磺胺甲唑对海水养殖废水处理过程中抗性细菌及抗性基因的富集作用

抗生素在海水养殖过程中大量使用,但仅有少部分被生物体利用,含有抗生素的废水进入水处理系统后,抗生素、抗性菌和抗性基因的响应过程尚不完全清楚.应用缺氧/好氧移动床生物膜反应器（A/O-MBBR）处理含磺胺甲唑（SMX）的海水养殖废水,探究在SMX选择压力下,反应器内抗生素和抗性基因丰度的变化规律,以及微生物群落和可培养的抗性细菌种群的响应.结果表明,在进水SMX浓度为500 μg·L^-1,水力停留时间为8 h,SMX加入初期会对NH₄⁺-N和NO₂^--N的去除率有轻微影响,随后逐步恢复;同时去除约32%的SMX,且78%以上SMX在缺氧区完成;抗性基因在缺氧区富集明显高于好氧区,在缺氧区磺胺类抗性基因（sul1）绝对丰度上升2.43 log,磺胺类抗性基因（sul2）上升1.71 log;而在好氧区,sul1 绝对丰度上升1.17 log,sul2 上升0.91 log.抗性平板培养结合高通量测序表明,假交替单胞菌属（Pseudoalteromonas）在反应器可培养抗性细菌中占最优势.高通量测序分析发现可培养的抗性细菌假单胞菌属（Pseudomonas）在反应器内占比最高.表明含SMX的海水养殖废水可促进水中抗性基因的富集,部分抗性细菌的数量显著增加.     

S-烯丙基-L-半胱氨酸缓解水稻种子幼根和幼芽镉胁迫机制

土壤Cd污染对水稻种子萌发和植株生长发育都具有毒害作用,最终可能导致稻谷产量下降及Cd含量超标.本文采用种子萌发试验研究了来源于大蒜提取物的天然含硫有机化合物S-烯丙基-L-半胱氨酸（SAC）缓解水稻种子幼根和幼芽Cd²⁺胁迫的潜在机制.本试验过程中选用我国南方稻区主栽水稻品种"中早35"种子作为试验材料,首先研究了SAC对水稻种子幼根和幼芽Cd²⁺胁迫的缓解效应;在此基础上,通过研究SAC对种子幼根和幼芽生理生化系统及Cd含量影响,探索了SAC缓解水稻种子幼根和幼芽Cd²⁺胁迫的生理机制;利用实时荧光定量PCR技术研究了SAC对水稻Cd转运蛋白编码基因表达水平的影响,探讨了SAC缓解Cd²⁺胁迫的分子机制.结果表明,当Cd²⁺胁迫浓度达到50 μmol·L^-1时水稻种子幼根和幼芽发育会受到显著抑制;当SAC添加浓度达到200 μmol·L^-1时,幼根的总根长、根表面积和根体积与50 μmol·L^-1Cd²⁺胁迫处理组相比分别增加173.5%、65.52%和37.04%;幼根和幼芽中CAT、SOD活性分别增加212.42%、110.76%和31.41%、47.31%;幼根和幼芽中MDA、GSH含量分别降低43.09%、34.12%和33.97%、35.74%;幼根和幼芽中Cd含量分别降低35.91%、28.86%.实时荧光定量PCR分析结果表明,添加SAC处理后Cd转运蛋白编码基因OsNramp5和OsHMA2的相对表达量与Cd²⁺胁迫处理组相比分别显著降低了33.38%和34.99%,OsHMA3的相对表达量与Cd²⁺胁迫处理组相比显著升高了33.96%.由以上试验结果可见,SAC降低水稻幼根和幼芽Cd²⁺胁迫的主要机制是SAC调控了Cd转运蛋白编码基因的表达水平,降低了Cd向幼根和幼芽中的转运,同时增加了Cd向液泡中的转运.     

