The novel regulator HdrR controls the transcription of the heterodisulfide reductase operon hdrBCA in Methanosarcina barkeri.

Methanogenic archaea play a key role in the global carbon cycle because these microorganisms remineralize organic compounds in various anaerobic environments. The microorganism Methanosarcina barkeri is a metabolically versatile methanogen, which can utilize acetate, methanol, and H2/CO2 to synthesize methane. However, the regulatory mechanisms underlying methanogenesis for different substrates remain unknown. In this study, RNA-seq analysis was used to investigate M. barkeri growth and gene transcription under different substrate regimes. According to the results, M. barkeri showed the best growth under methanol, followed by H2/CO2 and acetate, and these findings corresponded well with the observed variations in genes transcription abundance for different substrates. In addition, we identified a novel regulator, MSBRM_RS03855 (designated as HdrR), which specifically activates the transcription of the heterodisulfide reductase hdrBCA operon in M. barkeri. HdrR was able to bind to the hdrBCA operon promoter to regulate transcription. Furthermore, the structural model analyses revealed a helix-turn-helix domain, which is likely involved in DNA binding. Taken together, HdrR serves as a model to reveal how certain regulatory factors control the expression of key enzymes in the methanogenic pathway.IMPORTANCEThe microorganism Methanosarcina barkeri has a pivotal role in the global carbon cycle and contributes to global temperature homeostasis. The consequences of biological methanogenesis are far-reaching, including impacts on atmospheric methane and CO2 concentrations, agriculture, energy production, waste treatment, and human health. As such, reducing methane emissions is crucial to meeting set climate goals. The methanogenic activity of certain microorganisms can be drastically reduced by inhibiting the transcription of the hdrBCA operon, which encodes heterodisulfide reductases. Here, we provide novel insight into the mechanisms regulating hdrBCA operon transcription in the model methanogen M. barkeri. The results clarified that HdrR serves as a regulator of heterodisulfide reductase hdrBCA operon transcription during methanogenesis, which expands our understanding of the unique regulatory mechanisms that govern methanogenesis. The findings presented in this study can further our understanding of how genetic regulation can effectively reduce the methane emissions caused by methanogens.

Oleispirillum naphthae gen. nov., sp. nov., a bacterium isolated from oil sludge, and proposal of Oleispirillaceae fam. nov.

A microaerophilic, Gram-negative, motile, and spiral-shaped bacterium, designated Y-M2T, was isolated from oil sludge of Shengli oil field. The optimal growth condition of strain Y-M2T was at 25 °C, pH 7.0, and in the absence of NaCl. The major polar lipid was phosphatidylethanolamine. The main cellular fatty acid was iso-C17  :  0 3-OH. It contained Q-9 and Q-10 as the predominant quinones. The DNA G+C content was 68.1 mol%. Strain Y-M2T showed the highest 16S rRNA gene sequence similarity to Telmatospirillum siberiense 26-4bT (91.1 %). Phylogenetic analyses based on 16S rRNA gene and genomes showed that strain Y-M2T formed a distinct cluster in the order Rhodospirillales. Genomic analysis showed that Y-M2T possesses a complete nitrogen-fixation cluster which is phylogenetically close to that of methanogene. The nif cluster, encompassing the nitrogenase genes, was found in every N2-fixing strain within the order Rhodospirillales. Phylogeny, phenotype, chemotaxonomy, and genomic results demonstrated that strain Y-M2T represents a novel species of a novel genus in a novel family Oleispirillaceae fam. nov. in the order Rhodospirillales, for which the name Oleispirillum naphthae gen. nov., sp. nov. was proposed. The type strain is Y-M2T (=CCAM 827T=JCM 34765T).

Shumkonia mesophila gen. nov., sp. nov., a novel representative of Shumkoniaceae fam. nov. and its potentials for extracellular polymeric substances formation and sulfur metabolism revealed by genomic analysis.

