Methanoculleus Species Research Articles

Biogas-producing microbial composition of an anaerobic digester and associated bovine residues.

Influenced by feedstock type and microbial inoculum, different microbial groups must precisely interact for high‐quality biogas yields. As a first approach for optimization, this study aimed to identify through time the biogas‐producing microbial community in a 10‐ton dry anaerobic digester treating cattle manure by denaturing gradient gel electrophoresis (DGGE) and metagenomics. Moreover, the associated bovine residues or feedstocks (leachate, manure, oxidation lagoon water, rumen) were also characterized to determine their contribution. A diverse and dynamic community characterized by Bacteria (82%–88%) and a considerable amount of Archaea (8%–15%) presented profiles particular to each stage of biogas production. Eukaryotes (2.6%–3.6%), mainly fungi, were a minor but stable component. Proteobacteria represented 47% of the community at the start of the run but only 18% at the end, opposite to the Bacteroidetes/Chlorobi group (8% and 20%, respectively), while Firmicutes (12%–18%) and Actinobacteria (12%–32%) remained relatively constant. Methanogens of the order Methanomicrobiales represented by several species of Methanoculleus were abundant at the end of the run (77%) contrary to Methanosarcinales (11%) and Methanobacteriales (0.7%). Therefore, methanogenesis mainly occurred by the hydrogenotrophic pathway. Manure and oxidation lagoon water seemed to contribute key microorganisms, while rumen dominated by Methanobrevibacter (72%) did not proliferate in the digester. Manure particularly possessed Methanoculleus (24%) and uncultured methanogens identified by DGGE, whereas oxidation lagoon was exclusively abundant in Methanolinea (18%) and Methanosaeta (19%). Leachate, as the microbial inoculum from a previous run, adequately preserved the biogas‐producing community. These results could lead to higher biogas yields through bioaugmentation strategies by incorporating higher proportions or an enriched inoculum from the relevant feedstocks.

Enhanced anaerobic digestion of ammonia-rich swine manure by zero-valent iron: With special focus on the enhancement effect on hydrogenotrophic methanogenesis activity

Zero-valent iron (ZVI) supplementation for improving anaerobic digestion (AD) of ammonia-rich swine manure (initial ammonia-N ∼5000 mg/L) was tested. The addition of 5 g/L ZVI powder apparently accelerated the acidification process to produce more volatile fatty acids (VFAs) and optimized the fermentation type by contributing to a lower system oxidation–reduction potential (ORP) level of −181.7 to −250.0 mV favorable for ethanol-type and butyric-type fermentation during day 14–30, in comparison with that of −164.3 to −216.3 mV in the control group favorable for propionic-type. Overall, ZVI significantly decreased the proportion of propionic acid from 49.8% to 30.9% while increased the proportion of n-butyric acid from 6.8% to 18.7%. Microbial analysis revealed that fast growing and ammonia-tolerant hydrogenotrophic Methanoculleus species were enriched with ZVI, helping achieve a 54.2% higher CH4 yield relative to control. Results from this study demonstrated the potential of ZVI addition to enhance AD of ammonia-rich animal manure.

Hydrogen-Fueled Microbial Pathways in Biogas Upgrading Systems Revealed by Genome-Centric Metagenomics.

Biogas upgrading via carbon dioxide hydrogenation is an emerging technology for electrofuel production. The biomethanation efficiency is strongly dependent on a balanced microbial consortium, whose high- resolution characterization along with their functional potential and interactions are pivotal for process optimization. The present work is the first genome-centric metagenomic study on mesophilic and thermophilic biogas upgrading reactors aiming to define the metabolic profile of more than 200 uncultivated microbes involved in hydrogen assisted methanogenesis. The outcomes from predictive functional analyses were correlated with microbial abundance variations to clarify the effect of process parameters on the community. The operational temperature significantly influenced the microbial richness of the reactors, while the H2 addition distinctively alternated the abundance of the taxa. Two different Methanoculleus species (one mesophilic and one thermophilic) were identified as the main responsible ones for methane metabolism. Finally, it was demonstrated that the addition of H2 exerted a selective pressure on the concerted or syntrophic interactions of specific microbes functionally related to carbon fixation, propionate and butanoate metabolisms. Novel bacteria were identified as candidate syntrophic acetate oxidizers (e.g., Tepidanaerobacter sp. DTU063), while the addition of H2 favored the proliferation of potential homoacetogens (e.g., Clostridia sp. DTU183). Population genomes encoding genes of Wood-Ljungdahl pathway were mainly thermophilic, while propionate degraders were mostly identified at mesophilic conditions. Finally, putative syntrophic interactions were identified between microbes that have either versatile metabolic abilities or are obligate/facultative syntrophs.

