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。

芘降解菌在石油污染土壤修复过程中的代谢调控

沈抚灌区是我国面积最大、污灌历史最长的石油类污水灌溉区，土壤中大分子量多环芳烃污染严重，对当地粮食生产与生态安全造成严重危害。对此类污染土壤进行生物修复，对保证农产品的安全，实现当地人与自然的可持续发展具有重大的意义。 本研究以沈抚灌区污染土壤中大分子多环芳烃芘为主要研究对象，采用稳定同位素比率分析技术（IRMS），以磷脂脂肪酸（PLFA）为生物标记物，分析污染土壤参与芘降解的优势微生物类群；并以此为指导，采用分子生物学手段和传统微生物学分析方法，筛选土壤中的高效降解菌，并追踪其释放到土壤中后的动态变化与调控。 从沈抚灌区土壤富集培养芘的降解菌，经过双层平板法初筛和芘降解菌液体摇瓶复筛，获得5株以芘为唯一碳源生长的具有较高降解活性菌株。 将筛选的降解菌投加到污染土壤中，以13C标记的芘为代谢底物，以土壤微生物的磷脂脂肪酸为生物标记物，采用稳定同位素比率分析方法(GC-C-IRMs)，分析投加的降解菌在原位土壤中的降解作用。结果显示，与不加菌的对照土壤相比，富含13C的磷脂脂肪酸指纹图谱相似度较高的为投加了菌株B05和菌株B15的土壤，芘的降解效率也最高，表明这两株菌在原位土壤芘降解中发挥了重要作用。根据形态学观察、16项生理生化鉴定和16S rDNA序列分析结果，将菌株B05鉴定为 Aminobacter ciceronei，将菌株B15鉴定为 Microbacterium arabinogalactanolyticum。菌株B05初步确定为一株新的芘降解菌，并对菌株培养条件进行了优化。 采用PCR-DGGE方法，研究了筛选的5株降解菌在不同的营养条件下释放到土壤中后的数量和代谢活性的变化。PCR-DGGE图谱分析表明：投加初期外加菌在竞争中占据优势，但是随时间推移，营养物质的消耗，优势逐渐消失，PCR-DGGE的条带趋向于一致。菌株B05的稳定期相对较长，在DGGE图谱中的条带相对密度大，而且对芘的降解率最高，是一株具有潜在应用价值的高效降解菌。混合菌比单一菌降解率高，添加碳氮源有利于外加菌群更快更好的适应在污染土壤中生存，而且有助于对多环芳烃的降解。

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）。

嗜盐和嗜碱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意义和进展，分析了放线菌分类学研究特点及发展趋势；同时综述了嗜盐、嗜碱放线菌的研究进展，分析了嗜盐和嗜碱放线菌研究的重要性和意义。通过对采自渤海沿岸盐场、青海盐湖、张北盐湖、曲周、保定等地盐碱和高盐的样品进行嗜盐和嗜碱放线菌的分离，建立了适于嗜盐和嗜碱放线菌的分离培养条件；找到了广适性的嗜盐放线菌培养用培养基；发现了嗜盐放线菌多数耐碱的规律。采用形态培养特征、生理生化测定、细胞化学组分分析、DNA G＋C mol％测定以及DNA同源性分析和165 rDNA序列为基础的系统发育分析等相结合的多相分类技术对分离得到的部分典型菌株系统地进行了分类研究，从表型、基因型和系统发育三个层次对其分类地位进行了确定。本研究发现嗜盐放线菌新属新种1个，为波状盐场放线菌新属新种（Actinoosalterria undulata gen.nov．sp．nov），嗜盐放线菌新种4个，分别是：沧州拟诺卡氏菌(Nocardiopsl's cangzhouensis sp．nov．）、塘沽拟诺卡氏菌(Nocardiopsis tangguensis sp.nov）、茶卡拟诺卡氏菌(NocardioPs沽chakaensis)和盐场链霉菌(Streptomyces salinarum sP.nov.）；发现嗜碱放线菌新种2个，分别是；布尔津拟诺卡氏菌（NocardioPsis buerjinensis sp.nov．）和城墙拟诺卡氏菌（Nocardiopsis rampartis sp.nov．）；另有2株嗜碱放线菌鉴定为达松维尔拟诺卡氏菌，2株嗜碱放线菌鉴定为产气味拟诺卡氏。论文研究中还初步建立了放线菌磷酸类脂高效液相色谱测定的方法与测定条件。