A microaerophilic, mesophilic, chemoorganoheterotrophic bacterium, designated Y-P2T, was isolated from oil sludge enrichment in China. Cells of the strain were Gram-stain-negative, non-motile, non-spore-forming, rod-shaped or slightly curved with 0.8-3.0 µm in length and 0.4-0.6 µm in diameter. The strain Y-P2T grew optimally at 25 °C (range from 15 to 30 °C) and pH 7.0 (range from pH 6.0 to 7.5) without NaCl. The major cellular fatty acids were C16:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The main polar liquids of strain Y-P2T comprised phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). The respiratory quinone was Q-10. Acetate and H2 were the fermentation products of glucose. The DNA G + C content was 66.0%. Strain Y-P2T shared the highest 16S rRNA gene sequence similarity (90.3-90.6%) with species within Oceanibaculum of family Thalassobaculaceae in Rhodospirillales. Phylogenetic analyses based on 16S rRNA gene sequences and genomes showed that strain Y-P2T formed a distinct evolutionary lineage within the order Rhodospirillales. On the basis of phenotypic, phylogenetic and phylogenomic data, we propose that strain Y-P2T represents a novel species in a novel genus, for which Shumkonia mesophila gen. nov., sp. nov., within a new family Shumkoniaceae fam. nov. The type strain is Y-P2T (= CCAM 826 T = JCM 34766 T).

Serum folate mediates the associations of MTHFR rs1801133 polymorphism with blood glucose levels and gestational diabetes mellitus in Chinese Han pregnant women.

This study aimed to explore the mediation effects of one-carbon metabolism (OCM) related nutrients on the association between MTHFR rs1801133 polymorphism and gestational diabetes mellitus (GDM). Folate, vitamin B12 and homocysteine (Hcy) were measured in the serum of 1254 pregnant women. Linear and logistic regressions were used to estimate the associations of OCM nutrients and MTHFR rs1801133 polymorphism with blood glucose levels and GDM risk. Mediation analysis was applied to test the mediation effects of folate, vitamin B12 and Hcy on the association of MTHFR rs1801133 polymorphism with blood glucose concentrations and GDM. Pregnant women with MTHFR rs1801133 CC genotype had higher serum folate (10·75 v. 8·90 and 9·40 ng/ml) and lower serum Hcy (4·84 v. 4·93 and 5·20 µmol/l) than those with CT and TT genotypes. Folate concentrations were positively associated with fasting plasma glucose (FPG), 1-h plasma glucose (1-h PG), 2-h plasma glucose (2-h PG) and GDM risk. Vitamin B12 levels were negatively correlated with FPG and GDM. Although no direct association was found between MTHFR rs1801133 genotypes and GDM, there were significant indirect effects of MTHFR rs1801133 CC genotype on FPG (ß: 0·005; 95 % CI: 0·001, 0·013), 1-h PG (ß: 0·006; 95 % CI: 0·001, 0·014), 2-h PG (ß: 0·007; 95 % CI: 0·001, 0·015) and GDM (ß: 0·006; 95 % CI: 0·001, 0·014) via folate. In conclusion, serum folate mediates the effect of MTHFR rs1801133 on blood glucose levels and GDM. Our findings potentially provide a feasible GDM prevention strategy via individualised folate supplementation according to the MTHFR genotypes.

Description of a moderately acidotolerant and aerotolerant anaerobic bacterium Acidilutibacter cellobiosedens gen. nov., sp. nov. within the family Acidilutibacteraceae fam. nov., and proposal of Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov.

