BIOTECHNOLOGY AND BIOENGINEERING

VOL. XI, PAGES 683-699 (1969)

Use of pH and pE Measurements

during Methane Biosynthesis*

F. G. POHLAND, School of Civil Engineering, Georgia Institute

of Technology, Atlanta, Georgia, and K. H. MANCY, School of
Public Health, University of Michigan, Ann Arbor, Michyan

Summary
Methane biosynthesis within the anaerobic ecosystem results from the diphasic
conversion of complex organic substrates through a series of biologically mediated
oxidation-reduction reactions which are p H dependent. Routine parametric
analysis of the products emanating from the corresponding acid and methane
fermentation phases of the anaerobic digestion process provide a basis for deter-
mination of process condition and efficiency. Of these analyses, pH and electrode
potential of pE are intensive parameters of considerable significance but for which
interpretation is often difficult and misleading. Attention has been focussed on
the analogy and distinction between p H and pE determinations and their appli-
cation to the anaerobic ecosystem. Theoretical and practical limitations are
discussed and experimental evidence presented which supports the possible use
of p H and p E determinations for process evaluation and control.

INTRODUCTION
Methane biosynthesis is almost ubiquitous in nature. It occurs in
the digestive tracts of animals, the bottom deposits of marshes,
ponds, lakes and streams, and the anaerobic processes employed for
waste treatment. Fermentation in each of these environments has
been subjected to considerable study and some attention has been
focussed on either the optimum conditions required for effective
biosynthesis and substrate conversion or on changes in various
environmental parameters and their control when conditions are
other than optimum. This paper presents a conceptual and opera-
tional interpretation of such variations during methane biosynthesis
*Presented at the Third International Fermentation Symposium, Rutgers
University, September 2-6, 1968.
683
684 F. POHLAND AND K. MANCY

from waste organic substrates using the anaerobic digestion process

as a characteristic ecosystem and p H and pE as the master control
variables.
Process Description
Anaerobic digestion has become a common method for biological
treatment of both soluble and insoluble waste organic materials.
Various process modifications have been introduced, but the anaer-
obic contact. process, particularly suited for treatment of soluble
wastes, and stage digestion for insoluble sludges have become most
common. The essential features of these two process modifications
are shown schematically in Figure, 1.
During digestion, organic materials are sequentially and con-
tinuously converted to simpler intermediates and end-products with
the concomitant synthesis of biomass. Volatile organic acids are
the most frequently detected intermediates whereas methane, carbon
dioxide and stabilized sludge constitute the desirable end-products
issuing in quantity from the process. Based upon these products,
GAS

WASTE

EFFLUENT

LIOUID
EFFLUENT

SUPER NATANT

MIXED ANAEROBIC
EFFLUENT
CONTACT

SLUDGE RECYCLE SLUDGE

EFFLUENT

Fig. 1. Modifications of the anaerobic digestion process.

BIOTECHNOLOGY AND BIOENGINEERING, VOL. XI, ISSUE 4
 METHANE BIOSYNTHESIS 665

substrate conversion may be considered to be diphasic and con-

tingent on the growth and metabolism of two separate groups of
organisms. The capacity of each group is different, specific to its
respective phase and primary products, and responsive to varying
environmental stresses.
I n conventional practice it has become convenient to describe the
two phases of digestion as “acid fermentation” and “methane fer-
mentation,” respectively. Both phases coexist within the same
physical and chemical environment and process efficiency is therefore
determined by the rate limiting phase.
Because the methane formers do not grow rapidly and are more
sensitive to adverse environmental stresses, process regulation is
generally designed to provide an environment best suited to their
growth and proliferation. The regulation or adjustment required
to ensure such a condition is determined by routine evaluation and
control of certain system parameters or digestion indices. Gas pro-
duction and composition, pH, alkalinity, volatile acids, volatile
solids (V. S.) reduction or substrate conversion, and electrode
potential (E,) are the most frequently used parameters. Experience
has established the probable ranges for these indices during normal
and retarded digestion conditions as summarized in Table I.

