Nitrate-driven anaerobic oxidation of ethane and butane by bacteria.

The short-chain gaseous alkanes (ethane, propane, and butane; SCGAs) are important components of natural gas, yet their fate in environmental systems is poorly understood. Microbially mediated anaerobic oxidation of SCGAs coupled to nitrate reduction has been demonstrated for propane, but is yet to be shown for ethane or butane-despite being energetically feasible. Here we report two independent bacterial enrichments performing anaerobic ethane and butane oxidation, respectively, coupled to nitrate reduction to dinitrogen gas and ammonium. Isotopic 13C- and 15N-labelling experiments, mass and electron balance tests, and metabolite and meta-omics analyses collectively reveal that the recently described propane-oxidizing "Candidatus Alkanivorans nitratireducens" was also responsible for nitrate-dependent anaerobic oxidation of the SCGAs in both these enrichments. The complete genome of this species encodes alkylsuccinate synthase genes for the activation of ethane/butane via fumarate addition. Further substrate range tests confirm that "Ca. A. nitratireducens" is metabolically versatile, being able to degrade ethane, propane, and butane under anoxic conditions. Moreover, our study proves nitrate as an additional electron sink for ethane and butane in anaerobic environments, and for the first time demonstrates the use of the fumarate addition pathway in anaerobic ethane oxidation. These findings contribute to our understanding of microbial metabolism of SCGAs in anaerobic environments.

Histochemical and histomorphological evidence of the modulating role of 1-isothiocyanate-4-methyl sulfonyl butane on cisplatin-induced testicular-pituitary axis degeneration and cholesterol homeostasis in male Sprague-Dawley rats.

BACKGROUND: This study examine the histochemical and histomorphological effect of 1-isothiocyanato-4-methyl sulfonyl butane (SFN) on cisplatin (CP) induced testicular alteration and cholesterol homeostasis. MATERIALS AND METHODS: Ninety adult-male Sprague-Dawley rats were randomized into nine groups of ten (n=10) rats each. Group A (control) received normal saline, group B received a single dose of 10mg/Kg body weight (bwt) CP (i.p.), group C received 50mg/Kg bwt of SFN, group D received 100mg/Kg bwt of SFN, group E received 10mg/Kg bwt CP and 50mg/Kg bwt of SFN, group F received 10mg/Kg bwt CP and 100mg/Kg bwt of SFN, group G received 10mg/Kg bwt CP and 50mg/Kg bwt vitamin C, group H received 50mg/Kg bwt of SFN and 10mg/Kg bwt CP, group I received 100mg/Kg bwt of SFN and 10mg/Kg bwt CP. The procedure lasted for 56 days. Testicular histomorphology and histochemistry, testicular testosterone, sperm parameters, total antioxidant status (TSA), total oxidant status (TOS), oxidative stress index (OSI), and serum lipid profile were examined. RESULTS: Cisplatin decrease intra-testicular testosterone, sperm quality, and expression of glycogen and increases testicular TOS and OSI, serum lipid profile, collagen, and disruption of germinal epithelium. However, the intervention of SFN reversed the effect of CP on testes' weight and volume, DSP, ESP, testosterone production, TAS, TOS, and OSI. Histoarchitectecture showing normal seminiferous tubules and even distribution of glycogen and collagen fibers. CONCLUSION: Treatment with SFN ameliorate CP-induced testicular toxicity by reversing the cytotoxic mechanisms of CP.

Systems engineering of Escherichia coli for n-butane production.

