山西矿区复垦土壤中解磷细菌的筛选及鉴定

【目的】矿区复垦土壤贫瘠、 有效磷含量低。解磷细菌能够将有机磷和难溶性无机磷转化为可溶性磷,促进植物对磷素的利用。因此筛选和鉴定具有解磷能力的菌株,可为解决矿区生态恢复使用的微生物肥料提供菌种资源。【方法】采用平板分离法初筛菌株,得到D/d1.5的菌株,然后以磷酸钙为磷源,通过液体发酵试验复筛菌株,挑选出解磷率高于巨大芽孢杆菌(Bacillus megaterium)As1.223的菌株。以磷矿粉和卵磷脂为磷源,液体发酵试验测定菌株的解磷能力及磷酸酶活性。进行菌株的生长试验以测定菌株温度适宜性、 耐盐性及耐酸碱性。通过形态学、 基因序列分析及脂肪酸组成分析综合进行菌株鉴定。 菌落形态观察用营养琼脂平板培养基培养;菌体形态即细胞形态及其大小采用扫描电镜观察;基因序列分析采用16S rDNA序列测定,基因在线比对采用EzTaxon数据库;使用美国MIDI公司的Sherolock全自动细菌鉴定系统对菌株进行脂肪酸组成分析。【结果】利用无机磷和有机磷平板培养基,从山西省矿区复垦区土壤样品中筛选出19株解磷微生物,其中D/d1.5的有7株。在以磷酸钙为磷源的液体培养试验中,4株菌的解磷率高于巨大芽孢杆菌As1.223,解磷率为7.89%～12.61%,最高的为菌株Y14。4株菌对磷矿粉的解磷率为0.81%～1.21%,最高的为菌株Y14。在以卵磷脂为磷源的液体培养试验中,4株菌的解磷率与酸性磷酸酶活性分别为1.79%～3.07%和24.3～28.4U/L,均高于巨大芽孢杆菌As1.223; 碱性磷酸酶活性为11.9～50.2U/L;菌株Y14的解磷率与磷酸酶活性均最高。4株菌均有较强的环境适应能力,以Y14的适应性最强。H22、 Y11和Y34与假单胞菌属(Pseudomonas sp.)同源性在99%以上,Y14与泛菌属(Pantoea sp.)有99.79%的同源性; H22、 Y11和Y34的细胞脂肪酸组成特征峰与假单胞菌属(Pseudomonas sp.)相一致,Y14与泛菌属(Pantoea sp.)相一致;H22、 Y11和Y34被鉴定为假单胞菌(Pseudomonas sp.),Y14为泛菌属(Pantoea sp.)。【结论】分离、 筛选到4株高效解磷菌,对于磷酸钙和卵磷脂的解磷率均高于巨大芽孢杆菌As1.223。4株菌分别隶属于假单胞菌属(Pseudomonas sp.)和泛菌属(Pantoea sp.)。菌株Y14无机磷与有机磷平板的D/d值分别为3.28与1.59,降解磷酸钙、 磷矿粉、 卵磷脂的解磷率分别为12.61%、 1.21%、 3.07%,酸性与碱性磷酸酶活性分别为28.4 U/L和50.2 U/L,均为4株菌里最高的,且环境适应能力最强,生长温度为20～60℃,能耐受pH 4～11的酸碱梯度和2%～7%的盐分梯度,Y14被鉴定为泛菌属(Pantoea sp.)。4株菌均具有良好的解磷能力及较强的环境适应能力,可望进一步研发成为微生物肥料生产菌种。综合D/d值、 解磷率、 磷酸酶活性和生长试验,本试验最终确定适合山西矿区复垦农田推广的高效解磷菌菌株为Y14。     

