nifH sequences and nitrogen fixation in type I and type II methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N2 as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

Trichloroethylene oxidation by the membrane-associated methane monooxygenase in type I,type II and type X methanotrophs

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.Cultures actively oxidizing TCE were monitored for the presence of the soluble methane monooxygenase (sMMO) and membrane associated enzyme (pMMO). Transmission electron micrographs revealed the cultures always contained the internal membrane systems characteristic of cells expressing the pMMO. Naphthalene oxidation by whole cells, or by the cell free, soluble or membrane fractions was never observed. SDS denaturing gels of the membrane fraction showed the polypeptides associated with the pMMO. Cells exposed to ¹⁴C-acetylene showed one labeled band at 26 kDa, and this protein was observed in the membrane fraction. In the one strain examined by EPR spectroscopy, the membrane fraction of TCE oxidizing cells showed the copper complexes characteristic of the pMMO. Lastly, most of the strains tested showed no hybridization to sMMO gene probes. These findings show that the pMMO is capable of TCE oxidation; although the rates are lower than those observed for the sMMO.     

Assessing optimal fermentation type for bio-hydrogen production in continuous-flow acidogenic reactors

In this study, the optimal fermentation type and the operating conditions of anaerobic process in continuous-flow acidogenic reactors was investigated for the maximization of bio-hydrogen production using mixed cultures. Butyric acid type fermentation occurred at pH>6, propionic acid type fermentation occurred at pH about 5.5 with E(h) (redox potential) >-278mV, and ethanol-type fermentation occurred at pH<4.5. The representative strains of these fermentations were Clostridium sp., Propionibacterium sp. and Bacteriodes sp., respectively. Ethanol fermentation was optimal type by comparing the operating stabilities and hydrogen production capacities between the fermentation types, which remained stable when the organic loading rate (OLR) reached the highest OLR at 86.1kgCOD/m(3)d. The maximum hydrogen production reached up to 14.99L/d.     

Analysis of sMMO-containing type I methanotrophs in Lake Washington sediment

Methane-oxidizing bacteria (methanotrophs) containing soluble methane monooxygenase (sMMO) are of interest in natural environments due to the high co-metabolic activity of this enzyme with contaminants such as trichloroethylene. We have analysed sMMO-containing methanotrophs in sediment from a freshwater lake. Environmental clone banks for a gene encoding a diagnostic sMMO subunit (mmoX) were generated using DNA extracted from Lake Washington sediment and subjected to RFLP analysis. Representatives from the six RFLP groups were cloned and sequenced, and all were found to group with Type I Methylomonas mmoX, although a majority were divergent from known Methylomonas mmoX sequences. Direct hybridization of Lake Washington sediment DNA was carried out using a series of sMMO- and Methylomonas-specific probes to assess the significance of these sMMO-containing Methylomonas-like strains in the sediment. The total sMMO-containing population and the sMMO-containing Methylomonas-like population were estimated to be similar to previous estimates for total methanotrophs and Type I methanotrophs. These results suggest that the major methanotrophic population in Lake Washington sediment consists of sMMO-containing Methylomonas-like (Type I) methanotrophs. The whole-cell TCE degradation kinetics of such a strain, LW15, isolated from this environment, were determined and found to be similar to values reported for other sMMO-containing methanotrophs. The numerical significance of sMMO-containing Methylomonas-like methanotrophs in a mesotrophic lake environment suggests that these methanotrophs may play an important role in methanotroph-mediated transformations, including co-metabolism of halogenated solvents, in natural environments.     

Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation

The study of methane-oxidising bacteria (methanotrophs) is of special interest, because of their role in the natural reduction of methane emissions from many different sources. Therefore new probes were developed to detect specifically either type I (Methylococcaceae) or type II methanotrophs (Methylocystaceae). The probes have shown high specificity in fluorescence in situ hybridisations (FISH), as demonstrated by parallel hybridisation of target and reference strains as well as sequence data analysis. With these probes, methanotrophs were detected in soil and root samples from rice microcosms, demonstrating their applicability even in a complex environmental matrix.     

