Archaeal and bacterial community structures of rural household biogas digesters with different raw materials in Qinghai Plateau

The present study aims to investigate microbial community structures household biogas digesters with different raw materials in Qinghai Plateau rural. High-throughput 16S rRNA gene sequencing analysis revealed that Firmicutes, Bacteroidetes, and Proteobacteria are the most abundant bacterial phyla (64.08%). Prevotella group 7 was the most abundant genus in digester YL9 and YL10 (69.72% and 26.96%, respectively) using vegetable waste raw materials. Trichococcus exhibited the highest abundance (14.55%) in YL1 digester using sheep and pig manure. Clostridium sensu stricto 1 (13.89%) and Synergistaceae_uncultured (15.52%) comprised the highest abundances in digester YL5 with mixed raw materials (i.e., dairy manure, sheep manure, and human feces). In addition, Proteiniphilum and Pseudomonas exhibited the highest abundances among bacterial genera in YL4 digester using pig manure. Methanomicrobiales was the most dominant archaeal communities, ranging from 13.35% to 81.34% in abundance. Methanocorpusculum exhibited dominant abundances in all digesters using various raw materials. Methanogenium was the most abundant archaeal genera in YL4 and YL6 digesters, which consume pig manure as primary raw material. In addition, Methanosarcina and Methanosaeta exhibited the highest abundances in digester YL1 (55.03%) and YL9 (51.40%), respectively. Moreover, fermentation temperatures and pH both contributed to the archaeal and bacterial community structures in all the investigated digesters. Specially, fermentation temperature showed positive correlation with the abundances of Synergistaceae_uncultured, Methanogenium, and Methanosaeta, and pH was positively correlated with the abundances of Prevotella group 7 and Methanosarcina abundances.

     

Characterization of microbial communities in anaerobic bioreactors using molecular probes

The microbial community structure of twenty-one single-phase and one two-phase full-scale anaerobic sewage sludge digesters was evaluated using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens and sulfate-reducing bacteria. These probe results were interpreted in combination with results from traditional chemical analyses and metabolic activity assays. It was determined that methanogens in healthy mesophilic, single-phase sewage sludge digesters accounted for approximately 8–12% of the total community and thatMethanosarcinales andMethanomicrobiales constituted the majority of the total methanogen population.Methanobacteriales andMethanococcales played a relatively minor role in the digesters. Phylogenetic groups of mesophilic, Gram-negative sulfate-reducing bacteria were consistently present at significant levels:Desulfovibrio andDesulfobulbus spp. were the dominant sulfate-reducing populations,Desulfobacter andDesulfobacterium spp. were present at lower levels, andDesulfosarcina, Desulfococcus, andDesulfobotulus spp. were absent. Sulfate reduction by one or more of these populations played a significant role in all digesters evaluated in this study. In addition, sulfate-reducing bacteria played a role in favoring methanogenesis by providing their substrates. The analysis of the two-phase digester indicated that true phase separation was not accomplished: significant levels of active methanogens were present in the first phase. It was determined that the dominant populations in the second phase were different from those in the single-phase digesters.     

Air-side ammonia stripping coupled to anaerobic digestion indirectly impacts anaerobic microbiome

Air-side stripping without a prior solid–liquid phase separation step is a feasible and promising process to control ammonia concentration in thermophilic digesters. During the process, part of the anaerobic biomass is exposed to high temperature, high pH and aerobic conditions. However, there are no studies assessing the effects of those harsh conditions on the microbial communities of thermophilic digesters. To fill this knowledge gap, the microbiomes of two thermophilic digesters (55°C), fed with a mixture of pig manure and nitrogen-rich co-substrates, were investigated under different organic loading rates (OLR: 1.1–5.2 g COD l⁻¹ day⁻¹), ammonia concentrations (0.2–1.5 g free ammonia nitrogen l⁻¹) and stripping frequencies (3–5 times per week). The bacterial communities were dominated by Firmicutes and Bacteroidetes phyla, while the predominant methanogens were Methanosarcina sp archaea. Increasing co-substrate fraction, OLR and free ammonia nitrogen (FAN) favoured the presence of genera Ruminiclostridium, Clostridium and Tepidimicrobium and of hydrogenotrophic methanogens, mainly Methanoculleus archaea. The data indicated that the use of air-side stripping did not adversely affect thermophilic microbial communities, but indirectly modulated them by controlling FAN concentrations in the digester. These results demonstrate the viability at microbial community level of air side-stream stripping process as an adequate technology for the ammonia control during anaerobic co-digestion of nitrogen-rich substrates.     

