Populations In Anaerobic Digestion Research Articles

Absolute quantification and genome-centric analyses elucidate the dynamics of microbial populations in anaerobic digesters

Anaerobic digestion (AD) relies on myriads of functions performed by complex microbial communities in customized settings, thus, a comprehensive investigation on the AD microbiome is central to the fine-tuned control. Most current AD microbiome studies are based on relative abundance, which hinders the interpretation of microbes’ dynamics and inter-sample comparisons. Here, we developed an absolute quantification (AQ) approach that integrated cellular spike-ins with metagenomic sequencing to elucidate microbial community variations and population dynamics in four anaerobic digesters. Using this method, 253 microbes were defined as decaying populations with decay rates ranging from -0.05 to -5.85 d−1, wherein, a population from Flavobacteriaceae family decayed at the highest rates of -3.87 to -5.85 d−1 in four digesters. Meanwhile, 25 microbes demonstrated the growing trend in the AD processes with growth rates ranging from 0.11 to 1.77 d−1, and genome-centric analysis assigned some of the populations to the functional niches of hydrolysis, short-chain fatty acids metabolism, and methane generation. Additionally, we observed that the specific activity of methanogens was lower in the prolonged digestion stage, and redundancy analysis revealed that the feedstock composition and the digestion duration were the two key parameters in governing the AD microbial compositions.

Evaluating Different Quantitative PCR Assays to Enumerate Specific Microbial Populations in Anaerobic Digesters Treating Municipal Wastewater Solids

AbstractThe goal of this research was to use and to validate different quantitative polymerase chain reaction (qPCR) assays to quantify the pertinent microbial populations in full-scale anaerobic d...

Elucidating Syntrophic Butyrate-Degrading Populations in Anaerobic Digesters Using Stable-Isotope-Informed Genome-Resolved Metagenomics.

Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after growing on [13C]butyrate. Differential abundance analysis of recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in heavy [13C]DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (<95%) with any cultured representative species, indicating that it is a novel species that plays a significant role in syntrophic butyrate degradation within anaerobic digesters. The Methanothrix genome contained the complete pathway for acetoclastic methanogenesis, indicating that it was enriched in 13C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to identify in situ interspecies metabolic cooperation within syntrophic consortia important to anaerobic waste treatment as well as global carbon cycling.IMPORTANCE Predicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in situ metabolic activities of uncultured species may enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in situ function of mixed microbiomes by combining stable-isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C4 fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve "who is doing what" by obtaining confirmatory assimilation of the labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.

Solubilization of Organics Due to Thermal Hydrolysis Pretreatment and the Shift in Microbial Population in Anaerobic Digestion

A study was conducted to analyze the effect of solubilization due to thermal hydrolysis pretreatment on the microbial population responsible for anaerobic digestion at mesophilic temperature. Metagenomic and microbial population analysis was done on digesters receiving sludge with and without thermal hydrolysis pretreatment. The digester receiving thermal hydrolysis pretreatment was stable at lower detention times and showed a shift in microbial population with predominance of methane-producing population in comparison to conventional digesters receiving no thermal hydrolysis pretreatment. Among methanogens, Methanosarcina showed dominance in the thermal hydrolysis pretreatment digester. Digesters with no thermal hydrolysis pretreatment had a mixed culture of acid-producing bacteria and methane-producing archaea, with almost equal proportion of methane-producing Methanobacterium and Methanosarcina. Kinetic parameters showed high growth and substrate utilization rates in digester receiving thermal hydrolysis pretreatment. This article is protected by copyright. All rights reserved.

Specific quorum sensing signal molecules inducing the social behaviors of microbial populations in anaerobic digestion

A series of dominant functional floras involved in anaerobic digestion was obtained by subculturing anaerobic sludge with specific substrates, respectively, and their specific quorum sensing signals (AHLs) were identified. It was found that most of the AHLs were secreted in starvation of the functional floras. One or two AHLs were found to have a significant positive correlation with the behavior of hydrolytic-fermentative bacteria, homoacetogens, syntrophic propionate-oxidizing bacteria, syntrophic butyrate-oxidizing bacteria and aceticlastic methanogens, respectively. Hydrogenotrophic methanogens had a various self-organization means and broader relations with the other populations, which was induced by six AHLs at least. Performance of an upflow anaerobic sludge blanket in the startup process revealed that the dominant populations involved in anaerobic digestion would secret specific AHLs to induce K-strategy in resource-limited situation for reproduction and regulating their cooperation. This work presents a novel perspective on the social behaviors of microbial populations in anaerobic digestion.

