Acetotrophic Activity Facilitates Methanogenesis from LCFA at Low Temperatures: Screening from Mesophilic Inocula.

The inoculum source plays a crucial role in the anaerobic treatment of wastewaters. Lipids are present in various wastewaters and have a high methanogenic potential, but their hydrolysis results in the production of long chain fatty acids (LCFAs) that are inhibitory to anaerobic microorganisms. Screening of inoculum for the anaerobic treatment of LCFA-containing wastewaters has been performed at mesophilic and thermophilic conditions. However, an evaluation of inocula for producing methane from LCFA-containing wastewater has not yet been conducted at low temperatures and needs to be undertaken. In this study, three inocula (one granular sludge and two municipal digester sludges) were assessed for methane production from LCFA-containing synthetic dairy wastewater (SDW) at low temperatures (10 and 20°C). A methane yield (based on mL-CH4/g-CODadded) of 86-65% with acetate and 45-20% with SDW was achieved within 10 days using unacclimated granular sludge, whereas the municipal digester sludges produced methane only at 20°C but not at 10°C even after 200 days of incubation. The acetotrophic activity in the inoculum was found to be crucial for methane production from LCFA at low temperatures, highlighting the role of Methanosaeta (acetoclastic archaea) at low temperatures. The presence of bacterial taxa from the family Syntrophaceae (Syntrophus and uncultured taxa) in the inoculum was found to be important for methane production from SDW at 10°C. This study suggests the evaluation of acetotrophic activity and the initial microbial community characteristics by high-throughput amplicon sequencing for selecting the inoculum for producing methane at low temperatures (up to 10°C) from lipid-containing wastewaters.

Nitrate decreases ruminal methane production with slight changes to ruminal methanogen composition of nitrate-adapted steers.

BACKGROUND: This study was conducted to examine effects of nitrate on ruminal methane production, methanogen abundance, and composition. Six rumen-fistulated Limousin×Jinnan steers were fed diets supplemented with either 0% (0NR), 1% (1NR), or 2% (2NR) nitrate (dry matter basis) regimens in succession. Rumen fluid was taken after two-week adaptation for evaluation of in vitro methane production, methanogen abundance, and composition measurements. RESULTS: Results showed that nitrate significantly decreased in vitro ruminal methane production at 6 h, 12 h, and 24 h (P < 0.01; P < 0.01; P = 0.01). The 1NR and 2NR regimens numerically reduced the methanogen population by 4.47% and 25.82% respectively. However, there was no significant difference observed between treatments. The alpha and beta diversity of the methanogen community was not significantly changed by nitrate either. However, the relative abundance of the methanogen genera was greatly changed. Methanosphaera (PL = 0.0033) and Methanimicrococcus (PL = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (PL = 0.0013). The population of Methanoculleus, the least frequently identified genus in this study, exhibited quadratic growth from 0% to 2% when nitrate was added (PQ = 0.0140). CONCLUSIONS: Correlation analysis found that methane reduction was significantly related to Methanobrevibacter and Methanoplanus abundance, and negatively correlated with Methanosphaera and Methanimicrococcus abundance.

Shifts of methanogenic communities in response to permafrost thaw results in rising methane emissions and soil property changes.

Permafrost thaw can bring negative consequences in terms of ecosystems, resulting in permafrost collapse, waterlogging, thermokarst lake development, and species composition changes. Little is known about how permafrost thaw influences microbial community shifts and their activities. Here, we show that the dominant archaeal community shifts from Methanomicrobiales to Methanosarcinales in response to the permafrost thaw, and the increase in methane emission is found to be associated with the methanogenic archaea, which rapidly bloom with nearly tenfold increase in total number. The mcrA gene clone libraries analyses indicate that Methanocellales/Rice Cluster I was predominant both in the original permafrost and in the thawed permafrost. However, only species belonging to Methanosarcinales showed higher transcriptional activities in the thawed permafrost, indicating a shift of methanogens from hydrogenotrophic to partly acetoclastic methane-generating metabolic processes. In addition, data also show the soil texture and features change as a result of microbial reproduction and activity induced by this permafrost thaw. Those data indicate that microbial ecology under warming permafrost has potential impacts on ecosystem and methane emissions.

Archaeal community in a human-disturbed watershed in southeast China: diversity, distribution, and responses to environmental changes.

