Novel aerobic benzene degrading microorganisms identified in three soils by stable isotope probing

The remediation of benzene contaminated groundwater often involves biodegradation and although the mechanisms of aerobic benzene biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible for benzene degradation in mixed culture samples or at contaminated sites. To address this knowledge gap, DNA based stable isotope probing (SIP) was utilized to identify active benzene degraders in microcosms constructed with soil from three sources (a contaminated site and two agricultural sites). For this, replicate microcosms were amended with either labeled (¹³C) or unlabeled benzene and the extracted DNA samples were ultracentrifuged, fractioned and subject to terminal restriction fragment length polymorphism (TRFLP). The dominant benzene degraders (responsible for ¹³C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled benzene (¹³C) amended samples to the controls (from unlabeled benzene amended samples). Two phylotypes (a Polaromonas sp. and an Acidobacterium) were the major benzene degraders in the microcosms constructed from the contaminated site soil, whereas one phylotype incorporated the majority of the benzene-derived ¹³C in each of the agricultural soils (“candidate” phylum TM7 and an unclassified Sphingomonadaceae).     

Methyl coenzyme M reductase (<Emphasis Type="Italic">mcrA</Emphasis>) gene based phylogenetic analysis of methanogens population in Murrah buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The aim of the present study was to decipher the diversity of methanogens in rumen of Murrah buffaloes so that effective strategies can be made in order to mitigate methane emission from these methanogens. In the present study diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India was evaluated by using mcr-A gene library obtained from the pooled PCR product from four animals and by using MEGA4 software. A total of 104 clones were examined, revealing 26 different mcr-A gene sequences or phylotypes. Of the 26 phylotypes, 16 (64 of 104 clones) were less than 97% similar to any of the cultured strain of methanogens. Seven clone sequences were clustered with Methanomicrobium mobile and three clone sequences were clustered with Methanobrevibacter gottschalkii during the phylogenetic analysis. Uncultured group of methanogens comes out to be the major component of the methanogens community structure in Murrah buffaloes. Methanomicrobium phylotype comes out to be major phylotype among cultured methanogens followed by Methanobrevibacter phylotype. These results help in making effective strategies to check the growth of dominant communities in the rumen of this animal which in turn help in the reduction of methane emission in the environment and ultimately helps us in fighting with the problem of global warming.     

Activities and distribution of methanogenic and methane‐oxidizing microbes in marine sediments from the Cascadia Margin

We investigated methane production and oxidation and the depth distribution and phylogenetic affiliation of a functional gene for methanogenesis, methyl coenzyme M reductase subunit A (mcrA), at two sites of the Integrated Ocean Drilling Program Expedition 311. These sites, U1327 and U1329, are respectively inside and outside the area of gas hydrate distribution on the Cascadia Margin. Radiotracer experiments using ¹⁴C‐labelled substrates indicated high potential methane production rates in hydrate‐bearing sediments [128–223 m below seafloor (mbsf)] at U1327 and in sediments between 70 and 140 mbsf at U1329. Tracer‐free experiments indicated high cumulative methane production in sediments within and below the gas hydrate layer at U1327 and in sediments below 70 mbsf at U1329. Stable tracer experiments using ¹³C‐labelled methane showed high potential methane oxidation rates in near‐surface sediments and in sediments deeper than 100 mbsf at both sites. Results of polymerase chain reaction amplification of mcrA in DNA were mostly consistent with methane production: relatively strong mcrA amplification was detected in the gas hydrate‐bearing sediments at U1327, whereas at U1329, it was mainly detected in sediments from around the bottom‐simulating reflector (126 mbsf). Phylogenetic analysis of mcrA separated it into four phylotype clusters: two clusters of methanogens, Methanosarcinales and Methanobacteriales, and two clusters of anaerobic methanotrophic archaea, ANME‐I and ANME‐II groups, supporting the activity measurement results. These results reveal that in situ methanogenesis in deep sediments probably contributes to gas hydrate formation and are inconsistent with the geochemical model that microbial methane currently being generated in shallow sediments migrates downward and contributes to the hydrate formation. At Site U1327, gas hydrates occurred in turbidite sediments, which were absent at Site U1329, suggesting that a geological setting suitable for a gas hydrate reservoir is more important for the accumulation of gas hydrate than microbiological properties.     

