The anaerobic digestive process is a natural biological process in which an interlaced community of bacteria cooperates to obtain a stable and auto-regulated fermentation through assimilation, transformation and decomposition of the residual organic matter present in waste and wastewater into biogas. This is a complex multistep process in terms of chemistry and microbiology, where the organic material is degraded to basic constituents to obtain methane gas under the absence of an electron acceptor such as oxygen. The common metabolic pathway and process microbiology of anaerobic digestion is shown in Fig. 1 (Khanal, 2008).

Generally, the anaerobic digestion process consists of four stages; the first one is called hydrolysis (or liquefaction), it consists in the transformation of complex organic matter such as proteins, carbohydrates and lipids into simple soluble products like sugars, long-chain fatty acids, amino acids and glycerin, this stage is carried out by the action of extracellular enzymes excreted by the fermentative (group 1) (Khanal, 2008).

In the second step, called the acidogenic stage fermentative bacteria use the hydrolysis products to form intermediate compounds like organic acids, including volatile fatty acids (VFA). Theses VFA along with ethanol are converted to acetic acid, hydrogen and carbon dioxide by other group of bacteria known as hydrogen-producing acetogenic bacteria (group 2) (Khanal, 2008).

Organic acids are oxidized partially by bacteria called acetogenic in the third stage, which produce additional quantities of hydrogen and acetic acid. The acetogenesis is regarded as thermodynamically unfavorable unless the hydrogen partial pressure is kept below 10-3 atm, pathway efficient removal of hydrogen by the hydrogen-consuming organisms such as hydrogenotrophic methanogens and/or homoacetogens (Zinder, 1988).

Finally, in the fourth stage, both acetic acid and hydrogen are the raw material for the growth of methanogenic bacteria, converting acetic acid and hydrogen to biogas composed mainly of methane, carbon dioxide and hydrogen sulfide (Khanal, 2008).

Acetate, H2 and CO2 are the primary substrate for methanogenesis. On chemical oxygen demand (COD) basis about 72% of methane production comes from the decarboxylation of acetate, while the remainder is from CO2 reduction (McCarty, 1964). The groups of microorganisms involved in the generation of methane from acetate are known as acetotrophic or aceticlastic methanogens (group 3). The remaining methane is generated

from H2 and CO2 by the hydrogenotrophic methanogens (group 4). Since methane is largely generated from acetate, acetotrophic methanogenesis is the rate-limiting step in anaerobic wastewater treatment. The synthesis of acetate from H2 and CO2 by homoacetogens (group 5) has not been widely studied. Mackie and Bryant (1981) reported that acetate synthesis through this pathway accounts for only 1-2% of total acetate formation at 40°C and 3-4% total solids at 60°C in a cattle waste digester.

1.1 Process microbiology

a. Fermentative Bacteria (group 1): This group of bacteria is responsible for the first stage of anaerobic processes. The anaerobic species belonging to the family of Streptococcaceae and Enterobacteriaceae and the genera of Bacteroides, Clostridium, Butyrivibrio, Eubacterium, Bifidobacterium and Lactobacillus are most commonly involved in this process (Novaes, 1986).

b. Hydrogen-Producing Acetogenic Bacteria (group 2): This group of bacteria metabolizes higher organic acids (propionate, butyrate, H2, etc.), ethanol and certain aromatic compounds (i. e. benzoate) into acetate, H2 and CO2 (Zinder, 1998). The anaerobic oxidation of these compounds is not favorable thermodynamically by hydrogen-producing bacteria in a pure culture, however in a coculture of hydrogen — producing acetogenic bacteria and hydrogen-consuming methanogenic bacteria, these exists a symbiotic relationship between these two groups of bacteria. It is important to point out that during anaerobic treatment of complex wastewater such as vinasses or slaughterhouse, as many as 30% of the electrons is associated with propionate oxidation. Thus, these chemical appears to be more critical than oxidation of other organic acids and solvents (Deublein and Steinhaunser 2008).

c. Homoacetogens Bacteria (group 3): Homoacetogenesis has attracted much attention in recent years because of its final product acetate, an important precursor to methane generation. The responsible bacteria are either autotrophs or heterotrophs. The autotrophic homoacetogens utilize a mixture of hydrogen and carbon dioxide, with CO2 serving as the carbon source for cell synthesis. The heterotrophics homoacetogens, on the other hand, use organic substrate such as formate and methanol as a carbon source while producing acetate as the end product (Eq. 1 to 4) (Khanal, 2008).

CO2 + H2 ^ CH3COOH + 2H2O (1)

4CO + 2H2O ^ CH3COOH + 2CO2 (2)

4HCOOH ^ CH3COOH + 2CO2 + 2H2O (3)

4CH3OH + 2CO2 ^ 3CH3COOH + 2CO2 (4)

Acetobacterium woodii and Clostridium aceticum are the two mesophilic homoacetogenic bacteria isolated from sewage sludge (Novaes1986). Homoacetogenic bacteria have a high thermodynamic efficiency; as result there is no accumulation of H2 and CO2 during growth on multicarbon compounds (Zeikus 1981).

d. Metanogenic Bacteria (group 4 and 5): Methanogens are obligate anaerobes and considered as a rate-limiting specie in anaerobic treatment of wastewater. Abundant methanogens are found in anaerobic environments rich in organic matter such as swamps, marches, ponds, lake and marine sediments, and rumen of cattle. Most methanogens can grow by H2 as a source of electrons via hydrogenase as shown in the follow reaction (Eq. 5) (Khanal, 2008):

4H2 + CO2 ^ CH4 + 2H2O (5)

The source of H2 is the catabolic product of other bacteria in the system, such as hydrogen — producing fermentative bacteria, especially Clostridia (group 1) and hydrogen-producing acetogenic bacteria (group 2). The hydrogenotrophic pathway contributes up to 28% of the

methane generation in an anaerobic treatment system. It bears mentioning that there are many H2-using methanogens that can use formate as a source of electrons for the reduction de CO2 to methane, as show in reaction (Eq. 6):

4HCOO — + 2H+ ^ CH4 + CO2 + 2HCO3- (6)