RDX degrading microbial communities and the prediction of microorganisms responsible for RDX bioremediation

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has caused significant soil and groundwater contamination. To remediate these sites, there is a need to determine which microorganisms are responsible for in situ biodegradation of RDX to enable the appropriate planning of bioremediation efforts. Here, studies are examined that have reported on the microbial communities linked with RDX biodegradation. Dominant microorganisms across samples are discussed and summarized. This information is then compared to current knowledge on RDX degrading isolates to predict which organisms may be responsible for RDX degradation in soils and groundwater. From the phyla with known RDX degrading isolates, Firmicutes and Proteobacteria (particularly Gammaproteobacteria) were the most dominant organisms in many contaminated site derived samples. Organisms in the phyla Deltaproteobacteria, Alphaproteobacteria and Actinobacteria were dominant in these studies less frequently. Notably, organisms within the class Betaproteobacteria were dominant in many samples and yet this class does not appear to contain any known RDX degraders. This analysis is valuable for the future development of molecular techniques to track the occurrence and abundance of RDX degraders at contaminated sites.     

Evidence for the existence of psychrophilic methanogenic communities in anoxic sediments of deep lakes

In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5 degrees C), slurries were first incubated at temperatures between 4 and 60 degrees C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6 degrees C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15 degrees C, with maximal rates in slurries kept at 6 degrees C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6 degrees C after preincubation at 30 degrees C.     

褐煤强化产甲烷菌群的群落分析及条件优化

【目的】以白音华褐煤为底物,利用从我国多地煤矿及污水处理厌氧罐中富集-混合-驯化得到的高效混合菌群进行产气,分析其群落组成并优化产气条件。【方法】采用Miseq高通量测序分析混合菌群结构,通过Plackett-Burman(P-B)和Box-Behnken(B-B)试验对褐煤产气影响因素和条件进行筛选和优化。【结果】本源和外源微生物样本混合样品(HN+MD+WT)经驯化后菌群产气效率最高。该样品菌群中细菌群落多样性丰富,以变形菌门的脱硫弧菌属Desulfovibrio(15.07%)、拟杆菌门的屠场杆状菌属Macellibacteroides(14.6%)、厚壁菌门的梭菌属Clostridiaceae(9.77%)、互营菌门的脱硫代硫酸盐弧菌属Dethiosulfovibrio(8.76%)以及热袍菌门Oceanotoga属(8.66%)为主。古菌全部为广古菌门,其群落多样性则较为单一,其中甲烷卵圆形菌属Methanocalculus(80.28%)占据绝对优势。Plackett-Burman(P-B)试验结果表明温度、CoCl_2添加量和NiCl_2添加量是影响褐煤产气的关键因素;Box-Behnken(B-B)试验结果表明最优产气条件为:温度36°C,CoCl_2添加量0.17 g/L,NiCl_2添加量0.02 g/L,最优条件下褐煤累计产甲烷量(周期20 d)达到159.33μmol/g。【结论】经过驯化可以得到高效的产气菌群,优化培养条件可使产气效率明显提高。     

Community structure of methanogenic archaea and methane production associated with compost-treated tropical rice-field soil

The diversity and density of methanogenic archaea and methane production were investigated ex situ at different growth stages of rice plant cultivated in compost-treated tropical rice fields. The qPCR analysis revealed variation in methanogens population from 3.40?×?10(6) to 1.11?×?10(7) copies?g(-1) dws, in the year 2009 and 4.37?×?10(6) to 1.36?×?10(7) copies?g(-1) dws in the year 2010. Apart from methanogens, a large number of bacterial (9.60?×?10(9) -1.44?×?10(10) copies?g(-1) dws) and archaeal (7.13?×?10(7) -3.02?×?10(8) copies?g(-1) dws) communities were also associated with methanogenesis. Methanogen population size varied in the order: flowering > ripening > tillering > postharvest > preplantation stage. The RFLP-based 16S rRNA gene-targeted phylogenetic analysis showed that clones were closely related to diverse group of methanogens comprising members of Methanomicrobiaceae, Methanosarcinaceae, Methanosaetaceae and RC I. Laboratory incubation studies revealed higher amount of cumulative CH(4) at the flowering stage. The integration of methanogenic community structure and CH(4) production potential of soil resulted in a better understanding of the dynamics of CH(4) production in organically treated rice-field soil. The hypothesis that the stages of plant development influence the methanogenic community structure leading to temporal variation in the CH(4) production has been successfully tested.     

