MIRCENJournal, 1987, 3, 169-178

Development of a culture collection and

data bank for biometallurgical processes in
India

Department of Microbiology, M.A.C.S. ResearchInstitute, Law CollegeRoad, Pune 411 004, India.

Received 4 April 1986; accepted 12 January 1987

Introduction
For developing nations, biometallurgical processing of minerals is gaining prominence
owing to the current energy crisis, the repercussions of which are felt pointedly by
such nations. For instance, the energy-intensive pyrometallurgical processes to obtain
metal values from ores, especially low grade, have suffered a great setback, which has
made these nations look for alternative processing methods. Biometallurgy appears to
be the technology of choice from the point of view of pollution as the pollutants are
contained during the controlled process. Besides, it has other advantages such as
comparatively inexpensive procedure which does not require a high degree of
sophistication and can be adopted in a labour-intensive situation. There are factors
such as climate, which is warm in many developing nations, favouring biometallurgical
processes governed by microorganisms. The use of these processes, it is believed,
would lead to an increase in the production of metals which have an escalating
demand in such countries where industrialization is taking place and the demand has
to be met by imports. The technology, therefore, would lead a nation to self-
sufficiency by means of producing metals from available low-grade and waste ores or
upgrading their quality, which can be termed as appropriate technology for the
country. This communication deals with the development of a data bank for this
branch of biotechnology in India.
In the Indian context, it was decided to concentrate on copper and manganese
minerals which were amenable to microbial attack. The reasons for selecting
microbial leaching of copper, a well-known technology (Brierley 1978), was on
account of the fact that out of the estimated demand for copper (160,000 tonnes) per
annum, only 30% is met by the domestic production (45,000 tonnes) and hence, it is
an import-dependent commodity in India (Times of India Directory I984). The copper
reserves in the country are estimated to be 360 million tonnes out of which nearly 80%
* To whom correspondence should be addressed.
9 Oxford UniversityPress 1987
170 K. M. Paknikar and A. D. Agate

represent the low-grade variety which cannot be utilized for metal extraction by
conventional techniques owing to economic reasons. Similarly, manganese, which was
one of the major exported commodities from India, has fallen to a low level, owing to
high mechanization and use of novel technologies in other manganese-producing
countries (Weiss 1977). It was felt, therefore, that if innovations such as microbial
conversion and beneficiation techniques (Agate and Deshpande 1977) are used, it
would help the country to restore the balance of trade.
With this view in mind, work was started in our laboratory on microbial leaching of
copper and conversion or beneficiation of Indian manganese ores through the funds
provided by the various Government of India agencies such as the University Grants
Commission, Council of Scientific & Industrial Research, and Department of Science
& Technology.

Methodology
Collection of samples
Out of the ten manganese and copper mining states in India, nine were visited by our
team and samples were collected from different ecosystems such as ores, wastes, soils,
acid mine waters, etc. The samples were transported to the laboratory and subjected
to the following analyses:

1. Chemical and physical examination:

The colour, pH, and other physical characteristics were noted and the chemical
examination consisted of elemental analysis of the samples on an atomic absorption
spectrophotometer (Perkin-Elmer 2380). This information was tabulated on an 'Ore
Card' specially designed for the purpose (Fig. 1).

2. Bacteriological examination:
The bacteriological examination consisted of isolation of beneficial microorganisms
like the iron- and sulphur-oxidizing bacteria belonging to the genus Thiobacillus. The
major organisms, T. ferrooxidans and T. thiooxidans, were isolated by enrichment
using, respectively, the 9K salts medium of Silverman and Lundgren (1959) and the
medium of Vishniac and Santer (1957). The manganese oxidizers, such as Arthrobacter
and Bacillus species, were isolated by enrichment in the medium of Bromfield and
Skerman (1950).

3. Quantification of activity:
The beneficial activities of the isolated cultures, namely iron oxidation by T.
ferrooxidans and manganese oxidation by manganese oxidizers, were quantified by
the methods described below and which were specially devised in our laboratory.