The response of freshwater bacterial community to anthropogenic disturbance has been well documented, yet the studies of freshwater archaeal community are rare, especially in lotic environments. Here, we investigated planktonic and benthic archaeal communities in a human-perturbed watershed (Jiulong River Watershed, JRW) of southeast China by using Illumina 16S ribosomal RNA gene amplicon sequencing. The results of taxonomic assignments indicated that SAGMGC-1, Methanobacteriaceae, Methanospirillaceae, and Methanoregulaceae were the four most abundant families in surface waters, accounting for 12.65, 23.21, 18.58 and 10.97 % of planktonic communities, whereas Nitrososphaeraceae and Miscellaneous Crenarchaeotic Group occupied more than 49 % of benthic communities. The compositions of archaeal communities and populations in waters and sediments were significantly different from each other. Remarkably, the detection frequencies of families Methanobacteriaceae and Methanospirillaceae, and genera Methanobrevibacter and Methanosphaera in planktonic communities correlated strongly with bacterial fecal indicator, suggesting some parts of methanogenic Archaea may come from fecal contamination. Because soluble reactive phosphorus (SRP) and the ratio of dissolved inorganic nitrogen to SRP instead of nitrogen nutrients showed significant correlation with several planktonic Nitrosopumilus- and Nitrosotalea-like OTUs, Thaumarchaeota may play an unexplored role in biogeochemical cycling of river phosphorus. Multivariate statistical analyses revealed that the variation of α-diversity of planktonic archaeal community was best explained by water temperature, whereas nutrient concentrations and stoichiometry were the significant drivers of ß-diversity of planktonic and benthic communities. Taken together, these results demonstrate that the structure of archaeal communities in the JRW is sensitive to anthropogenic disturbances caused by riparian human activities.

Effect of domestication on microorganism diversity and anaerobic digestion of food waste.

To accomplish the rapid start-up and stable operation of biogas digesters, an efficient inoculum is required. To obtain such an inoculum for food waste anaerobic digestion, we domesticated dairy manure anaerobic digestion residue by adding food waste every day. After 36 days, the pH and biogas yield stabilized signifying the completion of domestication. During domestication, the microbial communities in the inocula were investigated by constructing 16S rDNA clone libraries. We evaluated the effect of the domesticated inoculum by testing batch food waste anaerobic digestion with a non-domesticated inoculum as a control. The pH and methane yield of the digestion systems were determined as measurement indices. Domestication changed the composition and proportion of bacteria and archaea in the inocula. Of the bacteria, Clostridia (49.3%), Bacteroidales (19.5%), and Anaerolinaceae (8.1%) species were dominant in the seed sludge; Anaerolinaceae (49.0%), Clostridia (28.4%), and Bacteroidales (9.1%), in domestication sludge. Methanosaeta was the dominant genus in both of the seed (94.3%) and domestication (74.3%) sludge. However, the diversity of methanogenic archaea was higher in the domestication than in seed sludge. Methanoculleus, which was absent from the seed sludge, appeared in the domestication sludge (21.7%). When the domesticated inoculum was used, the digestion system worked stably (organic loading rate: 20 gVS/L; methane yield: 292.2 ± 9.8 mL/gVS; VS = volatile solids), whereas the digestion system inoculated with seed sludge failed to generate biogas. The results indicate that inoculum domestication ensures efficient and stable anaerobic digestion by enriching the methanogenic strains.

Significant performance enhancement of a UASB reactor by using acyl homoserine lactones to facilitate the long filaments of Methanosaeta harundinacea 6Ac.

Methanosaeta strains are frequently involved in the granule formation during methanogenic wastewater treatment. To investigate the impact of Methanosaeta on granulation and performance of upflow anaerobic sludge blanket (UASB) reactors, three 1-L working volume reactors noted as R1, R2, and R3 were operated fed with a synthetic wastewater containing sodium acetate and glucose. R1 was inoculated with 1-L activated sludge, while R2 and R3 were inoculated with 200-mL concentrated pre-grown Methanosaeta harundinacea 6Ac culture and 800 mL of activated sludge. Additionally, R3 was daily dosed with 0.5 mL/L of acetyl ether extract of 6Ac spent culture containing its quorum sensing signal carboxyl acyl homoserine lactone (AHL). Compared to R1, R2 and R3 had a higher and more constant chemical oxygen demand (COD) removal efficiency and alkaline pH (8.2) during the granulation phase, particularly, R3 maintained approximately 90 % COD removal. Moreover, R3 formed the best granules, and microscopic images showed fluorescent Methanosaeta-like filaments dominating in the R3 granules, but rod cells dominating in the R2 granules. Analysis of 16S rRNA gene libraries showed increased diversity of methanogen species like Methanosarcina and Methanospirillum in R2 and R3, and increased bacteria diversity in R3 that included the syntrophic propionate degrader Syntrophobacter. Quantitative PCR determined that 6Ac made up more than 22 % of the total prokaryotes in R3, but only 3.6 % in R2. The carboxyl AHL was detected in R3. This work indicates that AHL-facilitated filaments of Methanosaeta contribute to the granulation and performance of UASB reactors, likely through immobilizing other functional microorganisms.

