2.8　Methane-producing Bacteria and Their Classification

The main product of biogas fermentation is methane, which is produced by the action of methane-producing bacteria. Thus, methane-producing bacteria are the core of biogas fermenting microbes. The study of methane-producing bacteria is not only for investigating the mechanism of fermentation and biogas production but also of importance in studying the origin of life.

2.8.1　Classification and Identification of Methane-producing Bacteria

2.8.1.1　Characteristics of Methane-producing Bacteria

Methane-producing bacteria are a group of extremely specific microbes. They are very sensitive to oxygen and oxides, thus their anaerobiosis is very specific. They can only utilize simpler organic and inorganic compounds as their substrates. They grow rather slow.

Methane-producing bacteria are coupled with oxygen sensitive F420 factor, thus, even a small amount of oxygen present will certainly affect their action. Investigators found that when oxygen content in the fermenting aqua of 5mL reaches 0.8 mL, their growth will be damaged.

As stated above, methane-producing bacteria can only utilize simpler organic and inorganic compounds and as pointed out by Smith in 1980, under pure culture conditions, all of the methane-producing-bacteria can use H2and CO2as their substrates yielding CH4and most of them can also utilize formic acid for methane production. There is one species of them, i. e. methane-producing-sarcina, which can utilize methanol, methyl amine, and acetic acid, but can't use formic acid. As pointed out by many reports: in the natural biogas fermentation, acetic acid is a critical substrate for forming methane, amounting approximately for 74％ of the substrates.

The growth and reproduction of methane-producing bacteria are considerably slow. The time for doubling them is rather long, say, 4～6 d. So far as people known, the shortest doubling time for methane-producing bacteria is less than 3 h.

Owing to the difficulties for isolating, incubating and storing of methane-producing bacteria, development in study have long been advanced slowly. But, since the establishment of anaerobic techniques by Hungate, during the past two decades, especially recently, there is a great success in the study of methaneproducing bacteria. Up to now the pure species of methane-producing bacteria obtained are still few, say, 13. Again, there are some species, due to incompleteness of incubation methods, haven't been confirmed yet since the physiological and biochemical characteristics are not clear.

2.8.1.2　The Third Fashion of Life

A brilliant concept concerning methane-producing bacteria has been raised by a team headed by C. R. Woese, Illinois University, America. They thought that this kind of microbes present in anaerobic conditions may not be known previously and the microbes are of genetic ability—the third fashion of life.

For years, this kind of methane-producing microbes has been thought to belong to bacterium, though their size under electronic microscope are similar to commonly occurring bacteria but the structure of cell wall is different. Based on literature, it is found that they are different from neither bacteria nor higher living things but belong to a third catalogue (spectrum) which has no relationship with that of higher living things and unicellular living things, known as“Archaebacteria” spectrum. Actually, they are not bacteria but they originated from specific fashion of life more than 3.4 billion years (34 hundred million years) ago, i. e. the third fashion of life.

As pointed out by Younamupenum, authority of Chemistry Development, University of Maryland; “Life originates from anaerobic conditions” which coincide well with that mentioned above. 3 hundred million years ago, there was essentially no oxygen in the atmosphere around earth, instead there was plenty hydrogen and carbon dioxide, which was the favourite environment for the growth of methaneproducing bacteria.

In 1977, Archaebacteria, a brilliant concept, has been raised by a team, headed by Carl. R. Woese, Illinois University, America. These unusual species are genealogically neither prokaryotes nor eukaryotes. This discovery means there are not two lines of descent but three: the archarbacteria, the true bacteria and the eukaryotes. In 1992, Carl R. Woese used Archaebacteria instead of Archaeon (Fig.2.10).

2.8.1.3　Morphology and Classification of Methane-producing Bacteria

(1) Forms of methanobacteria

There are 4 forms of Methanobacterium:sarcina,bacillar,globular and spiral (Fig.2.11).

①Methanosarcina: the cell multiplication of Methanosarcina is regular, its size is similar like sand particles piled together, but not only its form but also its size is different from that of true Sarcina. Even in the same species of Methanosarcina,there exits difference in their envelops.

②Methanobacillus:Methanobacillus is bacillar,usually curved,like a chain or long filament while that of M.arbophilicum is short and straight,no filamentformation in liquid culture.

③Methanococcus: their globular cells are from round to oval, paired or chained.

④Methanospirillum hungatei: their cells appear regular, curved and finally form spiral.

(2) Classification of Methanobacterium

Regarding classification of Methanobacterium at present there is no betterintegrated idea. Barker (1956) summed up Methanobacteria in 1 family, i. e. Methanobactericeae,including bacilli and cocci.This family consists of 4 genera and 8 species.The members of Methanobacteria from a sludge digester are as follows.

①Formic acid Methane-producing bacteria≥1×107/mL.

②Ruminant Methane-producing bacteria≥1×107/mL.

③Methanosarcina Barkeri=1×106/mL.