Microbial effects of part-stream low-frequency ultrasonic pretreatment on sludge anaerobic digestion as revealed by high-throughput sequencing-based metagenomics and metatranscriptomics

BackgroundPart-stream low-frequency ultrasound (LFUS) was one of the common practices for sludge disintegration in full-scale anaerobic digestion (AD) facilities. However, the effectiveness of part-stream LFUS treatment and its effect on AD microbiome have not been fully elucidated.MethodsHere we testified the effectiveness of part-stream LFUS pretreatment by treating only a fraction of feed sludge (23% and 33% total solid of the feed sludge) with 20 Hz LFUS for 70 s. State-of-the-art metagenomic and metatranscriptomic analysis was used to investigate the microbial process underpinning the enhanced AD performance by part-stream LFUS pretreatment.ResultsBy pretreating 33% total solid of the feed sludge, methane yield was increased by 36.5%, while the volatile solid reduction ratio remained unchanged. RNA-seq of the microbiome at stable stage showed that the continuous dosage of easy-degradable LFUS-pretreated feed sludge had gradually altered the microbial community by selecting Bacteroidales hydrolyzer with greater metabolic capability to hydrolyze cellulosic biomass without substrate attachment. Meanwhile, Thermotogales with excellent cell mobility for nutrient capturing was highly active within the community. Foremost proportion of the methanogenesis was contributed by the dominant Methanomicrobiales via carbon dioxide reduction. More interestingly, a perceivable proportion of the reverse electron flow of the community was input from Methanoculleus species other than syntrophic acetate-oxidizing bacteria. In addition, metagenomic binning retrieved several interesting novel metagenomic-assembled genomes (MAGs): MAG-bin6 of Alistipes shahii showed exceptional transcriptional activities towards protein degradation and MAG-bin11 of Candidatus Cloacimonetes with active cellulolytic GH74 gene detected.ConclusionsIn summary, despite the unchanged sludge digestibility, the applied part-stream LFUS pretreatment strategy was robust in adjusting the microbial pathways towards more effective substrate conversion enabled by free-living hydrolyser and beta-oxidation-capable methanogens.

Enhancing methane production from U. lactuca using combined anaerobically digested sludge (ADS) and rumen fluid pre-treatment and the effect on the solubilization of microbial community structures

Methane production by the anaerobic digestion of seaweed is restricted by the slow degradation caused by the influence of the rigid algal cell wall. At the present time, there has been no study focusing on the anaerobic digestion of U. lactuca by co-fermentation and pre-treatment with rumen fluid. Rumen fluid can favor methane production from algal biomass by utilizing the diversity and quantity of bacterial and archaeal communities in the rumen fluid. This research presents a novel method based on combined ADS and rumen fluid pre-treatment to improve the production of methane from seaweed. Biochemical methane potential (BMP) tests were performed to investigate the biogas production using combined ADS and rumen fluid pre-treatment at varied inoculum ratios on the performance of methane production from U. lactuca biomass. Compared to the control (no rumen fluid pre-treatment), the highest BMP yields of U. lactuca increased from 3%, 27.5% and 39.5% to 31.1%, 73% and 85.6%, respectively, for three different types of treatment. Microbial community analysis revealed that the Methanobrevibacter species, known to accept electrons to form methane, were only detected when rumen fluid was added. Together with the significant increase in species of Methanoculleus, Methanospirillum and Methanosaeta, rumen fluid improved the fermentation and degradation of the microalgae biomass not only by pre-treatment to foster cell-wall degradation but also by relying on methane production within itself during anaerobic processes. Batch experiments further indicated that rumen fluid applied to the co-fermentation and pre-treatment could increase the economic value and hold promise for enhancing biogas production from different seaweed species.

Ammonia threshold for inhibition of anaerobic digestion of thin stillage and the importance of organic loading rate.

