Anaerobic Digestion

Water Research Pergamon Press 1969. Vol. 3, pp. 459-494 Printed in Great Britain.
REVIEW PAPER
ANAEROBIC DIGESTION
II. THE CHARACTERIZATION AND CONTROL OF
ANAEROBIC DIGESTION
J. P. KOTZ~., P. G. THIEL and W. H. J. HATTINGH
National Institute for Water Research of the South African Council for Scientific and Industrial
Research, P.O. Box 395, Pretoria, Republic of South Africa.
(Received 4 February 1969)
INTRODUCTION
ANAEROmC digestion is a biological process in which organic matter is converted to

methane and carbon dioxide in the absence of oxygen (ToERIEN and HATTINGH, 1969).
This process is widely used for the purification of wastes containing high concentra-
tions of organic material such as domestic sewage sludge and effluents from various
industries.
Although well-operating anaerobic digesters will always have a common feature in
that organic material is eventually converted to methane and carbon dioxide, digesters
can differ vastly one from another with regard to their bacterial populations, the
sequence of reactions giving rise to the end-products and the prevailing environmental
conditions. The eventual character of any digester is determined by the composition
of the substrate, the number of species of microorganisms present in the inoculum and
other factors such as temperature, loading rate, hydraulic residence time, sludge
retention time, method of mixing of the sludge in the digester and the design of the
container of the process.
It is necessary to control the process of anaerobic digestion carefully to ensure
efficient operation. In contrast to the digestion of domestic sewage sludge, the digestion
of many industrial effluents is difficult to control and conditions in which high con-
centrations of volatile fatty acids accumulate can easily develop, usually leading to a
failure of the process. The prevention of such a failure necessitates knowledge of the
characteristics of a digester treating a specific waste. By employing certain relevant
control parameters the monitoring of these characteristics can serve as indicators of
impending failure and motivate steps to prevent failure.
An attempt is made in this paper to describe those factors which determine the
nature of different anaerobic digesters, the parameters which can serve as indicators of
impending failure and the methods which can be employed to avert failure. The
characterization and control of anaerobic digesters described in this paper are mainly
applicable to the anaerobic treatment of industrial wastes which often prove difficult
to control. For a discussion of the factors affecting the disposal of domestic sewage
sludge, the reader is referred to the literature survey by POrILAND (1962) and the
Water Pollution Control Federation Manual o f Practice on anaerobic digestion (1968).
459
460 J.P. KOTZ~,P. G. TmELand W. H. J. HATIINGH
FACTORS DETERMINING THE NATURE OF ANAEROBIC DIGESTERS
The major factors determining the nature of a specific digester are the substrate
composition and loading, the temperature of operation, the design of container, the
mode of operation and the source of the inoculum of microorganisms required to
digest the substrate. In order to evaluate the contribution of the individual factors to
the final nature of a digester each of these factors will be discussed in more detail.
Composition of substrate
General review of compounds occurring in wastes. The composition of the substrate
is one of the major factors which determines the characteristics of the ecosystem in an
anaerobic digester. The organic and inorganic components in the substrate lead to a
selection of those bacteria which are able to metabolize these compounds. The inter-
mediate metabolites formed will lead to a further enrichment of those bacteria which
carry the process to the eventual end-products, methane and carbon dioxide. The
form in which the compounds are present in the substrate (in solution or in suspension)
and the nature of these compounds have a definite effect on the availability of these
compounds to the bacteria and consequently the rate at which they are converted to
methane and carbon dioxide. A knowledge of the composition of the substrate, to be
treated anaerobically, is therefore essential in the interpretation of the behaviour of a
digester.
Wastes to be treated anaerobically are mainly made up of varying ratios of the follow-
ing compounds: carbohydrates, polysaccharides, amino acids, proteins, fatty acids,
lipids, alcohols and a group of nitrogenous compounds originating from living cells.
The carbohydrates occurring in sewage and industrial effluents may consist of a large
variety of compounds including, amongst others, hexoses, pentoses, aldoses and
ketoses. Industrial effluents can also contain relatively large quantities of poly-
saccharides such as starch, glycogen, cellulose, hemicellulose, pectins, mannans and
xylan. Starch, glycogen and cellulose are all polymers of D-glucose. Starch and
cellulose originate from plant and microbial material while glycogen occurs in
animal and microbial cells. Hemicellulose, also from plant origin, yields D-glucuronic
acid and o-xylose on acid hydrolysis. Pectic acids are components of many plant
tissues, and fruit, and appear to be long chains of D-galacturonic acid units. The
pectic acids are components of plant materials named pectins which also contain
polysaccharides composed of galactose (galactans) or arabinose units (arabans). More
detailed information on the chemical structure of these compounds are given by
FauToN and SIMMONDS(1958). Mannan and xylan are structural polysaccharides of
yeast and plant tissues respectively. Mannan may constitute up to 16 per cent of the
dry weight of yeast and is a polymer of o-mannose (FALCONEand NICKERSON, 1956).
Xylan is associated with cellulose in wood and wheat and is a polymer of o-xylose
(WHISTLER, 1950).
TWO classes of compounds originating from wood, which may occur in effluents are
the lignins and tannins. Lignin, which usually accompanies cellulose in wood, is
believed to consist of p-hydroxy-phenyl-propanes. The phenyl groups often contain
methoxy groups (FREUDENBERG,1954). Tannins occur in the bark of trees and are used
in the leather industry. Tannins comprise three groups of substances namely
gallotannins and the flavan or stilbene derivatives (NuRSTEN, 1961).
Anaerobic Digestion--II. Characterizationand Control of Anaerobic Digestion 461
Proteins are present in practically all material of biological origin. In abattoir
wastes, proteins account for a large proportion of the total organic waste (BoRt~v,
1939). Because proteins are labile and the peptide bonds between the constituent
amino acids can be split by the action of extracellular hydrolytic enzymes (proteo-
lyric enzymes), free amino acids and peprides are also present in wastes of organic
origin. THmL and DU TOIT (1965) found that the concentration of free amino acids
in brewery effluent was of the order of 25-95 mg/l expressed as leucine.
Lipids are present in most material of biological origin and in some cases may con-
stitute a major fraction of the organic material in a waste. Domestic sewage can con-
tain as much as 25-2 per cent lipids on a dry weight basis (PoI4LAND, 1962). Lipids are
separated into several groups depending on their chemical and physical properties.
Simple lipids are esters containing only carbon, hydrogen and oxygen and yield, on
hydrolysis, only fatty acids and an alcohol. In the case of the so-called neutral fats and
oils, three fatty acid units are joined by ester linkages to the trihydric alcohol, glycerol
(FRuTON and SIMMONDS, 1958). The more complex lipids include substances such as
the phospholipids (FRUTON and SIMMONDS,1958). The fatty acids present in lipids can
include both straight chain and branched chain fatty acids and saturated and un-
saturated fatty acids containing from 2-26 carbon atoms, depending on the origin of
the particular lipid.
Free fatty acids can also occur in waste waters as the end-products of bacterial
metabolism or can be formed by enzymatic hydrolysis of lipids. TmEL and r~u TO1T
(1965) reported that the concentration of volatile fatty acids in brewery waste could
vary from 50 to 350 rag/1 expressed as acetic acid.
Another group of compounds which can form an important part of the organic
material in a waste is that originating from microbial cells. The importance of this
group is borne out by the fact that bacteria make up approximately 25 per cent of the
dry weight of faeces (RosEBURY, 1962), and the large amounts of yeast cells which, for
instance, may occur in brewery wastes (DmTRICn, 1960). These cells contain, apart
from some of the compounds discussed above, the nucleic acids DNA and RNA,
which occur in all living cells, poly-fl-hydroxybutyric acid which occurs in micro-
organisms as a storage product, and the components of bacterial cell walls. The
nucleic acids DNA and RNA are polynucleotides in which one of the two acidic
groups of the phosphoric acid residue is esterified by one of the sugar hydroxyls of
another mononucleoride. The carbohydrate fraction of DNA consists only of deoxyri-
bose and that of RNA only of ribose. Escherichia coli may contain 5-2 and 19.1 per
cent DNA and RNA respectively on a dry weight basis, while Pseudomonas aeruginosa
on the same basis may contain 4.9 and 4.4 per cent respectively (ZAHN, 1964). There-
fore waste waters containing large amounts of microbial cells will also contain con-
siderable quantities of DNA and RNA. Poly-fl-hydroxybutyric acid can account for
50 per cent of the dry weight of microorganisms under growth conditions where
nitrogen is deficient (STANIERe t al., 1964).
Bacterial cell wails contain rnucopeptides, teichoic acids, polysaccharides, proteins
and lipids (STANIERe t al., 1964). The mucopeptides are polymers of compounds such as
glucosamine, D- and L-alanine, D-glutamic acid and either L-lysine, DO-, EL-, or meso-
~-~-diaminopimelic acid, 2,4-diaminobutyric acid or either D- or L-ornithine (RoGERS,
1965). The teichoic acids are made up of ribitol phosphate, glucose and alanine. The
polysaccharides of bacterial cell walls contain amino sugars and/or simple mono-
462 J . P . KOTZ~, P. G. THIEL and W. H. J. HATHNGH
saccharides, while little is known about the composition of the lipids (STANIERe t al.,
1964).
The aforementioned goups of organic substrates will invariably be present in
effluents or, in special cases, comprise a significant part of the organic loading. There
are, of course, also many other organic substances which may occur in certain
effluents. Substances such as gibberellic acids, antibiotics, plant hormones, pesticides
and detergents may occur in effluents and have an effect on the anaerobic process but
since their concentrations are usually small, they are not discussed in this review.
The inorganic composition of a waste will also have a pronounced effect on the
eventual nature of a digester. Some inorganic ions between specific concentration
levels, are necessary for cell metabolism, while others may prove toxic above certain
concentrations. This review is mainly concerned with the organic substrates of waste
waters and a detailed account of the effect of inorganic ions on anaerobic digestion is
not given.
As already indicated, a wide variety of organic substances may occur in waste
waters. Heterogeneous microbial populations, such as that of anaerobic digestion, will
effect sequential substrate removal when complex substrates are degraded because
certain substrates are more easily degraded than others. Sequential substrate removal
in aerobic processes has already been studied by GAUDYet al. (1963). In complex sub-
strates certain components might be completely inavailable to the microorganisms
resulting in incomplete purification. The composition of a waste fed to a heterogeneous
culture determine the eventual combination of species of the system as well as the
relative enzyme activities of such a system.
The above-mentioned effects of complex substrates on the process of anaerobic
digestion indicate the necessity of a thorough knowledge of the composition of the
substrate to be used. The available information on the composition of domestic
sewage sludge is summarized in the next section as an example of the complexity of
such a waste. Detailed information on the composition of different industrial wastes
is not given in this article.
Composition of domestic sewage sludge. The solids concentration of sewage sludge
varies from 8 to 12 per cent (IMHOFFand FAIR, 1956). The volatile matter of the
undigested solids may vary from 60 to 80 per cent (RUDOLFSand GEHM, 1942; IMHOFr,
1956), and the inorganic material (ash) of undigested solids from 20 to 40 per cent
(POHLAND, 1962).
Sewage sludge contains rather large quantities of ether-soluble material. RUOOLFS
(1944) analysed sludges from various treatment plants and found that the ether-
extractable material or grease varied from 5.7 to 44.0 per cent of the dry solids with an
average of 13-1 per cent. Various other workers confirmed this range (POHLA~,U~,1962:
BUSWELL and NEAVE, 1930; HEUKELEKIAN,1958; RUDOLFSand GEHM, 1942). This
ether-soluble fraction of the dry solids was found to be largely destroyed by anaerobic
digestion as the percentage grease in digested solids was reduced on average from
25.2 per cent to 6-9 per cent (BuSWELL and NEAVE, 1930). The ether-extractable
material consists of many components including free fatty acids, lipids and esters.
HEUKELEKIANand Mt~rLr.R (1958) found that the grease from sewage solids con-
mined 40-60 per cent free fatty acids, 20-40 per cent esterified fatty acids and 15-20
per cent unsaponifiable material and that the fatty acids included palmitic, stearic,
and dienoic acids. HUNTER (1962) reported that the settleable solids in sewage con-
tained 1.35 per cent free fatty acids, 9.66 per cent glyceride fatty acids, 3.75 per cent
unsaponifiable material and very low concentrations of phenols and detergents.
About 80-90 per cent of the fatty acids were saturated acids and the rest comprised
the following unsaturated acids in order of decreasing concentrations: oleic, linoleic,
linolenic and arachidonic acids.
Dry fresh sewage solids contain 2.49 per cent alcohol-soluble material and 9.52 per
cent material which is soluble in hot and cold water (POHLAND,1962). POHLAND(1962)
mentioned that sugars, amino acids, organic acids, starches, tannins and pectic sub-
stances were extracted by hot and cold water while the alcohol-soluble fraction con-
tained waxes, resins, alkaloids and choline. HUNTER (1962) analysed the settleable
solids in sewage and found that it contained 7.71 per cent alcohol-soluble-ether-
insoluble material, which consisted of 16.9 per cent amino acids, 4.4 per cent sugars
and 8.3 per cent tannins.
Another major group of components of sewage sludge is the polysaccharides which
include substances such as starch, cellulose and hemicellulose. POHLAND (1962)
reported that dry sewage sludge contained 3.78 per cent cellulose and 3.20 per cent
hemicellulose. BUSWELLand NEAVE (1930) determined that sewage sludge contained
10.8 per cent crude fibre of which the major components must have been polysacchar-
ides. RUDOLFSand GEHM(1942) reported concentrations of cellulose and hemicellulose
of 3-8 and 3.2 per cent respectively, while HUNTER(1962) measured a concentration of
21.84 per cent cellulose on a dry weight basis in the settleable material in sewage.
The concentration of free carbohydrates in sewage sludge is apparently very low,
probably because they are rapidly taken up and metabolized by microorganisms.
HUNTER (1962) found only 0.34 per cent sugars (as a percentage of the total dry
solids) in the fraction which was soluble in alcohol but insoluble in ether.
POHLAND (1962) and RUDOLFSand GEHM (1942) reported that dry sewage sludge
contained 5.8 per cent lignin. Lignin is not readily decomposed by anaerobic digestion
and is therefore present in digested sludge in larger concentrations than in fresh solids
(HEUKELEKIAN, 1930).
Protein material is the second largest component of sewage sludge after the group
of ether-extractable compounds. The protein content of dry sludge vary from 19 to 28
per cent (RUDOLFS and GEHM, 1942; POHLAND, 1962; BUSWELLand NEAVE, 1930).