Persistence of Methanosaeta populations in anaerobic digestion during process instability.

Anaerobic digestion is a sustainable technology for the treatment of organic waste and production of biogas. Acetoclastic methanogenesis accounts for the majority of methane production in anaerobic digestion. Therefore, sustaining robust acetoclastic methanogens is important for stable process performance. Due to faster growth kinetics at high acetate concentrations, it has been considered that Methanosarcina would be more prevalent than Methanosaeta in unstable anaerobic digestion processes which frequently experience high acetate levels. Methanogen population dynamics were monitored in multiple continuous anaerobic digesters for 500 days. Results from quantitative polymerase chain reaction analysis show that Methanosaeta dominated over Methanosarcina in anaerobic digestion at high acetate levels up to 44 mM, suggesting the potential of Methanosaeta as a robust and efficient acetoclastic candidate for resilient anaerobic methane conversion. Further efforts are needed to identify mechanisms contributing to the unexpected competitiveness of these methanogens at high acetate levels observed in this study.

Optimized Cultivation and Syntrophic Relationship of Anaerobic Benzene-Degrading Enrichment Cultures under Methanogenic Conditions.

Current challenges in the anaerobic bioremediation of benzene are the lack of capable cultures and limited knowledge on the biodegradation pathway. Under methanogenic conditions, benzene may be mineralized by syntrophic interactions between microorganisms, which are poorly understood. The present study developed an optimized formula for anoxic medium to successfully promote the growth of the putative benzene degrader Deltaproteobacterium Hasda-A and enhance the benzene degradation activity of methanogenic enrichment cultures. Within 70| |d of incubation, the benzene degradation activity and relative abundance of Hasda-A in cultures in the new defined medium increased from 0.5 to >3| |mg L-1 d-1 and from 2.5% to >17%, respectively. Together with Hasda-A, we found a strong positive relationship between the abundances of superphylum OD1 bacteria, three methanogens (Methanoregula, Methanolinea, and Methanosaeta) and benzene degradation activity. The syntrophic relationship between these microbial taxa and Hasda-A was then demonstrated in a correlation analysis of longitudinal data. The involvement of methanogenesis in anaerobic benzene mineralization was confirmed by inhibition experiments. The high benzene degradation activity and growth of Hasda-A were quickly recovered in successive dilutions of enrichment cultures, proving the feasibility of using the medium developed in the present study to produce highly capable cultures. The present results will facilitate practical applications in bioremediation and research on the molecular mechanisms underlying benzene activation and syntrophic interactions in benzene mineralization.

Calcium effect on microbial activity and biomass aggregation during anaerobic digestion at high salinity.

The potential effect of different Ca2+ additions (150, 300, 450, 600 and 1000 mg/L) on microbial activity and aggregation, during anaerobic digestion at moderate (8 g/L Na+) and high salinity (20 g/L Na+) has been investigated. Batch tests were carried out in duplicate serum bottles and operated for 30 days at 37 °C. At 8 g/L Na+, methanogenic activity and protein degradation were comparable from 150 to 450 mg/L Ca2+, and a significant inhibition was only observed at a Ca2+concentration of 1000 mg/L. In contrast, at 20 g/L Na+, 150 to 300 mg/L were the only Ca2+ concentrations to maintain chemical oxygen demand (COD) removal, protein hydrolysis and methane production. Overall, increasing Ca2+ concentrations had a larger impact on acetotrophic methanogenesis at 20 g/L than at 8 g/L Na+. Increasing Ca2+ had a negative effect on the aggregation behaviour of the dominant methanogen Methanosaeta when working at 8 g/L Na+. At 20 g/L Na+ the aggregation of Methanosaeta was less affected by addition of Ca2+ than at 8 g/L Na+. The negative effect appeared to be connected with Ca2+ precipitation and its impact on cell-to cell communication. The results highlight the importance of ionic balance for microbial aggregation at high salinity, bringing to the forefront the effect on Methanosaeta cells, known to be important to obtain anaerobic granules.