基于ITS 序列和matK 基因序列的中国药用石斛及其混伪品的分子鉴定

为了从分子水平对中国药用石斛及其混伪品进行鉴定，本文选取了核rDNA ITS 序列和叶绿体DNA 的matK 基因序列进行研究。采用改良的CTAB 法提取石斛的基因组DNA，PCR 产物直接测序法对17 种（共32 份）药用石斛的核糖体内转录间隔区ITS 全序列进行测定，克隆测序法对12 种（共22 份）药用石斛的叶绿体的matK 基因序列进行测定，运用BioEd it，MEGA4.0 等生物软件分析了石斛属植物的rDNA ITS 序列及叶绿体的matK 基因序列的特征，比较了石斛属间、种间、种内不同居群（品种）间的序列碱基差异及遗传距离，应用邻接法构建分子系统树。主要研究结果如下： （1）建立了17 种（共32 份）药用石斛rDNA ITS 区碱基全序列数据库，其中，ITS1 的长度为228~234 bp，GC 含量为45.7%~53.0%，变异位点167 个，占总位点67.34%，信息位点106 个，占总位点42.74%，ITS2 长度为241~247 bp，GC含量为44.8%~55.7%，变异位点165 个，占总位点66.27%，信息位点115 个，占总位点46.18%。 （2）建立了12 种（共22 份）药用石斛的叶绿体matK 基因全序列数据库，叶绿体matK 基因长1410 bp，变异位点51 个，信息位点11 个。除了存在碱基替换的遗传变异外，还存在碱基的插入和缺失。 （3）通过ITS 序列比较分析了各材料间的遗传距离和碱基差异，属间的遗传距离为0.295，石斛种间的平均遗传距离为0.142，碱基相差2~156 个，种内各居群间的平均遗传距离为0.002，碱基相差1~2 个。属间的遗传距离大于种间的遗传距离，种间的遗传距离大于种内不同居群（品种）间的遗传距离。 （4）根据分析石斛叶绿体的matK 基因序列得到，外类群（密花石豆兰）与石斛属间最小遗传距离为0.027，石斛种间的平均遗传距离为0.008，种间最大的遗传距离0.014， 最小的遗传距离为0.003，碱基相差8~20 个。种内不同居群（品种）遗传距离为0.001，相差1~5 个碱基。 （5）利用17 种石斛的全序列数据库及遗传分析软件，通过对待检种rDNA I TS区进行序列测定，成功地对10 个待检种进行了鉴定，并且在原植物开花后得到了验证。 （6）运用12 种石斛的matK 基因全序列数据库及遗传分析软件，成功地对4个待检种进行了鉴定，同样在原植物开花后得到了验证。 （7）本文利用石斛的核糖体内转录间隔区ITS 序列和叶绿体的matK 基因序列数据库分别构建了NJ 树，外类群与石斛属间石斛种间以及种内不同居群（品种）间均能在NJ 树中明显分化开来，二者构建的分子系统树一致，为石斛的分子鉴定提供了依据。 In order to identify Chinese Herba Dendrobii and its adulterant species on molecular level, we studied the sequences of rDNA ITS and chloroplast matK gene. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materia ls) were purified and then sequenced. The PCR products of chloroplast matK gene of Dendrobium (22 materia ls) were purified, cloned and then sequenced. The characteristic of the sequences and the genetic dista nce were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies, and different populations. Phylogenetic trees were constructed using the NJ method by the biology softwares including BioEd it, MEGA4.0 etc. The ma in results as follows: (1) It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materia ls). The ITS1 was 228~234 bp, the GC content accounting for 45.7%~53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241~247 bp, the GC accounting for 44.8%~55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. (2) The database of the chloroplast matK gene sequences was built up, which contained 12 species of Herba Dendrobii (22 materia ls). The matK gene sequences were about 1410bp in length. There were 51 variable sites and 11 Parsim-Informative sites. And there were nucleotides insertions and deletions in some species , in addition to the nucleotides substitutions. (3) The rDNA ITS sequences were compared and analyzed by the biology softwares. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was 0.295. The avera ge genetic dista nce was 0.142 between Dendrobium species, and there were 2~156 variable nucleotides. The avera ge genetic dista nce between different populations was 0.002, and there were 2~156 variable nucleotides. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was greater tha n that of Denrobium interspecies. Meanwhile, the genetic dista nce between Denrobium species was also greater tha n that of different populations (variaties). (4) The characteristics of the chloroplast matK gene sequences were obtained after analyzing by the biology softwares. The minima l genetic dista nce was 0.027 between Bulbophyllum odoratissimum and Dendrobium . The ma xima l genetic dista nce was 0.014 between Dendrobium species, and there were 20 variable nucleotides. The minima l genetic dista nce between populations was 0.003, and there were 8 variable nucleotides.The genetic dista nce between populations was 0.001, and there were 1~5 variable nucleotides. (5) The molecular Phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then we authenticated 10 materia ls on molecular level. What’s more, they had been proved when these pla nts flowered. (6) The molecular Phylogeny tree was built up on the database of chloroplast matK gene sequences of 12 species of Herba Dendrobii with the biology softwares.Then 4 materia ls were authenticated on molecular level. Moreover, they had also been proved when these pla nts were in flower. (7) The Phylogenetic trees were separately constructed on the sequences of rDNA ITS and chloroplast matK gene B. odoratissimum and Dendrobium all could be distinguished on the Phylogenetic trees. Meanwhile, the Phylogenetic trees based on two groups of sequences were coincident. rDNA ITS and matK gene sequence could be used as molecular markers for authentication of Herba Dendrobii.