HEUKELEKIANand BALMAT(1959) identified 14 amino acids in sewage sludge with
alanine, aspartic acid, glutamic acid, leucine, phenylalanine, methionine, valine, glycine,
histidine, cystine, serine, threonine, tyrosine and lysine listed in order of decreasing
concentration. The concentration of free amino acids in sewage sludge is however low
compared to the total crude protein in sewage sludge. HUNTER(1962) found only 1.3
per cent free amino acids in the dry settleable material of sewage.
Various other compounds may occur in low concentrations in sewage. It is known
that vitamins such as Vitamin C (RUDOLFS and HEINEMANN,1939)and Vitamin B12
(AURICH et aL, 1958) are present in sewage. The presence of indole and skatole in
sewage has been demonstrated by RUDOLFSand CHAMBERLIN(1932) and by RUDOLES
and INGOLS(1938) and BUSWELLand NEAVE (1930) found uric acid, xantine, creatine
and creatinine. Much has also been published on the presence and role ofalkyl benzene
sulphonates (synthetic detergents) in the biological treatment of sewage. CORDONet al.
(1968), CROFT and FAUST (1954), CULP and STALTENBERG(1953) and MANGANELLI
et al. (1960) reported on the degradation of detergents by sewage microorganisms.
464 J.P. KOTZI~,P. G. THIELand W. H. J. HATTINGH
The great variety of compounds present in sewage sludge is an indication of the
versatility of the anaerobic digestion process towards the removal of various organic
compounds from polluted waters. The value of anaerobic digestion for the treatment
of organically polluted industrial effluents is clear. Whereas the main compounds in
sewage sludge are fats, polysaccharides, and proteins, these compounds also occur in
high concentrations in many industrial effluents.
Brewery waste, for instance, contains high concentrations of polysaccharides while
the concentration of proteins is relatively low (THIELand Do TOlT, 1965). Yeast waste
on the other hand, contains relatively high concentrations of nitrogenous material
(KOTZ1~e t al., 1968).
Nutritional factors in the substrate. To maintain satisfactory digestion it is necessary
to ensure that optimum biological growth is maintained at all times. For optimum
biological growth various nutritional factors are normally necessary--these include,
for example, inorganic elements such as magnesium, calcium, potassium, sodium, zinc,
iron, cobalt, copper, molybdenum, manganese etc., which are cofactors necessary for
enzyme reactions in microorganisms.
Many other organic growth factors such as branched chain fatty acids (BRYANT,
1965), biotin, p-amino benzoic acid and amino acids, may be essential for the growth
of certain bacteria, as they cannot be synthesized by some bacteria and are necessary
either as cofactors in enzymic reactions or are used as intermediates in the synthesis of
cell material.
Because most of the inorganic elements and different organic growth factors are
usually present in domestic sewage, a lack of these factors seldom occurs in practice.
The major nutrients are nitrogen, carbon, sulphur and phosphorus. These four
elements are normally contained in excess in raw domestic sludge. Therefore admix-
ture of domestic sewage to industrial effluents offers a means of correcting nutrient
deficiencies in industrial wastes. The overall demand for nitrogen is in excess of the
microbial demand since part of the nitrogen is required in the control of the pH of the
system. It is generally assumed that alkalinity is mainly provided by NH4 + and I-ICO3'
ions. A reduction in the NH4 + content of a digester leads to a decrease in alkalinity and
perhaps pH, thus illustrating that nitrogen is essential both as nutritional element and
in the ammonium form for its buffering capacity. The phosphorus requirements on
the other hand would normally be met by the phosphorus content of the wastes to be
digested.
KAPLOVSKY (1952) found it necessary to add sufficient ammonium carbonate to
give a C :N ratio of 40:1 to achieve digestion of white water. SANDERSand BLOODGOOD
(1965) reported the minimum C :N ratio of anaerobic digestion to be 16:1.
Substrate loading
The rate at which organic material (substrate) is supplied to the microorganisms
participating in the degradation of the substrate is of prime importance in maintaining
stable digestion conditions. Different loadings can be achieved by either altering the
rate of flow through a digester, which will affect the volumetric residence time in the
digester, or by altering the concentration of the organic load of the feed. In a com-
pletely mixed system short residence times may result in the discharge of a large
quantity of unmetabolized substrate. Longer residence times, on the other hand, will
result in more complete degradation of the substrate. In practice a compromise must
be reached between the efficiency of long residence times and the economy of short
residence times. The load to a digester can seldom be altered in practice by changing
the concentration of the organic load as the composition of the waste water depends
entirely on the process in the specific industry. Therefore the most common practice of
changing the load to a digester is to change the flow rate. Excessive loads are known
to create unbalanced conditions where volatile fatty acids accumulate.
For the disposal of domestic sludge, a loading parameter of weight of volatile
solids/volume/day seems to be most generally applied. This loading parameter does
not take into account the number of microorganisms or the biomass present in the
volume participating in the degradation of the organic material and assumes that
sufficient organisms will be available. It became necessary when industrial effluents
had to be treated anaerobically, to develop other loading parameters which take into
account the amount of biologically active organisms present. Two problems arose in
the development of these loading parameters. One was the assessment of the amount
of organic material that might be biologically degraded and the other was to assess the
amount of biomass present in the digester.
The chemical oxygen demand (COD) determined by the dichromate reflux method,
appears to be the most favoured method of expressing the amount of oxidizable
material contained in the waste. The COD method, however, does not give any
indication of the amount of material that might not be biodegradable in the waste or
the presence of reducing substances such as Fe 2+, S" etc. The development of methods
to determine organic carbon in solution by means of oxidation of an aqueous sample
followed by infra-red detection of the evolved CO2, offers a more rapid and concise
method for the determination of organic carbon. The latter method still does not
distinguish between organic carbon amenable to biological degradation and organic
carbon resistant to degradation.
The determination of the amount of biomass present in anaerobically digesting
sludge is more difficult. Chemical methods to assess the weight of biomass have gained
favour. The main obstacle is to choose a parameter of biomass that will closely
measure the weight of microorganisms under all circumstances. The nucleic acid,
DNA, was chosen as such a parameter because it is only synthesized by living cells and
is one of the least variable compounds of living cells. Subsequent work revealed that
the DNA content of anaerobic sludge is significantly correlated to the volatile sus-
pended solids (VSS) content of a digester (AGARDYe t al., 1963; HATTINGHet al.,
1967a). Therefore volatile suspended solids represent a meaningful measure of biomass
and are used to assess biomass. In cases where the feed to a digester contains high
concentrations of suspended organic material, VSS will however not be a direct
measure of biomass. The loading rate parameter advocated for effluents with low
concentrations of suspended organic material is therefore the ratio of the weight of
COD or organic carbon to be treated to weight of cells in the digester per unit time.
This may be expressed as g COD/g VSS/time. At present it seems as if prolonged stable
anaerobic conditions can only be maintained if the loading rate is less than 0.25 g
COD/g VSS/day at 35°C.
Temperature
The reactions taking place in an anaerobic digester result from the activity of
the heterogenous bacterial population. The effect of temperature on the process
466 J.P. KOTZ~,P. G. THIELand W. H. J. HATTINGH
as a whole is therefore a reflection of the behaviour of the bacteria at different tem-
peratures.
Since living organisms consist mainly of water, the limits for biological activity in
water in a liquid state, range from slightly below 0°C to about 100°C. The upper limit
of the temperature growth region is determined by the thermal lability of the con-
stituents of living matter, the proteins and nucleic acids. These substances are rapidly
inactivated at temperatures between 50°C and 90°C. Exposure of microorganisms to
temperatures above these ranges result in rapid death, except in the special case of
heat-resistant spores (STANIERe t al., 1964). Temperatures below 0°C are not necessarily
lethal to bacteria and many bacteria survive temperatures as low as that of liquid air
(STANIERet al., 1964).
Although the total temperature range for the development of microorganisms
extends from - 5 ° C to about 80°C, specific microorganisms often have relatively
narrow temperature ranges in which they will multiply. For instance, a common
sewage bacterium, Escherichia coli, grows in the range 10-46°C but the optimum
temperature for its growth lies between 37°C and 45°C (STANIERe t al., 1964).
The temperature ranges for the optimal growth of microorganisms can conveniently
be divided into three temperature regions namely the psychrophilic (<20°C), the
mesophilic (20-45°C) and the thermophilic region (> 45°C) (STANIERe t al., 1964). A
particular bacterial species can then be described as a psychrophilic, mesophilic or
thermophilic bacterium depending on the temperature region in which optimal growth
is obtained. The growth temperature range of a specific bacterium is often used as a
taxonomic criterium. However, certain bacteria, known to have a specific temperature
optimum, can be adapted to other temperature ranges (ALLEN, 1953; MEEFERD and
CAMPBELL, 1952; KLUYVERand BAARS,1932). Abrupt changes in temperature are
often lethal to bacteria. Escherichia coli, grown at 45°C can be killed for example by
rapid cooling to 10°C (95 per cent) while gradual cooling over 30 min causes no death
(SHERMANand CAMERON,1934).
The temperature at which a bacterium is grown has a definite effect on the chemical
composition of the cell (INGRAHAM,1962), the activities of enzymes in the cell (BROWN
et al., 1957; ALFORD, 1960) and bacterial nutrition ([NGRAHAM,1962).
Because of these pronounced effects of temperature in single species of bacteria, it is
clear that the temperature at which a heterogeneous population of bacteria is grown
will have a definite effect on the species and numbers of bacteria occurring in the
heterogeneous population. Thus the temperature, which can be controlled externally,
is active in selecting the ultimate population that will prevail in a digester.
For the economical use of anaerobic digestion it is necessary that the highest
efficiency of the overall process is obtained. It is a well-known fact that by increasing
the temperature by 10°C, the rate of a chemical reaction can be increased approximately
two-fold. In narrow temperature ranges this is also roughly true for biological activities
which depend on the action of enzymes to bring about ordinary chemical changes.
This indicates the necessity of determining the temperature at which anaerobic
digestion of a specific substrate will proceed at the highest possible rate.
BUSWELL(1957) reported that production of methane could be achieved from 0°C
to 55°C. The economical applications of anaerobic digestion, however, take place only
in the mesophilic and thermophilic temperature ranges. Much controversy exists on
the advantages and disadvantages of mesophilic and thermophilic digestion. RUDOLFS
and HEUKELEKIAN(1931) reported that the rate of digestion of sewage sludge could be
increased by changing from mesophilic to thermophilic digestion (50°C). For the
digestion of board mill sludge, mesophilic digestion (36°C) was, however, more effective
than thermophilic digestion (50°C). Although thermophilic digestion was more
efficient for sewage sludge digestion, it was never generally accepted for conven-
tional sewage treatment, because of the development of foul odours, heating
difficulties (LOHMEYER,1959) and difficulties in dewatering the digested sludge (LUMs,
1941).
Sudden temperature changes are detrimental to anaerobic digestion. BROWN and
KINCHOSKY(1965) concluded that a digester normally operated at about 32°C could
be upset when the temperature increased to 40°C, as the mesophilic conditions were
drastically changed to thermophilic conditions and fatty acids accumulated. FISHER
and GREENE(1945) found during plant-scale studies of thermophilic digestion, that a
temperature drop of 2.7°C or more, during a relatively short period, greatly affected
digestion at 46-53°C. BUSWELL(1957) agreed with these results and stated that a
sudden change of as little as one or two degrees centigrade, completely interrupted
methane fermentation with the resultant accumulation of fatty acids. GARBER(1954)
on the other hand, concluded that when once established, thermophilic digestion
(49°C) was quite stable and resistant to upset and also that temperature decreases of
5°C in 48 hr produced no unusual changes but that prolonged lower temperatures,
changed the system to mesophilic conditions.
HEUKELEKIANand KAPLOVSKY(1948) reported that in a 20°C range between the
mesophilic and thermophilic temperature regions, the rate of digestion was decreased
by a rise in temperature. The rate of digestion only increased when the temperature was
increased to the optimal range in the thermophilic region. GOLUEKE(1958) however
found that changes in temperature from 35°C to 60°C had no effect on the rate of
fermentation. STANDERet al. (1967) found that optimum digestion of wine waste took
place at 35°C.
From these conflicting observations no general criteria can be layed down as to
the temperature at which anaerobic digestion should be practised. The optimum
temperature for the digestion of each individual waste should be determined experi-
mentally.
lnoculum
Only those bacteria which enter the digester via the inoculum and/or the substrate
and are adaptable to the environment will eventually have a chance of survival in the
digester. The effluents normally treated anaerobically are not sterile, with the result
that bacterial species absent in the original inoculum may, at a later stage, enter the
system via the substrate. Regardless of the origin of the inoculum the bacteria that will
eventually predominate will be those for which the substrate and environmental
conditions are most suitable.
Anaerobic digesters cannot be switched from one substrate to a widely different one
in a matter of hours or days. Time is required to adjust to the new substrate, which is
termed the adaptation period. The precise length of the adaptation period has not been
determined as yet and may even vary from substrate to substrate. HATTINGHet al.
(1967a) presented evidence that in a specific case adaptation was not completed even
after 60 days. During adaptation, the microbial population best suited to the substrate
468 J.P. KOTZI~,P. G. THIELand W. H. J. HATTIN~H
and environmental conditions is selected from the original inoculum. Therefore if the
original inoculum contains small numbers of a specific microorganism capable of
degrading a specific compound in the substrate, the length of time required to build up
a suitable population will depend on the growth rate of the microorganisms and the
mode of operation of the digester. Apart from the selection of microorganisms certain
non-lethal mutations can take place enabling certain organisms to adapt themselves
more efficiently than others to the existing conditions. Both the selection and mutation
processes can be the explanation for the increase in the stability of digesters (STANDER,
1968) as well as the changes in the metabolic patterns during prolonged operation
(HATTINGH et al., 1967a; KOTZ~ et al., 1968).
Mode of operation of a digester

