Anaerobic digestion II. The characterization and control of anaerobic digestion
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Water Research Pergamon Press 1969. Vol. 3, pp. 459-494 Printed in Great Britain.
ANAEROBIC DIGESTION II. THE CHARACTERIZATION AND CONTROL OF
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)
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 of Practice on anaerobic digestion (1968).
460 J.P. KOTZ~, P. G. TmEL and 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 (FALCONE and 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. Characterization and 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 (STANIER et al., 1964).
Bacterial cell wails contain rnucopeptides, teichoic acids, polysaccharides, proteins and lipids (STANIER et 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 (STANIER et 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 GAUDY et 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 (IMHOFF and FAIR, 1956). The volatile matter of the undigested solids may vary from 60 to 80 per cent (RUDOLFS and 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; RUDOLFS and 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. HEUKELEKIAN and 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-
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 463
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. BUSWELL and NEAVE (1930) determined that sewage sludge contained 10.8 per cent crude fibre of which the major components must have been polysacchar- ides. RUDOLFS and 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 RUDOLFS and 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; BUSWELL and NEAVE, 1930). HEUKELEKIAN and 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 RUDOLFS and CHAMBERLIN (1932) and by RUDOLES and INGOLS (1938) and BUSWELL and 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. CORDON et 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. THIEL and 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 (THIEL and Do TOlT, 1965). Yeast waste on the other hand, contains relatively high concentrations of nitrogenous material (KOTZ1~ et 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-ICO 3' 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. SANDERS and 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
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 465
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 (AGARDY et al., 1963; HATTINGH et 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 35C.
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. THIEL and 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 0C to about 100C. 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 50C and 90C. Exposure of microorganisms to temperatures above these ranges result in rapid death, except in the special case of heat-resistant spores (STANIER et al., 1964). Temperatures below 0C are not necessarily lethal to bacteria and many bacteria survive temperatures as low as that of liquid air (STANIER et al., 1964).
Although the total temperature range for the development of microorganisms extends from -5C to about 80C, 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 10-46C but the optimum temperature for its growth lies between 37C and 45C (STANIER et al., 1964).
The temperature ranges for the optimal growth of microorganisms can conveniently be divided into three temperature regions namely the psychrophilic ( 45C) (STANIER et 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; KLUYVER and BAARS, 1932). Abrupt changes in temperature are often lethal to bacteria. Escherichia coli, grown at 45C can be killed for example by rapid cooling to 10C (95 per cent) while gradual cooling over 30 min causes no death (SHERMAN and 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 10C, 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 0C to 55C. 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
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 467
and HEUKELEKIAN (1931) reported that the rate of digestion of sewage sludge could be increased by changing from mesophilic to thermophilic digestion (50C). For the digestion of board mill sludge, mesophilic digestion (36C) was, however, more effective than thermophilic digestion (50C). 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 32C could be upset when the temperature increased to 40C, 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 2.7C or more, during a relatively short period, greatly affected digestion at 46-53C. 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 (49C) was quite stable and resistant to upset and also that temperature decreases of 5C in 48 hr produced no unusual changes but that prolonged lower temperatures, changed the system to mesophilic conditions.
HEUKELEKIAN and KAPLOVSKY (1948) reported that in a 20C 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 35C to 60C had no effect on the rate of fermentation. STANDER et al. (1967) found that optimum digestion of wine waste took place at 35C.
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. HATTINGH et 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. THIEL and 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~R et 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~R et al., 1964). Undissociated molecules can penetrate more readily into bacterial cells than the ionic forms of the organic acids (STANIER et 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 (STANIEg et 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~EF~ 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. THIEL and 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 + H20 + NH 3 + CH4 (1)
CO2 +H20 +NH3~NH4 + +HCOa-. (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; RUDOLFS and 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 as HCO 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
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 471
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; SCHULZE and RA~O, 1958; HINDIN and DUNSTAN, 1959, 1960; MOELLER et 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 HATTINGH et 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), HEUKELEKIAN and KAPLOVSKY (1949), KAPLOVSKY (1951, 1952), HINDIN and 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, MCCARTY et 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. BUSWELL et al.
472 J.P. KOTZ~, P. G. THIEL and 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 of 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
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 473
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 (SIEBERT et al., 1968).
Biomass content can also be assessed by more indirect methods (analytical tech- niques) such as DNA 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 (HATTINGH et 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 DNA content of sludge (AGARDY et 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 DNA content in anaerobic sludge (AGARDY et al., 1963; HATTINGH et al., 1967a).
ATP is closely associated with biological activity and also with the energy status of living matter and like DNA 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; HOLMSEN et 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 DNA 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. THIEL and 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 HATTINGH et al. (1967a), KOTZ~ et al. (1968) and THmL et al. (1968). HATTINGH et 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 of 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 of 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
The metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and the Entner -Doudoro f f pathway and pentose phosphate shunt for the degradation of
carbohydrates have been found to be operative in anaerobic digesters (HATTINGH et al., 1967a; Koxzl~ et al., 1968 and 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 (HATTINGH et al., 1967a; KOTZ~, 1967; KoIz~ et al., 1968; and THIEL et al., 1968).
A new enzyme 1-PFK has recently been found in Aerobacter aerogenes by HANSON and AN- DERSON (1966), in Bacteroides symbiosus by REEVES et 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). JONES and 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), HATTINGH et al. (1967a), KOTZI~ et al. (1968) and THIEL et 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; HATTINGH et al., 1967a; KOTZI~ et al., 1968 and THIEL et 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 (SLATER and 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, WARRINGA and GUIDITTA (1958) and WARRINGA et 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. THIEL and 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 dehydrogenase from Ascaris lumbricoides (SEn)MAN and ENTNER, 1961).
