Biogeography of sulfate-reducing prokaryotes in river floodplains

In this study, a large-scale field survey was conducted to describe the biogeography of sulfate-reducing prokaryotes (SRPs) in river floodplains. Fingerprints obtained with three methods, i.e. 16S rRNA gene-based oligonucleotide microarray, dsrB-based denaturing gradient gel electrophoresis (DGGE) and polar lipid-derived fatty acid (PLFA) analyses, were used as a proxy to describe the SRPs community diversity. Each set of profiles was subjected to a combined multivariate/correlation analysis in order to compare SRP community profiles and to highlight the environmental variables influencing the SRPs distribution along environmental gradients. Floodplain soils harbored distinct SRP communities displaying biogeographic patterns. Nearly all profiles from the tidal sites consistently separated from the nontidal sites, independently from the screening method and the multivariate statistics used. The distribution of the microarray/DGGE/PLFA-based fingerprints in the principal component plots could be correlated to eight soil variables, i.e. soil organic matter, total nitrogen, total phosphorous and total potassium, and extractable ammonium, nitrate, phosphate and sulfate, as well as seven pore water variables, i.e. phosphate, sulfate, sulfide, chloride, sodium, potassium and magnesium ions. Indication of a salinity- and plant nutrient-dependent distribution of SRPs related to Desulfosarcina, Desulfomonile and Desulfobacter was suggested by microarray, DGGE and PLFA analyses.     

Phylogenetic and functional diversity of bacteria in biofilms from metal surfaces of an alkaline district heating system

District heating systems (DHS) are extreme aqueous environments characterized by high temperatures, high pH (9.5-10.0), and low nutrient availability. Culture-independent and culture-dependent techniques showed that DHS may nevertheless harbour geno- and phenotypically diverse bacterial biofilm communities. Approximately 50% of the cells in biofilms growing on mild steel coupons in rotortorque reactors connected to the return line (40 degrees C) of a Danish DHS were detectable by FISH analysis and thus were probably metabolically active. A bacterial 16S rRNA gene clone library generated from the biofilms was dominated by proteobacterial phylotypes (closely related to known aerobic species) and by phylotypes affiliated to the anaerobic class Clostridia. Anoxic enrichment cultures derived from biofilms primarily contained 16S rRNA gene and dsrAB (encoding major subunits of dissimilatory sulfite reductase) phylotypes affiliated to the latter class. Alkalitolerant and neutrophilic anaerobic bacteria were isolated from the DHS, including novel Gram-positive and deltaproteobacterial sulfate-reducers and sulfite-reducers constituting novel Gram-positive lineages. In total, 39 distinct 16S rRNA gene phylotypes representing ten classes were identified. The detection of several alkalitolerant, sulfide-producing, and, thus, potentially biocorrosive species underlines the need to maintain a high water quality in the DHS in order to prevent the proliferation of these species.     

Monitoring exogenous and indigenous bacteria by PCR-DGGE technology during the process of microbial enhanced oil recovery

A field experiment was performed to monitor changes in exogenous bacteria and to investigate the diversity of indigenous bacteria during a field trial of microbial enhanced oil recovery (MEOR). Two wells (26-195 and 27-221) were injected with three exogenous strains and then closed to allow for microbial growth and metabolism. After a waiting period, the pumps were restarted and the samples were collected. The bacterial populations of these samples were analyzed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rRNA fragments. DGGE profiles indicated that the exogenous strains were retrieved in the production water samples and indigenous strains could also be detected. After the pumps were restarted, average oil yield increased to 1.58 and 4.52 tons per day in wells 26-195 and 27-221, respectively, compared with almost no oil output before the injection of exogenous bacteria. Exogenous bacteria and indigenous bacteria contributed together to the increased oil output. Sequence analysis of the DGGE bands revealed that Proteobacteria were a major component of the predominant bacteria in both wells. Changes in the bacteria population in the reservoirs during MEOR process were monitored by molecular analysis of the 16S rRNA gene sequence. DGGE analysis was a successful approach to investigate the changes in microorganisms used for enhancing oil recovery. The feasibility of MEOR technology in the petroleum industry was also demonstrated.     

