极端环境放线菌的分离及多相分类研究

本文综述了放线菌分类学研究的目的和作用，分析了放线菌分类学的历史和现状，介绍了当前放线菌多相分类研究中所采用的技术方法及适用范围。同时还重点介绍了极端高温、低温、高盐放线菌分离及分类研究的进展。从云南采集高温温泉水样、火山口土样，从云南、新疆等地采集雪山土样，从新疆、青海等地采集盐碱土样进行放线菌分离，对不同极端环境下的放线菌分离方法进行探讨，并对分离到的部分典型放线菌菌株采用形态特征、培养特征，生理生化测定，细胞化学组份分析，DNA G+C mol%和DNA同源性测定，以及16SrDNA全序列分析等相结合的多相分类技术进行系统的分类研究。从表型、基因型及系统发育三个不同层次对其分类地位进行了最终确定。其中，分离自云南洱源温泉的菌株YIM60013和腾冲火山口的菌株YIM60032分别确定为高温放线菌属的两个新种：白色高温放线菌（Thermoactznomyces albus sp. nov.）和云南高温放线菌（Termoactomyces yunnanensis sp. nov.）；分离自新疆北疆地区的一株低温放线菌菌株，结合其形态特征、细胞化学组份及16S rDNA序列分析将其鉴定为链霉菌的一个新种，北疆链霉菌（Streptomyces beijiangensis sp. nov.）；来自新疆盐碱土样的6株嗜盐放线菌菌株YIM90001-90006中，菌株YIM90001被命名为嗜盐普氏菌新种(Prauserella halophila sp. nov.），菌株YIM90005被 命名为脱卤普氏菌新种（Prauserella dehalogenans sp. nov.），菌株YIM90002和YIM90003鉴定为拟诺卡氏菌科中的链单抱菌新属Streptomonospora gen. nov.）和它的两个新种：菌株YIM90002定为盐生链单抱菌新种(Streptomonospora saline sp. nov.），菌株YIM90003定为白色链单抱菌新种(Streptomonospora alba sp. nov.）；菌株YIM90004和YIM90006分别被确定为拟诺卡氏菌属的一个新种和一个亚种：新疆拟诺卡氏菌新种（Nocardopsi sxiniangensis sp. nov.）和嗜阿拉伯糖新疆拟诺卡氏菌亚种( Noocardiopsi sxiniangensis subsp．arabicus subsp．nov，）。

调节LDLR及VCAM-1基因表达的微生物代谢产物的研究

为了筛选对靶基因LDLR和VCAM-1的表达具有调节作用的生物活性物质，建立了两个基于重组人细胞系的高通量的筛选模型，使用荧光素酶在96-孔版上来筛选对上述靶基因的表达具有调节作用的微生物代谢产物。模型之一是来自于人肝HepG2细胞系的重组L39细胞，用于筛选增加LDLR报告基因表达的生物活性物质，以期发现新的具有降胆固醇作用的药物。筛选之二为来源于细胞系ECV304的重组细胞株Nl-14,用于筛选抑制VCAM-1基因表达的活性物质，以期发现治疗风湿性关节炎等免疫性疾病治疗的药物。上述筛选系统均是稳定转染的细胞系，分别含有与荧光素酶报告基因相融合的LDLR或VCAM-1基因的转录调节元件。通过对6300株微生物的总计12600个样品的筛选，共发现和分离了17个活性化合物并进行了结构解析。其中两个被命名为Cladospolede D和Zelkovamycin的化合物被确定为新的化合物。由真菌 FO-6605的发酵液提取得到的一个化合物对LDLR报告基因的表达具有很强的上调作用，其SC200为1 Onmol/L a使用荧光标记的LDL检测到该化合物对于HepG2细胞膜上LDLR具有剂量依赖的增强作用。由真菌FO-5897的发酵液中分离到了一个已知的化合物Ascofuranone，该化合物曾经被报道具有降血脂抗肿瘤的活性。值得注意的是我们首次发现了该化合物同时具有抑制 VCAM-1报告基因表达和增强LDLR报告基因表达的作用，该发现有可能会对其降血脂作用的深入研究提供帮助。由海洋真菌FT-0012产生的化合物Cladospolede D为一个12-员环的大环内酷类的化合物，该化合物对两个测活系统均显示出无选择性的抑制作用形态学研究显示该真菌属于Cladosporiun属。另外一个由土壤放线菌K96-670产生的新化合物为一个环八肤类的化合物，经~1H~1-H COSY,~(13)C-H COSY,~(13)C-~1H HMQC, ~(15)N-~1H HMQC,~(15)-~1N HHMBC等波谱学研究得知该化合物的分子结构中含有六个非普通的氨基酸和两个普通氨基酸。该化合物对VCAM-I报告基因的表达显示出非常好的选择性的抑制活性，其IC50值为9.5ug/ml.形态学的研究表明该菌株属于链霉菌属。 在筛选过程中从来源于云南省西双版纳的土壤中分离到了一株编号为YIM1272的放线菌，经包括形态学、生理一生化和16S rDNA在内的分类学研究，确定该菌株为链霉菌属的一个新种，被命名为佩版纳链霉菌，(Streptomyces.bannaensis．sp.nov）。

