发光细菌法测定环境中金属毒性的研究进展

在环境污染物的毒性评价和监测中,发光细菌法是一种具有快速、灵敏和廉价等优点的直接生物测试方法.本文简要回顾了发光细菌法的测定原理及其在水环境中的应用;总结了发光细菌法测定水环境中金属毒性的主要影响因素(如pH、有机无机配体和交互作用);重点评述了发光细菌法在土壤样品金属毒性测定方面的应用、不同提取方法的优缺点以及土壤金属毒性与形态之间的关系;提出今后应加强土壤中金属对发光细菌的毒害机理、土壤环境中发光细菌法标准化以及发光细菌法与其他测试方法的关系等研究.快速、廉价、标准化发光细菌法的建立对土壤环境中金属风险评价和监测具有重要的意义.     

发光细菌JMU07的分离鉴定及其在生物毒性测定的初步研究

从乌贼表皮通过分离纯化得到一株发光细菌JMU07。该菌的菌落形态呈典型细菌菌落特征;显微镜下观察其为球杆状菌,革兰氏染色阴性。用荧光分光光度计测定其发光波长在420-650 nm之间,最大发光波长为477 nm。16S rDNA法测序,构建系统进化树,初步鉴定发光细菌JMU07为鳆发光杆菌(Photobacterium leiog-nathi)。生长发光曲线测定表明,发光细菌JMU07发光强度最高出现在对数中后期,相比明亮发光杆菌,JMU07具有发光强度高,持续发光时间长的特点。根据国标GB/T15441-1995研究HgCl2浓度与发光细菌JMU07发光强度抑制率的关系得到:HgCl2浓度与发光细菌JMU07发光强度抑制呈良好线性关系;JMU07 EC50为0.11 mg/L,略低于明亮发光杆菌的0.14 mg/L,表明JMU07对HgCl2的毒性更敏感。因此新分离得到的发光细菌JMU07有希望用于环境检测、食品卫生与安全等领域综合毒性的快速检测。     

硝化基质和产物对发光细菌的急性毒性

摘要：【目的】对硝化基质和产物对硝化过程的影响进行初步研究。【方法】采用发光细菌法,在pH=7.0的条件下,测定了氨、羟胺、亚硝酸和硝酸对发光细菌的急性毒性（15min-半抑制浓度（the half inhibitory concentration,IC50））。【结果】单一物质的毒性试验结果表明,硝化基质和产物对发光细菌的毒性随浓度的升高而增大,且具有较好的线性关系;氨、羟胺、亚硝酸和硝酸的IC50分别为2180.2 mg/L、6.2740 mg/L、1207.2 mg/L和3140.3 mg/L;其毒性大小顺序为：羟胺 >亚硝酸 >氨 >硝酸。按等效浓度混合法测定硝化基质和产物的联合毒性,结果表明：氨与羟胺、氨与亚硝酸、羟胺与亚硝酸对发光细菌的联合毒性呈相加作用;氨与硝酸、羟胺与硝酸、亚硝酸与硝酸对发光细菌的联合毒性呈独立作用;氨、羟胺、亚硝酸、硝酸四元混合物的联合毒性也呈相加作用。【结论】根据硝化基质和产物对发光细菌和硝化细菌抑制浓度的相关性,可用发光细菌发光强度的变化指示硝化基质和产物的抑制作用。     

细菌发光的分子生物学及发光基因的应用

本对发光细菌生物发光的分子生物学、发光基因在转染细胞中的表达、发光基因在分子生物这中的应用以及基因工程发光菌的应用进行了综述。     

发光细菌lux报告基因系统的评价及应用

细菌生物发光基因（ｌｕｘ）的成功分离和表达极大地促进了其在应用和基础研究上的进展,几乎所有的发光细菌可归于３个属（发光杆菌属（Ｐｈｏｔｏｂａｃｔｅｒｉｕｍ）;弧菌属（Ｖｉｂｒｉｏ）;异短杆菌属（Ｘｅｎｏｒｈａｂｄｕｓ））,ｌｕｘ基因是编码和调控发光细菌生物发光的操纵子,与其他报告基因相比,具有快速、简便、灵敏和对细胞无伤害的优点,在分子生物学、临床微生物、以及生化检测中呈现潜在应用价值。本文就ｌｕｘ报告基因系统的优缺点及其与常用的几个报告基因系统进行比较和评价,并就ｌｕｘ报告基因系统在基因表达、…     

发光细菌法快速监测和评价工业废水综合毒性

以汞、镉、铅、砷和六价铬的混合后对发光细菌的综合毒性为参照标准,对工业废水进行快速监测和毒性分级的研究,实验得出上述混合毒物与发光细菌的发光强度的相关关系是y＝40.9131-23.7200lnx,r-0.995。根据工业废水的相对发光强度,建议将工业废水划分为微毒,低毒、中等毒,高毒和剧毒五个毒性级别,并可根据上述线性关系,推算出有毒物质的浓度范围和相当于国家标准的倍数。应用此法对22份工业废水进行了实际测定与评价;还对四个企业工业废水处理前后的毒性进行了测定与毒性比较,结果表明处理后较处理前的毒性的毒性明显降低。     

