Biological Formation of Methane

microorganisms. Particularly in the late summer when aquatic vegetation begins to decline because of a shortening of the days and the surrounding wate...
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H. A. BARKER University of California, Berkeley, Calif.

Biological Formation of Methane

The material presented here shows that we have acquired some knowledge of the biology, physiology, and biochemistry of methane-producing bacteria. tunately this knowledge is still incomplete and superficial in many respects.

UnforMuch

time and effort will be required to bring our understanding of this group to a level comparable with that of many other groups of microorganisms.

METHANE

is a common and abundant product of the bacterial decomposition of organic materials under anaerobic conditions. In nature, methane formation is most conspicuous where plants die and decompose under water, which acts as a blanket to exclude oxygen and favor the development of many types of anaerobic microorganisms. Particularly in the late summer when aquatic vegetation begins to decline because of a shortening of the days and the surrounding water is still warm. anyone who swims or fishes in inland waters cannot fail to notice the abundant gas that escapes from the bottom of lakes and ponds. This gas is mainly methane mixed with a smaller amount of carbon dioxide. Methane is also formed in conspicuous amounts in the digestive tracts of many animals, particularly ruminants. The rumen of the cow, for example, provides a n ideal place for the growth of anaerobic bacteria. It is well supplied with food, well buffered close to neutrality, warm, and almost free of oxygen. Consequently methane-producing bacteria develop rapidly and commonly form from 100 to 500 liters of methane/day/animal. From 5 to 10% of the caloric value of the feed may be lost as methane. Fortunately the cow is provided with a rather

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effective belching mechanism for getting rid of this large volume of gas. If this mechanism fails, which sometimes occurs when the animal eats a n excessive amount of green feed, the cow soon becomes bloated and frequently dies unless counter measures are quickly applied. Characteristics of Methane Bacteria The formation of methane in decomposing organic materials is the result of the action of a specialized physiological group of bacteria, which is often referred to as the “methane-producing” bacteria. These methane-producing bacteria (“methane bacteria,” for short) are entirely different from the aerobic methane-oxidizing bacteria. The former produce methane from various organic and inorganic compounds; the latter oxidize methane to carbon dioxide and water. Enrichment Cultures and Pure Cultures. The methane bacteria have not been studied so extensively as most other groups of bacteria of comparable scientific and practical importance. The reason for this is readily apparent. In order to study the biology and biochemistry of bacteria most effectively, it is to necessary to use pure cultures-i.e.,

INDUSTRIAL AND ENGINEERING CHEMISTRY

study one species a t a time. Unfortunately, with the methane bacteria this elementary but basic requirement has been difficult and in many instances impossible to achieve. Until 1936 all attempts to isolate pur? cultures or even to obtain growth or colonies in solid media were unsuccessful (2). Consequently all the early studies of methane bacteria and many of the more recent studies were of necessity carried out with enrichment cultures--Le., cultures in Lvhich the substrate and environmental conditions were chosen in such a way as to favor strongly the development of certain species of methane bacteria without, however, excluding a substantial and frequently significant number of other bacterial species, both methane-producing and -nonproducing types. By the use of enrichment cultures it was possible to obtain considerable information about the existing morphological types of methane bacteria, the general environmental conditions that favor their development, the kinds of substrates attacked, and the over-all chemical reactions that occur: but many points of biology, nutrition, and biochemistry could not be elucidated with these cultures ( 7 , 2). Since 1936, four species of methane

WATER P U R I F I C A T I O N bacteria-Methanobacterium omelianskii ( 4 ) ) .Mb. formicicum ( 7 6 ) ,Methanosarcina barkerii ( 7 6 ) , and Methanococcus oannielii (27)belonging in three different genera have been isolated in strictly pure culture. .4n additional four species-Mb. suboxydans (79)> M b . sohngenii ( 2 ) ) M s . methanica, and M c . marei-have been purified by isolation and reisolation of colonies from agar media until they were free of other species of methane-producing bacteria, but they may have been contaminated by other anaerobic bacteria. The complete or partial purification of these eight species indicates that the isolation of pure cultures of other species should also be possible. It must be emphasized, however, that the isolation of methane bacteria is generally a difficult and time-consuming task requiring great patience and considerable experience with anaerobic techniques. As far as the author is aware, pure cultures have been obtained until now in only three laboratories. Even after a pure culture has been isolated its preservation is no easy problem. Most of the pure cultures have been lost after a few years. Only one species, M b . omelianskii, has been kept in pure culture for a long period of time. Because of the difficulties encountered in the isolation and maintenance of pure cultures of methane bacteria only a rather limited amount of information has been obtained concerning the biological and physiological characteristics of the group as a whole. h'evertheless it is possible to make some tentative generalizations that are useful in defining the group and indicating the probable behavior of its members under a variety of circumstances. Relation to Oxygen. All the species that have been studied are strictly anaerobic-that is, they develop only in the absence of oxygen and in the presence of a suitable reducing agent. The methane bacteria are much more sensitive to oxygen or other effective oxidizing agent? such as nitrate, than most other anaerobes. For this reason it is much easier to grow them in liquid or semisolid media than on the surface of a n agar medium. Even in liquid media not fully protected from air, sufficient oxygen may leak in to inhibit cultures that have passed the peak of their activity. The beneficial effect on the growth of methane bacteria, caused by the addition of solid sediments, including shredded asbestos ( 6 ) , to a liquid medium may be partially attributable to a mechanical shielding of the bacteria from dissolved oxygen, although other explanations are possible. Satisfactory reducing agents for pure cultures are sodium sulfide and hydrosulfite. Care must be used in adjusting the concentration of these substances

