用于分子生态学研究的堆肥DNA提取方法

分子生态学为堆肥微生物的研究提供了新的技术手段,DNA的提取是该技术的基础,但由于腐殖酸类物质的污染,增加了堆肥微生物总DNA的提取难度.采用了3种不同的方法(溶菌酶法、超声波破碎法和蛋白酶K-CTAB法)从堆肥中提取微生物的总DNA,使用核酸和蛋白质分析仪检测后表明3种提取方法获得的DNA产量均较高;琼脂糖凝胶电泳结果表明其长度约为23 kb;使用细菌16S rRNA基因通用引物(27F和1 495R)对总DNA进行PCR扩增,都获得了几乎全长的16S rDNA序列(约1.5 kb);利用限制性内切酶(Hae Ⅲ和AluⅠ)对纯化后的PCR产物进行RFLP分析,结果表明3种方法提取的DNA反映了比较一致的微生物多样性.虽然3种方法各有优缺点,但其提取的DNA都可以用于堆肥微生物的分子生态学研究,可以根据实际需要选用某一种方法用于提取堆肥总DNA.     

一株氧化亚铁硫杆菌的筛选及生长条件研究

从广东省大宝山矿区尾矿中分离纯化得到一株嗜酸细菌,对其进行DNA提取和16S rRNA扩增后测序,将测序结果与国际基因数据库Genbank中已有相关菌株序列进行相似性比对后,发现该菌株与氧化亚铁硫杆菌(Acidithiobacil-lus ferrooxidans)Tf-49菌位于系统发育树的同一分支中,二者相似度高达99%以上,确定其为氧化亚铁硫杆菌并命名为DBS-8菌。另外,通过设计5因素4水平的正交实验,研究了接种量、培养温度、硫酸铵浓度、初始pH和初始亚铁浓度对该菌株生长状况的影响。结果表明适宜该菌株生长的最佳初始条件为:20%的接种量、3.0 g/L硫酸铵、9.0 g/L初始亚铁、pH=2.0和28℃培养,各初始条件对细菌生长影响的顺序为:pH初始亚铁浓度接种量温度硫酸铵浓度。     

夏季太湖梅梁湾水体中细菌的群落结构

利用高通量测序技术,选择16S rRNA V6区作为目标片段,对夏季太湖梅梁湾水体中的细菌群落结构进行了分析,结果表明,共产生了101 427条优质序列,细菌为100 935条,占99.5%;在蓝藻暴发期间,共检测到14门,55属,610个操作分类单位。优势门类为蓝藻门(39.7%),放线菌门(27.2%)和变形菌门(23.4%),微囊藻属(21.0%)和聚球藻属(15.9%)为主要优势种。     

Aerobic thermophilic bacteria enhance biogas production

The enhancing effect of aerobic thermophilic (AT) bacteria on the production of biogas from anaerobically digested sewage sludge (methanogenic sludge) was investigated. Sewage sludge (5%, w/w) was incubated at 65°C with shaking for a few months to prepare the AT seed sludge. AT sludge was prepared by incubation of the AT seed sludge (5%, v/v) and sewage sludge (5%, w/w) at 65°C with shaking. The addition of this AT sludge (1.2% ± 0.5% of total volatile solids) to methanogenic sludge enhanced the production of biogas. The optimum volume of the addition and the pretreatment temperature of the AT sludge for optimum biogas production were 5% (v/v) and 65°C. Batch-fed anaerobic digestion was covered with the addition of various AT sludges. The AT sludge prepared with the AT seed sludge improved the biogas production by 2.2 times relative to that from the sewage sludge addition. The addition of sludge without AT seed sludge weakly enhanced biogas production. An aerobic thermophilic bacterium (strain AT1) was isolated from the AT seed sludge. Strain AT1 grew well in a synthetic medium. The production of biogas from the anaerobic digestion of sewage sludge was improved by the addition of 5% (v/v) AT1 bacterial culture compared with that from the sewage sludge addition. The addition of AT1 culture reduced the volatile solids by 21%, which was higher than the 12.6% achieved with the sewage sludge addition. The AT1 bacterial culture enhanced the biogas production more than the AT seed sludge. The phylogenetic analysis of the 16S rRNA gene revealed that strain AT1 is closely related to Geobacillus thermodenitrificans (100% sequence similarity). The improvement in the production of biogas with the AT sludge could be caused by thermophilic bacterial activity in the AT sludge.     