Intensive Control Parameters

Theoretical Consideration
Analytical parameters used for the assessment of the physico-
chemical characteristics of a particular ecosystem may be categorized
as either intensity or capacity factors. Among the measurements
indicated in Table I, pH and electrode potential are intensive
parameters indicative of the respective proton and electron activities
associated with the biochemical transformations within the system.
By definition, pH is a measure of the average proton free energy per
proton and depends on the relative population and energy values of
the levels present. Likewise, electrode potentials measure the
average free energy per electron transferred.
An inert metal indicator electrode in equilibrium with a redox sys-
tem will exhibit a potential that is invariant with time and thermo-
dynamically related to the composition of the solution. The meas-
ured potential, E , is a function of the free energy of the electrons in
+3 ?
M TABLE I
0
5: g
2 Comparison of Digestion Parameters during Normal and Retarded Conditions
8
Gas Production Total Volatile Electrode LiU
2 Alkalinity Total Organic Solids Potential*
5 Digestion cu ft/#V. S. co2 mg/l as Acids, mg/l Conversion
t3 Condition added % PH CaC03 as CHICOOH
EC LiU
% mv.
E
0 .R
2 Normal 8-10 25-35 6.8-7.2 1500-5000 50-300 50-60 z-525
2 Retarded <7 35-45 <6.6 1000-3000 >2000 <40 <-500 z?-
3m *Measured electrode potential referenced to the standard calomel electrode. 24
E

P
 METHANE BIOSYNTHESIS 687

the metal electrode, and this potential is an intensive factor inde-

pendent of the quantities of the electroactive species. Instead of
using E expressed in volts, it is perhaps more convenient to use the
simpler notation, pE, as introduced by Jorgensenl and extended by
S U n 2 and Morris and S t ~ m m . ~The , ~ analogy between both pE
and pH may be illustrated by considering the proton and electron
transfer potentials for an organic substrate, S, as indicated in Table
11. Hence, in strongly alkaline solutions the pH is high (low proton
activity) and in strongly oxidizing solutions, the pE is similarly high
and positive (low electron activity).
Both pH and pE are particularly significant in analysis of the
anaerobic ecosystem since biochemical transformations often involve
the direct coupling of electron transfer with proton transfer in an
oxidation-reduction sequence. In addition, the oxidation or reduc-
tion of electroactive organic compounds on a metal indicator electrode

TABLE I1
Proton and Electron Transfer Potentials for Acid-Base and Oxidation-
hduction Reactions.

Transfer Equation:
SH + H20 + S- + H30+ S + 1/2H2 + H+ + S-
Transfer Equilibrium:

K -
IHsO+I [S-I
K -~
WI [S-I
- [SH] [HtO] - [S] [Hz]"~

Scale Definition:
pH = - log [H+] pE = - log [e-]

pH = pKp + log-ISHI
[S-I
pE = pE" + log [S-I
-
[SI

Transfer Potential:
Acidity Potential Redox Potential
2.3RT [SH] 2.3R T [S-]
E=E"+- log ~ E=E"+- log -
F [S-I F [Sl

a S = organic substrate; E" = standard potential at unit activity; F = Fara-

day's constant; R = gas constant; T = absolute temperature, "K
688 F. POHLAND AND K. MANCY

may also involve the coupling of electron transfer and proton transfer
from the aqueous solvent as illustrated by the following example.
Oxidation Reduction
SH -* SH+ + e- SH + e- + SH- (1)
SH+ + 2H2O + SHOH + H30+ SH- + H30+ + SHz + HzO (2)

This dependency of organic electrode processes on p H has been

given considerable attention in the literature pertaining t o organic
e l e c t r o ~ h e m i s t r y . ~The
~ ~ kinetic effect on the redox system is two-
fold. First, p H will control the availability or withdrawal of protons
indicated by Equation 2 and will thereby influence the reversibility
of the oxidation-reduction system. Secondly, the p H of the medium
is an important factor determining the nature of the electroactive
species. For example, the pH will dictate whether a weak acid will
exist in the undissociated molecular or dissociated ionic forms, both
of which differ in electroactivity.
The pH effect can be further illustrated by considering the oxida-
tion-reduction reaction of a dibasic organic acid (e.g., maleic or
fumaric acids) :
So + 2e- :Sr2- (3)

where So and Sr2- are the respective oxidized and reduced species.
The equilibrium potential for this reaction is :

