石油烃污染对稻田土壤微生物生态系统的影响

由于含油污水灌溉、原油储存运输和石油冶炼过程中导致的泄漏事故等原因，石油类物质已经成为环境中的主要污染物之一。石油烃污染物对土壤微生物遗传多样性和群落结构的影响在国外已有一些报道，但有关于石油烃污染对稻田土壤微生物生态系统的影响一直以来一直没有一个全面和系统的认识。本论文首次采用传统微生物培养方法、常规生化分析方法与PC树DGGE等现代分子生态学研究方法相结合的手段系统评价了长期石油污水灌溉对中国最大的石油类污水灌区-沈抚灌区的稻田土壤微生物种群数量、细菌群落组成、多样性及代谢活性的影响。结果表明，总石油烃（Total Petloleum hydrocarbon／TPH）在灌区干渠和支渠中的积累和分布趋势大体上是上游地区较严重，下游地区较轻，并且与土壤中有机质含量呈显著正相关。在目前的污染程度下，石油污水能够刺激土壤好氧异养细菌（Aerobic heterotrophic bacteria／AHB）和真菌的生长，其数量与TPH含量呈显著正相关。细菌基因多样性与TPH含量呈显著负相关，细菌群落中的优势菌群为变形细菌β-亚群和γ-亚群的菌种。土壤脱氢酶、过氧化氢酶、多酚氧化酶活性和土壤底物诱导呼吸（substrate-induced respiration／SIR）与土壤中TPH含量呈显著正相关，而土壤脉酶活性则与之呈显著负相关。实验室110天不同浓度石油烃胁迫模拟试验结果表明，当石油烃胁迫浓度低于1000mgk扩时可以被定义为轻度污染，土壤中AHB大量增殖，细菌多样性和群落结构可在处理第30天和第50天得到恢复；当石油烃胁迫浓度为5000mg·kg~(-1)-10000mg·kg~(-1)时可以被定义为中度污染，AHB数量显著增加，放线菌和真菌数量显著降低，土壤细菌多样性在培养前巧天显著降低，至培养结束时有逐渐恢复的趋势，细菌群落结构发生明显改变，土壤脱氢酶、多酚氧化酶、脉酶的活性及土壤SIR受到一定程度的抑制，但能够显著刺激土壤过氧化氢酶活性的提高。当石油烃胁迫浓度为10000mg·kg~(-1)-50000mg·kg~(-1)时可以被定义为重度污染，对AHB生长的刺激作用更为显著，土壤细菌多样性在培养前15天显著降低并不可恢复，群落组成与对照差异很大，所有土壤酶和SIR均受到严重抑制。从沈抚灌区上游地区旱田土壤、灌渠底泥和实验室高浓度石油烃胁迫土壤中筛选出了4株TPH生物降解率在60％以上的高效石油烃降解菌并鉴定其归属，这些菌株均能够利用正构烷烃、单环和多环芳烃及环烷烃。

高浓度CO_2下红松和长白赤松土壤微生物活性研究

大气CO_2浓度升高已经成为全球生态坏境研究中的热点，木文主要研究了长白赤松和红松根际及非根际土壤微生物数量、生物量C、土壤酶活性以及根际／非根际细菌群落结构对高浓度CO_2的响应规律，得出的主要研究结论如下：（1）高浓度CO_2对红松和氏白赤松根际及非根际土壤细菌数量影响显著，而对真菌和放线菌数量的影响却不明显；（2）700μmol·mol~(-1) CO_2对红松和长白赤松土壤微生物生物量碳的影响较500μmolmol~(-1)CO_2显著，且主要表现为抑制效应；（3）对红松土壤酶活性来说，高浓度CO_2对蛋白酶、服酶、磷酸酶、过氧化氢酶、多酚氧化酶以及脱氢酶活性主要表现出了促进作用，而对淀粉酶和转化酶的活性主要是抑制作用；对长白赤松说，高浓度CO_2对脉酶、转化酶、淀粉酶、过氧化氢酶、多酚氧化酶以及脱氢酶活性主要表现出了显著的促进作用，而对蛋白酶和磷酸酶的活性却主要起着抑制作用。此外，土壤酶活性的月动态规律在不同程度上也受到了高浓度CO_2的影响；（4）红松和长白赤松土壤酶活性之间、土壤酶活性与微生物生物量C以及微生物数量之间的相关性受到了高浓度CO_2的影响，但不同浓度CO_2其影响规律也不同：（5）不同树种其土壤酶活性、微生物数量以及微生物生物量C对高浓度CO_2的响应规律不同，即土壤生化活性与所研究的树种有关；（6）大气CO_2浓度升高对红松和长白赤松根际和非根际土壤细菌群落结构产生了较大的影响，主要表现为部分新的物种的出现／或原有物种数量的丰富以及原有物种的消失或数量被削弱，但主要建群种却未发生改变，此外，细菌群落结构在700μmol·mol~(-1) CO_2条件和500μmol·mol~(-1) CO_2下的变化规律亦不同。

杉木连栽土壤中毒与真菌毒素的研究

本论文报道了杉木连栽土壤中毒与真菌毒素的研究结果。研究结果表明：杉木连栽土壤致害真菌随杉木连栽代数的增加而显著增加，在国内外首先提出了杉木连栽土壤尖抱镰刀菌及其分泌的毒素对杉木生长的危害，是造成杉木连栽障碍的重要原因，并据此提出了克服杉木连栽障碍的PGPR生物调控技术措施，见到了可喜的苗头。以绿豆、小麦、白菜为指示植物对杉木连栽土壤进行的毒性检测结果，随杉木连栽代数的增加，杉木土壤对指示植物一绿豆、小麦、白菜的抑制作用呈现加强的趋势，表明杉木连栽导致土壤毒性物质的积累。为了探明土壤毒性物质来源，我们调查了不同连栽代数杉木土壤微生物区系变化情况，发现随连栽代数的增加，细菌、放线菌数量明显减少；而真菌数量随连栽代数的增加而显著增加，不同连栽代数杉木土壤真菌经PCR-DGGE分析显示：杉木连栽对土壤真菌多样性、群落结构、丰富度影响较大。随连栽代数的增加，土壤真菌生物多样性降低，镰刀菌等一些重要致害真菌类群急剧富集。用生物检测的方法从连栽杉木土壤中分离得到5株对杉木具有抑制作用的菌株，对两株毒性最强的菌株采用经典和现代分类鉴定方法，鉴定结果表明：SFZ为尖抱镰刀菌萎蔫专化忆仁（Fusariumoxysporumf.sp.vasinfectum）；SF31为正青霉（Eupenicilliumbrefeldianum），这两株真菌中SF2对杉木的毒害作用尤为突出，其发酵液在64倍稀释浓度下仍能强烈抑制杉木种子的发芽和生长，表明尖抱镰刀菌萎蔫专化性及其分泌的毒素是造成杉木连栽障碍的重要原因。我们对致害真菌SFZ发酵液经预处理、萃取、硅胶柱层析、制备薄层层析等步骤进行分离纯化得到了毒素粗结品，并进行了质谱检测，与已知的镰刀菌毒素一丁烯酸内酉爵目比较，推测质核比244为丁烯酸内酷的一种。连栽杉木土壤中毒的生物调控初步研究结果表明：519、B25和MB-97三种抗真菌混合PGPR菌剂在杉木林间微区实验应用4个月，能显著促进连栽土壤杉木幼苗的生长，缓解杉木连栽土壤中毒症状。对PGPR定殖能力检测显示：6个月后能检测到大量519存在于杉木土壤中，说明在这种特定的环境下，S19能生存并取得优势地位，有很好的定殖能力，表明S19可能对调控杉木土壤中毒具有良好的应用前景。同时，这一实验结果也是连栽杉木土壤中毒性物质来源于土壤真菌的有力佐证。

