Aerobic biodegradation of hexachlorobenzene by an acclimated microbial community

The aerobic degradation of hexachlorobenzene (HCB) by an acclimated microbial community which isolated from a contaminated site and acclimated in our laboratory was investigated. The enriched microbial community was capable of biodegrading HCB when cultivated in minimal salts medium and supplied HCB as the sole carbon source. The efficiencies of microbial community in the degradation of HCB under different pH and temperatures were examined. The phylogenetic analysis for the nearly complete sequences of 16S rDNA demonstrated that the bacteria assemblage in the microbial community was dominated by Azospirillum and Alcaligenes groups.

First description of a novel Weissella species as an opportunistic pathogen for rainbow trout Oncorhynchus mykiss (Walbaum) in China

Six strains of Gram-positive, catalase-negative, non-motile, irregular short rod-shaped Weissella bacteria, with width and length of 0.5-0.6 and 1.2-2.7 mu m were isolated from diseased rainbow trout Oncorhynchus mykiss (Walbaum) in winter of 2007 at a commercial fishery in Jingmen, Hubei province, China. The diseased rainbow trout exhibited hemorrhage in eyes, anal region, intestine and abdomen wall, petechia of liver, some fish with hydrocele in stomach. Six isolates had identical biochemical reactions, phylogenetic analysis of 16S rDNA sequences, amplified ribosomal DNA restriction analysis (ARDRA), enzymatic profile analysis and antimicrobial susceptibility results, indicating as a single clonal outbreak. But all were different from any other validated twelve Weissella species in the term of physiological and biochemical characters. It is indicated that isolates are phylogenetically closer to Weissella halotolerans, Weissella viridescens and Weissella minor on 16S rDNA phylogenetic analysis result, than to W halotolerans and W viridescens on the result of ARDRA study and enzymatic profile analysis. Antimicrobial susceptibility testing was used to scan effective drugs for the therapy of this disease. Experimental infection assays with one isolate were conducted and pathogenicity (by intraperitoneal injection) was demonstrated in rainbow trout O. mykiss (Walbaum) and crucian carp (Carassius auratus gibelio) fingerlings. Because no Weissella was detected in fish feedstuffs and pond water, the source of this pathogen remains unknown, and Weissella isolates were regarded as an opportunistic pathogen for rainbow trout. This is the first report of Weissella strains which can cause disease of cultured fish in the world. (C) 2009 Elsevier B.V. All rights reserved.

Genetic Diversity of the Plankton Community and Its Relation to Taxonomic Composition and Environmental Factors in Lake Xiliang

Genetic diversity of the plankton community in Lake Xiliang was depicted by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting. Seventy-seven bands (33 of 16S rDNA and 44 of 18S rDNA) were detected, sixty-two planktonic taxa were identified in six sample stations in November 2007. The most common taxa were Ceratium hirundinella, Bdelloidea, Keratella cochlearis, Polyarthra trigla, and copepod nauplii. Based on environmental factors, taxonomic composition, and PCR-DGGE fingerprinting, unweighted pair-group method using arithmetic averages clustering and principal components analysis were used to analyze habitat similarities. There was distinct spatial heterogeneity in Lake Xiliang, and the genetic diversity of the plankton community was closely related to taxonomic composition and environmental factors.

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCB1 for identification purposes. The 90.6% of the clones were affiliated with the two phyla Bacteroidetes (50%) and Proteobacteria (40%), and beta-, -gamma-, and delta-Proteobacteria accounted for 7.8%, 28.1%, and 4.7%, respectively. Minor portions were affiliated with the Actinobacteria and Firmicutes (both 3.1%). Only 6 out of 64 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species, which indicated that a substantial fraction of the clone sequences were derived from unknown taxa. Rarefaction analysis of operational taxonomic units (orrUs) clusters demonstrated that 150 clones screened were still insufficient to describe the whole bacterial diversity. Measurement of water quality parameter demonstrated that performance of the SMBR maintained high level, and the SMBR system remained stable during this study.