E = E" + RT
- In
2F
~
(Sol
is,'-] (4)

Assuming activity coefficients t o be unity and the presence of only

one species of oxidant, Co = [So], three reduced species can be recog-
nized, the sum of the molar concentrations of which will be:
Cr = [SrHz] + [SrH-l + [Sr*--l (5)
KaK,'Cr
[S,Z-] =
[H+lz + K , [H+l + K J L ' (6)

If K , and K,' are the apparent ionization constants for the dibasic
acid, the Nernst expression results, or

BIOTECHNOLOGY AND BIOENGINEERING, VOL. XI, ISSUE 4

 METHANE BIOSYNTHESIS 689

where
RT
E o = E” - -In K,K,’ (8)
2F
I n a well buffered solution t.he last term in Equation 7 is constant
and may be combined with Eo to give the operational constant, E’, or
RT Co
E = E‘ + 2F
-In -
C,
Therefore, i t is relevant t o this discussion to emphasize that there
must be a range of p E and pH for thc optimum performance of the
organic redox system considered in Table 11.
Limitations of Application
Thermodynamic calculations of p E from appropriate tables has
very little t o do with the experimentally measured values since the
degree of electroactivity within the system is not required. The
same holds for pH measurements although techniques for measure-
ment of p H and its interpretation have become more
and further discussions of its limitations will not be presented here.
However, limitations on pE measurements and their interpretations
are not as well recognized and some discussion of these limitations
was considered warranted.
Oxidation-reduction potential measurements in a heterogeneous
biochemical system seem t o be deceptively easy. Determinations of
the potential difference between an inert metal indicator electrode
(e.g., gold, platinum, . . .) and a reference electrode immersed in the
medium do not necessarily represent Nernst potentials and therefore
often are subject to considerable misuse and misinterpretation.
This had led to considerable controversy, often because i t was not
recognized that measured potential values are solely dependent on
the electrochemical reactions occurring a t the electrode surface and
may not be representative of what is predominant in the bulk of the
system.
I n a complex heterogeneous system such as the one under con-
sideration, there are a number of interrelated oxidation-reduction
sequences progressing simultaneously a t different rates. The
measured potential established will be determined by the kinetics of
both the oxidation and reduction reactions occurring a t the electrode
surface and is, therefore, a “mixed potential.” Furthermore, interpre-
690 F. POHLAND AND K. MANCY

tation of the system response and its performance characteristics

based on such potential measurements are gravely limited by the
diverse effects of various inherent electroactive as well as surface
active interferences, the degree of reversibility of the reactions
involved and the electrochemical characteristics of the indicator
electrode.
The effect of non-electroactive but surface active organic matter
is dependent on the indicator electrode potential. Adsorption of
surface active agents on the indicator electrode may result in partial
or complete coverage of the electrode surface. This adsorbed film
may significantly alter or reduce the kinetics of the oxidation-reduc-
tion reactions indicated by Equations 1 and 2. Potential measure-
ments under such conditions will be unstable and may drift only
slowly toward equilibrium.
Most organic reactions are highly specific with regard to the nature
and preparation of the indicator electrode surface. This is partly
due to the mechanism of adsorption characteristic of the electrode
surface €or both reactants and products. The electrode material
may also act catalytically involving reactions with a transient metal
oxide species prior to the electrochemical step.
+
H20 M + MOH + H+ + e- (10)
MOH + S -SOH + M (11)
Ideally speaking, one would hope for a potential established at the
electrode surface by one or more fast (reversible) redox couples which
serve as “electrochemical indicators” capable of following the bio-
logically mediated reactions of the ecosystem. Such an indicator
could be added to the system in a manner similar to the addition of
acid-base color indicators to aqueous samples or may already be an
inherent component of the system. This potentiometric indicator
should also act as a “mediator)’ or as a charge carrier between the
indicator electrode and the rest of the system. It is also essential
for the indicator system and the biologically mediated system to be
at equilibrium in order that the potential set up at the indicator
electrode may be capable of representing the total system. More-
over, the potentiometric indicator added should be at a concentration
much lower than that of the system and should be selected to have an
equilibrium potential near that expected to be attained by the
system under investigation. 6 , lo, l 1
BIOTECHNOLOGY A N D BIOENGINEERING, VOL. XI, ISSUE 4
 METHANE BIOSYNTHESIS 69 1