Effect of physical irradiation and chemical mutagen treatment on methane production by methanogenic bacteria

Biomethanation is one of the desirable options for obtaining clean fuel from abundant renewable biomass resources. Improvement of biomethane production may be achieved by using improved strains of microbes, particularly the terminal microbes – the methanogens. Attempts have been made to improve the efficiency of the methanogens isolated from local sources by subjecting the methanogens to mutagenic changes by physical (by irradiation, neutron bombardment) or chemical (by addition of chemicals like acridine orange, colchicine) means. The effects of the treated methanogens on biomethanation were studied. Irradiation or neutron bombardment mutagenesis was dose-dependent and time-dependent. High doses proved to be lethal but methanogens were found to be to some extent radiation resistant when subjected to irradiation at small doses for short duration (5–10 s). No or marginal improvement of methane production occurred for the two strains TDM and TRM. Improvement of methane production occurred from successive transfers of radiation treated strain SSM. Chemical mutagens invariably inhibited biomethanation and the inhibition was dose dependent.     

Comparative analysis of transposable elements in the melanogaster subgroup sequenced genomes

Transposable elements (TEs) are indwelling components of genomes, and their dynamics have been a driving force in genome evolution. Although we now have more information concerning their amounts and characteristics in various organisms, we still have little data from overall comparisons of their sequences in very closely-related species. While the Drosophila melanogaster genome has been extensively studied, we have only limited knowledge regarding the precise TE sequences in the genomes of the related species Drosophila simulans, Drosophila sechellia and Drosophila yakuba. In this study we analyzed the number and structure of TE copies in the sequenced genomes of these four species. Our findings show that, unexpectedly, the number of TE insertions in D. simulans is greater than that in D. melanogaster, but that most of the copies in D. simulans are degraded and in small fragments, as in D. sechellia and D. yakuba. This suggests that all three species were invaded by numerous TEs a long time ago, but have since regulated their activity, as the present TE copies are degraded, with very few full-length elements. In contrast, in D. melanogaster, a recent activation of TEs has resulted in a large number of almost-identical TE copies. We have detected variants of some TEs in D. simulans and D. sechellia, that are almost identical to the reference TE sequences in D. melanogaster, suggesting that D. melanogaster has recently been invaded by active TE variants from the other species. Our results indicate that the three species D. simulans, D. sechellia, and D. yakuba seem to be at a different stage of their TE life cycle when compared to D. melanogaster. Moreover, we show that D. melanogaster has been invaded by active TE variants for several TE families likely to come from D. simulans or the ancestor of D. simulans and D. sechellia. The numerous horizontal transfer events implied to explain these results could indicate introgression events between these species.     

寺河矿煤地质产甲烷微生物菌群的保藏和产甲烷性能

【背景】煤地质产甲烷微生物菌群可以代谢煤基质产生甲烷,对于实现煤层气资源的再利用具有重要意义。【目的】检测产甲烷菌群在保藏过程中群落结构的动态变化以及在产气实验中甲烷气的生成情况,以验证保藏方法的可行性,同时为煤层气的微生物增产奠定基础。【方法】分别于不同温度条件下比较3种菌种保藏方法,即甘油/L-半胱氨酸法、富营养法和煤基-基础盐法。通过产气实验检测不同保藏条件下产甲烷菌群的活力。同时,采用454高通量测序技术测定16S r RNA基因序列,分析25°C条件下煤基-基础盐菌种保藏过程中微生物群落结构的变化。【结果】比较了9组菌种保藏方法,发现菌种最佳保藏条件为25°C的煤基-基础盐保藏。在该条件下保藏的产甲烷菌群活性最高,甲烷生成量最大。以无烟煤为碳源进行产气实验时甲烷生成量为12%-25%,而以褐煤为碳源时甲烷生成量可达24%-73%。在25°C的煤基-基础盐菌种保藏条件下,保藏初期细菌的主要优势菌为假单胞菌属(Pseudomonas),而古菌的主要优势菌为甲烷八叠球菌属(Methanosarcina)。随着保藏时间的增加,细菌的群落结构变化显著,发酵细菌及产氢产乙酸细菌成为优势细菌,古菌的群落结构则相对稳定。【结论】菌种保藏的最佳条件为25°C的煤基-基础盐,保藏的产甲烷菌群能长期维持在较高的活性状态,具有较好的产甲烷能力。     