Genetic diversity of plankton community as depicted by PCR-DGGE fingerprinting and its relation to morphological composition and environmental factors in lake donghu

To collect information about the genetic diversity of the plankton community and to study how plankton respond to environmental conditions, plankton samples were collected from five stations representing different trophic levels in a shallow, eutrophic lake (Lake Donghu), and investigated by PCR-DGGE fingerprinting. A total of 100 bands (61 of 16S rDNA bands and 39 of 18S rDNA bands) were detected. The DGGE bands unique to any single station accounted for 38% of the total bands, whereas common bands detected at all five stations accounted for only 11%. Using UPGMA clustering and MDS ordination of DGGE fingerprints, stations I and II were found to initially group together into one cluster, which was later joined by station V. Stations III and IV were isolated into two separate groups of one station each. Some differences in grouping relationships were found when analysis was completed on the basis of chemical characteristics and morphological composition, with zooplankton composition showing the greatest variability. However, the most similar stations (I and II) were always initially grouped into one cluster. Moreover, stations that exhibited the same or similar trophic level (stations III and IV), but different concentrations of heavy metals, were further differentiated by the DGGE method. Results of the present study indicated that PCR-DGGE fingerprinting was more sensitive than the traditional methods, as other studies suggested. Additionally, PCR-DGGE appears to be more appropriate for diversity characterization of the plankton community, as it is more canonical, systematic, and effective. Most importantly, fingerprinting results are more convenient for the comparative analyses between different studies. Therefore, the use of the described fingerprinting analysis may provide an operable and sensitive biomonitoring approach to identify critical, and potentially negative, stress within an aquatic ecosystem.

Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 165 rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204(T)), Planktothrix rubescens (type strain CCAP 1459/22(T)) Planktothrix pseudagardhii sp. nov. (type strain T1-8-4(T)), Planktothrix mougeotii (type strain TR1-5(T)), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207(T)), Tychonema bourrellyi (type strain CCAP 1459/11B(T)) and Limnothrix redekei (type strain NIVA CYA 277/1(T)).

Isolation and Characterization of a Methomyl-Degrading Paracoccus sp mdw-1

Methomyl, an extremely toxic pesticide, is widely used in agriculture. A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study. It could utilize methomyl as the sole carbon or nitrogen source. The optimal temperature and medium pH for its growth and methomyl biodegradation were 30 degrees C and 7.0, respectively. It was identified as a Paracoccus sp. according to its morphological features, physiological and biochemical characteristics, and phylogenetic analysis based on the sequence of 16S rDNA. Gas chromatography-mass spectrometry (GC-MS) analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1.

铬污染土壤修复过程中土壤细菌群落多样性的RFLP分析

对污染土壤修复过程中土壤细菌群落多样性的变化进行研究。【方法】以淹水培养后的模拟铬污染土壤为供试材料,通过直接提取土壤中总细菌DNA,利用细菌专一引物克隆细菌16S rDNA片段,分别建立克隆文库。利用PCR-RFLP技术,分析比较了土壤淹水10 d(对照,S1)、添加Cr(Ⅵ)淹水10 d(S2)、添加Cr(Ⅵ)和Fe(OH)3淹水10 d(S3)及20 d(S4)4个处理中土壤细菌群落的变化。【结果】用专一引物克隆细菌16S rDNA片段,分别建立了克隆文库;用限制性内切酶RsaⅠ进行细菌16S rDNA PCR-RFLP分析,分别得到123,120,97和69个酶切类型,库容值分别为54.92%,55.43%,65.33%和76.60%;Shannon-Wiener指数、Gini指数、物种丰富度指数(dMa)和物种均匀度指数(Jgi)均表现为S1>S2>S3>S4,以上4个指数的变异系数分别为11.51%,1.84%,23.64%和1.55%;基于细菌多样性参数的聚类分析结果,将对照S1和添加Cr(Ⅵ)处理的S2归于一类,而2个添加Fe处理的土壤S3和S4聚为一类。【结论】经过10 d淹水处理,...

