d " by
humidifying the s o i l samples and keeping them for three days in a
thermostat at 30°C.

0,1 gram batches of the above s o i l samples were then suspend­


ed in 1,000 ml of water p r e v i o u s l y deionized and s t e r i l i z e d , and
the suspensions thus obtained were used as inocula of petri dishes
containing " s i l i c a t e agar" according to Alecsandrof (Table I I I ) .

TABLE III Silicate Agar According to Alecsandrof (17)

Agar-agar (purified of potassium) 2.00%


Saccharose 0.60%
0.10%
0.20%
0.05%
(1 drop of 1% solution per 100 ml of aluminum and
potassium silicate)
Bioextractive Applications and Optimization 307

From the colonies thus developed,two were selected which had a


l i q u i d gelatinous appearance with a prominent cupola and were
labeled Al and A2. The s t r a i n Al organisms are squat r o d s , about
1 ym long, characterized by a subterminal clostridium-shaped
deformation and provided with a capsula about 6 ym in diameter.
The s t r a i n A2 organisms are s i m i l a r in s i z e to those of s t r a i n A l ,
but have no capsula (aerobic Bacillus s p . ) .

C. The Cultures

For the experiments with mixtures of l e u c i t e and p y r i t e the


9K Silverman and Lundgren medium was used ( 4 3 ) .

For the experiments with Scopulariopsis brevicaule, Penicil-


lium expansion and Aspergillus niger a culture medium proposed for
bioleaching t e s t s on a l u m i n o s i l i c a t e s by Goni, Gugalski and Sima
(13) was used, which i s described in Table IV.

F i n a l l y , f o r the l e u c i t e bioleaching t e s t s with bacteria


i s o l a t e d from I t a l i a n l e u c i t i c s o i l s , the Alecsandrof medium was
used (17) and i t s composition i s reported in Table V.

TABLE IV Culture medium of Goni, Gugalski and Sima (13)

Deionized Water 1,000 g


Maltose 10 g
Peptone 1g

TABLE V Culture medium of Alecsandrof (17)

Distilled water 1,000 g


Saccharose 0.75 g
Ammonium sulphate 0.15 g
Disodium phosphate 0.30 g
Magnesium sulphate 0.75 g
Ferric chloride (1 drop of 1% soln.)
308 Giovanni Rossi

In order to evaluate the extent of the l e u c i t e degradation in


the presence of organic acids produced by certain microorganisms
( l i k e Aspergillus niger) a 50-50 s t e r i l e mixture of M/10 o x a l i c
acid and M/10 c i t r i c acid s o l u t i o n s were prepared.

A l l the t e s t s were c a r r i e d out in 2,000 ml Florence f l a s k s ,


each containing 1,000 ml of culture medium (or a c i d i c s o l u t i o n )
and 3 grams of ground l e u c i t e . The bottles were kept in a thermo-
s t a t i c chamber at 32°C and samples of about 5 ml were taken
a s e p t i c a l l y at regular time i n t e r v a l s .

D. The A n a l y t i c a l Methods

The potassium content of the samples was determined using a


Gio De Vita "Flammelectron 1 1 " flame photometer; analyses for
aluminum were effected by means of a Perkin-Elmer 306 atomic
absorption spectrophotometer. The potassium, aluminum and s i l i c a
content of the dried biomasses were determined by means of a
P h i l i p s PW1212 automatic X-ray fluorescence spectrometer; some
dried biomasses were a l s o i n v e s t i g a t e d using a General Electric
XRD5 X-ray u n i t .

The pH of the media were measured with a Beckman Century


S-S pH-meter.

IV. RESULTS

A. The Experiments with P y r i t e and Leucite Mixtures

The microorganism of the genus T h i o b a c i l l u s was not capable


of leaching p y r i t e in presence of l e u c i t e and, one month a f t e r the
inoculum, i t appeared i n h i b i t e d ; in the meantime the culture
medium pH had s l i g h t l y increased from the i n i t i a l value of 2.35 to
2.68.
Bioextractive Applications and Optimization 309

B. The Experiments with Moulds

The moulds grew in the respective f l a s k s in d i f f e r e n t ways:


the Aspergillus niger and PeniQilliumexpansum s t r a i n s formed
aggregates, up to some centimeters in s i z e , which, already at the
end of the second month, had covered the whole free surface of
the culture medium without spreading downwards and coming
into d i r e c t contact with the mineral powder which had s e t t l e d on
the bottom; on the contrary the Soopulariopsis brevioaule formed a
bulky biomass, constituted of several superposed layers (at l e a s t
seven), the surface of each layer being continuous and p r a c t i c a l l y
covering the whole c r o s s - s e c t i o n of the f l a s k perpendicular to i t s
v e r t i c a l a x i s at the level at which i t was formed.

Direct contact of the biomass with the mineral powder thus


occurred. No deposit on the inner wall of the f l a s k was noticed.

The i n i t i a l and f i n a l pH of the culture media are summarized


in Table V I . The increase in pH i s , t h u s , a feature common to a l l
of the culture media: i t seems, however, remarkably more pro-
nounced in the inoculated media and in the s t e r i l e mixture of
organic a c i d s , whereas the opposite appears to take place in the
s t e r i l e control. I t was p r e f e r a b l e , i n order to evaluate the
evolution with respect to time of the l e u c i t e bioleaching process,
to assay the culture media f o r potassium rather than f o r aluminum,
since the l a t t e r , being amphoteric, forms hydroxides the s o l u b i l -
i t y of which i s pH-dependent, as shown by the diagram in F i g . 3,
taken from a paper by Correns ( 4 4 ) .

The r e s u l t s of the assays are i l l u s t r a t e d by the curves of


F i g s . 4, 5, and 6 in which the curves of the s t e r i l e control as
well as those of the organic acids mixture were a l s o p l o t t e d .

During the f i r s t month, the l e u c i t e s o l u b i l i z a t i o n proceeded


in a s i m i l a r way both in the acids mixture and in the c u l t u r e s ,
since the rate of increase of potassium ion in s o l u t i o n was
p r a c t i c a l l y 2.65 mg per l i t e r per day f o r a l l of them.
310 Giovanni Rossi

TABLE VI Variation of the pH during the


time of the experiments

Flask contents Initial pH Final pH

A s p e r g i l l u s niger 5.65 6.85


S c o p u l a r i o p s i s brevicaule 5. 75 8.10
P e n i c i I l i u m expansum 6.00 7.98
Sterile control for the
cultures of moulds 7.20 5.81
Sterile organic acids mixture 1.62 1.65
Culture Al 3.98 5.91
Culture A2 5.42 4.62
Sterile control for cultures
Al and A2 4.35 6. 72

Fig. 3. Solubility of aluminum hydroxide gel as a function


of pH (after Correns).
Bioextractive Applications and Optimization 311

Fig. 4. Potassium solubilization from leucite vs. time -


Curve 1: sterile acids mixture. Curve 2: P e n l c i I l i u m expansum
culture. Curve 3: sterile control.

The s o l u b i l i z a t i o n rate underwent an abrupt slowing down at


the end of t h i s i n i t i a l p e r i o d , and the curves then followed
d i f f e r e n t trends: those of the acids mixture and of the f l a s k
inoculated with Penicilliumexpansum ( F i g . 4) tended to become
p a r a l l e l to the a x i s of the a b s c i s s a e ; the potassium contents of
the media thus tend asymptotically to values constant versus time.

The curves of the potassium contents in the media inoculated


with Aspergillus niger ( F i g . 5) and Scopulariopsis brevicaule
( F i g . 6 ) , a f t e r having reached a maximum at the 45th and 55th day
r e s p e c t i v e l y , started decreasing. This decrease was more marked
and permanent in the case of the second s t r a i n . The s t e r i l e
312 Giovanni Rossi

Fig. 5. Potassium solubilization from leucite vs. time -


Curve 1: sterile acids mixture. Curve 2: A s p e r g i l l u s niger.
Curve 3: sterile control.

control curve runs constantly lower than those of the c u l t u r e s ,


though showing a s l i g h t l y increasing trend with time.

At the end of the experiment the f l a s k s were emptied, care


being taken to separate the biomasses from the residual mineral
powder; t h i s operation was quite easy for the Aspergillus niger
andPenicilliumexpansum c u l t u r e s , whereas i t was p r a c t i c a l l y
impossible f o r the Scopulariopsis brevicaule, which, as pointed
out e a r l i e r had come into close contact with the s o l i d s which had
s e t t l e d on the bottom of the f l a s k .
Bioextractive Applications and Optimization 313

Fig. 6. Potassium solubilization from leucite vs. time -


Curve 1: sterile acids mixture. Curve 2: S c o p u l a r i o p s i s
brevicaule culture. Curve δ: sterile control.

The three biomasses were dried and assayed for potassium


contents. The amount of s o l i d residue of Scopulariopsis brevi­
caule was s u f f i c i e n t l y large to be analyzed by X-ray d i f f r a c t i o n ,
which showed the presence of c r y s t a l l i n e l e u c i t e .

Reliable X-ray d i f f r a c t i o n patterns could not be obtained f o r


the s o l i d residues of the other two biomasses as they were too
s m a l l ; they were therefore analyzed by means of the fluorescence
spectrometer. They revealed the presence of potassium but not of
aluminum or s i l i c a . This r e s u l t confirmed the observation made
during the experiment, i . e . , that these biomasses never came into
contact with the l e u c i t e .
314 Giovanni Rossi

From the diagrams i t appears that the potassium s o l u b i l i z e d


i s comprised between at l e a s t 27% in the case of the Scopulariop-
sis brevioaule culture and 22% in the case of the Penioillium
expansum c u l t u r e . These f i g u r e s are c o n s e r v a t i v e , since the
potassium assimilated by the organisms was not taken into account,
due to the uncertainty r e l a t i n g to Soopulariopsis bvevicaule.

C. Experiments with Microorganisms I s o l a t e d from Leucite S o i l s

In the f l a s k s inoculated with the s t r a i n s labeled Al and A2,


i s o l a t e d from l e u c i t i c s o i l s according to the procedure suggested
by Alecsandrof, the l e u c i t e biodegradation occurred at a remark-
ably slower rate than in the f l a s k s inoculated with moulds; a f t e r
150 days, the potassium s o l u b i l i z e d was of the same order of mag-
nitude as that of the s t e r i l e c o n t r o l s of the l a t t e r , even though
the t u r b i d i t y of the culture media, which was much more pronounced
than that of the s t e r i l e c o n t r o l s , was i n d i c a t i v e of a f l o u r i s h i n g
microbial flora.

The experiment was stopped a f t e r 24 months and during the


f i n a l 19 months no samples were taken in order to not excessively
a f f e c t the environmental c o n d i t i o n s . In both f l a s k s , but p a r t i c -
u l a r l y in the f l a s k containing the s t r a i n A l , the s e t t l e d slime
had formed c l o t s of various s i z e s , which were not present in the
sterile controls.

Furthermore, on the inner wall of the f l a s k the formation of


a white-gray deposit was noticed, which extended from the meniscus
down to the bottom of the Florence f l a s k . This phenomenon was not
observed on the walls of the s t e r i l e control flasks.

The f i l t r a t e s of both the inoculated f l a s k s and the s t e r i l e


controls assayed 41 p.p.m. of potassium. The amount of
deposit formed on the inner wall was very small (about 200 mg) and
i t was not p o s s i b l e to prevent i t s mixture with the very f i n e
leucite fractions. A n a l y s i s of t h i s deposit was only p o s s i b l e by
Bioextractive Applications and Optimization 315

means of the X-ray fluorscence spectrometer, but the r e s u l t s were


not c o n c l u s i v e , as the presence of A l , S i , and Κ was observed.
The i n i t i a l and f i n a l pH are shown in Table V I .

V. COMMENTS AND CONCLUSIONS

The experiments indicated that the u t i l i z a t i o n of the genus


T h i o b a c i l l u s and of p y r i t e f o r l e u c i t e i s impracticable since
the pH of 9K medium did not drop below l e v e l s incompatible with
the v i a b i l i t y of the organism. I t must be concluded that some
unknown soluble component of the l e u c i t e mineral tested i s t o x i c ,
even in very s l i g h t amounts, f o r the organism.

The r e s u l t s of the bioattack experiments with microorganisms


i s o l a t e d from l e u c i t i c s o i l s are l i k e w i s e negative: leucite is,
n e v e r t h e l e s s , not t o x i c f o r these organisms and i t i s quite
probable that other s t r a i n s y i e l d a better l e u c i t e d i s s o l u t i o n .

The r e s u l t s of the experiments c a r r i e d out with moulds


were very encouraging; among the l a t t e r , Scopulariopsis brevi­
caule appeared to be the most e f f i c i e n t , having produced a
notable potassium s o l u b i l i z a t i o n . The other two moulds, even
though they produced a l e s s pronounced s o l u b i l i z a t i o n , have
contributed to throwing l i g h t to a c e r t a i n extent on the p o s s i b l e
mechanism of ( b i o ) s o l u b i l i z a t i o n and to determine i t s present
limitations. A f i r s t s i g n i f i c a n t indication i s constituted
by the fact that in the i n i t i a l phase the d i s s o l u t i o n rates were
the same f o r both the moulds and the organic acids mixture
whereas, a f t e r one month, they were p r a c t i c a l l y reduced to zero
o r , as shown by F i g s . 5 and 6, the potassium contents of the
s o l u t i o n s showed a decrease.

The continuous slowing down of the potassium s o l u b i l i z a t i o n


rate observed in both the c u l t u r e s and the acid mixture might
denote that (a) l e u c i t e d i s s o l u t i o n takes place by i n d i r e c t
316 Giovanni Rossi

chemical attack, as already assumed by Goni, et a l . (17) and by


Keil^ in the case of biodegradation of other minerals in the
presence of heterotrophs and (b) the s o l u b i l i z a t i o n rate i s pro­
g r e s s i v e l y slowed down by the formation of a protective (inhibit­
ing) coating on the surfaces of the mineral p a r t i c l e s . The l a t t e r
explanation i s c o n s i s t e n t with the r e s u l t s of a research by Kruger
( 4 5 ) , who c a r r i e d out decomposition experiments with l e u c i t e in an
e l e c t r o d i a l y s i s apparatus, demonstrating that the potassium ions
at f i r s t go rapidly into s o l u t i o n and, a f t e r a protective r e s i d u e -
layer has been formed, migrate only very slowly. According to
Correns ( 4 6 ) , t h i s thin decomposition layer of S i C ^ and A ^ O ^
measures, at pH 3 with p a r t i c l e s of radius 1 ym, some 0.03 ym and
does not increase as the experimental time i s prolonged. Further­
more, since r e l a t i v e l y l i t t l e aluminum, as compared to potassium
and s i l i c o n , i s d i s s o l v e d o u t , Correns assumes that the residue
layer should be r i c h in aluminum.

The biodegradation process would thus s u f f e r , in t h i s case,


from the same drawbacks which have so f a r rendered useless the
e f f o r t s of the i n v e s t i g a t o r s who endeavoured to develop a chemical
process of l e u c i t e e x p l o i t a t i o n .

I t cannot be excluded that Scopulariopsis brevicaule gives


r i s e to d i r e c t attack. However, attempts made so f a r to ascertain
that t h i s kind of attack e x i s t s have up to now been u n s u c c e s s f u l ,
because of the intimate mixture of the biomass with the mineral
particles.

The maximum shown by the curves of F i g s . 5 and 6 and the


subsequent decreasing trends might be explained by the subtraction
of potassium from the s o l u t i o n s by the organisms. Experiments
are being c a r r i e d out presently to illuminate t h i s subject,
even though i t i s reasonable to believe t h a t , from the viewpoint

2
Paper to be published in the Proceedings of the "Round Table
on Leaching"' Braunschweig1977.
Λ
Bioextractive Applications and Optimization 317

of the a p p l i c a t i o n of the process on an i n d u s t r i a l s c a l e , large


volumes of biomass might create p r a c t i c a l problems.

As f a r as the drawback represented by the slowing down in the


s o l u b i l i z a t i o n rate i s concerned, a remedy can be found by r e -
grinding the ore in order to d i s e n c r u s t the surfaces of the p a r t i -
cles and to create new fresh s u r f a c e s . I t must not be f o r g o t t e n ,
however, that every comminution step increases s u b s t a n t i a l l y the
overall processing c o s t .

I n v e s t i g a t i o n s are presently under way aimed at throwing


l i g h t on these problems. Research aimed at the i s o l a t i o n of s i l i -
cobacteria capable of l e u c i t e and, more g e n e r a l l y , feldspar b i o -
degradation, i s a l s o being continued.

