2.2　Microbes in Three-step of Biogas Fermentation

2.2.1　Biogas Microbes in Nature

Biogas fermentation is a common phenomenon and a typical process in nature and an important component of Material Circle (Fig.2.4).

Biogas fermenting microbes are widely distributed in nature, especially in lakes, manure pit, sewage and various organized sludge. They are resources for obtaining utilizable biogas microbes for human.

Methane bacteria are also found in some advanced plants and animals, among which the stomach of ruminants is a typical organ of biogas fermentation. By studying microbes in ruminant stomach, men obtain good knowledge concerning bacteria. There are plenty of biogas bacteria in ruminant stomach where methane and carbon dioxide are produced. In the ruminant stomach of a milk cow, there are 100L cellulose fermenting material, which can produce more than 200L methane which is discharged during breathing.

2.2.2　Groups and Actions of Non-methane-producing Microbes

Non-methane-producing microbes are series of microbes which convert complex organics into simpler, smaller molecular compounds (Fig.2.5), i.e. the fermenting bacteria and hydrogen-producing bacteria mentioned previously. There is a large aumount of microbes involved in this step, which include anaerobes and facultative anaerobes. Their variety and amount change with the variety and quantity of fermenting materials.

Although at present, the basic understanding of the physiological groups of various microbes in non-methane-producing step is achieved and the study of isolation and identification of some pure strains of some bacteria is performed by some investigators, the microbiology of non-methane producing step is still unclear and needs further study.

2.2.2.1　Variety of Non-methane-producing Microbes

By classification, the variety of non-methane-producing microbes can be divided into three groups, namely, bacteria, fungi and protozoa, among which bacteria is the most important one.

(1) Bacteria

There are many types of non-methane-producing bacteria and the amount of them is large too. But those microbes with hydrolytic activity only accounts for a small group out of all the colonies, among which the obligate anaerobes are 100～200 times more than those of the facultative anaerobes and aerobes. The obligate anaerobes involved in non-methane-producing step which play an important role in the process, include clostridium butyricum, bacillus lactam, Gram's positive cocci and so on. In the 1950s and 1960s, the study of isolation and identification of non-methane-producing bacteria was performed by many investigators. The result of anaerobic digesting microbes in pig dung showed that there were three clonies of anaerobic and facultative anaerobes: the family of facultative cocci,43％～74％;the family of Bacteroides,20％～80％;other anaerobic bacteria, mainly clostridium, with a small number of curved Gram's negative bacillus.

According to the data, the non-methane-producing bacteria isolated and studied have reached 50 species in 18 genera (Table 2.1).

Based on their physiological groups, non-methane-producing bacteria are again divided into 7 groups, namely: cellulose-splitting, semi-cellulose-splitting, protein-splitting, fat-splitting, hydrogen-producing bacteria and other specific microbes, such as thio-vibro and lactic acid-utilizing ones. Certain colony group of bacteria can also have several functions, for example, some cellulose-splitting bacteria may be capable of catabolizing proteins.

(2) Fungi

In the 1950s and 1960s, Cooke found a great number of moulds and yeasts in anaerobic digestion and isolated 36 genera of moulds, among which there were plenty of half-known microbes and some Zygo-rhynchus. Through artificial cultivation and recovery detection test, he thought that moulds and yeasts might take part in digestion process, from which they got nutrients and grew.

(3) Protozoa

Protozoa are also detected during biogas fermentation process. Lackey(1952) and Liebmann (1936) pointed out that protozoa detected were mainly plasmodium,flagellate and amoeba etc., totally 18 species, in a small amount, so Lackey considered that they might play a minor role in the process.

(4) The amount of non-methane-producing bacteria

In anaerobic digestion, bacteria are among the most of non-methaneproducing microbes. Bacterial counting was performed by many investigators but due to the counting techniques used and some other reasons, the results vary a lot (Table 2.2).

2.2.2.2　Amount of Non-methane-producing Microbes

Based on the technique of incubation of microbes from the ruminant stomach, a counting non-methane-producing bacteria in biogas fermenting system was developed and the amount of bacteria in several digesters are: 39×107～15×109/mL for obligate anaerobic non-methane-producing bacteria, 8×105～1×108/mL for aerobes and facultative aerobes; the amount of anaerobic bacteria is often 100 times more than aerobic ones. A similar result was obtained by Post et al in 1967, Kotz's et al in 1968 and Thiel et al in 1968. This indicates that in biogas fermentation, the species of non-methane producing bacteria in specific anaerobic conditions is the most important part of the overall colonies of microbes.

In addition, the physiological amount of some microbes in biogas fermentation was detected separately by many investigators. Data reported by Hungate in 1950 showed anaerobic cellulose-splitting bacteria in digesting sludges was (0.8～2.0)×103/mL, that for domestic wastes was 4×105/mL; the amount of semi-cellulosesplitting bacteria were similar to that of cellulose-splitting bacteria, with 104/mL or a bit more. Using dilution technique, the amount of protein-splitting Gram's positive bacteria in digesting sludges detected by Harkness was 7×104/mL. Sulfer-reducing bacteria detected from digesting sludges by Toerien was (3～5)× 104/mL, lactate-utilizing bacteria in the digested material of pig dung was 3×107/mL. The total amount of aerobic bacteria in digesters of several laboratories and large-scale-production units reported by Kotz's in 1968 was (3～300)×106/mL;aerobic protein-splitting bacteria was (1～90)×105mL and aerobic fat-splitting bacteria was (2～16)×104/mL.