The mechanism of action of a potentiometric indicator can be

illustrated by considering the following slow redox system.
S+e-eP (12)

Both the organic constituents S and P are considered nonelectro-

active or, a t most, slightly electroactive as far as the indicator elec-
trode is concerned. Meaningful potential measurements for such a
system are not possible. However, if a small quantity of the electro-
active substance M is added and undergoes the following reversible
reaction a t the indicator's electrode surface,
M+e-eM- (13)
potential measurements for this system will give stable equilibrium
values in accordance with the following expression:
RT [MI
E = E" + - In __
F [M-I
The substance M may also undergo a fast chemical reaction with
the substrate, S, or
M+SCMS (15)
where

Then, the presence of small quantities of A4 in the system will produce

a fast electroactive redox system
MS +e-2 M- +S (17)
which will give an equilibrium potential value according to the
following Nernst expression :
EM = El + RT
-
F
ln-
[MS]
[M-I [Sl
The standard potentials E" and El (equilibrium potentials when all
activities are equal to unity) are related by the equation
RT
E l = E" +-
F
In K M

This relationship is independent of the rates of the electrochemical

and chemical reactions and hence is valid in all cases. Furthermore,
692 F. POHLAND AND K. MANCY

the equilibrium potential measured by the indicator electrode will

depend on S, or
[MS] RT
EM = El + RT
- In - -
F [M-1 F
In [S]- (20)

I n case of [S] >> [M], and the system MS +

e- e S + NI- is faster
than the system S + e- S P, the ratio [nllS]/[hil-] is constant and
eq. (20) reduces to
RT
EM = b - -In
F
[S] (21)

It is evident, therefore, that the presence of small quantities of a

potentiometric indicator in a slow nonelectroactive redox system,
S + P, will serve t o mediate the electrode reaction thereby resulting
in Nernstian potential measurements indicative of the redox system
given by eq. (21). Equation (21) is applicable provided that the
concentration of t h e potentiometric indicator system, 31, is constant
and does not undergo any reaction with other constituents in the
medium. Several authors have studied the application of potentio-
metric indicators t o complex biochemical system^^^^^'^ and studies are
progressing in this laboratory on the use of potentiometric indicators
during methane biosynthesis.
Process Applications
Experimental Studies
I n spite of the limitations noted previously several investigators
have shown that the p H and electrode potential measured within the
anaerobic ecosystem are often good indicators of process condition
and resultant conversion effi~iency.’~-’~ The effect of p H on the
methane fermentation phase of anaerobic digestion, both in terms of
substrate utilization and biomass concentration, has been well docu-
mented and it is generally agreed that the majority of the methane
formers are more efficient in a very limited range around neutral pH.
Similarly, the electrode potential must also be optimum for efficient
methane biosynthesis and is also dependent upon internal mediation
although attempts have been made to “poise” the potential within the
desirable range indicated in Table I.
Several studies concentrating particularly on the behavior of p H
and electrode potential responses during anaerobic fermentation of
BIOTECHNOLOGY A N D BIOENGINEERING, VOL. X I , ISSUE 4
 METHANE BIOSYNTHESIS 693

