海南西海岸四种真红树根系土壤放线菌物种多样性及其延缓衰老活性初筛

为更好地开发利用海洋微生物资源积累丰富的放线菌菌种,该文以海南西海岸潮间带区域四种真红树根系土壤为研究对象,分析了红树林根系放线菌物种多样性组成及其代谢产物活性。该研究以9种不同培养基为分离介质,采用纯培养方法和三区划线法分离纯化菌株,结合放线菌形态学特征及其16S rRNA基因序列结果开展多样性分析。放线菌发酵液经乙酸乙酯萃取,利用秀丽隐杆线虫(Caenorhabditis elegans)测试其延缓衰老活性。结果表明:(1)共分离获得22株放线菌,隶属于4目7科9属,其中链霉菌属(Streptomyces)为优势菌群,并初步确认IMDGX 6012、IMDGX 6028、IMDGX 6118、IMDGX 6326、IMDGX 61195株放线菌可能为潜在的新物种。(2)发酵产物延缓衰老研究结果表明,8株放线菌的代谢产物可显著延长线虫寿命(P<0.05);其中,IMDGX 6028和IMDGX 6118作为拟无枝酸菌属(Amycolatopsis)和短小杆菌属(Curtobacterium)的潜在新物种,具有极显著延缓衰老活性(P<0.01),与空白组比较,可分别延长线虫寿命的22.2%和26.6%。综上结果表明海南西海岸真红树根系土壤具有丰富的可培养放线菌资源,具有发现放线菌新物种和延缓衰老活性菌株的潜力。

TMAH浓度对生物硝化系统中微生物群落多样性的影响

本研究以高科技产业常用化学品氢氧化四甲基铵(Tetramethylammonium hydroxide, TMAH)作为研究目标,以生物处理方法进行TMAH生物急毒性减量,期能解决高科技产业废水生物急毒性议题。针对含TMAH的工艺废水,以好氧(Aerobic)反应槽进行探讨,设计TMAH浓度分别为0、1000、3000 mg∙L−1三个反应槽;实验结果,TMAH 0 mg∙L−1组呈现良好的硝化反应,微生物结构组成中浮霉菌门 (Planctomycetes) Gemmata属的比例比其他两组高。TMAH 1000 mg∙L−1组与TMAH 0 mg∙L−1组相比,TMAH 1000 mg∙L−1组增加了TMAH浓度的变异性,因而带入新的真菌物种(如:变形菌门(Proteobacteria) Brachymonas属、拟杆菌门(Bacteroidetes) Flavobacterium属),所以菌种丰富度也明显提高。TMAH 3000 mg∙L−1组菌种分布受到诸多因素(TMAH浓度高、硝化作用抑制)的综合制约影响,物种总量和丰度具有特异性,其中以变形菌门(Proteobacteria)中的Reyranella属数量明显比TMAH 0 mg∙L−1组及TMAH 1000 mg∙L−1组高。

复合系WSC-6的菌种组成特性及其木质纤维素分解能力

利用平板分离法与变性梯度凝胶电泳(DGGE)法研究了高效分解木质纤维素的微生物复合系WSC-6的菌种组成,平板法分离得到了9株好气性的细菌,它们与Pseudoxanthomonas taiwanensis、Tepidiphilus margaritif er、Bacillus sp.E53-10、Proteobacterium S072、Beta proteobacterium HMD444、Rhizobiaceae str.M100、Petrobacter succinimandens BON4、Bacillus thermoamylovorans、Paenibacillus sp.SAFN-016的相似率分别达到了97.5%,99.0%,98.4%,100%,98.9%,99.3%,98.1%,99.5%,99.8%。利用DGGE分析表明,复合系中还存在利用平板法难以培养的5株厌氧或者兼性厌氧细菌,它们的16SrDNA V3区的序列与Ureibacillus thermosphaericu、Clostridium thermosuccinogenes、Clostridium thermopalmarium、Uncultured Clostridium sp.clone A1-3、Uncultured bacterium tbr4-24具有很高的相似率。在50℃静置培养条件下,接种3d后复合系WSC-6可以分解添加稻秆总量的81.3%。好氧菌与厌氧菌共存于复合系中,复合系表现出了高的细菌组成多样性,从而保证了复合系具有强的稳定性和多菌协同分解木质纤维素的能力,明确菌种组成多样性对于研究复合系高效分解木质纤维素的机理意义重大。

多功能细菌复合系NSC-7的菌种组成多样性

NSC-7是一组具有降解纤维素和林丹双重功能的细菌复合系.为系统了解复合系的菌种组成,在有氧条件下,利用传统的平板画线法分离到11株单菌,将11株单菌按体积比1∶1重新组合并不具备分解纤维素的能力,利用单层和双层平板滤纸法检测NSC-7的分解能力,发现只有双层平板上的滤纸变黄且降解,说明复合系内纤维素降解的关键菌是厌氧或微好氧菌.利用现代分子生物学技术对NSC-7构建克隆文库,获得了195个16SrDNA片断,经DGGE筛选获得25个代表克隆,其序列数据库比对结果中有60%的近缘种为已知菌,分别归属于Clostridium、Petrobacter、Bacteria、Paenibacillus、Proteobacterium 5个属,其余40%的近缘种为难培养菌株.