饲料原料中有毒有害物质的综合毒性测定法初探

目的建立发光细菌综合毒性测定法,对实验动物饲料原料中有毒有害物质进行初筛.方法用发光细菌作指示生物,测定金属离子Pb2+,Zn2+,Cr+,Hg2+和毒性物质苯酚,灭害灵;以Hg2+为毒性对照物,测定10种饲料原料和3种人用食品.结果发光菌对有毒有害物质和金属离子敏感.在所测定的范围内,其发光强度随浓度增加而减弱,即相对抑光率增强,呈负线性相关,其相关系数在0.92以上;以Hg2+为毒性对照物,对10种饲料原料和3种人用食品进行测定,其中脱水蔬菜相对抑光率最大,为39.74%,相当于1.85×10-5%Hg2+的表征毒性,提示该样品可能受到农药和化肥等较严重的污染.结论发光细菌综合毒性测定法,可用于饲料原料中有毒有害物质的初筛,是一种快速、经济、有效的方法.     

PCR检测霍乱弧菌RTX吸毒素基因

建立了一种扩增最近发现的霍乱弧菌RTX毒素基因的PCR方法。在世界各地引起流行和散发霍乱病例的166株临床和环境霍乱弧菌分离物中,用PCR和Hep—2细胞细胞毒性试验发现它们都是产毒的。而在相关基因簇中有缺失的古典生物型标准株用这两种方法结果都是阴性。这是第一个用于鉴别O1群霍乱弧菌古典生物型菌株和E1　Tor生物型菌株以及包括O139血清型的其它非O1群菌株的快速基因分型方法。建立的PCR方法也可特异性地检测霍乱弧菌中的RTX毒素基因,因为用此RTX毒素特异性PCR以及Hep—2细胞毒性试验时副溶血弧菌、致腹泻性大肠杆菌、气单胞菌和邻单胞菌的临床分离株都是阴性。这些结果使霍乱弧菌中RTx毒素的特性明显了。其在细菌致病中的作用需要进一步研究。     

生物发光协助净化环境

对释放入环境的工业污染物数量的关注激励科研人员研制用于检测毒素,特别是重金属的简便而又精确的方法。微生物学家现已提供了一种新的检测毒素和重金属的途径,即利用依据其所处环境中的金属浓度发光或不发光的生物发光细菌。 David Holmes及其在Clarkson大学(Potsdam,NY)的同事将一种生物发光海洋细菌费氏弧菌(Vibrio fischeri)的发光基因与其它相应于存在铜或汞的生物基因融合,制备了检测铜和汞的遗传工     

PCR技术在植物病原物和植物抗病研究中的应用

着重结合植物病原物的群体遗传、毒性变异和分子检测,以及植物抗病机制和抗病基因定位等领域,综述了聚合酶链反应技术在植物病理研究中的应用现状和潜力。     

Bioluminescence intensity difference observed in luminous bacteria groups with different motility

Aims:  The aim of this study was to compare the luminescent intensity of bioluminescence from marine luminous bacteria with different motility.
Methods and Results:  Luminescent bacteria were separated according to their motility using a microfluidic device. The cell densities of the separated samples were measured using a counting plate. The luminescent intensity of the separated samples was measured using a luminometer. The luminescent intensity per cell was calculated, and the values from the mobile (swimmers) and the nonmobile cells (nonswimmers) per single cell were compared; as a result, the former were proved to be larger than the latter.
Conclusions:  Microfluidics were shown to be effective for the separation of bioluminescent bacteria and the bioluminescent intensity difference per cell was recognized with this experiment.
Significance and Impact of the Study:  This study introduced for the first time a method to examine the individual cell function of Photobacterium kishitanii .     

Fabrication of a bio-MEMS based cell-chip for toxicity monitoring

A bio-MEMS based cell-chip that can detect a specific toxicity was fabricated by patterning and immobilizing bioluminescent bacteria in a microfluidic chip. Since the emitted light intensity of bioluminescent bacteria changed in response to the presence of chemicals, the bacteria were used as the toxicity indicator in this study. A pattern of immobilized cells was successfully generated by photolithography, utilizing a water-soluble and negatively photosensitive polymer, PVA-SbQ (polyvinyl alcohol-styrylpyridinium) as an immobilization material. Using the recombinant Escherichia coli (E. coli) strain, GC2, which is sensitive to general toxicity, the following were investigated for the immobilization: an acceptable dose of long-wavelength UV light, the biocompatibility of the polymer, and the effect of the chip-environment. We found that 10 min of UV light exposure, the toxicity of polymer (SPP-H-13-bio), and the other chip-environment did not inhibit cell metabolism significantly for making a micro-cell-chip. Detection of a specific toxicity was demonstrated by simply immobilizing the bioluminescent bacteria, DK1, which increased bioluminescence in the presence of oxidative damage in the cells. An injection of hydrogen peroxide of 0.88 mM induced 10-fold increase in bioluminescent intensity confirming the capability of the chip for toxicity monitoring.     