一株耐辐射考克氏菌的分离与鉴定

从10kGy60Coγ射线辐照处理的新疆戈壁土壤中分离到一株耐辐射微生物I-7R,该菌菌落为橙红色革兰氏阳性球菌。最适生长温度为30℃,(G+C)mol%含量为67.5%,不具有氨苄青霉素、氯霉素、四环素、卡那霉素、潮霉素、利福平及壮观霉素等抗性。UV辐射生存曲线显示,I-7R菌株与大肠杆菌K-12相比,具有较强的紫外辐射抗性。16S rDNA序列比较分析表明I-7R菌株与Kocuria rosea、K.erythromyxa以及K.polaris同源性达到99%。结合I-7R菌株生理生化试验结果,该株菌归属于考克氏菌属(Kocuria),并与Kocuria rosea最为相近,暂命名为K.rosea I-7R。     

一株苯酚降解菌的筛选、鉴定及其降解特性

本研究采用逐量分批驯化的方法,从造纸废水中分离得到一株能够以苯酚为唯一碳源生长的苯酚降解菌株F5-1。经形态观察、生理生化特性鉴定及16S rDNA序列分析,将该菌株鉴定为克雷伯菌（Klebsiella sp.）。该菌株能够在7h时完全降解初始浓度为100mg/L的苯酚,降解苯酚主要发生在生长对数期;在pH5.0～9.0,NaCl浓度0～80g/L,温度20～40℃范围内,菌株F5-1均可有效降解初始浓度为100～1200mg/L的苯酚;能够耐受的最大苯酚浓度为1500mg/L。本研究结果表明,F5-1菌株对处理环境条件复杂的含酚废水具有潜在的应用前景。     

一株耐碳酸盐Bacillus属细菌的鉴定与特性分析

本研究从松嫩平原pH值在10.0以上、植物难以生存的斑块状裸地中分离到一株细菌SA-5,经鉴定该菌株为革兰氏染色阴性,杆状,接触酶阳性,氧化酶阴性,菌落呈橙黄色的好氧细菌,可水解淀粉。抗逆特性分析显示,菌株SA-5可在高达275mmol/L Na2CO3或pH 12.0以及2.0mol/L NaCl培养基中生长,中性条件下生长明显受到抑制,其中对碳酸盐的耐受性远高于普通植物。根据16SrDNA序列等分析结果显示,菌株SA-5属于Bacillus属,仅与菌株Bacillus aurantiacus K1-5T相似性最高,为99%,与其余已发表菌株均低于97%。由以上结果可见,菌株SA-5是Bacillus属中一株嗜碱耐盐菌,并且菌株SA-5对耐碳酸盐基因的开发具有潜在的价值。     

广西荔浦槟榔芋软腐病病原鉴定

从广西荔浦槟榔芋栽培区患芋软腐病的发病植株中分离纯化获得一株优势明显的菌株。电镜显微下,该菌菌体为短杆状,无芽孢产生,具鞭毛。致病性测定试验发现该菌株发病症状与田间芋软腐病症状一致。对该菌株进行生理生化分析,发现其符合胡萝卜果胶杆菌Pectobacterium carotovorum subsp. carotovorum的特性。16S rDNA、icdA、mdh、mtld、proA多基因联合分析结果表明该菌株与P. carotovorum subsp.carotovorum PC1 (GenBank Accession No.CP001657.1)相似。综合该菌株致病性、形态学、生理生化等特征特性及多基因序列分析,将引起槟榔芋软腐病的病原菌鉴定为Pectobacterium carotovorum subsp. carotovorum。     

一株极端耐辐射奇异球菌的分离和初步鉴定

从放射性污染的土壤样品中分离出1株具有极端耐辐射的微生物BR501,该菌最适生长温度为30℃,不具有氨苄青霉素、氯霉素、四环素、卡那霉素和利福平等抗性。UV、γ辐射生长曲线的结果显示BR501菌株具有极强的辐射抗性。BR501菌株的16S rDNA序列与多株Deinococcus属菌株有很高的同源性,其中,与D.radiodurans菌株的16S rDNA相似性高达99%。结合BR501菌株的表型和Deinococcus属具有代表性的D.RadioduransR1菌株的特征,将该菌株归属为Deinococcus属,初步命名为Deinococcus sp.BR501。     