VI. ACKNOWLEDGMENTS

The encouragement of Prof. Mario C a r t a , Director of the M i n -


ing and Mineral Dressing I n s t i t u t e of the U n i v e r s i t y of C a g l i a r i ,
i s g r a t e f u l l y acknowledged. The author would a l s o l i k e to thank
Prof. Paolo P i g a , Director of the Mining I n s t i t u t e of Rome Univer-
s i t y f o r the d i s c u s s i o n s which led to the i n i t i a t i o n of the i n v e s -
t i g a t i o n and f o r supplying the l e u c i t e concentrate samples. It is
furthermore a great pleasure to thank Prof. Antonio Spanedda, of
the Microbiology I n s t i t u t e of the U n i v e r s i t y of C a g l i a r i , who,
throughout the years of i n v e s t i g a t i o n , gave the author the support
of h i s knowledge and encouragement. Thanks are a l s o due to Prof.
Giovanni A l f a n o , Mining and Mineral Dressing I n s t i t u t e of C a g l i a r i
U n i v e r s i t y , f o r performing the X-ray fluorescence spectrometer
a n a l y s e s , to Prof. Antonio Massidda, I n d u s t r i a l and Applied Chem-
i s t r y I n s t i t u t e , U n i v e r s i t y of C a g l i a r i , f o r performing the X-ray
d i f f r a c t i o n determinations, and to Antonella Rossi f o r the coop-
eration in performing a n a l y t i c a l work. The i n v e s t i g a t i o n was
financed by the C o n s i g l i o Nazionale d e l l e Ricerche, Centro Studi
Geominerari e M i n e r a l u r g i c i , Engineering F a c u l t y , U n i v e r s i t y of
Cagliari.
318 Giovanni Rossi

VII. REFERENCES

1. Deer, W., Howie, R.A., and Zussman, M.A., "Rock forming


m i n e r a l s " , V o l . 4 , p. 276. Longmans, Green & Co., L t d . ,
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3. Washington, H.S., "The Roman comagmatic r e g i o n " . The
Carnegie I n s t i t u t i o n , Washington, 1906.
4. Washington, H.S., Met. Chem. Eng., 18, 65 (1918).
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Perugia, 1927.
10. De G r a z i a , S . , and Camiola, G . , Le stazioni sperimentali
agrarie italiane, 39, 829 (1906).
11. B a s s a l i k , Κ., Zeitschrift fur Garungsphysiologie, 2 , 1 (1913).
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13. Goni^ J . , G u g a l s k i , T . , and Sima, M., Bull. Bur. Reoh. Geol.
Minieres (Fr)., Sect. 4, 1 , 31 (1973).
14. Boyle, J . R . , V o i g t , G.K., and Sawhney, B.L., Science, 155,
193 (1967).
15. V e r n a d s k i i , V . I . , "Ocierchi geochimii" ("A t r e a t i s e on Geo­
chemistry") in R u s s i a n , Moscow, 1934.
16. Alecsandrof, V . G . , T e s i s i dokladov na 5 - i i naucnoy conferen-
z i i Kuybicescova s . - x . i n - t a . 27-30 I a n v a r i i a 1937 g.
Kuybiscev, 1947.
17. Alecsandrof, V . G . , Dok. Vses. Akad. Sel skokhoz. Nauk., 3,
f

34 (1949).
18. Alecsandrof, V . G . , Dok, Vses. Akad. Sel skokhoz. Nauk., 12,
r

12 (1949).
19. Alecsandrof, V . G . , and Zac, G.A., Mikrobiologiya, 19, 97 (1950).
20. Norchina, S . P . , and Pumpianscaia, L.V., Dok. Vses. Akad.
Sel'skokhoz. Nauk., 27 (1956).
21. Surman, K . J . , Byul. Nauchn.-Tekhn. Inform, po s-ch Mikrobiol.,
2 (1956).
22. Surman, K . J . , Docl. Vaschnil., 4 (1958).
Bioextractive Applications and Optimization 319

23. Surman, K . J . , Byul. Nauchn.^Tekhn. Inform, po s-chMikrobial.,


5 (1958).
24. Surman, K . J . , Sb. Bacterialni udobriva, Kiev, 1959.
25. Surman, K . J . , Sb. Bacterialni udobriva, Kiev, 1961.
26. Surman, K . J . , Avtoreferat D i s s e r t . , Moscow, 1962.
27. T e s i c , Z . , and Tudorovic, M., Zemljiste i biljka, 1 . 1 , 3
(1952).
28. T e s i c , Z . , and Todorovic, M., in " A t t i Congr. I n t e r n .
M i c r o b i o l . 2°-Rome, 1953", 6, 356 (1955).
29. T e s i c , Z . , and Todorovic, Μ., Zemljiste i biljka, V I I I , 233
(1958).
30. Alecsandrof, V . G . , Tr. Konf. po Vopr. Poch, Microbiol.
Kompleksa Dokuchaeva-Kostycheva-Vil'yamsa, Inst. Mikrobiol.,
Akad. Nauk SSSR, Moscow, 1 9 5 1 , 271 (1953).
31. Alecsandrof, V . G . , Tr. Sovesh. po Vopr. Bacter. Udobr., 1956,
Kiev, I z v . Akad. Nauk SSSR, 62 (1958).
32. Alecsandrof, V . G . , Burangulova, MN., and S o l o v ' e v a , E.P.,
Tr. Odessk. Sel'skokhoz. Inst., 13, 91 (1958).
33. Alecsandrof, V . G . , and Ternovskaia, M . J . , "A methodic manual
f o r l i q u i d preparation of s i l i c a t e b a c t e r i a " , Tr. Odessk.
Sel'skokhoz. Inst., 14 (1961).
34. Alecsandrof, V . G . , Ternovskaia, M . J . , and B l a g o d i r , R.N.,
Vest. Sel'skokhoz. Nauki, Vses. Akad. Sel'skokhoz. Nauk SSSR,
1 1 , 95 (1963).
35. Alecsandrof, V . G . , and Kodra, V . A . , Dokl. Akad. Sel'skokhoz
Nauk SSSR, 1 , 10 (1965).
36. Heinen, W., Arch. Mikrobiol., 37, 199 (1960).
37. Heinen, W., Arch. Mikrobiol., 45, 172 (1967).
38. V e n t r i g l i a , U., Ind. Mineraria (Rome), 1 , 371 (1950).
39. P i g a , P., Boll. Teen. Ing. e Arch. Sardi, 3-4 (1967).
40. Fontanive, F., and M a s s a c c i , P., Ind. Mineraria (Rome), 9,
279 (1968).
41. R o s s i , G., Resoconti Ass. Miner. Sarda, ( I g l e s i a s ) , 76, 5 (1971).
42. R o s s i , G . , Resoconti Ass. Miner. Sarda, ( I g l e s i a s ) , 74, 5 (1969).
43. Silverman P., and Lundgren, D . G . , J . Bacterid., 77, 642 (1959).
44. Correns, C.W., Kolloid-Z, 34, 341 (1924).
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OPTIMUM CONDITIONS FOR LEACHING OF URANIUM AND OXIDATION OF LEAD

SULFIDE WITH Thiobaoillus ferrooxidans AND RECOVERY OF METALS FROM

BACTERIAL LEACHING SOLUTION WITH SULFATE-REDUCING BACTERIA

Noboru Tomizuka
Mitsuo Yagisawa

Fermentation Research I n s t i t u t e
Chiba, Japan

The optimum conditions for leaching of uranium and oxidation


of lead sulfide in the presence of T. ferrooxidans and the recovery
of metals from bacterial leaching solution with sulfate-reducing
bacteria were investigated on a laboratory scale.
The optimum leaching of uranium occurred when the oxidation-
reduction potential increased to above 0. 75V and the pH decreased
to below 2.0. The continuous culture and the continuous leaching
of uranium were carried out in an apparatus for single stage
continuous culture. 'Wash out' and the highest output of cells
and ferric irons were observed at the dilution rate 0.10/hr and
0.062/hr respectively and over 80% of the uranium could be leached
at a dilution rate below 0.011/hr.
In the virtual absence of iron, the microbiological oxidation
of galena was limited. With addition of iron to the medium, the
rate of oxidation and the percentage oxidation could be increased
until other factors became limiting. Iron in amounts correspond-
ing to 10% of the galena on a molar basis was found to be most
effective.
A mixed culture of sul fate-reducing bacteria exhibited
superior ability to recover metals from bacterial leaching solu-
tion. A recovery treatment employing continuous culture was
effective. Batch culture was impossible. The optimum pH was 6.0,

321
322 Noboru Tomizuka and Mitsuo Yagisawa

which agreed with that for growth. The maximum rate of metal
removal for copper, zinc and iron was 47\5mg/i/hr, 18. lmg/l/hr and
25.7mg/i/hr, respectively. Content of copper, zinc and iron in
the black precipitate was about 6.9, 38.0 and 1.0 times that of
the original sulfide ore, respectively.

I. INTRODUCTION

Heap or dump leaching and in-place leaching have become today


standard methods i n e x t r a c t i v e metallurgy. Hydro-metallurgical
processes are playing an i n c r e a s i n g l y important role in the e x t r a c -
t i o n of both the common and the rare metals. On the other hand i t
i s now widely recognized that c e r t a i n kinds of microorganisms play
an important role i n d i s s o l u t i o n of metals from ore and in p r e c i p i -
t a t i o n of metals from metal s o l u t i o n .

The microorganisms involved i n these phenomena are c u r r e n t l y


used or have been suggested f o r use i n two hydro-metallurgical
processes, namely, the d i s s o l u t i o n of the desired metal from ore
into s o l u t i o n and the recovery of the desired metal from the metal
s o l u t i o n or mine drainage. The f i r s t of those processes c a l l e d
bacterial leaching i s used in the f o l l o w i n g e x t r a c t i v e process-
es of metallurgy: 1) in-place l e a c h i n g , 2) dump or heap l e a c h i n g ,
3) leaching i n s t i r r e d or agitated r e a c t o r s .

The present paper i s a summary of a number of published


a r t i c l e s and l a r g e l y concerned with the establishment of the o p t i -
mum conditions f o r bacterial uranium leaching and bacterial lead
oxidation on a laboratory s c a l e . B r i e f mention of recovery of
metals from a bacterial leaching s o l u t i o n with sulfate-reducing
bacteria i s a l s o made.

II. METHODS, RESULTS,DISCUSSION,AND CONCLUSION

A. Bacterial Leaching of Uranium (1,2)

The i n v e s t i g a t i o n was i n i t i a t e d i n order to f i n d those micro-


organisms which are able to leach s i g n i f i c a n t q u a n t i t i e s of
Bioextractive Applications and Optimization 323

Table I Isolation of Iron and/or Sulfur


Oxidizing Microorganisms

Strain No. 4Fe No. UFe No. 19Fe No. 2S No. US

Size (micron) 0.5 X 1.5 0.5 X 2.0 0.5 X 1.5 0.5 X 1.0 0.5 X 2.0
Opt. temp. CO 27 27 - 30 30 27 27 - 30
Opt. initial pH 2.5 - 3.0 2.5 2.0 - 3.0 3.0
Gram reaction — — — —
Flagellum — — + +
NaNO + + + - ++
(NHJJSOj.
4 & 4 +++ +++ +++ +++ +++
Liquia medium
FeSO. 7HJD + + + + + - -
S - ++ + ++++ ++++
Solid medium
FeSO 7H 0 + 2 + +
Na
2 2°3
S
^ 2°H
+ + + + +

uranium from i t s ores and to c l a r i f y which of them are applicable


to in-place leaching of uranium.

The Ningyo-toge uranium deposit used f o r t h i s study i s of the


sedimentary type and i s composed of conglomerate and arkose sand-
stone j u s t above the unconformity plane. The ore contains a t e t r a -
valent uranium mineral c a l l e d ningyoite ( C a U i P O ^ nh^O ) which
occurs coating the surface of pebbles and cementing the matrices
of the conglomerates. P y r i t e i s intimately associated i n concent-
r a t i o n with ningyoite and i s u s u a l l y contained at l e s s than 1.0%.
The grade of the uranium ore i s g e n e r a l l y 0.03 to 0.15% U^Og.

1. Isolation and Selection of a Microorganism which Has Efficient


and Stable Uranium Leaching Ability

I s o l a t i o n of s u l f u r and/or ferrous i r o n o x i d i z i n g microorga-


nisms from ores and mine waters of the Ningyo-toge mine was mainly
c a r r i e d out i n a modified 9K medium ( composition of medium
( g/L ): F e S 0 « 7 H 0 , 5 0 . 0 ; K H P 0 , 0 . 5 ; MgS0
4 2 2 4 4 7 H 0 , 0 . 5 ; KC1, 0 . 1 ;
2

( N H ) S 0 , 3.0; C a ( N 0 ) , 0 . 0 1 ; pH 3.0 ).
4 2 4 3 2 Some of the properties
of the f i v e s t r a i n s which were i s o l a t e d are shown i n Table I . No.
U F e had the most e f f i c i e n t and stable uranium leaching a b i l i t y
among them and exhibited many c h a r a c t e r i s t i c s which were s i m i l a r
324Nobor uTomizuk
aan dMitsu
oYagisaw
a

0.95

>

0.75
ω

0.55

"0 2 4 6 8 10 12 14
Leaching time (days)

Fig. 1. Uranium leaching by T. ferrooxidans at 25'C. The


incubation flask contained lOg ore and 100ml of Q.03% ammonium
sulfate solution ( pH 3.0 ) and was shaken on a rotary shaker.

to those of Thiobaoillus ferrooxidans.

Figure 1 shows a t y p i c a l uranium leaching curve of s t r a i n No.


Π Fe on a medium i n which 0.03% ammonium s u l f a t e alone was d i s s o l ­
ved.

2. Role of the Microorganism on Uranium Leaching

As shown i n F i g . 1 , over 90% of the uranium was leached when


the pH decreased to below 2.0 and the oxidation-reduction potential
increased to above 0.75V. According to the Eh - pH diagram of
U - 0 - H 0 - ( S - N a - C 0 ) , the area of the pH and the Eh i s that of
2 2 2

uranyl s u l f a t e .

D i a l y s i s c u l t i v a t i o n was c a r r i e d out to t e s t whether d i r e c t


microbiological action on the ores was necessary f o r uranium l e a c h ­
ing or not. In t h i s c u l t i v a t i o n , no microorganisms but only the
metabolic products were allowed to d i f f u s e through a c e l l u l o s e
membrane from the culture chamber to the ore containing r e s e r v o i r .
As shown i n F i g . 2 , the pH was s l i g h t l y decreased by the addition
of s u l f u r i c acid to a range s u i t a b l e f o r bacterial growth. The
decrease i n the pH caused by the metabolic product was accompanied
Bioextractive Applications and Optimization 325

Fig. 2. Uranium leaching on the modified 9K medium by T.


ferrooxidans in the dialysis flask. The culture chamber contained
100ml medium ( pH 4.5 ) and was inoculated with 2.0ml of T.
ferrooxidans maximum culture. The ore containing reservoir
contained 80g ore and 700ml of energy source-free medium ( pH
4.5 ). By the addition of sulfuric acid the pH was slightly
decreased to a suitable pH range ( below pH 4.0 ) for the growth.
Arrows show the points at which sulfuric acid was added. Cellu-
lose membrane was used as the semipermeable membrane.
0.15r

Fig. 3. Effect of pH on specific growth rate. Cultivation


was conducted on the modified 9K medium at 30°C and at 8.5 X 10 -7
g mole O^/ml min atm.
326 Nobor
uTomizuk
aandMitsu
oYagisaw
a

by an increase i n the leached uranium concentration of the s o l u ­


tion. This r e s u l t shows that the microorganism does not d i r e c t l y
attack uranium mineral, but rather that i t sets up chemical c o n d i ­
t i o n s s u i t a b l e f o r the d i s s o l u t i o n of uranium.

From these r e s u l t s i t i s suggested that the microorganism


plays an i n d i r e c t role i n the supply of f e r r i c i r o n and s u l f u r i c
acid and these o x i d i z i n g agents oxidize tetravalent uranium to the
a c i d - s o l u b l e hexavalent s t a t e . Thus, the leaching processes of
uranium mineral from the Ningyo-toge ore i s thought to be as
follows:

CaU(P0 ) 4 2 + Fe (S0 ) 2 4 3 + H S02 4 + 2H 0 2

— ^ U0 S02 4 + 2FeS0 + 2 H P 0 4 3 4 + CaS0 4

4FeS 2 + 150 2 + 2H 0 S £ ^ £ l
2
B a
2Fe (S0 )
2 4 3 + 2H S0
2 4

4FeS0 + 2 H S 0
4 2 4 + 0 2
B a
££££L a
2Fe (S0 )
2 4 3 + 2H 0 2

3. Optimum Culture Conditions and Continuous Culture of T,


ferrooxidans

A l l experiments were c a r r i e d out i n a 2 - l i t e r fermentor with


1-1 i t e r working volume on the modified 9K medium.

In batch culture i t was observed that there was a p a r a l l e l


r e l a t i o n s h i p between the increase of f e r r i c iron concentration and
of c e l l numbers. As shown i n F i g s . 3, 4 and 5, f o r the s p e c i f i c
growth r a t e , the optimum pH, temperature and aeration were between
2.3 and 2.7, between 29 and 34°C and above 5.0 Χ 1 0 " 7
g mole 0 /ml
2

min atm, r e s p e c t i v e l y . Ammonium s a l t s , urea and c e r t a i n kinds of


amino acids were used as nitrogen sources. Of these, ammonium
s u l f a t e was the most e f f e c t i v e f o r the growth and i t s optimum con­
centration was 0.3%. The optimum concentration of ferrous s u l f a t e
( FeS0 4 7H 0 ) as the energy source was 5%.
2 The organism was
capable of growing on glucose i n the presence of an inorganic
energy source. The growth was i n h i b i t e d by e x t e r n a l l y added keto
acids having r e l a t i v e l y small pKa value such as pyruvic a c i d , o ( -
Bioextractive Applications and Optimization 327

0.201

o.io
h

a>o.o3h

0.01
3.1 3.2 3.3 3.4 3.5 3.6
Temperature (l/TXlO^/T ) 1

Fig. 4. Effect of temperature on specific growth rate.


Cultivation jjas conducted on the modified 9K medium at pH 2.7 and
at 8.5 X 10" g mole 0^/ml min atm.

0.15

I 0.10
?
Ο
u
ο 0.05

1 2 5 10 20 50
Volumetric oxygen-transfer coefficient (kdXlQ)
7

Fig.5. Effect of aeration on specific growth rate. Cultiva­


tion was conducted on the modified 9K medium at pH 2.7 and at 30'C.

k e t o - g l u t a r i c acid and maleic a c i d .

Continuous culture was conducted i n a s i n g l e stage system.


The fermentor was f i r s t run batchwise f o r a few days and then was
s h i f t e d to continuous operation. F i g . 6 presents the e f f e c t of
the d i l u t i o n rate on c e l l growth and the oxidation of ferrous i r o n .
'Wash o u t ' and the highest output of c e l l s and f e r r i c iron were
observed at the d i l u t i o n rate of 0.10/hr and 0.062/hr, r e s p e c t i v e ­
ly. The curves i l l u s t r a t e d i n F i g . 6 show that i t i s not p o s s i b l e
to predict continuous culture behavior from batch culture data.
328 Noboru Tomizuka and Mitsuo Yagisawa

Dilution rate (hr *)

Fig. 6. Effect of dilution rate on cell growth and oxidation


of ferrous sulfate. Cultivation was conducted on the modified 9K_,
medium. Temperature, pH and aeration were 30° C, 2.7 and 8.5 X 10
g mole 0Jml min atm, respectively. # concentration of cells;
Ο tput of cells; • concentration of ferrous iron;Μ
ou

output of ferric iron.