厌氧复合菌系的筛选及含纤维素原料预处理复合菌剂的研究

本文从不同厌氧生境中获得7组(C-2、Y-2、L-2 、NZ、H-3、CZ、L-3)具有纤维素降解能力的复合菌系。经过不断传代、淘汰纤维素降解能力降低的菌系，最后得到一组高效、传代稳定的厌氧纤维素分解复合菌系L-3。该菌系可使滤纸在42 h内溃烂，并能在分解纤维素的同时产氢气。对L-3复合菌系的产酶条件进行了研究，结果表明，在实验范围内该菌系的产酶最适条件为：pH 6.5，温度37 ℃，接种量5 %，最佳碳源为滤纸，最佳氮源为硫酸铵。第10天测得羧甲基纤维素酶(CMCase)、滤纸酶(FPA)、外切葡聚糖酶(C1)、β-葡聚糖苷酶(β-glucodase)的酶活分别为0.216 U/ml、0.101 U/ml、0.132 U/ml、0.002 U/ml，滤纸失重率70.6 %。发酵代谢产物乙醇和丁酸含量分别可达1378 mg/L 、2695 mg/L，发酵产生的气体中氢气含量最高可达70.2 %。DGGE结果表明该菌系主要由14种菌组成，其中有三株菌在发酵前后菌数发生了明显的变化，说明在以滤纸为底物的降解过程中，这三株菌起到了重要作用，对这三株菌进行了分子生物学鉴定，初步定为Clostridium phytofermentans、Clostridium cellulovorans、Desulfovibrio sp。 利用实验室分离得到的纤维素降解菌，最终配制出由10、X-1、X-13、ST-13、L-3组成的好氧-厌氧纤维素降解复合菌剂。以秸秆为发酵底物，菌剂接种量1%，利用复合菌剂预处理后的秸秆，发酵总产气量相对于对照提高了71.62%，甲烷含量最高可达70.08%。 A group of microbial consortia L-3 was isolated from the anaerobic fermentation residue of corn stalk, which could degrade cellulose and produce hydrogen. The CMCase, FPA, C1 and β-glucosidase activity of L-3 could reach to 0.216 U/ml, 0.101 U/ml, 0.132 U/ml and 0.002 U/ml, respectively. In the filter degrading process, the filter paper collapsed in the liquid culture within 42 h and the filter degrading rate could reach to 70.6% in the 13 days, meanwhile, hydrogen was determined and the highest hydrogen content was 70.2%. The optimum cellulase-degrading conditions were filter papaer as the carbon source, (NH4)2SO4 as the nitrogen source, 37 ℃ and pH 6.5 in this experiment. DGGE results showed that the microbial consortia L-3 mainly included 14 strains. The amount of 3 strains were changed during the fermentation. These strains were identified as Clostridium phytofermentans、Clostridium cellulovorans、Desulfovibrio sp by 16S rDNA sequence analysis. The cellulose- degrading microbial agent was composed by 10, X-1, X-13, ST-13, L-3 which were isolated in the laboratory. The straw pretreated by cellulose-degrading microbial agent was used to ferment, the total biogas production increased by 72% comparing to the control. The content of methane could reach to 70.08%。

