Abstract
This study aimed at the investigation of the mechanisms of acidogenesis, which is a key process during anaerobic digestion. To expose possible bottlenecks, specific activities of the key enzymes of acidification, such as acetate kinase (Ack, 0.23–0.99 U mg−1 protein), butyrate kinase (Buk, < 0.03 U mg−1 protein) and butyryl-CoA:acetate-CoA transferase (But, 3.24–7.64 U mg−1 protein), were determined in cell free extracts of biogas reactor content from three different biogas reactors. Furthermore, the detection of Ack was successful via Western blot analysis. Quantification of corresponding functional genes encoding Buk (buk) and But (but) was not feasible, although an amplification was possible. Thus, phylogenetic trees were constructed based on respective gene fragments. Four new clades of possible butyrate-producing bacteria were postulated, as well as bacteria of the genera Roseburia or Clostridium identified. The low Buk activity was in contrast to the high specific But activity in the analysed samples. Butyrate formation via Buk activity does barely occur in the investigated biogas reactor. Specific enzyme activities (Ack, Buk and But) in samples drawn from three different biogas reactors correlated with ammonia and ammonium concentrations (NH3 and NH4+-N), and a negative dependency can be postulated. Thus, high concentrations of NH3 and NH4+-N may lead to a bottleneck in acidogenesis due to decreased specific acidogenic enzyme activities.
Highlights
The biochemical process of methane formation in biogas reactors can be divided into four phases: (i) hydrolysis of complex polymers, (ii) acidogenesis, (iii) acetogenesis and (iv) methanation (Weiland, 2010)
The most widespread acetate-producing pathway comprises the enzymes phosphotransacetylase (Pta) and acetate kinase (Ack), which convert acetyl-CoA to acetate (Ljungdahl, 1986). This pathway can be found in many different prokaryotes, e.g. Roseburia sp. (Duncan et al, 2002), Clostridium acetobutylicum (Winzer et al, 1997) or Methanosarcina thermophila (Gorrell and Ferry, 2007)
Butyryl-CoA is transformed to butyrate by a number of butyryl-CoA transferases, e.g. butyryl-CoA:acetate-CoA transferase (But), which uses acetate as co-substrate (Duncan et al, 2002)
Summary
The biochemical process of methane formation in biogas reactors can be divided into four phases: (i) hydrolysis of complex polymers, (ii) acidogenesis, (iii) acetogenesis and (iv) methanation (Weiland, 2010). In the first and second steps, the main products sugars and amino acids are formed, as well as volatile fatty acids (VFA) such as acetate, butyrate and propionate. These VFAs play a key role in biogas reactors. Butyrate-producing bacteria can be found, e.g., in marine sediments (Sørensen et al, 1981), and the human colon (Pryde et al, 2002; Louis et al, 2004; 2010), e.g. species related to Eubacterium, Roseburia or Faecalibacterium These bacteria use one of the four major bacterial butyrate synthesis pathways – acetyl-CoA, glutarate, lysine and 4-aminobutyrate pathway – which all have in common crotonyl-CoA as an intermediate (Vital et al, 2014). ButyrylCoA can be phosphorylated and transformed to butyrate by butyrate kinase (Buk) with simultaneous production of ATP (Diez-Gonzalez et al, 1999)
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