DNA-SIP Reveals That Syntrophaceae Play an Important Role in Methanogenic Hexadecane Degradation

The methanogenic degradation of linear alkanes is a common process in oil-impacted environments. However, little is known about the key players involved in this process. Here, the hexadecane-degrading organisms in a methanogenic, hexadecane-degrading consortium designated M82 obtained from Shengli oilfield and maintained at 35°C for over 4 years, were identified by DNA-stable isotope probing with UL-¹³C-hexadecane, followed by density-resolved terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and phylogenetic analysis of 16S rRNA gene fragments. Compared to the fractions of the ¹²C treatment, the relative abundance of two phylotypes significantly increased in the heavy fractions of the ¹³C-hexadecane incubated microcosm. One belongs to a uncultured member of the bacterial family Syntrophaceae, which show 95–97% rRNA sequence identity with Smithella propionica, and the other is affiliated with Methanoculleus receptaculi (>99% sequence identity). The results of the present study prove the significant role of uncultured Syntrophaceae in degradation of hexadecane, probably through syntrophic interactions with hydrogenotrophic methanogens.     

Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.     

Colonization of rice roots with methanogenic archaea controls photosynthesis‐derived methane emission

The methane emitted from rice fields originates to a large part (up to 60%) from plant photosynthesis and is formed on the rice roots by methanogenic archaea. To investigate to which extent root colonization controls methane (CH₄) emission, we pulse‐labeled rice microcosms with ¹³CO₂ to determine the rates of ¹³CH₄ emission exclusively derived from photosynthates. We also measured emission of total CH₄ (¹²⁺¹³CH₄), which was largely produced in the soil. The total abundances of archaea and methanogens on the roots and in the soil were analysed by quantitative polymerase chain reaction of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase respectively. The composition of archaeal and methanogenic communities was determined with terminal restriction fragment length polymorphism (T‐RFLP). During the vegetative growth stages, emission rates of ¹³CH₄ linearly increased with the abundance of methanogenic archaea on the roots and then decreased during the last plant growth stage. Rates of ¹³CH₄ emission and the abundance of methanogenic archaea were lower when the rice was grown in quartz‐vermiculite with only 10% rice soil. Rates of total CH₄ emission were not systematically related to the abundance of methanogenic archaea in soil plus roots. The composition of the archaeal communities was similar under all conditions; however, the analysis of mcrA genes indicated that the methanogens differed between the soil and root. Our results support the hypothesis that rates of photosynthesis‐driven CH₄ emission are limited by the abundance of methanogens on the roots.     

Identification of Methanogenic archaea in the Hyporheic Sediment of Sitka Stream

Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit (mcrA) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH₄ cycling in rivers.     

Characterization of methanogenic and prokaryotic assemblages based on mcrA and 16S rRNA gene diversity in sediments of the Kazan mud volcano (Mediterranean Sea)

The diversity of the methyl‐coenzyme reductase A (mcrA) and 16S rRNA genes was investigated in gas hydrate containing sediment from the Kazan mud volcano, eastern Mediterranean Sea. mcrA was detected only at 15 and 20 cm below seafloor (cmbsf) from a 40‐cm long push core, while based on chemical profiles of methane, sulfate, and sulfide, possible anaerobic oxidation of methane (AOM) depth was inferred at 12–15 cmbsf. The phylogenetic relationships of the obtained mcrA, archaeal and bacterial 16S rRNA genes, showed that all the found sequences were found in both depths and at similar relative abundances. mcrA diversity was low. All sequences were related to the Methanosarcinales, with the most dominant (77.2%) sequences falling in group mcrA‐e. The 16S rRNA‐based archaeal diversity also revealed low diversity and clear dominance (72.8% of all archaeal phylotypes) of the Methanosarcinales and, in particular, ANME‐2c. Bacteria showed higher diversity but 83.2% of the retrieved phylotypes from both sediment layers belonged to the δ‐Proteobacteria. These phylotypes fell in the SEEP‐SRB1 putative AOM group. In addition, the rest of the less abundant phylotypes were related to yet‐uncultivated representatives of the Actinobacteria, Spirochaetales, and candidate divisions OP11 and WS3 from gas hydrate‐bearing habitats. These phylotype patterns indicate that AOM is occurring in the 15 and 20 cmbsf sediment layers.     

Microbial diversity and community structure of a highly active anaerobic methane‐oxidizing sulfate‐reducing enrichment

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. Here we describe the microbial analysis of an enrichment obtained in a novel submerged‐membrane bioreactor system and capable of high‐rate AOM (286 μmol g_{dry weight}^?1 day^?1) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, we showed that the responsible methanotrophs belong to the ANME‐2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate‐reducing bacteria commonly found in association with other ANME‐related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. Fluorescent in situ hybridization analyses showed that the ANME‐2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with ¹³C‐labelled methane showed substantial incorporation of ¹³C label in the bacterial C₁₆ fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl‐archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment.     

