Institute for Microbiology and Wine Research of the Johannes Gutenberg University at Mainz
Of the dissertation:
Microbiology of fermentation in Renewable Raw Materials biogas plants paying particular attention to the degradation of propionic acid (written in German)
Renewable Raw Materials biogas plants can be acidified, because of bacterial formation of volatile fatty acids from easily fermentable carbon sources e. g. propionic acid. Methanogenic Archaea don’t grow at lower pH values. Therefore, the process of bacterial biogas formation is stagnating. This causes financial losses for operators of biogas plants. The objective of this dissertation was to identify the bacterial population that can degrade propionic acid to acetate and hydrogen in biogas plants. This anaerobic process is an endergonic process and works only with a low partial pressure of hydrogen. This task is performed by methanogenic Archaea. These so-called secondary fermenting bacteria exist in syntrophic culture with methanogenic Archaea.
In this task enrichment cultures of propionic acid degrading microorganisms from four Renewable Raw Materials biogas plants were identified and their substrates and products have been analysed. Enrichment cultures were provided by the Prüf- und Forschungsinstitut e. V. in Pirmasens. Stable mixed cultures were analysed by sequencing 16S rDNA. The mixed cultures contained Clostridiales, but also Bacteroides sp., δ-, ε-, γ-Proteobacteria, Spirochaetes, Synergistales and unusually Thermotogales. From propionic acid degrading mixed cultures and fermenters from biogas plants bacteria and methanogenic Archaea were cultured and isolated. Pure cultures of Clostridium sartagoforme strain Ap1a520 and Proteiniphilum acetatigenes strain Fp1a520 were identified by 16S rDNA analysis. From three fermenters and a secondary fermenter of Renewable Raw Materials biogas plants and additional two laboratory fermenters of the Leibniz-Institute for Agricultural Engineering Potsdam-Bornim (ATB) pure cultures were isolated. The pure cultures were identified as Methanobacterium formicicum, Methanoculleus bourgensis, Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina sp., Methanosaeta concilii and Methanomethylovorans sp.. So amongst others the typical methanogens were cultured and isolated from different fermenters, which previously were identified only by molecular biological methods. The specifically amplified polymorphic DNA-PCR (SAPD-PCR) was emerged as the appropriate method to distinguish between two different strains of the same species. The formation of methane by methanogenic pure cultures was analysed gaschromatographically. It has shown that the hydrogenotrophic methanogenesis is the more efficient way. Measurements of the methane formation and the determination of the cell number at the same time of isolate Methanoculleus bourgensis strain TAF1.1 showed that the formation of methane does not necessarily correlate. For the use in cultures, self-made reactor filtrate from fermenters contained plant fibres, acetate, the essential amino acid valine and the sugar alcohol glycerol. Defined co-cultures of secondary fermenting bacteria with self-isolated hydrogenotrophic methanogens showed that the methanogens have supported the bacteria. These bacteria fermented substrates or formed products, they were not able to transform in pure culture without the hydrogenotrophic methanogenic Archaea under these conditions.