Time (hr)

Fig. 7. Relationship between ferrous oxidation and uranium


leaching. Leaching of uranium was conducted on the modified 9K
medium with 10% ore at 30°C. Ο oxidation of ferrous iron;
• pH; # concentration of uranium leached.
Bioextractive Applications and Optimization 329

Fig. 8. Effect of pH on specific oxidation rate and specific


teaching rate. Leaching of uranium was conducted on the modified
9K medium with 10% ore at 30°C. # specific leaching rate of
uranium; Ο specific oxidation rate of ferrous iron.

0.15

-c

u 0.10
c
)xida

0.05

oi ι ι ' » ι
0 2.5 5.0 7.5 10.0
Pulp density {%)

Fig. 9. Effect of pulp density on specific oxidation rate


of ferrous iron. Leaching of uranium was conducted on the
modified 9Κ medium at 30°C and at pH 2.0.

The reason has not y e t been determined.

4. Optimum Conditions for Bacterial Leaching of Uranium and


Continuous Leaching of Uranium

The r e l a t i o n s h i p between the c h a r a c t e r i s t i c s of the uranium


ore and the necessary amount of p y r i t e or f e r r i c i r o n f o r uranium
leaching could not be determined. The ore used i n t h i s study
contained l e s s p y r i t e than necessary i n order to carry out e f f i -
330 Noboru Tomizuka and Mitsuo Yagisawa

cient uranium leaching i n a short incubation time. In general the


addition of 5%( W/V ) ferrous s u l f a t e ( F e S 0 * 7 H 0 ) was s u f f i c i e n t
4 2

to induce bacterial uranium leaching from the Ningyo-toge uranium


ore. Therefore, the modified 9K medium was used throughout the
tests. F i g . 7 shows the time course curves of uranium leaching
and ferrous o x i d a t i o n . The increase of leaching r a t i o was not
d i r e c t l y related to the r a t i o of ferrous o x i d a t i o n , but r a t h e r ,
as already demonstrated i n F i g . 1 , i t was found that low pH and
high oxidation-reduction potential are e s s e n t i a l to the uranium
leaching.

Effect of pH on s p e c i f i c oxidation rate of ferrous iron and


s p e c i f i c leaching rate of uranium i s i l l u s t r a t e d i n F i g . 8. The
optimum pH f o r uranium leaching was about 2.0 and there was absence
of a p a r a l l e l r e l a t i o n between uranium leaching and ferrous o x i d a -
t i o n in the terms of s p e c i f i c rate.

Effect of pulp density on s p e c i f i c oxidation rate of ferrous


i r o n i s presented i n F i g . 9. At low pulp d e n s i t i e s , uranium ore
contributed i n a p o s i t i v e manner to the oxidation of ferrous i r o n
added. The reason why the addition of ore has t h i s promoting
e f f e c t i s not c l e a r .

Continuous leaching of uranium was c a r r i e d out with 10% ore


at pH 2.0 and at 30°C. Data on the continuous leaching of uranium
at d i f f e r e n t d i l u t i o n rates i s shown i n F i g . 10. The highest o u t -
put of uranium leached was observed at the d i l u t i o n rate of about
0.060/hr, which was numerically, nearly equal to the d i l u t i o n rate
of the highest output of c e l l s and f e r r i c i r o n s ( F i g . 6 ), But,
to leach over 80% of the uranium contained i n ore employed, i t was
necessary to operate the continuous leaching at a d i l u t i o n rate of
under 0.011/hr. However, from F i g s . 6 and 8, i t i s seen that
continuous leaching with high recovery r a t i o of uranium at higher
d i l u t i o n rate may be f e a s i b l e by conducting the continuous leaching
in two-vessels with the continuous culture c a r r i e d out i n a f i r s t
vessel and the continuous leaching in a second v e s s e l .
Bioextractive Applications and Optimization 331

1001 1 1 1 r

Dilution rate (hr *)

Fig. 10. Continuous leaching of uranium on the modified 9K


medium. Leaching of uranium was conducted with 10% ore at pH 2.0
and at 30° C. m ratio of uranium leached; ο output of uranium
leached; φ concentration of ferrous iron; Ο output of ferric
iron.

5. Conclusion

I t has been demonstrated that Thiobacillus ferrooxidans,


which has e f f i c i e n t and stable uranium leaching a b i l i t y and i s
n a t u r a l l y present i n the Ningyo-toge uranium d e p o s i t , i s capable
of continuously leaching uranium i n s t i r r e d and/or agitated reac­
tors. However, no consideration has yet been given to the problem
of l a r g e - s c a l e operation. At any r a t e , i t i s not believed that
in-place leaching and dump ( heap ) leaching are p r a c t i c a l l y a p p l i ­
cable to Ningyo-toge uranium ore. The reasons are as f o l l o w s ;
(1) The Ningyo-toge uranium deposit i s of small s c a l e .
(2) The ore contains l e s s p y r i t e than necessary i n order to
carry out e f f i c i e n t uranium leaching.
(3) There are many sandstone l a y e r s i n the uranium deposit
which are impervious to the leaching s o l u t i o n .
(4) The ore i s so r i c h i n clay that i t i s d i f f i c u l t to supply
a i r i n t o the deposit or an a r t i f i c i a l ore heap.
(5) At low pH there was observed decomposition of the clay
332 Noboru Tomizuka and Mitsuo Yagisawa

cementing cracks of the unconformity plane. This means


the leaching s o l u t i o n would escape through the cracks.

B. M i c r o b i o l o g i c a l Oxidation of a Lead S u l f i d e (3)

The oxidation of lead s u l f i d e ( galena ) in the presence of


T. ferrooxidans has been i n v e s t i g a t e d . Information regarding the
b i o l o g i c a l oxidation of lead s u l f i d e i s scarce and c o n f l i c t i n g
conclusions have been reported.

I n t h i s study, lead s u l f i d e ( galena ) was obtained from the


carboniferrous limestone deposits of South Wales ( U. K. ) and
was crushed to pass a 300-mesh B. S. screen. The sample was found
to contain 84.5 - 86.2% lead and l e s s than 0 . 1 % i r o n . Oxidation
t e s t s were c a r r i e d out i n Erlenmeyer f l a s k s on 100ml of the
modified 9K medium with lead s u l f i d e replacing ferrous s u l f a t e as
an energy source. The temperature was maintained at 35 C and the
pH was manually c o n t r o l l e d by d a i l y adjustment back to an i n i t i a l
pH value with s u l f u r i c acid or sodium hydroxide during the o x i d a -
t i o n process. Oxidized lead was measured by atomic absorption
spectrophotometry a f t e r i t had been d i s s o l v e d i n t o the aqueous
phase by t r e a t i n g samples with 10% diethylenetriamine ( DETA )
s o l u t i o n at room temperature.

1, Oxidation of Galena in an Iron-free Medium

The a b i l i t y of T. ferrooxidans to use galena as an energy source


i n a medium i n the v i r t u a l absence of i r o n was measured by moni-
t o r i n g the oxidation of galena and the b a c t e r i a l growth. F i g . 11
shows t y p i c a l r e s u l t s with d i f f e r e n t i n i t i a l concentrations of
bacteria. The r e s u l t s i l l u s t r a t e the a b i l i t y of T. ferrooxidans
to p a r t i c i p a t e i n the oxidation of galena and use i t as an energy
source f o r growth. I t can be seen that the rate of oxidation i s
growth associated and the oxidation curve shows a logarithmic
increase with culture time.

Optimum conditions f o r galena oxidation were as f o l l o w s :


Bioextractive Applications and Optimization 333

1001 1 1 1 1 1 | 1

4 6 8 10 12 14
Oxidation time(days)
Fig. 11. Oxidation of galena by T. ferrooxidans and its
growth in an iron-free medium. Reaction conditions were as fol­
lows: volume of medium, 100ml; galena, 2.5g; pH, 2.5; inoculum as
cellular nitrogen, • 0.49mg, Ο 0.98mg, · Omg.

(1) A p a r t i c l e s i z e f o r the galena of l e s s than 50 microns,


i . e. -300 mesh B. S. screen.
(2) A s o l i d s concentration lower than 4 grams/lOOml o f medium.
(3) A pH value f o r the t e s t s of 2.0.

As shown i n F i g . 12, the pH of the culture was found to have


a pronounced e f f e c t . At pH values lower than 1.3 the percentage
oxidation of galena was found to be the same i n the inoculated
f l a s k as i n the control.Although the inoculum s i z e could a l s o be
seen to have a d e f i n i t e e f f e c t on the oxidation p r o c e s s , the f i n a l
percentage oxidation was the same f o r a l l inoculum s i z e s .

Under these conditions when an inoculum of 0.0068mg/ml of


medium as c e l l u l a r nitrogen was used, the maximum percentage o x i ­
dation obtained was 60.0% and the c e l l y i e l d per gram of galena
oxidized was 1.6mg as c e l l u l a r n i t r o g e n . The doubling time of the
microbe was found to be 4.2 days and consequently the s p e c i f i c
334 Noboru Tomizuka and Mitsuo Yagisawa

1001

20h

I ι ι , , I
0 1 2 3 4
pH
Fig. 12. Effect of pH on final percentage oxidation in an
iron-free medium and in an iron added medium. Reaction conditions
were as follows: volume of medium, 100ml; galena, 2.5g; Ο in a
0.001 moles iron added medium with microbes ( as cellular nitrogen,
0.48mg ); Ο in an iron-free medium with microbes ( as cellular
nitrogen, 0.79mg ); φ in an iron-free medium without microbes.
Crosses show the results are those obtained after the incubation
for 8 days.

oxidation rate and the s p e c i f i c growth rate were 6.9 X 10 /hr.

2. Oxidation of Galena in an Iron Added Medium

The marked influence of i r o n on the oxidation response of


galena i s i l l u s t r a t e d by the oxidation curves i n F i g . 13. They
show t h a t , once, the maximum percentage oxidation of galena was
obtained i n the absence of i r o n , further oxidation of the galena
was produced when i r o n was added. Addition of ferrous s u l f a t e to
the control produced no increase i n the amount of galena o x i d i z e d .

The optimum concentration of i r o n was found to be 0.001 moles


of i r o n per 100ml of medium. Iron additions i n excess of the o p t i ­
mum concentration were i n h i b i t o r y . The reason may be t h a t , as the
Bioextractive Applications and Optimization 335

100

I ι ι ι I
0 4 8 12 16
Oxidation time(days)
Fig. 13. Effect of iron on oxidation of galena. Reaction
conditions were as follows: volume of medium, 100ml, q pH 2.0,
2.3g galena and 2.64mg microbes as cellular nitrogen; q pH 3.0,
2.5g galena and 0.66mg microbes as cellular nitrogen; φ pH 2.0,
and 2.3g galena without microbes. Arrows show the points at which
0.001 moles ferrous sulfate was added.

i r o n i s oxidized to the f e r r i c form, a large amount of i t p r e c i p i ­


tates as ammonium f e r r i c s u l f a t e which contributes to the oxide
coating of the galena surface l i m i t i n g the oxidation process.

Figure 14 i l l u s t r a t e s the r e l a t i o n s h i p observed between the


galena oxidation and the oxidation of i r o n from the ferrous to the
f e r r i c form. The rate of o x i d a t i o n of galena was enhanced and the
f i n a l percentage oxidation was much higher than i n corresponding
t e s t s without i r o n a d d i t i o n s . The amount of extracted lead was
found to increase l o g a r i t h m i c a l l y while ferrous i r o n was oxidized
with the i n c r e a s i n g oxidation rate but was not completely oxidized
u n t i l a f t e r the galena had ceased to o x i d i z e .

Typical galena oxidation curves and the growth curves on the


medium with i r o n a d d i t i o n s are shown i n F i g . 15. The oxidation of
336 Noboru Tomizuka and Mitsuo Yagisawa

Oxidation time(days)
Fig. 14. Relationship between galena oxidation and ferrous
oxidation. Reaction conditions were as follows; volume of medium,
100ml; galena, 2.3g; pH, 3.0; iron, 0.001 moles ferrous sulfate;
inoculum as cellular nitrogen, 0.44mg, Ο , Ο with microbes,
• * W without microbes.

galena was a l s o found to be associated with bacterial growth i n a


s i m i l a r manner to oxidation t e s t s performed with i r o n - f r e e medium.
The f i n a l percentage oxidation was approximately 82.0% f o r most of
the t e s t s performed.

The optimum conditions i n the i r o n added medium at 35*C were


nearly the same as observed i n the i r o n - f r e e medium, except that
optimum pH was about 3.0 as shown i n F i g . 12. For a t y p i c a l test
performed at pH 3.0 with 2.5g of galena and an inoculum s i z e of
0.0088mg of c e l l u l a r nitrogen per ml of s o l u t i o n i n the presence
of 0.001 moles of ferrous s u l f a t e , the maximum percentage o x i d a ­
t i o n obtained was 83.5%, the doubling time of the microbe was 1.6
days ( s p e c i f i c growth and oxidation rate were 1.8 X 10 /hr ) and
the c e l l y i e l d per gram of galena oxidized was 1.8 mg as c e l l u l a r
nitrogen.
Bioextractive Applications and Optimization 337

1001 1 1 1 , , , ,

ι i 1 ί- 1 ι ι I 0
0 2 4 6 8 1 0 12 14
Oxidation time(days)
Fig. 15. Oxidation of galena by T. ferrooxidans and its
growth on iron added medium. Reaction conditions were as follows:
volume of medium, 100ml; galena, 2.5g; pH, 3.0; iron, 0.001 moles
ferrous sulfate; inoculum as cellular nitrogen, α 0.43mg,
Ο 0.86mg, φ Omg.

3. Mechanism of Oxidation of Galena

As shown i n Table I I , the formation of s u l f u r as predicted by


equation (1) was noted by an e x t r a c t i o n ( with carbon d i s u l f i d e )
of residue obtained from control t e s t s with galena i n the presence

PbS + H S 0
2 4 + l/20 2 ^ PbS0 + H 0 + S
4 2 (1)

and absence of i r o n . The incomplete chemical oxidation of galena


i s probably due to the formation of lead s u l f a t e and s u l f u r reac­
t i o n l a y e r s masking the reactive s u l f i d e s u r f a c e . In oxidation
t e s t s performed upon galena i n the presence of bacteria under ex­
perimental conditions i d e n t i c a l to those of the control t e s t s , i t
was found that no s u l f u r was detected in the t e s t s without i r o n
additions and only very small q u a n t i t i e s i n the t e s t s with i r o n
additions.

The role of the bacteria i n the oxidation of galena i n the


338 Noboru Tomizuka and Mitsuo Yagisawa

Table II Formation of Sulfur During Oxidation of Galena


in the Presence and Absence of T. ferrooxidans

Reaction conditions Oxidation Sulfur


, 4 ( mg )
2 rat%o
Galena 1

Microbe Irort* pH ( % ) Expected Found

2.5g — — 2.0 23.6 19.8


2.5g + — 2.0 59.1 154.0 0
2.5g — + 3.0 25.4 42.9 32.6
2.5g + + 3.0 79.1 219.0 8.4

(1) Galena sample less than 45 micron ( minus 400-mesh ) in


particle size of which 12.3% had been oxidized to lead
sulfate during preparation.
(2) Inoculum size used in tests with microbes 0.88mg as cellular
nitrogen;
(3) Concentration of ferrous sulfate employed 0.001 moles/lOOml
medium;
(4) Incubation period, 10 days;
(5) Sulfur as calculated from equation (1) and (2).

absence of i r o n would appear to l i e i n bacterial oxidation of any


s u l f u r formed during the reaction expressed by equation (1) thus
maintaining the acid level and thus removing s u l f u r from the s u l -
fide surface allowing further oxidation of s u l f i d e to proceed.
Therefore the extent of f i n a l percentage o x i d a t i o n of galena
depends mainly on the acid reaction expressed by equation ( 1 ) .

The precise reason why the rate of oxidation and the f i n a l


percentage oxidation of galena i n the presence of i r o n with bacte-
r i a were much higher than those t e s t s without i r o n i s not immedi-
ately apparent. However, i t i s suggested that one of the r o l e s of
i r o n i n themediummay be explained as the role of enhancing chemi-
cal oxidation of the galena producing elemental s u l f u r , as

PbS + F e ( S 0 )
2 4 3 ^ PbS0 + 2FeS0 + S
4 4 (2)

expressed by equation ( 2 ) , and i t may a l s o be involved i n i n c r e a s -


ing the a c t i v i t y of the bacterial o x i d i z i n g enzyme system. It is
a l s o suggested that f e r r i c i r o n produced by the oxidation of f e r -
rous i r o n i n the medium acts as an 'oxygen c a r r i e r 1
which promotes
Bioextractive Applications and Optimization 3 3 9

the oxidation of the l e s s a c c e s s i b l e surface area of the galena


p a r t i c l e s which i s unable to react with s u l f u r i c acid and oxygen
i n the manner expressed by the reaction i n equation ( 1 ) .

4. Conclusions

I t has been demonstrated that T. ferrooxidans can be a c t i v e l y


employed i n the enhancement of the oxidation of lead s u l f i d e i n
r e l a t i v e l y weak acid s o l u t i o n s and that the presence of i r o n a l s o
improves both the oxidation rate and f i n a l percentage o x i d a t i o n .
I t i s p o s s i b l e that such reactions may be useful as a preliminary
step i n the recovery of lead from s u l f i d e concentrates v i a an
amine e x t r a c t i o n route.

C Recovery of Metals from a Bacterial Leaching S o l u t i o n with


Sulfate-reducing Bacteria (4)

The study i s related to the recovery of metals from bacterial


leaching s o l u t i o n and acid mine drainage, which are characterized
by high concentrations of s u l f a t e , metals and hydrogen i o n .