ENEBO and PEHRSON (1960) reported on the degradation of all the Krebs cycle intermediates by anaerobic digestion and concluded that the Krebs cycle must 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 glyoxylic acid cycle as indicated by the enzyme isocitratc lyasc, has been postulated by HATTINGH et 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-phosphogluconate aldolase (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; GIBBS and DEMoss 1951, 1954; D~Moss et al., 1951; SOKATerI and 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~H et aL, 1967a; Korz~ et al., 1968; Trm~L et 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-phosphate dehydro- genase activities observed by HAa~nN~n et aL (1967a) was an indication that the pentose phos- phate pathway must have been operative in the anaerobic bacteria present in the digester.
Degradation ofl ipids and fa t ty 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-rINGH et 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
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 477
amino acids all have characteristic pathways by which they are degraded. Amino acids may be uti l ized in different ways by different microorganisms (BARKER, 1961 and GREENBERG, 1961). Phenylalanine, for example, is degraded by some bacteria via homogenestic acid and by others via f l -oxaloadipic acid. Acetyl-coenzyme A is pro- duced in both routes, though the former also yields fumarate, whereas the latter route produces succinate, which in turn is a precursor of fumarate (KoRNBERG, 1965).
WRIGHT (1967) studied the metabol ism of peptides by rumen microorganisms. He observed that peptides were more efficiently converted into bacterial protein than amino acids. The many different pathways of amino acid degradat ion will not be discussed in detail. The more general processes of amino acid degradat ion are:
(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-ketoglutaric acid + NADH + H + + NH3 Sum: Amino acid + NAD + H20~--~ct-keto acid + NADH + H + + NH3
The enzyme glutamate dehydrogenase catalyzes the second reaction in the above sequence. According to the principle described by GOEBEL and 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 STAOTMAN et 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 TABLE 1. The end-products for a specific amino acid are not always the metabolic end-products of the specific microorganisms.
The end-products of amino acids are very similar to those of the degraded carbo- hydrates. Both carbohydrate and amino acid metabolism thus produce a common pool of end-products which are degraded further by common 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 known 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 3-amino valerate Arginine Citrulline + ADP + inorganic phosphate Ornithine Asparagine Aspartic acid
Cystine and cysteine
Homocysteine Methionine Glutamic acid
Tyrosine and phenylalanine
Ammonia, carbon dioxide, acetic acid, propionic acids Ammonia, acetic, propionic and valeric acids Ammonia, acetic, propionic and valeric acids, ornithine and citrulline Ammonia, carbon dioxide, acetic, propionic and valeric acids, ATP and ornithine Ammonia, acetic, propionic and valeric acids Ammonia, carbon dioxide, acetic and succinic acids Ammonia, carbon dioxide, acetic, butyric, lactic, fumaric, and malic acids, ethanol, butanol Ammonia, carbon dioxide, hydrogen sulphide, formic, acetic, propionic, butyric, valeric and pyruvic acids Ammonia, hydrogen sulphide and a-ketobutyric acid Ammonia, methylmercaptan and a-ketobutyric acid Ammonia, carbon dioxide, hydrogen, acetic, butyric, and lactic acids (inter- mediates mesaconate and citramalate) Ammonia, carbon dioxide, hydrogen, acetic and pyruvic acids Ammonia, carbon dioxide, hydrogen, formic, acetic, butyric, lactic and glutamic acids and formamide Can undergo the Stickland reaction to the branched chain fatty acid p-methyl- butyric acid (N.B. leucine not easily fermented) Can undergo the Stickland reaction to the branched chain fatty acid a-methyl- butyric acid. (N.B. isoleucine not easily fermented) Can undergo the Stickland reaction to the branched chain fatty acid a-methyl- propionic acid Ammonia, carbon dioxide, acetic, propionic and butyric acids Can undergo the Stickland reaction to 3-aminovalerate and can be further fermented (See tLaminovalerate) The degradation products are unknown Ammonia, carbon dioxide, hydrogen, formic, acetic, propionic, butyric, valeric and lactic acids, alanine and ethanol Ammonia, carbon dioxide, hydrogen, formic, acetic, propionic, butyric, valeric and a-ketobutyric acids, acetaldehyde and glycine Ammonia, carbon dioxide, pyruvic acid, indole propionic acid, indole pyruvic acid and indole Ammonia, carbon dioxide, hydrogen, phenol, p-cresol, p-hydroxy phenylacetic, p-hydroxy phenylacrylic, and p-hydroxy-phenyllactic acids
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 479
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. RABINOW1TZ and 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 SAGERS and GUNSALUS (1958) and RABINOWITZ and 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 THIEL and HATTINGH (1967), and KOTZ~ (1967). HATT1NGH et 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. THIEL and 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 (GREEN et al., 1967) and, to detect growth of Staphylococcus aereus in foods (CH~sBRo and 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 BURGESS and 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 TOERIEN and HAa'TINGH (I 969). Their review referred mainly to work carried out using intact cells of pure cultures of methane-
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 481
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 BUCHANAN and 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). KLUYVER and 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; RABINOWITZ and PRICER, 1963).
The enzyme system of M. barkeri produced methane from 5-methyltetrahydrofolate but less efficiently from free formaldehyde or methylcobalamin (BLAYLOCK and 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 (BRYANT et al., 1967, 1968; LANGENBERG et 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 (BLAYLOCK and STADTMAN, 1966; STADTMAN and BLAYLOCK, 1966). Transacetylase and acetate kinase have also been found to be active in M. barkeri (BLAYLOCK and 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 (WOLIN et al., 1963).
