Analysis of alkane-dependent methanogenic community derived from production water of a high-temperature petroleum reservoir

Microbial assemblage in an n-alkanes-dependent thermophilic methanogenic enrichment cultures derived from production waters of a high-temperature petroleum reservoir was investigated in this study. Substantially higher amounts of methane were generated from the enrichment cultures incubated at 55 °C for 528 days with a mixture of long-chain n-alkanes (C₁₅–C₂₀). Stoichiometric estimation showed that alkanes-dependent methanogenesis accounted for about 19.8% of the total amount of methane expected. Hydrogen was occasionally detected together with methane in the gas phase of the cultures. Chemical analysis of the liquid cultures resulted only in low concentrations of acetate and formate. Phylogenetic analysis of the enrichment revealed the presence of several bacterial taxa related to Firmicutes, Thermodesulfobiaceae, Thermotogaceae, Nitrospiraceae, Dictyoglomaceae, Candidate division OP8 and others without close cultured representatives, and Archaea predominantly related to uncultured members in the order Archaeoglobales and CO₂-reducing methanogens. Screening of genomic DNA retrieved from the alkanes-amended enrichment cultures also suggested the presence of new alkylsuccinate synthase alpha-subunit (assA) homologues. These findings suggest the presence of poorly characterized (putative) anaerobic n-alkanes degraders in the thermophilic methanogenic enrichment cultures. Our results indicate that methanogenesis of alkanes under thermophilic condition is likely to proceed via syntrophic acetate and/or formate oxidation linked with hydrogenotrophic methanogenesis.     

Improvement of sludge digestate biodegradability by thermophilic bioaugmentation

The sludge digestate stabilized by mesophilic anaerobic digestion was further degraded through thermophilic anaerobic digestion using 0–10 % (v/v) of thermophilic, proteolytic Coprothermobacter proteolyticus, and/or methanogenic granular sludge. The results demonstrated that the temperature shift to thermophilic condition promoted abiotic solubilization of proteins and reactivated the fermentative bacteria and methanogens indigenous in the sludge digestate, resulting in a final methane yield of 6.25 mmol-CH₄/g-volatile suspended solid (VSS) digestate. The addition of C. proteolyticus accelerated the hydrolysis and fermentation of proteins and polysaccharides in the digestate during the early stage of thermophilic anaerobic digestion and stimulated methane production by syntrophic cooperation with methanogenic granular sludge. In the treatment with granular sludge and inoculated with 10 % (v/v) of C. proteolyticus, a final methane yield of 7 mmol-CH₄/g-VSS digestate was obtained, and 48.4 % proteins and 27.0 % polysaccharides were degraded. The dissolved proteins were contributed by abiotic factor, C. proteolyticus, and indigenous digestate bacteria, respectively, by around 16, 28, and 56 %.     

Role of fatty acids in cold adaptation of Antarctic psychrophilic Flavobacterium spp.

Cold-loving microorganisms developed numerous adaptation mechanisms allowing them to survive in extremely cold habitats, such as adaptation of the cell membrane. The focus of this study was on the membrane fatty acids of Antarctic Flavobacterium spp., and their adaptation response to cold-stress. Fatty acids and cold-response of Antarctic flavobacteria was also compared to mesophilic and thermophilic members of the genus Flavobacterium. The results showed that the psychrophiles produced more types of major fatty acids than meso- and thermophilic members of this genus, namely C_15:1 iso G, C_15:0 iso, C_15:0 anteiso, C_15:1 ω6c, C_15:0 iso 3OH, C_17:1 ω6c, C_16:0 iso 3OH and C_17:0 iso 3OH, summed features 3 (C_16:1 ω7cand/or C_16:1 ω6c) and 9 (C_16:0 10-methyl and/or C_17:1 iso ω9c). It was shown that the cell membrane of psychrophiles was composed mainly of branched and unsaturated fatty acids. The results also implied that Antarctic flavobacteria mainly used two mechanisms of membrane fluidity alteration in their cold-adaptive response. The first mechanism was based on unsaturation of fatty acids, and the second mechanism on de novo synthesis of branched fatty acids. The alteration of the cell membrane was shown to be similar for all thermotypes of members of the genus Flavobacterium.     