Quenching of the luminescence of phosphorescent bacteria as a test for assessing the toxicity of the phenol components of sewage

The object of this work was to estimate whether the luminescence of luminescent bacteria could be used as a biological test for assessment of the toxicity of phenol compounds in sewage. The toxicity of phenol compounds for luminescent bacteria was compared in terms of three indices: the quenching of luminescence, the inhibition of dehydrogenase activity and the ability to grow. Among the three indices, the quenching of luminescence was characterized by the highest sensitivity and the most rapid response.     

Luminescent bacteria toxicity assay in the study of mercury speciation

The toxicities of solutions of 10 mercury compounds to luminescent bacteria were measured using the Microtox Toxicity Bioassay. The aim of this study was to assess the influence that the counter-ions have on the aquatic toxicity of mercury salts. The toxicities of these mercury compounds were very similar, except for mercurous tannate and mercuric salicylate. This can be attributed to differences in the ionization and speciation patterns of these compounds relative to the other compounds tested. In general, the toxicity of the solutions at pH 5 was not significantly different from the toxicity of these solutions at pH 6, but a clear reduction in toxicity was observed when the pH of the solution was adjusted to pH 9. Significant differences were found between the toxicity of Hg(I) and Hg(II) salts of the same anion at pH 9. When cysteine was added to a mercuric nitrate solution (at pH 6), a reduction in the toxicity was observed. This can be explained in terms of the strong binding of mercury to cysteine, thus reducing the concentration of mercury species available to cause an observable toxic effect to the bioluminescent bacteria.     

Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

A novel continuous toxicity test system using a luminously modified freshwater bacterium

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.     

The luminescent bacteria toxicity test: Its potential as an in vitro alternative

During the past several years, the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives for some of the whole animal toxicity assays. One assay proposed as an alternative to animal testing is the luminescent bacteria toxicity test (LBT), provided under the trade name of Microtox®. The sensitivity and specificity of the LBT was compared with two commonly used toxicity tests–-the L-929 Minimal Eùgle's Medium (MEM) elution cytotoxicity test and the Draize test. Cytotoxicity and LBT test data from 709 medical device and biomaterial extracts were compared using a positive/negative ranking system which provided a measurement of false positive and false negative results. These data were compiled from nine separate laboratories producing or using a wide variety of biomaterials and medical device products. The LBT was more sensitive than the tissue culture assay and displayed few false negatives. LBT EC₅₀ values were compared with eye irritancy categories for a group of 34 chemicals and 27 personal care products. As with tissue culture, the LBT was more sensitive and produced minimal false negatives. The data from this study indicate the LBT has potential as a rapid, simple method to screen biomaterials and personal care products for toxicity and irritancy.     

A whole cell bioluminescent biosensor for the detection of membrane-damaging toxicity

The recombinant bacteria strain DPD2540, containing afabA::luxCDABE fusion, was used to detect the toxicity of various chemicals in this study. Membrane damaging agents such as phenol, ethanol, and cerulenin induced a rapid bioluminescent response from this strain. Other toxic agents, such as DNA-damaging or oxidative-damaging chemicals, showed a delayed bioluminescent response in which the maximum peak appeared over 150min after induction. This strain was also tested for measurement of toxicity in field samples such as wastewater and river water effluents.     

Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

Solid-phase contact assay that uses a lux-marked Nitrosomonas europaea reporter strain to estimate toxicity of bioavailable linear alkylbenzene sulfonate in soil

Information about in situ toxicity of the bioavailable pools of adsorptive soil pollutants is a prerequisite for proper ecological risk assessment in contaminated soils. Such toxicity data may be obtained by assays allowing for direct exposure of introduced test microorganisms to the toxicants, as they appear in solid solution equilibria in the natural soil. We describe a novel sensitive solid-phase contact assay for in situ toxicity testing of soil pollutants based on a recombinant bioluminescent reporter strain of Nitrosomonas europaea. A slurry of the reporter strain and soil sample was shaken for 1 h, after which bioluminescence was measured either directly (soil slurry protocol) or in the supernatant obtained after centrifugation (soil extract protocol). The assay was validated for both protocols by using linear alkylbenzene sulfonate (LAS) as a toxic and adsorptive model compound in the soil samples. Interestingly, LAS showed the same toxicity to the reporter strain with either soil incubation (both protocols) or pure culture, suggesting that adsorbed LAS pools contributed to the observed toxicity. The solid-phase contact assay that used the reporter strain of lux-marked N. europaea was slightly more sensitive for the detection of LAS toxicity in soil than activity-based assays targeting indigenous nitrifiers and much more sensitive than assays targeting indigenous heterotrophic microbes. We conclude that the new solid-phase contact assay, which is based on direct interaction of the test microorganisms with bioavailable pools of the toxicants in soil, provides a most sensitive and relevant method for evaluating the in situ toxicity and assessing the risks of soil contaminants.