since above the optimal level they become toxic to some species. hlylroie and Hungate (73) have recently described the successful use of hydrogen and palladium chloride as reducing systems in media for Methanobacterium forrnicicum. The usefulness of this reducing system supports the conclusion that a low oxidation-reduction potential in the medium is more important than the presence of a high concentration of a reduced sulfur compound per se. Energy Metabolism. A second characteristic of methane bacteria is that

Fatty Acids Formic .4cetic Propionate n-Bu tyric n-Valeric n-Caproic

methane-producing bacteria exist cannot be excluded, but convincing evidence for such organisms is lacking. As a group the methane-producing bacteria appear to be restricted to the utilization of relatively simple organic and inorganic compounds, many of which are products of the better known types of bacterial fermentations. The oxidizable substrates that have been shoivn by studies with pure or nearly pure cultures to be decomposed by methane bacteria fall into the following three groups:

Alcohols

Gases

Methanol Ethanol Propanol, n-, is0 Butanol, n-, is0 1-Pentanol

Hydrogen Carbon monoxide ( 7 7, 76) Carbon dioxide

their energy metabolism is specialized for a process that produces methane as a major product. A little later we shall take a closer look a t the nature of the energy-yielding reactions. Kow, it is only important to emphasize that the ability to form methane is not a common and widely distributed property of anaerobic bacteria but is restricted to a specialized group. A few possible exceptions to this generalization are recorded in the scientific literature. particularly in the Tvork of Laigret (72). Laigret reported that Clostridium perfringens. which normally does not produce methane, can be induced to do so in a peptone-formate medium by the addition of a small amount of iodine. However, because of the absence of experimental details, it is impossible to judge the validity of Laigret's work. A reinvestigation of this problem will be necessary before a definite conclusion can be reached concerning the ability of Clostridium perfringens to form methane. Compounds Fermented. Methane bacteria specialize not only in the chemical mechanisms of energy metabolism but also in respect to the types of substrates which they can utilize. For some obscure reason, all the methane-producing bacteria whose substrate requirements have been studied effectively by the use of pure or nearly pure cultures are unable to decompose the more usual substrates for bacteria such as carbohydrates and amino acids. Early reports of cellulose or glucose fermenting methane bacteria have not been confirmed; probably they were based on observations with mixed cultures containing both carbohydrate-fermenting and methaneproducing species. Of course the possibility that still unknown species of carbohydrate- or amino acid-fermenting,

Some additional compounds that are probably decomposed by methane bacteria, judging from observations made Lvith enrichment cultures, are: Acids n-Caprylic n-Capric Isobutyric Stearic Oleic Benzoic Phenylacetic

Acids Hydrocinnatnic Cinnamic Oxalic Succinic Acetone 2,S-Butylene glycol

These have all been shown, mainly by Buswell and his collaborators (8),to be converted by crude cultures derived from sewage sludge more or less quantitatively into methane and carbon dioxide. Since these compounds are not known to be attacked by other anaerobic bacteria under the test conditions used, it is probable that they are directly attacked by the methane bacteria. In the literature concerned with anaerobic decomposition processes in nature or in selvage sludge it is often stated that many other compounds such as sugars, cellulose, proteins. purines, glycerol, and hydroxy acids like lactic. citric, and tartaric are also suitable substrates for a methane fermentation. This is quite true in the sense that these substrates are decomposed and methane and carbon dioxide are formed. However, since these compounds are rapidly decomposed by many common bacteria that do not produce methane, it is virtually certain that the conversion of these substrates to methane is a two- or multistage biological process in which some bacteria convert the substrates to volatile fatty acids, alcohols, and other common fermentation products Lvhich are then transformed by methane bacteria to their characteristic products VOL. 48, NO. 9

0

SEPTEMBER 1956

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Species Substrate Specificity. In addition to the apparently severe limitations of the methane bacteria as a group with respect to utiiizable substrates, each species characteristically is restricted to the use of a few compounds. Some examples of the substrate specificities of different species are : Species Methano bacterium Jormicicum Methanobacterium omelianskii Methanobacterium suboxydans Methanosarcina barkerii

Oxidizable Substrates HQ?CO, formate H2,ethanol, primary and secondary alcohols Butyrate, valerate, caproate H?, CO, methanol, acetate