Rising concerns about climate change and sustainable energy have attracted efforts towards developing environmentally friendly alternatives to fossil fuels. Biosynthesis of n-butane, a highly desirable petro-chemical, fuel additive and diluent in the oil industry, remains a challenge. In this work, we first engineered enzymes Tes, Car and AD in the termination module to improve the selectivity of n-butane biosynthesis, and ancestral reconstruction and a synthetic RBS significantly improved the AD abundance. Next, we did ribosome binding site (RBS) calculation to identify potential metabolic bottlenecks, and then mitigated the bottleneck with RBS engineering and precursor propionyl-CoA addition. Furthermore, we employed a model-assisted strain design and a nonrepetitive extra-long sgRNA arrays (ELSAs) and quorum sensing assisted CRISPRi to facilitate a dynamic two-stage fermentation. Through systems engineering, n-butane production was increased by 168-fold from 0.04 to 6.74 mg/L. Finally, the maximum n-butane production from acetate was predicted using parsimonious flux balance analysis (pFBA), and we achieved n-butane production from acetate produced by electrocatalytic CO reduction. Our findings pave the way for selectively producing n-butane from renewable carbon source.

Targeted metabolomics combined with network pharmacology to reveal the protective role of luteolin in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease that significantly endangers human health, where metabolism may drive pathogenesis: a shift from mitochondrial oxidation to glycolysis occurs in diseased pulmonary vessels and the right ventricle. An increase in pulmonary vascular resistance in patients with heart failure with a preserved ejection fraction portends a poor prognosis. Luteolin exists in numerous foods and is marketed as a dietary supplement assisting in many disease treatments. However, little is known about the protective effect of luteolin on metabolism disorders in diseased pulmonary vessels. In this study, we found that luteolin apparently reversed the pulmonary vascular remodeling of PAH rats by inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). Moreover, network pharmacology and metabolomics results revealed that the arachidonic acid pathway, amino acid pathway and TCA cycle were dysregulated in PAH. A total of 14 differential metabolites were significantly changed during the PAH, including DHA, PGE2, PGD2, LTB4, 12-HETE, 15-HETE, PGF2α, and 8-iso-PGF2α metabolites in the arachidonic acid pathway, and L-asparagine, oxaloacetate, N-acetyl-L-ornithine, butane diacid, ornithine, glutamic acid metabolites in amino acid and TCA pathways. However, treatment with luteolin recovered the LTB4, PGE2, PGD2, 12-HETE, 15-HETE, PGF2α and 8-iso-PGF2α levels close to normal. Meanwhile, we showed that luteolin also downregulated the gene and protein levels of COX 1, 5-LOX, 12-LOX, and 15-LOX in the arachidonic acid pathway. Collectively, this work highlighted the metabolic mechanism of luteolin-protected PAH and showed that luteolin would hold great potential in PAH prevention.

Identification of active gaseous-alkane degraders at natural gas seeps.

Natural gas seeps release significant amounts of methane and other gases including ethane and propane contributing to global climate change. In this study, bacterial actively consuming short-chain alkanes were identified by cultivation, whole-genome sequencing, and stable-isotope probing (SIP)-metagenomics using 13C-propane and 13C-ethane from two different natural gas seeps, Pipe Creek and Andreiasu Everlasting Fire. Nearly 100 metagenome-assembled genomes (MAGs) (completeness 70-99%) were recovered from both sites. Among these, 16 MAGs had genes encoding the soluble di-iron monooxygenase (SDIMO). The MAGs were affiliated to Actinobacteria (two MAGs), Alphaproteobacteria (ten MAGs), and Gammaproteobacteria (four MAGs). Additionally, three gaseous-alkane degraders were isolated in pure culture, all of which could grow on ethane, propane, and butane and possessed SDIMO-related genes. Two Rhodoblastus strains (PC2 and PC3) were from Pipe Creek and a Mycolicibacterium strain (ANDR5) from Andreiasu. Strains PC2 and PC3 encoded putative butane monooxygenases (MOs) and strain ANDR5 contained a propane MO. Mycolicibacterium strain ANDR5 and MAG19a, highly abundant in incubations with 13C-ethane, share an amino acid identity (AAI) of 99.3%. We show using a combination of enrichment and isolation, and cultivation-independent techniques, that these natural gas seeps contain a diverse community of active bacteria oxidising gaseous-alkanes, which play an important role in biogeochemical cycling of natural gas.

Genome and proteome analyses show the gaseous alkane degrader Desulfosarcina sp. strain BuS5 as an extreme metabolic specialist.