白腐菌选择性降解木质素的研究

对15株白腐真菌进行了以玉米秸秆为基质的初步筛选，从中获得一株选择性系数较高的菌株Y10，并对其降解玉米秸秆的情况进行了研究。结果表明，在30天的培养过程中菌株Y10对玉米秸秆降解的选择性系数都大于1，第15天选择性系数最高为3.88。对未经降解和降解过的玉米秸秆分别作了紫外光谱和红外光谱分析，结果表明，经该菌降解后玉米秸秆的化学成分发生了很大变化，且木质素的降解程度要大于纤维素的降解程度。对菌株Y10进行了ITS-5.8S rDNA序列鉴定，初步判定其为Cerrena sp.。 为了考查不同的外源添加物对菌株Y10降解玉米秸秆的影响，在以玉米秸秆为基质的固态发酵培养基中分别添加了7种金属离子、8种碳源、6种氮源。结果显示，这7种金属离子均能促进木质素的降解，并且一定浓度的某些离子明显抑制纤维素的降解；其中添加0.036%的MnSO4·H2O和0.36%的MgSO4·7H2O对纤维素降解的抑制作用比较强，降解率分别为0.96%和1.31%，木质素的选择性系数分别达到了34.40和20.17。8种碳源中除麦芽糖外都能促进木质素的降解，除微晶纤维素外都明显促进纤维素的降解。6种氮源中酒石酸铵、硫酸铵、草酸铵和氯化铵的添加都会使该菌生长变慢，而且氮源浓度越高菌丝生长越慢。外加碳源和金属离子对半纤维素降解和选择性系数的影响不大。 同时对菌株Y10在液态培养下产木质素降解酶的条件和培养基做了优化。结果表明，在初始产酶培养基中，菌株Y10的漆酶酶活在第10d达到最高，锰过氧化物酶酶活在第11d达到最高，基本上检测不到木质素过氧化物酶。菌株Y10产漆酶的最适温度为32℃，最适PH为6.0；产锰过氧化物酶的最适温度为32℃，最适PH为6.5。菌株Y10产漆酶的最佳碳源为甘露糖，最佳氮源为酒石酸铵，最适诱导剂VA浓度为3 mmol/L，最适表面活性剂TW-80浓度为1%。 利用响应面法对其产漆酶的培养基进行优化，优化后的培养基配方为葡萄糖10.00 g/L，酒石酸铵0.50 g/L，大量元素296.50 ml/L，微量元素100.00 ml/L，NTA 1.40 g/L，VA 5.00 mmol/L，吐温-80加入量为0.10%。进行了菌株Y10产漆酶的验证实验，实测酶活为5282.56 U/L，与预测酶活5162.73 U/L接近。在优化后培养基中，菌株Y10在第14 d达到生长的最高峰，第20 d时，漆酶酶活最高，为11325.00 U/L；第16 d时，锰过氧化物酶酶活最高，为30.77 U/L。 对菌株Y10的漆酶酶学性质做了初步的研究，结果显示，酶反应的最适温度为40℃-65℃，最适PH为3.0。在40℃，PH=3.0时，漆酶催化ABTS反应的米氏方程为 。 Fifteen white-rot fungi based on corn stalk were screened. One white-rot fungus Y10 with high selectivity value was obtained. The degradation of corn stalk was initially studied. The results indicated that the selectivity value was above 1 during the 30 day-cultivation and the highest was 3.88 after 15 days. The composition of untreated and treated stalk was analyzed through ultraviolet spectroscopy and infrared spectroscopy. It was found that the composition of treated stalk was greatly altered and the degree of the degradation of lignin is greater than the cellulose. Y10 was identified as Cerrena sp. by ITS -5.8S rDNA sequence analysis. The influence of metal ions, carbon sources and nitrogen sources on corn stalk degradation by white-rot fungus was studied. While all seven metal ions could promote lignin degradation, the cellulose degradation was best inhibited at certain ion concentrations. Notably, when 0.036% MnSO4·H2O and 0.36% MgSO4·7H2O were added into the medium, the