Determination of Iron-Oxidation Rate (IOR)

A 48-h culture of T. ferrooxidans was centrifuged first at 2500 x g to remove ferric
hydroxide/jarosite precipitates, washed twice with 9K medium (without ferrous
sulphate) and then centrifuged at 10,000 x g for 10 min at 4~ The cell suspension
thus obtained, containing 108 cells/ml, was used as an inoculum at the 10% v/v level.
The iron oxidation experiments were carried out in 500 ml Erlenmeyer flasks
containing 180 ml 9K medium with 75 mM ferrous sulphate and after inoculation,
 Culture collection and data bank for biometallurgical processes 171

A. Fig 1. Ore card (originally 12.5 X 20 cm). A.front; B, reverse

COLLECTEO FROM:
CHEMICAL FORMULA
DATE:

CHEMICAL ANALYSIS % BIOLOGICAL ANALYSIS

COPPER SODIUM ORGANISM COUNT MCM NO
IRON POTASSIUM IRON OXIDIZERS
SULPHUR SULPHATE Thiobacillus ferrooxidans
MANGANESE SULPHIDES
ALUMINIUM CARBONATE
NICKEL CHLORIDE SULPHUR OXIDIZERS'
I COBALT Thiobacillus thiooxidans
ZINC
SILICA
URANIUM SULPHATE REDUCERS
SILVER Oesulphovibrio
ORGANIC CARBON
LEAD Desulfotomaculum
ORGANIC NITROGEN
MERCURY
MOLYBDENUM
CERIUM MANGANESE OXIDIZERS
BARIUM Arthrobacter
Bacillus
CALCIUM
CODE NATURE

Be
TOTALAUTOTROPHS!TOTAL HETEROTROPHS A:H RATIO AMENABILITY

LEACHING STUDIES MEDIUM BEST COMBINATION OF ORGANISMS

ORE QTY. ORGANISM % REMOVAL DAYS

LEACHATE COMPOSITION

RECOVERY
172 K. M. Paknikar and A. D. Agate

were incubated at 30~ The flasks were aerated by means of spargers using a filtered
compressed air supply for 24 h. Ferrous iron content, which was not oxidized by the
bacteria, was determined at 30 min intervals by taking 1 ml of the sample from each
flask and titrating with 0.1 u potassium dichromate using N-phenylanthranilic acid as
an indicator. Iron-oxidation curves were plotted by subtracting the values of
unoxidized ferrous iron from initial ferrous iron content against time (h) and IOR of
the culture was then calculated as oxidation capacity of the culture per ml per hour.

Determination of manganese-oxidizing activity

The manganese-oxidizing activity of the bacteria was checked by estimating the
residual manganese content in the culture supernatant after full growth and
centrifugation to remove cells and precipitated manganese dioxide. The manganese
remaining in the supernatant was estimated by atomic absorption spectrophotometry
and the manganese-oxidizing activity was expressed as per cent manganese oxidized.
The 10% (v/v) inoculum of a cell suspension, having an optical density at 550 nm of
0.14, was used and the experiments were carried out in 500 ml flasks containing
200 ml Bromfield and Skerman's (1950) medium at 30~

Preservation
Routinely, the cultures were maintained by serial transfers after two months, on slants
of Bromfield and Skerman's (1950) medium for manganese oxidizers while the iron-
oxidizing thiobacilli were cultured in liquid 9K medium with 150 m i FeSO4 (200 ml
held in 500 ml capped plastic bottles). With a view to providing these cultures in an
active form to mining organizations who wish to use them on a large scale, various
methods of preservation such as lyophilization, liquid nitrogen storage and mixing
with inert carriers were tested. Information obtained by these studies was tabulated
on 'Preservation Cards'. This represented the beginning of our Microbiological
Resource Centre where cultures used in biohydrometallurgy were preserved and the
culture collection was affiliated to the World Federation of Culture Collections,
Australia.

Laboratory studies
These studies were carried out on the cultures possessing good iron- and manganese-
oxidizing activities. Starting from Erlenmeyer flasks and air-lift percolators, both the
processes of copper leaching and manganese bioconversion were scaled to 100 kg vats
and 300 kg leach tanks and columns. A PVC column series is shown in Fig. 2.
The leaching of copper was monitored by estimating the amount of copper in the
leached solution (often referred to as the bioleachate) by atomic absorption
spectrophotometry. The bioconversion of manganese was quantified by estimating
manganese dioxide content according to the method of Vogel (1961). The optimal
process parameters for copper leaching and manganese bioconversion were also
defined during these studies.