The mode of operation has a definite effect on the nature of a digester. Two extreme
methods of operating a flow-through system are the plug-flow method and the com-
pletely-mixed method. In a completely-mixed system the substrate entering the system
is immediately and uniformly distributed through the entire system whereas in the
plug-flow method no intermixing occurs during the passage of the substrate through
the system. In anaerobic digestion only the completely-mixed method finds application
although the degree of mixing can vary.
Mixing of anaerobic sludge accomplishes three major objectives, firstly the organisms
are kept continuously in contact with the substrate supply, secondly the substrate supply
is uniformly distributed and made available and thirdly the concentration of inhibitory
biological intermediates and end-products are maintained at minimum levels (WPCF
Manual, 1968). Efficient mixing also reduces formation of scum layers to a minimum.
The only disadvantage of the completely-mixed method is that unmetabolized sub-
strate is bound to leave the system; the quantity lost depending on the volumetric
residence time and the contact period necessary for the assimilation of the substrate.
Mixing may be brought about by gas recirculation, sludge recirculation or mechanic-
ally turned impeller blades. Mixing also provides a means of maintaining an even
temperature distribution throughout the digester. The importance of maintaining
evenly distributed temperatures has been indicated earlier in this paper.
The manner of applying the substrate to the digester has a definite effect on the
process. Continuous application of the substrate is preferable because slug doses can
result in temporary overloading of the system.
In order to achieve high rates of biological activity in digesters sludge can be re-
cycled from the effluent to the digester. In doing this the sludge residence time is
increased relatively to the volumetric residence time.
CHARACTERIZATION OF ANAEROBIC DIGESTION

