A GFP-based bacterial biosensor with chromosomally integrated sensing cassette for quantitative detection of Hg(II) in environment

A mercury biosensor was constructed by integrating biosensor genetic elements into E. coli JM109 chromosome in a single copy number, using the attP/attB recombination mechanism of λphage. The genetic elements used include a regulatory protein gene (merR) along with operator/promoter (O/P) derived from the mercury resistance operon from pDU1358 plasmid of Serratia marcescens. The expression of reporter gene gfp is also controlled by merR/O/P. Integration of the construct into the chromosome was done to increase the stability and precision of the biosensor. This biosensor could detect Hg(II) ions in the concentration range of 100-1700 nmol/L, and manifest the result as the expression of GFP. The GFP expression was significantly different (P ≤ 0.05) for each concentration of inducing Hg(II) ions in the detection range, which reduces the chances of misinterpretation of results. A model using regression method was also derived for the quantification of the concentration of Hg(II) in water samples.     

微生物全细胞传感器在重金属生物可利用度监测中的研究进展

传统的物理化学方法主要用于测定环境重金属的总量,微生物全细胞传感器可以对土壤及水体环境的重金属生物可利用度进行监测.此外,微生物全细胞传感器还具有操作简单、快速、经济的特点,适用于污染事件的应急监测.微生物全细胞传感器的生物学元件主要由MerR、ArsR、RS等家族的金属调控蛋白和gfp、lux、luc等报告基因组成.调控蛋白、报告基因与微生物全细胞传感器的灵敏度、特异性和监测特点有关.受pH、金属螯合物及检测条件等因素的影响,不同的环境条件下的重金属生物可利用度是不同的.增加重金属在微生物细胞内的累积,进行调控蛋白的分子生物学改造,优化检测条件是提高传感器灵敏度、特异性和准确性的可行方案.实现污染物的原位和在线监测是微生物全细胞传感器的主要发展方向.     

砷污染湿地生境下土壤微生物多样性及群落结构特征

为研究As（砷）污染对微生物多样性的影响,找寻有利于As污染修复的抗As菌种,利用高通量测序技术分析As胁迫〔w（As）分别为0、80、100、150、200和400 mg/kg,依次记为AT0组、AT80组、AT100组、AT150组、AT200组和AT400组〕对湿地生境中土壤微生物多样性及群落结构特征的影响.结果表明:As污染会引起微生物多样性和群落结构的变化,但并不是简单的负相关.在各处理组中,AT80组的土壤中微生物多样性最高,OTU（operational taxonomic units）数高达1 849,较未经As处理的AT0组增加了58.9%,说明低As胁迫在一定程度上会刺激As敏感微生物的生长繁殖,如Desulfovibrio（脱硫弧菌属）和Allobaculum等菌属,使群落结构更加复杂多样.高w（As）（400 mg/kg）对微生物有明显的抑制作用,导致某些物种消亡和多样性下降.As胁迫会诱导抗As微生物〔如Pseuomonas（假单胞菌属）〕成为优势类群,群落结构趋于稳定、单一.微生物群落结构对不同w（As）胁迫有明显的响应特征,可作为As污染湿地土壤质量评价的灵敏指标.在AT400组中存在大量的Pseudomonas veronii,可为As污染湿地微生物修复提供借鉴.研究显示,微生物对As污染具有较为敏感的响应,其中,Proteobacteria（变形菌门）和Firmicutes（厚壁菌门）为As污染生境中微生物优势门类并且与非专性吸附态As和谷胱甘肽含量呈正相关.      

Phylogenetic analysis and arsenate reduction effect of the arsenic-reducing bacteria enriched from contaminated soils at an abandoned smelter site

Microbial reduction of As(V)(i.e.,arsenate)plays an important role in arsenic(As)mobilization in aqueous environment.In this study,we investigated As(V)reduction characteristics of the bacteria enriched from the arsenic-contaminated soil at an abandoned smelter site.It was found that As(V)was completely reduced to As(III)(i.e.,arsenite)in 21 h.After 3-d incubation,a yellow solid was precipitated and the concentration of As(III)decreased sharply.After 150 h incubation,ca.65%of soluble arsenic was removed fro...     