溶藻菌群对铜绿微囊藻的溶藻效应及其作用机理研究

水华暴发是一个世界性的问题，近年来在发展中国家显得尤其严重。水华暴发给环境和公众健康带来巨大灾难，一些蓝藻产生的毒素可以造成鱼类、鸟禽和家畜的死亡，而臭名昭著的微囊藻产生的微囊藻毒素更是有强烈致癌效应。因此，寻找控制水华藻类的有效方法非常迫切。在利用物理和化学方法处理不甚理想的情况下，利用溶藻细菌控藻成为一个新的研究方向。溶藻细菌一般直接从富营养化水体中分离，杀藻活力对有害蓝藻具有较强的选择性而不危害其它生物，尤其适合在水华发生初期使用，可以在短时间内达到阻止藻类增殖的效果。本研究富集分离到一个高效溶解铜绿微囊藻的溶藻菌群，对其溶藻效应和溶藻机制进行了探索研究。 1溶藻菌群的富集筛选及其溶微囊藻效果 富集筛选得到一个有明显抑藻效果的菌群，它对铜绿微囊藻有显著溶藻效果。与对照组相比，加入富集的溶藻菌后，第4 d开始出现溶藻现象，6~8 d出现明显的溶藻效果，8 d后测得叶绿素去除率在85%以上。 2 溶藻菌群的作用范围及溶藻特性 富集分离到的溶藻菌群对铜绿微囊藻和念珠藻有显著溶藻作用，对水华微囊藻和其它几株受试微囊藻没有明显溶藻效应。该溶藻菌群不仅可以在液体中溶解铜绿微囊藻，生长在固体平板上的藻苔也有一定的溶藻效应，生成溶藻空斑。保证快速溶藻的最大稀释度可以达到1/100, 000。 3 环境因子对菌群溶藻效力的影响 试验发现，不同的pH、温度、和光照条件下，溶藻菌群溶藻效力明显不同，且不同种类的氮源对其溶藻作用也有一定影响。这些条件对该菌群溶藻作用的影响，在相当的程度上可能取决于它们对藻和细菌两者的生长状况的影响综合。 4 溶藻菌群的溶藻作用机理 溶藻菌液过滤除菌和煮沸灭菌处理后溶藻液，未见明显的溶藻效果，只有原液具有很好的溶藻效果。因此可初步确定，蓝藻细胞的溶解可能是由溶藻菌直接接触藻细胞产生的作用效果。显微镜观察发现，细菌在溶藻的过程中频繁地接触藻细胞并侵入藻细胞，破坏进而裂解杀死藻细胞。这也进一步说明了此溶藻菌是通过直接方式杀藻。 5 溶藻菌群的菌群结构解析 分离有溶藻效果的纯菌的多次尝试都没有成功。结合DGGE和16S rDNA文库综合分析发现：Rubritepida菌，假单胞菌和鞘氨醇单胞菌是存在于铜绿微囊藻中的三种伴生细菌。加入富集的溶藻菌群后，菌群结构发生明显的变化，Rubritepida菌、假单胞菌消失，混合菌群则包含未培养黄杆菌，鞘氨醇单胞菌和噬氢菌，其中黄杆菌是优势菌群，并且细菌种群结构的变化与藻细胞消亡之间有显著的相关性。通过菌种的分离鉴定与DGGE和16S rDNA文库的测序结果比较，一些未培养菌可能在溶藻过程中起重要调控作用。 6 溶藻细菌控藻应用基础 (1) 扩大规模的模拟水华实验进一步确定了细菌对微囊藻的强烈溶解作用。 (2) 铜绿微囊藻(Microcystis aeruginosa 905, zc)、微囊藻(Microcystis spp., zd)和溶藻菌群共培养试验表明，zc可以抑制zd生长，而溶藻菌群可以溶zc。 本研究是第一次报道混合菌群的溶藻效应。该溶藻菌群对带有藻际细菌的铜绿微囊藻具有高效的溶藻效力，表明它对自然界中存在的带菌铜绿微囊藻和其它一些蓝藻的生消具有一定的控制作用。对进一步研究菌藻关系与生态学作用，以及对富营养化湖泊和水库水体中蓝藻暴发的防控，该菌群具有一定的应用潜力。 Cyanobacterial blooms break out frequently all over the world, especially in developing countries. Blooms create enormous disasters to public health and to the environment. Some cyanobacterial blooms produce extremely toxic substances that have killed fish, domestic animals and birds. It has been well known that microcystins, a hepatoxin produced by Microcystis, can promote tumors in humans. So it is very important to find an effective method for controlling the growth of the bloom-forming algae. Measures for controlling such kind of algae include physical, chemic and biologic means, but the former two may damage the aquatic environment and require high-energy inputs. The alternative approach for the elimination of nuisance algae involves the application of algicidal bacteria. The algicidal bacteria, which are nontoxic to other organisms and most of which are isolated from the eutrophic lake in situ, may be potential microbial algaecides. In the initial stages of the water blooms, they are able to restrain the biomass or multiplication of the bloom-forming algae in a short time. In order to use algicidal bacteria to suppress blooms of M. aeruginosa, we isolated a bacterial culture capable of lysing the noxious cyanobacteria M. aeruginosa. In this paper we described some properties of the bacterial culture and its growth-inhibiting or algicidal effects on the growth of M. aeruginosa, and investigated its algicidal mechanisms. 1 Enrichment of a microbial culture that lyses Microcystis aeruginosa A mixed bacterial culture was isolated from a hypereutrophic pond and showed significant algicidal activity against the noxious Microcystis aeruginosa. Algae lysis would be seen obviously 4 days later when the algae culture was killed and became yellow contrast to no-addition controls, and chlorophyll a (chl-a) reduction went beyond 85% 8 days later. 2 The host range and some other algicidal feature of the mixed algicidal culture. Microcystis aeruginosa, Nostoc sp., were susceptible to the mixed algicidal culture, while the lytic effects of this mixed culture on Microcystis flos-aquae and some other tested Microcystis were feeble.The algicidal culture can not only lyse M. aeruginosa in liquid media, but aslo lyse M. aeruginosa lawns on soft agar plates and form plaques. The maximun dilution of the mixed culture required for rapid Microcystis lysis is 1/100, 000. 3 Influences of environmental factors such as pH, temperature, illumination, and the nitrogen source on the lytic activity of the mixed bacterial culture on Microcystis aeruginosa. In our investigations, it was shown that the lytic activity of the mixed bacterial culture on Microcystis aeruginosa was straightly correlated with pH, temperature, illumination, as well as the nitrogen source in the medium. The impacts of these environmental factors on the algicidal activity of the mixed bacterial culture, to a certain extent, may depend on both the algal and the bacterial growth rates under the tested environmental conditions. 4 The mechanisms of algal cell lysis by the algicidal bacteria Death was detected when the mixed bacterial culture was added to the algal culture, but not when only the culture filtrate or autoclaved bacterial culture was added. This indicates that the mixed bacterial culture did not release extracellular products inhibitory to Microcystis aeruginosa. In addition, under the microscope, we observed frequent contacts btween bacteria and algae cells, and some bacteria can even penetrate into target algal cells and destroyed them. These results may suggest that the bacterium kill the alga by direct contact. 5 Molecular Characterization of the algicidal bacterial culture Attempts for isolation of pure bacterium or bacteria from the enrichment culture responsible for Microcystis lysis have so far been failed. Based on PCR-DGGE (denaturing gradient gel electrophoresis) and 16S rDNA clone library analysis, Rubritepida sp., Pseudomonas sp. and Sphingomonas sp., as accompanying bacteria, were existed in M. aeruginosa. The bacterial community in M. aeruginosa showed significant change after adding the enrichment culture, where uncultured Flavorbacterium sp., Sphingomonas sp. and Hydrogenophaga sp. were observed, and the uncultured Flavorbacterium sp. became a dominant species. The obvious correlation can be seen between change of bacterial population and extinction of M. aeruginosa. Compared identification of pure bacterium with sequencing of DGGE bands and the clone distribution of the clone libraries, it was inferred that some uncultured bacteria were probably play an important role in controlling the growth and abundance of M. aeruginosa. This report is the first example of a mixed bacterial culture with the ability to lyse M. aeruginosa. 6 Further study for algae control by applications of algicidal bacteria (1) Algae lysis would be seen obviously 6 days later when the algae culture was killed and became yellow contrast to no-addition controls, and chlorophyll a (chl-a) was reducted to a low level 20 days later in the simulated water bloom experiments. (2) The growth of Microcystis sp. (zd) was restrained by Microcystis aeruginosa 905 (zc) when they were co-cultured together, and zc was lysed by the algicidal bacterial culture. This report is the first example of a mixed bacterial culture with the ability to lyse M. aeruginosa, and its algicidal activity remained high against non-axenic tested M. aeruginosa, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of M. aeruginosa and other cyanobacteria. Such bacteria could also potentially be used as agents to prevent the mass development of cyanobacteria in eutrophic lakes and reservoirs.