Ammonia biofiltration and community analysis of ammonia-oxidizing bacteria in biofilters

Biological removal of ammonia was investigated using compost and sludge as packing materials in laboratory-scale biofilters. The aim of this study is to characterize the composition of ammonia-oxidizing bacteria (AOB) in two biofilters designed to remove ammonia. Experimental tests and measurements included analysis of removal efficiency and metabolic products. The inlet concentration of ammonia applied was 20–100 mg m⁻³. Removal efficiencies of BFC and BFS were in the range of 97–99% and 95–99%, respectively. Periodic analysis of the biofilter packing materials showed ammonia was removed from air stream by nitrification and by the improved absorption of NH₃ in the resultant acidity. Nitrate was the dominant product of NH₃ transformation. Changes in the composition of AOB were examined by using nested PCR, denaturing gradient gel electrophoresis (DGGE) and sequencing of DGGE bands. DGGE analysis of biofilter samples revealed that shifts in the community structure of AOB were observed in the experiment; however, the idle phase did not cause the structural shift of AOB. Phylogenetic analysis revealed the population of AOB showed Nitrosospira sp. remains the predominant population in BFC, while Nitrosomonas sp. is the predominant population in BFS.     

云冈石窟石质文物表面及周边岩石样品中微生物群落分析

【目的】通过对云冈石窟石质文物表面及云冈石窟周边岩石样品中微生物的研究,建立可用于快速检测石质文物中微生物的方法。【方法】选取云冈石窟石质样品和云冈石窟周边岩石样品作为研究对象,应用PCR-DGGE技术对样品中的微生物群落结构进行了分析研究。【结果】根据系统发育树聚类分析,可以得出云冈石窟中检测出的微生物主要分为四大类群:γ-变形菌纲、鞘脂杆菌门、α-变形菌纲和放线菌纲;根据GenBank数据库中的序列比对结果,可以知道云冈石窟周边类似岩石样品中的微生物主要属于γ-变形菌纲、厚壁菌门和α-变形菌纲等。【结论】本实验成功检测出云冈石窟石质样品表面及云冈石窟周边岩石样品中的微生物类群,为云冈石窟的保护工作提供了有力依据,同时也证明了DEEG和分子克隆技术相结合的方法是检测石质文物中微生物群落结构的一种可操作性强、快速、准确的检测手段。     

Rapid specific detection and quantification of bacteria and archaea involved in mineral sulfide bioleaching using real-time PCR

A SybrGreen real-time PCR assay was developed to detect and quantify both total and selected 16S rDNA species of bacteria and archaea involved in the bioleaching of metals from sulfide ores. A set of specific and universal primers based on 16S rDNA sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of Acidianus brierleyi, Sulfolobus sp., Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, Acidithiobacillus caldus, and Leptospirillum ferrooxidans. An artificial sequence based on 16S rDNA was constructed to quantify total 16S rDNA in mixed DNA samples. The real-time PCR assay was further validated using a mixture of 16S rDNA amplicons derived from the six different species, each added at a known amount. Finally, the real-time PCR assay was used to monitor the change of 16S rDNA copies of four bioleaching strains inoculated into chalcopyrite airlift column reactors operated at different temperatures. The growth dynamics of these strains correlated well with the expected effects of temperature in the chalcopyrite-leaching environment. The suitability of this method for monitoring microbial populations in industrial bioleaching environments is discussed.     

Identification and quantification of arsC genes in environmental samples by using real-time PCR

The arsC gene is responsible for the first step in arsenate biotransformation encoding the enzyme arsenate reductase. The quantitative real-time PCR method was developed to quantify the abundance of the arsC genes in environmental samples contaminated with arsenic. Two sets of primers that showed high specificity for the target arsC gene were designed based on consensus sequences from 13 bacterial species. The arsC gene was used as an external standard instead of total DNA in the calibration curve for real-time PCR, which was linear over six orders of magnitude and the detection limit was estimated to be about three copies of the gene. Soil samples from arsenic contaminated sites were screened for arsC genes by using PCR and showed the presence of this gene. The copy numbers of the gene ranging from 0.88 x 10(4) to 1.56 x 10(5) per ng total DNA were found in eight arsenic contaminated samples. Soil samples from a bioreactor containing pulp mill biomass and high concentration of arsenate showed a tenfold higher count of arsC gene copies than soil samples collected underground from an arsenic-rich gold mine.     

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples

PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.     