Community analysis of ammonia-oxidizing bacteria in activated sludge of eight wastewater treatment systems

We investigated the communities of ammonia-oxidizing bacteria(AOB) in activated sludge collected from eight wastewater treatment systems using polymerase chain reaction(PCR) followed by terminal restriction fragment length polymorphism(T-RFLP),cloning,and sequencing of the α-subunit of the ammonia monooxygenase gene(amoA).The T-RFLP fingerprint analyses showed that different wastewater treatment systems harbored distinct AOB communities.However,there was no remarkable difference among the AOB TRFLP profiles from different parts of the same system.The T-RFLP fingerprints showed that a full-scale wastewater treatment plant(WWTP) contained a larger number of dominant AOB species than a pilot-scale reactor.The source of influent affected the AOB community,and the WWTPs treating domestic wastewater contained a higher AOB diversity than those receiving mixed domestic and industrial wastewater.However,the AOB community structure was little affected by the treatment process in this study.Phylogenetic analysis of the cloned amoA genes clearly indicated that all the dominant AOB in the systems was closely related to Nitrosomonas spp.not to Nitrosospira spp.Members of the Nitrosomonas oligotropha and Nitrosomonas communis clusters were found in all samples,while members of Nitrosomonas europaea cluster occurred in some systems.     

农田土壤自养微生物碳同化潜力及其功能基因数量、关键酶活性分析

研究农田土壤自养微生物碳同化潜力,对全面认识农田生态系统碳吸收和碳储存有着重要意义.选取6种典型农田土壤,通过14C连续标记示踪技术结合密闭系统模拟培养,量化了土壤自养微生物碳同化潜力及其向土壤活性碳库组分转化,同时结合分子生物学技术及酶学分析方法,探讨了不同土壤自养微生物细菌固碳功能基因(cbbL)丰度及关键酶(RubisCO)活性.结果表明,土壤自养微生物具有可观的CO2同化潜力,在本实验条件下,全球每年表层(0~20 cm)土壤通过自养微生物的同化作用可固定的碳为0.57~7.3 Pg.供试土壤的14C土壤有机碳(14C-SOC)含量范围为10.63~133.81 mg·kg-1,而14C可溶性有机碳(14C-DOC)、14C微生物生物量碳(14C-MBC)含量范围分别为0.96~8.10 mg·kg-1、1.70~49.16 mg·kg-1.土壤可溶解性有机碳(DOC)、微生物量碳(MBC)和SOC的更新率分别为5.07%~14.3%、2.51%~13.12%和0.09%~0.64%.土壤细菌cbbL丰度范围为2.40×107~1.9×108copies·g-1,且RubisCO酶活性(CO2/soil)范围为34.06~71.86 nmol·(g·min)-1.相关分析表明,土壤14C-SOC与14C-MBC及RubisCO酶活性均呈极显著正相关关系(P<0.01).说明土壤对大气CO2的同化作用主要是由自养微生物参与的同化过程,且较高的RubisCO酶活性意味着较高的自养微生物CO2同化潜力.     

Construction of WCB-11: A novel phiYFP arsenic-resistant whole-cell biosensor

The prediction and assessment of environmental pollution by arsenic are important preconditions of advocating environmental protection and human health risk assessment.A yellow fluorescent protein-based whole-cell biosensor for the detection of arsenite and arsenate was constructed and tested.An arsenic-resistant promoter and the regulatory gene arsR were obtained by PCR from the genome of Escherichia coli DH5α,and phiYFP was introduced into E.coli DH5α as a reporter gene to construct an arsenic-resistant whole-cell biosensor(WCB-11) in which phiYFP was expressed well for the first time.Experimental results demonstrated that the biosensor has a good response to arsenic and the expression of phiYFP.When strain WCB-11 was exposed to As 3+ and As 5+,the expression of yellow fluorescence was time-dependent and dose-dependent.This engineered construct is expected to become established as an inexpensive and convenient method for the detection of arsenic in the field.     

沼泽红假单胞菌高效产氢hupL缺失突变株的构建

利用湖底淤泥分离的沼泽红假单胞菌(Rhodopseudomonas palustris)CQU01作为出发菌株,构建吸氢酶大亚基基因hupL缺失突变株,以提高光合细菌菌株的产氢效率.以PCR扩增的hupL两侧hupS 和 hupC基因为同源重组双交换臂,连入pMD18-T载体;再将hupS, hupC 和Kmr基因与经SalⅠ和HindⅢ双酶切的pSUP202,构建靶向自杀载体pBPZ.经接合转移转化R. palustris CQU01菌株,成功获得沼泽红假单胞菌吸氢酶活性缺失突变株R. palustris CQU012.测定突变株的吸氢酶活性及生长和产氢特性,结果表明,突变株的产氢量比野生菌株提高了约50%,而生长特性与野生菌株没有显著差异.R. palustris CQU012 吸氢酶缺失突变株可望为工业废水的生物治理提供高效产氢工程菌株.     

