Isolation of a methyl-reducing methanogen outside the Euryarchaeota.

Methanogenic archaea are main contributors to methane emissions, and have a crucial role in carbon cycling and global warming. Until recently, methanogens were confined to Euryarchaeota, but metagenomic studies revealed the presence of genes encoding the methyl coenzyme M reductase complex in other archaeal clades1-4, thereby opening up the premise that methanogenesis is taxonomically more widespread. Nevertheless, laboratory cultivation of these non-euryarchaeal methanogens was lacking to corroborate their potential methanogenic ability and physiology. Here we report the isolation of a thermophilic archaeon LWZ-6 from an oil field. This archaeon belongs to the class Methanosuratincolia (originally affiliated with 'Candidatus Verstraetearchaeota') in the phylum Thermoproteota. Methanosuratincola petrocarbonis LWZ-6 is a strict hydrogen-dependent methylotrophic methanogen. Although previous metagenomic studies speculated on the fermentative potential of Methanosuratincolia members, strain LWZ-6 does not ferment sugars, peptides or amino acids. Its energy metabolism is linked only to methanogenesis, with methanol and monomethylamine as electron acceptors and hydrogen as an electron donor. Comparative (meta)genome analysis confirmed that hydrogen-dependent methylotrophic methanogenesis is a widespread trait among Methanosuratincolia. Our findings confirm that the diversity of methanogens expands beyond the classical Euryarchaeota and imply the importance of hydrogen-dependent methylotrophic methanogenesis in global methane emissions and carbon cycle.

Petroclostridium xylanilyticum gen. nov., sp. nov., a xylan-degrading bacterium isolated from an oilfield, and reclassification of clostridial cluster III members into four novel genera in a new Hungateiclostridiaceae fam. nov.

A rod-shaped, Gram-stain-positive, obligately anaerobic, xylan-degrading bacterium, SK-Y3T, was isolated from oily-sludge of Shengli oilfield, China. Optimum growth occurred at 50 °C, at pH 7.5 and without addition of NaCl. The predominant cellular fatty acids of strain SK-Y3T were iso-C15 : 0, anteiso-C15 : 0 and iso-C17 : 0, and the main polar lipids were glycolipids (GL), lipids (L), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG); no respiratory quinones were detected. The genomic DNA G+C content was 37.2 mol%. Phylogenetic analysis of 16S rRNA gene sequences showed that strain SK-Y3T belongs to clostridial cluster III, exhibiting 91-92% sequence similarity to the most closely related species, namely Clostridium clariflavum, Clostridium straminisolvens and Acetivibrio cellulolyticus. Based on distinct physiological and phylogenetic differences from the aforementioned described taxa, strain SK-Y3T (=DSM 103557T=ACCC 19952T) is proposed as the type strain of a novel species of a new genus, Petroclostridium xylanilyticum gen. nov., sp. nov. Furthermore, analysis through 16S rRNA gene, ribosomal protein and whole genome sequences indicated that clostridial cluster III members should be reclassified into four novel genera for which the names Hungateiclostridium gen. nov., Thermoclostridium gen. nov., Ruminiclostridium gen. nov. and Pseudoclostridium gen. nov. are proposed. In combination with the genera Anaerobacterium, Cellulosibacter, Ercella, Fastidiosipila, Mageeibacillus, Pseudobacteroides, Petroclostridium and Saccharofermentans, clostridial cluster III members formed a monophyletic clade within the order Clostridiales but that was clearly distinguished from other Ruminococcaceae members, which is proposed as a novel family, Hungateiclostridiaceae fam. nov.

[Characterization and microbial community shifts of rice strawdegrading microbial consortia].