The mixed culture of s u l f a t e - r e d u c i n g bacteria used i n t h i s


study was i s o l a t e d from a s o i l sample obtained near our I n s t i t u t e .
The basal medium was as follows ( g/L ): N a S 0 , 1.0; C a C l 2 4 2 6H 0,
2

0 . 1 ; M g S 0 , 1.0; ( N H ) S 0 , 1.0; F e S 0
4 4 2 4 4 7H 0, 0 . 1 1 ; ^-glycerophos-
2

phate, 0 . 1 ; yeast e x t r a c t , 0 . 1 . S u l f a t e was supplied by N a S 0 2 4

s a l t s o l u t i o n or the supernatant of bacterial leaching s o l u t i o n


which was prepared from copper s u l f i d e ore with T. ferrooxidans
( medium ( g/L ): ( N H ) S 0 , 1.0; K H P 0 , 0 . 1 ; MgS0 7 H 0 , 0 . 1 ;
4 2 4 2 4 4 2

KC1, 0.05 ). Sodium lactate was used as an energy source and the
amount thereof added to inoculated s o l u t i o n s was calculated from
the following r e a c t i o n :

2CH CH0HC00" + H S 0 "


3 4 HS" + 2CH C00~ + 2C0 + H 0
3 2 2

The c u l t u r e s were incubated i n a 1 - l i t e r fermentor with 0 . 5 - l i t e r


working volume at 30°C throughout the experimental work.
340 Noboru Tomizuka and Mitsuo Yagisawa

level controller pH c o n t r o l l e r

Fig. 16. Test procedure for continuous recovery of metals


from bacterial leaching solution.

1. Continuous Culture of Sulfate-reducing Bacteria

Batch culture of s u l f a t e - r e d u c i n g bacteria on the supernatant


of bacterial leaching s o l u t i o n was impossible, because the low pH
and high concentration of metals of the supernatant were o b s t r u c -
t i v e to growth. However, the bacteria grew in batch culture on
basal medium and the pH of the incubated s o l u t i o n gradually
increased during the growth.

Continuous culture was conducted in a s i n g l e stage system as


shown i n F i g . 16. The fermentor was f i r s t run batchwise with the
basal medium and then was s h i f t e d to continuous operation with the
supernatant of bacterial leaching s o l u t i o n . Namely, the addition
of the supernatant f o r the adjustment back to an i n i t i a l pH value
during the c u l t i v a t i o n means the continuous feeding of fresh
Bioextractive Applications and Optimization 341

Time (hr)
Fig. 17. Continuous recovery of metals from bacterial leach­
ing solution with sulfate-reducing bacteria. The supernatant
contained 55.0ppm Cu, 32.0ppm Fe and 20.5ppm Zn. Continuous
recovery was conducted at 30'C. Ο Fe; Cu; Jk» Zn.

medium to the incubated s o l u t i o n . A g i t a t i o n with a magnetic


s t i r r e r was c a r r i e d out only during the period of pH adjustment.
To maintain constant volume i n the reaction v e s s e l , the c e l l s and
products were removed at the same rate as the feed r a t e .

Figure 17 shows the r e s u l t s obtained i n one example of c o n t i ­


nuous c u l t u r e . The steady state was achieved w i t h i n 40 hours.
The maximum feed rate of bacterial leaching s o l u t i o n was
270.8ml/hr, which was calculated from the slope between A and B.

2. Effect of pH and Metal Concentration on Growth Rate and Rate


of Removal of Metals

The s p e c i f i c growth rate and the rate of removal of metals


from bacterial leaching s o l u t i o n were found to be s t r o n g l y i n f l u ­
enced by the pH of the c u l t u r e . As shown i n F i g . 18, the optimum
pH f o r metal removal was 6 . 0 , which agreed with the optimum pH
f o r s p e c i f i c growth.
342 Noboru Tomizuka and Mitsuo Yagisawa

30

0.75 i fa

HO.50 Jl

0 J
0
5.0 6.0 7.0
pH
Fig. 18. Effect of pH on metal removal rate and specific
growth rate. Cultivation was conducted at 30 C. The solutions
with different pH values each contained Na-lactate ( I0.3g/l ) and
yeast extract ( 0.1g/l ) and had a metal concentration of 55.0ppm
Cu, 32.0ppm Fe and 20.5ppm Zn. •,·,• rate of metal removal;
Ο specific growth rate.

Figure 19 shows the influence of metal concentration of


bacterial leaching s o l u t i o n on the rate of metal removal and the
s p e c i f i c growth rate. In the continuous culture at optimum pH,
the maximum rate of metal removal was obtained at 40% metal con­
centration of the o r i g i n a l bacterial leaching s o l u t i o n ( Cu,
270.0mg/l; Zn, 102.5mg/l; Fe, 135.0mg/l ) and f o r copper, zinc and
i r o n the maximum rate of metal removal was 47.5mg/l/hr,
18.1mg/l/hr and 25.7mg/l/hr, r e s p e c t i v e l y .

The maximum s p e c i f i c growth rate observed i n the continuous


culture was 0.541/hr. The value was f a s t e r than the value which
was observed i n batch c u l t u r e . The reason f o r t h i s phenomenon i s
not apparent. I t i s suggested, however, that the bacterial growth
rate was accelerated as a r e s u l t of the formation of harmless
metal s u l f i d e from hydrogen s u l f i d e and metal ions which exert a
deleterious influence on microorganisms and as a r e s u l t of the
fact that a mixed culture was used.
Bioextractive Applications and Optimization 343

% of original metal concentration


in bacterial leaching solution
Fig. 19. Effect of metal concentration of bacterial leaching
solution on metal removal rate and specific growth rate.
Cultivation was conducted at 30°C and each diluted solution
contained Na-lactate ( I0.3g/l ) and yeast extract ( O.lg/l ).
Metal concentration of original supernatant of bacterial leaching
solution was 270.0mg/l Cu, 102.5mg/l Zn and 135.0mg/l Fe.
• A O rate of metal removal; Ο specific growth rate.

3. Characteristics of Precipitate and Conclusion

There was obtained a black p r e c i p i t a t e containing copper,


zinc and i r o n at 19.96%, 6.13% and 10.95%, r e s p e c t i v e l y . The
r a t i o s among the metals i n the p r e c i p i t a t e were s i m i l a r to those
among the metals i n the supernatant of the bacterial leaching
s o l u t i o n and the concentrations of the metals i n the p r e c i p i t a t e
were about 6 . 9 , 38.0 and 1.0 times those of the o r i g i n a l copper
s u l f i d e ore.

Although no data was obtained concerning l a r g e r systems, i t


seems p o s s i b l e to conclude from these r e s u l t s t h a t , i f the problem
concerning energy source can be s o l v e d , i t should be p o s s i b l e to
use s u l f a t e - r e d u c i n g bacteria f o r the recovery of c e r t a i n kinds of
metals from bacterial leaching s o l u t i o n and acid mine drainage.
344 Noboru Tomizuka and Mitsuo Yagisawa

III. REFERENCES

1. Tomizuka, N., and Takahara, Y . , i n Proc. IV· I n t e r n . Ferment.


11

Symp. ( Kyoto ), Ferment. Techno!. Today " ( G. T e r u i , Ed. ),


p. 513-520. Society Fermentation Technology, Osaka, Japan,
1972.
2. Tomizuka, N., Yagisawa, Μ., Someya, J . , and Takahara, Y . ,
Agr. Biol. Chem., 40, 1019 (1976).
3. Tomizuka, Ν., Report of the Fermentation Research Institute,
48, 51 (1976).
4. Yagisawa, M., Murakami, Y . , Kato, Y . , Tomizuka, N., Yamaguchi,
Μ., and Ooyama, J . , Journal of the Mining and Metallurgical
Institute of Japan, 9 3 , 447 (1977).
BIOGENIC EXTRACTION OF URANIUM

FROM ORES OF THE GRANTS REGION

Corale L. Brierley

New Mexico Bureau of Mines and Mineral Resources


Socorro, NM 87801 U.S.A.

Uranium ores from the Anaconda Co. Jackpile and Paute


orebodies were leached using T h i o b a c i l l u s f e r r o o x i d a n s ,
T h i o b a c i l l u s t h i o o x i d a n s , enrichment cultures of mesophilic,
iron-oxidizing bacteria, Sulfolobus a c i d o c a l d a r i u s , and a
Sul folobus-££/ce organism. The latter two organisms are extremely
thermophilic acidophiles. The four test ores ranged between
0.13% and 0.56% U^0^ with uranium mineralization occurring as
coffinite, oxidized uranium complexes, uraninite, and organo-
uranium complexes. The ores varied in pyritic and organic
matter content with some acid-consuming gangue present. Leaching
tests were conducted in airlift percolator columns, batch reactor
flasks, and drip-leach columns in which biologically-generated
leach solutions were applied to the uranium ores. Eh measure-
ments, iron oxidation kinetics, and uranium dissolution suggested
that leaching was not enhanced by bacteria in direct contact
with the ore. Supplementing inoculated ores with ferrous iron
did not increase biogenic U\p^ dissolution. Growth and manome-
tric studies indicated that bacterial growth and function were
suppressed in the presence of some ore samples. Bacterial
suppression by the ores may have resulted from the presence of
toxic agents or the strong reducing environment.

345
346 Corale L Brierley

I. INTRODUCTION

Bacterial leaching of p y r i t i c uranium ores i s both f e a s i b l e


and economical. The r o l e of Thiobaoillus ferrooxidans i n the
extraction of uranium i s probably i n d i r e c t and confined to the
generation of the o x i d i z i n g agent, f e r r i c s u l f a t e , and the
s o l v e n t , s u l f u r i c a c i d , accordingly:

2FeS 2 + 7k0 + H 0
2 2 > ft^;, * * Λ

Reduced uranium i s oxidized by f e r r i c s u l f a t e to acid soluble


hexavalent uranium:

U0 2 + Fe (S0 )
2 4 2 > UO£0 4 + 2FeSO
4

T. ferrooxidans then regenerate f e r r i c s u l f a t e as f o l l o w s :

2FeS0 + k0 4 2 + H S02 4 > Fe (S0 )


2 4 + H02

This bacterial process has been used as a scavenger operation


( 1 , 2, 3, 4) to obtain uranium values from mined-out areas.
The Agnew Lake Mines Ltd. i n northern Ontario i s now using
t h i s bacterial process as the p r i n c i p a l means for in-situ
recovery of uranium ( 5 ) .

The uranium ore of the Grants uranium b e l t i n New Mexico


i s l e s s amenable to bacterial leaching because of the low p y r i t e
content. The primary m i n e r a l i z a t i o n i s c o f f i n i t e (USiO^.nl^O),
u r a n i n i t e (U02) 5 organo-uranium complexes, and some oxidized
forms of uranium ( 6 ) . These uranium deposits are associated
with h i g h l y i n s o l u b l e organic material (7) which has an i n f r a r e d
spectra resembling coal (W. L. Blankenship, unpublished d a t a ) .

This paper describes manometry experiments and leach studies


on uranium ores from the Anaconda Co. Jackpile and Paute Mines
near Grants, New Mexico. T. ferrooxidans, T. thiooxidans, an
iron-enrichment culture obtained from the Jackpile-Paute Mines,
Sulfolobus aoidooaldarius, and Sul folobus-1ike organisms were
Bioextractive Applications and Optimization 347

used i n the s t u d i e s . The l a t t e r two organisms are the extremely


thermophilic, a c i d o p h i l i c bacteria which i n h a b i t hot, acid
s p r i n g s (8, 9) and have been shown to enhance metals e x t r a c t i o n
from r e c a l c i t r a n t ores (10, 1 1 ) .

II. MATERIALS AND METHODS

A. Ores

Five ore types — J l l , J27, P6B, P92, and SP46 — from the
Anaconda Co. Jackpile and Paute Mines, located 35 miles east of
Grants, New Mexico, were s i z e d to -1 mm + 425 ym (-16 + 35 mesh)
and analyzed f o r U^Og content. J-designated ores were c o l l e c t e d
from the Jackpile Mine; P-designated ores were from the Paute
Mine, and the SP46 ore was c o l l e c t e d from a s t o c k p i l e of blended
Jackpile and Paute o r e s .

B. Bacteria

Used i n the s t u d i e s were Thiobacillus thiooxidans (American


Type Culture C o l l e c t i o n # 8 0 8 5 ) , Thiobacillus ferrooxidans
(ATCC # 19859), a mesophilic i r o n - o x i d i z i n g enrichment culture
from the Jackpile-Paute mining d i s t r i c t , Sulfolobus acidocaldarius
( 8 ) , and a Sulfolobus-Mke bacterium ( 9 ) .

C. Leach S o l u t i o n s

Bryner and Anderson medium (12) and 9K medium ( 1 3 ) , made


up with d i s t i l l e d water rather than i r o n s o l u t i o n , were used as
leach s o l u t i o n s . The i n i t i a l pH was 2.5.

Energy sources and supplements included: 78 mM flowers of


s u l f u r , s t e r i l i z e d separate from the medium by intermittent
steaming f o r 30 min on 3 consecutive days; 36 mM a c i d i f i e d ,
F e S 0 . 7 H 0 , s t e r i l i z e d separate from the medium at 18 p s i a ,
4 2

121°C for 15 min; and 0.02% and 0.2% yeast e x t r a c t (Difco) f o r


Sulfolobus-Mke bacteria and Sulfolobus acidocaldarius Λ

respectively.
348 Corale L. Brierley

Chemical control experiments were s t e r i l i z e d with 0.2 mM


panacide (2,2-methylene b i s (4-chlorophenol))(ICN).

D. Analyses

U^Og a n a l y s i s of the leached ore ( t a i l s ) was completed by


t r i b u t y l phosphate e x t r a c t i o n (TBP) ( 1 4 ) . Spectrophotometrie
interferences were encountered when leach s o l u t i o n s were analyzed
by the TBP method.

Ferrous i r o n was determined by dichromate t i t r a t i o n ( 1 5 ) ,


and total i r o n was assayed by a Perkin Elmer 303 atomic
absorption spectrophotometer.

pH and Eh were measured with a Corning Model 7 pH meter.

The Folin-phenol method (16) was used f o r protein determi-


n a t i o n , and bacterial numbers were estimated with the 3-tube
most probable number (MPN) technique ( 1 7 ) . Oxygen uptake was
measured with a Gilson D i f f e r e n t i a l Respirometer according to
standard methods ( 1 8 ) . One-g ore samples were reacted with 5 ml
leach s o l u t i o n for 8-12 hr before i n o c u l a t i o n and oxygen uptake
monitoring to allow for carbonate n e u t r a l i z a t i o n and CO^
dissipation. Ores and ores supplemented with 36 mM Fe ( I I ) were
tested.

E. Leaching Experiments

The l e a c h a b i l i t y of the ores was tested in 250-ml


Erlenmeyer f l a s k s with 100 ml leach s o l u t i o n and a 1% to 5% pulp
density of ore. Batch reactors were incubated at 25°C f o r the
mesophilic bacteria and 60°C f o r thermophilic bacteria up to
32 days. After the incubation period, the reactor contents were
examined m i c r o s c o p i c a l l y , and the pH and Eh values of the s o l u -
tions were measured. In some cases Fe ( I I ) concentrations were
determined, and l L 0 ft a n a l y s i s was completed on the t a i l s .
Bioextractive Applications and Optimization 349

Percolator columns, 3.8 cm by 25 cm with 100 g of u n s t e r i -


l i z e d ore, were operated at room temperature for experimental
studies. Recycled over the ore was 250 ml Bryner and Anderson
medium (12) with 36 mM F e ( I I ) . Test columns were inoculated with
the iron-enrichment c u l t u r e , and control columns were s t e r i l i z e d
with Panacide. Samples were collected weekly f o r Eh and pH
measurements and F e ( I I ) a n a l y s i s . A f t e r completion of the
experiment, the t a i l s were analyzed f o r residual U^Og.

Spent media from the growth of T. thiooxidans and T.


ferrooxidans on Bryner and Anderson medium (12) were applied i n
500-ml a l i q u o t s at an approximate rate of 1.0 ml per min to
100-g samples o f uranium ore i n 3.8 cm by 25 cm columns.
I n f l u e n t s o l u t i o n s (spent media) and e f f l u e n t s o l u t i o n s from the
columns were analyzed f o r Eh, pH, number of b a c t e r i a , and F e ( I I )
when iron was i n i t i a l l y used i n the medium. The t a i l s from the
" d r i p - l e a c h " columns were assayed f o r U^Og content.

III. RESULTS

A. Ores

The -1 mm + 425 urn (-16 + 35 mesh) ores had the following


U 0g content:
3 J l l , 0.28%; J27, 0.56%; P6B, 0.13%; P92, 0.20%;
and SP46, 0 . 4 1 % . Non-sulfate s u l f u r content ranged from 0.22%
f o r high carbon ores to 0 . 1 % f o r r e f r a c t o r y ores ( J . Grunig,
personal communication). Total i r o n content i n the ores was as
follows: J l l , 0.7%, J27, 1 . 1 % ; P6B, 0.4%; P92, 0.8%; and
SP46, 0.8%.

B. Manometry Experiments

Figure 1 shows Q Q ^ (yl oxygen uptake/mg protein/hr) by


T. ferrooxidans in the presence of J l l and P6B o r e s . Oxygen
uptake was g r e a t l y reduced when these ores served as s o l e energy
350 Corale L Brierley

5 00h

Jll J l l
+ P6B|
Fe +
Fe J27 P6BJ
+
Fe

Substrate
Fig. 1 Fig. 2
Fig. 1. The effect of uranium ores, Jll and P6B, and iron-
supplemented ores on oxygen uptake by T. f e r r o x i d a n s .

Fig. 2. The effect of uranium ores, J27 and P6B, and iron-
supplemented ores on oxygen uptake by S u l f o l o b u s .

sources. I f J l l and P6B ores were supplemented with F e ( I I ) ,


oxygen uptake by T. ferrooxidans i n the presence of P6B was
equivalent to oxygen uptake observed when F e ( I I ) was the energy
source. Oxygen uptake by T. ferrooxidans remained depressed
when J l l ore was supplemented with i r o n .

Figure 2 i l l u s t r a t e s r e s u l t s obtained on the oxygen uptake


of a mixed population of Sulfolobus acidocaldarius and
Sulfolobus-like organisms i n the presence of J l l and P6B o r e s .
When J27 served as the sole energy source, no oxygen uptake was
noted; however, when P6B ore was the sole energy source, some
oxygen uptake was observed. I f J27 and P6B ores were supple-
mented with F e ( I I ) , oxygen uptake was nearly equivalent to that
Bioextractive Applications and Optimization 351

observed with i r o n as the only energy source; the presence of


the ores did not suppress i r o n o x i d a t i o n .