A Gram-stain positive, moderately thermophilic, acidotolerant and aerotolerant anaerobic bacterium, designated JN-28 T, was isolated from the pit mud of Chinese strong-flavor liquor. Growth was observed at 25-50 °C and pH 5.5-8.0 in the presence of 0-25 g l-1 NaCl (optimally at 45 °C, pH 6.0, without NaCl). Strain JN-28 T was heterotrophic, requiring yeast extract for growth. The major cellular fatty acids were iso-C15:0 and C14:0. The DNA G + C content of genomic DNA was 33.54 mol%. The strain was resistant to vancomycin (10 mg l-1). Genome analysis revealed the presence of genes involved in the response to mild acid stress and oxidative stress, and resistance to vancomycin. 16S rRNA gene-based phylogenetic analysis showed that strain JN-28 T shares ≤ 89.3 % sequence similarity with its closest relatives Sporanaerobacter acetigenes DSM 13106 T and other members in the order Tissierellales. Based on phenotypic and phylogenetic characteristics, Acidilutibacter cellobiosedens gen. nov., sp. nov. is proposed for the new genus and novel species with the type strain JN-28 T (=CCAM 418 T = JCM 39087 T). Further phylogenetic and phylogenomic analyses suggested strain JN-28 T represents a novel family within the order Tissierellales, for which Acidilutibacteraceae fam. nov. is proposed. In addition, the family Tissierellaceae was reclassified, Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov. were formally proposed. Emended description of the family Tissierellaceae is also provided.

Hominibacterium faecale gen. nov., sp. nov., an anaerobic L-arginine-degrading bacterium isolated from human feces.

Two anaerobic, mesophilic bacteria SF3T and ASD5510 were isolated from human feces in two different countries. Strain SF3T shared 99.9% of 16S rRNA gene sequence similarity with strain ASD5510, and 92.8% similarity with the most similar strain Aminipila butyrica DSM 103574T. Strain SF3T was an anaerobic, Gram-stain-positive bacterium. Cells of strain SF3T were short rods with 0.3-0.4 µm in width × 2.0-2.4 µm in length and occurred mostly in pairs or short chains. Spore formation was not observed. The strain grew optimally at 35 °C (range from 20 to 45 °C), pH 7.5 (pH 6.0-8.5) and without NaCl addition (range from 0 to 20 g l-1 NaCl). Yeast extract was an essential growth factor for strain SF3T, L-arginine and γ-aminobutyrate were utilized as substrates for growth. The major cellular fatty acids were iso-C15:0 and C16:0 DMA. The main polar lipids were aminophospholipid (APL), diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The G + C content of the genomic DNA of the strain SF3T was 47.38 mol %. The paired genomic average amino acid identity (AAI) and percentage of conserved proteins (POCP) values showed relatedness of less than 61.0 and 39.4% with type strains of order Eubacteriales. On the basis of phenotypic, phylogenetic and phylogenomic evidence strain SF3T constitutes a novel species in a novel genus, for which the name Hominibacterium faecale gen. nov., sp. nov. is proposed. The type strain is SF3T (= CCAM 730T = JCM 34755T = KCTC 25324T).

Aminipila luticellarii sp. nov., an anaerobic bacterium isolated from the pit mud of strong aromatic Chinese liquor, and emended description of the genus Aminipila.

A novel mesophilic, aerotolerant anaerobic bacterium, designated JN-18T, was isolated from the pit mud of a strong aromatic Chinese liquor. According to a 16S rRNA gene sequence analysis, it had the highest sequence similarity to Aminipila butyrica DSM 103574T (95.69%). The G+C content of its genomic DNA was 43.39 mol%. The cells were Gram-stain-negative, slightly curved rods with flagella. Optimum growth was observed at 37 °C, pH 6.5 and without extra addition of NaCl. Strain JN-18Tutilized amino acids (l-alanine, l-arginine, l-asparagine, l-lysine, l-methionine, l-serine and l-threonine), malate and pyruvate, and used l-arginine and l-lysine to produce acetate, butyrate, H2, and CO2. The major cellular fatty acids of strain JN-18T were C14:0, C16:0 DMA and C18:1 cis-9 DMA. The carbohydrate composition of the cell wall predominantly included galactose, glucose and rhamnose. Based on its phylogenetic, phenotypic, physiological and biochemical characteristics, strain JN-18T was classified as a representative of a novel species within the genus Aminipila, for which the name Aminipila luticellarii sp. nov. is proposed. The type strain is JN-18T (=CCAM 412T=JCM 39126T).