自养氨氧化混合种群特性和共存机理初步研究

组特殊自养氨氧化混合种群，表现：无机环境种群生长迅速、生物量高；在一个完全无机的自养生长环境中，不仅保持高氨氧化速率，并出现丰富的异养微生物种群；该种群置于异养、厌氧环境中，迅速表现出产氢特征。对于这样一个特殊的生态体系，研究其共生机理，以及联接这些种群之间的碳源和能源问题，将具有非常重要意义。我们拟从种群特征、细胞表面分泌产物、游离体系产物多糖、蛋白和脂肪酸方面开展研究。 第一部分，自养氨氧化混合种群的基本特征。采用氨氧化培养基，进行种群氨氧化特征研究；采用扫描电镜观察自养混合种群的微观特征；沉降、离心去除微生物种群，分析水相中的总有机碳、糖类等物质；利用LB培养基进行种群的分离、纯化，并采用DGGE手段对微生物种群结构进行分析。结果表明，接入菌种后（2/5000（V/V）），培养液中氨（200mg/L）在3－5天内快速降解；亚硝酸盐与氨氮变化呈负相关趋势，仅有少量硝酸盐含量（< 30mg/L）。氨氧化种群的生物量增长与氨氧化趋势一致，初始生物量7.75 mg/L(蛋白含量)，3-5天后生物量快速增长，并达到最高63.06 mg/L（蛋白含量）。电镜图片显示，种群外包裹一层粘液。离心除去菌体后，检测培养液总有机碳和糖的含量，同样表现出与生物量增长相似的特征，分别由初始的3.73、2.35 mg/L，3－5内天迅速增加，并分别达到最大值35.19、27.45 mg/L。经初步分离、纯化并对纯化菌株进行测序，获得了10株异养微生物分别为布鲁氏菌科苍白杆菌属、纤维单孢菌、类芽孢菌属、黄杆菌属、无色杆菌、鞘脂单胞菌、嗜麦芽寡养单胞菌、噬氢菌属、硫红球菌、假单胞菌；DGGE显示，约有20分条离带，我们对其中的两条优势条带进行切割回收测序，鉴定为欧洲亚硝化单胞菌（Nitrosomonas eur）。 第二部分：混合种群自养-异养菌共生的可能机制。在对微生物种群特征初步分析基础上，针对胞外糖类组分可能被微生物代谢分解，我们重点对微生物细胞蛋白质与糖类进行分析。采用超声结合RIPA裂解液裂解，SDS-PAGE电泳分析混合种群总蛋白种类，并通过氨基酸分析仪及红外光谱法分析氨基酸组成及蛋白红外特征。采用超声破碎结合反复冻融对细胞样品进行处理，提取液采用醇沉、Sevage脱氮白，凝胶过滤方法脱盐和分级分离。对提取物的糖分析包括：紫外扫描，红外光谱，核磁共振，单糖组成分析；扫描电镜观察菌群破裂现象。SDS-PAGE分析结果表明：氨氧化种群不同生长阶段都显示出42kD蛋白表达量很高，d4时42kD蛋白表达已经很强，4-7d内一直持续这种过量表达，直到d8后表达开始减弱。说明42kD蛋白可能与氨氧化密切相关。红外光谱分析显示：细胞提取物的特征峰分布在3427.42cm-1、1718.18 cm-1和1681.72 cm-1、1160.07和1086.74 cm-1，分别对应为OH、 C=O、C－O－C基团，表明具有蛋白的典型特征；氨基酸分析显示蛋白中的Gly,Asp,Ala,Glu含量相对较高。 提取物中胞外多糖分离谱图得到不均一组分，共得到6个收集峰；紫外扫描在201－213 nm处有多糖吸收峰，同样表明多糖成分不均一性；多糖红外光谱特征峰主要分别在3400.49 cm-1、2920.28 cm-1、1154.54和1087.52 cm-1，对应OH、－CH2－ or CH 、C－O－H or C－O－C等多糖特征基团；多糖提取物核磁共振1H d4.3～5.9之间出现强吸收峰，这是1H中，多糖存在的明显证据，1H NMR中，其中O-乙酰基的甲基上的氢信号为d1.1～1.3之间。糖肟全苯甲酸酯衍生物的HPLC测定中，得到单一的单糖峰，由于时间问题，还未进行更深入的试验；电镜图片显示，种群中的细胞有大量的破裂现象。 实验表明，自养氨氧化混合种群显示出快速的氨氧化速率，氨氧化过程生物量和有机质的增加明显。微生物种群包裹粘液层，并分离纯化出大量的异养菌；去除菌体后的游离培养液中存在有机质（包括多糖）说明无机自养生长体系中存在异养菌生长、繁殖的二次碳源；细胞提取物中蛋白条带数目多、种类丰富；细胞多糖提取物具有明显的多糖特征，以及单糖的存在。结合种群的显微特征和游离体系中的有机质的检测结果，我们认为，无机自养生长体系中，种群细胞生长过程中发生的破裂现象可能是导致大量的蛋白、多糖释放到游离胞外，并成为其他异养菌生长的碳源和氮源。这可能是自养体系中，大量异养菌共生的可能机制，至于是什么原因引起种群生长过程中产生的破裂现象，还有待下一步深入研究。 A group of mixed autotrophic ammonia oxidizing populations, having much biological characteristic tested by concerned personnel for pilot test: Performed rapid population growth and obtained high biomass in inorganic environment; Not only maintained a high rate of ammoxidation, promoted a wealth of heterotrophic microbial populations growth in a totally inorganic and autotrophic growth environment; Placed in heterotrophic and anaerobic environment,had the performance characteristics that could rapidly produce hydrogen.For such a special ecological system, Study its symbiotic mechanism and the connection between these populations of carbon and energy issues, will have a very important significance. We intended from the characteristics of the population, the secretion product of cell surface, free substance in the liquid medium like polysaccharide, protein and fatty acids carrying out research. Part I: The basic features of mixed autotrophic ammonia oxidizing populations . Use inorganic liquid medium, processed study for ammonia oxidation characteristics of the population; we used scanning electron microscopy to get micro-features of autotrophic ammonia oxidizing populations .The medium was carried out settlement and centrifugal then removed the microbial populations, after all of that we analysis the water phase for total organic carbon(TOC), carbohydrate and other substances; Solid ammonia oxidizing medium was adopted to separation and purification of population, DGGE means was for structure analysis of microbial population. The results showed that after the inoculum of bacteria (2 / 5000 (V / V)), ammonia in the culture medium (200 mg / L) was rapid degradation in 3-5 days; ammonia and nitrite have the negative correlation between changes in the trend, then only a small amount of nitrate content (<30mg / L). The biomass growth of ammoxidation population in line with the trend of ammonia oxidation, the initial volume of it was 7.75 mg / L (protein content), in 3-5 days upto 63.06 mg / L (protein content). Electron microscope image showed, the populations were wrapped in a layer of mucus, including the a large number ruptted micorbe , Centrifuge to remove bacteria, then detected the medium for total organic carbon and sugar content, result took on the same characteristics with biomass growth, that were from the initial 3.73、2.35 mg / L respectively, in 3-6 days achieved rapid increase in the maximum to 35.19、27.45 mg / L respectively. After initial separation、 purification ,then processed sequencing to strains purified and got the result that there were 10 heterotrophic microorganisms : Brucella Branch pale bacillus, Cellu lomonas, Bacillus species category, a Flavobacterium, colorless Bacteria, Aeromonas sheath fat, little support maltophilia Aeromonas, macrophages species hydrogen, sulphur-MI, Pseudomonas bacteria spores; DGGE display, there were 20 separation bands approximately. Part II: Mixed populations that autotrophic - heterotrophic bacteria symbiotic mechanism. On the basis of preliminary analysis of microbial population characteristics, aiming at extracellular carbohydrate components might be decomposition by microbial, we focused on microbial cell protein and carbohydrate analysis. Using ultrasound combined with RIPA lysis cracking the cells, SDS-PAGE electrophoresis analysis the total protein species of the population, and through the amino acid analyzer studied the compositions of amino acid and infrared spectroscopy analysis of a protein infrared characteristics. Using ultrasound combined with repeatedly freezing and thawing to treated the cell sample, then took the means that alcohol precipitation, deproteinization by Sevage, gel filtration aimed at desalination and grade separation to deal with the lysates . The extraction of sugar analysis included: UV scanning, IR, NMR, single-sugar composition analysis. SDS-PAGE analysis showed that: 42 kD protein expression was very high at different growth stages of mixed autotrophic ammonia oxidizing populations , on the fourth day, 42 kD protein expression had been very strong, 4-7d, it had continued this excessive expression, then started to weaken after 7 days. 42 kD protein that might be closely associated with ammonia oxidation. Infrared spectral analysis showed that: cell extracts with the characteristic that the peak distribution in 3427.42 cm-1、1718.18 cm-1 and 1681.72 cm-1、1160.07 cm-1 and 1086.74 cm-1 corresponding to OH、C = O、C-O-C Groups which had the typical characteristics of protein; and analysis showed that amino acids including Gly, Asp, Ala, Glu ,the content in the protein is relatively high. Exopolysaccharide in the extracts had the separation map that it was uneven, received a total of six collection peaks by the detection mode of phenol-sulphruic acid method ; ultraviolet scan in the 201-213 nm department had polysaccharide absorbing peak, the same ingredients that polysaccharide heterogeneity; infrared polysaccharide spectral characteristics of the main peak at 3400.49 cm-1, 2920.28 cm-1, 1154.54 and 1087.52 cm-1, corresponding OH,-CH2-or CH, C-O-H or C-O-C;and other characteristics of polysaccharide group; 1H NMR of polysaccharide extract appeared absorption peak between d4.3 ~5.9, which is the apparent evidence of polysaccharide, In 1H NMR, the hydrogen signal of one of O-acetyl was between 1.1 to 1.3. The determination of Sugar oxime whole benzoate derivatives by HPLC, there was a single-sugar peak, as a matter of time, yet more in-depth test. Summary: Mixed autotrophic ammonia oxidizing populations show us that it had the ability in ammonia oxidizing and it was great, organic matter and biomass increased significantly in the process of ammonia oxidation. Microbial populations was wrapped up slime layer, the phenomenon of cell breakdown obviously, and there were a lot of separation and purification of the heterotrophic bacteria; a lot of organic matter (including polysaccharides)remined in the medium that removal of cell indicated the inorganic system existed secondary carbon sources that could be used by the heterotrophic bacteria ; there were a large number proteins bands of cell extract, rich variety; cell extracts of polysaccharide had obvious characteristics of polysaccharide, and the existence evidence of single-sugar. Combined population of microscopic characteristics and free of organic matter in the test results, we believe that the health of inorganic system, population growth occurred in the course of the breakdown of the phenomenon is likely to lead to a lot of protein and polysaccharide released into the extracellular free, And other heterotrophic bacteria use them to the growth as carbon and nitrogen. This may be autotrophic system, the large number of heterotrophic bacteria symbiotic mechanism.