cellulose degradation was restrained to the extents that the coefficients of lignin selectivity rose to 34.40 and 20.17 respectively. It was also found that all carbon sources except maltose can promote lignin degradation. The addition of carbon sources other than microcrystalline cellulose significantly promoted cellulose degradation. The addition of the nitrogen sources, ammonium tartrate, ammonium sulfate, oxalate, ammonium chloride, resulted in remarkable inhibition to mycelium growth; the larger the concentrations of nitrogen sources are, the slower the mycelium grew. The addition of carbon sources and metal ions had less impact on the degradation of hemicellulose and selectivity value. Meanwhile, we optimized the conditions and culture medium of the lignin-degrading enzyme production of strain Y10. The results showed that in the initial culture medium, the Lac activity was highest at the 10th day, the MnP activity was highest at the 11th day and the LiP could not be detected. The optimum condition of Lac was at temperature 32 and PH =6.0 and the optimum condition of MnP was at temperature 32 and PH =6.5. The optimum carbon source for Lac was seminose, the optimum nitrogen source was ammonium tartrate, the optimum content of VA was 3 mmol/L, the optimum content of TW-80 was 1%. PB and RSM were used to optimize the culture medium of laccase by white-rot fungus Y10. The optimum culture medium was consist of glucose 10.00 g/L, ammonium tartrate 0.50 g/L, macro elements 296.50 ml/L, trace elements 100.00 ml/L, NTA 1.40 g/L, VA 5.00 mmol/L, TW-80 0.10%. Under the optimal conditions, the activity of laccase was 5282.56 U/L and the experimental value agreed with the predicted value 5162.73 U/L. The biomass was highest at the 14th day, the Lac activity was highest at the 20th day, the MnP activity was highest at the 16th day. The results of the studies on the characteristics of Lac showed that the optimum temperature for Lac activity is 40℃-65℃ ; the optimum PH for Lac activity is 3.0 and under 40℃，PH=3.0, the Michaelis-menten equation of Lac catalized ABTS oxidation was .

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)。