Analysis of lipids from a hydrothermal vent methanogen and associated vent sediment by supercritical fluid chromatography

Lipids from a thermophilic methanogen and the associated hydrothermal vent sediment (Guaymas Basin, Gulf of California) were analyzed by gas chrmotography-mass spectrometry (GC-MS) and supercritical fluid chromatography (SFC). The neutral lipids of the thermophilic methagonen consisted of straight chain alkanes (nC₂₂ to nC₃₆), with nC₂₄, nC₂₈, nC₃₂ and nC₃₆ predominating and C₂₅, C₃₀ and C₃₅-isoprenoids and hydroisoprenoids. The squalene (C₃₀) series was the most abundant (95.6%). The backbone structure of the novel C₃₅-isoprenouds was tentatively identified as 2,6,10,14,19,23,27-heptamethyloctacosane. Polar lipids of the thermophilic methanogen were analyzed by SFC and consisted fo diphytanyl glyceril diether (61.6%), macrocyclic glycerol diether (15.3%), dibiphytanyl diglycerol tetraether (11.8%) and an unidentified component (11.4%).Biomarker analysis of the Guaymas Basin sediment revealed the presence of small amounts of polyunsaturated C₃₀-isoprenoids, with a distribution similar to the C₃₀-isoprenoids of the thermophilic methanogen. In addition, the sediment contained ‘free’ diphytanyl glycerol diether as predominant ether lipid. Low levels of polar ether lipids, indicative of ‘active’ archaebacteria, were also detected. Results suggest that Guaymas Basin sediment recently contained active microbial populations with a lipid profile similar to the isolated thermophilic methanogen.     

Chemical structure of the ether lipids of thermophilic acidophilic bacteria of the Caldariella group

The lipids of the Caldariella group of extremely thermophilic acidophilic bacteria are based on a 72-membered macrocyclic tetraether made up from two C₄₀ diol units and either two glycerol units or one glycerol and one nonitol. The C₄₀ components have the 16,16′-biphytanyl skeleton and the detailed structure of three of them is established.     

Prenyl transferases from Micrococcus luteus: Characterization of undecaprenyl pyrophosphate synthetase

Three prenyl transferases in Micrococcus luteus were recovered in the soluble fraction following cell disruption. Undecaprenyl pyrophosphate (C₅₅-PP) synthetase chromatographed on DEAE-cellulose independently from geranylgeranyl-PP and octaprenyl-PP synthetases. Further purification of C₅₅-PP synthetase resulted in an approximate 250-fold purification over the crude lysate. The molecular weight of the synthetase was estimated to be between 47,000 and 49,000 by Sephadex G-100 chromatography. The enzyme had a broad specificity toward the allylic pyrophosphate substrate. The reactivities of the allylic substrates increased with chain length, C₁₀ < C₁₅ < C₂₀, except for trans-solanesyl-PP, which was unreactive. Moreover, the enzyme was active on allylic substrates having both cis- and trans-stereochemistry. Although C₅₅-PP and C₅₀-PP were the major products, some shorter chain products were also produced, when t,t-farnesyl pyrophosphate and Δ³sopentenyl pyrophosphate (IPP) were used as substrates. The stereochemistries of the products formed with C₅₅-PP synthetase were established, using [¹⁴C]IPP and 2R-[2-³H] and 2S-[2-³H]IPP. Each new isoprene unit added had a cis-configuration. The enzyme was inactive in the absence of added effectors. It was stimulated by Triton X-100, egg lecithin, and a whole phospholipid extract from M. luteus. Cardiolipin and deoxycholate were poor activators of the enzyme. The product chain length distribution observed with the phospholipid-activated enzyme showed highly favored production of the C₅₅-PP product over the C₅₀-PP product.     

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C₅ and C₆ sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216^T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 10³ colonies per μg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C₅ and C₆ sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals.     

Anaerobic alkalithermophiles, a novel group of extremophiles

Although some anaerobic and aerobic mesophiles have long been known to grow at alkaline pH (above 9.5), little was known until recently about thermophilic alkaliphiles, termed now alkalithermophiles. This minireview describes presently known and recently validly described anaerobic alkalithermophilic bacteria (pH_opt ^55C > 8.5; T_opt > 55°C) and alkalitolerant thermophiles (pH_opt ^55C < 8.5 but pH_max ^55C above 9.0). Some of these are widely distributed, but others have been isolated (thus far) only from one specific location. This novel group of anaerobic bacteria is comprised of physiologically different genera and species which, so far, all belong to the Gram-type positive Bacillus-Clostridium phylogenetic subbranch. An interesting feature of these anaerobic alkalithermophiles is that most of the isolates have short doubling times. The fastest growing among them are strains of Thermobrachium celere, with doubling times as short as 10 min while growing above pH 9.0 and above 55°C. Received: January 22, 1998 / Accepted: February 16, 1998     

Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase

Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C₅₅-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C₅₅-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C₅₅-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C₅₅-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C₅₅-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C₅₅-PP molecules on the same (outer) side of the plasma membrane.     