Thermosynergistes pyruvativorans gen. nov., sp. nov., an anaerobic, pyruvate-degrading bacterium from Shengli oilfield, and proposal of Thermosynergistaceae fam. nov. in the phylum Synergistetes.

A strictly anaerobic, thermophilic, Gram-stain-negative bacterium, named as strain S15T, was isolated from oily sludge of Shengli oilfield in PR China. Cells of strain S15T were straight or slightly curved rods with 0.4-0.8 µm width × 1.4-3 µm length and occurred mostly in pairs or short chains. Endospore-formation was not observed. The strain grew optimally at 55 °C (range from 30-65 °C), pH 6.5 (pH 6.0-8.5) and 0-30 g l-1 NaCl (optimum with 10 g l-1 NaCl). Yeast extract was an essential growth factor for strain S15T. The major cellular fatty acid was iso-C15 : 0 (58.2 %), and the main polar lipids were amino phospholipid (APL), glycolipids (GLs) and phosphatidylethanolamine (PE). The G+C content of DNA of strain S15T was 52.2 mol%. Strain S15T shared 89.8 % 16S rRNA gene similarity with the most related organism Acetomicrobium hydrogeniformans DSM 22491T in the phylum Synergistetes. The paired genomic average amino acid identity (AAI) and percentage of conserved proteins (POCP) values showed relatedness of less than 58.0 and 39.7 % with type strains of the species in the phylum Synergistetes. On the basis of phenotypic, phylogenetic and phylogenomic evidences, strain S15T constitutes a novel species in a novel genus, for the name Thermosynergistes pyruvativorans gen. nov., sp. nov. is proposed. The type strain is S15T (=CCAM 583T=JCM 33159T). Thermosynergistaceae fam. nov. is also proposed.

Correlating subword articulation with lip shapes for embedding aware audio-visual speech enhancement.

In this paper, we propose a visual embedding approach to improve embedding aware speech enhancement (EASE) by synchronizing visual lip frames at the phone and place of articulation levels. We first extract visual embedding from lip frames using a pre-trained phone or articulation place recognizer for visual-only EASE (VEASE). Next, we extract audio-visual embedding from noisy speech and lip frames in an information intersection manner, utilizing a complementarity of audio and visual features for multi-modal EASE (MEASE). Experiments on the TCD-TIMIT corpus corrupted by simulated additive noises show that our proposed subword based VEASE approach is more effective than conventional embedding at the word level. Moreover, visual embedding at the articulation place level, leveraging upon a high correlation between place of articulation and lip shapes, demonstrates an even better performance than that at the phone level. Finally the experiments establish that the proposed MEASE framework, incorporating both audio and visual embeddings, yields significantly better speech quality and intelligibility than those obtained with the best visual-only and audio-only EASE systems.

Zhaonella formicivorans gen. nov., sp. nov., an anaerobic formate-utilizing bacterium isolated from Shengli oilfield, and proposal of four novel families and Moorellales ord. nov. in the phylum Firmicutes.

A novel obligately anaerobic, thermophilic and formate-utilizing bacterium K32T was isolated from Shengli oilfield of China. Cells were straight rods (0.4-0.8 µm × 2.5-8.0 µm), Gram-stain-positive, non-spore-forming and slightly motile. Optimum growth occurred with pH of 7 and 0.5 g l-1 NaCl under temperature of 55-60 °C. Nitrate could be reduced into nitrite, syntrophic formate oxidation to methane and carbon dioxide occurred when co-culturing strain K32T and Methanothermobacter thermautotrophicus ΔH. The main cellular fatty acids were iso-C15 : 0 (24.0 %), anteiso-C15 : 0 (21.7 %), C16 : 0 (12.7 %) and C14 : 0 (10.8 %), and the main polar lipid was phosphatidylglycerol. The G+C content of the genomic DNA was 46.3 mol%. The 16S rRNA gene sequence of K32T shared ≤90.4 % of sequence similarity to closest type strains of Desulfitibacter alkalitolerans, Calderihabitans maritimus and members of the genus Moorella. Based on the phenotypic, biochemical and genotypic characterization, Zhaonella formicivorans gen. nov., sp. nov. is proposed with K32T (=CCAM 584T =DSM 107278T=CGMCC1.5297T) as the type strain, which is the first representative of Zhaonellaceae fam. nov. In addition, the order Thermoanaerobacterales and family Peptococcaceae were reclassified, and three novel families in the novel order of Moorellales ord. nov. were also proposed.