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.

厌氧产氢菌的分离筛选、诱变选育及其产氢特性的研究

本文从四川绵竹酒厂、成都市龙泉长安垃圾填埋场以及四川大学荷花池底的厌氧污泥中先后分离得到63株厌氧产氢菌，其中H-8、H-61、HC-10等16株产氢细菌产氢能力较高，HC-10的产氢能力最高，最大产氢量和最大产氢速率分别达到2840 ml H2/L培养基和25.39 mmol H2/g drycell·h，对HC-10进行生理生化鉴定和分子生物学鉴定，判定其为clostridium sp.，对HC-10的产氢条件进行了研究，结果表明，该菌的最适生长温度为35 ℃，最适生长初始pH为7，以葡萄糖为最佳碳源，以蛋白胨为最佳氮源，不利用无机氮源，其产氢发酵液相产物以乙醇和乙酸为主，其发酵类型属于乙醇型发酵。此外，以酒糟废液作为底物，进行了菌株HC-10的生物强化试验，研究表明，投加了HC-10的强化系统其产氢量比对照高出40.32%。 同时为了获得厌氧产氢菌的高效突变株，分别以产氢菌H-8和H-61为原始菌株进行微波诱变处理，对微波诱变参数进行了优化，考察了突变株的遗传稳定性、产氢特性及耐酸性。菌株H-8经过微波诱变得到5株高产氢突变株HW7、HW33、HW181、HW184、HW195，经多次传代表明HW195是稳定的高产突变株。突变株HW195具有较好的耐酸性，在pH值为2.8时仍能生长。通过间歇发酵实验，其最大产氢量和最大产氢速率分别达到2460 mL/L培养基和27.97 mmol H2/g drycell·h，比原始菌分别提高了50.75%和41.7%。菌株H-61经过微波诱变后选育得到的突变株HW-18，其最大产氢量和最大产氢速率分别达到2190 mL/L培养基和25.86 mmol H2/g drycell·h，比原始菌分别提高了23.03%和31.00%。 为了对比各种诱变方式对产氢菌产氢能力的影响，以厌氧产氢菌H-61为原始菌株，先后经亚硝基胍(NTG)、紫外(UV)诱变，选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下，其产氢量为3024 mL/L培养基，比原始菌株提高了69.89%；其最大产氢速率为33.19 mmol H2/g drycell·h，比原始菌株提高了68.14%。经过多次传代实验，稳定性良好。其发酵末端产物以乙醇和乙酸为主，属于典型乙醇型发酵。其最适产氢初始pH为6.5，最适生长温度为36 ℃，以蔗糖为最佳碳源。与原始菌株相比，突变株HCM-23的产氢特性发生了改变，如生长延滞期延长，可利用无机氮源等。 From anaerobic activated sludge, 16 strains of hydrogen producing bacteria were newly isolated. One of them named as HC-10 had the highest hydrogen producing capability, under the batch fermentative hydrogen production condition, the maximal hydrogen yield and hydrogen production rate was 2840 mL/L culture and 25.39 mmol H2/g drycell·h. It was identified as clostridium sp.HC-10 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen producing of strain HC-10 were achieved as: initial pH 7.0, temperature 35 ℃, glucose as the favorite substrate, Moreover, using distiller's solubles wastewater as substrate, HC-10 strain was added in the biohydrogen producing system to research the bioaugmentation effection. The results showed that the hydrogen production of bioaugmentation system was 40.32% higher than the noaugmentation system. An anaerobic, hydrogen producing strain H-8 was irradiated by microwave to optimize the microwave mutagenesis condition, and to test the heredity, hydrogen-producing potential and aciduric of the mutants. An aciduric mutant named as HW195 with steady hydrogen-producing capability was obtained, which can grow at pH 2.8. Its capability of hydrogen production was tested in the batch culture experiments. The maximum hydrogen yield and hydrogen production rate was 2460 mL/L culture and 29.97 mmol H2/g drycell·h, which was 50.7% and 41.7% higher than those of the initial strain, respectively. When used the strain H-61 as original strain, a mutant named as HW18 was obtained. The maximum hydrogen yield and hydrogen production rate was 2190 mL/L culture and 25.86 mmol H2/g drycell·h, which was 23.03% and 31.00% higher than those of the initial strain, respectively. The results demonstrated that microwave mutagenesis could be used in the field of hydrogen producing microorganism. The hydrogen producing strain H-61 was used as an original strain which was induced by NTG and UV for increasing and the hydrogen production capability. One of the highest efficient H2-producing mutants was named as HCM-23 with its stable hydrogen production capability. which was tested in the batch culture experiments. With the condition of 10 g/L glucose, its cumulative hydrogen yield and hydrogen production rate was 3024 mL/L culture and 33.19 mmol H2/g drycell·h, 69.89％and 68.14％ higher than that of the original strain, respectively. The terminal liquid product compositions showed that the mutant HCM-23 fermentation was ethanol type, while the original strain H-61 fermentation was butyric acid type. Varieties of parameters of hydrogen production fermentation were studied, including time, carbon source, nitrogen source, glucose concentration, glucose utilization, initial pH and incubation temperature had been studied, indicated the optimum condition of hydrogen production for the mutant HCM-23 as initial pH6.5, temperature 36 ℃, and the favorite substrate was sucrose. The hydrogen production characters of the mutant and the original strain were different, such as, the growth lag phase and the utilization of inorganic nitrogen source, etc. This work shows a good application potential of NTG-UV combined mutation in the biohydrogen production. And the hydrogen production mechanism and metabolic pathway should be explored furthermore.