"blethanobacterium formicimm oxidizes only hydrogen, carbon monoxide: and formate ( 7 7 ) . Methanobacterium omelianskii cannot oxidize carbon monoxide or formate but specializes in the decomposition of primary and secondary shortchain aliphatic alcohols ( 3 ) . I t also uses hydrogen (5). Mb. suboxjdans cannot attack any of the previously mentioned compounds except possibly hydrogen which was not tested but specializes in the oxidation of C4 to C6 fatty acids to acetate and propionate (79). The latter compounds accumulate in the medium. Finally, Methanosarcina barkcrii is restricted to the decomposition of hydrogen: carbon monoxide, methanol, and acetate (76). This species does not attack longer chain alcohols or acids. If these species are representative, and there is reason to believe that they are. it is apparent that the methane bacteria show a rather extreme development of substrate specificity. This implies that several species of methane bacteria must be required for the complete fermentation of the variety of compounds that are present in sewage and decomposing plant materials. Even for the complete fermentation of so simple a compound as valeric acid? as many as three species of methane bacteria are required. Valerate is oxidized by *%lb.suboxjdans to acetate and propionate which are not further attacked by this organism (79). hlethane is formed in a coupled reduction reaction. .4 second species? M b . propionicum, converts the propionate to acetate: carbon dioxide, and methane (79). .Mb. propimicum cannot attack acetate. Therefore a third species such as hlethanosarcina methanica is required to ferment the acetate ( 7 ) . The need to establish a balanced population of bacteria to participate in this type of fermentation sequence undoubtedly explains why considerable time is always required to develop a culture capable of causing a rapid and complete fermentation of complex mixtures of organic compounds. When such a culture is built up, it is capable of maintaining itself more or less indefinitely when a fresh supply of organic materials is added continually, because the major products of the fer-

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mentation are gases which escape from the medium leaving behind very little in the way of potentially toxic by-products. Nutrition. The general nutrirional requirements of methane bacteria appear to be very simple, although this can be said bvith certainty only for those species which have been studied in pure culture.

These species \vi11 all pro\\. ivcll in media containing the usual nutritive salts. carbon dioxide, a reducing agent, a single oxidizable compound suitable for the organism, and with ammonium ion as a source of nitrogen (4, 73. 76). One species, h l b . omelianskii. has been shoun to utilize nitrogen gas (75). The general physiological properties of the methane bacteria suggest, by comparison with other nitrogen-fixing bacteria, that the ability to fix nitrogen may be a common characteristic of the group. The addition of extracts containing amino acids growth factors, and other nutritional supplements to synthetic media does not have a beneficial effect on the rate or magnitude of groivth of the few species that have been studied in this respect.

oxide. However, there is at least one exception to the generalization that an alkaline medium is unfavorable. The formate fermenting species, M c . aannielii, grows best between pH 8 and 9 (27). So far no methane bacteria preferring acid media have been reported. Taxonomy, Three genera of methane bacteria, based on morphology, have SO far been recognized. The rod-shaped organisms are placed in the genus Methanobacterium. The species vary in the size and arrangement of the cells, the presence or absence of flagella, and the occurrence or nonoccurrence of endospores. Ultimately it will undoubtedly be desirable to subdivide the rod-shaped methane bacteria between two generaliethanobacterium for those that do not form spores and Methanobacillus for the spore formers. The small spherical organisms ivhose cells occur singly or in irregular masses are put in the genus .Uethanococcus. Some of the species of this genus are motile, others are nonmotile; no endospores have been observed. Finally, the organisms with large, more-or-less spherical cells that occur in three-dimensional sarcina packets are included in the genus hlethanosarcina. So far a total of eight species, distinguished by biochemical characters, have been described in these three genera as shown;

I. .I~~tlianobacterium-rod-shaped cells 1. .Vb. formicicum Formate, CO: H2 2. M b . omelianskii Primary and secondary alcohols, H ? 3. M b . propionicum Propionate 4. ,Mb. sohngenii Acetate, butyrate 11. Jlethanococcus-spherical 1. M c . mazei 2 . M c . uannieiii

cells, not in sarcina arrangexnent Acetate, butyrate Formate, H ?

111, Methanosarcina-spherical

1. M s . methanica 2. M s . barkerii

cells in sarcina arrangement .4cetate, butyrate(?) Methanol. acetate, H2:CO

Special attention should be draivn to the carbon dioxide requirement of methane bacteria. Qualitatively this is not unusual since apparently all microorganisms require small amounts of carbon dioxide to initiate and maintain groivth. HoLvever, several species of methane bacteria need a very large amount of carbon dioxide because they use this compound as a major substrate. The specific role of carbon dioxide in the energy metabolism of methane bacteria is discussed later. pH Range. Another environmental factor that is important for the grolvth of methane bacteria is the hydrogen ion concentration, As a general rule these bacteria are most active in rhe pH range from 6.4 to 7.2. Below p H 6 and above p H 8 the growth rate of methane bacteria falls off rapidly. At the high pH this may be the result of the greatly diminished concentration of free carbon di-