The metabolic potential of the sulfate-reducing bacterium Desulfosarcina sp. strain BuS5, currently the only pure culture able to oxidize the volatile alkanes propane and butane without oxygen, was investigated via genomics, proteomics and physiology assays. Complete genome sequencing revealed that strain BuS5 encodes a single alkyl-succinate synthase, an enzyme which apparently initiates oxidation of both propane and butane. The formed alkyl-succinates are oxidized to CO2 via beta oxidation and the oxidative Wood-Ljungdahl pathways as shown by proteogenomics analyses. Strain BuS5 conserves energy via the canonical sulfate reduction pathway and electron bifurcation. An ability to utilize long-chain fatty acids, mannose and oligopeptides, suggested by automated annotation pipelines, was not supported by physiology assays and in-depth analyses of the corresponding genetic systems. Consistently, comparative genomics revealed a streamlined BuS5 genome with a remarkable paucity of catabolic modules. These results establish strain BuS5 as an exceptional metabolic specialist, able to grow only with propane and butane, for which we propose the name Desulfosarcina aeriophaga BuS5. This highly restrictive lifestyle, most likely the result of habitat-driven evolutionary gene loss, may provide D. aeriophaga BuS5 a competitive edge in sediments impacted by natural gas seeps. Etymology: Desulfosarcina aeriophaga, aério (Greek): gas; phágos (Greek): eater; D. aeriophaga: a gas eating or gas feeding Desulfosarcina.

Exploring the Use of Gas Chromatography Coupled to Chemical Ionization Mass Spectrometry (GC-CI-MS) for Stable Isotope Labeling in Metabolomics.

Isotopic-labeling experiments have been valuable to monitor the flux of metabolic reactions in biological systems, which is crucial to understand homeostatic alterations with disease. Experimental determination of metabolic fluxes can be inferred from a characteristic rearrangement of stable isotope tracers (e.g., 13C or 15N) that can be detected by mass spectrometry (MS). Metabolites measured are generally members of well-known metabolic pathways, and most of them can be detected using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS. In here, we show that GC methods coupled to chemical ionization (CI) MS have a clear advantage over alternative methodologies due to GC's superior chromatography separation efficiency and the fact that CI is a soft ionization technique that yields identifiable protonated molecular ion peaks. We tested diverse GC-CI-MS setups, including methane and isobutane reagent gases, triple quadrupole (QqQ) MS in SIM mode, or selected ion clusters using optimized narrow windows (∼10 Da) in scan mode, and standard full scan methods using high resolution GC-(q)TOF and GC-Orbitrap systems. Isobutane as a reagent gas in combination with both low-resolution (LR) and high-resolution (HR) MS showed the best performance, enabling precise detection of isotopologues in most metabolic intermediates of central carbon metabolism. Finally, with the aim of overcoming manual operations, we developed an R-based tool called isoSCAN that automatically quantifies all isotopologues of intermediate metabolites of glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.

Engineering nature for gaseous hydrocarbon production.

The development of sustainable routes to the bio-manufacture of gaseous hydrocarbons will contribute widely to future energy needs. Their realisation would contribute towards minimising over-reliance on fossil fuels, improving air quality, reducing carbon footprints and enhancing overall energy security. Alkane gases (propane, butane and isobutane) are efficient and clean-burning fuels. They are established globally within the transportation industry and are used for domestic heating and cooking, non-greenhouse gas refrigerants and as aerosol propellants. As no natural biosynthetic routes to short chain alkanes have been discovered, de novo pathways have been engineered. These pathways incorporate one of two enzymes, either aldehyde deformylating oxygenase or fatty acid photodecarboxylase, to catalyse the final step that leads to gas formation. These new pathways are derived from established routes of fatty acid biosynthesis, reverse ß-oxidation for butanol production, valine biosynthesis and amino acid degradation. Single-step production of alkane gases in vivo is also possible, where one recombinant biocatalyst can catalyse gas formation from exogenously supplied short-chain fatty acid precursors. This review explores current progress in bio-alkane gas production, and highlights the potential for implementation of scalable and sustainable commercial bioproduction hubs.