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

To study the structure of microbial communities in the biological hydrogen production reactor and determine the ecological function of hydrogen producing bacteria, anaerobic sludge was obtained from the continuous stirred tank reactor (CSTR) in different periods of time, and the diversity and dynamics of microbial communities were investigated by denaturing gradient gel electrophoresis (DGGE). The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day, and the ethanol type fermentation was established. After 28 days the structure of microbial community became stable, and the climax community was formed. Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria (Clostridium sp. andEthanologenbacterium sp.), β-proteobacteria (Acidovorax sp.), γ-proteobacteria (Kluyvera sp.), Bacteroides (uncultured bacterium SJA-168), and Spirochaetes (uncultured eubacterium E1-K13), respectively. The hydrogen production rate increased obviously with the increase ofEthanologenbacterium sp.,Clostridium sp. and uncultured Spirochaetes after 21 days, meanwhile the succession of ethanol type fermentation was formed. Throughout the succession the microbial diversity increased however it decreased after 21 days. Some types ofClostridium sp.Acidovorax sp.,Kluyvera sp., and Bacteroides were dominant populations during all periods of time. These special populations were essential for the construction of climax community. Hydrogen production efficiency was dependent on both hydrogen producing bacteria and other populations. It implied that the cometabolism of microbial community played a great role of biohydrogen production in the reactors.     

PCR-DGGE方法分析原油储层微生物群落结构及种群多样性

使用基于 16 S r DNA的 PCR- DGGE(变性梯度凝胶电泳 )图谱分析结合条带割胶回收 DNA进行序列分析 ,对新疆克拉玛依油田一中区注水井 (12 # 9- 11)和与该注水井相应的两个采油井 (12 # 9- 9S、13# 11- 8)井口样品微生物群落的多样性进行了比较并鉴定了部分群落成员。 DGGE图谱聚类分析表明注水井与两油井微生物群落的相似性分别为 30 %和 2 0 % ,而两油井间微生物群落结构的相似性为 5 4 %。DGGE图谱中优势条带序列分析表明注水井样品和油井样品中的优势菌群为未培养的环境微生物 ,它们与数据库中 α、γ、δ、ε变形杆菌 (Proteobacteria)和拟杆菌 (Bacteroidetes)有很近的亲缘关系。 DGGE与分子克隆相结合的分子生物学方法在研究微生物提高原油采收率 (MEOR)机理 ,以及指导 MEOR在油田生产中的应用有着重要的意义     

油气田土壤甲烷氧化菌实时荧光定量PCR检测技术的 建立与应用

【目的】建立快速检测甲烷氧化菌含量的SYBR GreenⅠ实时荧光定量PCR技术,用于油气微生物勘探。【方法】以含有甲烷氧化菌功能基因pmoA片段的重组质粒为标准品,优化实验条件,建立标准曲线,进行敏感性、重复性和特异性评价,并将该技术用于实际样品的检测。【结果】该技术标准曲线的相关系数R2为0.999 9,扩增效率为99.976%,检测范围为3.897×101-3.897×109 copies/μL,检出限约为40 copies/μL,重复性实验中CT值的变异系数优于3%,对12种非甲烷氧化菌均没有扩增,显示该技术具有很好的敏感性、重复性和特异性。利用该技术对气田、油田和非油气田土样进行了检测,发现气田具有明显的异常高值。【结论】为油气田的勘探建立了一种高效、特异、灵敏、准确的甲烷氧化菌荧光定量PCR检测技术,同时为其它指示菌检测技术的建立提供了参考。     

Enumeration and detection of acetic acid bacteria by real-time PCR and nested PCR

Acetic acid bacteria play a negative role in wine making because they increase the volatile acidity of wines. They can survive in the various phases of alcoholic fermentation and it is very important to control their presence and ulterior development. The main objective of the present work is to test fast, sensitive and reliable techniques such as real-time PCR (rt-PCR) and nested PCR for enumerating and detecting the presence of this bacterial group without plating. Primers were designed on the basis of the available 16S rRNA gene sequences and tested successfully with reference acetic acid bacteria strains. The usefulness of rt-PCR was demonstrated by comparing the results with traditional techniques (colony and microscope counting). The results were similar with all the techniques. Optimized rt-PCR enabled numbers between 10(7) and 10(1) cells mL(-1) to be enumerated, while nested PCR detected less than 10 cells mL(-1). Although this latter technique cannot be used for enumeration, it has several advantages in routine laboratory analysis.     