Novel membrane bioreactor: Able to cope with fluctuating loads, poorly water soluble VOCs, and biomass accumulation

We determined the effect of different mixing intensities on the performance, methanogenic population dynamics, and juxtaposition of syntrophic microbes in anaerobic digesters treating cow manure from a dairy farm. Computer automated radioactive particle tracking in conjunction with computational fluid dynamics was performed to quantify the shear levels locally. Four continuously stirred anaerobic digesters were operated at different mixing intensities of 1,500, 500, 250, and 50 revolutions per min (RPM) over a 260-day period at a temperature of 34 +/- 1 degrees C. Animal manure at a volatile solids (VS) concentration of 50 g/L was fed into the digesters daily at five different organic loading rates between 0.6 and 3.5 g VS/L day. The different mixing intensities had no effect on the biogas production rates and yields at steady-state conditions. A methane yield of 0.241 +/- 0.007 L CH(4)/g VS fed was obtained by pooling the data of all four digesters during steady-state periods. However, digester performance was affected negatively by mixing intensity during startup of the digesters, with lower biogas production rates and higher volatile fatty acids concentrations observed for the 1,500-RPM digester. Despite similar methane production yields and rates, the acetoclastic methanogenic populations were different for the high- and low-intensity mixed digesters with Methanosarcina spp. and Methanosaeta concilii as the predominant methanogens, respectively. For all four digesters, epifluorescence microscopy revealed decreasing microbial floc sizes beginning at week 4 and continuing through week 26 after which no microbial flocs remained. This decrease in size, and subsequent loss of microbial flocs did not, however, produce any long-term upsets in digester performance.     

Microbial rRNA gene expression and co‐occurrence profiles associate with biokinetics and elemental composition in full‐scale anaerobic digesters

This study examined whether the abundance and expression of microbial 16S rRNA genes were associated with elemental concentrations and substrate conversion biokinetics in 20 full‐scale anaerobic digesters, including seven municipal sewage sludge (SS) digesters and 13 industrial codigesters. SS digester contents had higher methane production rates from acetate, propionate and phenyl acetate compared to industrial codigesters. SS digesters and industrial codigesters were distinctly clustered based on their elemental concentrations, with higher concentrations of NH₃‐N, Cl, K and Na observed in codigesters. Amplicon sequencing of 16S rRNA genes and reverse‐transcribed 16S rRNA revealed divergent grouping of microbial communities between mesophilic SS digesters, mesophilic codigesters and thermophilic digesters. Higher intradigester distances between Archaea 16S rRNA and rRNA gene profiles were observed in mesophilic codigesters, which also had the lowest acetate utilization biokinetics. Constrained ordination showed that microbial rRNA and rRNA gene profiles were significantly associated with maximum methane production rates from acetate, propionate, oleate and phenyl acetate, as well as concentrations of NH₃‐N, Fe, S, Mo and Ni. A co‐occurrence network of rRNA gene expression confirmed the three main clusters of anaerobic digester communities based on active populations. Syntrophic and methanogenic taxa were highly represented within the subnetworks, indicating that obligate energy‐sharing partnerships play critical roles in stabilizing the digester microbiome. Overall, these results provide new evidence showing that different feed substrates associate with different micronutrient compositions in anaerobic digesters, which in turn may influence microbial abundance, activity and function.     