General characteristics
To control anaerobic digestion efficiently it is necessary to characterize a well-
operating digester. POHLANO(1962) listed a number of parameters most frequently
used to judge the status of the fermentation process. The following general factors are
most frequently used to characterize a digester: pH, alkalinity, volatile fatty acid
concentration, the rate of gas production and composition of the gas produced. The
importance of these factors for the characterization of anaerobic digestion is discussed
below.
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 469
pH, alkalinity and volatile fatty acid concentration. The pH, alkalinity and volatile
fatty acid concentration are accepted parameters for practical control of anaerobic
digestion. These three factors are however interdependent and their effect on the
process of anaerobic digestion will be discussed together.
The pH of the internal environment of all living cells is believed to be approximately
7 (STANI~Ret aL, 1964). Most bacteria are relatively insensitive to external concentra-
tions of hydrogen or hydroxyl ions. Many species can grow well at any pH value
between 6-0 and 9.0 which represents a thousand-fold variation in hydrogen ion
concentration. This is because the membranes of living cells are only slightly permeable
to hydrogen and hydroxyl ions (STANn~Ret al., 1964). Undissociated molecules can
penetrate more readily into bacterial cells than the ionic forms of the organic acids
(STANIERet al., 1964). Weak acids are poorly dissociated at low pH values and in their
undissociated forms they easily migrate into the cells and thus change the internal pH,
whereas strong acids, which are highly dissociated have a much less drastic effect.
The same principle applies to weak bases at high pH values. The weak bases and acids
are thus toxic at high and low pH values, respectively, but are relatively harmless at
neutral pH values (STANIEget al., 1964).
The heterogeneous bacterial population in an anaerobic digester comprises a wide
variety of bacterial species. Each specific species has an optimum pH range in which
growth is best and its metabolic processes function at optimum levels. In anaerobic
digestion, therefore, the optimum pH range is the integral result of the different con-
tributions by the different reactions taking place.
BARKER(1956) concluded that the pH range 6.4-7.2 was most effective for methane
production and that below pH 6.0 and above pH 8.0 production declined rapidly.
I ~ E F ~ and URTES (1963) reported that high hydrogen ion concentrations were
inimical to anaerobic digestion. They obtained efficient digestion of formic, acetic,
propionic and butyric acids at pH 6.8. ALBERTSON(1961), however, found that digesters
which were acclimatized to either acetate, butyrate or ethyl alcohol, contained the
highest number of bacteria when the pH of all three digesters was about 7.1. During
the anaerobic treatment of the waste of the potato industry together with sewage
sludge, HINOIN and DUNSTAN(1963) obtained efficient digestion at pH values varying
from 7.0 to 8.0 when the potato waste in the feed did not exceed 50 per cent. Higher
loads of potato waste caused the pH of the system to decrease drastically.
For a digester to operate efficiently, the pH should therefore be in the region
6.4-7.2. The eventual pH obtained in a specific anaerobic digester will however be
determined to a large extent by the substrate supplied to the digester. Whatever the pH
of a digester in equilibrium may be, any definite drop in pH value, as a result of the
production of volatile fatty acids, will be an indication of a disturbance of this
equilibrium and will necessitate some method of control.
In discussing the effect of pH it is necessary that factors which have an
influence on pH must also be considered. Two factors closely associated with pH are
the alkalinity of a system and, in anaerobic digestion, the concentration of volatile fatty
acids. The hydrogen ion concentration of a biological system can fluctuate con-
siderably during biological processes if the system is not well buffered. The alkalinity
of an anaerobic digester is a measure of the buffering capacity of the contents of the
digester. A high alkalinity could therefore be an indication that the system is safe-
guarded against pH fluctuations while a low alkalinity could indicate that a sudden
470 J.P. KOTZ£, P. G. THIELand W. H. J. HATTINGH
rise in volatile fatty acid concentration may have lowered the pH to such an extent that
biological activity is impaired.
It is known that COz and acids such as formic, acetic, propionic, butyric, lactic,
malic, fumaric, succinic, citric, glutamic etc., are formed when carbohydrate
and proteinaceous materials are degraded biologically (see later). Cations such as
Na ÷, K +, Ca 2 +, Mg 2+ and NH4 + and anions such as bicarbonate and phosphate and
the gas CO2 provide an efficient buffering system. The main buffering substance in
most digesters is NH4HCOa (SIMPSON, 1960). Healthy digesters will produce NH4 ÷
and HCO 3- from the end-products of digestion e.g.
COHNS ~ CO 2 + H 2 0 + NH 3 + CH4 (1)
(organic matter)
CO2 + H 2 0 + N H 3 ~ N H 4 + + H C O a - . (2)
The NH4 + and HCO3- ions remain in solution and contribute to the alkalinity of
the substrate (COULTER, 1953). An ammonia nitrogen content of at least 100 mg/1 in
the mixed liquor is assumed to be necessary for efficient digestion of carbonaceous
material. Greater NH4 + and HCO3- production has been associated with thermo-
philic digestion due to the more complete breakdown of proteins (GARBER, 1954;
RUDOLFSand SETTER, 1937; GOLUEKE, 1958).
Alkalinity is normally determined by titration to pH 8.3 (phenolphthalein) or to
4-5 (methyl orange) (Standard Methods, 1965). As most digesters have pH values lower
than 8-3 the alkalinity titrated to pH 8.3 is zero. The alkalinity titrated to pH 4.5 (the
total alkalinity) is an exaggerated measure of the buffering capacity as the pH of a
digester should not be allowed to decrease to such a low value. HATTINGH et al.
(1967a) advocated the use of an alkalinity titrated to pH 6.0 and expressed a s H C O 3 -
as a more realistic measure of the available buffering capacity of an anaerobic digester.
YENCKO (1955), BACKMEYER(1955) and GOULD 0959) considered a total alkalinity
(as CaCO 3) of 2000-3500 mg/l as a desirable characteristic of a well established
digestion process treating sewage sludge. Comparative studies on the alkalinities of
fresh and digested sewage sludge revealed respective values of 1000-2000 rag~1 and
2500-4000 mg/1 (COPELAND, 1955). From these observations it was concluded that if
the total alkalinity decreased to values less than 2500 rag/l, normal biological processes
of digestion were slowing down and the feed of raw sludge should be reduced.
Alkalinity was reported to increase parallel to the increase in loading rate, pre-
sumably due to the amount of nitrogenous material in the fresh solids (SAWYER and
RoY, 1955; HI~qDnq and DUNSTAN, 1959). GOLUEKE (1958) found that for similar
loadings, thermophilic digestion resulted in higher alkalinities than mesophilic
digestion.
COULTER (1953) and HASELTINE (1949) reported alkalinity values for fresh and
digested sludge of 400-1200 mg/l and 3000-5000 mg/l respectively. They also suggested
that an alkalinity between 2000 and 3000 mg/l would be beneficial for digestion.
HASELTINE(1949) and FOGARTY(1961) ascribed alkalinity concentration to the break-
down of nitrogenous material, while FOGARTY (1961) found that above pH 7.0 the
breakdown of proteinaceous material decreased.
MUELLER et al. (1959) and POHLAND (1962) emphasized the need for a balance
between alkalinity and volatile fatty acid concentration for normal digestion and
implied that variations in pH occurred only after the volatile acids--alkalinity balance
had been destroyed or seriously affected. It has recently been stated that if the ratio of
volatile fatty acids (expressed as mg/l acetic acid) to total alkalinity (expressed as mg/l
CaCO3) was lower than 0.8, unbalanced conditions in a digester were indicated
(WPCF, 1968).
It is a well-known fact in anaerobic digestion that when the volatile fatty acids
accumulate, the digestion process is in imminent danger of total inhibition. BOSWELL
and NEAVE (1930) and BOSWELL and HATFIELD (1939) were amongst the first to
emphasize the importance of the behaviour of volatile fatty acid concentration as an
indication of digestion. It has generally been accepted that volatile acids which
accumulated in excess of 2000-3000 mg/1 as acetic acid, interfered with normal
digestion (SCHLENZ, 1944, 1947; SCHULZEand RA~O, 1958; HINDIN and DUNSTAN,
1959, 1960; MOELLERet al., 1959).
BACKMEYER (1955) and COPELAND (1955) maintained that the limiting value of
volatile acids should be 300 mg/1 while SAWYER(1960) stated that for normal digestion
the volatile acid concentration should be in the range 100-200 mg/1 as acetic acid.
More recently HATTINGHet al. (1967a), KOTZI~et al. (1968) and THIEL et al. (1968)
reported that their synthetic substrate and sewage sludge digesters reached a stage of
unbalance when the total alkalinity decreased to below 1500 mg]l HCO z- and the
volatile acid concentration increased to above 200 mg/l (as acetic acid).
Although there is still much controversy on the permissible concentration of volatile
fatty acids in a digester, it can be assumed that a steady increase in volatile fatty acid
concentration will be an indication of possible failure of the process as a result of
unbalanced conditions.
MUELLER et al. (1959), HEUKELEKIANand KAPLOVSKY(1949), KAPLOVSKY(1951,
1952), HINDINand DUNSTAN(1959, 1960), and GOLOEKE(1958) reported that the main
volatile acids produced during digestion were acetic, propionic and butyric and
that lesser amounts of formic and valeric acids were also produced. McCARTY et al.
(1963), reported that acetic acid was the most prevalent acid in methane fermentation
of carbohydrates, proteins and fats while propionic acid was also thought to be an
important intermediate. Although both formic acid and butyric acid were rapidly
metabolized by different sludges, MCCARTYet al. (1963), did not consider them to be
important intermediates in the process as they did not accumulate in the process to the
same extent as acetic and propionic acids.
ENEBO and PEHRSON (1960) concluded that propionic and valeric acid originated
from proteinaceous material and found that the thermophilic process liberated acetic,
butyric and a small amount of formic acid. They also discussed the probability that
the even-numbered fatty acids were more easily degraded than the uneven-numbered
molecules by both the mesophilic and the thermophilic digestion processes.
Notwithstanding the data available on the types of volatile fatty acids occurring
during normal or unbalanced anaerobic digestion, the problem as to whether these
acids act as intermediates in the process of the formation of methane and carbon
dioxide, has not yet been solved. This problem is one of the most important to be
solved in order to obtain a better understanding of the mechanism of the process.
Efficiency o f substrate conversion to gas. The volume and composition of the gas
produced are characteristic of the substrate digested. Variations in substrate composi-
tion and concentration are reflected in the gas production of a digester. BUSWELLet al.
B W
472 J.P. KOTZ~,P. G. THIELand W. H. J. HATlrINGH
(1932) and POMEROY(1933, 1935) presented evidence that the CO2 content of digester
gas was 50 per cent, 30 per cent and 16 per cent when carbohydrates, fats and proteins,
respectively, were digested. They also indicated that the volume of gas produced
varied with the type of material digested. Therefore no dogmatic rule can be laid down
concerning a definite ratio of CH, to CO2 or the volume of gas which should be
produced. Serious deviation from the generally observed values must immediately
be investigated. An increase in the CO2 content of the gas forecast difficulties in
digestion (WPCF, 1968).
For control of anaerobic digestion, the CH4/CO2 ratio presents a rapid and
sensitive parameter. Any sudden marked change in this ratio is indicative of unbalanced
conditions.
Efficiency o f the process. It is necessary to assess the overall efficiency of anaerobic
digestion as this has a direct bearing on the quality of the final effluent and the
economy of the process. A decrease in efficiency would be indicative of failure of the
process and can therefore serve as a control parameter.
Efficiency of anaerobic digestion may be measured from a knowledge of the COD or
carbon content of the substrate supplied and the COD or carbon content of the final
effluent. Another parameter of the efficiency is the percentage conversion of applied
COD to gas. The values so calculated would be characteristic of the efficiency of
digestion of the particular substrate and any deviation of the values would be indica-
tive of the setting in of unbalanced conditions in the process.
KOTZ~ et al. (1968) reported on a biological and chemical study of several digesters
receiving widely different substrates. They indicated that the overall efficiency of COD
removal varied from 71 per cent for a digester treating a yeast waste to 98 per cent for
a digester receiving a synthetic substrate. The percentages of COD conversion to gas
varied from 47 per cent for the yeast waste digester to 78 per cent for the synthetic
substrate digester. The authors assumed that the fraction of COD calculated from the
difference in efficiency of overall COD removal and the amount of COD converted to
gas, was consumed for cell synthesis. This figure might be high since some carbon was
converted to carbon dioxide which left the system as bicarbonate.
Biomass content. The purification brought about by anaerobic digestion is mainly
the result of biological action. For an efficient control of anaerobic digestion it is
therefore necessary that the amount of living matter (biomass) is known. Several
methods exist to assess the amount of biomass present in anaerobic sludge.
The most direct procedure used to assess the biomass is to count the total number
of bacteria present. Problems are posed in assessing the actual number of micro-
organisms from anaerobic digesters. PRETORIUS(1968) observed that total anaerobic
bacterial counts on digester supernatant liquor as substrate, were many times greater
than equivalent counts on other substrates. These counts were performed on digester
supernatant liquor drawn from digesters similar to those on which the counts were to
be performed. In order to enumerate the incidence of certain physiological groups
of bacteria (acid-producing, methane-producing, sulphate-reducing, etc.) specific
selective media and conditions must be used.
TOERIEN et aL (1967) and MAH and SUSSMAN(1968) showed that the concept that
anaerobic digestion was brought about mainly by facultative bacteria, was open to
question. Counts of aerobic and facultative anaerobic bacteria were always many
times less than counts of obligate anaerobic non-methanogenic bacteria. Work on the
development of a method to count the methane-producing bacteria, revealed that this
group of bacteria was as numerous as the obligate anaerobic non-methanogenic
bacteria (SIEBERTet al., 1968).
Biomass content can also be assessed by more indirect methods (analytical tech-
niques) such as D N A content, protein content, VSS content, adenosine triphosphate
(ATP) content and enzyme activities. Pure bacterial mass consists of about 50 per cent
protein on a dry weight basis (LURIA, 1960), therefore by determining the protein con-
tent of anaerobic sludge the biomass in the sludge can be estimated (HATTINGHet al.,
1967b). The mixed liquor in a digester may, however, contain extracellular protein and
consequently mere determination of the protein content of the mixed liquor may be
misleading.
A reliable method of estimating biomass content is the determination of the D N A
content of sludge (AGARDYe t al., 1963; HATTINGH and SIEBERT,1967) as this sub-
stance is only synthesized by living material. Another convenient method used to
estimate biomass is the determination of the VSS content of the sludge as this value is
easly determined and correlated to the D N A content in anaerobic sludge (AGARDY
et al., 1963; HATTINGHet al., 1967a).
ATP is closely associated with biological activity and also with the energy status of
living matter and like D N A will give an estimate of the biomass content of sludge.
Various methods for the enzymatic determination of minute concentrations of ATP
exist (ADDANKI et al., 1966; HOLMSENet al., 1966; TAL et al., 1964).
Enzymes are responsible for all biological changes brought about by microorgan-
isms. Under specific conditions the activities of certain enzymes can serve as a measure
of amount of substrate flow (KoTz~, 1967). Substrate flow depends on the number of
microorganisms and therefore well chosen enzyme activities can serve as a measure of
biomass content.
The parameter chosen to assess biomass depends on the purpose for which it is to be
used and the relative ease with which the determination can be done. Each of the
above-mentioned parameters has certain limitations. Because D N A is agreed to be a
reliable parameter of biomass and also because of the relative ease with which VSS
can be determined, the use of the VSS content can be advocated as a rough measure of
biomass content. Further investigations on ATP content and enzyme activities might
prove them to be more informative and reliable than VSS as measures of biomass.
Microbiological characteristics
The microbiological nature of a digester is determined by its inoculum and the
environmental conditions. The microbial population determine the biochemical
characteristics of a digester.
TOERIEN and HATTINGH (1969) described the microbiology of anaerobic digestion
thoroughly, in the first of this series of papers.
Biochemical characteristics
Life cannot exist without the active participation of enzymes (biological catalysts).
Enzymes are active both in degradation of organic matter (catabolism) and synthesis
of new cellular constituents (anabolism). Enzymes occur both extracellularly and
intracellularly. The hydrolytic extracellular enzymes are enzymes that are active in the
initial hydrolysis of macromolecules to their smaller units, which are then transported
474 J.P. KoTz~,P. G. THIELand W. H. J. HATTINGH
into the cell to undergo further degradation or to be used as building blocks in the
synthesis of new cellular material. The activities of the enzymes are therefore a measure
of the activity of the biomass in the digester. The occurrence of certain key inter-
mediate enzymes also reveals a picture of the metabolic pathways operative in the
degradation of the added substrates.
The presence and activities of several extracellular and intracellular enzymes in
anaerobic digesters have been reported by HATTINGHet al. (1967a), KOTZ~et al. (1968)
and THmLet al. (1968). HATTINGHet al. (1967a) reported that the chemical parameters
such as pH, alkalinity, volatile fatty acid content, rate of COD removal and volume
and composition of gases produced did not indicate the biological changes that took
place in an anaerobic digester when it was slowly adapted to a new substrate. Bio-
chemical analyses indicated that the process of adaptation was not completed after
60 days of operation. Therefore a study of the biochemical characterististics is neces-
sary to characterize a stable anaerobic digester.
The biological characteristics of an anaerobic digester are described, amongst other
things, by the different enzymes present, their relative activities and their participation
in meauingful metabolic patterns or pathways for the degradation of the organic
matter supplied in the substrate. The enzyme activities are the integrated result of the
composition of the waste (substrate), the loading rate, the nature of the microbial
population and the environmental conditions such as temperature, pH, alkalinity and
degree of anaerobiosis.
HATTINGH et al. (1967a) reported that the reactions catalyzed by extracellular
hydrolytic enzymes (protease, cellobiase, amylase and phosphatase) would not easily
be rate-limiting steps in the hydrolysis of organic matter to smaller units in anaerobic
digestion. The intermediate enzyme activities determined by them revealed valuable
information on the changes that took place in the biological pattern of the digester.
KOTZf/ et al. (1968) have shown that digesters operated on different types of sub-
strates showed characteristic enzyme activity patterns and that these patterns may be
used to characterize digesters. For instance, it was observed that the enzyme activities
of digesters operated in the laboratory, on a continuous flow principle, with a synthetic
substrate, fluctuate within small limits. The daily fluctuations did not normally exceed
10 per cent under balanced conditions but the onset of unbalanced conditions was
manifested by large daily fluctuations (more than 10 per cent). TmEL et al. (1968)
reported further on the daily observations of enzyme activities with the onset of un-
balanced conditions.
TOERmN and HATTINGH(1969) divided the process of anaerobic digestion into the
non-methanogenic and methanogenic phases. The discussions of the biochemical
characteristics will also follow this broad division.
Metabolic pathways o f the non-methanogenic phase

