黄海北部不同站位海洋细菌群落分布特征

[目的]为揭示北黄海不同海域中真细菌群落分布的差异,[方法]采用16s rRNA基因文库和变性梯度凝胶电泳(DGGE)技术,对远海和近海两个站位的沉积物和水体中细菌群落特征进行了解析和评价.[结果]文库分析揭示海水及沉积物中微生物种类丰富,存在大量未被认知的类群.各站位中主要为变形菌门(Proteobacteria),沉积物中γ-Proteobacteria和δ-Proteobacteria亚门占优势,水中则以α-Proteobacteria亚门占优势,但各亚门微生物在两个站位中存在明显系统发育学分歧.DGGE图谱聚类分析显示,近海沉积物和海水中细菌群落优势类群相似性很高,而远海沉积物和海水中则相似性很低.[结论]研究结果表明,微生物种类在不同地理位置和生存介质中存在明显差异,环境因素对微生物的分布起主导作用.     

不同发育阶段杉木人工林对土壤微生物群落结构的影响

采用变性梯度凝胶电泳技术(DGGE),分析土壤细菌16S rDNA和土壤真菌28SrDNA特异性片段多态性,研究了不同发育阶段杉木人工林对土壤微生物群落结构的影响.结果表明:土壤微生物群落结构随着杉木人工林的发育年龄而改变,杉木人工林土壤微生物群落多样性和丰富度随杉木生长发育显著增加(P<0.05),但均显著低于次生阔叶林(P<0.05);聚类分析表明,不同发育阶段杉木人工林土壤真菌群落相似性均<60%,而土壤细菌群落相似性最高可达65%,由此可推测不同发育阶段杉木人工林土壤真菌群落结构变化较土壤细菌群落结构变化剧烈;相关性分析表明,不同发育阶段杉木人工林土壤速效氮、碳氮比与土壤微生物群落多样性显著相关(P<0.05).本研究表明,长期种植单一杉木人工林能够通过改变土壤理化性质来影响土壤微生物群落组成,进而影响森林生态系统养分循环,导致人工林林分生产力下降.     

不同DGGE谱带信息提取方法对分析结果的影响

自1993年Muyzer,et al.[1]将变性梯度凝胶电泳技术(Denaturing gradient gel electrophoresis,DGGE)引入到微生物生态学研究以来,DGGE已被广泛应用于各种生态系统(如淡水、海洋、土壤、动物消化道等)的微生物群落结构分析[2—6]。但对于DGGE凝胶的分析至今仍没有统一的方法,     

阿特拉津及其降解菌的使用对土壤微生物群落的影响

比较了阿特拉津及降解菌株BTAH1的使用对土壤微生物的影响.结果表明,在实验周期内阿特拉津对土壤微生物的代谢作用有较明显的刺激作用,与空白土壤(未施用阿特拉津和降解菌)相比,对照土壤(施用50mg·kg^-1土阿特拉津)呼吸强度显著增加,且土壤中的阿特拉津浓度对土壤NH₄⁺-N和NO₃^--N浓度的影响显著.降解菌BTAH1可在1周内降解土壤中98%以上的阿特拉津,从而使土壤呼吸强度有所下降,土壤中NH₄⁺-N和NO₃^--N的浓度基本与空白土壤持平,对微生物量C和微生物量N影响不显著;放线菌和真菌数量也基本与空白持平,细菌数量较高.对土壤细菌的16SrDNA文库的ARDRA分析发现,阿特拉津及其降解菌的使用对土壤细菌群落结构有一定程度的影响,阿特拉津的使用会降低细菌群落的多样性,而降解菌的使用会恢复土壤细菌的多样性.     

