Project description:The microbial diversity and metabolic potential of a methanogenic consortium residing in a 3785-liter anaerobic digester, fed with wastewater algae, was analyzed using 454 pyrosequencing technology. DNA was extracted from anaerobic sludge material and used in metagenomic analysis through PCR amplification of the methyl-coenzyme M reductase ? subunit (mcrA) gene using primer sets ML, MCR, and ME. The majority of annotated mcrA sequences were assigned taxonomically to the genera Methanosaeta in the order Methanosarcinales. Methanogens from the genus Methanosaeta are obligate acetotrophs, suggesting this genus plays a dominant role in methane production from the analyzed fermentation sample. Numerous analyzed sequences within the algae fed anaerobic digester were unclassified and could not be assigned taxonomically. Relative amplicon frequencies were determined for each primer set to determine the utility of each in pyrosequencing. Primer sets ML and MCR performed better quantitatively (representing the large majority of analyzed sequences) than primer set ME. However, each of these primer sets was shown to provide a quantitatively unique community structure, and thus they are of equal importance in mcrA metagenomic analysis.
Project description:We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA-DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.
Project description:Anaerobic digesters become unstable when operated at a high organi c loading rate (OLR). Investigating the microbial community response to OLR disturbance is helpful for achieving efficient and stable process operation. However, previous studies have only focused on community succession during different process stages. How does community succession influence process stability? Is this kind of succession resilient? Are any key microbial indicator closely related to process stability? Such relationships between microbial communities and process stability are poorly understood.In this study, a mesophilic anaerobic digester for treating food waste (FW) was operated to study the microbial diversity and dynamicity due to OLR disturbance. Overloading resulted in proliferation of acidogenic bacteria, and the resulting high volatile fatty acid (VFA) yield triggered an abundance of acetogenic bacteria. However, the abundance and metabolic efficiency of hydrogenotrophic methanogens decreased after disturbance, and as a consequence, methanogens and acetogenic bacteria could not efficiently complete the syntrophy. This stress induced the proliferation of homoacetogens as alternative hydrogenotrophs for converting excessive H2 to acetate. However, the susceptible Methanothrix species also failed to degrade the excessive acetate. This metabolic imbalance finally led to process deterioration. After process recovery, the digester gradually returned to its original operational conditions, reached close to its original performance, and the microbial community profile achieved a new steady-state. Interestingly, the abundance of Syntrophomonas and Treponema increased during the deteriorative stage and rebounded after disturbance, suggesting they were resilient groups.Acidogenic bacteria showed high functional redundancy, rapidly adapted to the increased OLR, and shaped new microbial community profiles. The genera Syntrophomonas and Treponema were resilient groups. This observation provides insight into the key microbial indicator that are closely related to process stability. Moreover, the succession of methanogens during the disturbance phase was unsuitable for the metabolic function needed at high OLR. This contradiction resulted in process deterioration. Thus, methanogenesis is the main step that interferes with the stable operation of digesters at high OLR. Further studies on identifying and breeding high-efficiency methanogens may be helpful for breaking the technical bottleneck of process instability and achieving stable operation under high OLR.