Process

Biochemical Process

Anaerobic microbiological degredation is a multistep process in which different kinds of microorganism communities are in relation with eachother, and they derive energy and sustain microbiological growth by metabolising organic substances in an oxygen-free environment. At the end of the interrelated activities of the microorganisms, methane and carbondioxide are produced with little amount of hydrogen, water vapor, ammonia and hydrogen sulfide. The anaerobic digestion process can be subdivided into the following four phases, each requiring its own characteristic group of microorganisms. The following figure presents the biogas production stages briefly.

Hydrolysis : In anaerobic digestion process, hydrolysis step is the first step and is described as the break down of the complex organic materials into simple ones as in solubilization and depolymerization of the polymers. There are three main substrates in the wastes which are carbohydrates, proteins and lipids. In hydrolysis step, these substrates are broken down to monosaccharides, amino acids, long chain fatty acids and glycerol respectively. Hydrolysis of these substrates is accomplished by extracellular enzymes that is either attached to the bacteria` outer cell wall or secreted to the bulk substrate solution which are produced by fermentative bacteria. Hydrolysis of carbohydrates may take a few hours while hydrolysis of protein and lipids may take a few days. It is a common remark that hydrolysis is the rate limiting step in anaerobic digestion process because of the substrates (lipids, lignin, cellulose) that hardly hydrolyse, and because of the particulate matter in the bulk solution and pH of the environment.

Acidogenesis : The soluble organic materials (monomers) are taken up by fermentative bacteria in the absence of an electron acceptor (O2, NO-3, SO-4) and degraded into the volatile fatty acids (VFA) or short chain organic acids such as butyric acid, propionic acid and valeric acid and  hydrogen, carbon dioxide, and alcohols. Monosaccharides and amino acids are the most abundant substrates in degraded organic materials and have more energy content compared to other monomers. Dominant bacteria type in acidogenesis is the Clostridia microorganisms which can degrade both monosaccharides and amino acids. 

Acetogenesis : The meaning of acetogenetic phase is the production of acetate with the reduction of CO2 literarily. VFAs (butyric acid, propionic acid) and alcohols formed at the end of the fermentaiton process are further used  by hydrogen producing acetogenic bacteria as substrate and are converted into acetic acid, hydrogen and carbondioxide. Because of the production of H2 at the end of this process, the microorganisms involved in this phase are called hydrogen producing bacteria. On the other hand, the other group of acetogenic bacteria that use H+ and CO2 for growth and energy derivation are called homo-acetogens or hydrogen utilizing acetogens. They compete with methanogenic archaea for H2 and formate. Homoacetogens use H+ and CO2 as electron acceptors are very sensitive to high hydrogen concentrations or hydrogen partial pressure. This phenomena only proceed when methanogens that use hydrogen as substrate are active in the environment. The syntrophic relationship between hydrogen producers (acetogens) and hydrogen consumers (methanogens) is called interspecies hydrogen transfer. The figure below shows the interspecies hydrogen transfer between acetogens and methanogens.

It is stated that about 30% of the entire CH4 production in the anaerobic treatment can be attributed to the reduction of CO2 by H2 as shown in the figure above. Also, acetogenic phase in the final step is said to be the limiting phase of the methanization. The table below shows the conversion of most abundant VFAs into acetate and methane and H2.

Methanogenesis : The final step of the anaerobic process is the methane production phase called methanogenesis. In this phase acetate, CO2 and H2 and other ingredients with one carbon molecule that are produced by the acetogen bacteria in the acetogenic phase are used as substrate by methanogens for growth and deriving energy and methane is formed as a byproduct of this process. Methane producing microorganisms belong to Archaea domain that is different from the other fermentative bacteria. They are strictly anaerobic microorganisms excessive in environment in which oxygen, sulphate, nitrate or other electron acceptors don`t exist. Three main pathways for methane formation in anaerobic environments are known: acetotrophic methanogenesis in which acetate is converted into methane and carbon dioxide, hydrogenotrophic methanogenesis in which carbon

dioxide is reduced to methane, and methylotrophic methanogenesis in which methylated compounds such as methanol, methylamines, methylmercaptopropionate, dimethylsulfide, etc. that has one carbon are converted into methane.

Most known methane producing microorganicms are Methanosarcina and Methanosaeta  groups. These two microorganism types are responsible of the methane production from acetate and the methane ratio they produce  contributes to almost 70-75% of the total methane produced in the process. This pathway of methane production is carried out by acetotrophic methanogens (aceticlastic methanogens). The remaining amount of the methane production (25-30%) is performed by the hydrogenetrophic methanogens which use CO2 and H2 as substrate mostly. Regarding to this issue, it is stated that only 1-2% of the acetate is generated by homoacetogens using CO2 and H2. According to these known facts, one can claim that most of the methane production arises from acetotrophic methanogens that use acetate. The reactions shown at the table below presents the methane production reactions of the methanogens and net energy yields of  the reactions.

The syntrophic relationship between homoacetogens and methanogens was explained earlier. In this regard, methanogens maintain a low hydrogen partial pressure by using the H2 in close environment of hydrogen producing acetogens. In practice, most of the of methane derived from hydrogen occurs in flocks or biofilms, making a direct transfer of hydrogen from the hydrogen-producing microorganism to the hydrogen consuming methanogens.

If the chemical compositon of the substrate is known; then the amount of methane formation can be calculated acoording to the following stoichiometric equation since the substrate and the water are the inputs of the reaction and methane and carbondioxide are the outputs of the reaction (N (nitrogen) in the substrate is not included ).

References
1-T.Z.D. de Mes, A.J.M. Stams, J.H. Reith and G. Zeeman, Methane production by anaerobic digestion of wastewater and solid wastes,
2-Irini Angelidaki, Dimitar Karakashev, Damien J. Batstone, Caroline M. Plugge, and Alfons J. M. Stams, 2011. Biomethanation and Its Potential, Methods in Enzymology, Volume 494, ISSN 0076-6879, DOI: 10.1016/B978-0-12-385112-3.00016-0
3-Samir K. Khanal, 2008. Anaerobic Biotechnology For Bioenergy Production, Principles and Applications. 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82346-1.