TABLE I pH, Eh, and Microscopic Data


from Control and T. ferrooxidans-Inoculated Uranium
Ores after 10-Day, Shake-Flask Test

Control T. ferrooxidans

Ore pH Eh(+mv) Micro a


pH Eh(-hmv) Microa

Jll 3.8 634 0 3.7 736 0


J27 3.4 656 0 3.4 744 0
P6B 3.5 574 0 3.5 736 0
P92 3.7 613 0 3.7 741 0

a
'Subjective microscopic evaluation — 0 to +4

C. Batch Reactor Studies

The f o l l o w i n g r e s u l t s summarize data collected from


stationary and s h a k e - f l a s k leach s t u d i e s .

1. T. ferrooxidans

Table I d i s p l a y s Eh, pH, and microscopic data c o l l e c t e d


a f t e r 10 days o f T. ferrooxidans growth on uranium ores i n shake
f l a s k s with Bryner and Anderson medium. The pH increased i n
both control and inoculated f l a s k s , and an increase i n Eh was
observed for inoculated o r e s . After 10 days of i n c u b a t i o n , no
T. ferrooxidans could be observed m i c r o s c o p i c a l l y i n inoculated
shake f l a s k s .

I f the uranium ores and Bryner and Anderson medium were


supplemented with F e ( I I ) (Table I I ) , a pH increase was again
observed, and higher Eh increases were noted i n T. ferrooxidans-
inoculated shake f l a s k s than i n comparable iron-poor o r e s . An
exception was the P92 o r e .
352 Corale L Brierley

TABLE II pH, Eh, and Microscopic Data


from Control and T. f'errooxidans-Inoculated, Iron-
Supplemented Uranium Ores after 10-Day, Shake-Flask Test

Control T. ferrooxidans

Ore pH Eh(+mv) Micro a


pH Eh(+mv) Micro 0,

Jll 3.4 563 0 3.6 817 +3


J27 3.2 564 0 3.4 819 +3
P6B 3.2 565 0 3.5 814 +3
P92 3.3 563 0 3.3 516 +3

Subjective microscopic evaluation — 0 to +4

A microscopic examination of inoculated leach s o l u t i o n s a f t e r 10


days of incubation revealed large numbers of organisms.

When p y r i t e was added as an energy supplement, T.


ferrooxidans growth was noted, and Eh increased over control
reactors. The pH values of both control and inoculated reactors
were between 3.5 and 4 . 0 .

TABLE III pH, Eh, and Microscopic Data


from Control and T. ferrooxidans-Inoculated, Pyrite-
Supplemented Uranium Ores after 10-Day, Shake-Flask Test

Control T. ferrooxidans

Ore pH Eh(-hmv) Micro a


pH Eh(+mv) Micro a

Jll 3.8 575 0 3.9 724 +2


J27 3.5 607 0 3. 7 756 +2
P6B 3.7 586 0 3.9 749 +2
P92 3.8 574 0 3.6 753 +3

'Subjective microscopic evaluation — 0 to +4


Bioextractive Applications and Optimization 353

TABLE IV pH, Eh, Fe(II) and Microscopic Data


from Control and Enrichment-Inoculated Uranium
Ores after 32-Day, Shake-Flask Test

Control Iron-enrichment

Ore pH Eh(-hmv) Micro a


Fe(II) pH Eh(+mv) Micro a
Fe(II)
(mM) (mM)

111 2.6 360 0 0.36 2.6 535 +2 0.36


J27 2.6 410 0 0.18 2.6 510 +1 0.36
P6B 2.6 370 0 0.36 2.6 535 +2 0.18
P92 2.8 345 0 0.54 2.7 425 +2 0.90

a
Subjective microscopic evaluation — 0 to +4

2. Iron-enrichment Culture

An i r o n - o x i d i z i n g enrichment c u l t u r e , obtained from the


Jackpile-Paute uranium d i s t r i c t , grew on uranium ores with no
supplemental i r o n added (Table I V ) . Eh values were only
somewhat higher than those observed f o r uninoculated c o n t r o l s ,
and s o l u b l e F e ( I I ) concentrations were s i m i l a r for inoculated
and uninoculated r e a c t o r s .

I f F e ( I I ) was added to the leach s o l u t i o n s and uranium


o r e s , good growth of the iron-enrichment culture occurred; the
Eh was g r e a t l y increased over control r e a c t o r s , and added i r o n
was oxidized (Table V ) .

3. Sulfolobus Organisms

Mixed c u l t u r e s of Sulfolobus acidocaldarius and Sulfolobus-


l i k e organisms were grown on uranium ores not amended with
additional energy sources (Table V I ) . The pH values were
v a r i a b l e f o r the ores with J l l e x h i b i t i n g the greatest pH i n -
crease. Eh values for inoculated reactors were not s u b s t a n t i a l l y
higher than Eh values f o r control r e a c t o r s . Uranium was not
extracted from J l l and P6B o r e s , and uranium e x t r a c t i o n values
354 Corale L Brierley

TABLE V pH, Eh, Fe(II), and Microscopic Data


from Control and Enrichment-Inoculated, Iron-Supplemented
Uranium Ores after 32-Day, Shake-Flask Test

Control Iron-enrichment

Ore pH Eh(-hmv) Micro a


Fe(II) pH Eh(+mv) Micro a
Fe (II)
(mM) (mM)

Jll 2.3 350 0 40.32 2.3 630 +3 1.08


J27 2.3 355 0 35.84 2.3 645 +3 0.18
P6B 2.3 355 0 39.07 2.3 640 +3 1.08
P92 2.3 350 0 40.50 2.3 655 +3 1.43

Subjective microscopic evaluation — 0 to +4

from J27 and P92 ores were nearly the same f o r inoculated and
control reactors.

When the ores and 9K medium were supplemented with F e ( I I ) ,


(Table V I I ) , the pH values of control and inoculated reactors
were maintained between pH 2.3 and 3 . 1 . Eh values did not
increase for inoculated r e a c t o r s , and F e ( I I ) concentrations were
s i m i l a r for both control and inoculated f l a s k s . The percents
U^Og extracted were nearly the same f o r both control and
Sulfolobus-ix\ocu\atea r e a c t o r s .

Additional leaching experiments with Sulfolobus organisms


included supplementing the uranium ores and s o l u t i o n s with
elemental s u l f u r , y e a s t e x t r a c t , yeast e x t r a c t and i r o n , p y r i t e ,
and yeast e x t r a c t , and p y r i t e . None o f these supplements or
combinations of supplements measurably enhanced b i o l o g i c a l
U 0
o o extraction.
Bioextractive Applications and Optimization 355

TABLE VI pH, Eh, Microscopic and U 0 3 8

Extraction Data from Control and Sulfo]obus-Inoculated


Uranium Ores after 10-Day, Stationary, Batch Reactor Test

Control Sulfolobus

Ore pH Eh(+mv) Micro a


pH Eh(-hmv) Micro 0,
%U
3°8 b %U
3°8 b

Jll 6.5 564 0 0 6.6 514 +2 0


J 27 2.6 604 0 33 2.6 614 +3 33
P6B 2.9 574 0 0 2. 7 624 +2 0
P92 3.0 544 0 10 3.5 544 +3 25

Subjective microscopic evaluation — 0 to +4


Percent U 0„ extracted from ore

D. Percolator Leach Column Studies

Uranium ores were leached i n percolator columns with


u n s t e r i l i z e d ore inoculated with the iron-enrichment c u l t u r e ;
control columns were s t e r i l i z e d with Panacide. When P6B ore was

TABLE VII pH, Eh, Fe(II), and U 0 Extraction Data 3 8

from Control and Sulfolobus-Inoculated, Iron-Supplemented


Uranium Ores after 10-Day, Stationary, Batch Reactor Test

Control Sulfolobus

Ore pH Eh(+mv) Fe (II) XUJDJ 1 pH Eh(+mv) Fe(II) %u o a

(mM) όΟ (mM) 0 0

Jll 3.1 529 mfi 11 3.0 549 NlP 18


J 27 2.5 574 1.21 55 2.5 574 1.16 56
P6B 2.4 564 1.48 39 2.3 564 1.31 23
P92 2.8 ND b
1.48 45 2.6 EDO 0.90 45

a
Percent U0 q extracted from ore
Not determined
356 Corale L Brierley

leached, i t was noted that iron was not oxidized by the e n r i c h ­


ment culture u n t i l approximately 32 days a f t e r the leaching
experiment was i n i t i a t e d ( F i g . 3 ) . Bacterial counts at day 21
3
indicated about 10 i r o n - o x i d i z i n g microbes per ml of s l u r r y .
At t h i s time Eh increased in the inoculated column. Iron was
not oxidized i n the control column, and the Eh remained at about
+600 mv for the e n t i r e experiment. The pH values for both c o l ­
umns were about 3.0. After 54 days of leaching the percents
U^Og extracted were 92% and 95% for control and inoculated
columns, r e s p e c t i v e l y . S i m i l a r r e s u l t s were noted f o r pH, Eh,
F e ( I I ) concentrations, and U^Og e x t r a c t i o n f o r control and
enrichment-inoculated P92 uranium ore.

4 0h

l 1ι Χ ­
ΙΟ2 03 04 0
Days

fig. 3 , Changes in ρΗ Eh, and ferrous iron concentrations


Λ

during column leaching of iron-supplemented P6B ore. Iron-


enrichment culture: A-Fe, # - p # ,Μ -Eh; Control: Δ-Fe, O-pH,
D-Eh.
Bioextractive Applications and Optimization 357

Figure 4 i l l u s t r a t e s the pattern f o r pH, Eh, and F e ( I I )


concentrations during control and enrichment- culture leaching
of SP46 uranium ore. The Eh values i n the control and inoculated
columns remained between +500 mv and +550 mv, and the pH
s t a b i l i z e d at about 3.0 f o r both columns. At day 9,counts
indicated that about 10 i r o n - o x i d i z i n g bacteria per ml were
present. The F e ( I I ) concentration decreased i n both columns at
day 14 with the decrease occurring at a s l i g h t l y lower rate i n
the control column. The f i n a l F e ( I I ) concentration i n both
columns was about 18 mM. The U^Og e x t r a c t i o n a f t e r 42 days of
leaching was the same for both columns - - 96%.

Η 70 0

6 00

5 00

Fig. 4. Changes in pH, Eh, and ferrous iron concentrations


during column leaching of iron-supplemented SP46 ore. Iron-
enrichment culture: A-Fe, +-pH, M-Eh control: Δ-Fe, O-pH, D-Eh.
358 Corale L Brierley

In leach column tests of 2 weeks duration s i m i l a r pH, Eh,


and F e ( I I ) o x i d a t i o n patterns were observed. The U^Og
d i s s o l u t i o n ranged from 65% to 95% with l i t t l e v a r i a t i o n between
inoculated and control columns.

E. " D r i p Leach" Column Tests

F e r r i c i r o n and s u l f u r i c a c i d leach s o l u t i o n s , generated by


T. ferrooxidans and T. thiooxidans, were applied to columns
containing 100 g of uranium o r e s . The r e s u l t s from these leach
experiments were compared with r e s u l t s from experiments i n which
the same volumes of uninoculated leach s o l u t i o n s were applied
to the o r e s .

Table V I I I , part A summarizes data collected on the


generated l i x i v i a n t s which were applied to two columns of J l l
ore. T. ferrooxidans generated a F e ( I I ) concentration of 18 mM,
and 1.6 χ 10 organisms per 100 ml (MPN) of s o l u t i o n were present.
The Eh i n the inoculated s o l u t i o n had not increased s u b s t a n t i a l l y .
No F e ( I I ) was present i n the control leach s o l u t i o n . Table V I I I ,
part Β summarizes data from the column e f f l u e n t a f t e r reaction of
the leach s o l u t i o n s with J l l ore and the percent U^Og extracted
from the ore. Most notable were the increases i n pH and the 100%
extraction of U^Og from the ore leached with the b i o l o g i c a l l y -
generated l i x i v i a n t .

Data from s i m i l a r experimentation with T. thiooxidans-


generated leach s o l u t i o n are summarized i n Table I X . The
inoculated l i x i v i a n t had a somewhat lower pH than the control
s o l u t i o n (Table I X , part A ) . Using the inoculated leach s o l u t i o n
41% of the U^Og was extracted from J l l ore as compared with 14%
when control medium was applied to the ore (Table I X , part B ) .

The a p p l i c a t i o n of T. ferrooxidans-generdited leach s o l u t i o n


with 34 mM F e ( I I ) (Table X, part A) to SP46 ore y i e l d e d 90% U 0g. 3

The release of U~0 Q from SP46 ore leached with control medium
Bioextractive Applications and Optimization 359

TABLE VIII Drip Leach Results (A) Chemical and


Biological Data of T. ferrooxidans-Generated and Control
Influent Leach Solutions, and (B) Chemical and Biological
Data of Column Effluent Solutions and Percent
UJ0 Extraction from Jll Ore
R

(A) pH Eh(-hmv) Fe(II) Fe(TOT) MPN/100 ml


(mM) (mM)

Control 2.3 610 36 36 0

T. ferrooxidans 2.5 644 18 36 1600

(B) pH Eh(-hmv) Fe(II) Fe(TOT) MPN/100 ml %U 0


(mM) (mM) 1

Control 3.6 555 31 52 0 39

T. ferrooxidans 2.8 660 8 31 15 100

a
Percent U^O^ extracted from ore

containing 2 mM F e ( I I ) was 63%.

IV. DISCUSSION AND CONCLUSIONS

Manometric studies with Sulfolobus on uranium ores indicated


limited or no oxygen uptake ( F i g . 2 ) , however, i f the ores were
amended with F e ( I I ) , oxygen uptake by Sulfolobus was equivalent
to that observed with F e ( I I ) o n l y . Although the ores appeared
to be d e f i c i e n t i n o x i d i z a b l e energy s o u r c e s , the ores were
apparently not t o x i c to these organisms i n s h o r t term experiments.
Sulfolobus did grow on unamended uranium o r e s , but uranium
e x t r a c t i o n was not enhanced (Table V I ) . This suggests that the
increased temperature and acid medium are s u f f i c i e n t to leach
some of the uranium. I f the ores were amended with F e ( I I ) , i t
was found that Sulfolobus cultures neither a c t i v e l y o x i d i z e d the
i r o n nor was uranium e x t r a c t i o n enhanced i n inoculated batch
360 Corale L Brierley

TABLE IX Drip Leach Results (A) Chemical and


Biological Data of T. thi00xidans-Generated and Control
Influent Leach Solutions and (B) Chemical and Biological
Λ

Data of Column Effluent Solutions and Percent


U' 0 Extracted from Jll Ore

(A) pH Eh(+mv) MPN/100 ml

Control 2.6 636 0

J. thiooxidans 2.3 629 250

(B) pH Eh(+mv) MPB/100 ml %U 0


7

Control 3.9 605 0 14

T. thiooxidans 3.8 539 10 41

Percent UJ)~ extracted from ore

TABLE X Drip Leach Results (A) Chemical and


Biological Data of T. ferrooxidans-Generated and Control
Influent Leach Solutions and (B) Chemical and Biological
3

Data of Column Effluent Solutions and Percent


U 0 Extracted from SP46 Ore
R

(A) pH Eh(-hmv) Fe(II)(mM) Fe (TOT) (mM)

Control 2.3 573 34 36

T. ferrooxidans 2.7 794 4 36

(B) pH Eh(-hmv) Fe(II)(mM) Fe(TOT) (mM) %U 0 a

Control 3.8 683 30 35 63

T. ferrooxidans 2.7 474 3 28 90

a
Percent UJ) R extracted from ore
Bioextractive Applications and Optimization 361

reactors (Table V I I ) . Since the Sulfolobus organisms are


mixotrophic, i t i s p o s s i b l e that s u f f i c i e n t o x i d i z a b l e organic
matter was present to provide growth, and i r o n was not oxidized
by the organisms.

Manometric studies with T. ferrooxidans indicated that J l l


and P6B ores did not contain a s u f f i c i e n t source of energy for
demonstrable oxygen uptake ( F i g . 1 ) . I f ferrous i r o n was added
to the o r e s , T. ferrooxidans showed oxygen uptake comparable to
oxidation of i r o n o n l y , but oxygen uptake with J l l ore was
greatly suppressed. This i n d i c a t e s that J l l ore may contain
substances i n h i b i t o r y to i r o n o x i d a t i o n by T. ferrooxidans.
I n a 10-day batch reactor study i n which ores were amended with
F e ( I I ) , T. ferrooxidans growth was observed (Table I I ) . It is
p o s s i b l e that i n long-term t e s t s the organisms do adapt to
conditions which may i n i t i a l l y be i n h i b i t o r y and would be
observed i n manometric s t u d i e s .

Iron-enrichment cultures which had been i s o l a t e d from


uranium mining areas were used i n leach s t u d i e s . Presumably
these organisms would be more adapted than the ATCC T.
ferrooxidans to uranium o r e s . These organisms i n c o n t r a s t to
T. ferrooxidans did grow on unamended uranium ores (Table I V ) .
When the ores were amended with i r o n , good growth was observed,
and i r o n was r e a d i l y oxidized (Table V ) . These batch reactor
studies were incubated for longer time periods than comparable
t e s t s with Τ. ferrooxidans, so d i r e c t comparison i s not p o s s i b l e .
The enrichment culture was used i n percolator column t e s t s . The
F e ( I I ) i n amended P6B ore was oxidized by the culture ( F i g . 3 ) ,
but i t required 32 days. Uranium e x t r a c t i o n s i n the control and
inoculated columns were the same, and s i m i l a r uranium e x t r a c t i o n
r e s u l t s were observed for SP46 ore. These r e s u l t s indicate that
leaching of uranium ores with the organisms associated with the
ore does not appear to enhance uranium e x t r a c t i o n . The
development of the organisms or t h e i r a b i l i t y to o x i d i z e a
362 Corale L Brierley

substrate i s suppressed.

When leach s o l u t i o n s , which had been generated by bacterial


a c t i v i t y , were applied to o r e s , uranium e x t r a c t i o n was increased
over that observed when uninoculated media were a p p l i e d . The
increased uranium extraction resulted from the increased F e ( I I )
content of T. ferrooxidans a c t i v i t y and from increased a c i d i t y
of T. thiooxidans growth.