Impact of ultrasonic pre-treatment on domestic sludge digestion performance and microbial community dynamics

ABSTRACTUltrasonication-assisted sludge digestion technology is a lately used alternative sludge treatment method in wastewater treatment plants (WWTPs). This study focused on determining the influence of ultrasonication on aerobic and anaerobic sludge digestion, two most commonly used sludge handling processes, as well as on the investigation of microbial community structure after digestion. The effect of ultrasonication as a pre-treatment technique prior to sludge digestion on microbial population dynamics was not yet investigated comprehensively. Sludge sample taken from the primary and secondary settling tanks of a domestic wastewater treatment plant was used during the experiments. Based on the relevant data, while applied ultrasonication did not improve the anaerobic digestion efficiency, progress was achieved in the sludge dewaterability characteristics at the end of aerobic digestion. According to the results of both denaturant gradient gel electrophoresis and pyrosequencing data, ultrasonic pre-treatment decreased the richness of the microbial population in aerobic digestion, whereas increased the biocomplexity of the population in anaerobic digestion. We revealed that sludge pre-treatment with ultrasonication does not always improve the digestion performance. Composition of the sludge was the main factor defining the digestion performance.

Solubilization of Organics Due to Thermal Hydrolysis Pretreatment and the Shift in Microbial Population in Anaerobic Digestion.

A study was conducted to analyze the effect of solubilization due to thermal hydrolysis pretreatment on the microbial population responsible for anaerobic digestion at mesophilic temperature. Metagenomic and microbial population analysis was done on digesters receiving sludge with and without thermal hydrolysis pretreatment. The digester receiving thermal hydrolysis pretreatment was stable at lower detention times and showed a shift in microbial population with predominance of methane-producing population in comparison to conventional digesters receiving no thermal hydrolysis pretreatment. Among methanogens, Methanosarcina showed dominance in the thermal hydrolysis pretreatment digester. Digesters with no thermal hydrolysis pretreatment had a mixed culture of acid-producing bacteria and methane-producing archaea, with almost equal proportion of methane-producing Methanobacterium and Methanosarcina. Kinetic parameters showed high growth and substrate utilization rates in digester receiving thermal hydrolysis pretreatment.

DNA-SIP based genome-centric metagenomics identifies key long-chain fatty acid-degrading populations in anaerobic digesters with different feeding frequencies

Fats, oils and greases (FOG) are energy-dense wastes that can be added to anaerobic digesters to substantially increase biomethane recovery via their conversion through long-chain fatty acids (LCFAs). However, a better understanding of the ecophysiology of syntrophic LCFA-degrading microbial communities in anaerobic digesters is needed to develop operating strategies that mitigate inhibitory LCFA accumulation from FOG. In this research, DNA stable isotope probing (SIP) was coupled with metagenomic sequencing for a genome-centric comparison of oleate (C18:1)-degrading populations in two anaerobic codigesters operated with either a pulse feeding or continuous-feeding strategy. The pulse-fed codigester microcosms converted oleate into methane at over 20% higher rates than the continuous-fed codigester microcosms. Differential coverage binning was demonstrated for the first time to recover population genome bins (GBs) from DNA-SIP metagenomes. About 70% of the 13C-enriched GBs were taxonomically assigned to the Syntrophomonas genus, thus substantiating the importance of Syntrophomonas species to LCFA degradation in anaerobic digesters. Phylogenetic comparisons of 13C-enriched GBs showed that phylogenetically distinct Syntrophomonas GBs were unique to each codigester. Overall, these results suggest that syntrophic populations in anaerobic digesters can have different adaptive capacities, and that selection for divergent populations may be achieved by adjusting reactor operating conditions to maximize biomethane recovery.