INDUSTRIAL AND ENGINEERING CHEMISTRY

Undoubtedly many more species \+ill be discovered as more adequate isolation methods are developed, and the organisms associated \vith different habitats are systematically investigated. The world of methane bacteria is well populated and many of its inhabitants remain to be identified. Chemistry of Methane Fermentations The early studies on the chemistry of methane fermentations were done entirely mith enrichment cultures containing a single substrate and a mixture of organisms. The main result of such studies was the demonstration that a variety of compounds can be more or less quantitatively converted to methane and carbon dioxide. Equations summarizing several of the observed reactions are :

WATER P U R I F I C A T I O N CH3COOH -P 4CHaCH2COOH

+ CO:

CHI

+ 2H20i C H 4 + 5COn +

+

~ C H ~ C H S C H ~ C O O H2H?O jCH, 3C0~ 2CH3CHzOH 3CH4 CO? CHsCOCH3 HpO 2CH4 COn

+

+

-

+ ++ +

The equations show that the fermentations of acetic. propionic, and butyric acids, ethanol, or acetone all give the same products. Only the ratio of methane to carbon dioxide changes Ivith the oxidation state of the substrate. The remarkable aspect of this result is that the nature of the products is independent of the structure of the substrate. An explanation for the formation of carbon dioxide was not difficult to find. Presumably it was formed by the complete oxidation of part of the substrate. But why should methane always be formed? For one substrate, acetate: a simple and reasonable explanation was suggestednamely, that the acid is decarboxylated forming carbon dioxide from the carboxyl group and methane from the methyl group. Unfortunately this explanation is of no help with propionate which according to the hypothesis should yield ethane on decarboxylation. Actually no ethane could be detected (70. 22). Another hypothesis is that the propionate is oxidized to acetate and carbon dioxide, and then the acetate is decarboxylated. But this hypothesis predicts the formation of only 1 mole of methanef’mole of propionate, whereas actually 1.75 moles of methane is formed. KO generally applicable chemical hypothesis for the origin of methane was developed until 1934, when van Niel suggested the so-called carbon dioxide reduction theory (7). This theory postulates that the organic compounds fermented by methane bacteria are oxidized completely to carbon dioxide, and this oxidation is coupled with a reduction of some or all of the carbon dioxide to methane. T h e application of this idea to the fermentation of acetate is as follows : Oxidation

CHaCOOH

+ COi

Reduction

8H

Net

CHaCOOH

+ 2H20

+

+ +2 H8H 20

2CO2

-c -P

CH4

CHI

+ COI

The oxidation of acetate gives 2 moles of carbon dioxide and 8 equivalents of hydrogen. One carbon dioxide is reduced to methane and water. The net result would be the conversion of 1 acetate to 1 methane and 1 mole of carbon dioxide. The main evidence for the theory a t the time it was developed was provided by the early observations of Sohngen (77) on the fermentation of a mixture of hydrogen and carbon dioxide. Sohngen

found that enrichment cultures could couple the oxidation of hydrogen with the reduction of carbon dioxide to methane according to 4H2

+ COS

CHd

-t

2 moles of butyrate to 4 moles of acetate is coupled with the reduction of 1 mole of carbon dioxide to methane (79). In this fermentation also tracer experiments have shown that a t least 987, of the methane is derived from carbon dioxide. In the preceding examples of methane fermentations involving carbon dioxide reduction essentially no carbon dioxide is formed in the oxidation of the organic substrate. The fermentation of propionate by ‘Mb. propionicum is somewhat more complicated because it involves both carbon dioxide formation and utilization (75’) :

+ 2H.0

The experimental observations Ivere later confirmed in several laboratories by the use of pure cultures (5, 7 7 ) . Here is a clear-cut example of carbon dioxide reduction to methane by means of a n inorganic reducing agent. Much of the subsequent Ivork on the chemistry of the fermentation has been concerned with finding out Lvhether the

+ 8H20 + 24H + 2H20

Oxidation 4CH3CHzCOOH Reduction 3COp Net 4CHaCH2COOH

carbon dioxide reduction theory also applies to fermentations of organic substrates. By now a considerable amount of information has accumulated which shows that the theory is correct for fermentations of many but not all substrates. Examples of the reduction of carbon dioxide to methane are provided by the fermentations of ethanol by M b . omelianskii: of butyrate by M b . suboxydans: and of propionate by M b . propionicum. IVith all these species, the interpretation of the results is greatly simplified by the fact that they oxidize their substrates to acetate which they are unable to decompose. M b . omelianskii oxidizes ethyl alcohol quantitatively to acetate (3) according to 2CHsCH20H