Acid consuming m a t e r i a l , lack of energy source, and slow


development or a c t i v i t y of the bacteria w i t h i n the ore a l l
indicate that bacteria cannot be used e f f e c t i v e l y for leaching of
the Grants uranium o r e s . When b i o l o g i c a l l y - g e n e r a t e d leach
s o l u t i o n s are applied to the Grants uranium o r e s , increases i n
U^Og s o l u b i l i z a t i o n are noted, but these laboratory studies are
preliminary and do preclude e x t r a p o l a t i o n to i n d u s t r i a l leach
systems. I t may be conceivable to heap or in-situ leach ores
with b i o l o g i c a l l y - g e n e r a t e d l i x i v i a n t s . Further study i s neces-
sary to determine i f recycled leach s o l u t i o n s would carry
i n h i b i t o r y factors and a cost a n a l y s i s would be necessary to
e s t a b l i s h the economics o f the system.

V. ACKNOWLEDGMENTS

The author would l i k e to express her appreciation to the


Anaconda Co. for t h e i r f i n a n c i a l support of t h i s study and t h e i r
permission to publish the r e s u l t s .

VI. REFERENCES

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2. MacGregor, R.A., Can. Min. Metall. Bull., 59, 583 (1966).
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Bioextractive Applications and Optimization 363

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Uranium Region, Mem. 1 5 , " p. 2 1 . New Mexico Bureau o f Mines
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R . J . , J. Biol. Chem., 193, 265 (1951).
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Minneapolis, 1972.
MICROBIAL LEACHING OF CU-NI SULFIDE CONCENTRATE

Η. M. Tsuchiya

U n i v e r s i t y of Minnesota
M i n n e a p o l i s , Minnesota, U.S.A.

Mutalism between T h i o b a o i l l u s ferrooxidans ATCC 19859 and


B e i j e r i n c k i a lacticogenes ATCC 19361 has an effect upon both the
rate and extent of leaching copper and nickel from ore concen­
trates of the Duluth Gabbro.
All living organisms are made up of carbon, hydrogen, oxygen,
nitrogen, sulfur, phosphorus, and the other elements that comprise
the protoplasm. Moreover, these occur in a definite stoichiome-
try; these are the minimal requirements of living systems. The
stoichiometry of the environment from which the microorganisms
selectively extract elements in the process of growth, differs
from that of the microbial protoplasm. All organisms must there­
fore have an energy source to maintain the concentration gradients
and the stoichiometry of the elements that make up the environment
and the protoplasm.
Most of the known copper and nickel reserves in the United
States are of the sulfide form. Numerous investigators have
established the fact that the microorganisms play a role in the
oxidation of insoluble sulfides to soluble sulfates. The thio­
bacilli produce the lixiviant. The more thiobacilli there are,
the more lixiviant is produced and the faster the rate of and
greater the extent of leaching. The addition of B e i j e r i n c k i a to
the leaching- liquor stimulates the fixation of nitrogen. It is
proposed that the additional nitrogenous compounds stimulate the
population density of thiobacilli. As the numbers of thiobacilli
are increased, the amount of lixiviant is increased.

365
366 Η. Μ. Tsuchiya

I. INTRODUCTION AND PROCEDURES

Results of leaching t e s t s on a mixed copper and nickel sul­


f i d e concentration from the Duluth Gabbro complex are reported
here. The r e l a t i v e e f f i c a c i e s of pure cultures of Thiobacillus
ferroxxidans and mixed c u l t u r e s T. ferrooxidans and Beijerinckia
lacticogenes are compared as p o s s i b l e leaching a i d s .

The s u l f i d e concentrate studied was a combined copper and


nickel rougher f l o t a t i o n concentrate supplied by the U.S. Bureau
of Mines, Twin C i t i e s Research Center, Twin C i t i e s , Minnesota.
The ore body i s located in northeastern Minnesota and i s c a l l e d
the Duluth Gabbro complex of low grade copper and nickel sulfides.
The principal minerals are c h a l c o p y r i t e , m i l l e r i t e , p y r r h o t i t e ,
and pentlandite. The concentrate assayed 4.22% copper and 0.90%
nickel and was ground to -270 mesh. Detailed chemical analysis
i s given in Table I .

TABLE I Chemical Analysis of the Copper-Nickel Concentrate

Element Percent Compound Percent

Cu 4.22 Si0 2
36.62
Ni 0.90 Al 0 2 2 10.54
Fe 17.13 MgO 8.65
S 7.17 CaO 5.80
C 1.33 Na 0 2 1.79
Co 0.04 Ti0 2
0.99
κο
2
0.42
other 5.40

The part played by microorganisms in metallurgy i s a well


known f a c t . Leached copper was probably f i r s t recovered in
r e l a t i v e l y large amounts from Spanish mines. I t was not u n t i l
1954, however, that the active r o l e played by bacteria i n t h i s
oxidation process was demonstrated. The bacteria involved in the
oxidation of i n s o l u b l e metal s u l f i d e s to soluble metal s u l f a t e s
belong to the genus t h i o b a c i l l u s .
Bioextractive Applications and Optimization 367

The bacterium most studied in context of i t s a p p l i c a t i o n in


mineral processing i s T. ferrooxidans. I t i s an aerobic, a u t o t r o -
p h i c , a c i d o p h i l i c , carbon d i o x i d e - f i x i n g microorganism which
requires s u l f u r or ferrous iron as energy source for i t s growth
and maintenance.

Vishniac and Santer (1957) ( 1 0 ) , Kuznetsov, et a l . (1963) ( 5 ) ,


Pings (1968) ( 7 ) , Beck (1969) ( 1 ) , Z a j i c (1969) ( 1 1 ) , Malouf (1970)
( 6 ) , and Duncan (1972) ( 4 ) , are among some who have given excellent
accounts of properties and c h a r a c t e r i s t i c s of t h i o b a c i l l i .

From a l l published accounts of microbial l e a c h i n g , i t can be


concluded that the following f a c t o r s are among those important in
leaching with the help of b a c t e r i a .

(a) An a v a i l a b l e source of substrate (mineral s u l f a t e or


ferrous iron or p y r i t e i f the ore body i s an oxide ore body).

(b) A supply of carbon dioxide which i s the sole source of


carbon f o r the t h i o b a c i l l i .

(c) A supply of oxygen f o r the oxidation process.

(d) A supply of e s s e n t i a l n u t r i e n t s including n i t r o g e n .

(e) Water as a c a r r i e r f o r the n u t r i e n t s , source of trace


elements, and as a solvent f o r the solvated metal.

(f) An a c i d i c environment.

I t i s unnecessary to point out that in dump l e a c h i n g , the


organisms get almost a l l of the e s s e n t i a l n u t r i e n t s e i t h e r from
the ore or from the r e c i r c u l a t i n g barren s o l u t i o n . In addition
to the above f a c t o r s , i t i s a l s o recognized that temperature, p a r t i c l e
s i z e (surface a r e a ) , and a d d i t i v e s e f f e c t the rate of leaching.

A l l l i v i n g organisms are made up of carbon, hydrogen, oxygen,


n i t r o g e n , s u l f u r , and phosphorous, and other trace elements that
comprise the protoplasm; these occur in a d e f i n i t e stoichiometry.
The stoichiometry i s shown in Table I I .
368 Η. Μ. Tsuchiya

TABLE II Approximate Elementary Composition of the


microbial cell. S. E. Luria in the Bacteria (I.C.
Gunsalus and R. Y. Stonier, eds.) Vol. 1 (New York,
Academic Press, 1960)

Element Percent of Dry Weight

carbon 60
oxygen 20
nitrogen 14
hydrogen 8
phosphorus 3
sulfur 1

These are minimal requirements of l i v i n g systems. The


stoichiometry of the environment from which organisms selectively
extract elements i n the process of growth, d i f f e r s from that of
the protoplasm.

Since we are interested i n the i n t e r a c t i o n s of microbial


forms and with t h e i r common environment, we considered the p o s s i ­
b i l i t y of f u r n i s h i n g an organism which would supply nitrogen to
thiobacillus.

Beijerinckia, an aerobic, a c i d o p h i l i c , nitrogen f i x i n g ,


heterotrophic organism, c h a r a c t e r i s t i c a l l y n o n - f a s t i d i o u s with
respect to i t s carbon source seemed to be the organism of choice.
The a c i d o p h i l i c nature of Beijerinckia would make i t s u i t a b l e f o r
the low pH conditions that e x i s t in leaching systems. Starkey
and De (1939)(9),and Becking (1961a and 1 9 6 1 b ) ( 2 , 3 ) , have
reported on the h a b i t a t , i s o l a t i o n , and c h a r a c t e r i s t i c s of these
organisms. I t seemed reasonable to believe that i f these two
organisms, a Beijerinckia and a Thiobacillus, could be grown
together, the former would f i x dinitrogen to s a t i s f y the need of
nitrogenous compounds f o r i t s e l f and t h i o b a c i l l u s and the l a t t e r
would f i x carbon dioxide for i t s and Beijerinckia s organic matter r

in a medium devoid of added carbon and nitrogen sources. This


hypothesis i s represented schematically in F i g . 1 .
Bioextractive Applications and Optimization 369

Both have total n u t r i e n t supply if


both are present.

Fig. 1. Proposed mutualistic interaction of T, ferrooxidans


and B. lacticogenes in a leaching environment
devoid of fixed carbon or fixed nitrogen.

In t h i s paper are our f i n d i n g s with T. ferrooxidans, ATCC


19859 and B. lacticogenes ATCC 19361. The pure cultures of
t h i o b a c i l l u s were maintained on the 9K medium of Silverman and
Lundgren ( 1 9 5 9 ) ( 8 ) . Stock c u l t u r e s of Beijerinckia were maintained
on agar medium, the composition of which i s given in Table I I I .

The Beijerinckia were s y s t e m a t i c a l l y adapted to grow at low


pH v a l u e s . This was accomplished by sequential t r a n s f e r s of
a c t i v e l y growing c e l l s in f l a s k s containing i n c r e a s i n g l y higher
concentrations of hydrogen i o n s .

Thiobacillus was a l s o adapted to grow on the concentrate in


question in a nutrient medium c o n s i s t i n g of the 9K basal s a l t s of
Silverman and Lundgren ( 8 ) , but not containing ( N H ^ S O ^ or
Ca(N0~) . 9 The s a l t s s o l u t i o n i s shown in Table IV.
370 Η. Μ. Tsuchiya

TABLE III Composition of Agar Medium for Maintaining


Stock Cultures of the Nitrogen-Fixing Bacteria

Component Concentration (g/l)

KE F02 4 0.5
MgS0 7E 0 4m 2 0.2
CaCl 2 0.1
FeCl 3 0.005
Naflo0 2H 0 4m 2 0.0005
Difco yeast extract 0.5
Sucrose 2.5
Agar 15.0
pH adjusted to 3.0

TABLE IV Composition of the Τ Medium Used in


Leaching Tests

Component Concentration (g/l)

K HP0
2 4 0.5
MgS0 7H2 4u 0 0.5
Na S0 2 4 0.15
KCl 0.1
CaS0 JH 0 4 2 0.01
Na Mo0 2H 0
2 4t 2 0.0005
pH adjusted to 2.5

Pure cultures of T. ferrooxidans and a mixed culture of T.


ferrooxidans and B. lacticogenes were thus obtained in f l a s k s
containing no organic carbon source (only inorganic C0£ of the
atmosphere) and no nitrogen (except of the atmosphere), NH^*
e x i s t i n g as trace impurities in chemicals, and NH^ absorbed by
the medium at pH 2.5.

The leaching t e s t s were c a r r i e d out in Erlenmeyer f l a s k s with


a 20% s o l i d suspension of concentrate of 70ml of the nutrient
medium of the composition shown in Table IV. 10 ml inocula of
pure and mixed cultures were employed. The inocula were t r a n s -
Bioextractive Applications and Optimization 371

ferred twice, p r i o r to the i n o c u l a t i o n , for 2 days each in the


same medium as was used in the t e s t so as to get a c t i v e l y growing
cultures. A l l t e s t s were run in duplicate and repeated to check
precision. Chemical controls containing medium, but not b a c t e r i a ,
were run stimultaneously. The t e s t f l a s k s were incubated on a
reciprocating shaker with a 5 inch stroke at 76 rpm at 26°C.

The experiments were monitored in leaching f l a s k s by removing


10 ml a l i q u o t s at regular i n t e r v a l s . Metal analyses were c a r r i e d
out on a Perkin Elmer Model 303 atomic absorption spectrophoto-
meter.

Intermittent microscopic examination of the leach f l a s k s


showed an increase of c e l l s .

II. RESULTS AND CONCLUSIONS

Shown in Table V i s the e f f e c t of % s o l i d s on leaching of


copper with T. ferrooxidans and T. ferrooxidans plus B. laeti-
cognenes. I t can be seen that the amount of copper extracted was
s u b s t a n t i a l l y heavier in the c u l t u r e s with Thiobaoillus and
Beijerinckia than in the c u l t u r e s with t h i o b a c i l l i alone.

Shown in Table V I i s the e f f e c t of s o l i d s on leaching of


nickel with T. ferrooxidans and T. ferrooxidans in concert with
B. lacticogenes. I t can be seen that the amount of nickel
extracted was s u b s t a n t i a l l y heavier in the culture with the two
organisms than i t was with one.

Leaching t e s t s were c a r r i e d out with unadapted pure and


mixed c u l t u r e s . In each case, e x t r a c t i o n s were l e s s than achieved
with adapted c u l t u r e s . The t e s t s were run in duplicate and
repeated. Chemical c o n t r o l s were a l s o c a r r i e d out.

T. ferrooxidans o x i d i z e s the i n s o l u b l e s u l f i d e s occurring in


ores to soluble s u l f a t e . The more the number of Thiobacillus,
owing to the nitrogen f i x e d by Beijerinckia, the greater the rate
and more extensive the amount of copper and nickel leached.
372 Η. Μ. Tsuchiya

TABLE V Effect of % Solids on Leaching of Copper with


T. ferrooxidans and T. ferrooxidans plus B. lacticogenes

% Solids 5 10 20 33

Time Thiob Thiob Thiob Thiob Thiob Thiob Thiob Thiob


(hours) (g/D + (g/D + (g/D + (g/D +
Beij Beij Beij Beij
(g/D (g/D (g/D (g/D

25 0.26 0.28 0.23 0.25 0.23 0.26 0.16 0.20


50 0.32 0.36 0.33 0.33 0.30 0.34 0.30 0.50
75 0.49 0.54 0.55 0.62 0.62 0.70 0.38 0.96
150 0.96 1.37 1.10 1.55 1.20 1.82 0.66 1.83
200 1.20 1.64 1.14 2.07 1.48 2.46 0.77 2.17
300 1.76 1.98 2.35 3.12 2.00 3.68 1.08 2.81

TABLE VT Effect of % Solids on Leaching of Nickel with


T. ferrooxidans and T. ferrooxidans plus B. lacticogenes

% Solids 5 10 20 33

Thiob Thiob Thiob Thiob Thiob Thiob Thiob Thiob


Time (g/D + (g/D + (g/D + (g/D +
(hours) Beij Beij Beij Beij
(g/D (g/D (g/D (g/D

25 0.20 0.22 0.27 0.03 0.30 0.34 0.31 0.50


50 0.25 0.26 0.34 0.37 0.40 0.47 0.58 0.81
75 0.33 0.35 0.50 0.54 0.63 0.62 0.82 1.17
150 0.44 0.45* 0. 74 0.80 0.90 1.07 1.23 2.08
200 0.45* 0.45* 0.77 0.87 0.96 1.30 1.65 2.50
300 0.45* 0.45 0.83 0.90* 1.14 1.64 2.57 3.66

100% extraction

III REFERENCES

1. Beck, J . V . , ^Thiobacillus ferrooxidans and i t s Relation to


the S o l u b i l i z a t i o n of Ores of Copper and I r o n " , Fermen. Adv.,
747-771 (1969).
Bioextractive Applications and Optimization 373

2. Becking, J . H . , " S t u d i e s on N i t r o g e n - F i x i n g Bacteria of the


Genus Beijerinckia. I . Geographical and Ecological D i s t r i ­
bution in S o i l s " , Plant and Soil, 14, 49-81 (1961a).
3. Becking, J . H . , " S t u d i e s on N i t r o g e n - F i x i n g Bacteria of the
Genus Beijerinckia. I I . Mineral N u t r i t i o n and Resistance to
High Levels of Certain Elements in Relation to S o i l Type,
Plant and Soil, 14, 297-322 (1961b).
4. Duncan, D.W. and Walden, C.C., " M i c r o b i o l o g i c a l Leaching
in the Presence of F e r r i c I r o n " , Develop. Ind. Microbiol.,
13, 66-75 (1972).
5. Kuznetsov, S . I . , Ivanov, M.V. and L y a l i k o v a , N.N., " I n t r o ­
duction to Geological M i c r o b i o l o g y " , p. 252. McGraw-Hill
New York, 1963.
6. Malouf, E.E., " B i o e x t r a c t i v e Mining. I n : SME Short Course
in B i o e x t r a c t i v e M i n i n g " . Society of Mining Engineers of
AIME, New York, p. 1-45, 1970.
7. P i n g s , W.B., " B a c t e r i a l Leaching", Colo. Sen. Mines Miner.
Ind. Bull., 1 1 ( 3 ) , 1-19 (1968).
8. Silverman, M.P. and Lundgren, D.G., "Studies on the
Chemoautotrophic Iron Bacterium Ferroobaoillus ferrooxidans , 11

J. Bacteriol., 77, 642-647 (1959).


9. Starkey, R.L. and De, P.K., "A New Species of Azotobacter , n

Soil Sci., 47, 329-343 (1939).


10. V i s h n i a c , W. and Santer, Μ., "The T h i o b a c i l l i , "Bacteriol.
Rev., 12, 196-213 (1957).
11. Z a j i c , J . E . , "Microbial Biogeochemistry", p. 345. Academic
P r e s s , New York, 1969.
COMPLEX LEAD SULFIDE CONCENTRATE

LEACHING BY MICROORGANISMS

A.E. Torma

New Mexico I n s t i t u t e of Mining and Technology


Socorro, New Mexico 87801 U.S.A.

Recent bacterial leaching studies indicate that zinc, copper,


and cadmium values can selectively be removed from off-grade lead
sulfide concentrates without producing any air pollution. The
bacterial leaching technique is based on the difference in the
solubility of the metal sulfates which are resulted in this
process. The leach residue is an up-graded lead sulfide concen-
trate which is partially oxidized to sulfate and can be used
directly in the classical smelting process for recovery of lead.
The preliminary economic assessment presented in this study is
encouraging to warrant further consideration.