耐酸产甲烷菌的分离及其复合菌剂的研究

本文从成都龙泉垃圾填埋场和宜宾造纸厂分离到耐酸性能优良的高温产甲烷菌RY3和中温产甲烷菌SH4，并将其与实验室现有的利用不同底物的产甲烷菌配伍组合成了复合菌剂。采用活性污泥作为固体附着物，研制出了固体产甲烷菌复合菌剂。 菌株RY3的pH耐受范围为5.5～10.5，最适生长pH 6.0～8.0。菌株RY3为革兰氏阳性，长杆状，多数单生，不运动；菌落浅黄色，形状近圆形；利用H2+CO2或甲酸盐作为唯一碳源生长，不利用乙酸盐，对氯霉素非常敏感。该菌最适生长温度为55℃～65℃，最适NaCl浓度为0～2%。根据形态和生理生化特性及16S rDNA序列分析将其初步定为热自养甲烷热杆菌（Methanothermobacter thermautotrophicus）。添加RY3菌液与仅添加厌氧污泥作为接种物相比一周内可使达到最大产甲烷速率所需时间缩短三分之二，甲烷总产量提高约1.8倍。菌株SH4的生长pH范围5.5～9.5，其对酸碱具有良好的适应性，培养3天后，在初始pH值为6.0～8.0的培养基中甲烷产量相差不大，且基本达到最大产量。SH4革兰氏染色阳性，短杆状，多数单生，不运动；菌落近圆形，微黄；利用H2+CO2或甲酸盐作为唯一碳源生长，不利用乙酸盐，对氯霉素非常敏感。SH4最适生长pH 为7.0，最适生长温度为35℃，最适NaCl浓度为0～1.5%。实验表明，添加SH4菌液与仅添加厌氧污泥作为接种物相比可使产甲烷启动时间缩短三分之一，甲烷总产量亦有大幅提高。从形态和生理生化特征以及16S rDNA序列分析表明SH4为嗜树木甲烷短杆菌（Methanobrevibacter arboriphilus）。 以活性污泥为附着物，与培养基和菌种经搅拌后厌氧发酵可得产甲烷菌固体复合菌剂。固体复合菌剂的pH耐受范围为5.5～9.5，温度耐受范围为15℃～65℃，表明其对环境的适应性较强。以猪粪为底物进行厌氧发酵，接种复合菌剂进行试验，以接种实验室长期富集的产甲烷厌氧污泥作为对照，在20℃时，发酵甲烷浓度与对照基本一致，但每日产气量优于对照，第15天时接种复合菌剂的发酵瓶每日产气量是对照的1.59倍；50℃时达到最大甲烷含量所需时间比对照缩短三分之二，三周内总产气量约为对照的2.7倍，甲烷总产量约为2.8倍。以不加接种物为对照，接种复合菌剂20℃时发酵甲烷含量达到50%约需2周，对照2周内甲烷含量最高仅为4.3%；50℃时接种复合菌剂发酵仅需约1周甲烷含量便可达50%，对照则至少需要2周。 In this paper, high-temperature Methanogen RY3 and middle-temperature SH4 were isolated from Chengdu Longquan refuse landfill and Yibin paper mill. They could be used to make compound inoculum that producing methane with the existing Methanogens utilized different substrate. With using anaerobic activated sludge be solid fixture, the process had been designed to produce solid compound inoculum. Strain RY3 possessed excellent capacity of acid and alkali-tolerant. The pH-tolerant scale of RY3 was 5.5～10.5 and its optimum pH value for growth was 6.0～8.0. RY3 was G+, long-rod shape, monothetic and nonmotile, the colony was pale yellow with suborbicular-shape. Formate or H2+CO2 but not acetate was utilized by RY3 as sole C-source, and it was very sensitive to chloramphenicol. Besides, strain RY3 grew fastest at 55℃～65 and 0℃～2% NaCl. Characteristics of modality and physiology with sequence analysis of the 16s rDNA gene of strain RY3 preliminarily showed that it was Methanothermobacter thermautotrophicus. The experiments indicated that the time which began to produce methane with the highest velocity could be shortened two third by adding RY3 in one week, and the total methane production also was 1.8 times than before. Strain SH4 possessed wide scale of growing pH（5.5～9.5）and excellent ability of acclimatizing itself to acid-alkali. The methane production had no