Community and Proteomic Analysis of Anaerobic Consortia Converting Tetramethylammonium to Methane.
ABSTRACT: Tetramethylammonium-degrading methanogenic consortia from a complete-mixing suspended sludge (CMSS) and an upflow anaerobic sludge blanket (UASB) reactors were studied using multiple PCR-based molecular techniques and shotgun proteomic approach. The prokaryotic 16S rRNA genes of the consortia were analyzed by quantitative PCR, high-throughput sequencing, and DGGE-cloning methods. The results showed that methanogenic archaea were highly predominant in both reactors but differed markedly according to community structure. Community and proteomic analysis revealed that Methanomethylovorans and Methanosarcina were the major players for the demethylation of methylated substrates and methane formation through the reduction pathway of methyl-S-CoM and possibly, acetyl-CoA synthase/decarbonylase-related pathways. Unlike high dominance of one Methanomethylovorans population in the CMSS reactor, diverse methylotrophic Methanosarcina species inhabited in syntrophy-like association with hydrogenotrophic Methanobacterium in the granular sludge of UASB reactor. The overall findings indicated the reactor-dependent community structures of quaternary amines degradation and provided microbial insight for the improved understanding of engineering application.
Project description:The effect of nickel deprivation from the influent of a mesophilic (30 degrees C) methanol fed upflow anaerobic sludge bed (UASB) reactor was investigated by coupling the reactor performance to the evolution of the Methanosarcina population of the bioreactor sludge. The reactor was operated at pH 7.0 and an organic loading rate (OLR) of 5-15 g COD l(-1) day(-1) for 191 days. A clear limitation of the specific methanogenic activity (SMA) on methanol due to the absence of nickel was observed after 129 days of bioreactor operation: the SMA of the sludge in medium with the complete trace metal solution except nickel amounted to 1.164 (+/-0.167) g CH(4)-COD g VSS(-1) day(-1) compared to 2.027 (+/-0.111) g CH(4)-COD g VSS(-1) day(-1) in a medium with the complete (including nickel) trace metal solution. The methanol removal efficiency during these 129 days was 99%, no volatile fatty acid (VFA) accumulation was observed and the size of the Methanosarcina population increased compared to the seed sludge. Continuation of the UASB reactor operation with the nickel limited sludge lead to incomplete methanol removal, and thus methanol accumulation in the reactor effluent from day 142 onwards. This methanol accumulation subsequently induced an increase of the acetogenic activity in the UASB reactor on day 160. On day 165, 77% of the methanol fed to the system was converted to acetate and the Methanosarcina population size had substantially decreased. Inclusion of 0.5 muM Ni (dosed as NiCl(2)) to the influent from day 165 onwards lead to the recovery of the methanol removal efficiency to 99% without VFA accumulation within 2 days of bioreactor operation.
Project description:Upflow anaerobic sludge blanket (UASB) reactor has served as an effective process to treat industrial wastewater such as purified terephthalic acid (PTA) wastewater. For optimal UASB performance, balanced ecological interactions between syntrophs, methanogens, and fermenters are critical. However, much of the interactions remain unclear because UASB have been studied at a "macro"-level perspective of the reactor ecosystem. In reality, such reactors are composed of a suite of granules, each forming individual micro-ecosystems treating wastewater. Thus, typical approaches may be oversimplifying the complexity of the microbial ecology and granular development. To identify critical microbial interactions at both macro- and micro- level ecosystem ecology, we perform community and network analyses on 300 PTA-degrading granules from a lab-scale UASB reactor and two full-scale reactors. Based on MiSeq-based 16S rRNA gene sequencing of individual granules, different granule-types co-exist in both full-scale reactors regardless of granule size and reactor sampling depth, suggesting that distinct microbial interactions occur in different granules throughout the reactor. In addition, we identify novel networks of syntrophic metabolic interactions in different granules, perhaps caused by distinct thermodynamic conditions. Moreover, unseen methanogenic relationships (e.g. "Candidatus Aminicenantes" and Methanosaeta) are observed in UASB reactors. In total, we discover unexpected microbial interactions in granular micro-ecosystems supporting UASB ecology and treatment through a unique single-granule level approach.