Methanogenic capabilities of ANME‐archaea deduced from 13C‐labelling approaches

Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME‐1 or ANME‐2. The ¹³C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate‐dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME‐1 and ANME‐2. Considerable methane production rates (MPRs) from CO₂‐reduction were observed, particularly from ANME‐2 dominated samples and in the presence of methane, which supports the hypothesis of a co‐occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through ¹³C‐assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the ¹³C‐uptake into lipids, ANME‐1 was found to thrive also under methane free conditions. Finally, C₃₅‐isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME‐1, most likely functioning as a hydrogen sink during methanogenesis.     

Methanogenesis and sulfate reduction in timber and drainage water from a gold mine

Biogenesis of methane in the heartwood of diseased trees has been shown, but never in timber in service. Studies were undertaken to establish whether methan‐ogens and sulfate‐reducers were present in wooden pit props and drainage water from underground sites in a gold mine. The predominant methanogen in the mine ecosystem was tentatively identified as Methanobacterium bryantii. The sulfate‐reducers comprised Desulfovibrio desulfuricans and Desulfotomaculum antarcticum. Most probable numbers (MPN) of bacteria indicated that 3.5 × 10⁵ methanogenic and 7.9 × 10³ sulfate‐reducing bacteria were present per milliliter of stagnant drainage water. MPN values per gram of timber were lower for methanogens but comparable for sulfate‐reducers. Laboratory model systems predicted a maximum rate of methan‐ogenesis of 2.3 mL methane/g wood per day; however, rates would never attain this value because of nutrient limitations and environmental restrictions. Analysis of gas samples extracted from sealed areas of the gold mine verified the presence of methane.     

Influence of temperature on the δ13C values and distribution of methanotroph‐related hopanoids in Sphagnum‐dominated peat bogs

Methane emissions from peat bogs are mitigated by methanotrophs, which live in symbiosis with peat moss (e.g. Sphagnum). Here, we investigate the influence of temperature and resultant changes in methane fluxes on Sphagnum and methanotroph‐related biomarkers, evaluating their potential as proxies in ancient bogs. A pulse‐chase experiment using ¹³C‐labelled methane in the field clearly showed label uptake in diploptene, a biomarker for methanotrophs, demonstrating in situ methanotrophic activity in Sphagnum under natural conditions. Peat cores containing live Sphagnum were incubated at 5, 10, 15, 20 and 25°C for two months, causing differences in net methane fluxes. The natural δ¹³C values of diploptene extracted from Sphagnum showed a strong correlation with temperature and methane production. The δ¹³C values ranged from ?34‰ at 5°C to ?41‰ at 25°C. These results are best explained by enhanced expression of the methanotrophic enzymatic isotope effect at higher methane concentrations. Hence, δ¹³C values of diploptene, or its diagenetic products, potentially provide a useful tool to assess methanotrophic activity in past environments. Increased methane fluxes towards Sphagnum did not affect δ¹³C values of bulk Sphagnum and its specific marker, the C₂₃ n‐alkane. The concentration of methanotroph‐specific bacteriohopanepolyols (BHPs), aminobacteriohopanetetrol (aminotetrol, characteristic for type II and to a lesser extent type I methanotrophs) and aminobacteriohopanepentol (aminopentol, a marker for type I methanotrophs) showed a non‐linear response to increased methane fluxes, with relatively high abundances at 25°C compared to those at 20°C or below. Aminotetrol was more abundant than aminopentol, in contrast to similar abundances of aminotetrol and aminopentol in fresh Sphagnum. This probably indicates that type II methanotrophs became prevalent under the experimental conditions relative to type I methanotrophs. Even though BHP concentrations may not directly reflect bacterial activity, they may provide insight into the presence of different types of methanotrophs.     

