Characterising biogas production from waste residues: understanding the role of microbial metabolism
Molaoa, Reitumetse Reabetswe
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Biogas production follows the normal anaerobic digestion process, this process can be divided into four major steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis. The components of biogas are primarily methane and carbon dioxide, but it may contain small amounts of hydrogen, nitrogen, hydrogen sulphide and moisture; the composition of biogas also depends upon feed material. Biogas is a low-cost energy source derived from renewable resources; this is because it can be produced from any organic waste, including household food waste, animal waste and agricultural residue. These are wastes that will continue to be produced as long as humans live on earth and keep livestock. Cow dung, for example, is known to contain the necessary microorganisms, such as acid and methane formers, for biogas production. The biogas can be harnessed (and made environmentally friendly) by converting it to a fuel. Biogas digester systems provide a residue organic waste, after the anaerobic digestion (AD), this effluent is called digestate and has superior nutrient qualities over normal organic fertilizer. Biogas digesters also function as waste disposal systems, and can therefore prevent potential sources of environmental pollution and the spread of pathogens and disease-causing bacteria. The first step of AD is hydrolysis, during which the complex organic matter (polymers) are degraded into smaller units (mono and oligomers). During hydrolysis, polymers like carbohydrates, fats and proteins are converted into glucose, lipids and amino acids. Hydrolytic microorganisms release hydrolytic enzymes, converting biopolymers into simpler and soluble compounds. A variety of microorganisms are involved in hydrolysis, these bacteria are mostly strict or facultative anaerobes such as Bacteriocides, Clostridia and Streptococci. The products produced from hydrolysis are further metabolized by the microorganisms involved in the subsequent step. During acidogenesis, the products of hydrolysis are converted by acidogenic (fermentative) bacteria into methanogenic substrates. Simple sugars, amino acids and fatty acids are converted into volatile fatty acids (VFA) such as acetic, propionic and butyric acid. In acetogenesis, VFA and alcohols are oxidised into methanogenic substrates like acetate, hydrogen and carbon dioxide. VFA, with carbon chains longer than two units and alcohols, with carbon chains longer than one unit, are oxidized into acetate and hydrogen. The production of hydrogen increases the hydrogen partial pressure. This can be regarded as a waste product of acetogenesis and inhibits the metabolism of the acetogenic bacteria. During methanogenesis, hydrogen is converted into methane. Acetogenesis and methanogenesis usually run parallel, as a result of the symbiosis of the two groups of microorganisms. Examples of acetogenic bacteria are Acetobacterium woodii and Clostridium aceticum. The production of CH4 and CO2 from intermediate products is carried out by methanogenic archaea. Methanogenesis is a critical step in the entire anaerobic digestion process, as it is most prone to imbalance. Methanogenesis is severely influenced by operation conditions like temperature, pH, composition of feedstock and feeding rate. Digester overloading, temperature changes or large entry of oxygen can result in termination of methane production. Other factors which highly influence the biogas production are the process conditions at which the digestion process is carried out, these include the retention time, nutrients available and intermediates which are generated during the digestion process A close interaction of all the involved microorganisms is of absolute importance for the biogas production. This is especially true for a well-balanced partial pressure of hydrogen. A too high concentration of hydrogen can hinder the metabolism of the acetogenic bacteria. Therefore, it is important that the hydrogen is constantly being used up by the methanogens in order to avoid a breakdown of the whole process. In this study, specific enrichment media was used in order to select for microorganisms participating in all four stages of the biogas production process, these are hydrolytic, acidogenic, acetogenic and methanogenic microorganisms. Proliferation of the enriched microorganisms was confirmed microscopically as well as amplification of 16S rRNA genes and mcrA genes. Basal media specific for acetogenic bacteria was used as inoculum for the upscaling and optimization using Spent Mushroom Substrate (SMS). Various feedstock including bran, hominy chop, paper pulp, molasses, cow and swine manure were characterized by nutritional and chemical composition, and the feedstock was also tested for biogas potential in 2 L digesters. Spent Mushroom Substrate (SMS) was used for the upscaling and optimization of CH4 in a 20 L bioreactor while monitoring pH and Dissolved Oxygen (DO). Biogas production was monitored and quantified by GC. The process was also monitored by studying product formation. Total carbohydrate assay was used to look at the production of sugars, BCA assay was used to quantify protein degradation while LCFA and VFA were monitored by HPLC. Out of all feedstock tested bran yielded the most biogas of 885,7 Nl/kg vDM, this would be expected as following nutritional composition bran contained 955,77 g/kg organic matter, thus over 95% of organic material. Bran also contained 99% vTS, this means that 99% energy value contained in bran. The results obtained during this research also show that when using the 2-stage bioreactor, there is better control of the respective steps of biogas production and consequently higher gas yields are achieved than using a 2 L digester. The digestates were screened for bacteria and archaea using specific primers and the analysis was done using Sanger sequencing and Next Generation Illumina Sequencing. The NGS data confirmed the presence of Firmicutes and Actinobacteria which participate in hydrolysis and acetogenesis respectively. Methanogenic species such as Methanosaeta, Methanobacterium and Methanosarcina were also detected.