Gudongella oleilytica gen. nov., sp. nov., an aerotorelant bacterium isolated from Shengli oilfield and validation of family Tissierellaceae.

A novel Gram-stain-positive, rod shaped and anaerobic bacterium, designated as W6T, was isolated from Shengli oilfield in China. Strain W6T was observed to grow from 20 to 45 °C with pH 6.5-9.0 (optimally at 40 °C and pH of 7.5) and without addition of NaCl. The major cellular fatty acids were iso-C15 : 0 (29.1%), C14 : 0 (27.0%) and C16 : 0 (12.2%), and the main polar lipids were lipids (L) and aminolipids (AL). The DNA G+C content is 42.9 mol%. Based on 16S rRNA gene sequence analysis, strain W6T showed highest similarities to Tissierella creatinini DSM 9508T (94.9%) and Soehngenia saccharolytica DSM 12858T (94.1%). The morphological, physiological, biochemical, phylogenetic and phylogenomic analyses demonstrated strain W6T (CCAM 534T=DSM 28124T=CGMCC 1.5291T) represents a novel species in a new genus, for which the name Gudongella oleilytica gen. nov. sp. nov. is proposed. The family Tissierellaceae is proposed as a new family containing the genera Anaerosalibacter, Gudongella, Keratinibaculum, Soehngenia, Sporanaerobacter, Tepidimicrobium, Tissierella, Urmitella and species Clostridium ultunense based on the phylogenetic and phylogenomic analyses.

Description of Biomaibacter acetigenes gen. nov., sp. nov., and proposal of Thermosediminibacterales ord. nov. containing two novel families of Tepidanaerobacteraceae fam. nov. and Thermosediminibacteraceae fam. nov.

A Gram-stain-negative, rod-shaped, chemoorganotrophic and anaerobic bacterium, strain SK-G1T, was isolated from oily sludge sampled at the Shengli oilfield in PR China. Growth occurred with 0-30 g l-1 NaCl, at 40-65 °C and at pH 6.0-8.5. The predominant fatty acids were C14 : 0 and C13 : 0, and the major cellular polar lipids were phosphatidylglycerol and phosphatidylethanolamine. No respiratory quinone was detected. The genomic G+C content was 43.9 mol%. The strain had highest 16S rRNA gene sequence similarity (93.2 % identity) to Tepidanaerobacter syntrophicus DSM 15584T. The phylogenetic, phenotypic and chemotaxonomic data showed that strain SK-G1T (=CCAM 530T=KCTC 15783T=JCM 33158T) represents a novel species of a new genus Biomaibacteracetigenes gen. nov., sp. nov. The results of phylogenetic and phylogenomic analyses indicated that the genera Biomaibacter, Caldanaerovirga, Fervidicola, Tepidanaerobacter, Thermosediminibacter, Thermovenabulum and Thermovoraxformed a clade with high bootstrap support distinguishing to other taxon within the order Thermoanaerobacterales. This clade is proposed as Thermosediminibacterales ord. nov. and includes Tepidanaerobacteraceae fam. nov. and Thermosediminibacteraceae fam. nov. Emended descriptions of the order Thermoanaerobacterales and family Syntrophomonadaceae are also provided.

Simultaneous enhancement of nonylphenol biodegradation and short-chain fatty acids production in waste activated sludge under acidogenic conditions.