氮肥缺失对旱地土壤细菌群落多样性的影响

本文旨在研究氮肥缺失对旱地土壤细菌群落多样性的影响。采用直接提取土壤微生物总DNA的方法,对不施肥(CK)、适量施肥(F1)、和缺氮施肥(F2)3种不同施肥水平土样DNA进行提取,扩增细菌16S rDNA基因片段,建立克隆文库。用限制性内切酶HhaI和RsaI进行PCR-RFLP分析,分别得到146、187、11个酶切类型。采用α多样性的测度对试验结果进行分析统计结果表明,不同处理间土壤细菌的多样性(H′、Ds和Dg)和物种丰富度(dMa、R2和E)均为F1>CK>F2;λ、dMa、E和H′指数在不同施肥处理间的变异系数达到56.96%~163.1%,尤其Simpson指数λ是非常敏感的指标,处理间的差异最大,表明氮肥缺失严重影响土壤细菌群落多样性,合理施肥有利于土壤细菌的多样性。

硫化叶菌病毒STFV、STSV1及其与宿主关系研究

硫化叶菌病毒表现出了极为独特、多样的形态学和基因组学特征，它们为研究硫化叶菌提供了很好的模型，同时也为构建可在硫化叶菌中进行分子操作的分子工具提供了有用的材料。 本研究从西藏热泉中分离出一株嗜酸热菌S3-3，S3-3的16S rDNA序列和腾冲硫化叶菌同源性最高，为96％，而和其它硫化叶菌同源性相对较低。基于16S rDNA序列进行的系统发育学分析同样证实了，S3-3和腾冲硫化叶菌亲缘关系最近，推测S3-3是硫化叶菌属的一新种。 从S3-3a中分离出一株硫化叶菌病毒，命名为西藏硫化叶菌丝状病毒（Sulfolobus Tibet Filamentous Virus, STFV）。STFV呈丝状，外面有一层脂膜包裹，病毒长约1.4 μm，病毒直径约20 nm，两端各有一约60 nm长的小尾巴。病毒的复制抑制宿主细胞生长，但并不引起宿主细胞的裂解。STFV的DNA为线性双链DNA，推测其DNA末端含共价闭合的发夹结构。已经完成了除病毒末端以外的基因组序列的测定，共测得序列29 568 bp。病毒基因组DNA的GC含量为32.70％，编码49个阅读框，其中包括四个解旋酶基因，四个糖基转移酶基因，一个核苷酸转移酶基因和一个磷酸转移酶基因。其中35个阅读框在已知硫化叶菌病毒中有同源基因。病毒含有两个主要的结构蛋白，分别由ORF162 和 ORF219所编码，这两个结构蛋白在Betalipothrixvirus中非常保守。基因组比对发现，STFV和Betalipothrixvirus的同源性很高。所有的证据表明，STFV为一新的病毒，属于硫化叶菌病毒Lipothrixviridae病毒科，Betalipothrixvirus病毒属。 腾冲硫化叶菌纺锤型病毒STSV1由向小宇博士分离。该病毒编码一个整合酶基因。但通过Southern杂交分析，未检测到病毒基因组整合至宿主基因组中。STSV1病毒颗粒的蛋白由5个阅读框所编码，而其主要的结构蛋白由ORF40编码。

长期重金属污染胁迫下农田土壤微生物特征研究

本论文以沈阳张士污灌区土壤为例，首次采用传统微生物生态学与现代微生物分子生态学相结合的研究方法系统地研究了污灌区长期重金属污染胁迫下原位农田土壤微生物特征。结果表明，虽然已经停止污灌十多年，张士灌区土壤耕作层（0～30 cm）仍然存在普遍的Cd污染，灌区土壤Cd含量高达1.75～3.89 mg kg-1。部分区域土壤Cd呈现向下迁移的趋势,且同时伴随有Cu、Zn复合污染。灌区土壤Cd含量较高时清水灌溉能降低土壤表层Cd含量，灌区土壤Cd含量下降到一定程度（约2 mg kg-1）后，清水灌溉对消除土壤表层Cd污染的作用消失。重金属元素中Cd对土壤微生物的影响最突出，在三个不同季节中土壤Cd与土壤微生物生物量（MBC）和微生物商（qM）呈显著负相关，与土壤微生物代谢商（qCO2）呈显著正相关。所检测的微生物指标中qM和qCO2与多种重金属元素呈显著相关性，可作为评价一定程度重金属污染的微生物指标。土壤营养元素（除P外）与微生物特征呈显著正相关性，土壤营养元素对微生物的刺激作用有可能在某种程度上掩盖了重金属对土壤微生物的负面影响。 用16S rDNA-PCR-DGGE方法，研究了不同浓度Cd胁迫下土壤Cd抗性细菌群落结构的动态变化，结果表明在Cd的胁迫下Cd抗性细菌多样性显著增加，不同土壤样品中Cd抗性细菌群落结构向相似的方向偏移，群落结构最终将可能趋向一致。Cd胁迫使敏感菌Pontibacter消失，而伯克氏菌（Burkholderia）、罗尔斯通氏菌（Ralstonia）、芽孢杆菌（Bacillus）和节杆菌（Arthrobacter）则富集成为优势菌。 从张士灌区Cd污染土壤中分离出32株Cd抗性细菌，研究了Cd抗性细菌和Cd抗性基因cadA的分布特征。这32株Cd抗性细菌分别归属于拟杆菌门(Bacteroidetes)（37.5%）、变形菌门(Proteobacteria) （37.5%）、放线菌门(Actinobacteria)（9.4%） 和厚壁菌门(Firmicutes)（15.6%）。在液体LB培养基中对Cd的抗性浓度都大于2 mmol L-1，对Zn抗性浓度介于5～13 mmol L-1。首次从Cetobacillus属的Cd抗性菌株S1基因组DNA中扩增出cadA基因的部分片断。在芽孢杆菌属(Bacillus)的4株菌N7，N9，N10和N11的基因组DNA中扩增出cadA基因的部分片断。序列分析结果表明这5株菌的cadA基因序列相似性为99％～93％，它们与坚强芽孢杆菌（Bacillus firmus） cadA 基因序列(M90750）相似性为94％～92％。系统发育分析结果表明这5株菌的cadA都与Bacillus firmus cadA 基因有着较近的亲缘关系。不同属的Cd抗性细菌间cadA基因的高度相似性揭示了cadA基因能在不同种属间转移的特性。