Microbial Oxidation of Hydrocarbons and Related Compounds by Whole-Cell Suspensions of the Methane-Oxidizing Bacterium H-2

Previously, a thermophilic obligate methane-oxidizing bacterium, H-2 (type I), was isolated in our laboratory. H-2 is a new type of methylotroph because of the G+C content of DNA; it uses both the ribulose monophosphate pathway and the serine pathway for carbon assimilation and possesses a new quinone. In addition, we found that resting cell suspensions of H-2 had the ability to oxidize a variety of compounds different from the other methane-oxidizing bacteria as follows. (i) C₁ to C₈n-alkanes are hydroxylated and further oxidized, yielding mixtures of the corresponding alcohols, aldehydes, acids, and ketones. Liquid alkanes are transformed through a different oxidative pathway from that of gaseous ones. (ii) Both gaseous (C₂ to C₄) and liquid (C₅, C₆) n-alkenes are oxidized to their corresponding 1,2-epoxides. (iii) Liquid monochloro and dichloro n-alkanes (C₅, C₆) are oxidized, yielding their corresponding acids or haloacids. (iv) Diethyl ether is oxidized to acetic acid; no ethanol and acetaldehyde are detected. (v) Cyclic and aromatic compounds are also oxidized. (vi) Secondary alcohols (C₃ to C₁₀) are oxidized to their corresponding methyl ketones.     

Hydrogen-Producing Microflora and Fe–Fe Hydrogenase Diversities in Seaweed Bed Associated with Marine Hot Springs of Kalianda, Indonesia

Microbial fermentation is a promising technology for hydrogen (H₂) production. H₂ producers in marine geothermal environments are thermophilic and halotolerant. However, no one has surveyed an environment specifically for thermophilic bacteria that produce H₂ through Fe–Fe hydrogenases (H₂ase). Using heterotrophic medium, several microflora from a seaweed bed associated with marine hot springs were enriched and analyzed for H₂ production. A H₂-producing microflora was obtained from Sargassum sp., 16S rRNA genes and Fe–Fe H₂ase diversities of this enrichment were also analyzed. Based on 16S rRNA genes analysis, 10 phylotypes were found in the H₂-producing microflora showing 90.0–99.5 % identities to known species, and belonged to Clostridia, Gammaproteobacteria, and Bacillales. Clostridia were the most abundant group, and three Clostridia phylotypes were most related to known H₂ producers such as Anaerovorax odorimutans (94.0 % identity), Clostridium papyrosolvens (98.4 % identity), and Clostridium tepidiprofundi (93.1 % identity). For Fe–Fe H₂ases, seven phylotypes were obtained, showing 63–97 % identities to known Fe–Fe H₂ases, and fell into four distinct clusters. Phylotypes HW55-3 and HM55-1 belonged to thermophilic and salt-tolerant H₂-producing Clostridia, Halothermothrix orenii-like Fe–Fe H₂ases (80 % identity), and cellulolytic H₂-producing Clostridia, C. papyrosolvens-like Fe–Fe H₂ases (97 % identity), respectively. The results of both 16S rRNA genes and Fe–Fe H₂ases surveys suggested that the thermophilic and halotolerant H₂-producing microflora in seaweed bed of hot spring area represented previously unknown H₂ producers, and have potential application for H₂ production.     

Sulfate permease ofPenicillium duponti

The thermophilic fungusPenicillium duponti has an active sulfate permease system derepressible by growth in cysteic acid or by sulfur starvation. The system is sensitive to ionic strength and is optimal at 45°C, pH 6.5. It is saturable with an apparent K_m for sulfate of 55 μM. It is activated strongly by divalent cations and inhibited by inhibitors of mitochondrial ATP production and the group VI oxyanions.     