垃圾渗滤液场外硝化－原位反硝化脱氮协同填埋场温室气体减排

垃圾卫生填埋是国内外城市垃圾的主要处置方法。垃圾渗滤液是渗入填埋场垃圾的降水混合垃圾降解过程中产生的物质而形成的混合物，是垃圾填埋场向环境排放的主要污染物。渗滤液由于其所含高浓度有机和无机污染物，且其中很多物质有生物毒性或难生物降解，难于治理。特别是到填埋晚期，渗滤液中高浓度的氨氮更是增加了治理的难度。渗滤液场外硝化－原位反硝化是填埋场氮管理的新途径。本文利用从环境中筛选出优势硝化功能菌对渗滤液中的高浓度氨氮进行生物硝化，经硝化后的渗滤液回灌至以垃圾柱模拟的生物反应器填埋场，在填埋场内实现原位反硝化。 上述目标通过以下两部分来实现： 第一部分：渗滤液场外硝化。首先从污水厂的硝化污泥中富集并筛选出硝化功能菌，在模拟氨氮废水中优化。将驯化的硝化功能菌接种于连续式完全混合反应器（CSTR）进行高氨氮渗滤液硝化研究。在200余天的连续运行中，反应器硝化和有机物去除效果良好。在最大氨氮负荷和有机物负荷分别为0.65 g N l-1 d-1 和3.84 g COD l-1 d-1时，氨氮和COD去除率分别高于99%和57%。实验过程中发现，游离氨（FA）和溶解氧（DO）浓度对反应器中亚硝酸盐的积累影响很大。 第二部分：渗滤液原位反硝化。本文利用一个垃圾填充柱模拟生物反应器填埋场，研究了硝化渗滤液回灌对垃圾降解的影响，和回灌的硝化渗滤液中TON（总氧化态氮）对填埋场生物反应器产甲烷作用的影响。最后利用变性梯度凝胶电泳（DGGE）分析了硝化渗滤液回灌对垃圾填埋场菌群结构的影响。结果表明：回灌的TON被完全还原，反硝化为主要反应，最大TON负荷为28.6 mg N kg-1 TS d-1。当垃圾柱TON负荷大于11.4 mg N kg-1 TS d-1时，出现了产甲烷抑制，抑制作用随TON负荷的增加而加强。在此过程中，反硝化逐渐代替产甲烷作用成为填埋场内垃圾降解的主要反应，且更多产生的是清洁的氮气，而非温室气体甲烷。直到实验结束时，回灌硝化渗滤液的垃圾柱的甲烷产量仅相当于对照的2.5%，并且回灌的硝化渗滤液还加速了填埋场垃圾的降解与稳定。通过DGGE进行菌群结构分析发现，由于TON对填埋场的长期作用，反硝化菌增多而产甲烷菌减少。 Landfill still remains the chief method for MSW management around the world. Leachate is a mixture of rainfall permeating through landfill and organic and inorganic matters generated during decomposition of the wastes in the landfills, characterized as highly complicated and refractory wastewater. Ex-situ nitrification and sequential in-situ denitrification represents a novel approach to nitrogen management at landfills. In the present paper, nitrification was carried out in a continuous stirred tank reactor (CSTR) inoculated with nitrifying bacteria which were isolated from municipal WWTP of Chengdu city. The nitrified leachate from CSTR was recirculated to a lab-scale municipal solid waste (MSW) column where in-situ denitrification took place. The above object was achived through two parts as following: First, ex-situ nitification of leachate. After acclimated in simulated wastewater for 3 month, nitrifying bacteria isolated from WWTP nitrifying sludge were added to the CSTR for nitrification. The results over 200 days showed that the maximum nitrogen loading rate (NLR) and the maximum organic loading rate (OLR) was 0.65 g N l-1 d-1 and 3.84 g COD l-1 d-1, respectively. The ammonia and COD removal was over 99% and 57%, respectively. Moreover, the effects of free ammonia (FA) and dissolved oxygen (DO) on nitrification were investigated. Second, in-situ denitrification was studied in a municipal solid waste (MSW) column. Variation of nitrified leachate and its effects on the decomposition of municipal solid waste (MSW) were studied in a lab-scale MSW column to which nitrified leachate was recirculated. Additionally, DGGE was employed to investigate the microbial community of both MSW columns. The results suggested: complete reduction of total oxidized nitrogen (TON) was obtained with maximum TON load of 28.6 mg N kg-1 TS d-1 and denitrification was the main reaction responsible. Methanogenesis inhibition was observed while TON load was over 11.4 mg N kg-1 TS d-1 and the inhibition was enhanced with the increase of TON load. Denitrification gradually took over methanogenesis to become the main reaction responsible for decomposition of MSW while nitrogen gas, a clean byproduct, was generated instead. Till the end of the experiment, the average weekly methane production in the denitrification column was as low as 2.5% of that of the control, and the rate of decompition and stability of MSW was accelerated by the recirculation of the nitrified leachate.Owing to long term exposure of nitrified leachate to landfill, denitrifying bacteria increased and methanogen decreased.