Effect of arsenic contaminated irrigation water on Lens culinaris L. and toxicity assessment using lux marked biosensor

Contamination of irrigation water represents a major constraint to Bangladesh agriculture, resulting in elevated levels in the terrestrial systems. Lux bacterial biosensor technology has previously been used to measure the toxicity of metals in various environmental matrices. While arbuscular mycorrhizal fungi have their most significant effect on phosphorus uptake, but showed alleviated metal toxicity to the host plant. The study examined the effects of arsenic and inoculation with an arbuscular mycorrhizal fungus, Glomus mosseae, on lentil (Lens culinaris L. cv. Titore). Plants were grown with and without arbuscular mycorrhizal inoculum for 9 weeks in a sand and terra-green mixture (50:50, V/V) and watered with five levels of arsenic (0, 1, 2, 5, 10 mg As/L arsenate). The results showed that arsenic addition above 1 mg/L significantly reduced percentage of mycorrhizal root infection. On further analysis a close relationship was established with the vegetative and reproductive properties of lentil (L. culinaris) plants compared to the percentage bioluminescence of the soil leachate. However, arbuscular mycorrhizal fungal inoculation reduced arsenic concentration in roots and shoots. Higher concentrations of arsenic (5, 10 mg As/L arsenate) reduced the mycorrhizal efficiency to increase phosphorus content and nitrogen fixation. Therefore, this study showed that increased concentration of arsenic in irrigation water had direct implications to the lentil (L. culinaris) plants overall performance. Moreover the use of bioassay demonstrated that mycorrhiza and clay particle reduced arsenic bioavailability in soil.     

工程菌及其固定化细胞对有机磷农药的降解

将抗性库蚊解毒酶酯酶B1基因片段引入融合表达载体pThioHisA中,转化入大肠杆菌DH5α,在IPTG诱导下,经过8h,酯酶B1在大肠杆菌中获得融合高效表达。重组质粒pThioHisA-B1表达的酯酶融合蛋白具有较高的酯酶B1活性,能高效降解酯酶的特异性底物α-乙酸萘酯(α-NA)和β-乙酸萘酯(β-NA),将工程菌固定化后,固定化细胞在3h内对1000mg/L的甲基对硫磷降解率>65%。     

The study of biological activity of transformation products of diclofenac and its interaction with chlorogenic acid

In the present work we compared the biological activity of DCF, 4′-OHDCF and 5-OHDCF as molecules of most biodegradation pathways of DCF and selected transformation products (2-hydroxyphenylacetic acid; 2,5-dihydroxyphenylacetic acid and 2,6-dichloroaniline) which are produced during AOPs, such as ozonation and UV/H₂O₂. We also examined the interaction of DCF with chlorogenic acid (CGA). CGA is commonly used in human diet and entering the environment along with waste mainly from the processing and brewing of coffee and it can be toxic for microorganisms included in activated sludge. In the present experiment the evaluation of following parameters was performed: E. coli K-12 cells viability, growth inhibition of E. coli K-12 culture, LC₅₀ and mortality of Chironomus aprilinus, genotoxicity, sodA promoter induction and ROS generation. In addition the reactivity of E. coli SM recA:luxCDABE biosensor strain in wastewater matrices was measured. The results showed the influence of DCF, 4′-OHDCF and 5-OHDCF on E. coli K-12 cells viability and bacteria growth, comparable to AOPs by-products. The highest toxicity was observed for selected, tested AOPs by-products, in comparison to the DCF, 4′-OHDCF and 5-OHDCF. Genotoxicity assay indicated that 2,6-dichloroaniline (AOPs by-product) had the highest toxic effect. The oxidative stress assays revealed that the highest level of ROS generation and sodA promoter induction were obtained for DCF, 4′-OHDCF and 5-OHDCF, compared to other tested compounds. We have also found that there is an interaction between chlorogenic acid and DCF, which resulted in increased toxicity of the mixture of the both compounds to E. coli K-12, comparable to parent chemicals. The strongest response of E. coli SM biosensor strain with recA:luxCDABE genetic construct in filtered treated wastewaters, comparable to control sample was noticed. It indicates, that E. coli SM recA:luxCDABE biosensor strains is a good tool for bacteria monitoring in wastewater environment. Due to toxicity and biological activity of tested DCF transformation products, there is a need to use additional wastewater treatment systems for wastewater contaminated with pharmaceutical residues.     

