甲烷氧化细菌Methylosinus trichosporium 3011甲醇累积条件的研究

本文考察了甲烷氧化细菌Methylosi nus trichosporium 3011的生理特性及反应条件对甲烷单加氧酶和甲醇累积的影响。M.3011菌株在4℃保存30～40天内,菌株的细胞生长量和甲烷单加氧酶活性均不受影响。在生长对数期收获细胞,其甲烷单加氧酶活力最高可达125nmol甲醇/mg(细胞干重)·min。在M.3011菌株的生长对数期后期收集细胞,将反应菌悬液浓度控制在0.15—0.3mg(细胞干重/ml,pH为6.7,反应温度为35℃,甲醇累积量可达3.1μmol甲醇/mg(细胞干重)·h。反应液的磷酸缓冲液的最适浓度为60mmol/L。     

Diversity of methanotrophs in urea-fertilized tropical rice agroecosystem

Laboratory experiments were conducted to study the population size, diversity and methane oxidation potential of methanotrophs in tropical rice agroecosystem under the influence of N-fertilizer. Results indicate that the diversity of methane oxidizing bacteria (MOB) is altered in fertilizer treated soils compared to untreated control. Nevertheless, Type I MOB still dominated in the fertilized soils whereas the diversity of Type II methanotrophs decreases. Control soils have higher MOB population and CH₄ oxidation capacity than fertilized soils. Rhizospheric soil is more populated than non-rhizospheric soil in both unfertilized and fertilized conditions. Variation in K_m and V_max of methane oxidation in soils appears to be due to variation in methanotrophic community. Experimental results indicate that methanotrophic community differs both quantitatively and qualitatively in unfertilized and fertilized soils.     

Thermophilic methane production and oxidation in compost

Methane cycling within compost heaps has not yet been investigated in detail. We show that thermophilic methane oxidation occurred after a lag phase of up to one day in 4-week old, 8-week old and mature (>10-week old) compost material. The potential rate of methane oxidation was between 2.6 and 4.1 micromol CH4(gdw)(-1)h(-1). Profiles of methane concentrations within heaps of different ages indicated that 46-98% of the methane produced was oxidised by methanotrophic bacteria. The population size of thermophilic methanotrophs was estimated at 10(9) cells (gdw)(-1), based on methane oxidation rates. A methanotroph (strain KTM-1) was isolated from the highest positive step of a serial dilution series. This strain belonged to the genus Methylocaldum, which contains thermotolerant and thermophilic methanotrophs. The closest relative organism on the basis of 16S rRNA gene sequence identity was M. szegediense (>99%), a species originally isolated from hot springs. The temperature optimum (45-55 degrees C) for methane oxidation within the compost material was identical to that of strain KTM-1, suggesting that this strain was well adapted to the conditions in the compost material. The temperatures measured in the upper layer (0-40 cm) of the compost heaps were also in this range, so we assume that these organisms are capable of effectively reducing the potential methane emissions from compost.     

Pancreatic islet response to hyperglycemia is dependent on peroxisome proliferator-activated receptor alpha (PPARalpha)

This study tests the hypothesis that islet peroxisome proliferator-activated receptor alpha (PPARalpha) influences insulin secretion. Freshly isolated islets of normoglycemic PPARalpha-null mice display no major alteration of glucose-stimulated insulin release. However, after 24 h of culture in high glucose, PPARalpha-null islets exhibit elevated basal insulin secretion and fail to increase insulin mRNA. 24-h culture with palmitate replicates this phenotype in wild-type islets. The data suggest that PPARalpha is needed to ensure appropriate insulin secretory response in situation of short-term hyperglycemia, likely by maintaining islet lipid homeostasis. As such, islet PPARalpha could contribute to delay the progression of type 2 diabetes.     

Butachlor inhibits production and oxidation of methane in tropical rice soils under flooded condition

In laboratory incubation experiments, application of a commercial formulation of the herbicide butachlor (N-butoxymethyl-2-chloro-2',6'-diethyl acetanilide) to three tropical rice soils, widely differing in their physicochemical characteristics, under flooded condition inhibited methane (CH4) production. The inhibitory effect was concentration dependent and most remarkable in the alluvial soil. Thus, following application of butachlor at 5, 10, 50 and 100 microg g(-1) soil, respectively, cumulative CH4 production in the alluvial soil was inhibited by 15%, 31%, 91% and 98% over unamended control. Since CH4 production was less pronounced in the sandy loam and acid sulfate soil, the impact of amendment with butchalor, albeit inhibitory, was less extensive than the alluvial soil. Inhibition of CH4 production in butachlor-amended alluvial soil was related to the prevention in the drop in redox potential as well as low methanogenic bacterial population especially at high concentrations of butachlor. CH4 oxidation was also inhibited in butachlor-amended alluvial soil with the inhibitory effect being more prevalent under flooded condition. Inhibition in CH4 oxidation was related to a reduction in the population of soluble methane monooxygenase producing methanotrophs. Results demonstrate that butachlor, a commonly used herbicide in rice cultivation, even at very low concentrations can affect CH4 production and its oxidation, thereby influencing the biogeochemical cycle of CH4 in flooded rice soils.     