The metabolic pathways participating in the non-methanogenic phase are mainly
concerned with the degradation of macromolecules to smaller units. Therefore the
pathways for the degradation of carbohydrates, lipids, fatty acids, proteins, amino
acids and heterocyclic nitrogen-containing compounds will be discussed in more detail.
Degradation o f carbohydrates. Literature surveys on the carbohydrate metabolism
of mixed cultures have been published by S~MPSON(1960) and in the related field of
rumen metabolism by HUNGATE(1966) and BALDWIN(1965).
Anaerobic Digestion.--II. Characterization and Control of Anaerobic Digestion 475
T h e m e t a b o l i c p a t h w a y s such as glycolysis, the tricarboxylic acid cycle, and the

E n t n e r - D o u d o r o f f p a t h w a y a n d p e n to s e p h o s p h a t e shunt for the d e g r a d a t i o n o f
c a r b o h y d r a t e s have been f o u n d to be operative in a n a e r o b i c digesters (HATTINGH et al.,
1967a; Koxzl~ et al., 1968 a n d THIEL et al., 1968).
Glycolysis
The presence of the following enzymes participating in glycolysis has been indicated:
Hexokinase, phosphoglucomutase, fructose-6-phosphate kinase (6-PFK), fructose-l-phosphate
k inase (1-PFK), aldolase, enolase, pyruvate kinase and lactate dehydrogenase (HATTINGHet al.,
1967a; KOTZ~, 1967; KoIz~ et al., 1968; and THIELet al., 1968).
A new enzyme 1-PFK has recently been found in Aerobacter aerogenes by HANSONand AN-
DERSON(1966), in Bacteroides symbiosus by REEVESet aL (1966) and in Clostridium pasteurianum
W 5 by KOTZ~(1968a, 1969). The enzyme has also been found to be active in anaerobic digesters
(KoTzi~, 1967; KOTZ~, 1968a, 1969; KOTZ~ et al., 1968 and TmEL et al., 1968) and in various
other clostridia and other facultative or obligate anaerobes such as Pseudomonas, Butyrovibrio
and a bifid-like organism (KoTz~, 1968a). The activity of 1-PFK was correlated to the numbers
of anaerobes present in anaerobic digestion (THIEL et al., 1968). The presence of 1-PFK in
obligate aerobes has not yet been reported. The relative activities of 6-PFK and I-PFK thus
apparently give an indication of the anaerobic or aerobic origin of an unknown mixed culture
of microorganisms.
Citric acid cycle

KREBS and LOEWENSTEIN(1960) reported that the tricarboxylic acid cycle functioned only
when oxygen was constantly supplied. WATSON(1954) indicated that pyruvate proceeded to
oxaloacetate under anaerobic conditions, but that the isocitric dehydrogenase step of the citric
acid cycle was inhibited by anaerobiosis. SOMMERVILLE(1968) reported that malic enzyme,
malate dehydrogenase and glutamateoxaloacetate transaminase (GOT) were active in the
obligate anaerobe Peptostreptococcus elsdenii and that the citric acid cycle operated from
oxaloacetate to the formation of glutamate via GOT. Therefore, both aconitase and isocitrate
dehydrogenase (ICDH) must have been active, which contradicted the findings of WATSON
(1954). JONESand KING (1968) reported that succinate and fumarate were oxidized to malate
by extracts of facultative organisms such as Klebsiella aerogenes, Escherichia coli and Proteus
vulgaris. The statements by KREBS and LOEWENSTHN(1960) and WATSON(1954) are often
wrongly interpreted in that the citric acid cycle does not function in anaerobic microorgan-
isms.
KoTzl~ (1967), HATTINGHet al. (1967a), KOTZI~et al. (1968) and THIELet al. (1968) reported
that NADP dependent ICDH was normally present in microorganisms in anaerobic disgestion,
a finding which is also contrary to the belief of WATSON(1954). Other citric acid cycle enzymes
which were found to be present in anaerobic digestion are succinic dehydrogenase (SDH),
fumarase, malate dehydrogenase and aconitase (KoTz~, 1967; HATTINGHet al., 1967a; KOTZI~
et al., 1968 and THIELet al., 1968).
It has been observed that the succinate dehydrogenase of a domestic sewage sludge anaerobic
digester showed different properties to that of the aerobic electron transport system. Potassium
fluoride (0.001 M) in the presence of 0"05 i phosphate, activated succinate dehydrogenase by
increasing the activity by 14 per cent (KOTZ~, 1968b) whereas SDH from aerobic systems was
synergistically inhibited by phosphate and fluoride (SLATERand BONNER, 1956).
The reduction of fumarate by various obligate and facultative anaerobes has been described.
SINGER and HAUBER(1965) isolated, purified and characterized the enzymes responsible for
fumarate reduction from yeast, WARRINGAand GUIDITTA(1958) and WARRINGAet al. (1958)
from Micrococcus lactiliticus. The different fumarate reductases, except for two forms from
yeast, were all capable of catalyzing the oxidation of succinate as well as the reduction of
fumarate. Fumarate reductase was acid sensitive, but azide, arsenite, atabrin or malonate did
not inhibit the enzyme (DEIBEL and KVETKAS,1964). AUE and DEIBEL (1967), described the
properties of a fumarate reductase obtained from Streptococcus faecalis, which did not contain
succinate dehydrogenase. Malonate (0.01 M) inhibited the succinate dehydrogenase by about
476 J.P. KOTZ~,P. G. THIELand W. H. J. HATTINGH
90 per cent. The succinate dchydrogenasc from anaerobic sewage digesters was inhibited by
about 20 per cent when measured by the formation of fumarate. Succinate dchydrogenasc
from anaerobic sewage digesters also appeared to be readily reversible. The reversible reaction
appeared to be coupled to ATP formation (KoTz~, 1968b). These results are similar to those
obtained with succinate dehydrogenasefrom Ascaris lumbricoides (SEn)MANand ENTNER,1961).
ENEBOand PEHRSON(1960) reported on the degradation of all the Krebs cycle intermediates
by anaerobic digestion and concluded that the Krebs cyclemust function in anaerobic digestion.
Thus of the 8 different enzymes participating in the citric acid cycle, at least seven were found
to be operative in anaerobic digestion.
The existence of the glyoxylicacid cycleas indicated by the enzyme isocitratc lyasc, has been
postulated by HATTINGHet al. (1967a) and KOTZ~et al. (1968).
Entner-Doudoroff pathway
The significance of the Entner-Doudoroff pathway in anaerobic digestion has not yet been
determined. BALDWIN (1965) suggested that the 2-keto-3-deoxy-6-phosphogluconatealdolase
(Entner-Doudoroff) may function in the rumen. Although the Entner-Doudoroff route was
found in aerobes, it was also shown to function in Pseudomonas lindneri under anaerobic con-
ditions (WOOD, 1961; GIBBSand DEMoss 1951, 1954; D~Moss et al., 1951; SOKATerIand
GUNSALUS,1957).
Pentose phosphate shunt