Objective: To study the relationship between microbial community and degradation rate of rice straw, we compared and analyzed cellulose-decomposing ability, microbial community structures and shifts of microbial consortia F1 and F2. Methods: We determined exoglucanase activity by 3, 5-dinitrosalicylic acid colorimetry. We determined content of cellulose, hemicellulose and lignin in rice straw by Van Soest method, and calculated degradation rates of rice straw by the weight changes before and after a 10-day incubation. We analyzed and compared the microbial communities and functional microbiology shifts by clone libraries, Miseq analysis and real time-PCR based on the 16S rRNA gene and cel48 genes. Results: Total degradation rate, cellulose, and hemicellulose degradation rate of microbial consortia F1 were significantly higher than that of F2. The variation trend of exoglucanase activity in both microbial consortia F1 and F2 was consistent with that of cel48 gene copies. Microbial diversity of F1 was complex with aerobic bacteria as dominant species, whereas that of F2 was simple with a high proportion of anaerobic cellulose decomposing bacteria in the later stage of incubation. In the first 4 days, unclassified Bacillales and Bacillus were dominant in both F1 and F2. The dominant species and abundance became different after 4-day incubation, Bacteroidetes and Firmicutes were dominant phyla of F1 and F2, respectively. Although Petrimonas and Pusillimonas were common dominant species in F1 and F2, abundance of Petrimonas in F2 (38.30%) was significantly higher than that in F1 (9.47%), and the abundance of Clostridiales OPB54 in F2 increased to 14.85% after 8-day incubation. Conclusion: The abundance of cel48 gene related with cellulose degradation rate and exoglucanase activity, and cel48 gene has the potential as a molecular marker to monitor the process of cellulose degradation. Microbial community structure has a remarkable impact on the degradation efficiency of straw cellulose, and Petrimonas, Paenibacillus, Bacillales, Clostridiales were vital species for microbial consortia F1 and F2 decomposing rice straw.

[Effects of selective methanogenic inhibitors on methanogenesis and methanogenic communities in acetate degrading cultures].

OBJECTIVE: We evaluated the role of syntrophic acetate oxidation coupled with hydrogenotrophic methanogens in three different methanogenic consortia. METHODS: Three methanogenic hexadecane degrading consortia named Y15, M82 and SK were taken from the same oily sludge of Shengli oil-field and enriched. They were incubated at 15, 35 and 55 °C, respectively. The consortia amended with acetate and inhibitors of NH4Cl or CH3F were further transferred and incubated at corresponding temperatures. The cultures atlate logarithmic phase were collected for terminal restriction fragment length polymorphism (T-RFLP) combined with cloning and phylogenetic analysis of 16S rRNA gene fragments. RESULTS: Gas chromatograph analysis showed that all of the consortia could grow and produce methane, but the lag phase was delayed and the growth rate was retarded in the cultures amended with inhibitor. Combination analysis of T-RFLP and clone library revealed the predominance of obligate aceticlastic Methanosaeta in the acetate cultures of Y15, M82 and SK. Under the mesophilic and thermophilic conditions, after add inginhibitor the relative abundance of aceticlastic methanogen decreased but hydrogenotrophic methanogen increased. CONCLUSION: Syntrophic acetate oxidation during methanogenic degradation of petroleum hydrocarbons occurs under mesophilic and thermophilic conditions, although the situation at low temperature seems uncertain.

Clostridium swellfunianum sp. nov., a novel anaerobic bacterium isolated from the pit mud of Chinese Luzhou-flavor liquor production.

A novel Gram-positive, strictly anaerobic, spore-forming, rod-shaped bacterium, designated strain S11-3-10(T), was isolated from the pit mud used for Chinese Luzhou-flavor liquor production. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a monophyletic clade with the closely related type strains of Clostridium cluster I and was most closely related to Clostridium amylolyticum JCM 14823(T) (94.38%). The temperature, pH, and NaCl range for growth was determined to be 20-45 °C (optimum 37 °C), 4.0-10.0 (optimum pH 7.3), and 0-3.0% (w/v), respectively. The strain was able to tolerate up to 7.5 % (v/v) ethanol. Yeast extract or peptone was found to be required for growth. Acids were found to be produced from glucose, mannose and trehalose. The major end products from glucose fermentation were identified as ethanol, acetate and hydrogen. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified phospholipids and polar lipids. The major fatty acids (>5%) were identified as iso-C(15:0), C(16:0), C(16:0)dma, C(14:0), anteiso-C(15:0) and iso-C(13:0). No respiratory quinone was detected. The diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid and the whole-cell sugars were found to include galactose and glucose as major components. The DNA G+C content was determined to be 36.4 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic evidence, the isolate is considered to represent a novel species of the genus Clostridium for which the name Clostridium swellfunianum sp. nov. is proposed. The type strain is S11-3-10(T) (=DSM 27788(T) = JCM 19606(T) = CICC 10730(T)).