Nonylphenol (NP) biodegradation in waste activated sludge (WAS) under anaerobic conditions is usually slow, and no information on NP biodegradation under acidogenic conditions is currently available. In this study, the simultaneous enhancement of NP biodegradation and short-chain fatty acids (SCFAs) accumulation in a WAS fermentation system under acidogenic conditions was accomplished by controlling pH 10 and adding sodium lauryl sulfate (SLS). The biodegradation efficiency of NP was found to be 55.5% within 8 d under acidogenic conditions, much higher than that in the control (24.6%). Meanwhile, the concentration of SCFAs under the same conditions for NP biodegradation was increased from 2234 mg COD/L (control) to 4691 mg COD/L (at pH 10 with SLS). Mechanism study revealed that the abundances of both NP-degrading microorganisms and acidogenic bacteria increased under acidogenic conditions. Altering the enzymatic activity and the quantity of functional genes in the acidogenic fermentation system were beneficial to NP biodegradation and SCFAs accumulation. Furthermore, organic substrates available for uptake by NP-degrading and acidogenic bacteria, i.e. NP, protein and carbohydrate, were released from WAS under acidogenic conditions. More importantly, intermediate substrates involved in acidogenic fermentation were advantageous to the cometabolic biodegradation of NP.

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.

Lactobacillus panisapium sp. nov., from honeybee Apis cerana bee bread.

A novel facultatively anaerobic, Gram-stain-positive, non-motile, non-spore-forming, catalase-negative bacterium of the genus Lactobacillus, designated strain Bb 2-3T, was isolated from bee bread of Apis cerana collected from a hive in Kunming, China. The strain was regular rod-shaped. Optimal growth occurred at 37 °C, pH 6.5 with 5.0 g l-1 NaCl. The predominant fatty acids were C18 : 1ω9c, C16 : 0 and C19 : 0 iso. Respiratory quinones were not detected. Seven glycolipids, three lipids, phosphatidylglycerol and diphosphatidylglycerol were detected. The peptidoglycan type A4α l-Lys-d-Asp was determined. Strain Bb 2-3T was closely related to Lactobacillus bombicola DSM 28793T, Lactobacillus apis LMG 26964T and Lactobacillus helsingborgensis DSM 26265T, with 97.8, 97.6 and 97.0 % 16S rRNA gene sequence similarity, respectively. A comparison of two housekeeping genes, rpoA and pheS, revealed that strain Bb 2-3T was well separated from the reference strains of species of the genus Lactobacillus. The average nucleotide identity between strain Bb 2-3T and the type strains of closely related species was lower than the 95-96 % threshold value for delineation of genomic prokaryotic species. The G+C content of the genomic DNA of strain Bb 2-3T was 37.4 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, strain Bb 2-3T is proposed to represent a novel species of the genus Lactobacillus, for which we propose the name Lactobacillus panisapium sp. nov. The type strain is Bb 2-3T (=DSM 102188T=ACCC 19955T).

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.

Neuropeptide S reduces propofol- or ketamine-induced slow wave states through activation of cognate receptors in the rat.

Intracerebroventricular injection of NPS reduces the duration of the ketamine- or thiopental-induced loss of the righting reflex in rats. But the specific EEG activities are unknown. We therefore sought to examine the effects of the NPS-NPSR system on anesthetic-induced characteristics of EEG power spectra and sleep-wake profiles. NPS alone or together with an NPSR antagonist was injected intracerebroventricularly, whereas the propofol (50mg/kg) or ketamine (100mg/kg) was administrated intraperitoneally. NPS (1 or 2nmol) significantly reduced the amount of propofol-induced EEG delta activity and slow wave states (SWS). NPS (1 or 5nmol) significantly reduced the amount of ketamine-induced SWS and EEG delta activity. Cortical EEG power spectral analysis showed that, in saline-pretreated rats, propofol induced a marked increase in delta (0.5-4Hz) activity, decrease in theta (4.5-8.5Hz) activity, and decrease in high frequency activity (14.5-60Hz), while, in rats pretreated with 1nmol of NPS, the duration of delta activity was reduced, while its spectral pattern was not changed. Whereas injection of ketamine into saline-pretreated rats induced a marked increase in delta (0.5-4Hz) activity, a moderate increase in theta (4.5-8.5Hz) activity, and a marked decrease in high frequency (14.5-60Hz) activity. However, delta activity was reduced while theta activity increased under pretreatment with 1nmol of NPS. The inhibitory effect of NPS on anesthetic-induced SWS was characterized by a reduced SWS episode duration with no significant change in either episode number or latency to SWS. [D-Val5]NPS, an NPSR antagonist (20nmol), significantly attenuated the arousal-promoting effect of 1nmol of NPS, but had no effect on SWS when injected alone. We speculate that NPS significantly reduces anesthetic-induced SWS and EEG slow activity by selective activation of the NPSR, which, in turn, would trigger subsequent arousal pathways.