Culturable actinobacteria isolated from marine sponge <Emphasis Type="Italic">Iotrochota</Emphasis> sp.

The study describes the diversity of actinobacteria isolated from the marine sponge Iotrochota sp. collected in the South China Sea. Species and natural product diversity of isolates were analyzed, including screening for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetase (NRPS), and 16S rRNA gene restriction fragment length polymorphism (RFLP). PKS and NRPS sequences were detected in more than half of the isolates and the different “PKS-I–PKS-II–NRPS” combinations in different isolates belonging to the same species indicated a potential natural product diversity and divergent genetic evolution. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to genera Streptomyces, Cellulosimicrobium, and Nocardiopsis. The majority of the strains tested belonged to the genus Streptomyces and one of them may be a new species. To our knowledge, this is the first report of a bacterium classified as Cellulosimicrobium sp. isolated from a marine sponge. Key Laboratory of Marine Bio-recourses Sustainable Utilization (LMB-CAS), Guangdong Key Laboratory of Marine Materia Medica (LMMM-GD), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China.     

基于甲烷氧化菌的城镇污水厂尾水极限脱氮系统构建及机制

好氧甲烷耦合反硝化（AME-D）在城镇污水厂尾水深度脱氮方面具有巨大的应用潜力,研究采用改良型反硝化生物滤池,利用低浓度甲烷构建出AME-D极限脱氮系统.研究发现该系统在间歇式运行方式下,出水中总氮和氨氮的平均浓度能达到1.05 mg·L^-1和0.54 mg·L^-1,其平均去除率分别为94.77%和93.30%.拉曼光谱分析结果显示,由NO₃^-对称伸缩引起的峰明显消失,由醇COH面外弯曲或C—H面外弯曲振动吸收引起峰明显增强,甲烷被氧化形成的中间产物可能主要为醇类物质.16S rRNA基因测序结果表明,系统中的甲烷氧化菌主要为Methylocystis（0.27%）、Methylosarcina（0.10%）和Methyloparacoccus（0.12%）,反硝化菌主要为Pseudomonas（56.92%）、Paenibacillus（3.52%）和Lysinibacillus（3.00%）,硝化菌主要为Nitrospira（0.1%）,说明AME-D极限脱氮系统的脱氮功能是由好氧甲烷氧化菌、反硝化菌和硝化菌协同实现.     

辽河口芦苇湿地细菌和古菌群落周期日变化特征

采用实时荧光定量PCR和Illumina高通量测序技术对秋季辽河口芦苇湿地水体中一昼夜细菌和古菌群落特征进行了研究.测序共获得52802条细菌和106091条古菌高质量序列,以97%的相似性聚类并抽平后共划分了530和979个OTU.结果表明,湿地水体中浮游细菌和古菌群落丰度均在早上达到最高值,夜晚达到最低值.细菌和古菌群落多样性指数均存在周期日变化,不同时间细菌和古菌群落结构和组成具有显著差异.此外,古菌群落组成特征表明辽河口芦苇湿地具有较高的潜在甲烷生成能力.本研究为进一步认识河口湿地细菌和古菌群落特征和功能奠定了基础.     