The glycolytic pathway, glyoxylic acid cycle and citric acid cycle were found to operate
normally in anaerobic digesters whilst the activity of the pentose phosphate pathway was
normally low, except when digesters were fed a substrate high in carbohydrate (HAT'nN~Het aL,
1967a; Korz~ et al., 1968; Trm~Let al., 1968).
HAI~a~ISON(1959) stated that the pentose phosphate pathway (Warburg-Horecker-Dickens
route) depended on a supply of oxygen to the cell. However, the presence of this pathway
under strictly anaerobic conditions such as in an anaerobic digester indicated that this was not
necessarily true for many obligate anaerobic bacteria. The high glucose-6-phosphatedehydro-
genase activities observed by HAa~nN~net aL (1967a) was an indication that the pentose phos-
phate pathway must have been operative in the anaerobic bacteria present in the digester.
Degradation oflipids and f a t t y acids. It is generally accepted that the main pathways
of fatty acid degradation involve fl-oxidation of long-chain fatty acids (SIMPSON, 1960).
It is also known that ~o-oxidation could be responsible for the formation of dicarboxy-
lic acids (WHITE et al., 1964). The role of oJ-oxidation of fatty acids in anaerobic
digestion is not known, fl-oxidation of long-chain even-numbered fatty acids leads to
acetic acid as an end-product whereas the uneven-numbered fatty acids will result
in the production of acetic and propionic acids on fl-oxidation.
It is common knowledge that anaerobic digesters always contain certain amounts
the lower volatile fatty acids. During unbalanced conditions volatile fatty acids such
as formic, acetic, propionic, butyric and valeric often accumulate. Acetic and butyric
acids form the most prevalent volatile fatty acids. The fatty acids are the end products
of carbohydrate, amino acid and fatty acid metabolism. During unbalanced conditions
in anaerobic digestion, the hydrogen flow to CH4 is impaired and it is possible that
the longer-chain fatty acids, being a reservoir of bound hydrogen, could accumulate
the hydrogen.
The initial degradation of lipids is a hydrolytic step in which glycerol is liberated.
The glycerol enters the glycolysis route via the enzyme, glycerol-l-phosphate de-
hydrogenase. The presence of this enzyme in anaerobic digesters was indicated by
HAa-rINGHet al. (1967a) and KOTZ~ et aL (1968).
Degradation o f proteins and amino acids. Proteins and peptides must be degraded to
small fragments such as the amino acids, before it is absorbed by microorganisms. The
a m i n o acids all have characteristic p a t h w a y s b y which they are degraded. A m i n o acids
m a y be utilized in different ways b y different m i c r o o r g a n i s m s (BARKER, 1961 a n d
GREENBERG, 1961). Phenylalanine, for example, is d e g r a d e d b y s o m e b a c t e r i a v i a
h o m o g e n e s t i c acid a n d b y others via fl-oxaloadipic acid. A c e t y l - c o e n z y m e A is p r o -
d u c e d in b o t h routes, t h o u g h the f o r m e r also yields f u m a r a t e , whereas the latter r o u t e
p r o d u c e s succinate, w h i c h in t u r n is a p r e c u r s o r o f f u m a r a t e (KoRNBERG, 1965).
WRIGHT (1967) studied the m e t a b o l i s m o f peptides b y r u m e n m i c r o o r g a n i s m s . H e
o b s e r v e d t h a t p e p t i d e s were m o r e efficiently c o n v e r t e d into bacterial p r o t e i n t h a n
a m i n o acids. T h e m a n y different p a t h w a y s o f a m i n o a c i d d e g r a d a t i o n will n o t b e
discussed in detail. T h e m o r e general processes o f a m i n o acid d e g r a d a t i o n a r e :
(a) Transamination
Transamination reactions produce ct-keto acids that may be converted to amino acids, for
instance:
(i) L-glutamic acid+oxaloacetic acid,--~-ketoglutaric acid+L-aspartic acid. This reaction is
catalyzed by the enzyme glutamateoxaloacetate transminase (GOT).
(ii) L-glutamic acid+pyruvic acid,--,a-ketoglutaric acid+L-alanine. This reaction is catalyzed by
the enzyme glutamate~pyruvate transaminase (GPT).
By means of the above two reactions the different amino acids are linked to pyruvate, the converging
point of carbohydrate and amino acid degradation, and to oxaloacetate and a-ketoglutaric acid, the
intermediates of the citric acid cycle. The significance of both glutamate-oxaloacetate transaminase
and glutamate-pyruvate transarninase in anaerobic digestion has been indicated by HATT~GH et al.
(1967a), Koxz~ (1967), KOTZ~ et al. (1968) and TmEL et al. (1968).
(b) Transamination followed by deamination

Transamination may also be accompanied by deamination viz.
(i) a-ketoglutaric acid + amino acid~--~glutamie acid+ a-keto acid.
(ii) glutamic acid+ NAD + HzO,---'a-ketoglutaricacid + NADH + H + + NH3
Sum: Amino acid + NAD + H20~--~ct-ketoacid + NADH + H + + NH3
The enzyme glutamate dehydrogenase catalyzes the second reaction in the above sequence.
According to the principle described by GOEBELand KL~E~rnERG (1964) for the different isocitric
dehydrogenases, the NADP-dependent isocitric dehydrogenase was reversible and involved in
anabolism, whereas the NAD-dependent enzyme was irreversible and implicated in catabolism.
The NAD-dependent glutamate dehydrogenase was also operative in deamination.
A NADP-dependent glutamate dehydrogenase has been found in anaerobic digestion (KOTz~
1968b). This enzyme may be responsible for NH3 fixation. The role of the NAD-dependent gluta-
mate dehydrogenase in anaerobic digestion has been described (TmEL et al., 1968). The reaction
product of glutamate dehydrogenase, a-ketogiutaric acid, thus links the fixation of NH3 to the
citric acid cycle.
(c) Oxidative deamination

Oxidative deamination of amino acids usually occurs under aerobic conditions. The general
reaction may be indicated as follows:
(i) R-CHNH2 - COOH + FAD,----~R-CO-COOH+ NH3
(ii) FADH2 + O2~---~FAD+ H202
It is doubtful whether this process plays a significant role in anaerobic digestion due to the
absence of 02.
(d) Stickland reaction

A number of clostridia are also capable of degrading amino acids by coupled oxidation-reduction
reactions of appropriate pairs of amino acids. This type of reaction is known as the Stickland
reaction. The principle was excellently illustrated by STAOTMANet al. (1958) for the oxidation-
reduction of alanine and glycine; e.g. alanine+ 2 giycine+ 2H2-----'3NH3+ 3CH3COOH+ CO2.
The possible end-products of amino acid fermentation by various species of bacteria, based on
478 J.P. KOTZ~, P. G. THEL and W. H. J. HATTINGH
data presented by BARKER(1961), are summarized in TABLE1. The end-products for a specific amino
acid are not always the metabolic end-products of the specific microorganisms.
The end-products o f amino acids are very similar to those o f the degraded carbo-
hydrates. Both carbohydrate and amino acid metabolism thus produce a c o m m o n pool
o f end-products which are degraded further by c o m m o n pathways.
The microorganisms fermenting amino acids belong mainly to two groups, the
anaerobic spore-formers (clostridia) and the anaerobic cocci (Micrococcus and
Diplococcus). Only one non-sporulating, obligately anaerobic, rod-shaped bacterium
is k n o w n to ferment amino acids viz. Fusobacterium nucleatum (BARKER, 1961). Other
TABLE l . THE FERMENTATION END-PRODUCTS OF AMINO ACID FERMENTATION BY VARIOUS MICROBIAL

SPECIES
Amino acid Fermentation products
Alanine Ammonia, carbon dioxide, acetic acid, propionic acids

3-amino valerate Ammonia, acetic, propionic and valeric acids
Arginine Ammonia, acetic, propionic and valeric acids, ornithine and citrulline
Citrulline + ADP + Ammonia, carbon dioxide, acetic, propionic and valeric acids, ATP and
inorganic phosphate ornithine
Ornithine Ammonia, acetic, propionic and valeric acids
Asparagine Ammonia, carbon dioxide, acetic and succinic acids
Aspartic acid Ammonia, carbon dioxide, acetic, butyric, lactic, fumaric, and malic
acids, ethanol, butanol
Cystine and cysteine Ammonia, carbon dioxide, hydrogen sulphide, formic, acetic, propionic,
butyric, valeric and pyruvic acids
Homocysteine Ammonia, hydrogen sulphide and a-ketobutyric acid
Methionine Ammonia, methylmercaptan and a-ketobutyric acid
Glutamic acid Ammonia, carbon dioxide, hydrogen, acetic, butyric, and lactic acids (inter-
mediates mesaconate and citramalate)
Glycine Ammonia, carbon dioxide, hydrogen, acetic and pyruvic acids
Histidine Ammonia, carbon dioxide, hydrogen, formic, acetic, butyric, lactic and
glutamic acids and formamide
Leucine Can undergo the Stickland reaction to the branched chain fatty acid p-methyl-
butyric acid (N.B. leucine not easily fermented)
Isoleucine Can undergo the Stickland reaction to the branched chain fatty acid a-methyl-
butyric acid. (N.B. isoleucine not easily fermented)
Valine Can undergo the Stickland reaction to the branched chain fatty acid a-methyl-
propionic acid
Lysine Ammonia, carbon dioxide, acetic, propionic and butyric acids
Proline Can undergo the Stickland reaction to 3-aminovalerate and can be further
fermented (See tLaminovalerate)
Hydroxy-proline The degradation products are unknown
Serine Ammonia, carbon dioxide, hydrogen, formic, acetic, propionic, butyric,
valeric and lactic acids, alanine and ethanol
Threonine Ammonia, carbon dioxide, hydrogen, formic, acetic, propionic, butyric,
valeric and a-ketobutyric acids, acetaldehyde and glycine
Tryptophane Ammonia, carbon dioxide, pyruvic acid, indole propionic acid, indole pyruvic
acid and indole
Tyrosine and Ammonia, carbon dioxide, hydrogen, phenol, p-cresol, p-hydroxy phenylacetic,
phenylalanine p-hydroxy phenylacrylic, and p-hydroxy-phenyllactic acids
species of the Bacteriaceae will probably also ferment amino acids (BARKER, 1961).
Most of the bacteria that ferment nitrogenous compounds are obligately anaerobic.
One facultative species E. coli probably ferments certain amino acids.
It is evident that the end-products of amino acid fermentation yield a variety of
acids, hydroxy acids and even-numbered carbon chain alcohols and ammonia (TABLE
1). The fermentation of these carbon fractions to CH4 and CO2 follows a common
pathway, for instance, the degradation of pyruvic acid (produced by various meta-
bolic routes) will be similar in all instances. The removal of the carbon fractions as
CH4 and CO2 leaves NH a as the sole end-product which may increase the pH of the
digester.
Degradation of heterocyclic nitrogen-containing compounds. The heterocyclic nitrogen
compounds such as purines, pyrimidines, allantoin, nicotinic acid and uric acid are
the degradation products of DNA, RNA and certain coenzymes. Purines are known
to be fermented by a number of clostridia, such as CL acidiurici, Cl. cylindrosporum,
CI. uracilicum, and Micrococci such as M. aerogenes, M. lactilyticus, M. allantoicus and
Zymobacterium oroticum (BARKER,1961). The nucleosides are firstly hydrolyzed to the
free bases. Degradation products of the purine bases by CL acidiurici are ammonia,
carbon dioxide and acetate. Other degradation products of purine bases by anaerobic
microorganisms are hydrogen, formic, propionic, lactic acids, and glycine, urea and
uric acid. RABINOW1TZand BARKER(1956) reported on the further degradation of
uric acid.
The role of tetrahydrofolic acid in the C1 metabolism of purine degradation was
indicated by SAGERSand GUNSALUS(1958) and RABINOWITZand PRICER(1958). The
end-products of pyrimidine degradation by anaerobic cocci and clostridia are am-
monia, carbon dioxide, acetic, pyruvic, fumaric and lactic acids, hydrogen and uracil
(BARKER, 1961). Allantoin is fermented by Streptococcus allantoicus to ammonia,
urea, oxamic acid, carbon dioxide and formic, acetic, glycolic and lactic acids.
Nicotinic acid is fermented to ammonia, carbon dioxide and formic and propionic
acids (HARARY,1957).
Significance of enzyme activities and enzyme activity ratios. Methods to determine
the hydrolytic enzymes, protease, phosphatase, cellobiase and amylase and the inter-
mediary enzymes were presented by THIELand HATTINGH(1967), and KOTZ~ (1967).
HATT1NGHet al. (1967a) reported that the hydrolytic enzymes would not easily become
rate-limiting in anaerobic digestion. They also presented evidence that the activities of
the intermediary enzymes increased as a digester was slowly adapted to a synthetic
substrate. The increase in the enzyme activities was accompanied by an increase in the
protein, DNA and volatile suspended solids content of the digester and purification
efficiency increased since the new substrate was more amenable to anaerobic degrada-
tion.
The relative activities of the different enzymes in anaerobic digesters are typical for
the substrate degraded. For instance, digesters treating a wine waste showed a high
lactate dehydrogenase/6-phosphofructokinase ratio (Koxz~ et al., 1968, and Koxz~,
1968a) whereas glucose starch waste and raw sludge digesters showed high glutamate
oxaloacetate transaminase/lactate dehydrogenase ratios. Yeast waste and raw sewage
sludge digesters showed high hexokinase/6-phosphofructokinase ratios (Koxz~ et al.,
1968).
KOTZ~ et al. 0968) reported that when a digester, adapted to a synthetic substrate
480 J.P. KOTZ~,P. G. THIELand W. H. J. I-IATT1NOH
over a period of 60 days and operated on the synthetic substrate for a further 90 weeks
was analysed on successive days, the results supported the proposal that a digester in
steady state should show relatively constant enzyme activities. When conditions of
physiological stress occurred in a steady state digester, fluctuations of 100-200 per
cent in enzyme activities have been observed by Trn~L et aL (1968). It was also ob-
served that under conditions of physiological stress (lowering of nitrogen content of
the feed), the ratios of the different enzymes one to another varied considerably.
Recent work on continuous cultures of Ruminococcus albus and a Butyrivibrio
revealed that the normally believed steady state conditions were not realized when the
enzyme activities were taken into account (KoTz~. and KISTNER, 1968). For instance, it
was found when enzyme activity determinations were done on a Butyrivibrio sp.
(strain CF 51), grown in a continuous culture process and analysed after at least 15
volumetric replacements, that the ratio of enzyme/bacterial dry weight decreased as
the dilution rate increased from 0-12 hr -I to 0.5 hr -1. This finding indicated that
although the glucose determinations in the continuous culture medium, the optical
density and the dry bacterial cell weight suggested that steady state conditions pre-
vailed, the intermediate enzyme activities still varied considerably. Therefore, when-
ever steady state is referred to the parameters whereby steady state is determined must
be clearly stated.
KOTZ~ et aL (1968) proposed that the microbial population of anaerobic digesters
in steady state should show relatively constant levels of enzyme activities and that
any departure from these levels would be an indication of the onset of unbalanced
conditions.
Enzyme patterns have also been used in the classification and taxonomy of bacteria
such as Mycobacteria (CANN and WILLOX, 1965), Streptococci (LUND, 1965) Bacillus
thuringiensis (NORRIS, 1964) and Leptospira (GREENet al., 1967) and, to detect growth
of Staphylococcus aereus in foods (CH~sBRoand AUBORN, 1967).
Production of carbon dioxide and hydrogen