Effect of Freeze-Thaw Cycles on Bacterial Communities of Arctic Tundra Soil

The effect of freeze-thaw (FT) cycles on Arctic tundra soil bacterial community was studied in laboratory microcosms. FT-induced changes to the bacterial community were followed over a 60-day period by terminal restriction fragment length polymorphism (T-RFLP) profiles of amplified 16S rRNA genes and reverse transcribed 16S rRNA. The main phylotypes of the active, RNA-derived bacterial community were identified using clone analysis. Non-metric multidimensional scaling ordination of the T-RFLP profiles indicated some shifts in the bacterial communities after three to five FT cycles at −2, −5, and −10°C as analyzed both from the DNA and rRNA. The dominating T-RFLP peaks remained the same, however, and only slight variation was generally detected in the relative abundance of the main T-RF sizes of either DNA or rRNA. T-RFLP analysis coupled to clone analysis of reverse transcribed 16S rRNA indicated that the initial soil was dominated by members of Bacteroidetes, Acidobacteria, Alpha-, Beta-, and Gammaproteobacteria. The most notable change in the rRNA-derived bacterial community was a decrease in the relative abundance of a Betaproteobacteria-related phylotype after the FT cycles. This phylotype decreased, however, also in the control soil incubated at constant +5°C suggesting that the decrease was not directly related to FT sensitivity. The results indicate that FT caused only minor changes in the bacterial community structure.     

Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions

The prototypical representatives of the Euryarchaeota—the methanogens—are oxygen sensitive and are thought to occur only in highly reduced, anoxic environments. However, we found methanogens of the genera Methanosarcina and Methanocella to be present in many types of upland soils (including dryland soils) sampled globally. These methanogens could be readily activated by incubating the soils as slurry under anoxic conditions, as seen by rapid methane production within a few weeks, without any additional carbon source. Analysis of the archaeal 16S ribosomal RNA gene community profile in the incubated samples through terminal restriction fragment length polymorphism and quantification through quantitative PCR indicated dominance of Methanosarcina, whose gene copy numbers also correlated with methane production rates. Analysis of the δ¹³C of the methane further supported this, as the dominant methanogenic pathway was in most cases aceticlastic, which Methanocella cannot perform. Sequences of the key methanogenic enzyme methyl coenzyme M reductase retrieved from the soil samples before incubation confirmed that Methanosarcina and Methanocella are the dominant methanogens, though some sequences of Methanobrevibacter and Methanobacterium were also detected. The global occurrence of only two active methanogenic archaea supports the hypothesis that these are autochthonous members of the upland soil biome and are well adapted to their environment.     

Enrichment of ANME‐2 dominated anaerobic methanotrophy from cold seep sediment in an external ultrafiltration membrane bioreactor

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is a microbially mediated unique natural phenomenon with an ecological relevance in the global carbon balance and potential application in biotechnology. This study aimed to enrich an AOM performing microbial community with the main focus on anaerobic methanotrophic archaea (ANME) present in sediments from the Ginsburg mud volcano (Gulf of Cadiz), a known site for AOM, in a membrane bioreactor (MBR) for 726 days at 22 (± 3)°C and at ambient pressure. The MBR was equipped with a cylindrical external ultrafiltration membrane, fed a defined medium containing artificial seawater and operated at a cross flow velocity of 0.02 m/min. Sulfide production with simultaneous sulfate reduction was in equimolar ratio between days 480 and 585 of MBR operation, whereas methane consumption was in oscillating trend. At the end of the MBR operation (day 726), the enriched biomass was incubated with ¹³C labeled methane, ¹³C labeled inorganic carbon was produced and the AOM rate based on ¹³C‐inorganic carbon was 1.2 μmol/(g_dw d). Microbial analysis of the enriched biomass at 400 and 726 days of MBR operation showed that ANME‐2 and Desulfosarcina type sulfate reducing bacteria were enriched in the MBR, which formed closely associated aggregates. The major relevance of this study is the enrichment of an AOM consortium in a MBR system which can assist to explore the ecophysiology of ANME and provides an opportunity to explore the potential application of AOM.     

Dominance of Methanomicrobium phylotype in methanogen population present in Murrah buffaloes (Bubalus bubalis)

Aims:  To study the diversity of rumen methanogens in Murrah buffaloes ( Bubalus bubalis ) from North India by using 16S rRNA gene libraries obtained from the pooled rumen content from four animals and using suitable software analysis.
Methods and Results:  Genomic DNA was isolated and PCR was set up by using specific primers. Amplified product was cloned into a suitable vector and the positive clones were selected on the basis of blue–white screening and sequenced. The resulting nucleotide sequences were arranged in the phylogenetic tree. A total of 108 clones were examined, revealing 17 different 16S rRNA gene sequences or phylotypes. Of the 17 phylotypes, 15 (102 of 108 clones) belonged to the genus Methanomicrobium , indicating that the genus Methanomicrobium is the most dominant component of methanogen populations in Murrah buffaloes ( Bubalus bubalis ) from North India. The largest group of clones (102 clones) was more than 98% similar to Methanomicrobium mobile . BLAST analysis of the rumen contents from individual animals also revealed 17 different phylotypes with a range of 3–10 phylotypes per animal.
Conclusion:  Methanomicrobium phylotype is the most dominant phylotype of methanogens present in Murrah buffaloes ( Bubalus bubalis ).
Significance and Impact of the Study:  Effective strategies can be made to inhibit the growth of Methanomicrobium phylotype to reduce the methane emission from rumen contents and thus help in preventing global warming.     