细菌对芘的降解特性及其降解性质粒研究

本研究通过富集培养，从沈抚灌区石油污染土壤中分离到两株以芘为惟一碳源和能源生长的细菌，分别命名为ZHL-4和B61。经过形态观察、生理生化实验与16S rDNA序列分析鉴定，ZHL-4和B61分别为苍白杆菌属（Ochrobactrum）和假单胞菌属（Pseudomonas）。 ZHL-4和B61均对芘具有较强的降解能力，在不添加任何其它碳源的情况下，分别在7d内将10mg•L-1芘降解了71.8%和67.4%，各加入500mg•L-1的葡萄糖和酵母膏作为共代谢底物后，7d内对芘的降解率分别提高到86.8%和89.9%。 经电泳检测，菌株ZHL-4和B61均存在内生质粒。质粒消除实验表明，消除质粒后的菌株ZHL-4和B61不能利用芘进行生长；将质粒转化入大肠杆菌中后，转化子获得了在芘固体培养基上生长的能力，初步证明两株细菌的内生质粒是与芘代谢有关的降解性质粒，其降解芘的基因位于质粒上，这与其它高分子量PAHs降解菌的降解基因位于染色体上不同。 通过设计引物、PCR扩增，菌株ZHL-4和B61并不具有已报道的芘降解基因nidA，这表明ZHL-4和B61具有可能不同于nidA基因的新的芘降解基因，有待于进一步研究。

乙草胺、甲胺磷胁迫下土壤抑真菌作用退化的微生物学机理

利用微生物分子生态学方法（16S rDNA-PCR-DGGE，clone library，ARDRA，sequencing），研究了乙草胺、甲胺磷胁迫对土壤抑真菌作用的影响及微生物学机理。首次构建了具有相同理化性质而抑真菌作用不同的土壤模型，报道了乙草胺和甲胺磷胁迫可降低土壤抑真菌作用，土壤细菌，尤其是假单胞菌群落结构以及phlD功能基因丰度和多样性与土壤抑真菌作用密切相关。 经100℃、110℃和121℃处理得到土壤抑真菌作用逐渐下降的系列土壤样品，经PCR-DGGE分析、克隆文库构建以及测序等证实了土壤细菌多样性和群落结构与土壤抑真菌作用密切相关，其中假单胞菌是重要的功能种群，酸细菌、α，β-变形细菌、节杆菌以及一些不可培养细菌可能存在潜在的抑真菌能力。 乙草胺（50、150、250 mg•Kg-1）处理可降低自然清洁土壤抑真菌能力。随着土壤抑真菌作用的逐渐下降，土壤细菌、真菌以及特异性功能种群假单胞菌和芽孢杆菌的群落结构均发生一定改变。乙草胺通过改变土壤微生物群落组成而降低土壤抑真菌作用。甲胺磷（50、150、250 mg•Kg-1）处理在低浓度时即可极大降低土壤抑真菌作用，且不同浓度处理间差异不显著。 乙草胺和甲胺磷胁迫（浓度均为50、150、250 mg•Kg-1）均能降低土壤中总的可培养假单胞菌和抗典型病原真菌立枯丝核菌（Rhizoctonia solani）的假单胞菌多样性，改变其群落结构。同时还能降低重要抗真菌抗生素2，4-DAPG合成的关键功能基因phlD的多样性。而phlD基因的丰度在甲胺磷胁迫下亦逐渐下降。上述影响在实验5周内持续存在，不可恢复。 研究结果表明乙草胺和甲胺磷胁迫可通过改变土壤细菌，尤其是假单胞菌群落结构及phlD功能基因多样性而降低土壤抑真菌作用，对土壤质量存在潜在生态风险。 此外，对土壤真菌DNA的提取方法进行了优化，得到了适于我国北方土壤类型且真菌多样性程度较高的真菌DNA提取方法。