The exact role of ATP, which is essential for methane synthesis by both M. omelian- skii and M. barkeri enzyme systems (WOLIN et aL, 1963; BLAYLOCK and STADTMAN, 1966) and for methylcobalamin synthesis from methanol by M. barkeri extracts, is still unknown (BLAYLOCK and STADTMAN, 1964). In crude extracts a number of other nucleotide triphosphates and often ADP can replace ATP (BLAYLOCK and 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 (WOLIN et 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-cobalamin formation 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 (BLAYLOCK and STADTMAN, 1964). LEZIUS and 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; LJUNGDAHL et aL, 1965; LINDSTRAND, 1965). Clostridium thermoaceticum has been shown to synthesize acetate from carbon dioxide through a cobalt-carboxymethyl corrinoid derivative (LJUNGDAHL et al., 1965, 1966; LJUNGDHAL and 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 (BLAYLOCK and STADTMAN, 1966). The concentration used to effect 100 per cent inhibition is (5 x 10-SM) which is much
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 483
higher than concentrations normally used to inhibit folate-dependent systems (10-aM). Aminopterin is a very powerful inhibitor of dihydrofolate reductase (MATHEWS et 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 (HIGHLEY et 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; STADTMAN and 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 ~Ozl"l (bound C02+ 2H dehydogenese H
I THF (H-donor) ~ ATP H CO-THF
THF Serine -- - - CHfTHF- glycine
I ,%, CH 3 -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. KOTZL P. G. THIEL and 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. HATTINGH et 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 (20-45C)with an optimum
at about 37C 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. SAWYER et aL (1954) reported
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 485
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. THIEL and 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 of 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 DNA contained only in living cells (AGARDY et al., 1963; HATTINGH et 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.
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
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 487
take into account the intrinsic biological nature of the bacteria responsible for the process. In an attempt to acquire a better appreciat ion of the biological nature of anaerobic digestion the existing knowledge of the biochemical characteristics of the system has been summarized. F rom this summary it is evident that there is a paucity of knowledge of the biochemistry of the process and that a much better understanding of the biochemistry of the process is imperative for the efficient characterizat ion and control of an essentially biological process such as anaerobic digestion.
The existing parameters for the control of anaerobic digestion have been described, but will in future need to be supplemented by well-chosen biological parameters. The present knowledge on anaerobic digestion is largely based, on empirical observations which give no explanation for the reasons behind certain phenomena, and it is there- fore evident that further fundamental research on the anaerobic process is of prime importance if this process is to be util ized to its full capacity.
ADDANKI S., SOTOS J. F. and REARICK P. D. (1966). Rapid determination of picomole quantities of ATP with a liquid scintillation counter. Analyt. Biochem. 14, 261-264.
AGARDY F. J., COLE R. D. and PEARSON E. A. (1963) Kinetics and activity parameters of anaerobic fermentation systems. San. Eng. Res. Lab. Rept. 63-2, Univ. of California, Berkeley, 50.
ALBERTSON O. E. (1961) Ammonia nitrogen and the anaerobic environment. Water Pollut. Control Fed. d. 33, 978-995.
ALEORD J. A. (1960) Effect of incubation temperature on biochemical tests in the genera Pseudomonas and Achromobacter. d. Bact. 79, 591-593.
ALLEN M. B. (1953) The thermophilic aerobic sporeforming bacteria. Bact. Rev. 17, 125-173. AUE J. and DEIBEL R. H. (1967) Fumarate reductase activity of Streptococcus faecalis. J. Bact. 93,
1770-1776. AURICH H., DUMMLER W. and MUCKE n. (1958) Growth substances in sewage and their significance
in biological purification. Wasserwirtschaft Tech. 8, 496, cited by HUNTER J. V. (1962) Ph.D. Thesis, Rutgers Univ.
BACKMEYER n. P. (1955) How to avoid sour digesters. Wat. Sewage WksJ. 102, 369-378. BALDWIN R. L. (1965) Pathways of carbohydrate metabolism in the rumen. In Physiology of Digestion
in the Ruminant (Edited by DOUGHERTY R. W., ALLEN R. S., BURROUGHS W., JACOBSON N. L. and McGILLIARO A. D.) Vol. 12, pp. 379-389. Butterworths, Washington.
BARKER H. A. (1956) Biological formation of methane. Ind. Engng Chem. 48, 1438-1442. BARKER H. A. (1961) Fermentation of nitrogenous organic compounds. In The Bacteria (Edited by
GUNSALES I. C. and STANIER R. Y.) Vol. II, pp. 151-207. Academic Press, New York. BARKER H. A. (1967) Biochemical functions of corrinoid compounds. Biochem. J. 105, 1-15. BAUCHOP T. (1967) Inhibition of rumen methanogenesis by methane analogues. J. Bact. 94, 171-175. BLAYLOCK B. A. (1967) Methane fermentation. Ann. Rev. Microbiol. 21, 121-142 cited by STADTMAN
T. C. (1967). BLAYLOCK B. A. and STADTMAN T. C. (1963) Biosynthesis of methane from the methyl moiety of
methylcobalamin. Biochem. biophys. Res. Commun. 11, 34-38. BLAYLOCK B. A. and STADTMAN T. C. (1964) Enzymic formation of methylcobalamin in Methano-
sarcina barkeri extracts. Biochem. biophys. Res. Commun. 17, 475-480. BLAYLOCK B. A. and STADTMAN T. C. (1966) Methane biosynthesis by Methanosarcina barkeri.
Properties of the soluble enzyme system. Arch. Biochem. Biophys. 116, 138-158. BORUFF C. S. (1939) Stabilization of paunch manures and packing screenings. In Anerobic Fermenta-
tions bulletin 32 (Edited by BUSWELL A. M. and HATFIELD W. D.) pp. 152-159. Urbana, Illinois, U.S.A.