Aerobic cometabolism of 1,4-dioxane by isobutane-utilizing microorganisms including Rhodococcus rhodochrous strain 21198 in aquifer microcosms: Experimental and modeling study.

Aerobic cometabolism of the emerging contaminant 1,4-dioxane (1,4-D) by isobutane-utilizing microorganisms was assessed in pure culture and aquifer microcosm studies. The bacterium Rhodococcus rhodochrous strain ATCC 21198 transformed low, environmentally-relevant concentrations of 1,4-D when grown on isobutane. Microcosms were constructed with aquifer solids from Fort Carson, Colorado, a site contaminated with 1,4-D and trichloroethene (TCE). Multiple additions of isobutane and 1,4-D over 300 days were transformed in microcosms biostimulated with isobutane and microcosms bioaugmented with strain 21198. Results showed that, over time and with sufficient inorganic nutrients, biostimulation of native microorganisms with isobutane was just as effective as bioaugmentation with strain 21198 to achieve 1,4-D transformation in the microcosms. The presence of TCE at 0.2 mg/L did not inhibit 1,4-D transformation, though TCE itself was not readily transformed. An iterative process was used to determine kinetic parameter values to fit Michaelis-Menten/Monod models to experimental data for simultaneous isobutane utilization, biomass growth, and cometabolic transformation of 1,4-D. Parameter optimization resulted in good model fit to the data over multiple transformations of isobutane and 1,4-D in both short- and long-term experiments. Results suggest low concentrations of 1,4-D studied in the microcosms were cometabolically transformed according to a pseudo first-order rate of 0.37 L/mg TSS/day of 21198. Isobutane consumption was modeled with a maximum rate of 2.58 mg/mg TSS/day and a half saturation constant of 0.09 mg/L. 1,4-D transformation was competitively inhibited by the presence of isobutane and transformation rates were significantly reduced when inorganic nutrients were limiting. Simulations of the repeated additions found a first-order microbial endogenous decay coefficient of 0.03 day-1 fit the alternating periods of active transformation and stagnation between isobutane and 1,4-D additions over approximately one year. The model fitting process highlighted the importance of determining kinetic parameters from data representing low concentrations typically found in the environment.

Rhodopseudomonas palustris wastewater treatment: Cyhalofop-butyl removal, biochemicals production and mathematical model establishment.

Simultaneous (SPW and cyhalofop-butyl) wastewater treatment and the production of biochemicals by Rhodopseudomonas palustris (R. palustris) was investigated with supplementation of soybean processing wastewater (SPW). Compared to control group, cyhalofop-butyl was removed and single cell protein, carotenoid, bacteriochlorophyll productions were enhanced with the supplementation of SPW. Cyhalofop-butyl removal reached 100% after 5 days under 4000 mg/L COD group. Cyhalofop-butyl induced chbH gene expression to synthesize cyhalofop-butyl-hydrolyzing carboxylesterase through activating MAPKKKs, MAPKKs, MAPKs genes in MAPK signal transduction pathway. The induction process took one day for R. palustris. However, lack of organics in original wastewater did not maintain R. palustris growth for over one day. The supplementation of SPW provided sufficient carbon source. This new method resulted in the mixed wastewater treatment and improvement of biochemicals simultaneously, as well as the realization of reutilization of R. palustris. High-order non-linear mathematical model of the relationship between Rchb, Xc, and Xt was established.

Divergent methyl-coenzyme M reductase genes in a deep-subseafloor Archaeoglobi.