apparent difference among those cultivated in different initial pH（6.0～8.0）after three days and equaled to the maximum production basically. Cells of SH4 were G+, short-rod sharp, monothetic and nonmotile. The colony was pale yellow with suborbicular-shape. Formate or H2+CO2 but not acetate was utilized by SH4 as sole C-source, and it was very sensitive to chloramphenicol. Besides, it grew fastest at pH 7.0，55 ℃～65 and 0℃～2% NaCl concentration. The experiment indicated the time that began to produce methane could be shortening one third by adding SH4. And the total methane production also rose apparently. Characteristic of modality and physiology with sequence analysis of the 16S rDNA gene of strain SH4 demonstrated it was Methanobrevibacter arboriphilus. The activated sludge was utilized as fixture, mixed with culture medium and inocolum, that the solid compound inoculum could be produced by anaerobic fermentation. The compound inoculum could grow between pH 5.5～9.5, 15℃～65. It demonstrated the compound inoculum ha℃ve great ability of adapting to circumstance. In the experiment that making pig manure be substrate and taking the anaerobic sludge producing methane that cultured in long term in laboratory to be comparison, the concentration of methane in fermentation added compound inoculum almost equal to the comparison at 20℃, but the volume of gas production could be a little higher. The gas production everyday inoculated compound inoculum was 1.59 times to comparison. The time that the concentration of methane to maximum could be shortening by two third by adding compound inoculum, and the total gas production was 2.7 times to comprison while the total methane production was 2.8 times. If take the no inoculum be the comprasion, anaerobic fermentation added compound inoculum made the concentration of methane to 50% in 2 weeks but the comparison only to 4.3% at 20℃. The time that the concentration of methane to 50% by adding compound inoculum only need 1 week, but the comparison need 2 weeks at 50℃.

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。

阿特拉津降解菌SYSA的分离筛选和鉴定

从长期施用阿特拉津的土壤中筛选到1株能够以阿特拉津为惟一碳源生长的菌株SYSA,经生理生化特性鉴定和16S rDNA序列分析,该菌为阴沟肠杆菌(Enterobacter cloacae)。对SYSA菌的生物学特性研究表明,pH 7~8,30℃时,在以阿特拉津(20 mg/L)为惟一碳源的培养基上经146 h培养,降解率为87%。

产纤溶酶海洋微生物B5815菌株的筛选及鉴定

采用酪素平板、纤维蛋白平板的初筛和摇瓶复筛的方法从205株海洋微生物中筛选得到4株纤溶酶活性较强的菌株,其中菌株B5815产纤溶酶活性最高,平均达258 IU/mL。通过对菌株B5815的形态特征、生理生化特性的测定及16S rDNA序列分析,综合鉴定其为短小芽胞杆菌(Bacillus pumilus)。