Results
The state-wise distribution of samples collected and the beneficial microorganisms
isolated from these ecosystems are recorded in Tables 1 and 2. The representative
 Culture collection and data bank for biometallurgical processes 173

Fig. 2 Specially designed PVC columns for expanded-scale copper leaching (from Agate et aL 1984)

cultures having high IOR and manganese-oxidizing activity, which were screened and
selected for preservation studies are listed in Table 3 along with their activity checks
during the selected methods of preservation and storage for 12 months (Gupta and
Agate 1986). It was evident that T. ferrooxidans could be preserved without much loss
in activity by mixing with chalcopyrite ore. This method was also convenient for large-
scale supply of the cultures to interested mining organizations. The manganese-
oxidizing bacteria could be best preserved by the conventional technique of
lyophilization (S. G. Gupta, personal communication). Tables 4 and 5 show the
typical results of copper leaching and manganese-bioconversion processes (Agate et
al. 1984; Ghosh 1983), which indicated that both the processes are technologically
feasible on a large scale in India.
The data collated on iron oxidation, manganese oxidation, copper leaching,
manganese bioconversion, and methods of preservation for all the cultures were
recorded on 'Culture Cards' (Fig. 3). The Culture Cards and Ore Cards were stored in
a 'Kardex' filing system after cross-indexing the relevant information on ores and
cultures for easy retrieval when needed. This formed the data bank for these two
biometallurgical processes developed for Indian ores.
174 K. M. Paknikar and A. D. Agate

Table 1 Survey of Indian mines for collection of samples (ores, wastes, acid mine waters,
soils, etc.)

No. of mines visited No. of samples collected

State Copper Manganese Copper Manganese

Andhra Pradesh 1 9 1 14
Bihar 7 0 49 0
Goa 0 10 0 40
Gujarat 0 1 0 2
Karnataka 2 2 22 10
Madhya Pradesh 1 2 8 3
Maharashtra 1 8 4 39
Orissa 0 11 0 44
Rajasthan 5 0 24 0
Total 17 43 108 152

Table 2 Microorganisms isolated from Indian copper and manganese mines

Culture No. of isolates

Thiobacillus ferrooxidans 117

Thiobacillus thiooxidans 28
Bacillus spp. 22
Arthrobacter spp. 36
Pseudomonas spp. 4
Total 207

Table 3 Activity check on selected cultures preserved by different methods and using
different suspending media/carriers after storage for 12 months. Activity is expressed in per cent
loss in iron-oxidation rate or manganese-oxidation efficiency

Bacteria Lyophilization Mixing with inert carriers

Liquid N2
Sucrose + storage Chalcopyrite
Sucrose mannitol (glycerol) or pyrolusite ore Lignite

T. ferrooxidans
MCM B-8 70.3 70.3 28.4 2.82 51.8
MCM B-175 -* 64.1 20.2 5.12 46.2
MCM B-185 75.6 57.8 26.2 5.12 51.8
MCM B-191 56.7 54.2 31.7 5.12 40.6
MCM B-231 73.6 51.8 16.3 5.12 51.8
Arthrobacter spp. 2.0 2.0 2.0 5.12 23.4
Bacillus spp. 2.0 2.0 2.0 5.12 23.4

* Not possible to revive after storage for four months.

 Culture collection and data bank for biometallurgical processes 175

Table 4 Optimization of parameters for copper leaching. The data are for a copper extraction
efficiency of 58% in 14 days

Ore Chandmari chalcopyrite ore, Chandmari, Rajasthan

Copper content 1.67%
Particle size 100 mesh
Pulp density 10%
Organism Thiobacillusferrooxidans MCM B-203
IOR of T. ferrooxidans 0.116 mg/ml/h
Inoculum size 6% v/v of fully-grown culture
Temperature 30~
pH 2.5
Eh 500 mV

Table 5 Optimization of parameters for manganese bioconversion (per cent conversion to

MnO2 = 67%)

Ore Hausmanite ore, Dandeli mines, Dandeli, Karnataka

manganese 48%
MnO2 23%
Particle size < 50 mesh
Pulp density 100%
Medium for conversion Bromfield and Skerman (1950)
Orgardsm Arthrobacter MCM B-23
Inoculum size 10% v/v of cell suspension
optical density 0.14 at 550 nm
Temperature 30oC
Aeration shaking at 250 rev/min
pH 6.2