一株具有固氮功能的烟草根际微生物的鉴定及其初步效应

应用16S rDNA序列分析构建系统发育树,结合生理指标、生化反应,对分离自烤烟根际的固氮菌菌株N05进行了分类鉴定,并通过小区试验探讨其对烤烟生产的效应。结果表明,自生固氮菌N05属于产碱菌属（Alcaligenes）,粪产碱菌（Alcaligenes faecalis）。将固氮菌N05制成菌肥,烤烟移栽时施入（30 kg/hm2）同时施用80% 的氮肥（B+80% N）,与全量施用氮肥（FN）相比,B+80% N烤烟根际固氮菌的数量平均提高3.6倍,放线菌的数量显著降低;圆顶期烤烟根际土壤中除Mg 元素的有效性略有降低外,P、K、Ca、Cu、Zn、Fe和 Mn 等元素的有效性均有不同程度的提高,提高幅度在 2.51%～ 46.08%。烤烟杀青样中 N 的平均含量也高于FN。在减施氮肥的情况下,应用固氮菌肥可提高烤烟根际固氮菌数量和矿质元素的有效性。     

产低温淀粉酶菌株的筛选、鉴定及酶学性质初步研究

对低温淀粉酶资源的开发是功能酶研究的新热点,本研究从东帕米尔高原江布拉克冰川冻土中分离获得多株高产低温淀粉酶菌株,最高产酶菌株D5-2经16S rDNA鉴定为假单胞菌属(Pseudomonas sp.)细菌.对D5-2所产淀粉酶性质进行初步分析,结果表明,其最适作用温度和pH值分别为25 ℃和6.5,酶的热稳定性较差,40 ℃处理1h后酶活急剧下降,至70℃酶活力仅存23％;在pH 5.5～8.0条件下,酶活力相对稳定;Mn2+和Mg2+对淀粉酶有激活作用,而乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)、Fe2+和Na+则抑制酶活.结果提示,菌株D5-2分泌的淀粉酶符合低温酶特性,应用空间较大,值得深入探究开发.     

利用高通量测序对三江平原小叶章湿地土壤细菌多样性的研究

利用黑龙江省科学院自然与生态研究所三江平原野外实验研究站内3个不同小叶章生态类型湿地土壤样品,直接提取土壤微生物总DNA,应用Miseq测序技术对16S rDNA进行序列测定和分析。结果表明:不同小叶章湿地土壤细菌群落结构发生了显著变化,土壤细菌多样性和丰富度随着土壤含水率的增加而降低。草甸化湿地和沼泽化草甸湿地优势种群为酸杆菌,变形菌次之;沼泽化湿地优势种群为变形菌,酸杆菌次之。土壤含水率的增加减少了酸杆菌的分布,而增加了变形菌的分布。16S rDNAheatmap分析则表明,湿地水位的变化对酸杆菌和变形菌的群落结构影响最大。     

养殖欧洲鳗鲡不同体位及其养殖水体中的菌群组成研究

为探明养殖欧洲鳗鲡不同体位及其养殖水体中可培养菌群的组成结构,本研究利用16S rDNA序列分析法对从精养池鳗鲡的鳃部、肠道和表皮及其养殖水体中分离得到的可培养细菌进行了分子鉴定并构建了系统发育树。研究结果显示,鳃部、肠道、表皮和水体的菌密度分别为1.6×10^6 cfu/g、2.2×10^7 cfu/g、1.4×10^4cfu/cm2和4.5×10^3 cfu/mL;分离菌株分别属于γ-变形菌纲的肠杆菌属、不动杆菌属、栖水菌属,β-变形菌纲的食酸菌属,芽孢杆菌纲的葡萄球菌属、气球菌属,黄杆菌纲的金黄杆菌属和放线菌纲的微球菌属等5大类8个菌属。其中,微球菌属、肠杆菌属和栖水菌属分布最广,各样品中均有检出;而气球菌属和食酸菌属仅在鳃部分布。各生态位中,鳃部菌群最为多样,含有葡萄球菌属之外的7个属;而水体菌群种类最少,只有4个属。此外,菌群组成含量的分析结果表明,鳗鲡鳃部以金黄杆菌属（28.3%）和肠杆菌属（26.1%）居多;而肠道、表皮和养殖水体都以微球菌属占绝对优势,分别为43.6%、53.5%和74.8%。     