New DGGE strategies for the analyses of methanotrophic microbial communities using different combinations of existing 16S rRNA-based primers

Methane-oxidising microbial communities are studied intensively because of their importance for global methane cycling. A suite of molecular microbial techniques has been applied to the study of these communities. Denaturing gradient gel electrophoresis (DGGE) is a diversity screening tool combining high sample throughput with phylogenetic information of high resolution. The existing 16S rRNA-based DGGE assays available for methane-oxidising bacteria suffer from low-specificity, low phylogentic information due to the length of the amplified fragments and/or from lack of resolving power. In the present study we developed new combinations of existing primers and applied these on methane-oxidising microbial communities in a freshwater wetland marsh. The designed strategies comprised nested as well as direct amplification of environmental DNA. Successful application of direct amplification using combinations of universal and specific primers circumvents the nested designs currently used. All developed assays resulted in identical community profiles in wetland soil cores with Methylobacter sp. and Methylocystis sp.-related sequences. Changes in the occurrence of Methylobacter-related sequences with depth in the soil profile may be related to the decrease in methane-oxidizing activity.     

Recovery of in-situ methanotrophic activity following acetylene inhibition

Methane (CH₄) is the second most important greenhouse gas after carbon dioxide (CO₂). To understand CH₄ cycling, quantitative information about microbial CH₄ oxidation in soils is essential. Field methods such as the gas push-pull test (GPPT) to quantify CH₄ oxidation are often used in combination with specific inhibitors, such as acetylene (C₂H₂). Acetylene irreversibly binds to the enzyme methane monooxygenase, but little is known about recovery of CH₄ oxidation activity after C₂H₂ inhibition in situ, which is important when performing several experiments at the same location. To assess recovery of CH₄ oxidation activity following C₂H₂ inhibition, we performed a series of GPPTs over 8 weeks at two different locations in the vadose zone above a petroleum hydrocarbon-contaminated aquifer in Studen, Switzerland. After 4 weeks a maximum recovery of 30% and 50% of the respective initial activity was reached, with a subsequent slight drop in activity at both locations. Likely, CH₄ oxidation activity and CH₄ concentrations were too low to allow for rapid recovery following C₂H₂ inhibition at the studied locations. Therefore, alternative competitive inhibitors have to be evaluated for application in conjunction with GPPTs, especially for sites with low activity.     

The genomic rate of adaptive amino acid substitution in Drosophila

The proportion of amino acid substitutions driven by adaptive evolution can potentially be estimated from polymorphism and divergence data by an extension of the McDonald-Kreitman test. We have developed a maximum-likelihood method to do this and have applied our method to several data sets from three Drosophila species: D. melanogaster, D. simulans, and D. yakuba. The estimated number of adaptive substitutions per codon is not uniformly distributed among genes, but follows a leptokurtic distribution. However, the proportion of amino acid substitutions fixed by adaptive evolution seems to be remarkably constant across the genome (i.e., the proportion of amino acid substitutions that are adaptive appears to be the same in fast-evolving and slow-evolving genes; fast-evolving genes have higher numbers of both adaptive and neutral substitutions). Our estimates do not seem to be significantly biased by selection on synonymous codon use or by the assumption of independence among sites. Nevertheless, an accurate estimate is hampered by the existence of slightly deleterious mutations and variations in effective population size. The analysis of several Drosophila data sets suggests that approximately 25% +/- 20% of amino acid substitutions were driven by positive selection in the divergence between D. simulans and D. yakuba.     

Paraffin oil as a “methane vector” for rapid and high cell density cultivation of <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

Slow growth and relatively low cell densities of methanotrophs have limited their uses in industrial applications. In this study, a novel method for rapid cultivation of Methylosinus trichosporium OB3b was studied by adding a water-immiscible organic solvent in the medium. Paraffin oil was the most effective at enhancing cell growth and final cell density. This is at least partially due to the increase of methane gas transfer between gas and medium phases since methane solubility is higher in paraffin than in water/nitrate minimal salt medium. During cultivation with paraffin oil at 5% (v/v) in the medium, M. trichosporium OB3b cells also showed higher concentrations of the intermediary metabolites, such as formic acid and pyruvic acid, and consumed more methane compared with the control. Paraffin as methane vector to improve methanotroph growth was further studied in a 5-L fermentor at three concentrations (i.e., 2.5%, 5%, and 10%). Cell density reached about 14 g dry weight per liter with 5% paraffin, around seven times higher than that of the control (without paraffin). Cells cultivated with paraffin tended to accumulate around the interface between oil droplets and the water phase and could exist in oil phase in the case of 10% (v/v) paraffin. These results indicated that paraffin could enhance methanotroph growth, which is potentially useful in cultivation of methanotrophs in large scale in industry. Bing Han and Tao Su contributed equally to this work.