Rice roots select for type I methanotrophs in rice field soil

Methanotrophs are an important regulator for reducing methane (CH₄) emissions from rice field soils. The type I group of the proteobacterial methanotrophs are generally favored at low CH₄ concentration and high O₂ availability, while the type II group lives better under high CH₄ and limiting O₂ conditions. Such physiological differences are possibly reflected in their ecological preferences. In the present study, methanotrophic compositions were compared between rice-planted soil and non-planted soil and between the rhizosphere and rice roots by using terminal restriction fragment length polymorphism (T-RFLP) analysis of particulate methane monooxygenase (pmoA) genes. In addition, the effects of rice variety and nitrogen fertilizer were evaluated. The results showed that the terminal restriction fragments (T-RFs), which were characteristic for type I methanotrophs, substantially increased in the rhizosphere and on the roots compared with non-planted soils. Furthermore, the relative abundances of the type I methanotroph T-RFs were greater on roots than in the rhizosphere. Of type I methanotrophs, the 79 bp T-RF, which was characteristic for an unknown group or Methylococcus/Methylocaldum, markedly increased in field samples, while the 437 bp, which possibly represented Methylomonas, dominated in microcosm samples. These results suggested that type I methanotrophs were enriched or selected for by rice roots compared to type II methanotrophs. However, the members of type I methanotrophs are dynamic and sensitive to environmental change. Rice planting appeared to increase the copy number of pmoA genes relative to the non-planted soils. However, neither the rice variety nor the N fertilizer significantly influenced the dynamics of the methanotrophic community.     

Biosorption of Pb(II) and Cu(II) by activated sludge in batch and continuous-flow stirred reactors

Biosorption of Pb(II) and Cu(II) ions in single component and binary systems was studied using activated sludge in batch and continuous-flow stirred reactors. In biosorption experiments, the activated sludge in three different phases of the growth period was used: growing cells; resting cells; dead or dried cells. Because of the low adsorption capacity of the non-viable activated sludge especially in the case of Pb(II) ions, biosorption of the Cu(II) and Pb(II) ions from the binary mixtures was carried out by using the resting cells. The biosorption data fitted better with the Freundlich adsorption isotherm model. Using a mathematical model based on continuous system mass balance for the liquid phase and batch system mass balance for the solid phase, the forward rate constants for biosorption of Pb(II) and Cu(II) ions were 0.793 and 0.242 1 (mmolmin)(-1), respectively.     

保护性耕作对土壤光合细菌和Ⅱ型甲烷氧化菌的影响

保护性耕作对土壤微生物具有明显的保护效应,但是其对土壤光合细菌和甲烷氧化菌的影响却鲜有报道.本文采用土壤宏基因组16S rDNA变性梯度凝胶电泳(DGGE)和荧光定量PCR技术比较了不同耕作模式(免耕和传统翻耕)和不同秸秆覆盖量(0、50％、100％)对潮土中光合细菌和Ⅱ型甲烷氧化菌数量和群落结构的影响.结果表明:免耕土壤中光合细菌的多样性(多样性指数H=2.47)显著高于传统翻耕土壤(多样性指数H=2.35),且与土壤总氮呈显著正相关,数量略低于传统翻耕土壤;光合细菌的数量和多样性虽均随着秸秆覆盖量的增加而有所增加,但不显著;虽然免耕和秸秆覆盖对Ⅱ型甲烷氧化菌数量和多样性产生了有益的影响,但是耕作模式、秸秆覆盖及二者互作对其影响均不显著;不同处理中光合细菌和Ⅱ型甲烷氧化菌的种群结构无明显变化,光合细菌优势种群以根瘤菌目(Rhizobiales)和鞘脂单胞菌目(Sphingomonadales)为主,Ⅱ型甲烷氧化菌优势种群主要为甲基孢囊菌科(Methylocystaceae)的细菌类群.     

Functional antagonism between type I and type II interferons on human macrophages

A three-day treatment with IFN-gamma enhanced up to 300% the capacity of human monocytes and macrophages to produce H2O2 during the respiratory burst. IFN-alpha or -beta (type I IFNs), which did not by themselves influence the burst, were found to antagonize the enhancing effect of IFN-gamma (type II IFN). The antagonism was concentration-dependent and required the presence of type I IFNs during the whole period of IFN-gamma pretreatment. These results suggest that the host defense function of mononuclear phagocytes may be controlled by the relative local concentrations of type I and type II IFNs.     

Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs

Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O₂. Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O₂ to . Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of to H₂O₂, and the reductant dependent reduction of H₂O₂ to H₂O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios.     

Methanotroph diversity in landfill soil: isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis.

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4. 8 versus 6.8), air/CH(4)/CO(2) headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1x nitrate minimal salts [NMS] versus 0.2x NMS). Screening of the isolates showed that the nutrient-rich 1x NMS selected for type I methanotrophs, while the nutrient-poor 0.2x NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.     

Factors affecting competition of three strains of rhizobia nodulating groundnut,Arachis hypogaea*

The nitrogen (N₂) fixing ability of three strains of rhizobia (NC 92, NC 43.3, and TAL 176) was compared in groundnut cv. Robut 33–1. The competitiveness of these strains in pot culture in a sand-vermiculite medium and with native rhizobia in the field was also investigated. In pot culture, NC 43.3 formed more nodules than TAL 176 and NC 92. Nodules formed by NC 43.3 and NC 92 fixed more N₂ (as measured by total N content in the plants at 42 days after sowing) than nodules formed by TAL 176. TAL 176 was a poor competitor compared with NC 92, NC 43.3, or with native rhizobia in the field. NC 92 when mixed with NC 43.3 (10⁶ cells seed^-1 of each strain) formed only 21% of the nodules, but when independently inoculated in the soil containing native rhizobia, the two-strains formed similar percentages of nodules. Thirty percent of the nodules in two strain combinations of NC 43.3 and NC 92 showed double occupancy. Strain NC 43.3 formed nodules earlier than NC 92 and TAL 176 and this may be one of the factors responsible for its better N₂-fixation and competitiveness. Nodules formed earlier by one strain (NC 92 or TAL 176) were found to have no effect on the subsequent nodulation by the other (TAL 176 or NC 92) strain. Although NC 92 and NC 43.3 were equally competitive with native rhizobia in the field and NC 43.3 fixed more N₂ than NC 92 in pot culture, earlier experiments indicated that only inoculation with NC 92 increased pod yield in field trials.     

Effect of pH on competition for nodule occupancy by type I and type II strains ofRhizobium leguminosarum bv.phaseoli

The effect of soil pH on the competitive abilities of twoRhizobium leuminosarum bv.phaseoli type I and one type II strains was examined in a nonsterile soil system.Phaseolus vulgaris seedlings, grown in unlimed (pH 5.2) or limed (pH 7.6) soil, were inoculated with a single-strain inoculum containing 1 × 10⁶ cells mL^–1 of one of the three test strains or with a mixed inoculum (1:1, type I vs. type II) containing the type II strain CIAT 899 plus one type I strain (TAL 182 or CIAT 895). At harvest, nodule occupants were determined. In a separate experiment, a mixed suspension (1:1, type I vs. type II) of CIAT 899 paired with either TAL 182 or CIAT 895 was used to inoculateP. vulgaris seedlings grown in sterile, limed or unlimed soil. The numbers of each strain in the rhizosphere were monitored for 10 days following inoculation. The majority of nodules (> 60%) formed on plants grown in acidic soil were occupied by CIAT 899, the type II strain. This pattern of nodule occupancy changed in limed soil. When CIAT 899 was paired with TAL 182, the type I strain formed 78% of the nodules. The number of nodules formed by CIAT 899 and CIAT 895 (56% and 44%, respectively) were not significantly different. The observed patterns of nodule occupancy were not related to the relative numbers or specific growth rates of competing strains in the host rhizosphere prior to nodulation. The results indicate that soil pH can influence which symbiotype ofR. leguminosarum bv.phaseoli will competitively nodulateP. vulgaris.     