玉米秸秆低温降解复合菌系降解能力及微生物组成研究

根据还田秸秆配施尿素的生产实际,对玉米秸秆低温高效降解复合菌系GF-20进行氮源培养基驯化,探明其菌种组成和功能多样性及其与菌源菌种结构差异,完善复合菌系筛选方法,促进其开发利用。本文以低温高效降解复合菌系GF-20为研究对象,在硫酸铵和尿素不同配比下连续继代培养10代,获得不同氮源菌系(硫酸铵处理N1,硫酸铵和尿素混合处理N2-N5,尿素处理N6),测定其玉米秸秆降解率,评价复合菌系秸秆降解效率;采用MiSeq高通量测序对菌源土壤样品及不同氮源下继代培养的复合菌系菌种组成和功能多样性进行研究。结果显示N2处理玉米秸秆降解率显著高于其他处理;菌源土壤的Alpha多样性指数显著高于继代培养后的复合菌系,不同处理间N2处理显著高于其他氮处理;菌源和复合菌间以及不同氮处理间菌种组成具有显著差异,N2处理菌种组成多样性较高,菌群结构更加丰富、均匀,且碳水化合物的代谢通路丰度较高。菌源经限制性继代筛选后得到了参与玉米秸秆降解过程的功能菌,能有效提高秸秆降解率,其在硫酸铵和尿素氮源为0.16%+0.04%的条件下,菌系的秸秆降解效率较高,这为复合菌的生产实际开发利用提供了理论依据。

Investigation of Macrofungi in Wula Mountain National Forest Park at Yinshan Mountains

[Objective] This study aimed to investigate the fungi in Wula Mountain National Forest Park. [Method] More than 180 fungal specimens were collected from Wula Mountain National Forest Park from 2009 to 2012 for primarily studying the fungal species diversity. [Results] According to the classification system presented by Ainsworth et al. and with reference to the China catalogue of Macrofungi in species diversity catalogue of Fungi at Wula Mountain National Forest Park was written, involving in 80 species belonging to 43 genera, 22 families, 5 orders and 2 classes in the Basidiomycotina, and 6 species belonging to 2 genera, 2 families, 2 orders and2 classes in Ascomycotina, totally from 86 species, 45 genera, 7 orders, 24 families and 2 subdivisions. Among them, 49 species were edible and 22 species were medicinal, and 18 species were both edible and medicinal, and 7 species were poisonous, and 32 species were wood-rotting, and 5 species were mycorrhizal fungi,and 3 species were newly-recorded ones in Inner Mongolia. [Conclusion] There are still some specimens that have not been identified yet because of lack of literature,thus requiring further study for supplement.

Isolation and Identification of Soil Actinobacteria from Mangrove forest in Beihai,Guangxi Province

[Objective] The aim was to identify the species diversity of Actinomycetes from Mangrove forest in Beihai,Guangxi Province. [Method] 10 strains of typical Actinomycetes were isolated from Mangrove forest soil,and the Actinomycetes genomic DNA was successful extracted. 16S rDNA was amplified by PCR and sequenced by Sanger dideoxy sequencing method. [Result] All the sequences were blasted in genbank,eight strains belonged to the genus of Streptomyces (80%),and two strains belonged to the genus of Nocardiopsis (20%). [Conclusion] There are many different Actinomycetes species in Mangrove forest soil samples in Beihai,Guangxi Province.

Genetic Diversity of JUNCAO Germplasms Detected by i PBS Markers

[Objective] This study aimed to carry out a preliminary analysis of genetic diversity of 47 JUNCAO germplasms. [Methods] Twenty-eight iPBS （Intel Primer Binding Site Amplification） primers were firstly used for PCR screening on a subset of four germplasms, of which 11 gave good amplification patterns and were then used for analyzing the DNA of 47 JUNCAO germplasms. [Result] A total of 208 polymorphic DNA fragments were scored among the 47 JUNCAO germplasms from the electrophoresis patterns of the 11 selected iPBS primers. By using the NTSYSpc 2.1 software combined with UPGMA clustering analysis method, the simple matching （SM） coefficient of similarity was calculated among all accessions and ranged from 0.58 to 0.99. The 47 JUNCAO germplasms were clustered into 10 categories at a genetic similarity coefficient of 0.67. All the 47 accessions were distinguished from each other. [Conclusion] Our results showed that iPBS markers could be effectively used for genetic diversity analysis of JUNCAO germplasms. This study provides a preliminary theoretical guidance and technical support for the efficient management and utilization of JUNCAO germplasm resources.