Effect of seed sludge and operation conditions on performance and archaeal community structure of low-temperature anaerobic solvent-degrading bioreactors.

Two laboratory-scale expanded granular sludge bed (EGSB) anaerobic bioreactors (R1 and R2) were inoculated with biomass from different mesophilic (37 degrees C) treatment plants, and used for the treatment of an organic solvent-based wastewater at 9-14 degrees C at applied organic loading rates (OLRs) of 1.2-3.6kg chemical oxygen demand (COD)m(-3)d(-1). Replicated treatment performance was observed at 10-14 degrees C, which suggested the feasibility of the process at pilot-scale. Stable and efficient COD removal, along with high methane productivity, was demonstrated at 9 degrees C at an applied OLR of 2.4kgCODm(-3)d(-1). Clonal libraries and fluorescence in situ hybridization (FISH) indicated that the seed sludges were dominated (>60%) by acetoclastic Methanosaeta-like organisms. Specific methanogenic activity (SMA) profiles indicated shifts in the physiological profiles of R1 and R2 biomass, including the development of psychrotolerant methanogenic activity. Acetoclastic methanogenesis represented the primary route of methane production in R1 and R2, which is in contrast with several previous reports from low-temperature bioreactor trials. A reduction in the abundance of Methanosaeta-like clones (R2), along with the detection of hydrogenotrophic methanogenic species, coincided with altered granule (sludge) morphology and the development of hydrogenotrophic SMA after prolonged operation at 9 degrees C.

Similar evolution in delta 13CH4 and model-predicted relative rate of aceticlastic methanogenesis during mesophilic methanization of municipal solid wastes.

Similar evolution was obtained for the stable carbon isotope signatures delta (13)CH(4) and the model-predicted relative rate of aceticlastic methanogenesis during mesophilic methanization of municipal solid wastes. In batch incubations, the importance of aceticlastic and hydrogenotrophic methanogenesis changes in time. Initially, hydrogenotrophic methanogenesis dominated, but increasing population of Methanosarcina sp. enhances aceticlastic methanogenesis. Later, hydrogenotrophic methanogenesis intensified again. A mathematical model was developed to evaluate the relative contribution of hydrogenotrophic and aceticlastic pathways of methane generation during mesophilic batch anaerobic biodegradation of the French and the Chinese Municipal Solid Wastes (FMSW and CMSW). Taking into account molecular biology analysis reported earlier three groups of methanogens including strictly hydrogenotrophic methanogens, strictly aceticlastic methanogens (Methanosaeta sp.) and Methanosarcina sp., consuming both acetate and H(2)/H(2)CO(3) were considered in the model. The total organic and inorganic carbon concentrations, methane production volume, methane and carbon dioxide partial pressures values were used for the model calibration and validation. Methane isotopic composition (delta (13)CH(4)) evolution during the incubations was used to independently validate the model results. The model demonstrated that only the putrescible solid waste was totally converted to methane.

Growth kinetics of Candidatus 'Methanoperedens nitroreducens' enriched in a laboratory reactor.

Recently it has been shown that Candidatus 'Methanoperedens nitroreducens', an anaerobic methanotrophic archaea (ANME), can reduce nitrate to nitrite using electrons derived from anaerobic oxidation of methane. In this study, the growth kinetics of 'M. nitroreducens' enriched in a laboratory reactor were studied. In the experimental concentration range (up to 16 mg CH4 L-1), anaerobic oxidation of methane by 'M. nitroreducens' was found to comply with first order kinetic model with a rate constant of 0.019 ±â€¯0.006 h-1 and a biomass-specific rate constant of 0.04-0.14 L h-1 g-1VSS. Meanwhile, the nitrate reduction to nitrite was well described by the Monod-type kinetic model with an affinity constant for nitrate of 2.1 ±â€¯0.4 mg N L-1, which is slightly higher than, but comparable to, that of most known denitrifying bacteria. This is the first time that the growth kinetics of 'M. nitroreducens' have been experimentally studied. The applicability of the kinetic model reported herein to this organism or similar organisms in natural or engineering systems requires further investigation.

Shifts in methanogen community structure and function associated with long-term manipulation of sulfate and salinity in a hypersaline microbial mat.