Evaluation of bacterial pathogen diversity, abundance and health risks in urban recreational water by amplicon next-generation sequencing and quantitative PCR

The microbial quality of urban recreational water is of great concern to public health.The monitoring of indicator organisms and several pathogens alone is not sufficient to accurately and comprehensively identify microbial risks.To assess the levels of bacterial pathogens and health risks in urban recreational water,we analyzed pathogen diversity and quantified four pathogens in 46 water samples collected from waterbodies in Beijing Olympic Forest Park in one year.The pathogen diversity revealed by 16 S r RNA gene targeted next-generation sequencing(NGS) showed that 16 of 40 genera and 13 of 76 reference species were present.The most abundant species were Acinetobacter johnsonii,Mycobacterium avium and Aeromonas spp.Quantitative polymerase chain reaction(q PCR) of Escherichia coli(uid A),Aeromonas(aer A),M.avium(16S r RNA),Pseudomonas aeruginosa(oaa) and Salmonella(inv A) showed that the aer A genes were the most abundant,occurring in all samples with concentrations of 10~(4–6) genome copies/100 m L,followed by oaa,inv A and M.avium.In total,34.8% of the samples harbored all genes,indicating the prevalence of these pathogens in this recreational waterbody.Based on the q PCR results,a quantitative microbial risk assessment(QMRA) showed that the annual infection risks of Salmonella,M.avium and P.aeruginosa in five activities were mostly greater than the U.S.EPA risk limit for recreational contacts,and children playing with water may be exposed to the greatest infection risk.Our findings provide a comprehensive understanding of bacterial pathogen diversity and pathogen abundance in urban recreational water by applying both NGS and q PCR.     

1株反硝化除磷菌的鉴定及其反硝化功能基因研究

研究反硝化除磷菌(denitrifying polyphosphate-accumulating organism,DPAO)的筛选、鉴定及反硝化功能基因.利用反硝化培养基分离得到菌株ZQN2,经好氧吸磷试验,硝酸盐还原产气试验并辅助异染颗粒和PHB颗粒染色,确定其为反硝化除磷菌.厌氧释磷/缺氧吸磷试验结果表明,菌株ZQN2在厌氧段释磷并合成PHB(poly-β-hydroxybutyrate),在缺氧段以NO3-为电子受体氧化PHB并过量吸磷,进行了同步反硝化除磷.对菌株ZQN2进行16S rRNA基因序列分析,并构建系统进化树,结果表明该菌株与GenBank数据库中多株Bacillus cereus菌株16S rRNA基因的同源性在99%以上.结合生理生化检测,判断菌株ZQN2为蜡状芽胞杆菌(Bacillus cereus).对其反硝化功能基因的研究表明,菌株ZQN2为nirS+和nirK-型,从分子生物学角度确认其具有反硝化功能;菌株ZQN2的nirS序列的系统发育分析结果表明,其nirS序列与多株Pseadomonas aeruginosa的亲缘关系最接近,这与基于16S rRNA的系统发育分析的结果不一致.     

皮氏罗尔斯通氏菌株DX-T3-01的耐镉性能及镉富集机理

从江西德兴铜矿土壤中分离筛选到一株具有较强耐镉(Cd)能力的细菌DX-T3-01,经生理生态及16S rRNA分析进行鉴定,研究其对不同浓度Cd2+的耐受特性及对重金属Zn2+、Cu2+、Ni2+的耐受性,进一步利用扫描电镜-能谱仪(SEM-EDX)和红外光谱(FTIR)探讨菌株对Cd2+累积去除的机理.经鉴定,耐镉细菌DX-T3-01为皮氏罗尔斯通氏菌(Ralstonia pickettii);Cd2+最高耐受浓度(MTC)为16 mmol L-1,最低抑制浓度(MIC)为4 mmol L-1,并可耐受一定浓度的Zn2+、Cu2+;菌体积累Cd2+后细胞表面有沉淀物附着并有镉元素检出,对Cd2+的累积主要依靠细胞壁上—PO43-、胺基中的—C—N—、—M—O(O—M—O)、C=O和酰胺基(—CO—NH—)基团.图5参16