The reactions and pathways of degradation of different substrates commonly
present in anaerobic digestion were presented here to illustrate their fate in the non-
methanogenic phase of anaerobic digestion. It is evident from the foregoing that
carbon dioxide and hydrogen are often among the end-products of fermentation.
Bacteria that produce hydrogen are known to be present in anaerobic digesters (viz.
clostridia). It is therefore most likely that hydrogen is produced in digesters but owing
to the fact that it is probably actively reabsorbed by other microorganisms, the net
effect is that no significant accumulation of hydrogen appears. The presence of small
quantities of H2 in digesters has been demonstrated by BURGESSand WOOD (1964).
Claims have recently been made that one bacterial species can degrade glucose to
methane (PRETOR1US,1968). The relative contribution of this mechanism in anaerobic
digestion has not yet been established.
Metabolic pathways directly involved in methane production

The microbiology of anaerobic digestion and of the bacteria participating in the
production of methane was discussed by TOERIENand HAa'TINGH(I 969). Their review
referred mainly to work carried out using intact cells of pure cultures of methane-
producing bacteria. The work reported in this article deals with information presently
available on the pathways of the biochemical methane formation by cell-free extracts
of methane-producing bacteria.
Cell-free methane formation. Cell-free studies were conducted on Methanosarcina
barkeri and Metanobacillus omelianskii and it was concluded that cell-free extracts
produced methane from carbon dioxide, formate, formaldehyde, methanol and serine
but not from acetate (STADTMAN, 1967).
Role of co-factors in cell-free methane formation. A number of co-factors have been
found to be necessary for the formation of methane by cell-free extracts. STADTMAN
(1967) reported that the reduction of carbon dioxide to methane could be augmented
by a ferredoxin-like substance. Ferredoxin has been isolated and characterized from
Methanobacterium omelianskii by BUCHANANand RABINOW1TZ(1964). When carbon
dioxide was absent, reduced ferredoxin, which was associated with substrate oxidation,
was free to react with the hydrogenase system present and molecular hydrogen
appeared as the reduced fermentation product (STADTMAN, 1967). The reduction of
carbon dioxide to methane is thermodynamically more favourable than the produc-
tion of free hydrogen, therefore, when both carbon dioxide and hydrogen are present,
hydrogen is consumed and methane formed (SCHNELLEN,1947).
BARKER (1967) presented evidence that some form of tetrahydrofolate and a cor-
rinoid substance seemed to participate in the conversion of carbon dioxide to methane.
RABINOWlTZ and PRICER (1963) detected tetrahydrofolate formylase activity in
Methanosarcina barkeri. It is not yet known if the subsequent reactions for the
formation of N-5-methyltetrahydrofolate are present. Methanobacterium omelianskii
did not reduce labelled formate in a hydrogen atmosphere, but labelled carbon
dioxide was evolved (Wood et al., 1965). KLUYVERand SCHNELLEN(1947) reported
the conversion of carbon monoxide and water to carbon dioxide and methane by
Methanobacteriumformicicum, but on the other hand no utilization of carbon mon-
oxide by M. omelianskii was reported.
On analogy with similar biochemical reactions it seems probable that the formate is
first "activated" by ATP-dependent reactions to N-10-formyltetrahydrofolate,formyl-
coenzyme A or formylphosphate, followed by reduction to the aldehyde. No experi-
mental evidence for the formation of formylphosphate by extracts of M. barkeri can
be found. Crude extracts of M. barkeri showed an active acetate kinase (STADTMAN,
1967; RABINOWITZand PRICER,1963).
The enzyme system of M. barkeri produced methane from 5-methyltetrahydrofolate
but less efficiently from free formaldehyde or methylcobalamin (BLAYLOCKand
STADTMAN, 1966).
WOOD et al. (1965) and WOOD and WOLFE (1965) reported on the role of folate
derivatives in the production of methane from formaldehyde, serine and methanol in
extracts of M. omelianskii. In view of the fact that M. omelianskii has been reported to
be a symbiotic pair of bacteria, it is difficult to ascribe the reactions found to a
specific bacterium and thus to formulate its exact role in methane formation
(BRYANTe t al., 1967, 1968; LANGENBERGet al., 1968).
Coenzyme A is essential for the formation of methane by cell-free extracts of M.
barkeri when pyruvate is used as source of reducing equivalent (BLAYLOCKand
STADTMAN,1966; STADTMANand BLAYLOCK,1966). Transacetylase and acetate kinase
have also been found to be active in M. barkeri (BLAYLOCKand STADTMAN,1966).
482 J.P. KOTZI~,P. G. TnI~L and W. H. J. I-[ATTINGH
In M. omelianskii, coenzyme A is only essential for methane formation when carbon
dioxide and hydrogen are the substrates (WOLINet al., 1963).
The exact role of ATP, which is essential for methane synthesis by both M. omelian-
skii and M. barkeri enzyme systems (WOLINe t aL, 1963; BLAYLOCKand STADTMAN,
1966) and for methylcobalamin synthesis from methanol by M. barkeri extracts, is still
unknown (BLAYLOCKand STADTMAN, 1964). In crude extracts a number of other
nucleotide triphosphates and often ADP can replace ATP (BLAYLOCKand STADTMAN,
1966; WOOD and WOLFE, 1966b). BLAYLOCK(1967) found that purified extracts
of M. barkeri were more specific for ATP.
A heat and acid stable, anionic substance, which is transparent in visible light, and
essential for the reduction of methanol to methane or the conversion of methanol to
methyl-cobalamin has been found in M. barkeri and Clostridium sticklandii (STADT-
MAN and BLAYLOCK,1966; BLAYLOCK,1967).
Extracts of M. barkeri actively converted the methyl-moiety of methyl-Blz to
methane when hydrogen and/or pyruvate were added as electron donor (BLAYLOCK
and STADTMAN, 1963, 1964, 1966). Extracts of M. omelianskii also produced methane
from methylcobalamin (WOLINet al., 1963). Thus the ability to produce methane from
methylcobalamin is not restricted to a single species of methane-producing bacteria.
Fractionated extracts of M. omelianskii and M. barkeri revealed that a minimum of
three proteins, namely a red corrinoid protein, a ferredoxin and an unidentified protein
are necessary to achieve methyl-cobalaminformation from methanol (BLAYLOCK,1967).
Further efforts to purify the remainder of the enzyme system necessary for methane
production by extracts of M. omelianskii or M. barkeri have been unsuccessful
(STADTMAN, 1967). Experiments by BLAYLOCK (1967) indicated that the factors
essential for methyl-cobalamin formation from methanol stimulated the production of
methane in extracts with low activities.
STADTMAN and BLAYLOCK(1966) found that crude and pure cultures of methane-
producing bacteria were exceptionally rich in corrinoid compounds. M. barkeri
cultures were found to contain such large amounts of corrinoid compounds that the
cells were pinkish in colour (BLAYLOCKand STADTMAN, 1964). LEZIUSand BARKER
(1965) systematically studied the corrinoids present in pure cultures of methane
organisms and found that vitamin forms of 5-hydroxybenzimidazolylcobamide were
the principal form of the corrinoid group present in these cultures. The methyl
derivative of the cobalamin was present in low quantities in the bacteria. Workers
succeeded only very recently in isolating small quantities of the methyl-cobalamin from
bacteria (IRION and LJUNGDAHL,1965; LJUNGDAHLet aL, 1965; LINDSTRAND, 1965).
Clostridium thermoaceticum has been shown to synthesize acetate from carbon dioxide
through a cobalt-carboxymethyl corrinoid derivative (LJUNGDAHLe t al., 1965, 1966;
LJUNGDHALand IRION, 1966). In this derivative, the acetate is said to be bound at the
methyl side of the molecule.
STADTMAN(1967) speculated on the possibility that in a reverse reaction, the forma-
tion of methane from such a bound carboxymethyl group could also be possible. Such
a mechanism would explain the observations that the carboxyl moiety of acetate is
converted to carbon dioxide and the methyl moiety to methane.
Role of inhibitors. The folate antagonist, aminopterin (4-amino-pteroylglutamate)
inhibits methane formation in extracts of M. barkeri (BLAYLOCKand STADTMAN,1966).
The concentration used to effect 100 per cent inhibition is (5 x 10-SM) which is much
higher than concentrations normally used to inhibit folate-dependent systems (10-aM).
Aminopterin is a very powerful inhibitor of dihydrofolate reductase (MATHEWSet al.,
1963).
Tetrahydroaminopterin inhibits methionine synthesis much less efficiently than
aminopterin (GUEST and WooDs, 1965). No explanation can be offered for the in-
hibitory effect of aminopterin on methane formation by M. barkeri.
In M. barkeri the glycoprotein, intrinsic factor (HIGHLEYet al., 1966) is an effective
inhibitor of methane formation from methanol, methylcobalamin, N-5-methyl-
tetrahydrofolate, formaldehyde, formate, carbon dioxide and pyruvate (BLAYLOCK
and STADTMAN,1966; STADTMANand BLAYLOCK,1966). The component in M. barkeri
extracts which is affected by the glycoprotein must be irreversibly bound since when
the substrate methylcobalamin is added in quantities 300-800 times the Vitamin B12
binding capacity of the intrinsic factor, methane formation is still inhibited.
Alkylhalides have been reported to inhibit the production of methane and methyl-
transfer in methionine synthesis (WOOD and WOLFE, 1966a; BROT and WEJSSaACrt,
1965 ; BAUCHOP, 1967 ; THIEL, 1969). However, WOOD and WOLFE (1966a) reported
that the inactivated enzyme system of M. omelianskii could be reactivated by photo-
lytic cleavage (photolysis) of the alkyl-enzyme bond.
Proposed scheme for methane formation. From the foregoing data the scheme pro-
posed by BARKER(1967) for the reaction sequence for the formation of methane from
carbon dioxide, methanol, serine or acetate appears to be justified. This scheme is
presented below:
formo~e
C02+ 2H dehydogenese H ~Ozl"l (bound
(H-donor) ITHF
~ATP
H CO-THF
THF
Serine -- - - CHfTHF- glycine
I ,%,
CH3 -THF
CH30H'~"~
ATP ,-~,.~ l Co BI2 - enzyme
CH3-Co--B=2 J CH3-CoBi2-enzyme
-COz [
CH3CO2H/ 2H
ATP
Ell4 +Co BI2 + enzyme
Importance of biochemical studies for anaerobic digestion. It is evident that our

knowledge of the biochemical pathways through which the complex substrates sub-
484 J.P. KOTZLP. G. THIELand W. H. J. HATTINGH
jetted to anaerobic digestion are eventually degraded to carbon dioxide and methane
is still meagre. This is particularly true of the mechanism by which methane is produced.
Evidence has been presented that enzyme analysis, i.e. quantity and activity of
enzymes participating in the degradation of the substrate may be used to characterize
the biological nature of a digester. The results from a careful study of the enzyme
spectrum may yield information about the onset of unstable conditions in a digester.
Investigations on the significance of the different metabolic pathways under specific
conditions, the effects of stress conditions, shock loads and the mean energy yield
during the process can give valuable information on the anaerobic process which will
result in a more efficient control of the system of anaerobic digestion.
CONTROL OF ANAEROBIC DIGESTION