新一代高通量测序与稳定性同位素示踪DNA/RNA技术研究稻田红壤甲烷氧化的微生物过程

[目的]利用新一代高通量测序技术分析复杂土壤环境中整体微生物群落结构的变化规律,研究特定功能微生物生理过程的分子机制;利用稳定性同位素示踪微生物核酸DNA/RNA,研究复杂土壤中关键元素转化的微生物调控机制.[方法]针对我国第四纪红色粘土母质发育的3种稻田红壤,围绕13C-甲烷好氧氧化的微生物过程,在DNA和RNA水平高通量测序土壤微生物群落16S rRNA基因和16S rRNA,通过超高速密度梯度离心土壤微生物总核酸获得13C-标记的DNA/RNA,进一步采用克隆文库技术研究稻田红壤甲烷好氧氧化的微生物作用者.[结果]新一代高通量测序结果表明,3种稻田红壤甲烷的好氧氧化过程中,甲烷好氧氧化菌占土壤整体微生物群落的丰度显著增加,RNA水平的增幅显著高于DNA水平,能够更为灵敏地反映土壤甲烷好氧氧化的微生物过程.3种稻田红壤甲烷的好氧氧化过程中,类型Ⅰ和类型Ⅱ甲烷好氧氧化菌在湖南古市土壤中显著增加,湖南桃源土壤中类型Ⅱ甲烷好氧氧化菌增加明显,而类型Ⅰ甲烷好氧氧化菌在广东雷州土壤中增幅最大.进一步利用13C-DNA和13C-RNA分别构建pmoA基因和16S rRNA克隆文库,发现类型Ⅰ甲烷好氧氧化菌主导了湖南古市和广东雷州稻田红壤甲烷的好氧氧化过程,类型Ⅱ甲烷好氧氧化菌主导了湖南桃源稻田红壤甲烷的好氧氧化过程.[结论]新一代高通量测序技术能够在整体微生物群落水平,清楚反映复杂土壤中特定功能微生物的生理生态过程,而RNA较DNA水平的分析更为灵敏;稳定性同位素示踪微生物核酸DNA/RNA技术能够准确地揭示复杂土壤重要过程的微生物作用者.     

Shifts of methanogenic communities in response to permafrost thaw results in rising methane emissions and soil property changes

Extremophiles - Permafrost thaw can bring negative consequences in terms of ecosystems, resulting in permafrost collapse, waterlogging, thermokarst lake development, and species composition...     

Assessment of metabolic properties and kinetic parameters of methanogenic sludge by on-line methane production rate measurements

This report presents a new approach to studying the metabolic and kinetic properties of anaerobic sludge from single batch experiments. The two main features of the method are that the methane production is measured on-line with a relatively cheap system, and that the methane production data can be plotted as rate vs time curves. The case studies of specific methanogenic activity, biodegradability and toxicity tests here presented show that very accurate kinetic data can be obtained. The method is specifically useful in experiments in which strong changes in methane production occur, and it is proposed as a powerful tool to study methanogenic systems. Furthermore, the method is simple and could be implemented by industry in the routine analysis of sludge.     

Molecular analysis of methanogenic archaeal communities in managed and natural upland pasture soils

Grassland management influences soil archaeal communities, which appear to be dominated by nonthermophilic crenarchaeotes. To determine whether methanogenic Archaea associated with the Euryarchaeota lineage are also present in grassland soils, anaerobic microcosms containing both managed (improved) and natural (unimproved) grassland rhizosphere soils were incubated for 28 days to encourage the growth of anaerobic Archaea. The contribution of potential methanogenic organisms to the archaeal community was assessed by the molecular analysis of RNA extracted from soil, using primers targeting all Archaea and Euryarchaeota. Archaeal RT‐PCR products were obtained from all anaerobic microcosms. However, euryarchaeal RT‐PCR products (of putative methanogen origin) were obtained only from anaerobic microcosms of improved soil, their presence coinciding with detectable methane production. Sequence analysis of excised denaturing gradient gel electrophoresis (DGGE) bands revealed the presence of euryarchaeal organisms that could not be detected before anaerobic enrichment. These data indicate that nonmethanogenic Crenarchaeota dominate archaeal communities in grassland soil and suggest that management practices encourage euryarchaeal methanogenic activity.     

Photobioreactors: production systems for phototrophic microorganisms

Microalgae have a large biotechnological potential for producing valuable substances for the feed, food, cosmetics and pharmacy industries as well as for biotechnological processes. The design of the technical and technological basis for photobioreactors is the most important issue for economic success in the field of phototrophic biotechnology. For future applications, open pond systems for large-scale production seem to have a lower innovative potential than closed systems. For high-value products in particular, closed systems of photobioreactors seem to be the more promising field for technical developments despite very different approaches in design.     

Molecular identification of the enzyme responsible for the mitochondrial NADH-supported ammonium-dependent hydrogen peroxide production

A homogeneous protein with a subunit apparent molecular mass of ∼50 kDa that catalyzes the previously described mitochondrial NADH-supported ammonium-stimulated hydrogen peroxide production (Grivennikova, V.G., Gecchini, G. and Vinogradov, A.D. (2008) FEBS Lett. 583, 1287–1291) was purified from the mitochondrial matrix of bovine heart. Chromatography of partially purified protein showed that the peaks of ammonium-stimulated NADH-dependent H₂O₂ production and that of NADH:lipoamide oxidoreductase activity coincided. The catalytic properties and mass spectrometry of the trypsin-digested protein revealed peptides that allowed identification of the protein as the Bos taurus dihydrolipoyl dehydrogenase.     