Project description:The effect of omitting zinc from the influent of mesophilic (30 degrees C) methanol fed upflow anaerobic sludge bed (UASB) reactors, and latter zinc supplementation to the influent to counteract the deprivation, was investigated by coupling the UASB reactor performance to the microbial ecology of the bioreactor sludge. Limitation of the specific methanogenic activity (SMA) on methanol due to the absence of zinc from the influent developed after 137 days of operation. At that day, the SMA in medium with a complete trace metal solution except Zn was 3.4 g CH4-COD g VSS(-1) day(-1), compared to 4.2 g CH4-COD g VSS(-1) day(-1) in a medium with a complete (including zinc) trace metal solution. The methanol removal capacity during these 137 days was 99% and no volatile fatty acids accumulated. Two UASB reactors, inoculated with the zinc-deprived sludge, were operated to study restoration of the zinc limitation by zinc supplementation to the bioreactor influent. In a first reactor, no changes to the operational conditions were made. This resulted in methanol accumulation in the reactor effluent after 12 days of operation, which subsequently induced acetogenic activity 5 days after the methanol accumulation started. Methanogenesis could not be recovered by the continuous addition of 0.5 microM ZnCl2 to the reactor for 13 days. In the second reactor, 0.5 microM ZnCl2 was added from its start-up. Although the reactor stayed 10 days longer methanogenically than the reactor operated without zinc, methanol accumulation was observed in this reactor (up to 1.1 g COD-MeOH L(-1)) as well. This study shows that zinc limitation can induce failure of methanol fed UASB reactors due to acidification, which cannot be restored by resuming the continuous supply of the deprived metal.
Project description:According to the U.S. Department of Energy and the European Union, tellurium is a critical element needed for energy and defense technology. Thus methods are needed to recover tellurium from waste streams. The objectives of this study was to determine the feasibility of utilizing upflow anaerobic sludge bed (UASB) reactors to convert toxic tellurite (TeIV) oxyanions to non-toxic insoluble elemental tellurium (Te0) nanoparticles (NP) that are amendable to separation from aqueous effluents. The reactors were supplied with ethanol as the electron donating substrate to promote the biological reduction of TeIV. One reactor was additionally amended with the redox mediating flavonoid compound, riboflavin (RF), with the goal of enhancing the bioreduction of TeIV. Its performance was compared to a control reactor lacking RF. The continuous formation of Te0 NPs using the UASB reactors was found to be feasible and remarkably improved by the addition of RF. The presence of this flavonoid was previously shown to enhance the conversion rate of TeIV by approximately 11-fold. In this study, we demonstrated that this was associated with the added benefit of reducing the toxic impact of TeIV towards the methanogenic consortium in the UASB and thus enabled a 4.7-fold higher conversion rate of the chemical oxygen demand. Taken as a whole, this work demonstrates the potential of a methanogenic granular sludge to be applied as a bioreactor technology producing recoverable Te0 NPs in a continuous fashion.
Project description:We studied the peptide-degrading anaerobic communities of methanogenic reactors from two mesophilic full-scale modified upflow anaerobic sludge blanket (UASB) reactors treating brewery wastewater in Colombia. Most probable number (MPN) counts varied between 7.1 x 10(8) and 6.6 × 10(9) bacteria/g volatile suspended solids VSS (Methanogenic Reactor 1) and 7.2 × 10(6) and 6.4 × 10(7) bacteria/g (VSS) (Methanogenic Reactor 2). Metabolites detected in the highest positive MPN dilutions in both reactors were mostly acetate, propionate, isovalerate and, in some cases, negligible concentrations of butyrate. Using the highest positive dilutions of MPN counts, 50 dominant strains were isolated from both reactors, and 12 strains were selected for sequencing their 16S rRNA gene based on their phenotypic characteristics. The small-subunit rRNA gene sequences indicated that these strains were affiliated to the families Propionibacteriaceae, Clostridiaceae and Syntrophomonadaceae in the low G + C gram-positive group and Desulfovibrio spp. in the class ?-Proteobacteria. The main metabolites detected in the highest positive dilutions of MPN and the presence of Syntrophomonadaceae indicate the effect of the syntrophic associations on the bioconversion of these substrates in methanogenic reactors. Additionally, the potential utilization of external electron acceptors for the complete degradation of amino acids by Clostridium strains confirms the relevance of these acceptors in the transformation of peptides and amino acids in these systems.
Project description:In this study, the long-term performance and microbial dynamics of an Upflow Anaerobic Sludge Blanket (UASB) reactor targeting sulfate reduction in a SOx emissions treatment system were assessed using crude glycerol as organic carbon source and electron donor under constant S and C loading rates. The reactor was inoculated with granular sludge obtained from a pulp and paper industry and fed at a constant inlet sulfate concentration of 250 mg S-SO<sub>4</sub><sup>2-</sup>L<sup>-1</sup> and a constant C/S ratio of 1.5 ± 0.3 g Cg<sup>-1</sup> S for over 500 days. Apart from the regular analysis of chemical species, Illumina analyses of the 16S rRNA gene were used to study the dynamics of the bacterial community along with the whole operation. The reactor was sampled along the operation to monitor its diversity and the changes in targeted species to gain insight into the performance of the sulfidogenic UASB. Moreover, studies on the stratification of the sludge bed were performed by sampling at different reactor heights. Shifts in the UASB performance correlated well with the main shifts in microbial communities of interest. A progressive loss of the methanogenic capacity towards a fully sulfidogenic UASB was explained by a progressive wash-out of methanogenic <i>Archaea</i>, which were outcompeted by sulfate-reducing bacteria. <i>Desulfovibrio</i> was found as the main sulfate-reducing genus in the reactor along time. A progressive reduction in the sulfidogenic capacity of the UASB was found in the long run due to the accumulation of a slime-like substance in the UASB.