Anaerobic digestion of waste organic matter, in particular industrial effluents
from fermentation processes, is widely used today. Studies on this process and its
application over a number of years have demonstrated that certain factors greatly
influence digestion. No single factor (parameter) can be used as a control measure of
the process of anaerobic digestion as the degradation of organic matter to methane
and carbon dioxide is brought about by a heterogeneous microbial population. It has
been indicated that various factors affect digestion and that a number of "general"
parameters such as pH, alkalinity, volatile fatty acid content, solids and volatile
solids content, COD of feed and effluent and rate and composition of gases produced
are normally used to control anaerobic digestion. HATTINGHet al. (1967a) indicated
that the above named "general" parameters were not sufficient to describe anaerobic
digestion and thus biological parameters were developed by them to assess the state of
biological activity. More study is needed to obtain an understanding of the process of
anaerobic digestion. To control anaerobic digesters, when these digesters are operated
on the continuous flow principile to treat industrial wastes, the following parameters
are suggested, all of which must be taken into account to ensure efficient digestion.
Temperature
Anaerobic digestion in the mesophilic temperature range (20-45°C)with an optimum
at about 37°C is advised. Thermophilic temperatures often bring about more efficient
digestion but control of temperature seems to be the greater problem.
pH, alkalinity and volatile fatty acid content

The parameters pH, alkalinity and volatile fatty acid content are related. A mere
knowledge of pH is of no value. It must be considered together with the alkalinity
figure (buffering capacity). For efficient digestion the pH should be in the range 6.0--8.0
with an optimum at pH 7.0. To ensure that sufficient buffering capacity is available
to counteract sudden increases in fatty acid content, the alkalinity titrated to pH 6.0
should be greater than 1000 mg HCO3-/I.
The pH value should be immediately adjusted, should it fall below 6.0. Much con-
troversy exists in literature on the advisability of liming to increase the pH. Uncon-
trolled liming practices may lead to pH values of 10.0 or above resulting in serious
shock to the digester. Carefully controlled additions of lime should do no harm,
if the lime is well distributed throughout the digester. SAWYERet aL (1954) reported
especially that the addition of ammonium bicarbonate was somewhat superior to that
of lime as an alkaline agent. The pH may also be controlled by the addition of well
digested sludge from an active digester.
The volatile fatty acid (VFA) content of the mixed liquor of a digester is one of the
most useful parameters in controlling digestion. Any sudden change from a constant
value is an indication of a disruption of equilibrium conditions. The "safe level" of
VFA content is a debating point amongst workers in the field of anaerobic digestion.
BUSWELL(1959) after many years of research, stated that the overall upper limit of
2000 mg VFA/1 was over emphasized. He preferred the use of sudden changes in a
constant value of the VFA content, as a control parameter, rather than setting levels
for "safe" digestion.
A volatile fatty acid content of less than 200 mg/l is generally preferred. Any sudden
change from a constant value should immediately be followed by remedial measures.
To date the most effective method used to restore more stable conditions has been a
reduction in substrate flow. If a source of digesting sewage sludge is available, a
volume suitable to restore balanced conditions should be transferred to the unbalanced
digester. Care must be exercized that the inoculum sludge is efficiently mixed with the
contents of the digester.
Rate of COD conversion to gas and composition of gas

Any decline in rate of gas production from a constant value accompanied by a
change in the methane/carbon dioxide ratio is indicative of unbalanced conditions. A
change in this ratio is partially caused by a release of carbon dioxide from the liquor
due to a rise in VFA concentration. The change in methane]carbon dioxide ratio seems
to take place approximately 24 hr before a rise in VFA concentration is noted. The
methane/carbon dioxide ratio is a sensitive parameter for controlling anaerobic
digestion and any sudden change in the ratio must be immediately investigated. With
the introduction of gas-chromatographic apparatus and techniques the determination
of the composition of the gas is a simple matter. VAN HtrVSSTEEN(1967) reported that
both methane and carbon dioxide can be determined on a single column packed with
a porous polymer. The determination of the gas composition is accomplished in about
3 min.
The methane/carbon dioxide ratio of the gas is also characteristic of the substrate
supplied, for instance, POMEROY(1933, 1935) presented evidence that the methane]
carbon dioxide ratios for the gas produced from carbohydrates, fats and proteins
would be 1.0, 2.3 and 5.3 respectively. A value of 1.9 for domestic sewage sludge
therefore is an average value for a substrate containing different ratios of carbo-
hydrates, fats and proteinaceous material.
Efficiency of the process

The efficiency of digestion is determined by the knowledge of the amount of carbon
(COD) entering and leaving the system. The efficiency of converting applied carbon
to gas may also be calculated from the volume of gas produced per day. Corrections
must be made in calculating the conversion of carbon to gas as some of the produced
carbon dioxide will dissolve in the mixed liquor in the digester and leave the digester
in the effluent. This is particularly evident when acidic industrial effluents (from
fermentation industries) containing no dissolved carbon dioxide are digested. There-
486 J.P. KOTZI~,P. G. THIELand W. H. J. HATTINGH
fore, the amount of carbon dioxide dissolved in the effluent from the digester must
be determined and added to the volume of gas produced.
The difference in efficiencies of total carbon (COD) removal and the amount o f
carbon (COD) appearing as gas may be taken as a measure of the amount of carbon
transformed to microbial cells. KOTZ~ et al. (1968) reported that about 20-30 per cent
of the applied COD was utilized for cell synthesis during the digestion of a synthetic
substrate, while 70-80 per cent appeared as gas. The efficiency of converting substrate
to gas is also a measure of the degradability of the substrate. If the COD content of the
substrate is known, any deviation from the expected gas production is a sign of un-
balanced conditions in the digester.
Volatile suspended solids content

The volatile content of the suspended solids (VSS) in a digester may be used as a
measure of biomass since it is significantly related to the D N A contained only in
living cells (AGARDYet al., 1963; HATTINGHet aL, 1967a). The volatile content of the
suspended solids in a raw sewage sludge digester is normally about 65 per cent while
in digesters operated on a continuous flow principle and receiving almost completely
soluble industrial wastes the figure may increase to about 80 per cent. The suspended
solids in digesters operated in the laboratory on a completely soluble synthetic sub-
strate contain about 95 per cent volatile matter (HAaO'I~GH et al., 1967a) which
approaches that contained in activated sludge. The determination of the VSS content
of anaerobic sludge is a relatively simple and rapid analysis and, since it is related to
microbial mass, it affects a rapid estimation of microbial mass for use in calculating
the loading parameter. By increasing the volatile suspended solids content of a
digester, the amount of substrate supplied may be increased with a resulting increase
in overall digestion capacity.
Biological parameters
In addition to these general control parameters discussed earlier, biochemical
determinations such as enzyme activity measurements and bacterial counts proved to
be useful in the control of anaerobic digesters (HATTINGrI et aL, 1967a). This approach
to anaerobic digestion has only recently been introduced. A further study of the
mechanism of the process, as a whole, will lead to a better understanding of these
parameters and their use as control parameters.
CONCLUSIONS
An attempt has been made in this article to summarize and describe those factors
which determine the eventual character of any digester. Of those factors described, the
composition of the substrate and the rate at which this substrate is supplied to the
digester have a more pronounced effect on the eventual nature of the system than
factors such as temperature, the original inoculum and the mode of operation of the
digester.
Reference has been made to the more generally accepted ways of characterizing a
digester such as pH, alkalinity, volatile fatty acid concentration, rate of gas produc-
tion, composition of gas, measures of efficiency, measures of biomass and nutritional
factors. These general parameters only superficially describe the system and do not
t a k e into a c c o u n t the intrinsic biological n a t u r e o f the b a c t e r i a responsible for the
process. I n a n a t t e m p t to acquire a better a p p r e c i a t i o n o f the biological n a t u r e o f
a n a e r o b i c digestion the existing k n o w l e d g e o f the b i o c h e m i c a l characteristics o f the
system has been s u m m a r i z e d . F r o m this s u m m a r y it is evident t h a t there is a p a u c i t y o f
k n o w l e d g e o f the b i o c h e m i s t r y o f the process a n d t h a t a m u c h better u n d e r s t a n d i n g
o f the b i o c h e m i s t r y o f the process is i m p e r a t i v e for the efficient c h a r a c t e r i z a t i o n a n d
c o n t r o l o f a n essentially biological process such as a n a e r o b i c digestion.
T h e existing p a r a m e t e r s for the c o n t r o l o f a n a e r o b i c digestion have been described,
b u t will in future need to be s u p p l e m e n t e d by well-chosen biological parameters. T h e
p r e s e n t k n o w l e d g e o n a n a e r o b i c digestion is largely based, o n empirical o b s e r v a t i o n s
which give n o e x p l a n a t i o n for the reasons b e h i n d certain p h e n o m e n a , a n d it is there-
fore evident t h a t further f u n d a m e n t a l research on the a n a e r o b i c process is o f prime
i m p o r t a n c e if this process is to be utilized to its full capacity.
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Anaerobic Digestion

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Anaerobic Digestion

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Water Research Pergamon Press 1969. Vol. 3, pp. 459-494 Printed in Great Britain.

J. P. KOTZ~., P. G. THIEL and W. H. J. HATTINGH

(Received 4 February 1969)

ANAEROmC digestion is a biological process in which organic matter is converted to

Mode of operation of a digester

CHARACTERIZATION OF ANAEROBIC DIGESTION

Metabolic pathways o f the non-methanogenic phase

T h e m e t a b o l i c p a t h w a y s such as glycolysis, the tricarboxylic acid cycle, and the

Citric acid cycle

Pentose phosphate shunt

(b) Transamination followed by deamination

(c) Oxidative deamination

(d) Stickland reaction

TABLE l . THE FERMENTATION END-PRODUCTS OF AMINO ACID FERMENTATION BY VARIOUS MICROBIAL

Amino acid Fermentation products

Alanine Ammonia, carbon dioxide, acetic acid, propionic acids

Production of carbon dioxide and hydrogen

Metabolic pathways directly involved in methane production

Importance of biochemical studies for anaerobic digestion. It is evident that our

CONTROL OF ANAEROBIC DIGESTION

pH, alkalinity and volatile fatty acid content

Rate of COD conversion to gas and composition of gas

Efficiency of the process

Volatile suspended solids content

ADDANKIS., SOTOSJ. F. and REARICKP. D. (1966). Rapid determination of picomole quantities of

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