聚乳酸/聚己二酸-对苯二甲酸丁二酯对土壤细菌群落结构的影响及其降解菌的筛选

【背景】近年来,聚乳酸/聚己二酸-对苯二甲酸丁二酯(polylactide/polybutyleneadipateco-terephthalate,PLA/PBAT)可降解地膜得到了广泛的使用,然而材料使用对土壤微生物的影响却鲜有报道。【目的】以新疆土壤为例,研究PLA/PBAT地膜的使用对土壤中微生物群落结构的影响;并从土壤中筛选可降解PLA/PBAT的菌株,为土壤环境的原位修复提供技术支持。【方法】采用高通量测序的方法对比使用PLA/PBAT地膜前后土壤中细菌群落的结构变化;采用筛选培养基从土壤中分离、鉴定PLA/PBAT的降解菌,通过改变不同培养条件研究菌株降解效果。【结果】使用PLA/PBAT地膜后,土壤中酸杆菌门、芽单胞菌门的相对丰度上升,变形菌门、放线菌门的相对丰度下降,这可能是地膜降解过程中其中间产物对土壤pH及微生物的抑制作用所致;并从土壤中分离出一株PLA/PBAT降解菌XJ11,初步鉴定为Delftiatsuruhatensis,在外加1.5%胰蛋白胨的PLA/PBAT (规格1×1×0.05 cm)筛选培养基中,接种菌液1 mL,在pH为7.2、37°C、130 r/min的条件下,7d内PLA/PBAT的降解率可达6.87%。【结论】PLA/PBAT地膜的使用可以改变土壤细菌群落结构,从环境中筛选出高效的PLA/PBAT降解菌成为解决地膜污染的有效措施。     

啶虫脒污染下土壤微生物多样性

避开传统的分离培养过程,采用现代分子生物学方法探讨了杀虫剂啶虫脒污染条件下旱地土壤微生物种群多样性.通过对不同培养时间、不同浓度啶虫脒污染下旱地土壤微生物进行DGGE基因多样性的分子指纹图谱分析,发现随着培养时间不同,各处理之间的土壤微生物基因多样性出现了一定的差异.但在整个试验过程中,正常田间使用浓度（0.5mg kg^-1干土）的啶虫脒对土壤微生物群落的影响不明显,DGGE图谱条带与对照没有明显差异,土壤微生物基因多样性没有明显下降,这说明在旱地中使用正常田间浓度的啶虫脒不会对微生物群落造成较大的影响,高浓度啶虫脒对土壤微生物群落基因多样性有一定的影响,但是影响时间不长.在培养第五周时,浓度为5 mg kg^-1干土的土样出现了特异性条带,为对照所没有,其他处理浓度染色暗淡.经序列比对分析,与来自土壤的Uncultured bacterium具有100﹪的相似率,可能为不可培养或未培养过的细菌种.     

杉木连栽土壤微生物多样性的比较研究

分析了位于湖南会同广坪镇1～4代人工杉木林根际土壤微生物数量变化情况,结果表明,随杉木连栽代数的增加,根际土壤中三大类群微生物数量发生显著变化,细菌和放线菌数量明显减少,真菌数量显著增加.利用PCR和DGGE技术分析了1～4代杉木林根际土壤细菌区系和真菌区系.结果表明,细菌生物多样性在不同连栽代数杉木根际土壤中变化不明显,各代杉木土壤之间细菌的遗传相似性为87%.而真菌随连栽代数的增加,DGGE图谱带逐渐减少,真菌生物多样性降低,各代杉木土壤之间真菌的遗传相似性也较低,仅为45%.对各代杉木土壤主要真菌类群的分析表明,随连栽代数的增加,病原真菌及产毒真菌增加显著.     