Proteomics for routine identification of microorganisms

The invention of MALDI-TOF-MS enormously contributed to the understanding of protein chemistry and cell biology. Without this technique proteomics would most likely not be the important discipline it is today. Besides 'true' proteomics, MALDI-TOF-MS was applied for the analysis of microorganisms for their taxonomic characterization from its beginning. This approach has since been developed as a diagnostic tool readily available for routine, high-throughput analysis of microbial isolates from clinical specimens by intact-cell mass spectrometry (ICMS), the direct analysis of whole bacterial cell without a preceding fractionation or separation by chromatography or electrophoresis. ICMS exploits the reproducibility of mass fingerprints for individual bacterial and fungal strains as well as the high similarity of mass fingerprints within a species. Comparison of mass spectral data to genomic sequences emphasized the validity of peak patterns as taxonomic markers. Supported by comprehensive databases, MALDI-TOF-MS-based identification has been widely accepted in clinical laboratories within only a few years.     

Novel bioresources for studies of Brassica oleracea: identification of a kale MYB transcription factor responsible for glucosinolate production

Plants belonging to the Brassicaceae family exhibit species‐specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health–promoting properties. Among them, glucoraphanin (aliphatic 4‐methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full‐length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild‐type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale.     

End product inhibition in methane fermentations: Effects of carbon dioxide and methane on methanogenic bacteria utilizing acetate

Summary The effects of pCO₂ and pCH₄ in the interval 0–1 bar on rates of acetate degradation and methane formation by methanogens as well as methane yields were studied in enrichment cultures in batch and continuous fermentations.In batch fermentations the rate of acetate utilization by methanogens was 1,000–1,500 mg/l · d at low levels of pCO₂. CO₂ was inhibitory and degradation rates were around 350 mg/l · d in 1 bar CO₂. The degradation of acetate was almost linear. In continuous culture maximum rates of acetate utilization around 2,500 mg/l · d were obtained and the acetate concentration in the substrate was reduced by 98–99%.The yields of methane on acetate substrates were close to the theoretical value (1 mole CH₄ per mole HAc) in the interval pCO₂-0–0.5 bar. In 1 bar CO₂ yields decreased by 20–30%.CH₄ was found to be only slightly inhibitory; the inhibiting effects of 1 bar CH₄ on acetate degradation rates were comparable to the effects of 0.3 bar CO₂. Also gas sparging and rapid mixing had small effects compared with a non-sparged, slowly mixed culture.The redox potential was usually around –200 mV during fermentations and no connection was found between acetate degradation rate, E_h and pCO₂.Acetate and propionate degradation were the reactions most sensitive to pCO₂ and to obtain maximum rates as well as maximum methane yields pCO₂-levels around 0.2 bar were found to be optimal.     

Comparative study of riboflavin production from two microorganisms: Eremothecium ashbyii and Ashbya gossypii

The conversion of the various substrates to riboflavin via fermentation by Eremothecium ashbyii and Ashbya gossypii was investigated. Optimum operation temperature and initial pH of the medium were determined as 30°C and 6.5 respectively for both microorganisms. In order to examine the effects of different substrates and their initial concentrations, glucose, glycerol, sunflower oil, whey and various combinations of these were utilized in the experiments. Maximum specific growth rates and riboflavin yields were obtained in the media which contained glucose and sunflower oil together as the substrate.     

Comparative analysis of genes encoding methyl coenzyme M reductase in methanogenic bacteria

Summary The sequence of the gene cluster encoding the methyl coenzyme M reductase (MCR) in Methanococcus voltae was determined. It contains five open reading frames (ORF), three of which encode the known enzyme subunits. Putative ribosome binding sites were found in front of all ORFs. They differ in their degrees of complementarity to the 3 end of the 16 S rRNA, which is discussed in terms of different translation efficiencies of the respective genes. The codon usage bias is different in the subunit encoding genes compared with the two other ORFs in the cluster and two other known genes of Mc. voltae. This is interpreted in terms of increased translational accuracy of the highly expressed MCR subunit genes. The derived polypeptide sequences encoded by the five ORFs of the MCR cluster were compared to those of the respective genes in Methanobacterium thermoautotrophicum Marburg and Methanosarcina barkeri. Conserved regions were detected in the enzyme subunits, which are candidates for factor binding domains. Conserved hydrophobic sequences found in the and subunits are discussed with respect to the membrane association of the enzyme.