Methanogenic population dynamics during start-up of anaerobic digesters treating municipal solid waste and biosolids

An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions. The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S. production rates. The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days. Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters. Chemical analyses were performed to evaluate digester performance. The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum. After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals. In contrast, the mesophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids increased dramatically and pH control was difficult. After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high. Methanogenic population dynamics correlated well with performance measures. Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed. Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up. The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively). Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester.     

Feedstocks Affect the Diversity and Distribution of Propionate CoA-Transferase Genes (pct) in Anaerobic Digesters

Anaerobic digestion (AD) is an attractive microbiological technology for both waste treatment and energy production. Syntrophic acetogenic bacteria are an important guild because they are essential for maintaining efficient and stable AD operation. However, this guild is poorly understood due to difficulties to culture them. In this study, we developed specific PCR assays targeting the propionate-CoA transferase genes (pct) to investigate their diversity and distribution in several mesophilic anaerobic digesters and a bench-scale temperature-phased AD (TPAD) system. Phylogenetic analysis of sequenced pct amplicons revealed the occurrence of Syntrophobacter fumaroxidans and six other clusters of putative pct genes. Principal coordinate analysis (PCoA) showed that pct diversity and abundance were largely correlated to the feedstocks of the digesters, while little difference was seen between the granular and the liquid fractions of each digester or between the two digesters of the TPAD system. Cluster-specific qPCR analysis revealed major impact of feedstocks and fractions on the abundance of pct genes. Readily fermentable substrates such as sugar- or starch-rich feedstocks selected for pct genes (Cluster I) related to Syntrophobacter, while manure feedstock selected for pct clusters related to pct of Clostridium spp. These results suggest that propionate metabolism can be affected by feedstocks and partition differently between solid and liquid phases in digesters. The PCR assays developed in this study may serve as a tool to investigate propionate-oxidizing bacteria in anaerobic digesters and other anaerobic environments.     

Quantifying electricity generation and waste transformations in a low-cost, plug-flow anaerobic digestion system

Methane production, electricity production, and wastewater transformations were quantified for a digestion system that combines biogas from a swine digester and dairy digester in Costa Rica. The low-cost, plug-flow digesters were not heated and were operated in the lower portion of the mesophilic range (25–27 °C).The dairy digester produced 27.5 m³/day of biogas with 62.6% methane and reduced organic matter (COD) by 86%. The swine digester produced 6.0 m³/day of biogas with 76.4% methane and reduced COD by 92%. Combining biogas from a swine and dairy digester, increased electricity production due to the higher biogas production rate of the dairy farm and the higher quality biogas obtained from the swine farm. The farm’s 2-h peak electricity demand (12.9 kW/day) was 81.8% met. The electricity was produced using manure equivalent to the quantity excreted by 5 dairy cows and 40 pigs remaining in corrals 100% of the time.The $21,000 capital cost of the digester project will be recovered in 10.1 years through electricity savings and reductions in wastewater fines. If the generator were more appropriately sized for the farm, the capital recovery time would have been 7.6 years.     

Comparative metagenomic analysis of bacterial populations in three full-scale mesophilic anaerobic manure digesters

While the use of anaerobic digestion to generate methane as a source of bioenergy is increasing worldwide, our knowledge of the microbial communities that perform biomethanation is very limited. Using next-generation sequencing, bacterial population profiles were determined in three full-scale mesophilic anaerobic digesters operated on dairy farms in the state of Vermont (USA). To our knowledge, this is the first report of a metagenomic analysis on the bacterial population of anaerobic digesters using dairy manure as their main substrate. A total of 20,366 non-chimeric sequence reads, covering the V1-V2 hypervariable regions of the bacterial 16S rRNA gene, were assigned to 2,176 operational taxonomic units (OTUs) at a genetic distance cutoff value of 5 %. Based on their limited sequence identity to validly characterized species, the majority of OTUs identified in our study likely represented novel bacterial species. Using a naïve Bayesian classifier, 1,624 anaerobic digester OTUs could be assigned to 16 bacterial phyla, while 552 OTUs could not be classified and may belong to novel bacterial taxonomic groups that have yet to be described. Firmicutes, Bacteroidetes, and Chloroflexi were the most highly represented bacteria overall, with Bacteroidetes and Chloroflexi showing the least and the most variation in abundance between digesters, respectively. All digesters shared 132 OTUs, which as a “core” group represented 65.4 to 70.6 % of sequences in individual digesters. Our results show that bacterial populations from microbial communities of anaerobic manure digesters can display high levels of diversity despite sharing a common core substrate.     

