Omics-based characterization of anaerobic metabolism in methanogenic system and chain elongation process

Abstract

The thesis is to explore possible improvement of anaerobic fermentation system and to reveal the underlying mechanism. Specifically, the microbial communities and interspecies interactions of two methanogenic systems were investigated. One is a novel staged anaerobic fluidized bed ceramic membrane bioreactor (SAF-CMBR) with granular activated carbon (GAC) as fluidized biofilm carriers for low-strength synthetic wastewater (250 mg COD/L Na-propionate and acetate) treatment, another is a SAF-CMBR with GAC and polyethylene terephthalate (PET) beads as fluidized biofilm carriers, respectively. Microbial communities of the two systems were examined for a comparative study between GAC and PET beads as carriers. By using GAC as carrier, anaerobic treatment was achieved mainly by microorganism grown on the GAC particles in which propionate-degrading syntrophs (Syntrophobacter and Smithella), acetoclastic methanogens Methanothrix and exoelectrogenic Geobacter dominated. Whereas PET beads are less selective environment for microorganisms associated with methane production. Notably, methanogenesis would be promoted by the syntrophic cooperation between methanogens with Geobacter via direct interspecies electron transfer (DIET) for increased methane production. The conductive GAC could facilitate DIET and resulted in relatively high efficiency and methane yield, but not the non-conductive PET. Metagenomic and metatranscriptomic analyses were performed to further decipher the microbial interactions on the granular activated carbon (GAC) fluidizing media. Metabolic pathway reconstructions and metatranscriptomics mapping revealed that the syntrophic propionate oxidizing bacteria (SPOB) degraded propionate into acetate, which was further converted into methane and CO₂ by M concilii via the acetoclastic methanogenesis pathway. Concurrently, G. lovleyi oxidized acetate into CO₂ and released electrons into the extracellular environment. By accepting these electrons through direct interspecies electron transfer (DIET), M. concilii was capable to perform CO₂ reduction for further methane formation. Most notably, our study has, for the first time, showed that an alternative RuBisCO-mediated CO₂ reduction (the reductive hexulose-phosphate (RHP) pathway) is transcriptionally-active in M. concilii. The RHP pathway enables M. concilii to gain dominance and energy. Moreover, the RHP pathway could constitute a third methanogenesis route in M. concilii via a methyl-H4MPT intermediate. Further analysis verified that the acetoclastic methanogenesis, coupling of acetoclastic methanogenesis and CO₂ reduction pathways for methane formation are thermodynamically favorable even under very low substrate condition. Such tight interactions involving both mediated and direct interspecies electron transfer (MIET and DIET) promoted the overall efficiency of bioenergy processes. Another anaerobic mix-culture bioprocess, carboxylate chain elongation process in in which acetate is converted into valuable biochemicals, caproate, with ethanol as an electron donor, was studied. The feasibility of upgrading lignocellulosic ethanol (LE) to produce value-added chemical, caproate, via the chain elongation process was examined. Also, the effects of yeast extract and cellulose containing in the LE were evaluated separately. Fermentation performance showed that using LE as feedstock greatly shortened the lag phase of caproate production (4 days), and the similar enhancement effects were observed in the experimental group with extra supplement of yeast extract (6 days), and cellulose (9 days) compared with the control group (17 days) without extra supplement. Depletion of ethanol limited further elongation into caproate, resulting in comparable caproate yields and carbon conversion ratios. Microbial community and microbial kinetics analysis revealed that yeast extract could be metabolized by protein-utilizing bacteria into short chain carboxylates (SCCs), which facilitated biological chain elongation. Meanwhile, yeast extract boosted microbial growth by serving as nitrogen and other nutrient sources. Furthermore, cellulose was utilized and further converted into SCCs, or even caproate, by cellulolytic bacteria. Together, caproate production was enhanced with high microbial activities and intermediates formation using LE. Yeast extract was commonly added as a supplement in the CE process. In this study, the effects of casamino acids, the main composition in the yeast extract, and the conductive GAC particles on medium chain carboxylates (MCCs) production via CE process were evaluated, respectively. The results showed that the addition of casamino acids greatly shortened the lag phase for caproate production. While the addition of GAC extended the lag phase for the butyrate production (the first step of chain elongation), presumably because microorganisms needed longer time to adapt and enrich on GAC particles than these without such interference. But once the community was well-enriched, the microorganisms cooperated and functioned efficiently and resulted in shorter lag phase of caproate production compared to the Control. Further microbial analysis indicated that the reactors Control and AA showed high similarity of community structure over time. Whereas the communities of the reactor GAC showed great variations, suggesting that the addition of GAC induced adaptation and reformation of microbial consortia. While after 51 days of fermentation, the communities of three reactors converged and became similar after cultivation and enrichment. A sample was taken from the reactor AA (with casamino acids supplement) on Day 14 (significant caproate production) for further interspecies metabolic interactions exploration via metagenomics and transcriptomics analysis. The high-quality genome bins that phylogenetically identified to be closely related to the genomes of Clostridium kluyveri, Proteiniphilum acetatigenes and Clostridium aminophilum were recovered, which together represented the majority of the microbial community in the reactor AA. The complete ethanol-acetate fermentation pathway for caproate production via the reversed β-oxidation pathway was fully recovered in the genome bin closely related to C. kluyveri. Also, metatranscriptomics analysis confirmed that the genes involved in this pathway were actively-transcribed and contributed to the caproate prodution in the CE process. Moreover, in the genome bin of Proteiniphilum acetatigenes and Clostridium aminophilum, the pathways of amino acids, such as serine and glycine to produce butyrate were revered and found to be transcriptionally-active. It indicated that amino acids could not only support microbial growth, but also be directly involved in the CE metabolism and attributed to increased efficiency of the process.