Biogas production from nitrogen-rich feedstock results in release of ammonia (NH3), causing inhibition of the microbial process. The reported threshold ammonia value for stable biogas production varies greatly between studies, probably because of differences in operating conditions. Moreover, it is often difficult to separate the effect of ammonia inhibition from that of organic loading rate (OLR), as these two factors are often interrelated. This study attempted to distinguish the effects of ammonia and OLR by analysis of two laboratory-scale biogas reactors operating with thin stillage and subjected to an increase in free ammonia (from 0.30 to 1.1 g L(-1)) either by addition of an external nitrogen source (urea) or by increasing the OLR (3.2-6.0 g volatile solids L(-1) d(-1)). The results showed that ammonia concentration was detrimental for process performance, with the threshold for stability in both processes identified as being about 1 g NH3-N L(-1), irrespective of OLR. Analysis of the methanogenic community showed limited differences between the two reactors on order level and a clear increase in the abundance of Methanomicrobiales, particularly Methanoculleus sp., in response to increasing ammonia concentration. Further comprehensive molecular analysis revealed that diverse Methanoculleus species dominated in the reactors at a given ammonia level at different OLR. The acetogenic community was clearly affected by both ammonia concentration and OLR, suggesting that the volatile fatty acid load in relation to the higher OLR was important for the dynamics of this community.

Insights into the annotated genome sequence of Methanoculleus bourgensis MS2T, related to dominant methanogens in biogas-producing plants

The final step of the biogas production process, the methanogenesis, is frequently dominated by members of the genus Methanoculleus. In particular, the species Methanoculleus bourgensis was identified to play a role in different biogas reactor systems. The genome of the type strain M. bourgensis MS2T, originally isolated from a sewage sludge digestor, was completely sequenced to analyze putative adaptive genome features conferring competitiveness within biogas reactor environments to the strain. Sequencing and assembly of the M. bourgensis MS2T genome yielded a chromosome with a size of 2,789,773bp. Comparative analysis of M. bourgensis MS2T and Methanoculleus marisnigri JR1 revealed significant similarities. The absence of genes for a putative ammonium uptake system may indicate that M. bourgensis MS2T is adapted to environments rich in ammonium/ammonia. Specific genes featuring predicted functions in the context of osmolyte production were detected in the genome of M. bourgensis MS2T. Mapping of metagenome sequences derived from a production-scale biogas plant revealed that M. bourgensis MS2T almost completely comprises the genetic information of dominant methanogens present in the biogas reactor analyzed. Hence, availability of the M. bourgensis MS2T genome sequence may be valuable regarding further research addressing the performance of Methanoculleus species in agricultural biogas plants.

Methanogenic biodegradation of two-ringed polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAH) are widespread in methane-rich subsurface environments, such as oil reservoirs and fuel-contaminated aquifers; however, little is known about the biodegradation of these compounds under methanogenic conditions. To assess the metabolism of PAH in the absence of electron acceptors, a crude oil-degrading methanogenic enrichment culture was tested for the ability to biodegrade naphthalene, 1-methylnaphthalene (1-MN), 2-methylnaphthalene (2-MN), and 2, 6-dimethylnaphthalene (2, 6-diMN). When methane was measured as an indicator of metabolism, nearly 400 μmol of methane was produced in the 2-MN- and 2, 6-diMN-amended cultures relative to substrate-unamended controls, which is close to the amount of methane stoichiometrically predicted based on the amount of substrate added (51-56 μmol). In contrast, no substantial methane was produced in the naphthalene- and 1-MN-amended enrichments. In time course experiments, metabolite analysis of enrichments containing 2-MN and 2, 6-diMN revealed the formation of 2-naphthoic acid and 6-methyl-2-naphthoic acid, respectively. Microbial community analysis by 454 pyrosequencing revealed that these PAH-utilizing enrichments were dominated by archaeal members most closely affiliated with Methanosaeta and Methanoculleus species and bacterial members most closely related to the Clostridiaceae, suggesting that these organisms play an important role in the methanogenic metabolism of the substituted naphthalenes in these cultures.