Methanogenesis was characterized in hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico both in situ and after long-term manipulation in a greenhouse environment. Substrate addition experiments indicate methanogenesis to occur primarily through the catabolic demethylation of non-competitive substrates, under field conditions. However, evidence for the coexistence of other metabolic guilds of methanogens was obtained during a previous manipulation of sulfate concentrations. To fully characterize methanogenesis in these mats, in the absence of competition for reducing equivalents with sulfate-reducing microorganisms, we maintained microbial mats for longer than 1 year under conditions of lowered sulfate and salinity levels. The goal of this study was to assess whether observed differences in methane production during sulfate and salinity manipulation were accompanied by shifts in the composition of methanogen communities. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogen assemblages. Clone libraries from mats sampled in situ or maintained at field-like conditions in the greenhouse were exclusively composed of sequences related to methylotrophic members of the Methanosarcinales. Increases in pore water methane concentrations under conditions of low sulfate correlated with an observed increase in the abundance of putatively hydrogenotrophic mcrA, related to Methanomicrobiales. Geochemical and molecular data provide evidence of a significant shift in the metabolic pathway of methanogenesis from a methylotroph-dominated system in high-sulfate environments to a mixed community of methylotrophic and hydrogenotrophic methanogens under low sulfate conditions.

Microbial dynamics in anaerobic enrichment cultures degrading di-n-butyl phthalic acid ester.

Although anaerobic biodegradation of di-n-butyl phthalic acid ester (DBP) has been studied over the past decade, only little is known about the microorganisms involved in the biological anaerobic degradation pathways. The aim of this work is to characterize the microbial community dynamics in enrichment cultures degrading phthalic acid esters under methanogenic conditions. A selection pressure was applied by adding DBP at 10 and 200 mg L(-1) in semi-continuous anaerobic reactors. The microbial dynamics were monitored using single strand conformation polymorphism (SSCP). While only limited abiotic losses were observed in the sterile controls (20-22%), substantial DBP biodegradation was found in the enrichment cultures (90-99%). In addition, significant population changes were observed. The dominant bacterial species in the DBP-degrading cultures was affiliated to Soehngenia saccharolytica, a microorganism described previously as an anaerobic benzaldehyde degrader. Within the archaeal community, there was a shift between two different species of the genus Methanosaeta sp., indicating a highly specific impact of DBP or degradation products on archaeal species. RNA-directed probes were designed from SSCP sequences, and FISH observations confirmed the dominance of S. saccharolytica, and indicated floccular microstructures, likely providing favourable conditions for DBP degradation.

Characterization of anaerobic granular sludge developed in UASB reactors that treat ethanol, carbohydrates and hydrolyzed protein based wastewaters.

In this work, granules developed from UASB reactors that treat different types of wastewaters (ethanol, carbohydrates and protein-based synthetic wastewaters) were studied. Granule parameters (size distribution; density; settlement characteristics; elemental composition; acidogenic and methanogenic activities) were analyzed along with micro-organisms identified by FISH to better understand granule behavior and its formation process. Micro-organisms distributions in anaerobic granules are highly dependent on the type of treated wastewater. Granules developed in a UASB reactor that treats wastewater with a high content of carbohydrates presented high acidogenic bacteria colonization. Members of Methanosaetaceae were the dominant methanogens in the studied granules, and Methanobacteriales appear to be co-dominant in the granules developed with carbohydrates and protein-based wastewaters.

The hunt for the most-wanted chemolithoautotrophic spookmicrobes.

Microorganisms are the drivers of biogeochemical methane and nitrogen cycles. Essential roles of chemolithoautotrophic microorganisms in these cycles were predicted long before their identification. Dedicated enrichment procedures, metagenomics surveys and single-cell technologies have enabled the identification of several new groups of most-wanted spookmicrobes, including novel methoxydotrophic methanogens that produce methane from methylated coal compounds and acetoclastic 'Candidatus Methanothrix paradoxum', which is active in oxic soils. The resultant energy-rich methane can be oxidized via a suite of electron acceptors. Recently, 'Candidatus Methanoperedens nitroreducens' ANME-2d archaea and 'Candidatus Methylomirabilis oxyfera' bacteria were enriched on nitrate and nitrite under anoxic conditions with methane as an electron donor. Although 'Candidatus Methanoperedens nitroreducens' and other ANME archaea can use iron citrate as an electron acceptor in batch experiments, the quest for anaerobic methane oxidizers that grow via iron reduction continues. In recent years, the nitrogen cycle has been expanded by the discovery of various ammonium-oxidizing prokaryotes, including ammonium-oxidizing archaea, versatile anaerobic ammonium-oxidizing (anammox) bacteria and complete ammonium-oxidizing (comammox) Nitrospira bacteria. Several biogeochemical studies have indicated that ammonium conversion occurs under iron-reducing conditions, but thus far no microorganism has been identified. Ultimately, iron-reducing and sulfate-dependent ammonium-oxidizing microorganisms await discovery.