Effect of feeding mode and dilution on the performance and microbial community population in anaerobic digestion of food waste

The effect of feeding mode and dilution was studied in anaerobic digestion of food waste. An upflow anaerobic digester with a settler was fed at six different organic loading rates (OLRs) from 4.6 to 8.6kgCOD/m3/d for 200days. The highest methane productivity of 2.78LCH4/L/d was achieved at 8.6kgCOD/m3/d during continuous feeding of diluted FW. Continuous feeding of diluted food waste showed more stable and efficient performance than stepwise feeding of undiluted food waste. Sharp increase in propionate concentration attributed towards deterioration of the digester performances in stepwise feeding of undiluted food waste. Microbial communities at various OLRs divulged that the microbial distribution in the continuous feeding of diluted food waste was not significantly perturbed despite the increase of OLR up to 8.6kgCOD/m3/d, which was contrast to the unstable distribution in stepwise feeding of undiluted food waste at 6.1kgCOD/m3/d.

Unexpected competitiveness of Methanosaeta populations at elevated acetate concentrations in methanogenic treatment of animal wastewater.

Acetoclastic methanogenesis is a key metabolic process in anaerobic digestion, a technology with broad applications in biogas production and waste treatment. Acetoclastic methanogenesis is known to be performed by two archaeal genera, Methanosaeta and Methanosarcina. The conventional model posits that Methanosaeta populations are more competitive at low acetate levels (<1mM) than Methanosarcina and vice versa at higher acetate concentrations. While this model is supported by an extensive body of studies, reports of inconsistency have grown that Methanosaeta were observed to outnumber Methanosarcina at elevated acetate levels. In this study, monitoring of anaerobic digesters treating animal wastewater unexpectedly identified Methanosaeta as the dominant acetoclastic methanogen population at both low and high acetate levels during organic overloading. The surprising competitiveness of Methanosaeta at elevated acetate was further supported by the enrichment of Methanosaeta with high concentrations of acetate (20mM). The dominance of Methanosaeta in the methanogen community could be reproduced in anaerobic digesters with the direct addition of acetate to above 20mM, again supporting the competitiveness of Methanosaeta over Methanosarcina at elevated acetate levels. This study for the first time systematically demonstrated that the dominance of Methanosaeta populations in anaerobic digestion could be linked to the competitiveness of Methanosaeta at elevated acetate concentrations. Given the importance of acetoclastic methanogenesis in biological methane production, findings from this study could have major implications for developing strategies for more effective control of methanogenic treatment processes.

Cellular adhesiveness and cellulolytic capacity in Anaerolineae revealed by omics-based genome interpretation.

BackgroundThe Anaerolineae lineage of Chloroflexi had been identified as one of the core microbial populations in anaerobic digesters; however, the ecological role of the Anaerolineae remains uncertain due to the scarcity of isolates and annotated genome sequences. Our previous metatranscriptional analysis revealed this prevalent population that showed minimum involvement in the main pathways of cellulose hydrolysis and subsequent methanogenesis in the thermophilic cellulose fermentative consortium (TCF).ResultsIn further pursuit, five high-quality curated draft genomes (>98 % completeness) of this population, including two affiliated with the inaccessible lineage of SBR1031, were retrieved by sequence-based multi-dimensional coverage binning. Comparative genomic analyses revealed versatile genetic capabilities for carbohydrate-based fermentative lifestyle including key genes catalyzing cellulose hydrolysis in Anaerolinea phylotypes. However, the low transcriptional activities of carbohydrate-active genes (CAGs) excluded cellulolytic capability as the selective advantage for their prevalence in the community. Instead, a substantially active type VI pili (Tfp) assembly was observed. Expression of the tight adherence protein on the Tfp indicated its function for cellular attachment which was further testified to be more likely related to cell aggregation other than cellulose surface adhesion. Meanwhile, this Tfp structure was found not contributing to syntrophic methanogenesis. Members of the SBR1031 encoded key genes for acetogenic dehydrogenation that may allow ethanol to be used as a carbon source.ConclusionThe common prevalence of Anaerolineae in anaerobic digesters should be originated from advantageous cellular adhesiveness enabled by Tfp assembly other than its potential as cellulose degrader or anaerobic syntrophs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0524-z) contains supplementary material, which is available to authorized users.