BROT N. and WEISSBACn H. (1965) Enzymatic synthesis of methionine. Chemical alkylation of the enzyme-bound cobamide. J. biol. Chem. 220, 3064-3070.
488 J.P. KOTZ~I, P. G. THIEL and W. H. J. HATTINGH
BROWN D. K., MILITZER W. and GEORG1 C. E. (1957) The effect of growth temperature on the heat stability of a bacterial Pyrophosphatase. Archs. Biochem. Biophys. 70, 248-236.
BROWN J. M. and KrNCH~SKY B. (1963) Digester indigestion from high temperature. Water Pollut. Control Fed. J. 37, 416-417.
BRYANT M. P. (1965) Rumen methanogenic bacteria. In Physiology of Digestion in the Ruminant (Edited by DOUGRERTY B. S.) Waverly Press, Baltimore.
BRYANT M. P., WOLIN E. A., WOLIN M. J., WOLFE R. S. and MANDEL M. (1967) Methanobacillus omelianskii. A symbiotic association of two bacterial species. Bact. Proc. 19.
BRYANT M. P., McBRIDE B. C. and WOLFE g. S. (1968) Hydrogen-oxidizing methane bacteria--I. Cultivation and methanogenesis. 3". Bact. 95, 1118-1123.
BUCHANAN B. B. and tL~iNowrrz J. C. (1964) Some properties of Methanobacterium omelianskii ferredoxin. J. Bact. 88, 806-807.
BURGESS S. G. and WOOD L. B. (1964) The properties and detection of sludge gas. J. Inst. Sewage Purif Part 1, 24-46.
BUSWELL A. M. (1957) Fundamentals of anaerobic treatment of organic wastes. Sewage ind. Wastes 29, 717-721.
BUSWELL A. i . (1959) Interpretation of sewage treatment plant tests--panel discussion. SAWYER C. N. (Moderator). Sewage ind. Wastes 31, 92-97.
BUSWELL A. i . and HATFIELD W. D. (1939) Anaerobic Fermentations. State of Illinois Department of Registration and Education Bulletin No. 32.
BUSWELL A. M. and NEAVE S. L. (1930). Laboratory studies of sludge digestion. Illinois St. War. Surv. Bull. 30, 84 pp.
BUSWELL A. M., BORUFF C. S. and WIESMAN C. K. (1932) Anaerobic stabilization of milk waste. Ind. Engng Chem. 24, 1423-1425.
CANN D. G. and WILLOX M. E. (1965) Analysis of multimolecular enzymes as an aid to the identifica- tion of certain rapidly growing mycobacteria, using starch gel electrophoresis. J. appl. Bact. 28, 165-173.
CHESBRO W. R. and AUBURN K. (1967) Enzymatic detection of the growth of Staphylococcus aureus in foods. Appl. Microbiol. 15, 1150-1159.
COPELAND W. R. (1955) Basic elements of digester operation. Sewage ind. Wastes 27, 224-226. CORDON T. C., MAURER E. W., NU~z-PoNzoA M. V. and STIRTON A. J. (1968) Metabolism of some
anionic tallow-based detergents by sewage microorganisms. J. appl. ~Iicrobiol. 16, 48-52. COULTER R. G. (1953) Environment for anaerobic destruction of organic material. Pub. Wks iV. Y. 84,
No. 5 cited by FOGARTY W. J. (1961) M.S. Thesis Purdue University. CROFt D. K. and FAUST S. I). (1954) More data on syndets in municipal sewage. Wat. Sewage Wks J.
101, 286-287. CULl' R. L. and STALTENBERG H. (1953) Synthetic detergent pollution in Kansas. Am. Wat. Wks Ass.
J. 45, 1187-1195. DEmEL R. H. and KVETKAS M. J. (1964) Fumarate reduction and its role in the diversion of glucose
fermentation by Streptococcasfaecalis. J. Bact. 88, 858--864. DE Moss R. D., BARD R. C. and GUNSALUS I. C. (1951) The mechanism of the heterolactic fermenta-
tion: a new route of ethanol formation. J. Bact. 62, 499-511. DmTRICH K. R. (1960). Ablaufverwertung und Abwasserreinigung in der biochemischen Industrie. In
Biochemie und Technologic Dr. Alfred Hiitig, Heidelberg. ENEEO L. and PEHRSON S. O. (1960) Thermophilic digestion of a mixture of domestic sewage sludge
and cellulose materials. Acta polytech, scand. 281, 1-36. FALCONE G. and NICKERSON W. J. (1956) On the biosynthesis of the porphyrinlike moiety of Vitamin
B12. Science N. Y. 124, 272-273. FISCHER A. J. and GREENE R. A. (1945) Plant scale tests on thermophilic digestion. Sewage WksJ. 17,
718-729. FOGARTY W. J. (1961) Alkalinity and ammonia in anaerobic digestion. M.S. Thesis University of
Purdue. FREUDENnERG K. (1954) Neuere Ergebnisse auf dem Gebiete des Lignins und der Verholzung. In
Fortschritte der Chemic der organischen Naturstoffe (Edited by ZECHMEISTER L.) Vol. II, pp. 43-82. Springer, Wien.
FRUTON J. S. and SIMMONDS S. (1958) General Biochemistry 2nd ed. John Wiley, New York.
Anaerobic Digestion--I I . Characterization and Control of Anaerobic Digestion 489
GARBER W. F. (1954) Plant-scale studies of thermophilic digestion at Los Angeles. Sewage Ind. Wastes 26, 1202-1216.
GAUDY A. F., KOMOLRIT K. and BHATLA M. N. (1963) Sequential substrate removal in heterogeneous populations. Wat. Pollut. Control Fed. J. 35, 903-922.