Defluviitalea raffinosedens sp. nov., a thermophilic, anaerobic, saccharolytic bacterium isolated from an anaerobic batch digester treating animal manure and rice straw.

A thermophilic, anaerobic, fermentative bacterium, strain A6T, was obtained from an anaerobic batch digester treating animal manure and rice straw. Cells were Gram-stain-positive, slightly curved rods with a size of 0.6-1×2.5-8.2 µm, non-motile and produced terminal spores. The temperature, pH and NaCl concentration ranges for growth were 40-60 °C, 6.5-8.0 and 0-15.0 g l-1, with optimum growth noted at 50-55 °C, pH 7.5 and in the absence of NaCl, respectively. Yeast extract was required for growth. d-Glucose, maltose, d-xylose, d-galactose, d-fructose, d-ribose, lactose, raffinose, sucrose, d-arabinose, cellobiose, d-mannose and yeast extract were used as carbon and energy sources. The fermentation products from glucose were ethanol, lactate, acetate, propionate, butyrate, valerate, iso-butyrate, iso-valerate, H2 and CO2. The G+C content of the genomic DNA was 36.6 mol%. The predominant fatty acids were C16 : 0, iso-C17 : 1, C14 : 0, C16 : 1ω7c, C16 : 0 N-alcohol and C13 : 0 3-OH. Respiratory quinones were not detected. The polar lipid profile comprised phosphoglycolipids, phospholipids, glycolipids, a diphosphatidylglycerol, a phosphatidylglycerol and an unidentified lipid. Phylogenetic analyses of the 16S rRNA gene sequence indicated that the strain was closely related to Defluviitalea saccharophila DSM 22681T with a similarity of 96.0 %. Based on the morphological, physiological and taxonomic characterization, strain A6T is considered to represent a novel species of the genus Defluviitalea, for which the name Defluviitalea raffinosedens sp. nov. is proposed. The type strain is A6T (=DSM 28090T=ACCC 19951T).

[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.

Enrichment and Characterization of a Psychrotolerant Consortium Degrading Crude Oil Alkanes Under Methanogenic Conditions.

Anaerobic alkane degradation via methanogenesis has been intensively studied under mesophilic and thermophilic conditions. While there is a paucity of information on the ability and composition of anaerobic alkane-degrading microbial communities under low temperature conditions. In this study, we investigated the ability of consortium Y15, enriched from Shengli oilfield, to degrade hydrocarbons under different temperature conditions (5-35 °C). The consortium could use hexadecane over a low temperature range (15-30 °C). No growth was detected below 10 °C and above 35 °C, indicating the presence of cold-tolerant species capable of alkane degradation. The preferential degradation of short chain n-alkanes from crude oil was observed by this consortium. The structure and dynamics of the microbial communities were examined using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting and Sanger sequencing of 16S rRNA genes. The core archaeal communities were mainly composed of aceticlastic Methanosaeta spp. Syntrophaceae-related microorganisms were always detected during consecutive transfers and dominated the bacterial communities, sharing 94-96 % sequence similarity with Smithella propionica strain LYP(T). Phylogenetic analysis of Syntrophaceae-related clones in diverse methanogenic alkane-degrading cultures revealed that most of them were clustered into three sublineages. Syntrophaceae clones retrieved from this study were mainly clustered into sublineage I, which may represent psychrotolerant, syntrophic alkane degraders. These results indicate the wide geographic distribution and ecological function of syntrophic alkane degraders.