The methyl-coenzyme M reductase (MCR) complex is a key enzyme in archaeal methane generation and has recently been proposed to also be involved in the oxidation of short-chain hydrocarbons including methane, butane, and potentially propane. The number of archaeal clades encoding the MCR continues to grow, suggesting that this complex was inherited from an ancient ancestor, or has undergone extensive horizontal gene transfer. Expanding the representation of MCR-encoding lineages through metagenomic approaches will help resolve the evolutionary history of this complex. Here, a near-complete Archaeoglobi metagenome-assembled genome (MAG; Ca. Polytropus marinifundus gen. nov. sp. nov.) was recovered from the deep subseafloor along the Juan de Fuca Ridge flank that encodes two divergent McrABG operons similar to those found in Ca. Bathyarchaeota and Ca. Syntrophoarchaeum MAGs. Ca. P. marinifundus is basal to members of the class Archaeoglobi, and encodes the genes for ß-oxidation, potentially allowing an alkanotrophic metabolism similar to that proposed for Ca. Syntrophoarchaeum. Ca. P. marinifundus also encodes a respiratory electron transport chain that can potentially utilize nitrate, iron, and sulfur compounds as electron acceptors. Phylogenetic analysis suggests that the Ca. P. marinifundus MCR operons were horizontally transferred, changing our understanding of the evolution and distribution of this complex in the Archaea.

Comparison of postmortem butane distribution between two fatal butane poisoning cases.

Fatal accidents during butane abuse frequently occur in Japan and in many countries around the world. Although analytical data about butane concentration in postmortem samples is being accumulated, when using the data to determine the cause of death, careful interpretation is required because the gas is easily diffused. Two fatal butane poisoning cases were encountered, and butane quantification of autopsy samples obtained from left and right heart blood, femoral blood, kidney, liver, lung, brain and fatty tissues was performed. In both cases, butane concentration in the left heart blood was lower than in the right heart blood or the femoral blood, despite gas inhalation. These findings may indicate that the deceased individuals interrupted gas inhalation and inhaled room air immediately before their death, therefore ruling out asphyxia due to anoxia as the mechanism of death. Case 1, which was suspected to be a not acute death, showed a very high butane concentration ratio of fatty tissues to femoral blood of over 70. Case 2 was considered an acute death, and the butane concentration ratio of fatty tissues to femoral blood was 8.2. These results are consistent with previously reported findings showing that much higher ratios of fatty tissues to blood are compatible with long survival time. In conclusion, the comparison of butane concentration among different samples, including left heart blood versus right heart blood and fatty tissues versus blood, is useful when interpreting the result of postmortem butane analysis to examine the mechanism of death and survival time.

Recent insights into the microbial catabolism of aryloxyphenoxy-propionate herbicides: microbial resources, metabolic pathways and catabolic enzymes.

Aryloxyphenoxy-propionate herbicides (AOPPs) are widely used to control annual and perennial grasses in broadleaf crop fields and are frequently detected as contaminants in the environment. Due to the serious environmental toxicity of AOPPs, there is considerable concern regarding their biodegradation and environmental behaviors. Microbial catabolism is considered as the most effective method for the degradation of AOPPs in the environment. This review presents an overview of the recent findings on the microbial catabolism of various AOPPs, including fluazifop-P-butyl, cyhalofop-butyl, diclofop-methyl, fenoxaprop-P-ethyl, metamifop, haloxyfop-P-methyl and quizalofop-P-ethyl. It highlights the microbial resources that are able to catabolize these AOPPs and the metabolic pathways and catabolic enzymes involved in their degradation and mineralization. Furthermore, the application of AOPPs-degrading strains to eliminate AOPPs-contaminated environments and future research hotspots in biodegradation of AOPPs by microorganisms are also discussed.

In vitro metabolism of the synthetic cannabinoid 3,5-AB-CHMFUPPYCA and its 5,3-regioisomer and investigation of their thermal stability.