GIBBS M. and DE Moss R. D. (1951) Ethanol formation in Pseudomonas lindneri. Archs Biochem. Biophys. 34, 478-479.
GIBBS M. and DE Moss R. D. (1954) Anaerobic dissimilation of t4C-labelled glucose and fructose by Pseudomonas lindneri. J. biol. Chem. 207, 689-694.
GOEBEL H. and KLINGENBERO M. (1964) DPN- spezifische Isocitrat-Dehydrogenase der Mito- chondrien--I. Kinetische Eigenschaften, Vorkommen und Funktion der DPN-spezifischen Isocitrat-Dehydrogenase, Biochem. Z. 340, 441-464.
GOLUEKE C. G. (1958) Temperature effects on anaerobic digestion of raw sewage sludge. Sewage ind. Wastes. 30, 1225-1232.
GOULD R. H. (1959) Economical practices in the activated sludge and sludge digestion processes. Sewage ind. Wastes 31, 399-405.
GREEN S. S., GOLDBERG H. S. and BLENDEN D. C. (1967) Enzyme patterns in the study of Leptospira. Appl. Microbiol. 15, 1104-1113.
GREENBERG D. M. (1961) Carbon catabolism of amino acids. In Metabolic Pathways (Edited by GREENBERG D. M.) Vol. II, pp. 79-172. Academic Press, New York.
GUEST J. R. and WOODS D. D. (1965) The interaction of folio acid derivatives in the methylation of homocysteine. Biochem. J. 97, 500-512.
HANSON T. E. and ANDERSON R. L. (1966) D-Fructose-l-phosphate kinase, a new enzyme instru- mental in the metabolism of D-fructose. J. biol. Chem. 241, 1644-1648.
HARARY I. (1957) Bacterial fermentation of nicotinic acid. J. biol. Chem. 227, 815-822. HASELTINE T. R. (1949) Preconditioning and digestion of sewage sludge. Part III. Nature of digestion:
acidity control. Wat. Sewage Wks 96, 271-274. HARRISON K. (1959) A Guide-book to Biochemistry Cambridge University Press. HAX'rINGH W. H. J. and SIEBERT M. L. (1967) Determination of the deoxyribonucleic acid (DNA)
content of anaerobic sludge. Water Research 1, 197-203. HATTINGH W. H. J., KOTZE J. P., THIEL P. G., TOERIEN D. F. and SIEBERT M. L. (1967a) Biological
changes during the adaptation of an anaerobic digester to a synthetic substrate. Water Research 1, 255-277.
HA'rrINGH W. H. J., THIEL P. G. and StEBERT M. L. (1967b) Determination of protein content of anaerobic digesting sludge. Water Research 1, 185-189.
HEUKELEKIAN H. (1930) Certain organic constituents of fresh and ripe sewage sludge. New Jers. agric Exp. Stn Bull. 502, 31-36.
HEUKELEKIAN H. (1958) Basic principles of sludge digestion. In Biological Treatment of Sewage and Industrial Wastes (Edited by MCCABE B. J. and ECKENFELDER W. W.) Vol. 2, pp. 25-43. Reinhold, New York.
HEUKELEKIAN H. and BALMAT J. L. (1959) Chemical composition of the particulate fractions of domestic sewage. Sewage ind. Wastes. 31,413-423.
HEUKELEKIAN H. and KAPLOVSKY A. J. (1948) Effect of change of temperature on thermophilic digestion. Sewage Wks J. 20, 806-815.
HEUKELEKIAN H. and KAPLOVSKY A. J. (1949) Improved method of volatile acid recovery from sewage sludges. Sewage Wks J. 21,974-983.
HEUKELEKIAN H. and MUELLER P. (1958) Transformation of some lipids in anaerobic sludge digestion. Sewage ind. Wastes 30, 1108-1120.
HIGHLEY D. R., DAVIES M. C. and ELLEt, rBOGEN L. (1966) Chemical and physical properties of intrinsic complex factor and Vitamin B~2--intrinsic factor. Fedn Proc. Fedn Am. Socs exp. Biol. 25, 277.
HINDIN E. and DUNSTAN G. H. (1959) Some aspects of sludge digestion. Water Sewage Wks 106, 457460.
HINDIN E. and DUNSTAr~ G. H. (1960) Effect of detention time on anaerobic digestion. Wat. Pollut. Control Fed. Y. 32, 930.
HINDIN E. and DtmSTAN G. H. (1963) Anaerobic digestion of potato processing wastes. Wat. Pollut. Control Fed. J. 35, 486-492.
490 J .P. KOTZI~, P. G. TmEL and W. H. J. HATTINGH
HOLMSEN H., HOLMSEN I. and BERNHARDSEN A. (1966) Microdetermination of adenosine diphosphate and adenosine triphosphate in plasma with the firefly luciferase system. Analyt. Biochem. 17, 456--473.