waste substrates are worthy of special mention. The results of

thermophilic (55°C) batch fermentation of potato starch production
wastes, by Maslova and Pantskhava” have been reproduced on
Figure 2 . A sharp drop in redox potential was observed after initial
inoculation followed by an increase t o a maximum and a subsequent
decrease to a range considered optimum for methane biosynthesis.
Increases in potential corresponded t o increases in volatile acids
accumulations, reduction in pH and gas production and general
inhibition of the process. The fall in potential during the first few
days was considered related to the development of bacteria capable of
converting carbohydrate t o volatile acids, hydrogen and carbon
dioxide. This increase in volatile acids was accompanied by increases
in redox potential t o a maximum as shown in Figure 2 . The eventual
decrease in potential and volatile acids concentrations was attributed
to the activities of facultative ammonifers. These organisms
apparently prepared the environment for the development of the
obligate anaerobes responsible for the production of methane by
decreasing the electrode potential and increasing the pH to optimum
ranges.
The optimum potential ranges for anaerobic mesophilic digestion
of waste sewage sludges have been studied in detail by Grune and
Chueh14 and Dirasian et a1.15 Figure 3 has been prepared as an
example of this work and illustrates the results of anaerobic fer-
mentation in the dialysis-type fermentation system reported by
Borchardt.’* The unique feature of this system is that it permits the
volatile acids produced during the acid fermentation phase in one
reactor t o be extracted through a dialysis membrane and serve as a
substrate for the methane fermentation phase in a second reactor.
By this technique, the volatile acids content of the fermenting
medium in the first unit decreased in concentration for a particular
organic loading and the pH and electrode potential tended toward
the values already established in the second unit as optimum for
methane biosynthesis.
I n all of the studies referenced herein, the normal digestion condi-
tions described in Table I were not achieved until the pH and redox
potential values (E,) approximated ranges near neutral and - 500
to - 600 mv. respectively. Moreover, an apparent relationship
existed between the pH, electrode potential and volatile acids con-
694 F. POHLAND AND K. MANCY

700 - 1.4 -I
600 - m 1.2 -

Experimental Period , days

Fig. 2 Variations in digestion parameters during methane biosynthesis from

potato-starch production wastes (after Maslova and Pantskhava”).
BIOTECHNOLOGY A N D BIOENGINEERING, VOL. XI. ISSUE 4
 METHANE BIOSYNTHESIS 695

-
-.-
0
c
-
P
1000-

FERMENTOR N0.I -
FERMENTOR N0.2 ----

g -500-

Fig. 3. Internal adjustment of pH and electrode potential by volatile acids

dialysis and utilization during anaerobic sludge digestion (after Borchardtls).

centration existing during either normal or retarded digestion

conditions.
Conceptual Interpretation and Discussion
Since the anaerobic digestion process has been described as a
diphasic system requiring a symbiotic relationship between acid and
methane fermentation, the data chosen and presented herewith
suggest that the pH and pE eventually established during normal
digestion are optimum for the rate limited methane fermentation
step. Environmental requirements for its beneficial existence must
therefore be represented by these optimum values and would naturally
tend toward these optima by internal regulation unless inhibited by
abnormal influences; e.g., toxic materials, system overload, etc.
Moreover, since the potential responses observed are reaction inter-
696 F. POHLAND AND K. MANCY

related, they should also display deviations of varying magnitudes as

the predominating reactions and processes change from normal to
retarded conditions.
With either normal or retarded digestion conditions, both pH and
pE are apparently “buffered” a t values characteristic of prevailing
conditions. If the reactions responsible for anaerobic conversion of
complex organic materials can be represented by a simple oxidation to
volatile acids and subsequent conversion to methane, the scheme
illustrated in Table I11 may be conveniently used as a simplified
description of the process.

TABLE I11
Microbially Mediated Oxidation-Reduction Sequences Representative of
Acid and Methane Fermentation within the Anaerobic Ecosystem

Acid Fermentation (oxidation) :

4C31&OzNS + 8Hz0-,4CH3COOH + 4 C 0 2 + 4NH3 + 4H2S + 8HC + 8e-
Methane Fermentation (reduction) :
8HC + 8e- + YCH3COOH + COz -,4CH4 + 3coz + 2 H 2 0
Anaerobic Fermentation (oxidation & reduction) :
4C3H702NS + 6HzO + 4CH4 + 6C02 + 4NH3 + CH3COOH + 4Hzs

