磁性纳米颗粒介导分离技术筛选土壤中多氯联苯降解菌及其降解特性

【背景】磁性纳米颗粒介导分离(magnetic nanoparticle-mediated isolation, MMI)技术是近年来发展起来的一种无须底物标记就能从复杂菌群中分离活性功能微生物的方法,目前尚无研究报道该技术应用于难降解污染物3,3′,4,4′-四氯联苯(3,3′,4,4′-tetrachlorobiphenyl, PCB77)。【目的】从土壤中筛选PCB77活性降解菌并研究其污染物降解特性。【方法】利用磁性纳米颗粒(magnetic nanoparticles, MNPs)富集原位活性PCB77降解菌群,通过高通量测序分析细菌群落变化,经平板筛选得到PCB77降解菌,并研究其对多氯联苯和多溴联苯醚的降解特性。【结果】基于MMI技术获取的富集培养液能够高效地转化PCB77,与对照组相比底物降解效率从6%提升至79.3%,同时该富集培养液中细菌物种多样性显著降低,群落组成发生明显变化。从对照组和MMI处理组中分别筛选到PCB77降解菌红球菌CT2和类芽孢杆菌MT2,发现红球菌为对照组中唯一的优势物种,而MMI处理组的优势物种由红球菌和类芽孢杆菌共同组成。菌株MT2对PCB...     

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

AIMS: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.     

Extensive profiling of a complex microbial community by high-throughput sequencing

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.     

Taxonomical and functional microbial community selection in soybean rhizosphere

This study addressed the selection of the rhizospheric microbial community from the bulk soil reservoir under agricultural management of soybean in Amazon forest soils. We used a shotgun metagenomics approach to investigate the taxonomic and functional diversities of microbial communities in the bulk soil and in the rhizosphere of soybean plants and tested the validity of neutral and niche theories to explain the rhizosphere community assembly processes. Our results showed a clear selection at both taxonomic and functional levels operating in the assembly of the soybean rhizosphere community. The taxonomic analysis revealed that the rhizosphere community is a subset of the bulk soil community. Species abundance in rhizosphere fits the log-normal distribution model, which is an indicator of the occurrence of niche-based processes. In addition, the data indicate that the rhizosphere community is selected based on functional cores related to the metabolisms of nitrogen, iron, phosphorus and potassium, which are related to benefits to the plant, such as growth promotion and nutrition. The network analysis including bacterial groups and functions was less complex in rhizosphere, suggesting the specialization of some specific metabolic pathways. We conclude that the assembly of the microbial community in the rhizosphere is based on niche-based processes as a result of the selection power of the plant and other environmental factors.     

Design of long oligonucleotide probes for functional gene detection in a microbial community

MOTIVATION: Analysis of the functions of microorganisms and their dynamics in the environment is essential for understanding microbial ecology. For analysis of highly similar sequences of a functional gene family using microarrays, the previous long oligonucleotide probe design strategies have not been useful in generating probes. RESULTS: We developed a Hierarchical Probe Design (HPD) program that designs both sequence-specific probes and hierarchical cluster-specific probes from sequences of a conserved functional gene based on the clustering tree of the genes, specifically for analyses of functional gene diversity in environmental samples. HPD was tested on datasets for the nirS and pmoA genes. Our results showed that HPD generated more sequence-specific probes than several popular oligonucleotide design programs. With a combination of sequence-specific and cluster-specific probes, HPD generated a probe set covering all the sequences of each test set. AVAILABILITY: http://brcapp.kribb.re.kr/HPD/     

Development of functional gene microarrays for microbial community analysis

Functional gene arrays (FGAs) are a special type of microarrays containing probes for key genes involved in microbial functional processes, such as biogeochemical cycling of carbon, nitrogen, sulfur, phosphorus and metals, virulence and antibiotic resistance, biodegradation of environmental contaminants, and stress responses. FGAs have been demonstrated to be a specific, sensitive, and quantitative tool for rapid analysis of microbial communities from different habitats, such as waters, soils, extreme environments, bioreactors, and human microbiomes. In this review, we first summarize currently reported FGAs, and then focus on the FGA development. We will also discuss several key issues of FGA technology as well as challenges and directions in future FGA development.     