HLrNGATE R. E. (1966) The Rumen andits Microbes Academic Press, New York. HUNTER J. V. (1962) The organic composition of various domestic sewage fractions. Ph.D. Thesis,
Rutgers University. INGRAHAM J. L. (1962) Temperature relationships. In The Bacteria (Edited by GUNSALUS I. C. and
STANIER R. Y.) Vol. IV, pp. 265-296. Academic Press. New York, London. IMHOFF K. and FAIR G. M. (1956) Sewage Treatment 2nd ed. John Wiley, New York. IRION W. and LJUNGDAHL L. (1965) Isolation of Factor IIIm Coenzyme and Cobyric Acid Coenzyme
plus other B12 Factors from Clostridium thermoaceticum. Biochemistry N. Y. 4, 2780-2790. JONES M. and KING H. K. (1968) Oxidation of dicarboxylic acids by strict aerobes and facultative
anaerobes. Biochem. d. 108, 119. KAPLOVSKY A. J. (1951) Volatile acid production during the digestion of seeded, unseeded, and limed
fresh solids. Sewage ind. Wastes 23, 713-721. KAPLOVSKY A. J. (1952) Volatile acid production during digestion of several industrial wastes. Sewage
ind. Wastes 24, 194-201. KEEFER C. E. and URTES H. C. (1963) Digestion of volatile acids. Wat. Pollut. Control Fed. J. 35,
334-343. KLUYWR A. J. and BAARS J. K. (1932) On some physiological artefacts. Konikl. Akad. v. Wetenschap.
te Amsterdam Vol. 35, pp. 370-378. Proc. of the Section of Sciences. KL~JVWR A. J. and SCHNELL~N C. G. T. P. (1947) On the fermentation of carbon monoxide by pure
cultures of methane bacteria. Archs. Biochem. 14, 57-70. KORNBERG H. L. (1965) The co-ordination of metabolic routes. In Function and Structure of Micro-
organisms (Edited by POLLOCK M. R. and RICHMOND M. H.)pp. 8-31. Cambridge University Press. KoTz~ J. P. (1967) Methods for the determination of intermediary enzymes in mixed cultures used for
the purification of organic polluted waters. Water Research 1, 351-365. KOTZ~ J. P. (1968a) 1-phosphofructokinase from Clostridium pasteurianum W 5 S.A. J. agric. Sci.
11, 349-362. KOTZ~ J. P. (1968b) Unpublished results. KOTZ~ J. P. (1969) The role of the enzyme 1-phosphofructokinase in the metabolism of Clostridium
pasteurianmn W 5. Water Research 3, 83-91. KOTZ~ J. P. and KISTNER A. (1968) Unpublished results. KoTz[ J. P., THmL P. G., TOERmN D. F., HAT'rrNGH W. H. J. and SIEaERT M. L. (1968) A biological
and chemical study of several anaerobic digesters. Water Research 2, 195-213. KREBS H. A. and LOEWENSTEIN J. M. (1960) The tricarboxylic acid cycle. In Metabolic Pathways
(Edited by GREENBERG D. M.) Vol. 1, pp. 129-203. Academic Press, New York. LANGEBERG K. F., BRYANT M. P. and WOLFV R. S. (1968) Hydrogen-oxidizing methane bacteria--II.
Electron microscopy. J. Bact. 95, 1124-1129. LEzivs A. G. and BARKER H. A. (1965) Corrinoid compounds of Methanobacillus omelianskii--I.
Fractionation of the corrinoid compounds and identification of Factor III and Factor III- Coenzyme. Biochemistry, N. Y. 4, 510-518.
LINDSTRAND K. (1965) Crystalline methylcobalamin from a microorganism. Acta chem. scand. 19, 1762-1763.
LJUNGDAa-IL L. and IRION E. (1966) Photolytic and reductive cleavage of co-carboxymethylcobalamin. Biochemistry, N. Y. 5, 1846-1850.
LJON~DAI~L L., IRION E. and WOOD H. G. (1966) Role of corrinoids in the total synthesis of acetate from CO2 by Clostridium thermoaceticum. Fedn Proc. Fedn Am. Socs exp. Biol. 25, 1642-1648.
LJUN~DA~L L., IRION E. and WOOD H. G. (1965) Total synthesis of acetate from CO2--1. Co-methyl- cobyric acid and co-(methyl)-5-methoxybenzimidazolylcobamide as intermediates with Clostri- dium thermoaceticum. Biochemistry, N.Y. 2771-2780.
LOHMEYER G. T. (I 959) A review of sludge digestion. Sewage ind. Wastes 31, 221-235. L~B C. (1941) Some investigations into the digestion and drying of humus and activated sludges.
Surveyor 100, 91 : cited by POHLAND F. G. (1962) Ph.D. Thesis, Purdue University. LtrND B. M. (1965) A comparison by the use of gel electrophoresis of soluble protein components
and esterase enzymes of some group D Streptococci. J. gen. Microbiol 40, 413-419.
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 491
LUmA S. E. (1960) The bacterial protoplasm. Composition and organization. In The Bacteria (Edited by GtrNSALUS I. C. and STAMER R. Y.) Vol. 1, pp. 1-34. Academic Press, New York.
MAH R. A. and SUSSMAN C. (1968) Microbiology of anaerobic sludge fermentation I. Enumeration of the non-methanogenic anaerobic bacteria. Appl. Microbiol. 16, 358-361.
MATIJEWS C. K., SCRIM6EOUR K. C. and HtrE~EKENS F. M. (1963) Dihydrofolic reductase. In Methods in Enzymology (Edited by COLOWICK S. P. and KAPLAN N.O.) Vol. 6, pp. 364-368. Academic Press, New York and London.
MANOA~,mLLI R., ORFORD H. and HENOERSON C. (1960) Levels of detergents in sewage treatment processes. Presented at the 32nd Annual Meeting, New York Sewage Ind. Wastes Ass.
MCCARTY P. L., JERIS J. S. and MURDOCI-I W. (1963) Individual volatile acids in anaerobic treatment. Water Pollut. Control Fed. J. 35, 1501-1515.
MEFrORD R. B. and CAMPBELL L. L. (1952) Studies on a thermophilic variant of Bacillus globigii. Tex. Rep. Biol. Med. 10, 419-420.
MUELLER L. E., HINDIN E., LtmSFORD J. V. and DUNSTAN G. H. (1959) Some characteristics of anaerobic sludge digestion--I. Effect of loading. Sewage ind. Wastes 31, 669-677.