污水处理体系中的微生物群落特征研究

污水生物处理系统本质上是一种人工强化的工程化微生态系统。污水处理过程往往由多个功能互补的反应单元协同完成，例如因对污水中有机碳、氮和磷兼具良好的去除功能而在城市污水处理中被广泛应用的Anoxic / Oxic（A/O）生物处理工艺。不同反应单元特有的微生物群落之间的相互关系和相互作用与处理系统的稳定性和处理效率密切相关。所以对污水处理系统中微生物群落进行系统分析非常重要。研究系统中微生物群落的时空演替对于优化处理系统的设计和操作具有重要意义。但是，以往对于污水处理系统中微生态系统的解析多数针对实验室规模的其中个别反应器独立进行，还缺乏从系统水平对实际大规模运行的整个污水处理过程中所有反应单元群落进行分析的研究。 悬挂链移动曝气系统是对A / O工艺的完善和发展。悬挂链曝气工艺的实现是依靠悬挂链移动曝气设备和完善的自动控制系统来完成的。可以在系统中实现类似多级A/O的可能性，水力停留时间较长，污泥龄达到15天以上，能够完全实现A / O 工艺。 目前正被广泛应用在各种行业的污水处理项目中。 本文应用基于细菌16S rRNA中的PCR扩增方法（Polymerase Chain Reactor），结合变性梯度凝胶电泳指纹分离技术（Denaturing Gradient Gel Electrophoresis, DGGE），对实际规模的运用Anoxic / Oxic（A/O）工艺并采用悬挂链式移动曝气技术的污水生物处理系统中微生物群落特征，主要对细菌组成结构和群落动态，细菌优势菌群的多样性以及与系统功能稳定性的关系进行了研究，拟为更全面了解活性污泥处理系统中的优势菌群特征，以及细菌群落结构和功能动态变化关系，实现对活性污泥处理的优化操作，对污染物降解功能菌群的筛选，为运用现代培养技术实现分离培养并运用于环境修复实践奠定方法和理论基础。 首先，对影响PCR-DGGE分析的重要前操作步骤进行了优化和筛选，包括两个方面：细菌基因组DNA的高效提取和纯化；不同16S rRNA靶序列对PCR-DGGE分析的影响。从中选出适合于活性污泥样品的细菌基因组DNA提取方法和PCR-DGGE分析的最优靶序列组合。 其次，运用PCR-DGGE指纹图谱技术分析了该污水处理系统中不同功能反应单元中活性污泥的细菌种群结构特征，探讨了系统运行过程中细菌种群时间和空间上的动态特征。并将图谱中所显示的优势条带进行切割回收，重复扩增，电泳检测，序列测定并与GenaBank数据库中的微生物类群进行同源性比对，探讨活性污泥中细菌种群多样性，了解污泥中可能含有的主要具有污染物降解功能的类群信息。 在整个处理过程中，同一功能反应单元中不同位置的活性污泥微生物菌群结构不同。执行不同功能的处理单元活性污泥细菌多样性和组成结构各有不同。 在系统稳定运行的状态下，细菌组成结构的时间变化动态不显著。但是在系统的不同操作条件下，主要处理池的微生物群落的DGGE遗传指纹图谱较独特。 对该处理系统污泥中优势菌群的序列测定和同源性比对表明，优势菌群所对应的细菌的16S rDNA序列可以被归属于以下四个主要的细菌系：α, β, γ- Proteobacteria 以及厚壁菌门 phylum Firmicutes （low G+C Gram-positive）。 该处理系统的优势菌群的DGGE条带拥有潜在的具有异养硝化/好氧反硝化的除 N / P 类群。该类菌群中的大多数属于Pseudomonas spp.。另外，回收到两个与已鉴定的具有异养硝化和好氧反硝化能力的Pseudomonas stutzeri 和 Pseudomonas borbori 最相似的菌株的条带。γ-变形菌纲门（γ- Proteobacteria）的微生物类群在该缺氧-好氧处理厂中分布较广泛，尤其是和 N / P 去除紧密相关的具有脱氮除磷能力的Pseudomonas 类群，而且在好氧曝气处理池中分布较广，这可能和系统中表现的好氧反硝化现象相关。 不同的操作状况下微生物群落结构有差异。增加污泥回流比，增加DO（Dissolved oxygen）浓度，COD去除率和NH4+-N去除率显著增加，总N和总P的去除率改变不显著。 最后，对整个处理过程中微生物群落结构在系统正常调控改变范围内的长期动态和稳定性进行了探讨。整个处理系统的长期稳定性与体系中的每个处理环节相关，而不是仅与其中的单个主要反应池相关。污水处理体系的功能稳定性与其中的微生物群落稳定性相关，微生物群落结构决定了生态功能，群落结构变化能反应系统的运行状况及其降解效率。