应用PCR-DGGE技术研究石莼、网地藻藻际微生物多样性

应用变性凝胶梯度电泳(PCR-DGGE)技术对石莼、网地藻藻际微生物多样性进行研究,并对其进行相似性、未加权聚类分析(UPGMA)及微生物多样性(Shannon)指数分析。结果表明,PCR-DGGE图谱显示,石莼、网地藻藻际微生物DGGE图谱有着明显的差异性,具体表现在条带数及密度值的不同。Quantity one图谱分析表明:石莼藻际微生物16S r RNA基因的V3区共分离得到25条DNA片段,网地藻为16条DNA片段。DGGE相似性及未加权聚类分析表明:网地藻藻际微生物间的相似性为77.78%,石莼藻际微生物细菌群落间的相似性为49.25%。Shannon指数分析表明,石莼藻际微生物多样性指数显著高于网地藻(P0.05)。石莼藻际微生物细菌群落较网地藻丰富。     

三株土壤解磷细菌的分离及其解磷效果分析

旨为解决农业面源污染问题,分离鉴定土壤解磷微生物并开发复合微生物菌剂。以有机磷农药及无机难溶磷作为筛选磷源,对土壤中具有解磷能力的微生物进行分离、鉴定并对其解磷效果进行分析。从土壤中分离得到3株解磷细菌,分别命名为菌株W、Y、B;3个菌株均是革兰氏阴性菌;W菌株对敌百虫的降解能力最强,达到17.39%,B菌株对毒死蜱的降解率最强,为23.06%;3个菌株对固态难溶磷的解磷效果显著,其中B菌株解磷量最高,为96.31 mg/L;复合菌的解磷效果明显优于单菌,另外复合菌对稻田、大棚土壤解磷的促进效果显著,分别增加18.38 mg/L、14.08 mg/L。分离得到3株有效土壤解磷细菌,有一定的有机磷农药降解能力,对无机磷溶解效果较强,构建的复合菌剂对土壤解磷的促进效果显著。     

不同坡向凋落物分解对土壤微生物群落的影响

采用空间与时间序列并用的方法,对贵州茂兰喀斯特森林阳坡和阴坡凋落物的分解特征及土壤微生物进行1年的野外调查和测定,探讨凋落物分解规律及其对土壤微生物群落的影响。凋落物在分解1年后的质量损失率约为72%,在分解过程中的碳、磷元素表现为释放,氮元素表现出富集;土壤微生物各群落磷脂脂肪酸(PLFA)在分解过程中的平均含量表现为:细菌(149.8 nmol/g)放线菌(63.9 nmol/g)真菌(31.3 nmol/g),阳坡和阴坡的微生物总PLFA在180d(559.2 nmol/g)和360d(513.6 nmol/g)的含量显著高于分解前;随分解时间的增加,阳坡细菌特征磷脂脂肪酸18:1ω7c的含量增加最大,其次是cy19:0和真菌特征磷脂脂肪酸18:1ω9c,阴坡土壤微生物PLFA含量表现出减少的趋势,细菌特征脂肪酸18:1ω7c减少量最大,约9.42 nmol/g,其次是i15:0和真菌特征脂肪酸18:1ω9c,约减少4.29—4.86 nmol/g;凋落物养分释放特征与微生物群落间的Spearman相关分析表明,真菌群落与碳元素释放率呈显著正相关关系,细菌群落与凋落物碳、磷元素的释放呈极显著正相关关系,且阳坡凋落物分解对微生物群落的影响高于阴坡。坡向和凋落物养分释放量对喀斯特森林凋落物的分解及土壤微生物群落的影响较显著,特征磷脂脂肪酸18:1ω7c、cy19:0、i15:0和18:1ω9c对环境变化的响应较为敏感。     

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。     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

用PCR-DGGE研究长期施用无机肥对种稻红壤微生物群落多样性的影响

以中国科学院红壤生态试验站的发育于第四纪红粘土的种稻红壤为研究对象,采用PCR-DGGE方法研究了长期施用无机肥对土壤微生物群落多样性的影响。在种植双季稻、连续13a施用不同无机肥后,土壤中细菌、古菌、放线菌和真菌的群落结构发生了较大的变化。未种植水稻的土壤与种稻土壤间四类微生物SSUrDNADGGE带谱相似性只有33%~66%。施磷肥的处理NP、PK、NPK之间微生物群落结构相似性较高,4类微生物的SSUrDNADGGE带谱相似性高达75%~81%。施氮钾肥(NK)、不施肥(CK)处理与施磷肥处理间土壤微生物群落结构的差异较大,其四类微生物的SSUrDNADGGE带谱相似性分别为69%~77%、55%~77%。研究的目的是深入地了解土壤中微生物群落的多样性,为科学施肥、合理利用土壤、保护微生物多样性和实现农业生态系统的可持续发展提供科学依据。     

Identification of cellulolytic bacteria in soil by stable isotope probing

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. ¹³C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the ¹³C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.     