Factors affecting the determination of total mercury in biological samples by continuous-flow cold vapor atomic absorption spectrophotometry

The acidic reduction of Hg using a continuous-flow analytical system was evaluated. With 25% SnCl₂ as the reductant, characteristic concentrations (sensitivities) of 0.44 μg/L (open cell) and 0.29 μg/L (flow-through cell) were obtained using inorganic Hg²⁺ standards in 1.5% HCl. When CH₃Hg⁺ standards were used, absorption signals were an order of magnitude lower, indicating that Sn(II) is incapable of producing Hg° from organic Hg in this acidic reduction system. Addition of CdCl₂ to the SnCl₂ reductant, as suggested by Magos (1) for the reduction of organomercurials under alkaline conditions, was without beneficial effect. Similarly, combining Sn, with another reducing agent (hydroxylamine hydrochloride), or a strong alkaline solution (40% NaOH), in the reaction coil of the flow-through system did not significantly enhance the Hg absorption signal for either inorganic or organic Hg. Recovery of Hg from spiked liver homogenates digested at 70–80°C using a HNO₃/H₂SO₄/HCl procedure and stabilized with 0.5 mM K₂Cr₂O₇ was >85%, using either inorganic Hg²⁺ or CH₃Hg⁺, indicating that this digestion procedure successfully breaks the C-Hg bond to form readily reducible Hg species. Usingl-cysteine to stabilize standards of inorganic Hg²⁺ in HCl caused significant depressions of the Hg absorption signal atl-cysteine concentrations >0.001% (≈0.5 mM); 0.1%l-cysteine caused total suppression of the Hg signal. These results indicate that: (1) acidic reduction of Hg by Sn in this continuous-flow system requires breakdown of organomercurials prior to analysis; (2) tissue digestion using HNO₃/H₂SO₄/HCl followed by the addition of K₂Cr₂O₇ to stabilize Hg²⁺ achieves this breakdown and allows good recovery of total Hg; and (3) use ofl-cysteine to complex and prevent losses of Hg should be avoided in systems using acidic reduction of Hg. Concentrations of endogenous tissue sulfhydryls are generally lower than those associated with depressed absorbance signals during the acidic reduction of Hg.     

Biological conversion of propane to 2-propanol using group I and II methanotrophs as biocatalysts

Journal of Industrial Microbiology & Biotechnology - Propane is the main component of liquefied petroleum gas and is derived from crude oil processing. Methanotrophic bacteria can convert...     

Structural basis for the functional differences between type I and type II human methionine aminopeptidases

Determination of the crystal structure of human MetAP1 makes it possible, for the first time, to compare the structures of a Type I and a Type II methionine aminopeptidase (MetAP) from the same organism. Comparison of the Type I enzyme with the previously reported complex of ovalicin with Type II MetAP shows that the active site of the former is reduced in size and would incur steric clashes with the bound inhibitor. This explains why ovalicin and related anti-angiogenesis inhibitors target Type II human MetAP but not Type I. The differences in both size and shape of the active sites between MetAP1 and MetAP2 also help to explain their different substrate specificity. In the presence of excess Co(2+), a third cobalt ion binds in the active site region, explaining why metal ions in excess can be inhibitory. Also, the N-terminal region of the protein contains three distinct Pro-x-x-Pro motifs, supporting the prior suggestion that this region of the protein may participate in binding to the ribosome.     

Long-term competition between sulfate reducing and methanogenic bacteria in UASB reactors treating volatile fatty acids

The competition between acetate utilizing methane-producing bacteria (MB) and sulfate-reducing bacteria (SRB) was studied in mesophilic (30 degrees C) upflow anaerobic sludge bed (UASB) reactors (upward velocity 1 m h-1; pH 8) treating volatile fatty acids and sulfate. The UASB reactors treated a VFA mixture (with an acetate:propionate:butyrate ratio of 5:3:2 on COD basis) or acetate as the sole substrate at different COD:sulfate ratios. The outcome of the competition was evaluated in terms of conversion rates and specific methanogenic and sulfidogenic activities. The COD:sulfate ratio was a key factor in the partitioning of acetate utilization between MB and SRB. In excess of sulfate (COD:sulfate ratio lower than 0.67), SRB became predominant over MB after prolonged reactor operation: 250 and 400 days were required to increase the amount of acetate used by SRB from 50 to 90% in the reactor treating, respectively, the VFA mixture or acetate as the sole substrate. The competition for acetate was further studied by dynamic simulations using a mathematical model based on the Monod kinetic parameters of acetate utilizing SRB and MB. The simulations confirmed the long term nature of the competition between these acetotrophs. A high reactor pH (+/-8), a short solid retention time (<150 days), and the presence of a substantial SRB population in the inoculum may considerably reduce the time required for acetate-utilising SRB to outcompete MB.