Plant functional group richness-affected microbial community structure and function in a full-scale constructed wetland

We examined the linkages between plant functional group richness and microbial community structure and functions in a full-scale vertical flow constructed wetland (VFCW), where five plant functional group richness levels (including zero (control), one, two, three and four functional groups) were applied. Most diagnostic phospholipid fatty acid (PLFA) abundances and enzyme activities were higher in planted treatments than in the control (p < 0.05). Among the diagnostic PLFAs determined, only the fungal PLFA (18:2ω6,9) abundance was related to plant functional group richness level (p < 0.05). For the enzyme activities determined, dehydrogenase, invertase, urease and acid phosphatase activities responded positively to plant functional group richness (p < 0.05). Redundancy analysis (RDA) identified that the Gram-positive and Gram-negative bacteria, actinomycetes and fungi abundances affected enzyme activities. Principal component analyses (PCAs) revealed that the enzyme profiles had greater resolving power in distinguishing plant functional group richness than the PLFA profiles. We conclude that plant functional group richness was closely linked to microbial community structure and functions in the full-scale VFCW we studied.     

复合菌对十溴联苯醚的降解特性研究

【目的】利用6株十溴联苯醚(decabromodiphenyl ether, BDE-209)降解细菌,探究复合菌对BDE-209的降解特性和降解路径,为BDE-209污染环境的生物修复提供科学依据。【方法】利用高效液相色谱法测定BDE-209的浓度,通过液相色谱-质谱联用仪分析鉴定BDE-209降解产物。【结果】短芽孢杆菌属(Achromobacter sp.) M1和无色杆菌属(Achromobacter sp.) M2的组合对BDE-209的降解效果最好,在30 ℃、pH值7.0、接种量15%的条件下,120 h后10 mg/L BDE-209的降解效率可达87.7%。相比于单一菌株,复合菌M(1+2)可以更有效、更快地降解BDE-209。在0.5-10 mg/L范围内,复合菌M(1+2)对BDE-209的降解率随着BDE-209初始浓度的增大而增大。通过液相色谱-质谱联用仪(liquid chromatograph-mass spectrometer, LC-MS)检测到11种BDE-209微生物降解产物,复合菌M(1+2)通过脱溴、羟基化、去质子化、醚键断裂和开环等反应对BDE-209进行降解。【结论】复合菌M(1+2)对BDE-209具有良好的降解能力,研究结果为进一步提高微生物对BDE-209污染环境的生物修复能力提供了良好的微生物资源。     

杀虫剂类POPs对土壤中微生物群落多样性的影响

农药类持久性有机污染物(POPs)如DDT和HCH在我国2 0世纪5 0年代到80年代曾广泛使用,在停止使用2 0 a后,在土壤中仍然可以检测到DDT和HCH的残留。利用BIOL OG微平板研究土壤微生物群落功能多样性,意在反映有机氯杀虫剂类POPs对土壤微生物群落多样性的影响。结果表明,加了HCH后土壤微生物群落的颜色平均变化值(AWCD)的变化速度和最终能达到的AWCD值要高于空白土壤,并且随着农药浓度的加大,AWCD值的变化速率也越来越快,最终能达到的最大值也呈比例增大。加了DDT后的土壤与空白土壤的AWCD变化速度和程度相差不大。方差分析结果表明:空白土壤、HCH0 .5mg/kg、HCH1.5 mg/kg各处理间土壤的AWCD值有显著性差异(p<0 .0 1) ,空白土壤、DDT0 .5 m g/kg、DDT1.5 m g/kg各处理间土壤的AWCD值达不到显著性差异的水平(p>0 .0 5 ) ,表层土壤的AWCD值要高于第2层土壤(p<0 .0 1)。从多样性指数的变化来看,当加入到土壤中的DDT和HCH含量稍低时,微生物会利用农药为碳源进行分解作用,从而刺激了微生物的生长,这时表现出丰富度、均匀性和多样性都呈增长趋势。但当农药的浓度进一步加大时,反而会抑制某些种的微生物生长,另外一些种则对加入到土壤中的农药有一定的耐受性,从而表现出群落的均匀性下降,而丰富度升高。在相同施用浓     

High-resolution metagenomics targets specific functional types in complex microbial communities

Most microbes in the biosphere remain unculturable. Whole genome shotgun (WGS) sequencing of environmental DNA (metagenomics) can be used to study the genetic and metabolic properties of natural microbial communities. However, in communities of high complexity, metagenomics fails to link specific microbes to specific ecological functions. To overcome this limitation, we developed a method to target microbial subpopulations by labeling DNA through stable isotope probing (SIP), followed by WGS sequencing. Metagenome analysis of microbes from Lake Washington in Seattle that oxidize single-carbon (C1) compounds shows specific sequence enrichments in response to different C1 substrates, revealing the ecological roles of individual phylotypes. We also demonstrate the utility of our approach by extracting a nearly complete genome of a novel methylotroph, Methylotenera mobilis, reconstructing its metabolism and conducting genome-wide analyses. This high-resolution, targeted metagenomics approach may be applicable to a wide variety of ecosystems.     