Project description:We herein analyzed the diversity of microbes involved in anaerobic sulfur oxidation in an upflow anaerobic sludge blanket (UASB) reactor used for treating municipal sewage under low-temperature conditions. Anaerobic sulfur oxidation occurred in the absence of oxygen, with nitrite and nitrate as electron acceptors; however, reactor performance parameters demonstrated that anaerobic conditions were maintained. In order to gain insights into the underlying basis of anaerobic sulfur oxidation, the microbial diversity that exists in the UASB sludge was analyzed comprehensively to determine their identities and contribution to sulfur oxidation. Sludge samples were collected from the UASB reactor over a period of 2 years and used for bacterial 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and next-generation sequencing analyses. T-RFLP and sequencing results both showed that microbial community patterns changed markedly from day 537 onwards. Bacteria belonging to the genus Desulforhabdus within the phylum Proteobacteria and uncultured bacteria within the phylum Fusobacteria were the main groups observed during the period of anaerobic sulfur oxidation. Their abundance correlated with temperature, suggesting that these bacterial groups played roles in anaerobic sulfur oxidation in UASB reactors.
Project description:Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates-ciliates frequently found in anoxic ecosystems-on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885-3,190 and 2,387-2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.
Project description:The redox-mediating capacity of magnetic reduced graphene oxide nanosacks (MNS) to promote the reductive biodegradation of the halogenated pollutant, iopromide (IOP), was tested. Experiments were performed using glucose as electron donor in an upflow anaerobic sludge blanket (UASB) reactor under methanogenic conditions. Higher removal efficiency of IOP in the UASB reactor supplied with MNS as redox mediator was observed as compared with the control reactor lacking MNS. Results showed 82% of IOP removal efficiency under steady state conditions in the UASB reactor enriched with MNS, while the reactor control showed IOP removal efficiency of 51%. The precise microbial transformation pathway of IOP was elucidated by high-performance liquid chromatography coupled to mass spectroscopy (HPLC-MS) analysis. Biotransformation by-products with lower molecular weight than IOP molecule were identified in the reactor supplied with MNS, which were not detected in the reactor control, indicating the contribution of these magnetic nano-carbon composites in the redox conversion of this halogenated pollutant. Reductive reactions of IOP favored by MNS led to complete dehalogenation of the benzene ring and partial rupture of side chains of this pollutant, which is the first step towards its complete biodegradation. Possible reductive mechanisms that took place in the biodegradation of IOP were stated. Finally, the novel and successful application of magnetic graphene composites in a continuous bioreactor to enhance the microbial transformation of IOP was demonstrated.
Project description:An ecogenomic analysis of the methanogenic microbial community in a laboratory-scale up-flow anaerobic sludge blanket (UASB) reactor treating soy sauce-processing wastewater revealed a synergistic metabolic network. Granular sludge samples were collected from the UASB reactor operated under psychrophilic (20°C) conditions with a COD removal rate >75%. A 16S rRNA gene amplicon sequencing-based microbial community analysis classified the major microbial taxa as Methanothrix, Methanobacterium, Pelotomaculaceae, Syntrophomonadaceae, Solidesulfovibrio, and members of the phyla Synergistota and Bacteroidota. Draft genomes of dominant microbial populations were recovered by metagenomic shotgun sequencing. Metagenomic- and metatranscriptomic-assisted metabolic reconstructions indicated that Synergistota- and Bacteroidota-related organisms play major roles in the degradation of amino acids. A metagenomic bin of the uncultured Bacteroidales 4484-276 clade encodes genes for proteins that may function in the catabolism of phenylalanine and tyrosine under microaerobic conditions. Syntrophomonadaceae and Pelotomaculaceae oxidize fatty acid byproducts presumably derived from the degradation of amino acids in syntrophic association with aceticlastic and hydrogenotrophic methanogen populations. Solidesulfovibrio organisms are responsible for the reduction of sulfite and may support the activity of hydrogenotrophic methanogens and other microbial populations by providing hydrogen and ammonia using nitrogen fixation-related proteins. Overall, functionally diverse anaerobic organisms unite to form a metabolic network that performs the complete degradation of amino acids in the psychrophilic methanogenic microbiota.