敌敌畏降解菌的分离鉴定及降解特性研究

目的:从受有机磷农药污染的土壤中分离能降解DDVP的菌株,对其进行鉴定和降解特性研究。方法:采用DDVP为惟一碳源和能源的无机盐培养基,通过富集培养、平板划线分离得到一株优势菌,编号为DDW-1,采用形态学、生理生化和16S-rDNA序列分析对其进行鉴定,采用气相色谱测定菌株DDW-1对DDVP的降解能力,并进行底物广谱性测试和降解酶定位实验。结果:该菌株鉴定为甲基杆菌属(Methylobacterium sp.)。降解特性试验结果表明,其最佳生长条件为温度28℃,初始pH为7.0,在该条件下,500mg·L-1DDVP经过DDW-1菌株代谢3d后,降解率达63.7%。结论:菌株DDW-1能降解DDVP,该菌株产胞内酶。

多环芳烃降解菌筛选及其降解特性

通过选择性富集培养,从辽河油田稠油污染土壤4号土样中,获得了能以高浓度菲(2000 mg·L-1)为唯一碳源和能源快速生长的优势菌系和优良菌株ZL5.16S rDNA核苷酸序列分析表明,ZL5菌株归类于鞘氨醇单胞菌属.分得的菌系和菌株有较强的降解菲能力,120 h混合菌系降解了投加菲的95.28％,菌株降解了69.24％,但它们对芘的降解能力均较低.外加碳源葡萄糖可提高菌系和菌株的菲、芘降解能力,加量多,提高幅度大,但超过一定量,降解速率开始下降,表现出抑制效应.所以,应用时需控制适宜的浓度.

高温放线菌属两菌株的化学分类和分子分类研究

从云南温泉和火山口分离出两株嗜热放线菌YIM60013和YIM60032。对其形态、生理生化特性、细胞壁化学组分以及16SrDNA序列进行了研究,并与高温放线菌的已知种进行了比较,初步认为这是高温放线菌属的两个新种,分别命名为白色高温放线菌(Thermoactinomycesalbussp.nov)和云南高温放线菌(Thermoactinomycesyunnanensissp.nov)。两个新种在气生菌丝和基内菌丝上均产生单孢子,纯细胞壁和全细胞糖分析表明含meso DAP和半乳糖、阿拉伯糖、木糖及少量的葡萄糖。菌丝体自溶或非自溶,气生菌丝白色或灰色,碳源利用类型有所不同。

高效石油烃降解菌的分离鉴定及降解特性

为获得更为丰富的石油降解微生物资源,从沈抚污灌区石油污染土壤和实验室高浓度柴油胁迫土壤中筛选出了4株高效石油烃降解菌SF-422、SF-428、SF-433和SYS-1.这4株菌总石油烃(Total petroleum hydrocarbon/TPH)生物降解率为67.4%~73.6%.经过16项生理生化特性实验和16S rDNA序列分析鉴定,SF-433,SF-428,SF-422和SYS-1分别为蜡状芽孢杆菌(Bacillus cereus),木糖氧化无色杆菌(Achromobacter xylosoxidans),施氏假单胞菌(Pseudomonas stutzeri)和洋葱伯克霍尔德氏菌(Burkholderia cepacia).纯烃降解定性实验表明所筛选出的4株高效降解菌均能够利用正十六烷、苯、菲和环己烷为唯一碳源生长,其中菌株SF-428和SYS-1显示了对芳烃及环烷烃较强的利用能力.

利用16SrDNA序列对十株根瘤菌系统发育的研究

为了确定本所保存的部分根瘤菌的种属地位,我们从中选出10株菌株,对其16S rDNA进行扩增并测序,利用分子生物学软件对序列进行分析,得到根瘤菌系统发育树状图,对比相应的生理生化结果,重新定位了它们在根瘤菌系统发育中的种属地位,将生物信息学应用用于根瘤菌的种属命名具有重要的科学意义。

利用DGGE指纹图谱技术考察不同营养条件下土壤微生物群落结构的变化

利用现代分子生物学技术,结合经典方法,克服传统的分离培养缺陷,探讨在不同营养条件下土壤微生物群落的基因多样性。经过直接从土壤中抽提总DNA,并对总DNA中16S rDNA及其中V6~V8可变区序列作PCR扩增、变性梯度凝胶电泳(DGGE)分析等,发现不同处理条件下的土壤微生物的基因多样性变化与土壤微生物量的波动并不一致,说明微生物群落多样性与微生物量的关系并非线形。同时发现秸秆的添加更有利于土壤微生物群落的稳定。