The amino acid, cysteine, was arbitrarily chosen to represent a

possible but specific combination of primary elements (CHONS)
commonly present in organic wastes. The reactions illustrate the
sequential conversion of organic carbon to either COZ or CH4 with the
associated release of the reduced forms of nitrogen and sulfur. The
oxidation-reduction sequence is mutually dependent ; organic carbon
is oxidized to COz and/or intermediate products such as volatile
organic acids (acetic acid) during the acid fermentation half-reaction
with the concomitant reduction to CH4during methane fermentation.
The sum of the two balanced half reactions describes the total
conversion process as defined and provides a valuable insight into the
origin and relative abundance of products routinely detected during
the course of anaerobic fermentation. The extent of oxidation or
BIOTECHNOLOGY AND BIOENGINEERING, VOL. XI, ISSUE 4
 METHANE BIOSYNTHESIS 697

reduction occurring during fermentation determines the prevailing

electrode potential or pE; the type and/or predominance of acidic or
basic products determines the pH; and, the p H and p E collectively
reflect the condition of the process.
Morris and Stumm3v4have provided a more detailed treatment of
possible reaction combinations representative of biochemical con-
version in aquatic ecosystems. By selecting various combinations
of bio!ogically mediated oxidation-reduction reactions, the thermo-
dynamic sequence or ecological succession in the presence of excess
organic material and incipient concentrations of 0 2 , N01-, Sod2-, and
HC03- was shown t o be : aerobic respiration, denitrification, nitrate
reduction, fermentation (acid), sulfate reduction and finally, methane
fermentation or biosynthesis. Dependent upon choice of half-
reaction pairs combined t o describe a particular event, the resultant
pE for reaction completion could be calculated and for the sequence
indicated, changed in decreasing order of magnitude from positive to
negative values.
Although the reactions chosen in Table I11 to represent the total
conversion process within the anaerobic environment are somewhat
hypothetical and exclusive of other oxidation-reduction sequences
proposed by S t ~ m mthey, ~ are consistant with present concepts and
yield similar products with corresponding potential requirements
consistent with those observed in practice and recorded in Figures 2
and 3 for either normal or retarded digestion conditions. If the
anaerobic digestion process was retarded and predominated by
reactions other than methane fermentation, the pE of the process
would be expected to be higher and characteristic of a particular
dominating mechanism. If this methanism was acid fermentation,
volatile acids would accumulate and both the pE and pH would re-
flect) their formulation and buffer capacity. The effects of the
accumulation of volatile acids on the pH and buffer capacity within
the anaerobic environment have been described in considerable
detail elsewhere.19920

It may be concluded from the previous discussion that p H and

electrode potential (or pE) can be beneficial for characterizing process
condition during anaerobic digestion of waste organic substrates.
However, a distinction must be -made between interpretation of such
measurements and their use. Whether it is possible or not to provide
698 F. POHLAND AND K. MANCY

more simplified and meaningful interpretations of pH and pE in

complex biological systems should not be confused with the practica-
bility of using these measurements for process characterization nor
hinder their application as control techniques. Such operational
application is presently warranted in spite of the difficulty in provid-
ing rigorous and definitive conceptual interpretations and could
provide the basis from which new insight may develop.
This report was prepared while the senior author was a visiting scholar at the
University of Michigan and supported in part by a special fellowship from the
Federal Water Pollution Control Administration of the U.S. Department of
Interior.
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1. H. Jorgenson, Redox-malinger, Gjellerup, Copenhagen, 1945.
2. L. G. Sill&, Chemical Equilibrium i n Analytical Chemistry, Interscience,
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3. J. C. Morris and W. Stumm, Equilibrium Concepts in Natural Water
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4. W. Stumm, Proceedings Third International Conference on Water Pollution
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5. M. J. Allen, Organic Electrode Processes, Reinhold Publishing Corp., New
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6. W. M. Clark, Oxidation-Reduction Potentials of Organic Systems, The
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 METHANE BIOSYNTHESIS 699

17. L. G. Maslova and E. S. Pantskhava, Appl . Bioch. and Microbiol., 1, 172

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Received January 20, 1969