生物传感细胞ADP1_pWHlux在水环境急性毒性检测中的应用

针对天然发光菌和以模式微生物为宿主构建的生物传感细胞在急性毒性检测应用中对测试条件要求苛刻等适用性问题,本研究将1株基因工程构建的生物传感细胞不动杆菌ADP1_p WHlux应用于急性毒性检测,建立检测方法,考察其灵敏度及检测范围.结果表明,ADP1_p WHlux发光受急性毒物的抑制,毒物剂量与发光抑制存在剂量效应关系.在4 mg·L-1Hg Cl2诱导下仅5 min可作出响应,暴露30~60 min后可以给出较为准确的结果.对Hg Cl2的检出限可达0.04 mg·L-1.对我国生活饮用水卫生标准的指标中Be2+、Ba2+、Cu2+、Ni2+检出效果明显,对Be2+、Ba2+、Cu2+的检测范围均在0.025~250 mg·L-1,对Ni2+的检测范围在0.002 5~250 mg·L-1,对Pb2+、Br O-3、Cl O-2的检出限均在0.002 5 mg·L-1,对Cl O-3检出限为0.025mg·L-1.采用ADP1_p WHlux生物传感细胞检测方法对北京市清河水环境急性毒性进行评价,表明ADP1_p WHlux生物传感细胞检测方法可用于污染水样检测.     

Arsenic removal from contaminated soil via biovolatilization by genetically engineered bacteria under laboratory conditions

In Rhodopseudomonas palustris, an arsM gene, encoding bacterial and archaeal homologues of the mammalian Cyt19 As(III) S-adenosylmethionine methytransferase, was regulated by arsenicals. An expression of arsM was introduced into strains for the methylation of arsenic. When arsM was expressed in Sphingomonas desiccabilis and Bacillus idriensis, it had 10 folds increase of methyled arsenic gas compared to wild type in aqueous system. In soil system, about 2.2%–4.5% of arsenic was removed by biovolatilization during 30 days. This study demonstrated that arsenic could be removed through volatilization from the contaminated soil by bacteria which have arsM gene expressed. These results showed that it is possible to use microorganisms expressing arsM as an inexpensive, efficient strategy for arsenic bioremediation from contaminated water and soil.     

ICP-AES连续氢化(还原)同时测定水、水生生物和沉积物样品中As、Sb、Bi和Hg

本文利用KBH_4的氢化(还原)作用,将酸性样品溶液中砷、锑、铋氢化和将汞离子还原为汞蒸气一同引入ICP(电感耦合等离子体)光源进行发射光谱测定,探讨了水、鱼、螺蚌类肉中As、Sb、Bi和Hg,沉积物样品中As、Sb和Bi的测定方法。As、Sb、Bi检出限为0.01ppm,Hg为0.001ppm。10次测定的RSD％A86.5、Sb6.8、Bi10.9、Hg3.6(5次)。进行多次加际回收试验和标准参考样的测定,得到了好的结果。该方法利用一套标准溶液同时适用于不同类样品的测定,具有简单、快速的特点,灵敏度能满足水环境监测(Ⅲ级地面水汞尚不足)的需要。     

Phylogenetic analysis and arsenate reduction e ect of the arsenic-reducing bacteria enriched from contaminated soils at an abandoned smelter site

Microbial reduction of As(V) (i.e., arsenate) plays an important role in arsenic (As) mobilization in aqueous environment. In this study, we investigated As(Ⅴ) reduction characteristics of the bacteria enriched from the arsenic-contaminated soil at an abandoned smelter site. It was found that As(Ⅴ) was completely reduced to As(Ⅲ) (i.e., arsenite) in 21 h. After 3-d incubation, a yellow solid was precipitated and the concentration of As(Ⅲ) decreased sharply. After 150 h incubation, ca. 65% of soluble arsenic was removed from the solution. The analysis of the precipitate by scanning electron microscopy and energy dispersive spectrometer (SEM-EDS) and X-ray diffraction (XRD) revealed that the main component was crystalline arsenic sulfide (ASS). Microbial mediated reduction and mobilization of adsorbed As(Ⅴ) on ferric hydroxide was also examined. In the microcosm slurry experiment, ca. 53% of the adsorbed As(V) was reduced to As(Ⅲ) by the bacteria, which resulted in an appreciable release of arsenic into aqueous phase. The released arsenic was present predominantly as As(Ⅲ). The microbial diversity was analyzed by 16S rDNA-dependent molecular phylogeny. A near-full-length 16S rDNA gene clone library was constructed. The 197 clones were analyzed using RFLP (restriction fragment length polymorphism) and 72 OTUs were obtained, which contributed 51% of the content for total clone number in six OTUs. Six bacterial clones in these six OTUs were selected for sequencing and the sequenced clones were found to belong to the group Caloramator, Clostridium, and Bacillus.     