NORRIS J. R. (1964) The classification of Bacillus thuringiensis. J. appl. Bact. 27, 439-447. NURSTEN H. E. (1961) Tannins and related substances. Biochemists' Handbook (Edited by LONG C. F.)
pp. 998-1018. E. & F. N. Spon, London. POHLAND F. G. (1962) Laboratory investigations on the thermological aspects and volatile acids--
alkalinity behaviour during anaerobic sludge digestion. Ph.D.Thesis, Purdue University. POMEROY R. D. (1933) Report on Digestion Studies. Co. San. Dists. of Los Angeles Co. Calif. Part I.
cited by War. Pollut. Control Fed. Manual of Practice No. 16. Anaerobic Sludge Digestion (1968).
POMEROY R. D. (1935) Report on Digestion Studies. Co. San. Dists. of Los Angeles Co. Calif. Part III. cited by Wat. Pollut. Control Fed. Manual of Practice No. 16. Anaerobic Sludge Digestion (1968).
PRETORIUS W. A. (1968) Personal communication. RAmNOWlTZ J. C. and BARKER H. A. (1956) Purine fermentation by Clostridium cylindrosporum--I.
Tracer experiments on the fermentation of guanine. J. biol. Chem. 218, 147-160. RABINOWlTZ J. C. and PRICER W. E. (1958) Crystallization of tetrahydrofolic formylase. Fedn Proc.
Fedn Am. Socs exp. Biol. 17, 293. RAmNOWITZ J. C. and PRICER W. E. (1963) Formyltetrahydrofolate synthetase. In Methods in Enzymo-
logy (Edited by COLOWICK S. P. and KAPLAN N. O.) Vol. 6, pp. 375-379. Academic Press, New York, London.
REEVES R. E., WAR~EN L. G. and Hsu D. S. (1966) 1-phosphofructokinase from an anaerobe. J. biol. Chem. 241, 1257-1261.
ROOERS H. J. (1965) The outer layers of bacteria. The biosynthesis of structure. In Function and Structure in Microorganisms (Edited by POLLOCK M. R. and RICHMOND M. H.) pp. 186-219. Cambridge University Press.
ROSEaURY T. (1962) Microorganisms Indigenous to Man McGraw-Hill, New York. RtroOLrS W. (1944) Decomposition of grease during digestion, its effect on gas production and fuel
value of sludges. Sewage Wks J. 16, 1125-1155. RLrDOLFS W. and CHAMBERLIN N. S. (1932) Indole and skatole in sewage. Ind. Engng Chem. 24,
111-112. RODOLFS W. and GEHM H. W. (1942) Chemical composition of sewage sludges, with particular
reference to their phosphoric acid contents. New Jets. agric. Exp. Stn. Bull. 699, 1-15. RUDOLFS W. and HEINEMANN B. (1939) Growth-promoting substances in sewage and sludge----III.
Vitamin C, carotene, amino acids, fatty acids and naphthyl compounds. Sewage Wks J. 11, 587-594.
RUDOLFS W. and HEUKELEKIAN H. (1931) Thermophilic digestion of sewage solids--III. Fresh solids and activated sludge. Ind. Engng Chem. 23, 67-71.
RODOLFS W. and INGOLS R. S. (1938) Growth-promoting substances in sewage and sludge--II. Indole and skatole Sewage Wks J. 10, 653-660.
RUDOLFS W. and SET'rER L. R. (1937) High and low temperature digestion experiments--III. Effect of certain organic wastes. Sewage Wks J. 9, 549-568.
SAGERS R. D. and GUNSALUS I. C. (1958) Bact. Proc. 119.
492 J .P. KOTZI~, P. G. THIEL and W. H. J. HATYINGH
SANDERS F. A. and BLOODGOOD D. L. (1965) The effect of nitrogen-to-carbon ratio on anaerobic decomposition. Wat. Pollut. Control Fed. J. 37, 1741-1752.
SAWYER C. N. (1960) Chemistry for Sanitary Engineers McGraw-Hill, New York. SAWYER C. N. and RoY H. K. (1955) A laboratory evaluation of high-rate sludge digestion. Sewage
ind. Wastes 27, 1356--1363. SAWYER C. N., HOWARD F. S. and PERSHE E. R. (1954) Scientific basis for liming digesters. Sewage ind.
Wastes 26, 935-944. SeHLENZ H. E. (1944) Controlled digestion. Sewage WksJ. 16, 504--526. SCHLENZ H. E. (1947) Important considerations in sludge digestion. Part I--Practical aspects.
Sewage Wks J. 19, 19-27. SCHNELLEN C. G. T. P. (1947) Onderzoekingen over de Methaangisting. Dissertation Techn. Univ
Delft De Maasstad. Rotterdam Publs. SCHULZE K. L. and Rmu B. N. (1958) Studies on sludge digestion and methane fermentation--lI.
Methane fermentation of organic acids. Sewage ind. Wastes 30, 164-184. SEIDMAN I. and ENTNER N. (1961) Oxidative enzymes and their rate in phosphorylation in sarcosomes
of adult Asearis lumbricoides. J. biol. Chem. 236, 915-919. SEERMAN J. M. and CAMEgON G. M. (934) Lethal environmental factors within the natural range of
growth. J. Bact. 27, 341-348. SIEBERT M. L., TOERIEN D. F. and HA'rTINGH W. n. J. (968) Enumeration studies on methanogenic
bacteria. Water Research 2, 545-554. SIMPSON J. R. (1960) Some aspects of the biochemistry of anaerobic digestion. In Waste Treatment
(Edited by ISAAC P. C. G.) pp. 31-51. Pergamon Press, Oxford. SINGER T. P. and HAUBER J. (1965) The fumarate reductases of yeast. Fedn Proc. Fedn Am. Socs exp.