耕作和施肥对不同深度黑土中细菌群落结构的影响

【目的】为探讨耕作和施加有机肥、化肥对黑土表层(0-30cm)、中层(100-130cm)及深层(200-230cm)细菌群落结构的影响,【方法】应用DGGE技术对相应土层中细菌群落结构进行了解析。【结果】结果表明,与对照相比,耕作和施加有机肥、化肥对表层黑土细菌群落结构影响较大,二者差异度为4%;而对中层和深层细菌群落结构影响较小,二者差异度为2%。对于细菌群落结构的垂向变化,中层和深层中细菌群落结构的相似性远远高于同表层的相似性。【结论】可见,耕作和施加有机肥、化肥仅对黑土表层土壤(0-30cm)具有一定的影响,而对100cm以下土壤细菌群落影响较小,细菌群落随土壤深度不同的垂向变化要远高于土壤管理措施造成的影响。     

Detection of stable reference genes for real-time PCR analysis in schizophrenia and bipolar disorder

Gene expression studies using postmortem human brain tissue are a common tool for studying the etiology of psychiatric disorders. Quantitative real-time PCR (qPCR) is an accurate and sensitive technique used for gene expression analysis in which the expression level is quantified by normalization to one or more reference genes. Therefore, accurate data normalization is critical for validating results obtained by qPCR. This study aimed to identify genes that may serve as reference in postmortem dorsolateral-prefrontal cortices (Brodmann’s area 46) of schizophrenics, bipolar disorder (BPD) patients, and control subjects. In the exploratory stage of the analysis, samples of four BPD patients, two schizophrenics, and two controls were quantified using the TaqMan Low Density Array endogenous control panel, containing assays for 16 commonly used reference genes. In the next stage, six of these genes (TFRC, RPLP0, ACTB, POLR2a, B2M, and GAPDH) were quantified by qPCR in 12 samples of each clinical group. Expressional stability of the genes was determined by GeNorm and NormFinder. TFRC and RPLP0 were the most stably expressed genes, whereas the commonly used 18S, POLR2a, and GAPDH were the least stable. This report stresses the importance of examining expressional stability of candidate reference genes in the specific sample collection to be analyzed.     

Evaluation of reference genes for quantitative real-time PCR in the brain,pituitary, and gonads of songbirds

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.     

三江源地区高寒草甸土壤氨氧化细菌多样性研究

目的:利用16S rDNA-PCR-DGGE方法首次对青藏高原核心区三江源自然保护区的高寒草甸样地的土壤氨氧化细菌进行了多样性研究。方法:从土壤中抽提微生物总DNA,运用巢式PCR扩增氨氧化细菌16S rRNA基因的V3可变区,结合DGGE(denaturing gradient gel electrophoresis)技术分析样品氨氧化细菌群落组成,并利用相关性分析探讨了群落结构多样性与环境因子的联系。结果:4个样地(玉树S1;囊谦S2;治多S3和S4)共检测到14个多态性条带,多样性指数为1.69～2.00。相关性分析显示多样性指数与海拔、C/N呈负相关,与NO3-呈显著正相关。结论:海拔、C/N及NO3-可能是影响土壤氨氧化细菌多样性的重要因子。     

Molecular characterization of sulfate-reducing bacteria in anaerobic hydrocarbon-degrading consortia and pure cultures using the dissimilatory sulfite reductase (dsrAB) genes

The characterization of sulfate-reducing bacteria (SRBs) is presented using the dissimilatory sulfite reductase (dsrAB) gene from various samples capable of mineralizing petroleum components. These samples include several novel, sulfidogenic pure cultures which degrade alkanes, toluene, and tribromophenol. Additionally, we have sulfidogenic consortia which re-mineralize benzene, naphthalene, 2-methylnaphthalene, and phenanthrene as a sole carbon source. In this study, 22 new dsrAB genes were cloned and sequenced. The dsrAB genes from our pollutant-degrading cultures or consortia were distributed among known SRBs and previously described dsrAB environmental clones, suggesting that many biodegradative SRBs are phylogenetically distinct and geographically wide spread. Specifically, the same dsrAB gene was discovered in independently established consortia capable of benzene, phenanthrene, and methylnaphthalene degradation, indicating that this particular SRB may be a key player in anaerobic degradation of hydrocarbons in the environment.