Methanoculleus hydrogenitrophicus sp. nov., a methanogenic archaeon isolated from wetland soil

An obligately anaerobic, methanogenic archaeon, strain HC(T), was isolated from soil of the Zoige wetland on the Tibetan plateau, China. The strain was isolated through construction of an artificial butyrate-degrading consortium in co-culture with a syntrophic bacterium, 'Syntrophomonas erecta subsp. sporosyntropha' JCM 13344. Cells of strain HC(T) were irregular coccoids, 0.8-2 mum in diameter, that occurred singly and utilized only H(2)/CO(2) for growth and methane production. Growth occurred at 18-45 degrees C (optimum around 37 degrees C). The pH for growth was 5.0-8.5 (optimal growth around pH 6.6). The G+C content of the genomic DNA was 60.2 mol%. 16S rRNA gene sequence analysis indicated that strain HC(T) was affiliated to the genus Methanoculleus, with sequence similarities of 94.8-97.2 % to existing members. However, strain HC(T) was distinguished from described Methanoculleus species by not using formate for growth or methane formation and not requiring acetate as a growth factor. On the basis of phylogenetic analysis and phenotypic characteristics, the novel species Methanoculleus hydrogenitrophicus sp. nov. is proposed, with strain HC(T) (=CGMCC 1.5146(T) =JCM 16311(T)) as the type strain.

Methanoculleus chikugoensis sp. nov., a novel methanogenic archaeon isolated from paddy field soil in Japan, and DNA-DNA hybridization among Methanoculleus species.

A strictly anaerobic, irregularly coccoid, methanogenic archaeon, strain MG62T (= JCM 10825T = DSM 13459T), was isolated from paddy field soil in Chikugo, Fukuoka, Japan. The cells stained gram-negative, were 1.0-2.0 microm in diameter, were lysed by SDS and hypotonic solutions and were flagellated. Motility was not observed. The strain was able to use H2/CO2, 2-propanol/CO2, formate, 2-butanol/CO2 and cyclopentanol/CO2 as substrates for methanogenesis, but did not utilize acetate, ethanol, methanol or methylamines. The optimum temperature and pH were 25-30 degrees C and 6.7-7.2. Analysis of lipid component parts (core lipids, phospholipid polar head groups and glycolipid sugar moieties) showed the characteristic pattern of members of the family Methanomicrobiaceae except for the absence of glucose as a glycolipid sugar moiety. The G+C content of the DNA was 62.2 mol %. Sequence analysis of the 16S rDNA revealed that the strain belonged to the genus Methanoculleus. The strain had DNA-DNA hybridization values of less than 50% with type strains of Methanoculleus species. On the basis of phenotypic, genotypic and phylogenetic characteristics, the name Methanoculleus chikugoensis sp. nov. is proposed for strain MG62T (= JCM 10825T = DSM 13459T). The DNA hybridization study also revealed the close relationships of three species, Methanoculleus olentangyi, Methanoculleus bourgensis and Methanoculleus oldenburgensis, among Methanoculleus species.

Methanoculleus palmolei sp. nov., an irregularly coccoid methanogen from an anaerobic digester treating wastewater of a palm oil plant in north-Sumatra, Indonesia.

Strain INSLUZT (= DSM 4273T) was isolated from a biogas-producing bioreactor treating wastewater of a palm oil mill on North-Sumatra (Indonesia). Cells of strain INSLUZT were highly irregularly coccoid, 1.25-2.0 microns in diameter, had a cell envelope consisting of the cytoplasmic membrane and an S-layer of hexagonally arranged glycoprotein subunits with an M(r) of 120,000, and were flagellated (motility was not observed). Cells were mesophilic and grew most rapidly at 40 degrees C on H2/CO2' formate, 2-propanol/CO2 2-butanol/CO2 and cyclopentanol/CO2 to give methane. Tungstate promoted growth on H2/CO2 with acetate as the solely required organic medium supplement. The G + C content of DNA was 59 mol% (Tm method) and 59.5 mol% (HPLC method). 16S rDNA analysis revealed a phylogenetic relationship to Methanoculleus species; the name Methanoculleus palmolei sp. nov. is therefore proposed for strain INSLUZT (= DSM 4273T).

Isolation and characterization of a novel mesophilic, fresh-water methanogen from river sediment Methanoculleus oldenburgensis sp. nov.

A new mesophilic, irregular coccoid methanogen isolated from a river sediment is described. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. For growth acetate is strictly required. Elevated levels of sodium chloride were not required and were inhibitory at concentrations above 1.5% (w/v). The optimal growth temperature was at 45°C. The DNA base ratio was 48.6±1 mol% G+C. The polar lipid pattern and the polyamine content were similar to that found in several Methanoculleus species. The new isolate CB-1 was assigned as Methanoculleus oldenburgensis (DSM 6216).