Environmental Mn(II) enhances the activity of dissimilatory arsenate-respiring prokaryotes from arsenic-contaminated soils

Many investigations suggest that dissimilatory arsenate-respiring prokaryotes (DARPs) play a key role in stimulating reductive mobilization of As from solid phase into groundwater, but it is not clear how environmental Mn(II) affects the DARPs-mediated reductive mobilization of arsenic. To resolve this issue, we collected soil samples from a realgar tailings-affected area. We found that there were diverse arsenate-respiratory reductase (arr) genes in the soils. The microbial communities had high arsenate-respiring activity, and were able to efficiently stimulate the reductive mobilization of As. Compared to the microcosms without Mn(II), addition of 10 mmol/L Mn(II) to the microcosms led to 23.99%-251.79% increases in the microbial mobilization of As, and led to 133.3%-239.2% increases in the abundances of arr genes. We further isolated a new cultivable DARP, Bacillus sp. F11, from the arsenic-contaminated soils. It completely reduced 1 mmol/L As(V) in 5 days under the optimal reaction conditions. We further found that it was able to efficiently catalyze the reductive mobilization and release of As from the solid phase; the addition of 2 mmol/L Mn(II) led to 98.49%-248.78% increases in the F11 cells-mediated reductive mobilization of As, and 70.6%-104.4% increases in the arr gene abundances. These data suggest that environmental Mn(II) markedly increased the DARPs-mediated reductive mobilization of As in arsenic-contaminated soils. This work provided a new insight into the close association between the biogeochemical cycles of arsenic and manganese.     

氧化铜纳米颗粒(CuO NPs)对土壤环境因子与砷生物有效性的影响

氧化铜纳米颗粒（CuO NPs）可以通过农药和肥料施用、意外泄露或污水灌溉进入As污染农田土壤,从而对土壤环境因子和As生物有效性产生影响.本试验选取两种不同类型土壤（安徽宿松黄棕壤和黑龙江海伦黑土）进行人工As污染,添加不同浓度的CuO NPs,探究90 d淹水-落干过程中CuO NPs对As污染农田环境因子和As生物有效态的影响.结果表明,CuO NPs进入土壤后12 h内快速溶解产生Cu²⁺,且在黄棕壤中的溶解速度较黑土迅速.CuO NPs可在短时间内降低土壤pH,提高土壤氧化还原电位（E_h）,降低土壤电导率（EC）,但随着培养时间增加土壤EC逐渐提高.一定时间内CuO NPs在两种类型土壤中可降低51.0%~82.5%土壤浸出液中的As和15.7%~66.5%的As生物有效性,减少淹水时Fe （II）的含量.但在土壤落干时期产生一定的“纳米效应”从而促进了Fe （II）的产生.研究表明,CuO NPs进入As污染农田改变了土壤环境因子,一定时间内降低了土壤As生物有效性.     

高砷煤矿废水灌溉区土壤砷的分布特征探究

通过对贵州兴仁县交乐村高砷煤矿废水灌溉区土壤总砷的分布特征进行研究。文章对土壤总砷、pH值及有机质含量进行了分析,结果表明,污灌区土壤砷的含量最高达237.55mg/kg,最低为24.20mg/kg,平均As含量为85.62mg/kg,均高于世界、中国及贵州黄壤中的平均水平;pH平均值为5.12,属酸性土壤;有机质含量普遍偏高;高砷煤矿废水灌溉导致了灌区土壤中砷的累积。灌溉方式影响了土壤中砷的迁移与分布情况,土壤中砷的含量,随着与灌溉水源距离的增加而急剧降低。研究区土壤中砷的迁移较缓慢,但有向下游逐渐扩散的趋势。土壤总砷含量受到了pH值和有机质影响,但与两者的相关性并不明显。     