④Methanobacterium sp.=1×108/mL.

⑤Methanobacterium sp.=1×107/mL.

⑥Methanococcus sp.=1×106/mL.

In the Bergy's Manual of Determinative Bacteriology, Methanobactericeae was summed up in 1 family, including 3 genera and 9 species.

Balch (1979) suggested a newer classification system, by which methanobacteria were grouped into 3 orders,9 families,7 genera and 13 species.Now it is added to 5 classes, 7 orders, 14 families, 33 genera and 101 species (Table 2.6).

2.8.2　Metabolic Substrates of Methane-producing Bacteria

The methane-producing bacteria can utilize H2, HCOOH, CH3OH, methylamine, acetic acid etc. as substrate to produce methane. Some special methanogen could can use of ethanol, propionic acid, iso-butyric acid to produce methane. About 2/3 of methane is originated from acetic acid splitting, while the other methane come from carbon dioxide reduction. In 1980, Wolfe studied metabolism of methane with pure cultivation. He found most methanogen can utilize H2and CO2to produce methane. Some chemical formulas of metabolic substrates of methane-producing bacteria are as follows:

H2reducing CO2:　　4H2+CO2=CH4+H2O　　(2-9)

Formic acid:　　4HCOOH=CH4+3CO2+2H2O　　(2-10)

Methanol:　　4CH3OH=3CH4+CO2+2H2O　　(2-11)

Acetic acid:　　CH3COOH=CH4+CO2　　(2-12)

Methylamine:　　4CH3NH+2H2O=3CH4+4NH3+CO2　　(2-13)

Dimethylamine:　　2(CH3)2NH+2H2O=3CH4+2NH3+CO2　　(2-14)

Trimethylamine:　　4(CH3)3N+6H2O=9CH4+4NH3+3CO2　　(2-15)

2.8.3　Theory of Methane Formation

2.8.3.1　Theory of Carbon Dioxide Reduction

One theory was proposed by Van Niel in the 1930s. He thought, under all conditions applied by different investigators, methane-producing organics must be converted to CO2and then reduced to form methane. It is seen from the following formulas:

4H2A=4A+8H　　(2-16)

8H+CO2=CH4+2H2O　　(2-17)

4H2A+CO2=CH4+2H2O+4A　　(2-18)

In the equation, H2A represents compounds capable of being used by biogas microbes, and CO2is generated from substrate oxidation. Studies of methane conversion from ethanol, butyrates and carbon monoxide all confirmed this theory. Up to now,all the known purely-cultured materials of methanobacteria are capable to reduce hydrogen to form methane.

2.8.3.2　Theory of Methyl Group Directly Convert to Methane

But in 1948, using isotope tracer, Buswell and Sollo stated completely opposite results compared to the theory mentioned above, hence a second theory was developed. According to their theory, methane can be generated directly from methyl group, not via CO2formation, as illustrated by results of experiments of labeled acetates:

It is also true in experiments of ethanol:

Through the experiments, it was known that the extra hydrogen of CH4is from water.

2.8.3.3　Barker's Hypothesis

At present, it is thought that methane is formed through two pathways.

During the process, methyl group of acetic acid is reduced directly from methane and carboxyl group of acetic acid is converted to CO2, and then the latter one is again reduced to form methane.

Based on the two theories mentioned above, Barker proposed a hypothesis as follow:

According to the theory, under anaerobic conditions, regardless of CO2, methanol or acetates, all should be converted to CH3X, and CH3X can further be reduced to form methane.

2.8.4　Methane Formation Metabolized by Acetate and Formate

In 1956, Barker et al. researched acetic acid reaction with Methanosarcina barkeri.It meant methyl forms methane,and carboxyl forms CO2.The formula is as follow.

14CH3COOH=14CH4+CO2　　(2-24)

In 1976, J. G. Zeikus et al. observed that acetate marked by methyl can form 14CO2, which meant the methyl in acetate can be oxidized into carbon dioxide.

There are two possible ways for acetate to form carbon dioxide. One is methyl directly forming methane, or first forming carbon dioxide then methane. Another is carboxyl first forming carbon dioxide then methane.

The possible way of methane formed by metabolizing of formate could be formate first converted to hydrogen and carbon dioxide by hydrogenase, then to methane.

In 1983, Ferguson and Mah researched formic acid metabolism forming methane with radioactive carbon dioxide. The reaction are as follows:

4HCOOH=4H2+4CO2　　(2-25)

4H2+CO2=CH4+2H2O　　(2-26)

Total reaction:

4HCOOH=CH4+3CO2+2H2O　　(2-27)

2.8.5　Methane Original

Most methanogen can reduct hydrogen to carbon dioxide to generate methane, while different methanobacteria can produce methane by using acetate, carbon oxide, ethanol, methanol, methyl amine and methyl sulfide. About 72％ of methane is originated from acetic acid, while 28％ of methane is from hydrogen reduction carbon dioxide (Fig.2.12).