Recently, the pyrazole-containing synthetic cannabinoid N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-3-(4-fluorophenyl)-1H-pyrazole-5-carboxamide (3,5-AB-CHMFUPPYCA) has been identified as a 'research chemical' both in powdered form and as an adulterant present in herbal preparations. Urine is the most common matrix used for abstinence control and the extensive metabolism of synthetic cannabinoids requires implementation of targeted analysis. The present study describes the investigation of the in vitro phase I metabolism of 3,5-AB-CHMFUPPYCA and its regioisomer 5,3-AB-CHMFUPPYCA using pooled human liver microsomes. Metabolic patterns of both AB-CHMFUPPYCA isomers were qualitatively similar and dominated by oxidation of the cyclohexylmethyl side chain. Biotransformation to monohydroxylated metabolites of high abundance confirmed that these species might serve as suitable targets for urine analysis. Furthermore, since synthetic cannabinoids are commonly administered by smoking and because some metabolites can also be formed as thermolytic artefacts, the stability of both isomers was assessed under smoking conditions. Under these conditions, pyrolytic cleavage of the amide bond occurred that led to approximately 3 % conversion to heat-induced degradation products that were also detected during metabolism. These artefactual 'metabolites' could potentially bias in vivo metabolic profiles after smoking and might have to be considered for interpretation of metabolite findings during hair analysis. This might be relevant to the analysis of hair samples where detection of metabolites is generally accepted as a strong indication of drug use rather than a potential external contamination. Copyright © 2016 John Wiley & Sons, Ltd.

Mechanistic Study of the Stereoselective Hydroxylation of [2-2 H1 ,3-2 H1 ]Butanes Catalyzed by Cytochrome P450 BM3 Variants.

Engineered bacterial cytochrome P450s are noted for their ability in the oxidation of inert small alkanes. Cytochrome P450 BM3 L188P A328F (BM3 PF) and A74E L188P A328F (BM3 EPF) variants are able to efficiently oxidize n-butane to 2-butanol. Esterification of the 2-butanol derived from this reaction mediated by the aforementioned two mutants gives diastereomeric excesses (de) of -56±1 and -52±1 %, respectively, with the preference for the oxidation occurring at the C-HS bond. When tailored (2R,3R)- and (2S,3S)-[2-2 H1 ,3-2 H1 ]butane probes are employed as substrates for both variants, the obtained de values from (2R,3R)-[2-2 H1 ,3-2 H1 ]butane are -93 and -92 % for BM3 PF and EPF, respectively; whereas the obtained de values from (2S,3S)-[2-2 H1 ,3-2 H1 ]butane are 52 and 56 % in the BM3 PF and EPF systems, respectively. The kinetic isotope effects (KIEs) for the oxidation of (2R,3R)-[2-2 H1 ,3-2 H1 ]butane are 7.3 and 7.8 in BM3 PF and EPF, respectively; whereas KIEs for (2S,3S)-[2-2 H1 ,3-2 H1 ]butanes are 18 and 25 in BM3 PF and EPF, respectively. The discrepancy in KIEs obtained from the two substrates supports the two-state reactivity (TSR) that is proposed for alkane oxidation in cytochrome P450 systems. Moreover, for the first time, experimental evidence for tunneling in the oxidation mediated by P450 is given through the oxidation of the C-HR bond in (2S,3S)-[2-2 H1 ,3-2 H1 ]butane.

[Changes of microbial abundance and functional genes in oil and gas under gaseous hydrocarbon condition].

Objective: Hydrocarbon microseepage is a natural phenomenon that hydrocarbon gases of subsurface petroleum accumulations migrate upward by reservoir pressure. The detection of the activity and distribution of these highly specialized populations can be used to forecast the existence of oil and gas deposits. However, the hydrocarbon-oxidizing bacterial population are usually not predominant in soil samples above the typical onshore oil and gas reservoirs. It is hard to assess the abundance of hydrocarbon-oxidizing bacteria. Methods: In this study, changes of microbial abundance and functional genes were studied. Results: Under gaseous hydrocarbon condition, changes of methane and butane oxidizing bacteria were different. Furthermore, changes of functional genes indicated that genome analysis was more proper for microbial anomalies detection. Conclusion: The profiling data of this study provide a comprehensive insight into gene expression profiles and lay the foundation for optimizing the microbial prospecting technology.

Thermophilic archaea activate butane via alkyl-coenzyme M formation.