A variety of glycolipids in green photosynthetic bacteria

The compositions of glycolipids in the following seven strains of green photosynthetic bacteria were investigated at the molecular level using LC–MS coupled with an evaporative light scattering detector: Chlorobium (Chl.) limicola strains Larsen (30 °C as the optimal cultivation temperature) and DSM245 (30 °C), Chlorobaculum (Cba.) tepidum strain ATCC49652 (45 °C), Cba. parvum strain NCIB8327 (30 °C), Cba. limnaeum strain 1549 (30 °C), Chl. phaeovibrioides DSM269 (30 °C), and Chloroflexus (Cfl.) aurantiacus strain J-10-fl (55 °C). Dependence of the molecular structures of glycolipids including the chain-length of their acyl groups upon bacterial cultivation temperatures was clearly observed. The organisms with their optimal temperatures of 30, 45, and 55 °C dominantly accumulated glycolipids possessing the acyl chains in the range of C₁₅–C₁₆, C₁₆–C₁₇, and C₁₈–C₂₀, respectively. Cba. tepidum with an optimal temperature of 45 °C preferred the insertion of a methylene group to produce finally a C₁₇-cyclopropane chain. Cfl. aurantiacus cultured optimally at 55 °C caused a drastic increase in the chain-length. Notably, the length of such acyl groups corresponded to that of the esterifying chain in the 17-propionate residues of self-aggregative bacteriochlorophylls-c/d/e, indicating stabilization of their supramolecular structures through hydrophobic interactions among those hydrocarbon chains. Based on the detailed compositions of glycolipids, a survival strategy of green photosynthetic bacteria grown in the wide range of temperatures is discussed.     

Stability curves of laboratory evolved thermostable mutants of a Bacillus subtilis lipase

Shape of the protein stability curves changes to achieve higher melting temperature. Broadly, these changes have been classified as upward shift (increased ?G_s), rightward shift (increase in T_s) and flattening of the stability curves (decrease in ?C_p). Comparative studies on homologous mesophilic–thermophilic protein pairs highlighted the differential contribution of these three strategies amongst proteins. But unambiguous way of identification of the strategies, which will be preferred for a protein, is still not achieved. We have performed comparative thermodynamic studies using differential scanning calorimeter (DSC) on thermostable variants of a lipase from Bacillus subtilis. These variants are products of 1, 2, 3 and 4 rounds of directed evolution and harbor mutations having definite contribution in thermostability unlike natural thermophilic proteins. We have shown that upward and rightward shift in stability curves are prime strategies in this lipase. Our results along with that from the other study on laboratory evolved xylanase A suggest that optimization of suboptimal thermodynamic parameters is having a dominant influence in selection of thermodynamic strategies for higher thermostability.     

Grevillea robusta seed oil: A source of ω-5 monoenes

The fatty acids from Grevillea robusta seed oil triglycerides contain 22.5 % ω-5 monoenes ranging in chain length from C₁₄ to C₂₈. C₁₆ to C₂₆ saturates (18 %), C₁₈ to C₂₄ ω-9 monoenes (55 %), C₁₈ diene (2.3 %) and C₁₈ triene (0.7 %) make up the remainder of the acids.     

Biological function of modified nucleotides T54 and Ψ55 of yeast tRNAAla

The 3′ half molecule of yeast tRNA^Ala (nucleotides 36–75) was hybridized with a DNA fragment (5′GGAATCGAACC 3′) and the hybrid was then digested withE. coli RNase H (from Boehringer). The enzyme can specifically cleave the 3′ half molecule at the 3′ side of nucleotide Ψ₅₅, thus a fragment C₃₆-Ψ₅₅ was prepared. The 3′-terminal T or TΨ of this fragment was removed by one or two cycles of periodate oxidation and β-elimination. The products were fragments C₃₆-T₅₄ and C₃₆-G₅₃. Three yeast tRNA_Ala fragments C₅₆-A₇₆, U₅₅-A₇₆ (with Ψ₅₅ replaced by U), U₅₄-A₇₆ (with T₅₄Ψ₅₅ replaced by UU) were synthesized and ligated with three prepared fragments (C₃₆-Ψ₅₅, C₃₆-T₅₄ and C₃₆-G₅₃) respectively by T4 RNA ligase. The products were further ligated with the 5′ half molecule (nu-cleotides 1–35). Using this method, one reconstituted yeast tRNA^Ala (tRNAr) and two yeast tRNA^ALa analogs: (i) tRNAa with U₅₅ instead of Ψ₅₅; (ii) tRNAb with U₅₄U₅₅ instead of T₅₄Ψ₅₅ were synthesized. The charging and incorporation activities of these three tRNAs were determined. In comparison with the reconstituted tRNA, the charging activity was 75% for tRNAa and 45% for tRNAb and the incorporation activity was 65% for tRNAa and 70% for tRNAb. These results suggest that the modified nucleotides T₅₄ and Ψ₅₅ play an important role in yeast tRNA^Ala function.     