+ COz

-*

2CHaCOOH

+

CHa

For each 2 moles of alcohol oxidized 1 mole of carbon dioxide is used up and 1 mole of methane is formed. Furthermore the oxidation of alcohol is dependent on the supply of carbon dioxide; when carbon dioxide is used up, the oxidation of alcohol stops. Now the equation, which closely represents the experimental data, strongly indicates that the acetate is derived from the alcohol, and methane is derived from carbon dioxide. T o confirm this interpretation by an independent method, unlabeled ethyl alcohol was incubated with C14-labeled carbon dioxide (78). At the end of the fermentation, the C“ content of the methane was found to be approximately equal to that of the carbon dioxide. This proved that the methane produced in this fermentation is derived mainly if not entirely from carbon dioxide, The fermentation of butyrate by .Mb. suboxydans is very similar in principle : 2CH3CH2CHzCOOH

+ 2Hp0 + 602

4CH3COOH

+

-

kH4

As the equation shoivs, the oxidation of

-* +

+ +

+ 24H + 3CH4

4CH8COOH 4co2 3CH4 6HiO 4CH3COOH COY

+

The observed reaction, shown in the third line, is a conversion of 4 moles of propionate to 4 moles of acetate, 1 mole of carbon dioxide, and 3 molrs of methane. Although only 1 mole of carbon dioxide accumulates, it is reasonable, on the basis of Lvhat is known about the oxidation of propionate in other biological systems, to assume that the oxidation initially results in the formation of equimolar quantities of acetate and carbon dioxide and the removal of 24 “hydrogens” or electrons as is shown in line 1. If these 24 hydrogens serve to reduce carbon dioxide, 3 moles would be converted to methane (line 2) which accounts for the observed yield of me thane. The postulated intermediate role of carbon dioxide was tested by experiments with C14-labeled carbon dioxide or propionate. IVithout going into the experimental details, the results may be interpreted to indicate that approximately 1 mole of carbon dioxide is formed per mole of propionate, in accordance with line 1, and that carbon dioxide is a precursor of most, if not all, of the methane as indicated on line 2. Buswell and his associates (7), have also done tracer experiments on the fermentation of propionate using enrichment cultures, presumably containing a mixture of species and capable of converting propionate completely to carbon dioxide and methane without the accumulation of acetate. These cultures were shown to use carbon dioxide and to convert all three carbon atoms of propionate to both methane and carbon dioxide in varying degrees. Part of these results can be interpreted in terms of the reactions already discussed in combination with a secondary decomposition of acetate. However, some additional reaction must be involved to account for the preferential conversion of the alpha carbon of propionate to methane and a substantial conversion of the beta carbon to carbon dioxide. A possible exVOL. 48, NO. 9

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SEPTEMBER 1956

144 1

planation for these results would be the formation from propionate of a symmetrical intermediate like succinate. This would cause a randomization of the alpha and beta carbons of propionate and therefore would account for the similarity in their behavior. Other explanations are also possible. This type of problem probably cannot be resolved until pure culturcs of all of the orgar.isn:s involved are available. The results mentioned, as ~ v c l l as others, show conclusively that with many substrates and a t least several spxies? methane is formed mainly or wholly by rcduction of carbon dioxide. However, there are a t least t\vo compounds, methanol and acetate, which are converted to methane by chemical pathways not involving carbon dioxide. Schnellen ( 7 6 ) : working in Kluyver's laboratory, showed that methyl alcohol is readily fermented by a species of Methanosarcina according to 4CHzOH

-t

3CH4

+

COz

+ 2H?O

The possibility existed a t that time that the complete oxidation of the alcohol was being coupled with the reduction of carbon dioxide. Hoivever this possibility was excluded by tracer experiments which showed that less than lYc of the methane was derived from carbon dioxide ( 7 8 ) . ll-ith acetate, which is decomposed by several species of methane bacteria: the situation is similar. The fermentation of acetate is equivalent to a decarboxylation. According to van Kiel's theory, acetate should be completely oxidized to carbon dioxide, half of which is simultaneously reduced to methane. This mechanism was first critically tested by Buswell and Sollo ( 9 ) in 1948 by the cse of CI4-labeled carbon dioxide. T h r y demonstrated that essentially none of the methane i s derived from carbon dioxide. Subsequently Stadtman and the author (78, 20) showed by the tracer method that the methane is derived entirely from the methyl carbon, and the carbon dioxide exclusively from the carboxyl carbon of acetate :

These results are inconsistent Lvith the carbon dioxide reduction theory since the methyl carbon of acetate is not oxidized to carbon dioxide and carbon dioxide is not a precursor of methane. After the fate of the carbon atoms of acetate was established, it became of interest to find out what happens to the hydrogen a t o m attached to the methyl group, in particular, whether some or all of these hydrogen atoms are removed by a n oxidative reaction during the course of the fermentation of acetate or whether the methyl group is incorporated intact into methane. This question

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was investigated by Pine (74) in experiments schematically represcnted by H20

CDaCOOH

--+ C:I),H

D,O CHJCOOH > C'H,U

+ CO? + CO?