Cheese whey anaerobic digestion — Effect of chemical flocculant addition

Summary Two stirred tank digesters were operated with biomass retention by internal settling during effluent removal. In one digester a flocculating agent was used to enhance microbial aggregation and settling. The unflocculated digester achieved a maximum stable loading rate of 8–8.5kg COD/m³. day and biomass density of 19.3g VSS/l compared to the flocculated digester which achieved a (non-maximal) stable loading rate of 12.3kg COD/m³. day and biomass density of 43.3g VSS/l. Both digesters had a COD conversion efficiency greater than 97%. Operation of a stirred tank digester in a mix/settle mode allows a significant increase in biomass levels over conventional continuously stirred digesters. The addition of a chemical flocculant significantly enhances this improvement.     

Examination of Thermophilic Methane-Producing Digesters by Analysis of Bacterial Lipids

Thermophilic methane-producing digesters were examined by the analysis of lipids to determine the microbial biomass, community structure, and nutritional status of the microbes within the digesters. The digesters received a daily feedstock of cattle feed and Bermuda grass, with some digesters receiving additional supplements of propionate, butyrate, or nitrate. Microbial biomass, measured as total extractable lipid phosphate, was decreased in slurries from digesters receiving continuous addition of the fermentation intermediates propionate or butyrate as compared with slurries from control digesters receiving the feedstock alone. In slurries from digesters that received continuous addition of nitrate, the microbial biomass was higher than in the slurries from control digesters. The control digesters had ca. 2.5 × 10¹¹ bacteria per g (dry weight) as determined from total extractable lipid phosphate. Shifts in microbial community structure were observed by analysis of ester-linked phospholipid fatty acids. Statistical analysis of the patterns of phospholipid fatty acids indicated that the digesters receiving different supplements could be distinguished from the control digester and from each other. Poly-β-hydroxybutyric acid, an indicator of metabolic stress, was detected in slurries from all the digesters. Slurries from the nitrate-amended digester had the highest concentration of poly-β-hydroxybutyric acid, whereas slurries from the propionate-amended digester had the lowest concentration. These chemical analyses offer a quantitative means to correlate shifts in microbial biomass, community structure, and nutritional status in complex fermentation systems to the production of a specific end product.     

Impact of bioaugmentation by compost on the performance and ecology of an anaerobic digester fed with energy crops

The influence of compost as inoculum during continuous anaerobic digestion of fodder beet silage was studied over 330 days. Two simultaneously driven mesophilic fermentors (Inoc-1/Inoc-2) were inoculated with manure and sewage sludge. Only the digester Inoc-2 was inoculated additionally with compost. After 160 days fermentor Inoc-2 reached a hydraulic retention time (HRT) around 15 days whereas Inoc-1 remained at a HRT of 40d. After changing the substrate feed from one to three times a day both digesters stabilised at a shorter HRT; Inoc-2 at 10 days and Inoc-1 around 20 days. An additional inoculation of fermentor Inoc-1 by compost shortened the HRT to 10 days and revealed a minor increased gas production of about 6%. Fluorescence in situ hybridization indicated that probably an archaeal population shift was responsible for the observed stimulations. An addition of compost induced a methanogenic community change towards hydrogenotrophic methanogens.     