有机碳对混合菌群亚硝酸盐氮氧化的影响

自养硝化过程在自然界氮素循环和污水处理系统脱氮过程中起着关键作用。因此，了解有机碳对硝化的影响和硝化菌与异养菌之间的竞争对微生物生态学和污水处理系统设计都很重要。目前对氨氧化到硝酸盐氮过程的研究文献很多，但对亚硝酸盐氧化过程在异养菌的存在下如何受到有机碳影响的研究甚少。本文从生理生化指标、基因组学、蛋白组学三方面考察了在实验室条件下有机碳（乙酸钠）对硝化细菌和异养菌组成的混合菌群的硝化性能、菌群结构及代谢功能的变化的影响。 全文分为两大部分： 第一部分为乙酸钠对游离态硝化混合菌群的硝化性能和菌群结构的短期影响。混合菌株先在自养条件下进行连续培养，两个月后硝化速率达到20 mg N/(L·d)；而后离心收集菌体进行批式实验。在批式反应器中，初始亚硝氮均为126mg N/ L，乙酸钠-C 与亚硝酸盐-N 的比分别为0，0.44，0.88，4.41，8.82。结果表明：在低C/N 比（0.44 和0.88）时，亚硝酸盐去除速率比C/N=0 下高，细菌呈现一次生长；而在高C/N 比（4.41 和8.82）时，出现连续的硝化反硝化，亚硝酸盐去除率仍比对照下高，细菌呈现二次生长。不同C/N 比下微生物群落明显不同，优势菌群从自养和寡营养细菌体系（包括亚硝酸盐氧化菌，拟杆菌门,α-变形菌纲,浮霉菌门和绿色非硫细菌下的一些菌株）过渡到异养和反硝化菌体系 （γ-变形菌纲的菌株尤其是反硝化菌Pseudomonas stutzeri 和P. nitroreducens 占主导）。 第二部分为乙酸钠对硝化混合菌群生物膜的硝化性能和菌群结构的长期影响。接种富集的硝化混合菌群于装有组合式填料的三角瓶中，于摇床中自养培养；两个月后填料上形成生物膜的硝化速率达到20 mg N/ (L·d)；而后进行长期实验，每12 小时更换混合营养培养基（亚硝氮约200 mg N/ L，C/N 比同上）。结果显示：相较于C/N 比=0 时的亚硝酸盐氧化反应来说，低C/N 比出现了部分的反硝化，而高C/N 比则是几乎完全的反硝化。与对照比，C/N=0.44 时亚硝酸盐氧化速率并未受乙酸钠的影响，反而上升了，但C/N=0.88 时亚硝酸盐氧化速率有所下降。菌群结构分析表明自养对照与混合营养下微生物群落的不同；PCR-DGGE未检测出混合营养下硝化杆菌的存在，而显示异养菌尤其是反硝化菌的大量存 在。荧光定量PCR 结果表明随C/N 比上升，硝化杆菌数量从2.42 × 104 下降到1.34× 103 16S rRNA gene copies/ ng DNA，反硝化菌由0 增加至2.51 × 104 nosZgene copies/ ng DNA。SDS-PAGE 的结果表明不同C/N 比下的蛋白组较为复杂且呈现一定的差异性。 有机碳对亚硝氮氧化及微生物群落的影响很复杂，本文分别讨论了对游离态和生物膜固定态两种状态的混合菌群相应的短期和长期影响研究。研究发现，有机碳并非一定带来硝化的负影响，如果控制在适当的C/N 比范围，有机碳是有利于亚硝氮氧化的。这些发现阐明了有机碳和硝化反硝化的关系，填补了硝化微生物生态学上的空白，对污水处理系统中减少异养菌的影响并提高氮去除率有一定理论指导意义。 Nitrification plays a key role in the biological removal of nitrogen in both nature and wastewater treatment plant (WWTP). So, understanding of the effect of organic carbon on nitrification and the competition between nitrifying bacteria and heterotrophic bacteria is important for both microbial ecology and WWTP design and operation. Despite the fact that the nitrification process of ammonia to nitrate has been extensively investigated, it is not known how the process of nitrite oxidization is affected by organic carbon when heterotrophic bacteria are present. By measuring different physiological and biochemical parameters, as well as using genomic DNA and proteome analysis, we investigated the influence of organic (acetate) on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria under laboratory conditions. The dissertation involves two parts: Part one deals with the effect of organic matter on functional performance and bacterial community shift of nitrite-oxidizing and heterotrophic bacteria under suspended state. The bacteria were prepared in a continuous-flow stirred reactor under autotrophic condition; after two months, the nitrification rate of the culture reached about 20 mg N/ (L·d); then the bacteria were harvested for the next batch experiments. The initial concentrations of nitrite were 126 ± 6 mg N/ L in all flasks, and sodium acetate (C) to nitrite (N) ratios were 0, 0.44, 0.88, 4.41, and 8.82, respectively. The results showed that at low C/N ratios (0.44 or 0.88), the nitrite removal rate was higher than that obtained under autotrophic condition and the bacteria had single growth phase, while at high C/N ratios (4.41 or 8.82), continuous aerobic nitrification and denitrification occurred besides higher nitrite removal rates, and the bacteria had double growth phases. The community structure of total bacteria strikingly varied with the different C/N ratios; the dominant populations shifted from autotrophic and oligotrophic bacteria (NOB, and some strains of Bacteroidetes, Alphaproteobacteria, Actinobacteria, and green nonsulfur bacteria) to heterotrophic and denitrifying bacteria (strains of Gammaproteobacteria, especially Pseudomonas stutzeri and P. nitroreducens). Part two describes the influence of acetate on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria in biofilms. Bacterial enrichments was transferred into flasks with polypropylene carriers and cultured under agitated and autotrophic condition. After two month, the biofilms grown on the carriers had a nitrification rate of about 20 mg N/ (L·h); then the biofilms were refreshed with mixotrophic medium (nitrite were 200 mg N/ L in all flasks, and C/N ratios was the same as above) every 12 h. the results show: normal nitrite oxidization reactions were performed when C/N = 0, but nitrite oxidization and partial denitrification occurred with low C/N ratios (0.44 or 0.88). At high C/N ratios (4.41 or 8.82), we mainly observed denitrification. In contrast to C/N = 0, the nitrite oxidization rate was unaffected when C/N = 0.44, but decreased with C/N = 0.88. The structure of bacterial communities varied significantly between autotrophic and mixotrophic conditions. Nitrobacter was hard to detect by PCR-DGGE while heterotrophs and especially denitrifiers were in the majority under mixotrophic conditions. Real-time PCR indicated that the Nitrobacter population decreased from 2.42 × 104 to 1.34 × 103 16S rRNA gene copies/ ng DNA, while the quantity of denitrifiers obviously increased from 0 to 2.51×104 nosZ gene