In the first experinienr (line 1 ) acptate labeled in the methyl group \vith deuterium was fermented and the amount of deuterium in the evolved methane \\-as compared \\-ith that in the substrate. The analysis showed that thr isotope contents of thr acetate and methane were the same. In the s x o n d experiment (line 2), unlabeled acetate \\-as fermented in the presence of D20. The data indicate that approximately one atom of deuterium per molecule of methane was derived from the solvent. These results demonstrate that the methyl group is transferred from acetate into methane as a unit, \Yithout the loss of attached hydrogen or deuterium. This result suggests that one or more transmethylation reactions may occur during the fermentation. The specific reactions involved in methane formation from carbon dioxide, acetate, or methanol are still largely a matter of speculation. The simplest mechanism proposed for the conversion of carbon dioxide to methane is a step\vise reduction involving formic acid or carbon monoxide, formaldehyde. and methanol as intermediates. HOWever, rncst of the evidrnce is against such a series of reactions. For example, Kluyver and Schnellen ( 7 7) have shown that several methane bacteria cannot use these postulated intermediates as substitutes for carbon dioxide when hydrogen is used as the reductant. 1,Vhen formate and carbon monoxide are ustd by methane bacteria they are usually first oxidized to carbon dioxide which is then reduced. Pine (personal communication) has recently obtained evidence for a more direct conversion of formate to methane by .Mb. omelianskii, but the rate of the process is slow compared to carbon dioxide reduction. Therefore it is virtually certain that the intermediates in methane formation are not one-carbon compounds. A schematic reprmntation of the possible pathkvays of carbon in methane formation is as folIo\vs: C:Oz

+ RH

CHiOH

+ KH

INDUSTRIAL AND ENGINEERING CHEMISTRY

Literature Cited Barker. H .4.,Arch .2fzkrobio1 7 , 40419 119361. (2) Ibid., pp. 420-38 (3) Barker, H. A , , J . Bzol. Chem. 137, 153-67 (1941). (4) Barker, H. .4.,Letuztenhoek 6, 201-20 (1940). ( 5 ) Barker, H. A., Proc. 2Vatl. clcad. Sci. V.S.29, 184-90 (1943). (6) Breden, C. R., Buswell. .4.51., J . Bacteriol. 26, 379-83 (1933). (7) Buswell, A. M., Fina, L., Mueller, H. F., Yahiro, A , , J . Am. Chem. Soc. 73, 1809-11 (1951). ( 8 ) Buswell, -4. M., Neave, S.L., Division of State Water Survey, Illinois, Bull. 30, 1930. [9) Buswell, A . M., Sollo, F. LV., J . A m . Chem. Soc. 7 0 , 1778-80 (1948). (10) Davis, J. B., Squires, R. M., Science 119, 381-2 (1954). (11) Kluyver, .4.J., Schnellen, C. G. T. P.? Arch. Biochem. 14, 57-70 (1947). (12) Laigret, J. C. R., Acad. Sci. 221, 353 i 1945). (13) Mylroiej R. L., Hungate, R. E., Can. J . Microbial. 1, 55-64 (1954). (14) Pine, hl. J., Barker, H. A,, Bacteiioi. Proc. 1954, p. 98. 115) Pine, M. J.. Barker, H. A , , J . Bacterbl. 68, i89-91 (1954). Schnellen, C. G. T. P., Dissertation Tech., Univ. Delft, Holland, 1947. Sohngen, N. L., Rec. traL8. chim. 29, 238-74 (1910). Stadtman, T. C., Barker, H. h.,Arch. Biochem. 21, 256-64 (1949). Stadtman, T. C . , Barker, H. h.,J . Bacteriol. 61, 67-80 (1951). Ibid., pp. 81-6. Ibld., 62, 269-80, 1951. Thayer, L. A , Bull. Am. Assoc. Petroleum Geologists 15, 441-53 (1931). i1j

'

R,COOH +2H R', CHO

4

+2H R CH?OH

1 1

+2H

CH3COOH

Carbon dioxide is postulated to combine with a n unidentified organic compound R H to form a carboxylated derivative of R ? which is then reduced by three successive steps to a methyl derivative of R which on reduction yields methane and regenerates the carbon dioxide acceptor. Methanol and acetic acid are postulated to react with RH to give the intermediate R.CH3 of the carbon dioxide reduction pathway. The direct formation of this intermediate from the organic methyl donors might well inhibit carbon dioxide utilization-for example, by lowering the concentration of RH available to reaction with carbon dioxide or in other ways. This scheme, Tvhich is only a working hypothesis, has the \'irtue ' of providing a unified theory of methane formation from various sources. A unified theory appears desirable because carbon dioxide, acetate, and methanol can all be used as sources of methane by a single species. I t is improbable that an organism would possess several types of chemical machinery for making methane from different sources. S o w it is only necessary to identify the hypothetical compounds postulated in this scheme.

+ RH + R

CH,

+ COP

+2H RH CHI

+

RECEIVED for review January 12, 1956 A C C E P T E D April 13, 1956

WATER PURIFICATION

DISCUSSION

.. .