Med. 24, 297. SLATER E. C. and BONNER W. D. (1956) The effect of fluoride on the succinic oxidase system. Biochem.
J. 52, 185-196. SOKATCH J. T. and GUNSALUS I. C. (1957) I. Pathway of carbon in an inducible gluconate fermentation
by Streptococcus faecalis. J. Bact. 73, 452-460. SOMERVILLE H. J. (1968) Enzymic studies on the Biosynthesis of amino acids from lactate by Pepto-
streptococcus elsdenii. Biochem. J. 108, 107-I 19. STADTMAN T. C. (1967) Methane fermentation. Ann. Rev. MicrobioL 21, 121-142. STADTMAN T. C. and BLAYLOCK B. A. (1966) Role of B12 compounds in methane fermentation. Fedn
Proc. Fedn Am. Socs exp. Biol. 25, 165%1661. STADTMAN T. C., ELLIOT P. and TIEMANN L. (1958) Studies on the enzymic reduction of amino acids--
III. Phosphate esterification coupled with glycine reduction. J. biol. Chem. 231, 961-973. Standard Methods for the Examination of Water and Waste Water (1965) 12th Edn. Am. Publ. Hlth
Ass., New York. STANDER G. J. (1968) Personal communication. STANDER G. J., CILLIE G. C., ROSS W. R. and BAILLm R. D. (1967) Treatment of wine distillery
wastes by anaerobic digestion. Proc, 22nd Ind. Waste Conf. Purdue Univ. pp 892-907. STANIER R. Y., DO~ROFF M. and ADELBERQ E. A. (1964) General Microbiology 2nd Edn. Mac-
millan, London. TALE., DIKSTEIN S. and SULMAN F. G. (1964) ATP determination with the tricarb scintillation counter.
Experientia 20, 652-653. TI-h'EL P. G. (1969) The effect of methane analogues on methanogenesis in anaerobic digestion.
Water Research 3, 215-223. Tm~L P. G. and DU TOIT P. J. (1965) The chemical composition of a brewery waste. I. Inst. Brew. 71,
509-514. THIEL P. G., TOERIEN D. F., HATTINGH W. H. J., KOTZI~ J. P. and SIEBERT, M. L. (1968) Interrelations
between biological and chemical characteristics in anaerobic digestion. Water Research 2, 393-408.
TOERIEN D. F. and HATTINGH W. H. J. (1969) Anaerobic digestion--I. Microbiology. Water Research 3, 385-416.
TOERIEN D. F., SIEBERT i . L. and HATIXNGH W. H. J. (1967) The bacterial nature of the acid-forming phase of anaerobic digestion. Water Research l , 497-507.
Anaerobic Digestion--II. Characterization and Control of Anaerobic Digestion 493
VAN HUYSSTEEN J. J. (1967) Gas chromatographic separation of anaerobic digester gases using porous polymers. Water Research 1, 237-242.
WARRIr~CA M. G. P. J., S~aIXH O. H., GnmrrrA A. and SIN~ER T. P. (1958) Studies of succinic de- hydrogenase--VIII. Isolation of succinic dehydrogenase-fumaric reductase from an obligate anaerobe. Y. biol. Chem. 230, 97-109.
WARRrNGA M G. P. J. and G~DrrrA A. (1958) Studies ofsuccinic dehydrogenase---IX. Characteriza- tion of the enzyme from Micrococcus lactilytieus. J. biol. Chem. 230, 111-123.
WATER POLLUTION CONTROL FEDERATION (1968) Anaerobic Sludge Digestion Manual of Practice No. 16.
WATSON P. G. (1954) Metabolic Integrations Heifer, Cambridge. WHISTLER R. L. (1950) Xylan. Adv. Carbohyd. Chem. 5, 269-290. WHITE A., HANDLER P. and SMITH E. I,. (1964) Principles of Biochemistry 3rd Edn. McGraw-Hill,
New York. WRIGHT D. E. (1967) Metabolism of peptides by rumen microorganisms. Appl. Microbiol. 15, 547-550. WOLIN E. A., WOLIN M. J. and WOLFE R. S. (1963) Formation of methane by bacterial extracts. J. biol.
Chem. 238, 2882-2886. WOOD W. A. (1961) Fermentation of carbohydrates and related compounds. In The Bacteria (Edited
by GUNSALUS I. C. and STANIER R. Y.) Vol. II, pp. 59-149. Academic Press, New York. WOOD J. M., ALLAM A. M., BRXLL W. J. and WOLFE R. S. (1965) Formation of methane from serine
by cell-free extracts of Methanobacillus omelianskii. J. biol. Chem. 240, 4564-4569. WOOD J. M. and WOLFE R. S. (1965) The formation of CH4 from NS--methyltetrahydrofolate mono-
glutamate be cell-free extracts of Methanobacillus omelianskii. Biochem. Biophys. Res. Commun. 19, 306-311.
WOOD J. M. and WOLEE R. S. (1966a) Alkylation of an enzyme in the methane-forming system of Methanobacillus omelianskii. Biochem. Biophys. Res. Commun. 22, 119-123.
WOOD J. M. and WOLFE R. S. (1966b) Components required for the formation of CH4 from methyl- cobalamin by extracts of Methanobaeillus omelianskii. J. Bact. 92, 696-700.
YENCHKO J. (1955) Digester operation problems in Pennsylvania. Sewage ind. Wastes 27, 94-99. ZArrN R. K. (1964) Biochemisches Taschenbuch (Edited by RAUEN H. M.) 2nd Edn, pp. 428-436.