Identifying key pathogenic mechanisms and potential intervention targets for recurrence after laryngeal cancer treatment through bioinformatics screening.

Background: Laryngeal cancer (LC), a prevalent malignant tumor of the head and neck, is characterized by a high rate of postoperative recurrence and significant treatment challenges upon recurrence, severely impacting patients' quality of life. There is a pressing need for effective biomarkers in clinical practice to predict the risk of LC recurrence and guide the development of personalized treatment plans. This study uses bioinformatics methods to explore potential biomarkers for LC recurrence, focusing on key genes and exploring their functions and mechanisms of action in LC recurrence. The aim is to provide new perspectives and evidence for clinical diagnosis, prognostic evaluation, and targeted treatment of LC. Methods: Gene expression profiles from the GSE25727 data set in the Gene Expression Omnibus database were analyzed to detect the differentially expressed genes (DEGs) between the tumor tissues of postoperative recurrent and non-recurrent early stage LC patients. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were also conducted. A protein-protein interaction (PPI) network and transcription factor (TF)-DEG-microRNA (miRNA) network were developed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, with key genes selected using the Molecular Complex Detection (MCODE) plugin. A Gene Set Enrichment Analysis (GSEA) was carried out to investigate the possible mechanisms of the key genes. A retrospective analysis was conducted using the clinical data of 83 LC patients. Immunohistochemical staining was used to examine the transcription level of the key genes in the LC tumor tissues and the factors affecting postoperative recurrence. Results: A total of 248 upregulated and 34 downregulated DEGs were identified in the GSE25727 data set. The PPI network analysis identified a significant module and five candidate genes (i.e., RRAGA, SLC38A9, WDR24, ATP6V1B1, and LAMTOR3). The construction of the TF-DEG-miRNA network indicated that ATP6V1B1 might be regulated by one TF and interact with 17 miRNAs. The KEGG and GSEA analyses suggested that ATP6V1B1 may influence LC recurrence through the involvement of pro-inflammatory and pro-fibrotic mediators, glutathione metabolism, matrix metalloproteinases, immune regulation, and lymphocyte interactions. The recurrence rate of the 83 LC patients included in the study was 19.3% (16/83). The immunohistochemistry results indicated that ATP6V1B1 was highly expressed in patients with recurrent LC. The univariate and multivariate logistic regression analyses revealed that tumor stage T3 (P=0.04), tumor stage T4 (P=0.01), and a high expression of ATP6V1B1 (P=0.02) were risk factors for recurrence after surgical treatment in LC patients. Conclusions: The key genes and signaling pathways identified through the bioinformatics screening provide insights into the potential mechanisms of the pathogenesis of LC. ATP6V1B1 may promote the recurrence of LC by weakening the immune phenotype. Our findings provide a theoretical basis for further research into clinical diagnostics and treatment strategies for LC.

Anaerobic hydrocarbon biodegradation by alkylotrophic methanogens in deep oil reservoirs.