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus 'Candidatus Syntrophoarchaeum', show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding ß-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

Syntrophic Interactions Within a Butane-Oxidizing Bacterial Consortium Isolated from Puguang Gas Field in China.

Butane oxidation by the hydrocarbon degradation bacteria has long been described, but little is known about the microbial interaction in this process. To investigate this interaction, the efficiency of butane oxidation was estimated in monocultures and co-cultures of six strains of butane-oxidizing bacteria (BOB) and a butanol-oxidizing strain. Results showed that the butane degradation velocity was at least 26 times higher in the co-culture of the seven strains (228.50 nmol h(-1)) than in the six individual monocultures (8.71 nmol h(-1)). Gas chromatographic analysis of metabolites in the cultures revealed the accumulation of butanol in the monocultures of BOB strains but not in the co-culture with the butanol-oxidizing strain. These results evidenced a novel syntrophic association between BOB and butanol-oxidizing bacteria in the butane oxidation. The BOB strains oxidized butane into butanol, but this activity was inhibited by the accumulated butanol in monocultures, whereas the removal of butanol by the butanol-oxidizing strain in co-culture could eliminate the suppression and improve the butane degradation efficiency. In the co-culture, both BOB and butanol-oxidizing bacteria could grow and the time needed for butane complete removal was shortened from more than 192 h to less than 4 h. The unsuppressed effect of the co-culture was also consistent with the results of reverse transcription quantitative real-time PCR (RT-qPCR) of bmoX gene because increased expression of this gene was detected during the syntrophic growth compared with that in monoculture, pointing to the upregulation of bmoX in the syntrophic interaction.

Unusual Butane- and Pentanetriol-Based Tetraether Lipids in Methanomassiliicoccus luminyensis, a Representative of the Seventh Order of Methanogens.

UNLABELLED: A new clade of archaea has recently been proposed to constitute the seventh methanogenic order, the Methanomassiliicoccales, which is related to the Thermoplasmatales and the uncultivated archaeal clades deep-sea hydrothermal vent Euryarchaeota group 2 and marine group II Euryarchaeota but only distantly related to other methanogens. In this study, we investigated the membrane lipid composition of Methanomassiliicoccus luminyensis, the sole cultured representative of this seventh order. The lipid inventory of M. luminyensis comprises a unique assemblage of novel lipids as well as lipids otherwise typical for thermophilic, methanogenic, or halophilic archaea. For instance, glycerol sesterpanyl-phytanyl diether core lipids found mainly in halophilic archaea were detected, and so were compounds bearing either heptose or methoxylated glycosidic head groups, neither of which have been reported so far for other archaea. The absence of quinones or methanophenazines is consistent with a biochemistry of methanogenesis different from that of the methanophenazine-containing methylotrophic methanogens. The most distinctive characteristic of the membrane lipid composition of M. luminyensis, however, is the presence of tetraether lipids in which one glycerol backbone is replaced by either butane- or pentanetriol, i.e., lipids recently discovered in marine sediments. Butanetriol dibiphytanyl glycerol tetraether (BDGT) constitutes the most abundant core lipid type (>50% relative abundance) in M. luminyensis We have thus identified a source for these unusual orphan lipids. The complementary analysis of diverse marine sediment samples showed that BDGTs are widespread in anoxic layers, suggesting an environmental significance of Methanomassiliicoccales and/or related BDGT producers beyond gastrointestinal tracts. IMPORTANCE: Cellular membranes of members of all three domains of life, Archaea, Bacteria, and Eukarya, are largely formed by lipids in which glycerol serves as backbone for the hydrophobic alkyl chains. Recently, however, archaeal tetraether lipids with either butanetriol or pentanetriol as a backbone were identified in marine sediments and attributed to uncultured sediment-dwelling archaea. Here we show that the butanetriol-based dibiphytanyl tetraethers constitute the major lipids in Methanomassiliicoccus luminyensis, currently the only isolate of the novel seventh order of methanogens. Given the absence of these lipids in a large set of archaeal isolates, these compounds may be diagnostic for the Methanomassiliicoccales and/or closely related archaea.