Biological Formation of Methane

s i n c e our laboratory has been concerned largely with developing the techniques or know-how for the application of anaerobic fermentation in reducing waste material to carbon dioxide and methane a t high rates, it appears appropriate to summarize this phase of the problem. The methane fermentation, as exp!ained by Barker, is unique in that it may be carried on with the aid of mixed or enriched cultures; hence it is possible to maintain this process on a large scale conrinuously, for apparently a n indefinite period. This is in contrast to many other types of fermentations which require sterile substrate and pure culture inoculations. A second unique characteristic of this fermentation is that with the exception of a few fundamental limitations, such as the necessary mineral salts and ions and the proper carbon-nitrogen ratio, the fermentation can be applied to any type of substrate. Lignin and mineral oil appear to be the only materials not fermentable. The third characteristic is that in a majority of casm the reaction is quantitative, converting the entire substrate to carbon dioxide and methane, according to a n empirical equation referred to by Barker and developed in this laboratory. Fourthly, no specific temperature limitation is observed. The fermentation can be carried out a t temperatures as lo\v as 0’ C. and as high as 55’ C. The rate a t higher temperatures is of course greatly increased. Holyever, once a culture has been acclimated to a given temperature-for example, 26’ or 37’ C. -a sudden change of as little as one or tivo degrees may completely interrupt the methane fermentation with the accumulation of acids. Fifthly, the necessity of asbestos in .certain pure compound culture work on a laboratory scale has been mentioned. The presence of inert solid material is equally- important in large-scale applications. Addition of straw or sawdust to industrial wastes may be required. Sixthly, Barker has indicated that the decomposition of more complicated or.ganic compounds passes through the

lolver aliphatic acids, characteristically acetic, and that they are the immediate precursors of carbon dioxide and methane. It has been observed empirically that, if the substrate concentration is too high, there is a tendency for these acids to accumulate faster than their conversion into methane occurs. Lnder this condition, methane formation may be arrested completely. This charactcristic of the process is regularly controlled through determination of the amount of volatile acids b>- a well-known modification of the Duclaux procedure. Empirically it has been found that if the volatile acids rise above 2000 to 3000 p.p.m. a condition may develop under which methane fermentation cannot get under way or will be stopped. The addition of alkali does not remedy this situation since it is not a p H effect. It can only be controlled by limiting the amount and rate of addition of substrate. (If scum is allowed to accumulate. it constitutes a zone of excessive amount of substrate and will result in the local production of excessive amounts of volatile acids. Mineral salts begin to inhibit the fermentation a t 4000 p.p.m. and SO p.p.m. of NOsS inhibits i t completely.) initiation and Control of the Methane Fermentation

The design of a plant for treatment of \+Tastes bv anaerobic fermentation requires a knowledge of the total volume to be treated per day, the content of organic matter, a sufficient chemical anahsis to indicate the carbon to nitrogen ratio. and thc presence of necessary mineral salts. The actual p H of the liquor to be treated is not so important as the knowledge of the materials contained therein which may affect the acid concentration during fermentation. Assuming the proper chemical and physical composition of the waste to be treated. the following rules must be observed : 1. I n starting the fermentation, the initial load must not exceed one tenth of the anticipated final load. This load is increased by 50 to 100$70 per day with

daily determination of volatile acids. If volatile acids begin to increase rapidly the rate of feed must be decreased. The rate of feed which results in a relatively constant volatile acid content below 2000 p.p.m. is considered a safe continuous load. 2. The temperature selected for the fermentation must be kept constant within 1 1 ’ or 2’ C. 3. T o prevent overheated or high concentration zones the tank must be provided with means for circulating from the bottom, middle, and top to the top, middle, and bottom. I n other words. complete circulation must be provided for. The amount of this circulation usually does not exceed a n hour a day in properly operating tanks. For safety, we prefer to operate this process in a series of three tanks provided with piping for complete intrrcirculation, thus avoiding the necessity of dumping a tank which may become sour because of poor operation. \2:here the procedures indicated are carefully observed, it has been found possible to load treatment tanks with as much as 1 6 kilograms per cubic meter per day (1 lb./cu.ft./day). Without such precautions, the loadings must be kept to one tenth to one twentieth of the figure cited. And finally in the writer’s cxxperience, the process finds its greatest economy in wastes running 1 to 3’3, digestible solids. Less than 1% solids is likely to result in a n undesirably large installation and in most cases over 3% solids \vi11 usually justify evaporation with the recovery of dry material for either food or fertilizer. As is no doubt well known, this process is in very general use, and the gas produced is employed either in internal combustion engines for the production of power or, after proper scrubbing, is sold to local gas companies and mixed with regular city gas supplies. An extensive bibliography of the process can be found in chapter 14 of “Industrial Fermentations,” vol. 2, Chemical Publishing Co., New York, 1954.

A. M. BUSWELL University of Florida, Gainesville, Fla. VOL. 48, NO. 9

SEPTEMBER 1956

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J. B. DAVIS Magnolia Petroleum Co., Dallas, Tex.