I. INTRODUCTION

Lead i s one of the e a r l i e s t known elements by man ( 1 ) . The


Egyptians used i t f o r g l a z i n g pottery as early as about 7 to 5
thousand B.C., the ancient Romans made water-pipes from i t and
the alchemists believed that i t was associated with the planet of
Saturn ( 2 ) . Today, there i s a v a r i e t y of lead products a v a i l a b l e
f o r use (3) including metal products ( a l l o y s , p i p e s , battery

375
376 Α. Ε. Torma

g r i d s ) , pigments (white and colored) and chemicals (gasoline a n t i ­


knock a d d i t i v e s ) .

The principal lead mineral i s galena PbS ( 4 ) . I t s ore deposits


are generally associated with varying q u a n t i t i e s of i r o n , z i n c ,
copper, s i l v e r , g o l d , antimony, arsenic and bismouth i m p u r i t i e s .
Other lead minerals are o x i d e s , PbO, s u l f a t e s ( a n g l e s i t e , PbSO^)
and carbonates ( c e r u s s i t e , PbCO^).

M e t a l l i c lead i s produced from f l o t a t i o n s u l f i d e bearing con­


centrates by the conventional smelting processes ( 2 ) , which are
based on the following r e a c t i o n s :

2PbS + 3 0 t 2PbO + 2 S 0
2 2 [1]

PbS + 2PbO t 3Pb° + S 0 2 [2]

PbS + 2 0 t PbS0 2 4 [3]

PbS + PbS0 t 2Pb° + 2 S 0 4 2 [4]

The thermodynamics of these e q u i l i b r i a i s well established (5,6).


I t i s a l s o p o s s i b l e to produce lead in one s t e p , which can be
derived from equations [1] and [ 2 ] :

PbS + 0 2 t Pb° + S 0 2 [5]

However, no i n d u s t r i a l method e x i s t s yet working with t h i s p r i n c i ­


ple.

I f lead i s to be produced from off-grade lead s u l f i d e concen­


trates the f i n e l y d i s s i m i l a t e d metal values ( z i n c , cadmium, and
copper) are transferred p r i n c i p a l l y into the s l a g during the
conventional smelting processes. The recovery of these elements
from the s l a g i s d i f f i c u l t and c o s t l y ( 7 - 9 ) . Many e f f o r t s have
been devoted to overcome these problems and a number of hydro-
metallurgical processes were suggested claiming such advantages as
f a c i l i t y f o r s e l e c t i v e recovery of the associated metal values and
avoiding S 0 - a i r p o l l u t i o n .
2 These processes include:
Bioextractive Applications and Optimization 377

- Pressure leaching of lead s u l f i d e s in a l k a l i n e ( 1 0 , 1 1 ) ,


s u l f u r i c acid ( 1 2 , 1 3 ) , and hydrochloric acid (14) media;
- Anodic d i s s o l u t i o n of lead s u l f i d e concentrates in hydro­
c h l o r i c and p e r c h l o r i c acid s o l u t i o n s ( 1 5 - 1 7 ) ; and
- Bacterial leaching of off-grade lead s u l f i d e concentrates
in s u l f u r i c acid media ( 1 8 , 1 9 ) .

II. MICROBIOLOGICAL LEACHING OF LEAD SULFIDES

When intergrowths of s u l f i d e s of l e a d , z i n c , cadmium, copper,


and iron occur together, the q u a n t i t a t i v e recovery of the i n d i v i d ­
ual minerals i s rendered impossible because of f i n e m i n e r a l i z a t i o n
and differences in g r i n d a b i l i t y . For t h i s type of ore the micro­
b i o l o g i c a l leaching appears to hold promise in recovery of the
associated metal values. The microorganisms involved in metal
s u l f i d e oxidation processes are i n t e n s i v e l y s t u d i e d , as i t i s the
case, f o r example, f o r the chemol i t h o t r o p h i c Thiobacillus ferro­
oxidans (21-27). For other type of microorganisms, which may
o x i d i z e i n s o l u b l e s u l f i d e substrates such as thermophilic organ­
isms ( 2 8 - 3 1 ) , many studies are undertaken to elucidate t h e i r r o l e
in these processes. The b a c t e r i a l leaching can be represented by
the s i m p l i f i e d equation as f o l l o w s :

bacteria
MS + 2 0 2 ^> MS0 4 [6]

where Μ i s a bivalent metal. Generally, the metal s u l f i d e s are


i n s o l u b l e in the aqueous media while the s u l f a t e s are s o l u b l e .
Hence, i s the metal d i s s o l u t i o n process. Evidence has been r e ­
ported that T. ferrooxidans i s able to o x i d i z e lead s u l f i d e s (35,
3 6 ) , which oxidation was found to be accelerated in presence of
iron ( 3 7 ) . However, the oxidation of lead s u l f i d e r e s u l t s in the
formation of i n s o l u b l e lead s u l f a t e . The difference in the s o l u ­
b i l i t y of heavy metal s u l f a t e s i n c i t e d i n v e s t i g a t o r s (32) to study
the p o s s i b i l i t y of s e l e c t i v e removal of copper from a lead b l a s t
furnace matte by microorganisms. This idea was pursued by several
researchers (33,34) who reported on s e l e c t i v e bacterial leaching
378 Α. Ε. Torma

of arsenic and copper from oxidized t i n concentrates. The a p p l i ­


c a b i l i t y of the microbiological leaching technique f o r s e l e c t i v e
extraction of cadmium.,copper and zinc values from an off-grade
lead s u l f i d e concentrate ( 1 8 , 1 9 ) , has been demonstrated. A
schematical presentation of t h i s method i s given i n F i g . 1 .

OFF-GRADE
Pb-CONC.

AIR* _
LEACHING NUTR.SOL.
co 2 -

HIGH-GRADE
S/L
PbS-CONC.

CaCO„ JHYDRO LYSIS • F e " RESIDUE

Zn • CEMENT. •Cd-Cu

MgO- PRECIPIT.

S/L

H SO
2 A Zn(0H) o

ELECTRO­
LYSIS

Zn

Fig. 1 . Schematical representation of a microbiological


leaching process of an off-grade lead sulfide concentrate.

In the process outlined in F i g . 1 , the f i n e l y ground lead


s u l f i d e concentrate i s contacted with a pH 2.3 i r o n - f r e e aqueous
nutrient medium (38) in proportion to produce a 14% (W/V) s o l i d
to l i q u i d suspension, which i s inoculated with an adapted active
culture of T. ferrooxidans. The leach suspension i s agitated and
aerated with a i r containing 0.2% (V/V) carbon dioxide (39) and
incubated at 35°C f o r four days. After s o l i d - l i q u i d separation,
Bioextractiv
eApplication
san dOptimizatio
n 379

lead i s recovered from the leach r e s i d u e , which can be considered


to be a high-grade Pb-concentrate and the d i s s o l v e d metals, copper,
cadmium and z i n c , from the f i l t r a t e . A f e a s i b i l i t y study of the
leach l i q u o r treatment (41) i n d i c a t e d , that f i r s t iron has to be
precipitated by increasing the pH to 3.0, i . e . with c a l c i t e :

3Fe (S0 ) +12H 0i2{H[Fe(S0 )


2 4 3 2 4 2 · 2Fe(0H) ]} + 5H S0
3 2 4 [7]

jarosite

The above p a r t i a l h y d r o l y s i s reaction of f e r r i c s u l f a t e r e s u l t s


mainly hydrogen j a r o s i t e , which i s easy to f i l t e r . Then, cadmium
and copper are recovered through cementation on zinc d u s t :

Cd + +
+ Zn > Cd + Z n + +
[8]

and

Cu + +
+ Zn > Cu + Z n + +
[9]

The f i n e Cd-Ni p a r t i c l e s are removed as a f i l t e r - c a k e and can be


shipped elsewhere f o r further treatment. The zinc remaining in
s o l u t i o n can be precipitated by i n c r e a s i n g the pH to about 7.5 by
addition of magnesium oxide:

Zn + +
+ 20H" Ϊ Z n ( 0 H ) 2 [10]

The zinc hydroxide can be d i s s o l v e d separately by s u l f u r i c acid


and recovered by e l e c t r o l y s i s . The e l e c t r o l y t e may be used in a
c y c l i c process in t h i s system.

After zinc recovery the leach s o l u t i o n i s recycled to the


nutrient r e s e r v o i r and adjusted f o r pH with s u l f u r i c acid and f o r
the nutrient concentrations, before being reused in the leaching
step.

Semi-industrial studies (40) c a r r i e d out with 301 leach s u s ­


pensions resulted in overall e x t r a c t i o n s higher than 94%. These
studies included releaching of the leach r e s i d u e , which has been
regrounded to 1iberate new substrate s u r f a c e s . I t has been found
that, when about 50% of extraction was achieved, the substrate
380 Α. Ε. Torma

surface has been covered with basic f e r r i c hydroxides and lead


s u l f a t e p r e c i p i t a t e s , which impeded the bacterial a c t i v i t y . At
the end of the second leaching the leach residue has been analyzed.
I t contained lead in form of PbS and PbSO^ in proportions of about
one to one. This one to one r a t i o i s of technological importance
in the s i n t e r i n g and b l a s t furnace smelting, where exothermic
(according to equations [ 1 ] and [ 3 ] ) and endothermic (according to
equations [ 2 ] and [ 4 ] ) reactions take place. I f the r i g h t propor­
t i o n between PbS and PbSO^ e x i s t e d i n the feed, then the heat
liberated by the exothermic reactions would provide the heat
necessary f o r the endothermic r e a c t i o n s . Hence, the presence of
s u l f a t e i n the feed would reduce the c y c l i c load used to d i s s i p a t e
the excess heat liberated by the exothermic reactions during con­
ventional sintering.

III. OPTIMUM LEACH CONDITIONS

The optimum conditions f o r the microbiological leaching of


off-grade lead s u l f i d e concentrates are s i m i l a r to those which are
derived f o r leaching of other metal s u l f i d e s by T. ferrooxidans.
These conditions are summarized i n Table I .

TABLE I Optimum Conditions for Lead Sulfide


Concentrates Leaching

Factors Optimum Conditions Reference No.

1. Temperature 35°C 42
2. pH 2.3 42
3. Eh <500 mV 27,44
4. Substrate concentrate 14-20% (W/V)
(-400 mesh)
5. Bacterial inoculum 7% 18
6. Nutrients: (NHJ SO 3g/l 38,42,43
4 2 4 n A

K HP0
2 4 O.Sg/l 38,42,43
CO2 in air 0.2% (V/V) 39

°2 an intensive aeration 27
is required
7. Substrate particle size -325 to -400 mesh 18
Bioextractive Applications and Optimization 381

IV. ECONOMICAL ASPECTS

The preliminary cost estimate described in t h i s section deals


with the microbiological leaching of an off-grade lead s u l f i d e
concentrate containing 42.9% l e a d , 29.6% s u l f u r , 16.7% i r o n , 7.7%
z i n c , 2.4% copper and 0.02% cadmium. In t h i s process 100,000 kg
of lead s u l f i d e concentrate w i l l be leached in suspensions con­
t a i n i n g 20% pulp density at pH 2.3 and temperature 35°C during
f i v e days. The minimum recovery f o r z i n c , copper and cadmium i s
projected to be 95%.

This leaching process can be c a r r i e d out in conjunction with


an e x i s t i n g pyrometallurgical lead smelting p l a n t . The high grade
lead concentrate, containing PbS and PbSO^, produced in t h i s
process can be mixed with other concentrates to provide an ideal
feed f o r the smelter (or s i n t e r ) .

The capital cost of t h i s process i s estimated to be


$7,300,000 (45-47). This includes the costs of leach tanks (con­
c r e t e ) , pumps, a e r a t i o n , r e g r i n d i n g equipments,thickeners, f i l t e r s ,
dryer, e l e c t r o l y s i s equipment, t h e i r i n s t a l l a t i o n s , and the
building.

The y e a r l y metal production i s shown in Table I I .

TABLE II

Metal PbS Cone. Metal Con­ Recovery d/Y Yearly Metal


kg tent % % Production
kg/Y

Zn 100,000 χ 7.7/100 X 95/100χ 365 * 2,700,000


Cu 100,000 χ 2.4/100 X 95/100χ 365 830,000
Cd 100,000 χ 0.02/100 X 95/100χ 365 7,000

The y e a r l y metal production values are rounded up to s i m p l i f y


the c a l c u l a t i o n . The y e a r l y market value of metals are given in
Table I I I .
382 Α. Ε. Torma

TABLE III

Metal Metal Production Selling Price Market Value Reparti­


$/kg $/y tion %

Zn 2,700,000 χ 0. 76 2,050,000 59.3


Cu 830,000 χ 1.62 1,350,000 39.0
Cd 7,000 χ 8.36 60,000 1.7

Total Market Value: 3,460,000 100.0

The market values are rounded-up and the r e p a r t i t i o n percentages


are based on the total market value.

The y e a r l y expenses are given in Table IV.

TABLE IV

Direct Costs:
Administration
Labor
Reactives (oxygen, limestone, hydrated lime,
magnesia, H SO , K HPO , (NH ) S0.
0 0 and zinc
o

powder)
Electricity $800,000/year

Indirect Costs:
Maintenance
Electricity
Water
Depreciation at 10%
Interest at 10%
Insurance $1,600,000/year

Total: $2, 400, 000/year

The r e p a r t i t i o n of the y e a r l y expenses per metal produced i s


given in Table V.
Bioextractive Applications and Optimization 383

TABLE V

Metal Total Leaching Repartition Leaching Expenses


Expenses $/y (Table 3) % $/y
Zn 2, 400, 000 χ 59.3/100 1,420,000
Cu 2,400,000 χ 39.0/100 940,000
Cd 2,400,000 χ 1.7/100 40,000

Total.: 2,400,000

The cost of metal produced with respect to the leaching


expenses i s given in Table V I .

TABLE VI

Metal Leaching Expenses Metal Production Cost of Metal


$/y (Table 2) kg/y Produced $/kg

Zn 1,420,000 / 2,700, 000 0.53


Cu 940,000 / 830,000 1.13
Cd 40,000 / 7,000 5.71

The gain ($/kg) of metal produced i s given in Table V I I .

TABLE VII

Costs of metal production $/kg


Zn Cu Cd

Costs of mining concentration


(estimated) 0.11 0.14 0.39
Costs of leaching, regrinding,
cementation, electrowinning
(Table 6) 0.53 1.13 5. 71

Total expenses (A) 0.64 1.27 6.10

Selling price (B) 0. 76 1.62 8.36

Gain (B-A) 0.12 0.35 2.26


384 Α. Ε. Torma

The estimated costs (48) for mining and concentration do not i n ­


clude any expenses f o r lead which make up the major part of the
costs.

The r e a l i s a b l e y e a r l y p r o f i t of the leaching plant i s given


in Table V I I I .

TABLE VIII

Metal Metal produced Gain realized Profit


(Table 2) kg/y (Table 7) $/kg $/y

Zn 2, 700, 000 χ 0.12 324,000


Cu 830,000 χ 0.35 - 290,000
Cd 7,000 χ 2.26 16,000

Total: 630,000

The y e a r l y return of revenue of about $6.3 χ 10° f o r an


investment of $7.3 χ 1 0 6
appears acceptable. The above revenue
should be increased with the amount of money which would be paid
f o r the lead concentrate produced by t h i s leaching process.

V. CONCLUSION

The preliminary economic assessment presented in t h i s study


indicates that the microbiological leaching technique may present
an economically v i a b l e a l t e r n a t i v e f o r treatment of off-grade lead
s u l f i d e concentrates. The leach residue of t h i s process i s an
upgraded lead concentrate which can be used in the c l a s s i c a l
smelting process f o r recovery of lead. The d i s s o l v e d metal values
can be recovered by producing a reasonable p r o f i t .

Further, the microbiological leaching technique requires


r e l a t i v e l y low capital investment and low operating c o s t . I t can
be b u i l t near the mining s i t e and used as a complementary t r e a t ­
ment p r i o r to the smelting process. The microbiological leaching
treatment does not require high temperature nor pressure. It is
Bioextractive Applications and Optimization 385

easy to operate and c o n t r o l . This process, i f properly operated


does not compromise in any way the q u a l i t y of the environment.