Lebetimonas natsushimae sp. nov., a novel strictly anaerobic,moderately thermophilic chemoautotroph isolated from a deep-sea hydrothermal vent polychaete nest in the Mid-Okinawa Trough

A moderately thermophilic, strictly anaerobic, chemoautotrophic bacterium, designated strain HS1857^T, was isolated from a deep-sea hydrothermal vent at the Noho site in the Mid-Okinawa Trough. Strain HS1857^T grew between 35 and 63 °C (optimum 55 °C), in the presence of 10–55 g l^?1 NaCl (optimum 25 g l^?1), and pH 5.5–7.1 (optimum 6.4). Growth occurred with molecular hydrogen as the electron donor and elemental sulfur, nitrate, or selenate as the electron acceptors. Formate could serve as an alternative electron donor with nitrate as an electron acceptor. During growth with nitrate as the electron acceptor, strain HS1857^T produced ammonium and formed a biofilm. CO₂ was utilized as the sole carbon source. The G + C content of the genomic DNA was 33.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HS1857^T is a member of the order Nautiliales, showing a sequence similarity of 95.0% with Lebetimonas acidiphila Pd55^T. The fatty acid composition was similar to that of L. acidiphila, which was dominated by C_18:0 (47.0%) and C_18:1 (23.7%). Based on the genomic, chemotaxonomic, phenotypic characteristics, the name Lebetimonas natsushimae sp. nov., is proposed. The type strain is HS1857^T (= NBRC 112478^T = DSM 104102^T).     

Anaerobic oxidation of long-chain n-alkanes by the hyperthermophilic sulfate-reducing archaeon,Archaeoglobus fulgidus

The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain VC-16 (DSM 4304), which is known to oxidize fatty acids and n-alkenes, was shown to oxidize saturated hydrocarbons (n-alkanes in the range C₁₀–C₂₁) with thiosulfate or sulfate as a terminal electron acceptor. The amount of n-hexadecane degradation observed was in stoichiometric agreement with the theoretically expected amount of thiosulfate reduction. One of the pathways used by anaerobic microorganisms to activate alkanes is addition to fumarate that involves alkylsuccinate synthase as a key enzyme. A search for genes encoding homologous enzymes in A. fulgidus identified the pflD gene (locus-tag AF1449) that was previously annotated as a pyruvate formate lyase. A phylogenetic analysis revealed that this gene is of bacterial origin and was likely acquired by A. fulgidus from a bacterial donor through a horizontal gene transfer. Based on three-dimensional modeling of the corresponding protein and molecular dynamic simulations, we hypothesize an alkylsuccinate synthase activity for this gene product. The pflD gene expression was upregulated during the growth of A. fulgidus on an n-alkane (C₁₆) compared with growth on a fatty acid. Our results suggest that anaerobic alkane degradation in A. fulgidus may involve the gene pflD in alkane activation through addition to fumarate. These findings highlight the possible importance of hydrocarbon oxidation at high temperatures by A. fulgidus in hydrothermal vents and the deep biosphere.     

Methane fermentation of xylan by mesophilic and thermophilic methane sludges

The degradation of xylan during methane fermentation proceeded as a first-order reaction. The rate constants were calculated to be 0.40–0.09 day^–1 at 37° C and 0.341 day^–1 at 55° C. From calculations based on the experimental data, K _A and C _A values in the expression of the velocity of xylose consumption changed as the fermentation progressed. In the mesophilic fermentation, the degradation of xylan slowed down after 2 days of incubation, but the rate of consumption of xylose increased between days 3 and 4 of incubation and slow again at the 5th day of incubation. In the thermophilic fermentation, the degradation of xylan proceeded at a constant rate and the rate of consumption of xylose increased slightly on the 3rd day of incubation. When the velocity of gas evolution was determined, the C _G value for acetate at 55° C was about 1.8 times larger than the value at 37° C.