宏基因组研究及其应用研究进展

传统的微生物培养方法损失了近乎99%的微生物资源,而宏基因组研究能够直接从环境中提取全部微生物基因组DNA,通过构建宏基因组文库并结合适当的筛选方法,得到目的基因及其表达蛋白,该研究能够最大限度地开发微生物的序列空间。文章简要概述了宏基因组研究的产生、原理及其在生态学方面的发展和对新基因资源的开发和利用。     

低温1,2,4-TCB降解菌的选育、降解特性及邻苯二酚1,2-双加氧酶基因表达水平

从长期受1,2,4-三氯苯(1,2,4-TCB)污染的地下水中筛选出一株低温寡营养降解菌A2,对A2菌进行革兰氏染色鉴定和16S rDNA鉴定,研究了不同pH、温度、盐度等因素对A2菌降解1,2,4-TCB效果以及对邻苯二酚1,2-双加氧酶基因表达的影响,并进行了正交实验。结果表明,A2菌为革兰氏阴性短杆细菌,初步鉴定为假单胞菌;在pH值为7、培养温度30℃、盐度0.8%、培养时间6 d时,A2菌对1,2,4-TCB降解效果最好,降解率达到88.14%,同时该条件下邻苯二酚1,2-双加氧酶基因相对表达水平最高;培养温度为10℃时,A2菌对1,2,4-TCB降解率可达到85.3%,同时邻苯二酚1,2-双加氧酶基因也有较高的相对表达水平。以上结果说明,将A2菌应用于低温寡营养地下水的生物修复是可能的。     

Toxic effects of acetochlor, methamidophos and their combination on nifH gene in soil

Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis （DGGE） and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity （both in gene richness and diversity index values） and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patterns showed that nifH gene composition which had been affected by low concentration of acetochlor （50 mg/kg） recovered firstly. Methamidophos reduced nifH gene richness that except at 4 weeks. The medium concentration of methamidophos （150 mg/kg） caused the most apparent changes in nifH gene diversity at the first week while the high concentration of methamidophos （250 mg/kg） produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes （bands） responded differently to the impact of agrochemicals： four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha- and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.     

基于16S rRNA基因测序法分析北京霾污染过程中PM2.5和PM10细菌群落特征

2013年1月8~14日,北京出现了严重的霾污染.霾污染时高浓度的大气颗粒物增加了暴露人群的健康风险,而大气中的微生物也可能带来一些风险,但目前对霾污染时大气中微生物组成了解较少.本研究选取了2013年1月8~14日北京7d的PM2.5和PM10采样样本,通过对细菌16S rRNA基因V3区扩增和Mi Seq测序,得到PM2.5和PM10中的细菌群落结构特征,并将结果与相同采样样本的宏基因组测序结果及三项国外基于16S rRNA基因测序方法的大气中细菌研究结果进行了比较.研究发现7 d连续采样条件下,PM2.5中细菌群落结构在门和属的水平上均差别不大.在属级别上,节杆菌属(Arthrobacter)和弗兰克氏菌属(Frankia)是北京冬季大气中细菌群落的主要类群.16S rRNA基因测序与宏基因组测序结果对比分析发现,在属级别上,两种分析方法中有39个相同的属类群(两种分析方法丰度前50的细菌类群合并所得),弗兰克氏菌属(Frankia)和副球菌属(Paracoccus)在16S rRNA基因测序分析结果中相对含量较多,而考克氏菌属(Kocuria)和地嗜皮菌属(Geodermatophilus)在宏基因组测序结果中相对含量较高.在门和属的水平上,PM2.5和PM10中细菌群落结构特征呈现出相似的规律.在门水平上,放线菌门(Actinobacteria)在PM2.5中的相对百分比较大,而厚壁菌门(Firmicutes)在PM10中的相对百分比较大.在属水平上,梭菌属(Clostridium)在PM10中的相对百分比较大.与三项国外基于16S rRNA基因测序研究结果对比发现,尽管在采样地点和采样时间上有较大差异,大气中普遍存在一些相同的细菌类群,且近地面大气细菌群落结构特征相似度较高,区别于高空对流层中细菌群落结构特征.