VI. REFERENCES

1. Anonymous, in "McGraw-Hill Encyclopedia of Science and


Technology", V o l . 7, p. 424. McGraw-Hill Book Co., I n c . ,
New York, 1960.
2. McKay, J . E . , in " K i r k Ottmer Encyclopedia of Chemical
Technology", Vol. 12, p. 207. John Wiley & Sons, I n c . , New
York, 1967.
3. Moulds, D.E., U.S. Dept. I n t e r . Bur. M i n . , Washinqton, D.C.,
1965.
4. Dana, E.S.,and Ford, W.E., "A Textbook of M i n e r a l o g y " ,
p. 415. John Wiley & Sons, I n c . , New York, 1932.
5. K e l l o g , H.H., and Basu, S . K . , Trans. Met. Soc. AIME, 218, 70
(I960).
6. K e l l o g , H.H., Trans. Met. Soo. AIME, 220, 1622 (1962).
7. Yurko, G.A., in "AIME, World Symposium on Mining and
Metallurgy of Lead and Z i n c " , (C.H. C o t t e r i l l and J.M. Cigan,
E d s . ) , Vol. 2, p. 330. AIME P u b l i c a t i o n , New York, 1970.
8. Hancock, G.C., Hart, D.H., and Pelton, L.A.H., in "AIME,
World Symposium on Mining and Metallurgy of Lead and Z i n c " ,
(C.H. C o t t e r i l l and J.M. C i g a n , E d s . ) , V o l . 2 , p. 790. AIME
P u b l i c a t i o n , New York, 1970.
9. Woods, S . E . , and Temple, D.A., The Present Status of Imperial
Smelting Process. Presented at the 8th Commonwealth Min.
Met. Congr. in Melbourne, Feb. 27 - Apr. 4 , 1965.
10. V i z s o l y i , Α . , Veltman, H., and Forwards, F.A., in "Unit
Process in Hydrometallurgy", (M.E. Wadsworth and F.T. D a v i s ,
E d s . ) , p. 336. Gordon and Breach Science P u b l i s h e r s , New
York, 1964.
11. Gerlach, J . , Pawlek, F., and T r a u l s e n , K., Erzbergb.
Metallhuttenw. , 18, 605 (1965).
12. V i z s o l y i , A . , Veltman, H., and Forwards, F.A., Trans. Met.
Soo. AIME, 227, 215 (1963).
13. Mackiw, V . N . , and Veltman, H., Can. Min. Met. Bull., 60, 80
(1967).
14. S c o t t , P.D., and N i c o l , M . J . , Inst. Min. Met. Trans. Seet.C,
85, C40 (1976).
15. Skewes, H.R., Proo. Aust. Inst. Min. Met., 244, D e c , 35
(1972).
386 Α. Ε. Torma

16. Terayama, Κ., I z a k i , Τ . , and A r a i , Κ., J. Jap. Inst. Met.,


36, 591 (1972).
17. K r u e s i , P.R., A l l e n , E . S . , and Lake, J . L . , Can. Min. Met.
Bull., 66, 81 (1973).
18. Torma, A . E . , and Subramanian, K.N., Internat. J. Miner.
Process., 1 , 125 (1974).
19. Torma, A . E . , Can. Pat. Appl. No. 203,547 (1974).
20. E h r l i c h , H.L., and Fox, S . I . , Biotechnol. Bioeng., 9, 471
(1967).
21. LeRoux, N.W., New Scientist, 43, 12 (1969).
22. Z a j i c , J . E . , "Microbial Biogeochemistry", Academic P r e s s ,
New York, 1969.
23. Trudinger, P.Α., Miner. Sci. Eng., 3, 13 (1971).
24. Karavaiko, G . I . , Kuznesov, S . I . , and Golomzik, A . E . ,
" B a c t e r i a l Leaching of Metals from O r e s " , Nauka, Moscow,
1972.
25. Tuovinen, O.H., and K e l l y , D.P., Internat. Metal. Rev., 19,
21 (1974).
26. Schwartz, W., Bild Wiss., 13, 60 (1976).
27. Torma, A . E . , Adv. Biochem. Eng., 6, 1 (1977).
28. Brock, T . D . , Brock, K.M., B e l l y , R.T., and Weiss, R.L.,
Arch. Mikrobiol., 84, 54 (1972).
29. B r i e r l e y , C.L., and B r i e r l e y , J . Α . , Can. J. Microbiol., 19,
183 (1973).
30. deRosa, M., Gambacorta, Α . , and B u ' l o c k , J . D . , J. Gen.
Microbiol., 86, 156 (1975).
31. Murr, L.E., and Berry, V.K., Hydromet., 2, 11 (1976).
32. C o r r i c k , J . D . , and Sutton, J . Α . , U.S. Bur. M i n . , R . I . No.
7126, 1968.
33. Pol k i n , S . I . , Karavaiko, G . I . , Tauzhayanskaya, Z.A., and
Panin, V.V., in "Proceedings of the 9th International
Mineral Processing C o n g r e s s " , (N. A r b i t e r , E d . ) , V o l . 1 ,
p. 347. Gordon and Breach Science P u b l i s h e r , New York, 1970.
34. P o l k i n , S . I . , Panin, V.V., Adamov, E.V., Karavaiko, G . I . , and
Chernyak, A . S . , Presented at the 11th I n t e r n a t . Miner.
Process. Congr. in C a g l i a r y , I t a l y , 1975.
35. L y a l i k o v a , N.N., S h l a i n , F.B., Unanova, O.G., and Anisimova,
L . S . , Izv. Akad. Nauk SSR. Ser. Biol., No. 4, 564 (1972).
36. S i l v e r , Μ., and Torma, A . E . , Can. J. Microbiol., 20, 141
(1974).
Bioextractive Applications and Optimization 387

37. Tomizuka, N., Report of the Fermentation Research Institute,


Japan, No. 48, 51 (1976).
38. Silverman, M.P., and Lundgren, D.G., J. Bacteriol., 77, 642
(1959).
39. Torma, A . E . , Walden, C.C., Duncan, D.W., and Branion, R.M.R.,
Biotechnol. Bioeng., 14, 777 (1972).
40. Torma, A . E . , CRM-Internal Report, Q.D.N.R., May 17, 1973.
41. Kougioumoutzakis, D., Torma, A.E.., O u e l l e t , R., and
L e H o u i l l i e r , R., Presented at the Ann. Congr. Assoc. Can.
Franc. Adv. S c i . in Quebec, May 8-10, 1974.
42. Torma, A . E . , Walden, C.C., and Branion, R.M.R., Biotechnol.
Bioeng., 12, 501 (1970).
43. Tuovinen, O.H., Niemela, S . I . , and Gyllenberg, H.G.,
Biotechnol. Bioeng., 13, 517 (1971).
44. Moss, E.T., and Andersen, J . E . , Proc. Aust. Int. Min. Met.,
No. 225, 15 (1968).
45. Guthrie, K.M., Chem. Eng., March 24, 114 (1969).
46. Leibson, I . , and Trischman, C.A., Chem. Eng., May 3 1 , 69
(1971).
47. P a r k i n s o n , E.A., and Mular, A . L . , "Mineral Processing
Equipment Cost E s t i m a t i o n " , published by Can. I n s t . Min.
Met., Montreal, 1972.
48. Torma, A . E . , O u e l l e t , R., and Gabra, G.G., Presented at the
Ann. Conf. Chem. I n s t . Can. in Regina, Saskatchewan in June
2 - 5 , 1974.
MICROBIOLOGICAL LEACHING OF CARBONATE-RICH

GERMAN COPPER SHALE

K. Bosecker

Bundesanstalt fur Geowissenschaften und Rohstoffe


3000 Hannover 5 1 , Federal Republic of Germany

D. Neuschutz
U. Scheffler

F r i e d r i c h Krupp GmbH., Krupp F o r s c h u n g s i n s t i t u t


4300 Essen, Federal Republic of Germany

Bacterial leaching techniques have been tested to extract the


copper from German copper shale. Laboratory tests were carried
out in airlift-percolators and in shaking flasks. Pilot plant
units were constructed using glass-fibre columns filled with 1.4
tons of copper shale. Pure cultures of T. ferroxidans and T. t h i o -
oxidans were used as leaching agents. Because of the high car-
bonate content of the ore, bacterial leaching was feasible only
after previous neutralization of the carbonate or with simulta-
neous addition of acid. Within 380 days 31% of the copper and
55% of the zinc were extracted. After optimization an extraction
yield of 70% Cu and 80% Zn seems to be feasible. Some factors
influencing the leaching process and a preliminary evaluation of
the economy of bacterial leaching of the copper shale are dis-
cussed.

389
390 Κ. Bosecker ef al.

I. INTRODUCTION

Within the l a s t few decades there has been a continously


increasing world-wide demand f o r m e t a l l i c raw m a t e r i a l s . This
demand can be covered in the future only by the discovery of new
d e p o s i t s , the improvement of conventional processing methods, and
the development of new technical operations for recovering v a l u a ­
ble metals from low-grade ores and a l s o from waste m a t e r i a l s . The
German Federal Republic i s the 4th l a r g e s t copper consumer in the
world ranging a f t e r the USA, Japan, and the USSR. In 1973,20.8%
of the world copper production was mined in the USA whereas the
German copper consuming i n d u s t r y , which depends mainly on foreign
copper resources, had to import 1.2 m i l l i o n tons of copper. Fig.
1 shows the imports of copper during the l a s t few decades and
gives an impression of the increasing c o s t s .

German copper shale - the most important copper ore in the


GFR - contains about 12 m i l l i o n tons of copper. Conventional
mining of copper shale was stopped in 1955 because of u n p r o f i ­
table production costs r e s u l t i n g from the low copper content and
from the small thickness of the copper-bearing layer.

Low-grade ores may be extracted by microbiological leaching


and these biohydrometallurgical methods are already being used
commercially for the recovery of copper and uranium ( 1 - 6 ) . On
the whole, the b i o l o g i c a l and chemical reactions which occur
during the leaching process are known ( 7 ) . But the e f f i c i e n c y of
such processes depends mainly on the chemical and mineralogical
c h a r a c t e r i s t i c s of the individual ore and therefore the leaching
conditions must be e s t a b l i s h e d f o r each type of ore. In the pre­
sent paper, the a p p l i c a b i l i t y of bacterial leaching in the
extraction of copper from the German copper shale has been i n v e s ­
tigated.
Bioextractive Applications and Optimization 391

10001 Mio.DM

3280

3000

2000
1800

UOO
1181 1200
1000 1000

600 477
309
200

1953 1963 1973

Fig. 1. German copper imports between 1953 and 1973

in tons H I and values

II. MATERIALS AND METHODS

Laboratory t e s t s were c a r r i e d out in a i r - l i f t - p e r c o l a t o r s and


in shaking Erlenmeyer f l a s k s . The copper shale used in these
experiments was obtained from old mining dumps and contained
2 . 1 % Cu, 3.0% Fe, 0 . 0 1 % Zn, 2.5% S, and 1 8 . 1 % C 0 . 2 The main ore
mineral was chalcopyrite followed by c h a l c o c i t e , b o r n i t e , and
pyrite.

Copper shale (200 - 500 g, crushed to a p a r t i c l e s i z e of


about 10 mm) was i r r i g a t e d in a i r - l i f t - p e r c o l a t o r s with 150 ml of
nutrient broth inoculated with pure cultures of Thiobacillus
ferrooxidans, T. thiooxidans, or with mixed cultures of both
strains.
392 Κ. Bosecker ef al.

Shaking Erlenmeyer f l a s k s were used for submerged fermenta­


t i o n of f i n e - g r a i n e d ore. D i f f e r e n t amounts of copper shale with
a p a r t i c l e s i z e of 63 - 200 μ were suspended in 90 ml of culture
medium, inoculated with 10 ml of a c t i v e l y growing t h i o b a c i l l i ,
and incubated on a rotary shaker at 200 rpm. Samples were taken
p e r i o d i c a l l y from the leach suspensions to determine pH-values,
metal content, and presence of bacteria. The metal content was
determined by atomic absorption spectroscopy, s u r v i v i n g bacteria
were i d e n t i f i e d a f t e r i n o c u l a t i o n into fresh culture medium.

The pure cultures of T. ferrooxidans and T. thiooxidans used


in t h i s study were o r i g i n a l l y i s o l a t e d from acid mine water in the
Harz Mountains and are c u l t i v a t e d in our laboratory. T. thiooxi­
dans i s maintained in the medium described by Starkey (8) using
elemental s u l f u r as an energy source. T. ferrooxidans i s c u l t i ­
vated in the culture medium described by Leathen, et a l . ( 9 ) .

A l l laboratory experiments were c a r r i e d out at a temperature


of 30°C. The laboratory t e s t s were t r a n s f e r r e d to the semi-tech­
nical stage in p i l o t plant u n i t s , constructed of g l a s s - f i b r e
columns of 2.5 m in height and 0.8 m in diameter. Each column
was f i l l e d with 1.4 tons of copper shale which had been crushed
into a g r a i n s i z e of 40% < 10 mm. The material was a l s o gathered
from old mining dumps but was d i f f e r e n t in the metal content.
This ore contained 0.76% Cu, 2.3% Fe, 0.6% Zn, 1.6% S, and 1 9 % C 0 . 2

At the beginning of the experiments the ore was i r r i g a t e d with


d i l u t e d s u l f u r i c acid adjusting the pH in the i n f l u e n t s o l u t i o n s
to 1.5, 1.7, and 2.0. After reaching pH-values of 2.0 to 2.5 in
the e f f l u e n t s o l u t i o n , the columns were inoculated with mixed
cultures of T. ferrooxidans and T. thiooxidans, and the pH in the
i n f l u e n t suspension was maintained within a range of 1.9 to 2.1 in
one t e s t and 2.0 to 2.2 in another with the addition of s u l f u r i c
acid. The c i r c u l a t i n g leach suspension (about 800 to 900 1) was
collected in a big tank at the bottom of the column, pumped up
Bioextractive Applications and Optimization 393

and sprayed again on top of the f i l l . The tank could be aerated


separately.

III. RESULTS AND DISCUSSION

A. Leaching in A i r - L i f t Percolators

Because of the high carbonate content of the copper s h a l e ,


the pH-values in the leach suspension continuously increased. The
decrease in pH f o r d i f f e r e n t leaching experiments a f t e r i n o c u l a ­
t i o n with pure and mixed c u l t u r e s of T. thiooxidans and T. ferro­
oxidans i s shown in F i g . 2. S u l f u r i c acid was added to lower the
pH, but in case of T. ferrooxidans ( F i g . 2 b) the pH-values
always rose to 7.5 followed by a complete l o s s of bacterial acti­
vity. A f t e r repeated a d d i t i o n of a c i d , only T. thiooxidans was
able to maintain a pH of 4 in the leach suspension. T. ferro­
oxidans survived when mixed c u l t u r e s were used ( F i g . 2 c ) . Both
s t r a i n s grew rather well in the mixed culture but in s p i t e of
better growth only 0.17% of the copper was leached within 100
days. In the same period 27% of the copper was extracted by
chemical leaching with 1 Ν H^SO^ ( F i g . 3 ) . In regard to these
r e s u l t s , bacterial leaching of the copper shale seemed to be
f e a s i b l e only a f t e r previous n e u t r a l i z a t i o n of the carbonate or
with simultaneous a d d i t i o n of a c i d . Strong acid conditions
(6 Ν H^SO^) accelerated the process of n e u t r a l i z a t i o n of the o r e ,
however the leaching process was stopped because the forma­
t i o n of mud in the leaching system. Using low acid concentrations
(for example 1 Ν r ^SO^) the n e u t r a l i z a t i o n was very slow and
afterwards the surface of the ore was covered by a gypsum p r e c i ­
pitate which i n h i b i t e d a d i r e c t bacterial contact with the ore
and diminished the leaching efficiency.

B. Leaching in P i l o t Plants

A i r - l i f t - p e r c o l a t o r s were used as a model for dump and heap


leaching. However, r e s u l t s obtained at a laboratory scale may be
394 Κ. Bosecker ef al.

pH

6HM M ι

Th. t h i o o x i d a n s

Days

Days
PH

6 a in
4

Th.thiooxidans •
2
Th.t*rrooxidans

20 40 Days

Fig. 2. Bacterial leaching of carbonate-rich German copper


shale in air-lift-percolators. pH-trend after inoculation with
pure cultures of T. thiooxidans (a), T. ferrooxidans (b), and
mixed cultures of T. thiooxidans and T. ferrooxidans (c) ^ addi­ 3

tion of 1 Ν Η SO .

applied on an i n d u s t r i a l scale only with r e s e r v a t i o n . Therefore,


i n v e s t i g a t i o n s in p i l o t plant u n i t s seemed to be necessary. The
columns we used were considered as a cut out of a mining dump in
which the conditions for optimizing the leaching process were
tested. Because of the high carbonate content of the copper s h a l e ,
the most important f a c t s which were taken into consideration to
achieve optimum copper extraction rates are the pH-value of the
leach suspension and a s u f f i c i e n t supply of oxygen in the f i l l .

The r e s u l t s of the percolator experiments indicated that


bacterial leaching would be p o s s i b l e only a f t e r previous neutra­
l i z a t i o n of the carbonate. Complete n e u t r a l i z a t i o n produces
Bioextractive Applications and Optimization 395

50

AO

30

20

10

20 60 80 100 120

Fig. 3. Chemical leaching of carbonate-rich German copper


shale in air-lift-percolators with 1 Ν H^SO^. pH-trend and
copper extraction.

about 600 kg Ca^SO^ · 2 h^O per ton of copper shale which clogs
the f i l l and stops the leaching process. Partial neutralization
i s s u f f i c i e n t provided that the pH in the leach suspension i s
within a s u i t a b l e range f o r bacterial growth. T. ferrooxidans,
which i s more s e n s i t i v e to pH-changes than T. thiooxidans, i s
most active in the pH range of 1.8 to 2.5 ( 1 0 - 1 2 ) . In order not
to i n h i b i t the bacterial a c t i v i t y , the pH in the i n f l u e n t leach
suspension was maintained in the two experiments within a range
of 1.9 to 2.1 and 2.0 to 2.2. At maximum flow r a t e , which depends
on the permeability of the f i l l , the pH of the e f f l u e n t increased
by about 0.3 as a r e s u l t of the p a r t i a l n e u t r a l i z a t i o n of the
carabonate during p e r c o l a t i o n . At lower flow r a t e s , the d i f f e r ­
ence in the pH between the i n f l u e n t and e f f l u e n t leaching s o l u ­
t i o n s increased considerably. However, the oxygen supply within
the f i l l , which i s necessary f o r a d i r e c t bacterial oxidation of
metal s u l f i d e s , i s reduced at maximum i r r i g a t i o n . At maximum
flow there i s no d i r e c t oxygen supply v i a holes and pores in
396Κ .Bosecke
r et al.

the f i l l and therefore only the oxygen d i s s o l v e d in the l i q u i d


phase can be used by bacteria. An oxygen demand of 37.2 kg 0^
per ton of copper shale has been calculated f o r the d i r e c t bac­
t e r i a l oxidation of metal s u l f i d e s . Without a r t i f i c i a l aeration
several years would be needed to transport the oxygen into the
fill. In view of these data, d i r e c t bacterial oxidation of metal
s u l f i d e s (reaction a) would seem to be n e g l i g i b l e , and the bac­
t e r i a l a c t i v i t y i s mainly r e s t r i c t e d to an i n d i r e c t oxidation of
s u l f i d e s via reoxidation of chemically reduced f e r r i c iron (reac­
t i o n b and c ) .

Me S + 2 0 - f^ooxidans
2
T
> ^ ^ ( a )

Me S + F e ( S 0 )
2 4 3 > Me S 0 + 2 Fe S 0 + S°
4 4 (b)

2 F e S 0 + H S 0 + l/2 0
4 2 4 2
T
' f^oxidans > ^ { $ 0 ^ ^ ^ ( c )

In order to accelerate the bacterial oxidation of ferrous


iron to f e r r i c iron the e f f l u e n t leaching suspension was aerated.
P r e c i p i t a t i o n of f e r r i c iron was avoided by taking care that the
leachin