Comparative study of reactor performance and microbial community in psychrophilic and mesophilic biogas digesters under solid state condition.

Psychrophilic (15°C) and mesophilic (35°C) reactor performance and microbial community dynamics were compared when the biogas fermenters were performed at high altitude and solid state condition using animal manure and highland barley straw as substrate. Longer biogas fermentation time, higher peak methane content and lower volatile fatty acids (VFA) accumulation were found at psychrophilic condition compared to that of at mesophilic condition although the biogas production in both temperature conditions was similar. The cumulative biogas production at 35°C and 15°C were 246 (±5) and 225 (±7) ml/g volatile solids, respectively. The highest total VFA concentration under 35°C was 10,796 (±310) mg/kg total solid, while it only reached to 2346 (±87) mg/kg total solid at the condition of 15°C. Additionally, the variation of pH, soluble chemical oxygen demand and total ammonia nitrogen during the anaerobic digestion under psychrophilic condition were much smaller than that of under mesophilic condition. Polymerase chain reaction and denaturing gradient gel electrophoresis analysis followed by 16S rDNA sequencing showed that bacteria of genera Bacillus and Clostridium and archaea of genera Methanosarcina and Methanosaeta played a pivotal role during the biogas production.

Growth kinetics and competition between Methanosarcina and Methanosaeta in mesophilic anaerobic digestion.

Methanosarcina species with a high maximum specific growth rate (mumax) and high half-saturation coefficient (KS) and Methanosaeta species with a low mumax and low KS are the only known aceticlastic methanogens. Because of Methanosaeta's low KS, the low acetate concentrations in conventional, mesophilic anaerobic digestion yield Methanosaeta dominance. However, Methanosarcina absorbs increases in acetate more efficiently and thus promotes more stable digestion. This paper tests the hypothesis that decreasing digester feeding frequencies can increase Methanosarcina predominance. Two acetate-fed reactors were established at a 17-day solids retention time. One reactor was fed hourly, and one was fed once daily. Microscopic and molecular methods were used to verify that the hourly fed reactor enriched for Methanosaeta, while the daily fed reactor enriched for Methanosarcina. Growth and substrate-use kinetics were measured for each reactor. A digester overload condition was simulated, and the Methanosarcina-enriched reactor was found to perform better than the Methanosaeta-enriched reactor. These findings indicate that Methanosarcina dominance can be achieved with infrequent feedings, leading to more stable digestion.

Biochar alleviates combined stress of ammonium and acids by firstly enriching Methanosaeta and then Methanosarcina.

This investigation evaluated the effectiveness of biochar of different particle sizes in alleviating ammonium (NH4(+)) inhibition (up to 7 g-N/L) during anaerobic digestion of 6 g/L glucose. Compared to the control treatment without biochar addition, treatments that included biochar particles 2-5 mm, 0.5-1 mm and 75-150 µm in size reduced the methanization lag phase by 23.9%, 23.8% and 5.9%, respectively, and increased the maximum methane production rate by 47.1%, 23.5% and 44.1%, respectively. These results confirmed that biochar accelerated the initiation of methanization during anaerobic digestion under double inhibition risk from both ammonium and acids. Furthermore, fine biochar significantly promoted the production of volatile fatty acids (VFAs). Comparative analysis on the archaeal and bacterial diversity at the early and later stages of digestion, and in the suspended, biochar loosely bound, and biochar tightly bound fractions suggested that, in suspended fractions, hydrogenotrophic Methanobacterium was actively resistant to ammonium. However, acetoclastic Methanosaeta can survive at VFAs concentrations up to 60-80 mmol-C/L by improved affinity to conductive biochar, resulting in the accelerated initiation of acetate degradation. Improved methanogenesis was followed by the colonization of the biochar tightly bound fractions by Methanosarcina. The selection of appropriate biochar particles sizes was important in facilitating the initial colonization of microbial cells.

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA.

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82% and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.