Microbia Decomposition of Hywocarbons Academic and applied aspects of microbial decomposition of hydrocarbons are being increasingly appreciated. The variety of microorganisms which decompose hydrocarbons including bacteria, actinomycetes, filamentous fungi, and yeasts indicate that this form of organic material is readily utilized. Potential applications of microbial decomposition of hydrocarbons include products formation, waste disposal, and oil prospecting.

MICROBIOLOGISTS

have found that hydrocarbons are utilized by certain bacteria, actinom)-cetes, filamentous fungi, and yeasts, although they are not so readily decomposed, generally, as are carbohydrates, proteins, or fats. H!-drocarbons are produced in relatively small quantities in the biosphere. This fact. in addition to their innate chemical stability, may influence the abundance and prevalence of microbial forms capable of utilizing them for carbon and as a source of energy. However, the adaptability of microorganisms in nature, due either to short-term enzymic change or to mutagenic evolutionary change, results in a remarkable potentiality for microbial decomposition processes. The soil microbiologist considers any naturally produced organic compound susceptible to microbial decomposition. Presumably, the only organic matter that escapes decomposition is that which is buried in sedimentary rocks. Contrary to belief that accumulations such as petroleums and coals resulted primarily from their resistance to microbial decomposition, it is more likely that the prototype organic material fortuitously escaped microbial decomposition, and subsequent physicochemical processes converted it into comparatively stable fuels, each characteristic of its own peculiar geologic history. Various petroleums (crude oils) vary widely in their composition. Petroleum is essentially a complex mixture of paraffinic, naphthenic, and aromatic hydrocarbons, and of oxygen, nitrogen, and sulfur derivatives of these hydrocarbons. The microbial decomposition of these various compound types or fractions may be studied in bulk or as a single component representative of the respective fraction. The mechanism of hydrocarbon oxidation by microorganisms is not adequately known and little

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sustained effort has been made in this direction, possibly because hydrocarbons are considered of small biochemical significance. The applied aspects of microbial decomposition of hydrocarbons are consequently preceding fundamental scientific knowledge, after the pattern of the early fermentation industries. It is presumed that successful industrial applications of microbial hydrocarbon decompostion will stimulate adequate, academic effort in this field. ZoBell (36) in 1950 and Beerstecher ( 7 ) in 1954 comprehensively reviewed the general subject of hydrocarbon decomposition by microorganisms. Before discussing something of )chat is knoivn about microbial decomposition of hydrocarbons, some aspects m-hich are presently being evaluated or projected by industry may be of interest. First, natural gas, consisting chiefly of methane, is one of the cheapest sources of organic carbon. Bacteria, actinomycetes; and fungi isolated from soil readily utilize the hydrocarbons of natural gas which they convert principally into cell material and carbon dioxide. The cell material is of little practical value at present and no extracellular products of immediate consequence have been reported. Hoxvever: the potential should yield to research. Secondly, hydrocarbon-oxidizing soil microorganisms, as indexes of hydrocarbons emanating from petroleum deposits, are being exploited in the U.S..4. and in Russia for exploration purposes. Finally, a knowledge of microbial decomposition of crude oil and its products is being sought by the petroleum industry because of storage, disposal, and pollution problems.

Microbial Decomposition of Gaseous Aliphatic Hydrocarbons Interest in microbial decomposition of gaseous hydrocarbons was first stimulated

INDUSTRIAL AND ENGINEERING CHEMISTRY

by the ivork of Sohngen (27) and others (74: 76. 30) \vho isolated methaneoxidizing bacteria from soil and lvatcr. The organism described by Sohngen was later named .Mcthanomonas methanica by Orla-Jensen (IS). I t has been convenient, although probably inaccurate, to classify the bacteria studied by these different workers as strains of .Methanomonas meihanica. -Ifethanomonas methanica is probably the thick, rod-shaped, motile bacterium which can be isolated readily from pond mud and which characteristically forms a thick, pink pellicle on the supernatant of methane enrichment cultures. Sohngen described his bacterium more or less in this way. Ethane, propane, and butane are not utilized by this organism. There are methaneoxidizing bacteria, however, which d o not fit this description. Hutton and ZoBell (17) more recently reported methane-oxidizing bacteria which occur commonly in marine sediments and in soil, some of which also utilize ethane and propane. The methane-utilizing bacteria that they isolated from marine and soil environments Lvere all morphologically similar and would not utilize common bacteriological media such as peptone or glucose. I n 1949 Nechaeva ( 77) reported isolating two mycobacteria from soil tvhich utilized methane. Strawinski (23) reported in 1955 the isolation of methane. oxidizing bacteria from agitated soil enrichment cultures. These also are very probably not h'ethanomonas rnethanica. Critical descriptive, taxonomical studies of methane-oxidizing microorganisms \could be a desirable contribution. Dlvorkin discussed this point in his thesis (7). I n 1954, Bokova (2) reported isolating from soil nvo mycobacteria which utilized gaseous hydrocarbons with the exception of methane. Here is evidence