An approach to improve methanogenesis through the use of mixed cultures isolated from biogas digester

Biogas production has been shown to be inhibited by branched chain fatty acids (isobutyric, isovaleric) produced in the digester by cellulolytic organisms. Performance of these mixed cellulolytic cultures isolated at 25°C (C₂₅) and at 35° (C₃₅) in a batch digester using cattle manure confirmed that C₃₅, which forms mainly straight chain fatty acids from cellulose was more suitable for use as an inoculum than C₂₅ which formed predominantly branched chain fatty acids. Reconstitution of cellulolytic culture C₃₅ and mixed methanogens M₃₅ almost doubled both the amount and rate of methane production. Cellulolytic culture was useful in pretreatment of water hyacinth prior to its use as a substrate for methane generation A method for preservation and transportation of mixed cellulolytic culture for use as an inoculum in the digester is described.     

Robustness of archaeal populations in anaerobic co-digestion of dairy and poultry wastes

The objective of this study is to investigate the responses of methanogen populations to poultry waste addition by comparing the archaeal microbial populations in continuous anaerobic digesters with or without the addition of poultry waste as a co-substrate. Poultry waste was characterized as an organic/nitrogen-rich substrate for anaerobic digestion. Supplementing dilute dairy waste with poultry waste for anaerobic co-digestion to increase organic loading rate by 50% resulted in improved biogas production. Elevated ammonia derived from poultry waste did not lead to process inhibition at the organic loadings tested, demonstrating the feasibility of the anaerobic co-digestion of dairy and poultry wastes for improved treatment efficiency. The stability of the anaerobic co-digestion process was linked to the robust archaeal microbial community, which remained mostly unchanged in community structure following increases in organic loading and ammonia levels. Surprisingly, Crenarchaeota archaeal populations, instead of the Euryarchaeota methanogens, dominated the archaeal communities in the anaerobic digesters. The ecological functions of these abundant non-methanogen archaeal populations in anaerobic digestion remain to be identified.     

Effects of ammonia nitrogen of H2 and CH4 production during anaerobic digestion of dairy cattle manure

A number of researchers have verified the inhibitory effects of elevated H2 concentrations on various anaerobic fermentation processes. The objective of this work was to investigate the potential for using hydrogen gas production to predict upsets in anaerobic digesters operating on dairy cattle manure. In an ammonia nitrogen overload experiment, urea was added to the experimental digesters to obtain increased ammonia concentrations (600, 1,500, or 3,000 mg N/l). An increase in urea concentration resulted in an initial cessation of H2 production followed by an increase in H2 formation. Additions of 600, 1,500, or 3,000 mg N/l initially resulted in the reduction of biogas H2 concentrations. After 24 h, the H2 concentration increased in the 600 and 1,500 mg N/l digesters, but production remained inhibited in the 3,000 mg N/l digesters. Both methane and total biogas production decreased following urea addition. Volatile solids reduction also decreased during these periods. The digester effluent pH and alkalinity increased due to the increased NH4 formed with added urea. Based on these results, changes in H2 concentration could be a useful parameter for monitoring changes due to increased NH3 in dairy cattle manure anaerobic digesters.     

An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors.

An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test. The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make the test well suited for routine analysis of large numbers of samples and thus for process surveillance during operation of biogas digesters.     

Influence of H(2) stripping on methane production in conventional digesters

Hydrogen is a central metabolite in the methanization process. In this study the partial pressure of hydrogen in the gas phase of laboratory manure digesters was monitored over extensive periods of time and found to vary between 50 and 100.10(-6) atm. By sparging the gas phase of the digester through an auxiliary reactor, hydrogenotrophic methanogens were allowed to develop at the expense of hydrogen and carbon dioxide present in the biogas, independently of the liquid or cell residence time in the main reactor. By scrubbing ca. 100 volumes of biogas per liter reactor per day through an auxiliary reactor, hydrogen concentration could be decreased maximally 25%. This resulted in an increase in the gas production rate of the main digester of ca. 10% and a concomitant improved removal of volatile fatty acids from the mixed liquor. The results obtained indicate that considerable stripping of hydrogen from the digester could be achieved at acceptable energy expenditure. However, the microbial removal of the hydrogen at these low concentrations is extremely slow and limits the applicability of this approach.