Distinctive non-methanogen archaeal populations in anaerobic digestion.

Methanogens define the archaeal communities involved in anaerobic digestion. Recently, non-methanogen archaeal populations have been unexpectedly identified in anaerobic digestion processes. To gain insight into the ecophysiology of these uncharacterized archaeal populations, for the first time, a phylogenetic analysis was performed on a collection of non-methanogen archaeal 16S rRNA gene sequences from anaerobic digesters of broad geographic distribution, revealing a distinct clade formed by these sequences in subgroup 6 of the Miscellaneous Crenarchaeotal Group in the newly proposed archaeal phylum Bathyarchaeota. This exclusive phylogenetic assemblage enabled the development of a real-time quantitative PCR (qPCR) assay specifically targeting these non-methanogen archaeal populations in anaerobic digestion. Application of the qPCR assay in continuous anaerobic digesters indicated that these archaeal populations were minor constituents of the archaeal communities, and the abundance of these populations remained relatively constant irrespective of process perturbations. Analysis of the archaeal populations in methanogenic communities further revealed the co-occurrence of these non-methanogen archaea with acetoclastic methanogens. Nevertheless, the low abundance of non-methanogen archaea as compared with acetoclastic methanogens suggests that the non-methanogen archaeal populations were not major players in animal waste-fed methanogenic processes investigated in this study and the functions of these archaeal populations remain to be identified.

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 500days. Results from quantitative polymerase chain reaction analysis show that Methanosaeta dominated over Methanosarcina in anaerobic digestion at high acetate levels up to 44mM, 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.

Development of Methanoculleus -specific real-time quantitative PCR assay for assessing methanogen communities in anaerobic digestion

To develop a Methanoculleus-specific real-time quantitative PCR (RT-qPCR) assay with high coverage and specificity for the analysis of methanogenic populations in anaerobic digestion. A Methanoculleus-specific primer/probe set for RT-qPCR was designed in this study based on all Methanoculleus 16S rRNA gene sequences in Ribosomal Database Project (RDP) according to TaqMan chemistry. The newly designed primer/probe set was shown to have high coverage and specificity by both in silico and experimental analyses. Amplification efficiency of the Methanoculleus-specific primer/probe set was determined to be ideal for RT-qPCR applications. Subsequent field testing on anaerobic digesters showed that results from RT-qPCR were consistent with those from clone library analysis, validating the accuracy of the RT-qPCR assay. The Methanoculleus-specific RT-qPCR assay designed in this study can serve as a rapid and effective tool for the quantification of Methanoculleus populations in anaerobic digestion. Methanoculleus populations represent important members of archaeal communities in methanogenic processes, necessitating the need to develop effective tools to monitor Methanoculleus population abundance. The RT-qPCR developed in this study provides an essential tool for the quantification of Methanoculleus populations in anaerobic digestion and for the understanding of the functions of these methanogens in anaerobic biotransformation.

Efficiency of RNA extraction from selected bacteria in the context of biogas production and metatranscriptomics

Understanding the microbial population in anaerobic digestion is an essential task to increase efficient substrate use and process stability. The metabolic state, represented e.g. by the transcriptome, of a fermenting system can help to find markers for monitoring industrial biogas production to prevent failures or to model the whole process. Advances in next-generation sequencing make transcriptomes accessible for large-scale analyses. In order to analyze the metatranscriptome of a mixed-species sample, isolation of high-quality RNA is the first step. However, different extraction methods may yield different efficiencies in different species. Especially in mixed-species environmental samples, unbiased isolation of transcripts is important for meaningful conclusions. We applied five different RNA-extraction protocols to nine taxonomic diverse bacterial species. Chosen methods are based on various lysis and extraction principles. We found that the extraction efficiency of different methods depends strongly on the target organism. RNA isolation of gram-positive bacteria was characterized by low yield whilst from gram-negative species higher concentrations can be obtained. Transferring our results to mixed-species investigations, such as metatranscriptomics with biofilms or biogas plants, leads to the conclusion that particular microorganisms might be over- or underrepresented depending on the method applied. Special care must be taken when using such metatranscriptomics data for, e.g. process modeling.