Isolation and identification of bacteria associated with the surfaces of several algal species

We conducted this study to assess the diversity of bacteria associated with the surfaces of algae based on 16S rDNA sequence analyses.Twelve strains of bacteria were obtained from the surfaces of the following four species of algae:Gracilaria textorii,Ulva pertusa,Laminaria japonica,and Polysiphonia urceolata.The isolated strains of bacteria can be divided into two groups:Halomonas and Vibrio,in physiology,biochemical characteristics and 16S rDNA sequence analyses.The phylogenetic tree constructed based on 16S rDNA sequences of the isolates shows four obvious clusters,Halomonas venusta,Vibrio tasmaniensis,Vibrio lentus,and Vibrio splendidus.Isolates from the surface of P.urceolata are more abundant and diverse,of which strains P9 and P28 have a 16S rDNA sequence very similar(97.5%-99.8%) to that of V.splendidus.On the contrary,the isolates from the surfaces of G.textorii,U.pertusa and L.japonica are quite simple and distribute on different branches of the phylogenetic tree.In overall,the results of this study indicate that the genetic relationships among the isolates are quite close and display a certain level of host species specificity,and alga-associated bacteria species are algal species specific.

Intraspecific Diversity of Aureobasidium pullulans Strains from Different Marine Environments

Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nine strains among these marine yeasts belong to Aureobasidium pullulans, although the morphologies of their colonies are very different. The marine yeasts isolated from different marine environments indicate that A. pullulans is widely distributed in different environmental conditions. These Aureo-basidium pullulans strains include A. pullulans 4#2, A. pullulans N13d, A. pullulans HN3-11, A. pullulans HN2-3, A. pullulans JHSc, A. pullulans HN4.7, A. pullulans HN5.3, A. pullulans HN6.2 and A. pullulans W13a. A. pullulans 4#2 could produce cellulase and single cell protein. A. pullulans N13d could produce protease, lipase, amylase and cellulase. Both A. pullulans HN3-11 and A. pullulans HN2-3 were able to produce protease, lipase and cellulase. A. pullulans JHSc could secrete cellulase and killer toxin. Both A. pullulans HN4.7 and A. pullulans HN5.3 could yield lipase and cellulase. A. pullulans W13a was able to secrete extracellular amylase and cellulase while A. pullulans HN4.7 and A. pullulans N13d could produce siderophores. This means that different A. pullulans strains from different marine environments have different physiological characteristics, which may be applied in many different biotechnological industries.

复合菌系BYND-8的种群组成及其对沼气产量的影响

为了明确1组在中温下(30℃)高效分解木质纤维素的复合菌系的菌群组成,研究复合菌系预处理秸秆对沼气发酵的影响,利用平板分离法和变性梯度凝胶电泳(DGGE)法研究了中温木质纤维素降解复合菌系BYND-8的菌种组成多样性,通过添加该复合系菌液到以牛粪为原料的沼气发酵体系,研究了添加秸秆降解液对沼气产量的影响.利用平板法分离得到了6株细菌,它们与Serratia sp.PSGB 13、Serratia marcescens strain UFLA-25LS、Serratia marcescens strain DAP33、Alcaligenes sp.YcX-20、Stenotrophomonas maltophilia strain C6和Bacillus cereus isolate BRL02-71的相似率分别达到了99%、100%、96%、100%、100%和99%.同时利用DGGE方法还检测到了1株利用平板分离法没有获得到的细菌,它的16S rDNA V3区的序列与Uncultured bacterium clone ATB-KS-1446具有100%的同源性.稻秆经复合菌系BYND-8预处理后应用于沼气发酵中,在发酵的前15d内,累积产气量达到13 167 mL,甲烷产量达到7 248 mL,比对照分别提高了44.5%和95.3%.复合菌系具有较高的菌种组成多样性,将复合菌系应用于沼气发酵的原料预处理过程中,可以将产气时间提前,并提高产气量.

Selection of a DNA barcode for Nectriaceae from fungal whole-genomes

A DNA barcode is a short segment of sequence that is able to distinguish species. A barcode must ideally contain enough variation to distinguish every individual species and be easily obtained. Fungi of Nectriaceae are economically important and show high species diversity. To establish a standard DNA barcode for this group of fungi, the genomes of Neurospora crassa and 30 other filamentous fungi were compared. The expect value was treated as a criterion to recognize homologous sequences. Four candidate markers, Hsp90, AAC, CDC48, and EF3, were tested for their feasibility as barcodes in the identification of 34 well-established species belonging to 13 genera of Nectriaceae. Two hundred and fifteen sequences were analyzed. Intraand inter-specific variations and the success rate of PCR amplification and sequencing were considered as important criteria for estimation of the candidate markers. Ultimately, the partial EF3 gene met the requirements for a good DNA barcode: No overlap was found between the intra-and inter-specific pairwise distances. The smallest inter-specific distance of EF3 gene was 3.19%, while the largest intra-specific distance was 1.79%. In addition, there was a high success rate in PCR and sequencing for this gene (96.3%). CDC48 showed sufficiently high sequence variation among species, but the PCR and sequencing success rate was 84% using a single pair of primers. Although the Hsp90 and AAC genes had higher PCR and sequencing success rates (96.3% and 97.5%, respectively), overlapping occurred between the intraand inter-specific variations, which could lead to misidentification. Therefore, we propose the EF3 gene as a possible DNA barcode for the nectriaceous fungi.