Discussion

It can be seen from the results that a culture collection and data bank has been created
for India with regard to the potentially useful microorganisms for the leaching of
copper and manganese from various ores/wastes. It was felt that this was an urgent
need as such a collection of information did not previously exist for these important
minerals. In many developed nations, such information on important deposits does
exist, but is in an isolated form belonging to private sources or mining organizations.
This is therefore the first systematic attempt to have a centralized facility, where not
only laboratory data on such biometallurgical processes is kept, but the laboratory is
able to provide cultures used in the technology in an active, ready-to-use form. It is
anticipated that this will aid the development of biometallurgy in India.
As a result of our studies, two copper-mining industries in India, The Hindustan
Copper Ltd and the Hutti Gold Mines Ltd (formerly Karnataka Copper
Consortium Ltd), have already shown interest and the latter has decided to explore
the possibilities of microbial leaching of copper on a large scale at their mining sites
using the cultures and data available from our laboratory. Similarly, the manganese-
mining industry in India, which is beset with problems of pollution and disturbance of
the environment, has also indicated an interest in this technology for dealing with
176 K. M. Paknikar and A. D, Agate
A. Fig. 3 Culture card (originally 12-5 X 20 cm). A. front; B, reverse
ISOLATED BY GROWTH MEDIUM PRESERVATION
ECOSYSTEM
DATE
METABOLISM RATE CULTURECHARACTERISTICS METAL TOLERANCE
IRON OXIDATION ALUMINIUM MOLYBDENUM
IRON REDUCTION COPPER CHROMIUM
SULPHUR OXIDATION IRON POTASSIUM
SULPHATE REDUCTION MORPHOLOGY MANGANESE SODIUM
S~O3OXIDATION NICKEL CALCIUM
$203 REDUCTION ZINC BARIUM
TETRATHIONATE URANIUM
H2S CARBON SOURCES COBALT
Mn OXIDATION LEAD
Mn REDUCTION MERCURY
P SOLUBILIZATION SILVER
NITROGEN SOURCES LEA( HING
COPPER
URANIUM
ENERGYSOURCES MANGANESE
IRON

CODE NAME SPECIAL

B.
BIOCHEMICAL CHARACTERISTICS RESULTS CARBOHYDRATE UTILIZATION RESULTSI
INDOLE ARABINOSE
M.R. CELLULOSE
V.P. FRUCTOSE
CITRATE GALACTOSE
OXIDASE GLUCOSE
CATALASE LACTOSE
UREASE MANNITOL
HAEMOLYSIN STARCH
COAGULASE XYLOSE
NITRATE (AEROBIC)
NITRATE (ANAEROBIC)
LECITHINASE
PHOSPHATASE
NITRITE
UPASE
GELATIN
 Culture collection and data bank for biometallurgical processes 177

these problems: a private manganese-mining company in Goa has shown interest in

dealing with the problem of removal of manganiferrous clay from their washing plant
using microorganisms. These are additional signs that this technology is poised for
development, if p r o p e r support and incentives are offered.

Acknowledgements
The authors are grateful to the Director, M.A.C.S. Research Institute, Pune, for his
keen interest and facilities provided during the course of these investigations. We also
wish to thank the personnel from various mining organizations in India, without
whose spontaneous help this work would not have been possible.

References
AGATE, A.D. & DESHPANDE,H.A. (1977) Leaching of manganese ores using Arthrobacter
species. In: Conference Bacterial Leaching, ed. Schwartz, W. GBF Monograph No. 4,
pp. 243-250. Weinheim, New York: Verlag Chemie.
AGATE, A.D., PAKNIKAR,K.M., KmNVASARA,N.J. & GUPTA,S.G. (1984) Microorganisms
associated with biometallurgy of copper in India. 7th International Biotechnology Symposium,
pp. 483-484, New Delhi, India.
BRIERLEY,C.L. (1978) Bacterial leaching. CRC Critical Reviews in Microbiology, 6, 207-262.
BROMFIELD,S.M. & SKERMAN,V.B.D. (1950) Biological oxidation of manganese in soils. Soil
Science, 69, 337-348.
GUPTA, S.G. & AGATE, A.D. (1986) Preservation of Thiobacillus ferrooxidans and T.
thiooxidans cultures with activity check. Antonie van Leeuwenhoek 52, 121-127.
GHosr~, J.S. (1983) Optimization studies on beneficiation of manganese ores, Ph.D. thesis,
University of Poona, Pune.
SILVERMAN,M.P. & LUNDGREN,D.G. (1959) Studies on the chemoautotrophic bacterium
Ferrobacillus ferrooxidans. I. An improved medium and a harvesting procedure for securing
high cell yields, Journal of Bacteriology, 77, 642-647.
VISnhqAC, W. & SANTER,M. (1957) The thiobacilli. Bacteriological Reviews, 21,195-213.
VOGEL, A.I. (1961) Quantitative Inorganic Analysis. London: Longmans Green.
WEIss, S.A. (1977) Manganese--the other uses, Garden City Press, Letchworth, Metal Bulletin
Books Ltd.