copies/ ng DNA with an increasing C/N ratio. SDS-PAGE indicated the complexity of and a certain difference between the proteome of nitrite-oxidizing and heterotrophic bacteria at different C/N ratios. We conclude that the influence of organic matter on nitrite oxidation and the community structure of NOB and heterotrophic bacteria is complex. In this dissertation, we focused on how sodium acetate influenced the system both under suspended state and in biofilms. We observed that acetate did not necessarily have a negative impact on nitrification. Instead, an appropriate amount of acetate benefited both nitrite oxidization and denitrification. These findings provide a greater understanding about the relationship between organics and nitrification; they fill the gaps in the field of microbial ecology of nitrifying bacteria; they also provide insight into how to minimize the negative impact of heterotrophic bacteria and maximize the benefit of nitrogen removal in biological treatment systems.

功能菌强化应急处理苯胺突发污染事故研究

近年来各种环境污染事故频发，据统计仅2001~2003年间，发生的各类环境污染事故就高达5606次，其中水污染事故3235次，占全部的57.7%。这些事故不仅给人民生命财产造成巨大损失，也给生态环境造成严重的破坏。因此开发安全高效的应急处理技术迫在眉睫。本研究以筛选高效苯胺降解菌为基础，通过对高效菌降解性能的研究指导将高效菌作为功能郡主投加到已有生物处理系统强化应急处理苯胺突发污染事故废液，取得了良好的效果。 苯胺高效降解菌AN-P1为红球菌（Rhodococcus sp.），其通过间位途径降解苯胺，AN-P1利用苯胺生长和降解的最佳pH为6，最适浓度为2000 mg/L，最适温度为30 ℃，最佳接种量为0.3‰。AN-P1降解含500 mg/L、1000 mg/L、2000 mg/L苯胺的培养物分别经过28 h、24 h、32 h降解，出水苯胺含量能达到《污水综合排放标准》（GB8978-1996）一级标准。但由于苯胺降解过程中释放了大量氨氮，出水氨氮仍较高未能达标排放。而常规SBR系统应急处理效果较差，苯胺和COD去除率均低于10%，出水未能达标排放。活性碳吸附后的回收和后续处理也会带来操作不变和二次污染问题，且处理后出水往往难于达标排放，尚需进行进一步处理。 生物处理系统应急处理后恢复运行处理效果监测和PCR-DGGE图谱分析显示，用AN-P1菌强化应急处理系统后不仅能快速高效的去除苯胺，而且可以有效保障处理系统对污染物的净化性能，有效的保护系统中的功能微生物免受苯胺毒害。 研究结果表明，从实际处理效果、对原有生物系统性能保护及实际应用操作等多方面考虑，用AN-P1菌强化应急处理苯胺突发污染事故在技术上都是可行的。本研究为应急处理苯胺突然污染事故废液提供了新的方法。 Recent years, environment pollution accidents happened frequently, the data showed that there are 5606 accidents between 2001 and 2003, including 3235 water environment accidents, which is 57.7% of all. These accedents not only caused money lost and life lost but also caused serious damage to the ecologicl environment. So exploring highly-effective and secure methods to solve these accidents is an urgent mission. We screened a highly-effective aniline-degrading bacterium and did some researches on its ability to degrade aniline, in order to guide the emergency treatment of aniline containing wastewater that caused by sudden accident pollution with bioaugmentation. A highly-effective aniline-degrading bacterium AN-P1 was isolate and characterized as Rhodococcus sp. It degrades aniline through meta-cleavage pathway. The optimal pH and temperature for cell growth and aniline degradation were 6 and 30 ℃, respectively, and the opitimal concentration of aniline was 2000 mg/L, the optimal inoculation amount was 0.3‰.It took bacterium AN-P1 only 18 h, 24 h and 32 h, respectively, for the treatment of MSB containing 500 mg/L, 1000 mg/L, 2000 mg/L aniline to meet the first grade of national some of the NH4+-N which caused by aniline degradation. It took bacterium AN-P1 only 10 h, 20 h and 32 h, respectively, for the treatment of wastewater containing 500 mg/L, 1000 mg/L, 2000 mg/L aniline to meet the first grade of national integrated wastewater discharge standard. The bacterium AN-P1 can also remove some of the NH4+-N which caused by aniline degradation. It took bacterium AN-P1 only 10 h, 20 h and 32 h, respectively, for the treatment of wastewater containing 500 mg/L, 1000 mg/L, 2000 mg/L aniline to meet the first grade of national integrated wastewater discharge standard. By combing AN-P1 with regular SBR system, it took only 36 h for the emergency treatment of wastewater containing 2000 mg/L aniline under simulating engineering conditions to meet the discharge standard. While the NH4+-N of effluent can not meet the standard because of the high amount NH4+-N caused by aniline degradation. The regular SBR system was not good at aniline and COD removal. The removal efficiency of which are less than 10%. It cost 67.8 g activated carbon to absorbed 1000 mg aniline. It is inconvenient to transport and use it for the emergency treatment of aniline when the sudden pollution accident happened. Meanwhile, it was complex ad hard to recycle the activated carbon and treat the aniline wastewater get from activated carbon recycling too. Hard to meet the effluent standard was also a problem of activated carbon absorption method. According to the PCR-DGGE profile and removal efficiency of pollutants and COD when the systerm recover from emergency treatment, AN-P1 can efficiently protect the microbial community of regular activated sludge system against the aniline. It proved that combing AN-P1 with regular biological system is a feasible strategy for emergency treatment of aniline sudden pollution accident. The research offered a new way for emergency treatment of aniline sudden pollution accident.