Effect of Application of Rice Straw and Compost on the Bacterial Communities Associated with Moina sp. in the Floodwater of a Paddy Soil Microcosm: Estimation Based on DGGE Pattern and Sequence Analyses

Bacterial communities associated with Moina sp. in the floodwater of a paddy field microcosm were examined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA. Eighteen out of 20 eubacterial DGGE bands were sequenced. The associated eubacterial communities mainly consisted of the Cytophaga-Flavobacterium-Bacteroides group and α-, β-, and γ-Proteobacterial groups, irrespective of the application of rice straw and rice straw compost. The effect of the application of rice straw and compost on the communities was not appreciable, compared with host specificity. An uncultured Cytophagales bacterium was estimated to be specifically associated with Moina sp. Presence of bacteria that are specific to rice straw treatment was also estimated.     

Structural differences in the rhizosphere communities of legumes are not equally reflected in community-level physiological profiles

The relationship of structural diversity and differences in the functional potentials of rhizosphere communities of alfalfa, common bean and clover was investigated in microcosms. PCR-SSCP (single strand conformation polymorphism) analysis of 16S rRNA genes revealed significant differences in the composition of the leguminous rhizosphere communities at the shoot stage of plants grown in the same soil. Sequencing of dominant SSCP-bands indicated the presence of plant specific organisms. The partial rRNA gene sequences were related to members of the α- and γ-Proteobacteria, Bacteroidetes and Actinobacteria. Besides the plant species, the soil also affected the structural diversity in rhizospheres. The dominant bacterial populations of alfalfa grown in soils with different agricultural histories were assigned to different taxonomic groups. Addressing the functional potentials, community-level physiological profiles (CLPP) were generated using BIOLOG GN^®. The three leguminous rhizosphere communities could be differentiated by principle component analysis, though the overall analysis indicated that the metabolic potential of all rhizosphere samples was similar. The functional variation examined in rhizospheres of alfalfa was minor in response to the soil origin and was found not to be significant different at different growth stages. The results indicate that similar functional potentials may be provided by structurally different bacterial communities.     

Molecular Monitoring of SRB Community Structure and Dynamics in Batch Experiments to Examine the Applicability of in situ Precipitation of Heavy Metals for Groundwater Remediation (15 pp)