In subsurface biodegraded oil reservoirs, methanogenic biodegradation of crude oil is a common process. This process was previously assigned to the syntrophy of hydrocarbon-degrading bacteria and methanogenic archaea. Recent studies showed that archaea of the Candidatus Methanoliparum named as alkylotrophic methanogens couple hydrocarbon degradation and methane production in a single archaeon. To assess the geochemical role of Ca. Methanoliparum, we analyzed the chemical and microbial composition and metabolites of 209 samples from 15 subsurface oil reservoirs across China. Gas chromatography-mass spectrometry analysis revealed that 92% of the tested samples were substantially degraded. Molecular analysis showed that 85% of the tested samples contained Ca. Methanoliparum, and 52% of the tested samples harbored multiple alkyl-coenzyme M derivatives, the intercellular metabolites of alkylotrophic archaea. According to metagenomic and metatranscriptomic analyses, Ca. Methanoliparum dominates hydrocarbon degradation in biodegraded samples from the Changqing, Jiangsu, and Shengli (SL) oilfields, and it is persistently present as shown in a 15-year-long sampling effort at the Shengli oilfield. Together, these findings demonstrate that Ca. Methanoliparum is a widely distributed oil degrader in reservoirs of China, suggesting that alkylotrophic methanogenesis by archaea plays a key role in the alteration of oil reservoirs, thereby expanding our understanding of biogeochemical process in the deep biosphere.

Thermodynamic restrictions determine ammonia tolerance of methanogenic pathways in Methanosarcina barkeri.

Ammonia is a ubiquitous potential inhibitor of anaerobic digestion processes, mainly exhibiting inhibition towards methanogenic activity. However, knowledge as to how ammonia affects the methanogens is still limited. In this study, we cultured a multitrophic methanogen, Methanosarcina barkeri DSM 800, with acetate, H2/CO2, and methanol to evaluate the influence of ammonia on different methanogenic pathways. Aceticlastic methanogenesis was more sensitive to increased ammonia concentrations than hydrogenotrophic and methylotrophic methanogenesis. Theoretical maximum NH3 tolerances of M. barkeri fed with acetate, H2/CO2, and methanol were calculated to be 39.1 ± 9.0, 104.3 ± 7.4, and 85.7 ± 1.0 mg/L, respectively. The order of the ΔG range of M. barkeri under three methanogenic pathways reflected the order of ammonia tolerance of M. barkeri. Our results provide insights into the role of the thermodynamic potential of methanogenesis on the tolerance of ammonia stress; and shed light on the mechanism of ammonia inhibition on anaerobic digestion.

Coexistence and competition of sulfate-reducing and methanogenic populations in an anaerobic hexadecane-degrading culture.

BACKGROUND: Over three-fifths of the world's known crude oil cannot be recovered using state-of-the-art techniques, but microbial conversion of petroleum hydrocarbons trapped in oil reservoirs to methane is one promising path to increase the recovery of fossil fuels. The process requires cooperation between syntrophic bacteria and methanogenic archaea, which can be affected by sulfate-reducing prokaryotes (SRPs). However, the effects of sulfate on hydrocarbon degradation and methane production remain elusive, and the microbial communities involved are not well understood. RESULTS: In this study, a methanogenic hexadecane-degrading enrichment culture was treated with six different concentrations of sulfate ranging from 0.5 to 25 mM. Methane production and maximum specific methane production rate gradually decreased to 44 and 56% with sulfate concentrations up to 25 mM, respectively. There was a significant positive linear correlation between the sulfate reduction/methane production ratio and initial sulfate concentration, which remained constant during the methane production phase. The apparent methanogenesis fractionation factor (αapp) gradually increased during the methane production phase in each treatment, the αapp for the treatments with lower sulfate (0.5-4 mM) eventually plateaued at ~1.047, but that for the treatment with 10-25 mM sulfate only reached ~1.029. The relative abundance levels of Smithella and Methanoculleus increased almost in parallel with the increasing sulfate concentrations. Furthermore, the predominant sulfate reducer communities shifted from Desulfobacteraceae in the low-sulfate cultures to Desulfomonile in the high-sulfate cultures. CONCLUSION: The distribution of hexadecane carbon between methane-producing and sulfate-reducing populations is dependent on the initial sulfate added, and not affected during the methane production period. There was a relative increase in hydrogenotrophic methanogenesis activity over time for all sulfate treatments, whereas the total activity was inhibited by sulfate addition. Both Smithella and Methanoculleus, the key alkane degraders and methane producers, can adapt to sulfate stress. Specifically, different SRP populations were stimulated at various sulfate concentrations. These results could help to evaluate interactions between sulfate-reducing and methanogenic populations during anaerobic hydrocarbon degradation in oil reservoirs.