DNA指纹图谱技术在土壤微生物多样性研究中的应用

土壤中的微生物多样性是十分丰富的,传统培养方法对土壤微生物多样性的研究有很大局限性。近年来,各种基于16S rDNA基因的指纹图谱分析技术取得了长足的进步,并广泛应用于土壤微生物多样性的研究。这些技术主要有变性梯度凝胶电泳(DGGE)/温度梯度凝胶电泳(TGGE)、单链构象多态性(SSCP)、随机引物扩增多态性DNA(RAPD)、限制性片段长度多态性(RFLP)和扩增核糖体DNA限制性分析(ARDRA)等。对这些技术近年来在土壤微生物多样性研究领域的应用予以简短综述,并初步探讨未来几年土壤微生物分子生态学发展的方向。

阿特拉津微污染原水生物活性炭处理系统种群动态演替分析

采用降解菌柱、土壤悬液柱和对照柱3种活性炭柱处理阿特拉津微污染原水,考察了阿特拉津去除效率,并利用PCR-DGGE技术分析炭柱运行期间微生物菌群动态变化。结果表明140d后对照柱去除率下降至30%～40%,而降解菌柱的去除效率保持在65%～75%。DGGE分析表明3个炭柱中都有自来水中微生物生长,土壤悬液柱中微生物在贫营养状态时种群多样性降低,实验室分离的阿特拉津降解菌接入降解菌柱后在运行期间可以保持相对优势,延长了炭的使用时间。

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16SrDNA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。

长白赤松和红松土壤微生物活性对高浓度CO_2的响应(英文)

研究了2003 年夏季长白赤松和红松土壤微生物活性对高浓度CO2的响应规律。结果表明,长白赤松和红松土壤细菌数量受高浓度CO2影响显著(p < 0.05)减少;与对照箱(350 μmol ·mol-1 CO2)和裸地(350 μmol ·mol-1 CO2)相比,红松土壤淀粉酶和转化酶活性降低,而长白赤松土壤淀粉酶和转化酶活性却表现为增加;同时发现受700 μmol ·mol-1 CO2处理的红松和长白赤松土壤微生物生物量碳均表现为显著降低。DGGE 结果表明:受高浓度CO2 的影响,长白赤松和红松土壤细菌群落结构发生了明显的变化。研究结果表明土壤微生物对高浓度CO2的响应规律与所研究的的树种有关。图2 表2 参29。

长期石油和重金属污染对农田土壤假单胞菌种群多样性及结构的影响

采用PCR-DGGE方法,对我国东北地区长期石油和重金属污染农田土壤中的假单胞菌多样性及其种群结构进行研究.结果表明:石油污染区土壤中假单胞菌多样性指数显著高于重金属污染区;石油污染区旱田土壤假单胞菌多样性接近于对照清洁土壤,但低于相似污染程度的石油污染区水田土壤,表明污染物类型与耕作管理方式是影响土壤中假单胞菌多样性的主要因素.经16SrRNA的V6/V7区测序,Pseudomonas mendocina、P.stutzeri和P.aerugi-nosa是该石油和重金属污染区土壤中的优势类群,说明在长期污染胁迫下,这3种假单胞菌分别得到了不同程度的富集,推测与石油烃的自然降解及假单胞菌的重金属抗性有关.

长期有机污水灌溉对土壤固氮细菌种群的影响

固氮细菌是土壤生态系统中十分重要的功能群之一,在土壤氮素循环中发挥着不可替代的作用。本文通过传统培养方法和固氮基因(nifH)PCR-DGGE指纹图谱分析方法,从微生物功能群角度,研究了长期有机污水灌溉对土壤表层和亚表层固氮细菌种群数量和多样性的影响。结果表明,土壤表层固氮细菌数量随土壤污染的增高而不同程度减少,亚表层自生固氮菌数量无明显变化。从DGGE指纹图谱结果来看,对照清洁土壤的固氮细菌种群多样性较丰富,其Shannon指数在表层为2.4674,在亚表层为2.8210,分别高于受有机污水灌溉不同年限的土壤固氮细菌种群shannon指数,且种群的相似程度有所差异,说明种群结构发生了一定的变化。改清灌3a和改清灌30a土壤固氮细菌种群shannon指数分别略有增加,但种群结构并没有得到完全恢复。污水灌溉对土壤固氮细菌种群的影响具有长期的生态效应,是导致土壤生态与结构功能改变的重要因素之一。