Biodiversity and multifunctionality in a microbial community: a novel theoretical approach to quantify functional redundancy

Ecosystems have a limited buffering capacity of multiple ecosystem functions against biodiversity loss (i.e. low multifunctional redundancy). We developed a novel theoretical approach to evaluate multifunctional redundancy in a microbial community using the microbial genome database (MBGD) for comparative analysis. In order to fully implement functional information, we defined orthologue richness in a community, each of which is a functionally conservative evolutionary unit in genomes, as an index of community multifunctionality (MF). We constructed a graph of expected orthologue richness in a community (MF) as a function of species richness (SR), fit the power function to SR (i.e. MF = cSR^a), and interpreted the higher exponent a as the lower multifunctional redundancy. Through a microcosm experiment, we confirmed that MF defined by orthologue richness could predict the actual multiple functions. We simulated random and non-random community assemblages using full genomic data of 478 prokaryotic species in the MBGD, and determined that the exponent in microbial communities ranged from 0.55 to 0.75. This exponent range provided a quantitative estimate that a 6.6–8.9% loss limit in SR occurred in a microbial community for an MF reduction no greater than 5%, suggesting a non-negligible initial loss effect of microbial diversity on MF.     

Changes in functional abilities of the microbial community during composting of manure

The objective of this study was (a) to detect changes of the functional abilities of the microflora during composting of manure as a result of windrow turning frequency and (b) to detect differences between distinct zones within the windrows. Biolog GN microtiter plates containing 95 different carbon sources were inoculated with diluted suspensions of compost material containing 15,000 microorganisms per well (120 l). We found a dramatic shift in functional microbial community structure during the 8-week composting process. The shift was more rapid when the compost windrows were turned. The substrate use pattern in the outer, well-aerated zone of the unturned windrow was similar to that of the turned windrows. Microbial biomass and respiration decreased more rapidly in the turned than in the unturned windrows, indicating a different pace of compost maturation. The data suggest that the Biolog assay may be a suitable approach to determine compost maturity. Correspondence to: H. Insam     

Metasecretome-selective phage display approach for mining the functional potential of a rumen microbial community

Background

In silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects.

Results

By combining secretome-selective phage display and next-generation sequencing, we focused the sequence analysis of complex rumen microbial community on the metasecretome component of the metagenome. This approach achieved high enrichment (29 fold) of secreted fibrolytic enzymes from the plant-adherent microbial community of the bovine rumen. In particular, we identified hundreds of heretofore rare modules belonging to cellulosomes, cell-surface complexes specialised for recognition and degradation of the plant fibre.

Conclusions

As a method, metasecretome phage display combined with next-generation sequencing has a power to sample the diversity of low-abundance surface and secreted proteins that would otherwise require exceptionally large metagenomic sequencing projects. As a resource, metasecretome display library backed by the dataset obtained by next-generation sequencing is ready for i) affinity selection by standard phage display methodology and ii) easy purification of displayed proteins as part of the virion for individual functional analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-356) contains supplementary material, which is available to authorized users.     

Comparison of CE-SSCP and DGGE for monitoring a complex microbial community remediating mine drainage

Capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) is a promising high-throughput tool for profiling complex bioremediation communities, but has not been well characterized with respect to other methods such as denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to compare CE-SSCP with DGGE with respect to: 1) overall representation of the community in terms of the dominant species identified and corresponding Shannon diversity indices; 2) reproducibility and resolution; and 3) artifacts, using a complex sulfate-reducing community remediating mine drainage as a model system. Some of the dominant microorganisms were detected by both methods, but there were also differences in the reported community compositions, and more phylogenetic groups were detected by CE-SSCP. CE-SSCP Shannon diversity indices were slightly higher than those determined from DGGE data, and differed in terms of the time point at which the community was reported to have the highest diversity. Both methods had high reproducibility, but CE-SSCP resolution was higher in terms of the total number of peaks resolved, reduced co-migration of distinct DNA sequences, and length and legibility of the DNA sequencing data of clones used to identify peaks. Ten double bands in the same lane representing the same species were found by DGGE, whereas only one such artifact was observed by CE-SSCP. Finally, less overall sample preparation and analysis time was required for CE-SSCP than for DGGE. The results suggest that CE-SSCP offers several advantages over DGGE, especially for high-throughput monitoring.     

Succession of sulfur-oxidizing bacteria in the microbial community on corroding concrete in sewer systems

Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O3(2-)). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below.