Biomethanization from sulfate‐containing municipal solid waste: effect of molybdate on microbial consortium

AbstractBACKGROUNDThe effect of molybdate on a microbial consortium of dry thermophilic anaerobic digestion (AD) from sulfate‐containing municipal solid waste was studied.RESULTSThe study showed molybdate inhibition affecting hydrolysis, acidogenesis, and methanogenesis, but the main microbial populations in AD can sustain the molybdate load over a short period of time. Molybdate was not a bactericide for all members of the principal groups, only a few of them are affected. Although all the microbial populations decreased, the weakest groups were sulfate reducing bacteria (SRBs) and butyrate‐utilising acetogens (BUAs) and the most resistant group was propionate‐utilising acetogens (PUAs). Prior to using molybdate, the relative percentages were: Eubacteria:Archaea 80:20; acetogens 25% (BUAs 11% and PUAs 14%) and SRBs 17%. After molybdate addition the relative percentages were: Eubacteria:Archaea were 83:17, acetogens remained over 28% (BUAs 8% and PUAs 20%) and SRBs around 11%. The relation between acetate‐utilising methanogens (AUMs) and hydrogen‐utilising methanogens (HUMs) changed slightly from 55:45 to 53:47.CONCLUSIONAll biochemical reactions in AD were inhibited and an increase in food:microorganism(F:M) ratio was observed. The microorganism's removal rates and growth inhibition rates are indicators of inhibitory effect in all analysed populations by inhibitor addition. © 2013 Society of Chemical Industry

Impact of substrate overloading on archaeal populations in anaerobic digestion of animal waste

To characterize adaptive changes in methanogenic microbial community in response to substrate overloading and identify potential linkages between process performance and microbial community composition. Triplicate continuous anaerobic digesters were developed as model anaerobic digestion processes, which were subsequently disrupted by substrate overloading. The clone library analysis of archaeal communities experiencing substrate overloading showed that populations related to Methanosaeta were the dominant methanogens before and after substrate overloading, suggesting the functional importance of these acetoclastic methanogens in balanced anaerobic digestion processes characterized with low organic acids concentrations. Population redundancy in Methanosaeta increased following substrate overloading with the emergence of additional populations of Methanosaeta. More importantly, the methanogenic community responded to process imbalance with greater functional diversity with increased abundance of functionally distinct hydrogenotrophic and acetoclastic methanogens, which likely enhanced the functional stability of anaerobic digestion during disruptions in the anaerobic food web under process perturbation. Crenarchaeota were identified as persistent constituents of the archaeal communities in anaerobic digestion, warranting further efforts to identifying the functions of these phylogenetically distinct populations in anaerobic digestion. Substrate overloading in anaerobic digestion resulted in an increased functional diversity of the methanogenic community, which enhanced the capacity to overcome subsequent occurrences of process perturbations without performance disruption, providing a potential strategy to maintain process stability in anaerobic digestion. Anaerobic digestion is a sustainable option for waste treatment and renewable energy production. However, process instability resulting from variations in substrate loading has been one of the obstacles to the broader adoption of anaerobic digestion technology. Insight into the linkages between process performance and microbial community gained in this study is valuable for developing strategies for the mitigation of the impact of substrate overloading on anaerobic digestion processes.

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.

Separation and quantification of cofactors from methanogenic bacteria by high-performance liquid chromatography: optimum and routine analyses

Four assays were developed, employing high-performance liquid chromatography, which gave optimal detection and separation of derivatives of 7-methylpterin, coenzyme F 420, factor F 430 or vitamin B 12. In addition an assay was developed in which thirteen of these cofactors can be separated and quantified simultaneously and which can be used in routine analysis of methanogenic populations in anaerobic digesters. The application of the different assays is demonstrated by analyses of extracts of pure cultures of Methanobacterium thermoautotrophicum and Methanosarcina barkeri and of sludge from a methanogenic fluidized bed reactor. Mixtures of authentic methanogenic cofactors were used in reference analyses and a relative peak area method was employed to identity the various cofactors in the extracts.