Summary
Biometallurgical processing of minerals is a fast-developing science and is of considerable
importance especially to nations like India where rapid industrialization has increased the
domestic demand for metallic raw material which has to be satisfied by heavy imports. One of
the ways of leading the country to self-sufficiency is to attempt to produce the metals from
indigenously available low-grade/waste ores or beneficiate their quality using an appropriate
technology like biometallurgy. We have developed a culture collection and data bank in respect
of this technology in India for microbial leaching of copper and bioconversion of manganese. In
this programme, 207 potentially-useful micro-organisms such as iron-, sulphur- and manganese-
oxidizing bacteria were isolated from 60 copper and manganese mines in the country, and they
were used for leaching copper and converting manganese to a better variety. These two
processes were scaled-up to the 300-kg level and the isolated microorganisms were stored in an
active, ready-to-use form by devising suitable preservation methods. Such a compilation of
information on ores and cultures, with a centralized facility for supply of useful cultures, was
referred as a 'culture collection and data bank'. Various Indian mining industries have shown
interest in trying these processes on a large scale at their mines, using the cultures and the data
available from us.
178 K. M. Paknikar and A. D. Agate

R~sum6
Ddveloppement d'une collection de cultures et d'une banque de donndes pour les
proc~d~s bio-mdtallurgiques en Inde
Le traitement bio-m6tallurgique des minerals est une Science en d6veloppement rapide. II
pr6sente une importance consid6rable, notamment pour les pays, comme l'Inde, dont
l'industrialisation a rapidement accru la demande int6rieure de m6taux bruts, devant ~tre
import6s ~ un prix 61ev6. Un des moyens d'atteindre l'auto-suffisance nationale est la
production de m6taux ~ partir de minerais ~ basse teneur ou de rebut, ou encore l'am61ioratiou
de la qualit6 des minerais par une technologie appropri6e, telle que la bio-m6tallurgie. En Inde,
nous avons cr66 darts ce domaine une collection de cultures et une banque de donn6es
concernant le lessivage du cuivre et la bio-conversion du mangan6se. Dans le cadre de ce
programme, 207 micro-organismes potentiellement utilisables, dont des bact6ries oxydant le
fer, le soufre et le mangan6se, ont 6t6 isol6s h partir de 60 mines nationales de cuivre et de
manganese. Ces micro-organismes ont 6t6 employ6s pour le lessivage du cuivre et pour la
conversion du mangan6se sous une forme mieux utilisable. Les deux proc6d6s ont 6t6
d6velopp6s ~ l'6chelle de 300 kg et les micro-organismes isol6s ont 6t6 conserv6s sous forme
active et disponible, grace ~t la raise au point de m6thodes de pr6servation appropri6es. Cette
compilation d'information sur les minerais et les cultures, assortie d'un organisme centralis6
pour la diffusion des cultures, construe une 'collection de cultures et banque de donn6es'.
Diverse firmes indiennes ont entrepris d'essayer ces proc6d6s ~ grande 6chelle dans leurs mines,
en utilisant nos cultures e t n o s donn6es.

Resumen
Desarrollo de una colecci6n de cultivos y de un banco de datos para procesos
biometalurgicos en la India
El procesado biometalurgico de los minerales es una ciencia en rfipido desarrollo y de
considerable importancia sobretodo en naciones como la India donde la r~ipida industrializaci6n
ha incrementado la demanda interna de minerales que ha de set si~tisfecha mediante costosas
importaciones. Una de las formas de conseguir la autosuficiencia es intentar producir estos
metales a partir de minerales aut6ctonos de baja graduaci6n o de desecho, o bien mejorar su
calidad, utilizando una tecnologia adecuada tal como la biometalurgia. Se ha desarrollado una
colecci6n de cultivos y un banco de datos para esta tecnologia en India aplicable al lavado de
cobre y a la bioconversi6n del manganeso. En este programa se hart aislado de sesenta minas del
pais 207 microorganismos potencialmente titiles, tales como bacterias oxidadoras de sulfuros y
de manganeso. Estas bacterias se utilizaron para el lavado del cobre y para meiorar la calidad
del manganeso. Ambos procesos se realizaron a una escala de 300kg y los microorganismos
aislados se almacenaron en forma activa, lista para su utilizaci6n mediante el disefio de sistemas
de conservaci6n adecuados. Esta compilaci6n de informaci6n sobre menas y cultivos con un
servicio centralizado para proporcionar los cultivos que puedan set utiles se ha denominado
colecci6n de Cultivos y Banco de Datos. Varias compafiias mineras indias se han mostrado
interesadas en probar estos procesos a gran escala en sus minas utilizando para ello los cultivos y
los datos que les podemos proporcionar.