Background, Aims and Scope   Sulfate-reducing bacteria (SRB) are known for their capacity to reduce and precipitate heavy metals (HM) as metal sulfides, offering the opportunity to create an in situ reactive zone for the treatment of heavy metal-contaminated groundwater, a process called in situ metal precipitation (ISMP). The applicability of the ISMP technology first has to be investigated at a laboratory scale before going into an on site application. The evaluation and optimization of the ISMP process is facilitated when physical/chemical analysis techniques are combined with molecular tools that specifically monitor the abundance, diversity and dynamics of the indigenous sulfate reducing microbial community. In this study, batch experiments were conducted in order to investigate the feasibility of ISMP as a groundwater remediation strategy for an industrial site contaminated with elevated levels of Zn, Cd, Co and Ni. Methods   The potential of different types of carbon source/ electron donor (lactate, acetate, methanol, ethanol, Hydrogen Release Compound?, molasses) to stimulate the sulfate reduction and metal precipitation activity of the naturally present (or indigenous) SRB community was explored. In addition, the effect of amending vitamin B12 and yeast extract was evaluated. The ISMP process was monitored by combining analytical analyzes of process parameters (SO42-concentration, heavy metal concentrations, pH, Eh) with molecular tools such as SRB subgroup and genus specific PCR, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis of clone sequences, based on either the 16S rRNA or the dsr (dissimilatory sulfite reductase) gene. Results and Discussion   The efficiency of different carbon-sources to stimulate the ISMP process followed the order HRC 〉 molasses 〉 methanol 〉 lactate 〉 ethanol 〉 acetate. Within 10 weeks, the highest sulfate and metal removal efficiencies ranged from 85% to 99%. Addition of yeast extract boosted the ISMP process, whereas vitamin B12 negligibly affected SRB activity. Analysis of the sulfate reducing population by SRB subgroup and genus specific PCR demonstrated that members of the genus Desulfosporosinus dominated in all batch tests, while 16S rDNA DGGE profiles additionally revealed the presence in the microbial communities of non-sulfate reducing bacteria within the family Clostridium and the -proteobacteria. The dsrB-based DGGE profiles allowed us to assess the diversity and dynamics of the sulfate reducing community and added to a better understanding of the effects of different batch conditions on the ISMP process. Remarkably, all dsrB sequences affiliated with the dsrB gene sequence cluster found in Desulfotomaculum, which received their xenologous dsrB gene from the -proteobacteria. Conclusions   The batch experiments, which aimed at stimulating the activities of the indigenous SRB communities, demonstrated that these communities were present and that their activities could be used to obtain efficient in situ precipitation of the contaminating heavy metals. This opens the possibility to test this concept in the future as an on site demonstration as part of the groundwater strategy for the heavy metal contaminated site. Although batch setups are suitable for preliminary feasibility studies for ISMP, they do not reflect the in situ situation where sulfate and heavy metal and metalloid polluted groundwater are supplied continuously. A sulfate reducing strain JG32A was isolated from whose 16S rRNA gene affiliated with the genus Desulfosporosinus, while its dsrB gene sequence clustered with Desulfotomaculum dsrB gene sequences, which received their xenologous dsr genes from -proteobacteria. Therefore we hypothesize that the batch experiments enrich members of the Desulfosporosinus genus that possess a non-orthologous dsrB gene. Recommendation and Perspective   The next step towards an on site pilot test for ISMP will be the setup of a series of column experiments, with process conditions that are selected based on the above mentioned results. This will allow to define optimal ISMP process conditions and to test its long-term efficacy and sustainability before going into an on site bioremediation application. By applying the described molecular tools together with physical-chemical analyzes, it can be investigated whether the same SRB community is enriched and which type of C-source is most effective in promoting and sustaining its growth and sulfate-reduction activity.     

Distribution and biodiversity of soybean rhizobia in the soils of Shennongjia forest reserve,China

Soil samples were collected at an altitude of 500, 1,060, 1,500, 1,950, 2,400 and 3,100 m, respectively, from Shennongjia, a forest reserve in Hubei province (central China). Their corresponding pHs were 5.50, 4.91, 5.64, 5.28, 5.49 and 4.60. By using a plant trap method, a total of 25 soybean rhizobia were isolated from the soil above an altitude of 1,500 m and all identified to be Sinorhizobium fredii. Their genetic biodiversity was characterized by 16S–23S rDNA internally transcribed spacer (ITS) region polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and random amplification DNA (RAPD) analysis. All the tested strains produced a 2.1 kb 16S–23S rDNA ITS fragment. After digestion with three restriction endonucleases (HaeIII, MspI and CfoI), respectively, great variations in 16S–23S rDNA ITS PCR-RFLP patterns were observed. The tested strains could be differentiated into 11 ITS genotypes. The genotypes of rhizobia were not related to geographical location. Twelve primers were applied to RAPD analysis and a dendrogram was obtained, showing that all the strains (including reference strain S. fredii USDA205) were divided into two diverging groups. Moreover, each group could be further divided into two subgroups. Both RAPD and 16S–23S rDNA ITS PCR-RFLP analysis indicated that a high degree of genetic diversity existed among S. fredii strains isolated from Shennongjia virgin soils. Since Shennongjia is an unexploited forest region in central China and the gene centre of soybean is located in China, the symbiotic genes harboured by these strains may be of great importance and the rich diversity of these strains might contribute to the adaptation of soybean to an alpine environment.     

西藏妥昌公路K351滑坡形成机制及危险性评价

对滑坡成因机制及稳定性分析是一个由表及里、由微观到宏观分析的过程。通过对K351滑坡的表面形态特征、滑坡内部岩性结构面的分析,从滑坡的微观形态（如裂缝）、宏观形态（如滑坡后壁、滑坡前缘）出发,结合当地的地形地貌条件,对K351滑坡的形成特征、运动特征进行了探讨。并运用滑坡危险度判别模型对滑坡的危险程度进行了评价。