深圳市土壤砷的背景含量及其影响因素研究

为摸清深圳市土壤砷的背景含量现状,以全市不受或很少受人类活动影响的基本生态控制线区域作为调查范围,布设土壤表层样点450个、土壤典型剖面样点50个,采用多点增量采样法采集表层样品500个、深层样品100个.结果表明,深圳市表层土壤砷的背景含量范围为0.07~202mg/kg,算术平均值为11.3mg/kg,95%分位值为55.9mg/kg,显著高于“七五”深圳土壤环境背景调查结果砷的95%分位值（35.4mg/kg）.不同土类中赤红壤、红壤和黄壤砷背景含量的算术平均值相近,95%分位值的高低顺序为：红壤 > 赤红壤 > 黄壤.不同母质母岩发育的土壤砷背景含量的算术平均值高低顺序为：灰色灰岩 > 砂砾页岩 > 凝灰熔岩 > 变质岩 > 花岗岩 > 片麻岩,95%分位值的高低顺序为：灰色灰岩 > 砂砾页岩 > 凝灰熔岩 > 变质岩 > 片麻岩 > 花岗岩.不同剖面层次砷背景含量的平均值和95%分位值差异较大,高低顺序为：底层 > 中层 > 表层,随着深度增加,砷的背景含量逐渐增加.土壤pH值、砂粒含量与砷背景含量呈显著负相关,黏粒、粉粒含量与砷背景含量呈显著正相关.土壤母质母岩和机械组成是影响深圳市土壤砷背景含量的主要因素.研究结果可为南方典型区域砷的土壤环境背景值研究提供参考与借鉴.     

接合子细胞强化对生物反应器降解2，4-D效应研究

研究了接合性质粒pJP4在2株纯菌E．coliDH5α、Alcaligenes sp．及好氧颗粒污泥混合菌群中的水平转移情况,并以获得pJP4质粒的接合子Alcaligenes sp．：：pJP4为强化菌种,考察了接合子细胞强化对好氧颗粒反应器和生物膜反应器中难降解有机物2,4-二氯苯氧乙酸（2,4-D）的去除效应．结...     

泥蚶对砷的积累效应及风险评价

通过对2013年12月到2015年6月养殖的泥蚶做跟踪调查,约每5个月采集一次样品进行总砷和无机砷的测定,计算泥蚶中总砷和无机砷的积累效应,并对其进行风险评价。结果表明:泥蚶随着养殖时间的增长对总砷的积累较强,呈正相关关系,对无机砷的积累整个过程无明显变化;泥蚶的肥满度与总砷含量呈显著正相关,与无机砷含量无显著关系。按照《海洋生物质量》标准进行评价,除苗蚶外均受到总砷的污染,按照《无公害食品水产品中有毒有害物质限量》标准,泥蚶均未受到无机砷的污染。根据联合国粮农组织和世界卫生组织下的食品添加剂联合专家委员会提出的暂定每周耐受摄入量（provisional tolerable weekly intake,PTWI）无机砷为15 μg/kg·bw。泥蚶中无机砷的每周评估摄入量（estimated weekly intake,EWI）值均小于PTWI,仅占PTWI的5.5%~6.2%,其消费引起的潜在致癌风险均为可接受风险。     

An E. coli SOS-EGFP biosensor for fast and sensitive detection of DNA damaging agents

An E. coli SOS-EGFP biosensor which expresses enhanced green fluorescent protein as a reporter protein under the control of recA gene promoter in SOS response was constructed for detection of DNA damage and evaluation of DNA damaging chemicals. The chemicals that may cause substantial DNA damage will trigger SOS response in the constructed bacterial biosensor, and then the reporter egfp gene under the control of recA promoter is stimulated to express as a fluorescent protein, allowing fast and sensitive fluorescence detection. Interestingly, this biosensor can be simultaneously applied for evaluation of genotoxicity and cytotoxicity. The SOS-EGFP bacterial biosensor provides a sensitive, specific and simple method for detecting known and potential DNA damaging chemicals.     

共面多氯联苯抗体的制备及其用表面等离子共振方法检测的应用研究

根据文献方法获得抗共面多氯联苯(Co-PCBs)的重组抗体的轻链、重链基因序列,并用柔性连接肽(Gly4Ser)3进行连接.然后将获得的重组抗体基因序列通过克隆、表达、纯化、复性等手段得到纯度大于95%的Co-PCBs抗体.最后将Co-PCBs抗体组装到金膜表面,制备成检测用的蛋白芯片,并应用表面等离子共振技术(SPR)对共面多氯联苯中的PCB126进行了检测.结果显示,得到的Co-PCBs抗体特异性强,而且SPR检测方法测定PCB126的检测限为5×10-4pg·L-1,达到环境中Co-PCBs的超痕量检测水平要求.整个检测过程方便快捷、样品用量少、灵敏度高,可为环境中多氯联苯的实时检测研究提供指导.