Bioremediation of the tobacco waste-contaminated soil by Pseudomonas sp. HF-1: nicotine degradation and microbial community analysis

The highly effective nicotine-degrading bacterium Pseudomonas sp. HF-1 was augmented into the tobacco waste-contaminated soil to degrade nicotine and evaluate the effect of the bioremediation. Comparing with non-adding (NA) systems, the treatments with addition of strain HF-1 (TA) exhibited considerably stronger pollution disposal abilities and higher stability of pH value and moisture content, especially in groups containing a large quantity of tobacco waste. The denaturing gradient gel electrophoresis (DGGE) profiles showed that the Shannon–Wiener index decreased with increasing wastes in the NA treatments, while a gradual increase was found in the TA groups. A comparison of sequences from DGGE bands demonstrated that there were differences in the dominant microbial species between the two treatments, suggesting that strain HF-1 could persist in the soil and enhance the efficiency of tobacco waste disposal. The results of real-time fluorescence quantitative PCR (RT-qPCR) also indicated that strain HF-1 existed in the TA systems and grew with relative high quantities. In conclusion, the nicotine-degrading strain HF-1 played a leading role in the bioremediation of the tobacco waste-contaminated soil and influenced the dynamics and structure of the microbial community.     

Effect of soil ammonium concentration on N2O release and on the community structure of ammonia oxidizers and denitrifiers

The effect of ammonium addition (6.5, 58, and 395 microg of NH4+-N g [dry weight] of soil(-1)) on soil microbial communities was explored. For medium and high ammonium concentrations, increased N2O release rates and a shift toward a higher contribution of nitrification to N2O release occurred after incubation for 5 days at 4 degrees C. Communities of ammonia oxidizers were assayed after 4 weeks of incubation by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the small subunit of ammonia monooxygenase. The DGGE fingerprints were invariably the same whether the soil was untreated or incubated with low, medium, or high ammonium concentrations. Phylogenetic analysis of cloned PCR products from excised DGGE bands detected amoA sequences which probably belonged to Nitrosospira 16S rRNA clusters 3 and 4. Additional clones clustered with Nitrosospira sp. strains Ka3 and Ka4 and within an amoA cluster from unknown species. A Nitrosomonas-like amoA gene was detected in only one clone. In agreement with the amoA results, community profiles of total bacteria analyzed by terminal restriction fragment length polymorphism (T-RFLP) showed only minor differences. However, a community shift occurred for denitrifier populations based on T-RFLP analysis of nirK genes encoding copper-containing nitrite reductase with incubation at medium and high ammonia concentrations. Major terminal restriction fragments observed in environmental samples were further described by correspondence to cloned nirK genes from the same soil. Phylogenetic analysis grouped these clones into clusters of soil nirK genes. However, some clones were also closely related to genes from known denitrifiers. The shift in the denitrifier community was probably the consequence of the increased supply of oxidized nitrogen through nitrification. Nitrification activity increased upon addition of ammonium, but the community structure of ammonium oxidizers did not change.     

Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.     