长期施肥黑土微生物群落特征及其季节变化

研究长期施肥黑土微生物群落特征及其季节变化，可准确揭示施肥对黑土肥力质量的影响。本文以黑土肥料试验基地10个有机肥和无机肥处理0—20 cm表层土壤为研究对象，以不施肥和休闲处理为对照，分别经过8次季节取样，分析长期施肥与季节更替条件下黑土微生物群落特定磷脂脂肪酸（PLFA）含量、微生物量碳（氮）含量、r-k策略菌群数量、土壤酸（碱）磷酸酶活力以及16S rDNA的PCR-DGGE条带构成等微生物特征及其变化规律，探讨长期施肥与季节更替对黑土微生物群落的影响，明确限制各微生物群落生长的主要养分因子。 不同季节、多种施肥处理之间的土壤微生物指标多重比较表明，施肥与季节更替显著影响黑土微生物各菌群生长与活力，且施肥×季节更替交互作用显著。 与无机肥处理相比，有机肥施用能显著提高黑土各菌群特征PLFA与微生物量碳（氮）含量、r-k策略细菌或真菌数量以及磷酸酶活力；在一定时期、一定程度上改变土壤细菌PCR-DGGE条带构型，提高细菌群落多样性。不同有机肥-化肥配施处理之间各微生物学指标比较发现，有机肥+磷肥（MP）处理中的可培养细菌数量、细菌与放线菌PLFA含量、微生物量碳含量以及碱性磷酸酶活力普遍较低；高量有机肥（M2CK）处理中的各菌群特征性PLFA、微生物量碳（氮）含量以及酸性磷酸酶活力等则有一定提高。单施化肥各处理，尤其是氮磷钾（NPK）处理，在一定程度上抑制了黑土各菌群生长与活力。各处理PLFA因子分析表明，施用有机肥对黑土放线菌、G+与G-细菌等菌群影响较大。另外，休闲处理真菌特征PLFA含量、r-策略真菌数量、碱性磷酸酶活力及微生物量碳（氮）含量等均处较高水平。 季节更替对微生物群落各指标影响亦达显著水平，且各指标季节变化趋势存在一定差异。黑土各菌群特征性PLFA含量、微生物量氮、G+/G-比值及r-策略细菌等指标，均自春至夏有显著升高趋势；而磷酸酶活力、真菌/细菌比值及k-策略细菌等指标则自春至夏有明显的下降趋势。各处理PLFA因子分析表明，夏秋季节对土壤单烯不饱和脂肪酸影响较大。 多元线性回归分析表明，碱解氮、有机碳、有效磷等养分几乎是影响所有已测微生物学指标的最关键养分因子，较次要的微生物生长限制因子是速效钾与pH值。