Biomarker Evidence for Widespread Anaerobic Methane Oxidation in Mediterranean Sediments by a Consortium of Methanogenic Archaea and Bacteria

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in ¹³C (δ¹³C values are as low as −95‰). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of ¹³C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H₂-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [¹³C]propionate (<10 mM) and the oxidation of ~30 μmol of ¹³C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were ¹³C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” ¹³C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of ¹³C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [¹³C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.     

Carbon isotopic composition of lipid biomarkers from an endoevaporitic gypsum crust microbial mat reveals cycling of mineralized organic carbon

Microbial mats that inhabit gypsum deposits in ponds at Guerrero Negro, Baja California Sur, Mexico, developed distinct pigmented horizons that provided an opportunity to examine the fixation and flow of carbon through a trophic structure and, in conjunction with previous phylogenetic analyses, to assess the diagenetic fates of molecular δ¹³C biosignatures. The δ¹³C values of individual biomarker lipids, total carbon, and total organic carbon (TOC) were determined for each of the following horizons: tan‐orange (TO) at the surface, green (G), purple (P), and olive‐black (OB) at the bottom. δ¹³C of individual fatty acids from intact polar lipids (IPFA) in TO were similar to δ¹³C of dissolved inorganic carbon (DIC) in the overlying water column, indicating limited discrimination by cyanobacteria during CO₂ fixation. δ¹³C_TOC of the underlying G was 3‰ greater than that of TO. The most δ¹³C‐depleted acetogenic lipids in the upper horizons were the cyanobacterial biomarkers C₁₇ n‐alkanes and polyunsaturated fatty acids. Bishomohopanol was 4 to 7‰ enriched, relative to alkanes and intact polar fatty acids (IPFA), respectively. Acyclic C₂₀ isoprenoids were depleted by 14‰ relative to bishomohopanol. Significantly, ?[δ¹³C_TOC ? δ¹³C_∑IPFA] increased from 6.9‰ in TO to 14.7‰ in OB. This major trend might indicate that ¹³C‐enriched residual organic matter accumulated at depth. The permanently anoxic P horizon was dominated by anoxygenic phototrophs and sulfate‐reducing bacteria. P hosted an active sulfur‐dependent microbial community. IPFA and bishomohopanol were ¹³C‐depleted relative to upper crust by 7 and 4‰, respectively, and C₂₀ isoprenoids were somewhat ¹³C‐enriched. Synthesis of alkanes in P was evidenced only by ¹³C‐depleted n‐octadecane and 8‐methylhexadecane. In OB, the marked increase of total inorganic carbon δ¹³C (δ¹³C_TIC) of >6‰ perhaps indicated terminal mineralization. This δ¹³C_TIC increase is consistent with degradation of the osmolyte glycine betaine by methylotrophic methanogens and loss of ¹³C‐depleted methane from the mat.     

Methanosarcina as the dominant aceticlastic methanogens during mesophilic anaerobic digestion of putrescible waste

Taking into account isotope ¹³C value a mathematical model was developed to describe the dynamics of methanogenic population during mesophilic anaerobic digestion of putrescible solid waste and waste imitating Chinese municipal solid waste. Three groups of methanogens were considered in the model including unified hydrogenotrophic methanogens and two aceticlastic methanogens Methanosaeta sp. and Methanosarcina sp. It was assumed that Methanosaeta sp. and Methanosarcina sp. are inhibited by high volatile fatty acids concentration. The total organic and inorganic carbon concentrations, methane production, methane and carbon dioxide partial pressures as well as the isotope ¹³C incorporation in PSW and CMSW were used for the model calibration and validation. The model showed that in spite of the high initial biomass concentration of Methanosaeta sp. Methanosarcina sp. became the dominant aceticlastic methanogens in the system. This prediction was confirmed by FISH. It is concluded that Methanosarcina sp. forming multicellular aggregates may resist to inhibition by volatile fatty acids (VFAs) because a slow diffusion rate of the acids limits the VFA concentrations inside the Methanosarcina sp. aggregates.