Impact of pesticides on soil microbiological parameters and possible bioremediation strategies

Intensive agriculture is spectacularly successful since last couple of decades due to the inputs viz; fertilizers and pesticides along with high yielding varieties. The mandate for agriculture development was to feed and adequate nutrition supply to the expanding population by side the agriculture would be entering to into new area of commercial and export orientation. The attention of public health and proper utilization natural resources are also the main issues related with agriculture development. Concern for pesticide contamination in the environment in the current context of pesticide use has assumed great importance [1]. The fate of the pesticides in the soil environment in respect of pest control efficacy, non-target organism exposure and offsite mobility has been given due consideration [2]. Kinetics and pathways of degradation depend on abiotic and biotic factors [6], which are specific to a particular pesticide and therefore find preference. Adverse effect of pesticidal chemicals on soil microorganisms [3], may affect soil fertility [4] becomes a foreign chemicals major issue. Soil microorganisms show an early warning about soil disturbances by foreign chemicals than any other parameters. But the fate and behavior of these chemicals in soil ecosystem is very important since they are degraded by various factors and have the potential to be in the soil, water etc. So it is indispensable to monitor the persistence, degradation of pesticides in soil and is also necessary to study the effect of pesticide on the soil quality or soil health by in depth studies on soil microbial activity. The removal of metabolites or degraded products should be removed from soil and it has now a day’s primary concern to the environmentalist. Toxicity or the contamination of pesticides can be reduced by the bioremediation process which involves the uses of microbes or plants. Either they degrade or use the pesticides by various co metabolic processes.     

一株溴氰菊酯降解菌的分离鉴定及其降解条件优化

【背景】拟除虫菊酯类农药的降解已成为食品安全和环境卫生领域的研究热点,而生物降解被认为是一种绿色高效的解决方法。【目的】从长期受拟除虫菊酯类农药污染的草莓根系土壤分离一株溴氰菊酯(deltamethrin,DM)降解菌,并优化其培养基及降解条件,从而提高DM降解菌的降解效率。【方法】采用富集驯化、分离纯化法筛选DM降解菌,通过形态学和生理生化特征,以及16S rRNA基因序列分析进行鉴定。通过Plackett-Burman因素筛选试验、最陡爬坡试验和Box-Behnken试验优化菌株降解条件。【结果】筛选获得一株DM降解菌LH-1-1,96h对DM(100mg/L)的降解率为53.43%,经鉴定为琼氏不动杆菌(Acinetobacter junii);通过优化后,在DM浓度75mg/L、胰蛋白胨3 g/L、pH值6.8、硫酸铵1.5 g/L、氯化铁0.01 g/L、接种量为5%(体积比)、菌龄12 h、培养温度30℃条件下,菌株LH-1-1对DM降解率达82.36%,较未优化前提高了28.93%。【结论】A. junii LH-1-1具有较高的DM降解能力,该菌可为生物修复受DM或拟除虫菊酯类农药污染的环境提供优良的微生物资源。     

Effect of bioaugmentation and biostimulation on sulfate-reducing column startup captured by functional gene profiling

Sulfate-reducing permeable reactive zones (SR-PRZs) depend upon a complex microbial community to utilize a lignocellulosic substrate and produce sulfides, which remediate mine drainage by binding heavy metals. To gain insight into the impact of the microbial community composition on the startup time and pseudo-steady-state performance, functional genes corresponding to cellulose-degrading (CD), fermentative, sulfate-reducing, and methanogenic microorganisms were characterized in columns simulating SR-PRZs using quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). Duplicate columns were bioaugmented with sulfate-reducing or CD bacteria or biostimulated with ethanol or carboxymethyl cellulose and compared with baseline dairy manure inoculum and uninoculated controls. Sulfate removal began after ~?15?days for all columns and pseudo-steady state was achieved by Day 30. Despite similar performance, DGGE profiles of 16S rRNA gene and functional genes at pseudo-steady state were distinct among the column treatments, suggesting the potential to control ultimate microbial community composition via bioaugmentation and biostimulation. qPCR revealed enrichment of functional genes in all columns between the initial and pseudo-steady-state time points. This is the first functional gene-based study of CD, fermentative and sulfate-reducing bacteria and methanogenic archaea in a lignocellulose-based environment and provides new qualitative and quantitative insight into startup of a complex microbial system.