Syntrophic degradation of amino acids by thermophilic methanogenic consortia

Waste water usually contains large amounts of different organic compounds. A variety of microbial processes are involved in the anaerobic methanogenic treatment of waste water, such as hydrolysis of lipids, polysaccharides and proteins, fermentation of sugars and amino acids, acetogenic conversion of fatty acids and methanogenesis from acetate and H ₂ /CO ₂ . The ultimate end product from these microbial processes is biogas, which consists of methane and carbon dioxide. To optimise anaerobic treatment detailed knowledge of the different microorganisms and their metabolic interactions must be obtained.This thesis describes the fate of glutamate in moderate thermophilic methanogenic systems. Glutamate is a major constituent of protein and can be degraded by a variety of microorganisms and via different pathways.To get more insight into glutamate degradation in mesophilic and moderate thermophilic methanogenic granular sludge, anaerobic glutamate-degrading microorganisms were quantified. We found that the major part of glutamate was degraded by microorganisms that grow in syntrophic consortia with methanogensSlow-growing thermophilic glutamate-degrading microorganisms were enriched in a special designed reactor system. Several novel organisms were isolated from it. Caloramator coolhaasii sp. nov., an anaerobic glutamate-converting bacterium, was capable of complete glutamate conversion to acetate, H ₂ , CO ₂ , NH ₄⁺ and traces of propionate. In the presence of a hydrogen scavenging methanogen, glutamate was converted to the same products, but the growth rate was 4-fold higher. Methanosaeta strain A is a methanogenic acetoclastic archaeon. This organism formed granules, which were difficult to disintegrate, under all growth conditions tested. The property to form granules could give this organism an extra advantage in upflow anaerobic sludge bed reactors.A glutamate- and propionate-degrading enrichment was found to consist of two organisms: a glutamate-degrading and a propionate-oxidising bacterium. Both organisms were not capable of growth without the presence of a methanogenic archaeon.Gelria glutamica gen. nov., sp. nov., could convert glutamate only in the presence of a methanogen to propionate, H ₂ , CO ₂ and ammonia. Traces of succinate were found in the degradation of glutamate. Desulfotomaculum thermosyntrophicum sp. nov.,a moderate thermophilic spore-forming syntrophic propionate-oxidising bacterium, wass only capable of growth on propionate in the presence of a methanogen. Propionate is degraded to acetate, H ₂ and CO ₂ . In pure culture, the organism can convert pyruvate, lactate, fumarate and H ₂ /CO ₂ . Remarkable is that TPO is capable of benzoate fermentation to acetate, propionate and an unknown compound, without the production of hydrogen. TPO is also capable of sulphate reduction and is phylogenetically related to other spore forming thermophilic sulphate reducers.The glutamate metabolism of the two previously mentioned glutamate degraders and Thermanaerovibrio acidaminovorans was studied in detail. Caloramator coolhaasii and Th. acidaminovorans converted glutamate to acetate through theβ-methylaspartate pathway. Propionate formation occured through direct oxidation of glutamate via the intermediates succinyl-CoA and methylmalonyl-CoA. This pathway also occurred in Gelria glutamica for propionate formation from glutamate.The arginine metabolism of Th. acidaminovorans was studied in detail. In pure culture arginine was converted by Th. acidaminovorans to citrulline and ornithine, whereas in coculture with a methanogen acetate, propionate, H ₂ , CO ₂ and ammonia was formed. The arginine deiminase pathway was used for arginine degradation. Although there is an ATP-generating conversion in this pathway, yield studies did not confirm this. At higher arginine concentrations the molar growth yield decreased.The results that were presented in this thesis improved our knowledge of the glutamate-degrading microbial population in thermophilic anaerobic methanogenic bioreactors. The anaerobic degradation of glutamate as performed by microorganisms studied in this thesis can be put in a reaction scheme that is depicted in Fig. 1.<div align="center"><img src="/wda/abstracts/i2977.gif" width="600" height="420" alt="Fig. 1" border="0"/><strong>Fig. 1.</strong> The anaerobic degradation of glutamate in thermophilic methanogenic environments. C.c., Caloramator coolhaasii ; T.a., Thermanaerovibrio acidaminovorans ; G.g., Gelria glutamica ; D.t., Desulfotomaculum thermosyntrophicum ; Strain A, Methanosaeta thermophila strain A; R43, Methanobacterium sp. R4