| With the economic and social development,combustion and utilization of fossil energy,as well as sewage irrigation,leakage,poor governance,the situation of the mercury and phenanthrene pollution become more serious than ever.In addition to a small portion of the Hg,PHE entering the global atmospheric circulation in the form of gas,over 90%of those pollutants settled and deposited in the soil environment,and jeopardized the soil ecosystem.Traditional analysis may qualitatively and quantitatively determine Hg and PHE,however,the actual bioavailable fraction of those contaminants as well as their effectiveness on soil microorganism are difficult to evaluate,due to the complex nature of soil environment.As a helpful supplement to traditional methods of chemical identification,biosensors,especially genetically engineered microbial whole-cell biosensors(EMWCB),have been developed and are considered to be an effective method for the environmental assessment.EMWCBs can not only qualitatively evaluate specific toxins under a sub-lethal dose,but also semi-quantitatively assess their chronic effects instead of the acute impact of 'Global'contamination.With high specificities,EMWCBs can inform us about the bioavailable fraction of certain chemicals,thus provide more biologically relevant information than traditional analytical or toxicological evaluations.At present,there is still a blank for the use of EMWCBs to evaluate the bioavailability of combined mercury and phenanthrene in soil samples.Furthermore,although EMWCBs can be considerably more time-and cost-effective,the problem of how to improve the genetic stability of microbial sensors,how to maintain cell viability whereas decrease the minimum detection limit,and how to apply those sensor cells on actual soil samples with combined pollution are still need to be solved.Therefore,based on previous work on pollutants degradation and screening of resistant strains,this study mainly focused on the soil with single or combined Hg/PHE pollution and the evaluation of their bioavailabilities with specific luminescent microbial biosensors.The reliability and applicability of the technique were expounded from various angles and the factors affecting the bioavilabilities of each pollutant were discussed.Firstly,A luminescent reporter gene system merR-PmerT-egfp was constructed by fusing the mercury-inducible promoter,PmerT,and its regulatory gene,merR with a promoterless reporter gene EGFP.A stable and non-antibiotic whole-cell reporter(BMB-ME)was created by introducing the system cassette into the chromosome of Pseudomonas putida strain.Another genetically engineered reporter BMB-PL,which based on the aromatic compounds regulator gene phnR and specific promoter Pphns connecting a luciferase gene luxCDABE cassette,were used to determine bioavailable phenanthrene.Both reporter gene system on chromosome DNA or in plasmid vector could express green fluorescent protein or luciferase in response to Hg or PHE,and a strong fluorescent or spontaneous excitation signal are produced respectively.BMB-ME and its sensory strain series specifically recognize Hg2+ and express strong fluorescence signal,while other divalent heavy metal cations at Nano molar level did not interfere with the measurement.The signal intensity of its expression was significantly higher than other heavy metal ions(P<0.01).Reporter BMB-PL showed responses to low ring PAH:naphthalene,anthracene,phenanthrene and their metabolites,after treated with 50mg·L-1 cytochrome P450 6h.It was found that bioavailability of all those pollutants increased,and relative luminescent unit(RLU)of PHE was significantly higher than the other low ring PAHs(P<0.05).All the reporters were treated with calcium alginate and embedded the sensor cells in gel beads.Filter paper adsorption immobilization were also used to keep cell viabilities and stabilized in one-week storage by both methods.This is the first study to construct 'Light-on'biosensors,which based on chromosome and plasmid system.The improved signal intensity,enhanced genetic stability,optimized substrate specificity and increased cell stability laid the foundation for the practical application of the EMWCBs technology.Secondly,the typical red soil from Jiangxi China was used as a model,and the bioavailability of mercury,phenanthrene and their combined pollutants were evaluated.Immobilized BMB-ME and BMB-PL were test in the simulated in situ Hg and PHE contaminated conditions.The total concentration of carbon sources,induction time,water content and temperature were set as four main influencing factors.Orthogonal experiment were then designed with three levels differences were set for each factor,and the optimum detection conditions were determined.At the same time,bioavailability of single and combined PHE/Hg in red soil were detected.Results showed that single spiked soil with 0-60 mg kg-1 PHE had different characteristic curve with combined PHE/Hg spiked samples.Whereas,bioavailability of Hg,which concentration ranged from 0 to 240 ?g kg-1,changed a little either in single or in combined pollutions.Therefore,red soil were extracted and both cold atomic absorption and BMB-ME were used for quantitative Hg fractions determination.It was found that the residual mercury was the main form of mercury pollution in the soil,and its content was about 30%of the total amount.Yet,the bioavailability of soil Hg is determined by the BA and water soluble fractions,which were respectively less than 10%and 15%of its total.In addition,bothl5 days and 30 days aging time decreased mercury bioavailability,either treated with high dose(100 g-1 kg)or low dose(10 g-1 kg)HgCl2.We also compared the CVAAS,RP-HPLC and the EMWCB method for the determination of mercury and phenanthrene.In a certain concentration range,the report bacteria BMB-ME could make a quantitative determination of mercury pollution in red soil,and the detection rate of mercury had no difference at level of 0.05.BMB-PL can be better used for reflecting the concentration of PHE in a range of 0-240 ?g kg-1.However,its detection ability still showed a big gap comparing with the analysis method,and the detection rate is only 10-20%.These findings optimized a in situ conditions detection of combined Hg/PHE bioavailablity and established the technical system of the assesment by EMWCBs.They also gave a better insight into the bioavailable Hg/PHE interaction under a combined form.Thirdly,For the monitoring of mercury and phenanthrene pollutants in real soils,it is very important for the method to inform us bioavailable pollutants in key areas and let us know the spatial distribution of those contaminants.Therefore,sigmoid curves of bioavailable Hg and PHE in standard GSF-1 soil samples were constructed by exploiting BMB-ME 1 and BMB-PL reporters and the threshold for Hg or PHE analysis were also calculated(19.6?111.63 ?g·kg-1 b-Hg;21.5?110.9?g·kg-1 b-PHE).This ability of the system at these thresholds makes it possible to analyze combined b-Hg and b-PHE in real environmental samples.Then,Large scale and fast screening of soil contamination across Yangtze River Delta(YRD)was conducted and soils from three large refinery facilities(NJ,SH and NB)were tested.Bioavailability of both Hg and PHE were evaluated by sensor strains and results were verified by GC-MS and CVAAS analysis.Results showed strong correlations between analytical and sensor strains method,suggesting a 10%consistency between the bioavailable and total pollutants.In addition,b-Hg detection rate of 35%and 80%was resulted in NJ and NB refinery sites,respectively.The highest b-Hg concentration was 84.4 ?g·kg-1 in NJ,which was significantly higher than the SH and non-refinery surrounding soil.The detection rate of b-PHE in SH sites was 60%,and its highest concentration was 63?g·kg-1 g,while the content of b-Hg was relatively lower.Detection rates of b-Hg and b-PHE in 25 soil samples from random collected Yangtze River Delta were 35%and 0%,respectively.This suggested high toxicity and more serious situation of Hg contamination than PHE,which is mainly concentrated in surrounding soil of heavy chemical industrial facilities.Because of the migration of mercury and phenanthrene in the environment,as well as great impact of human activities on those pollutants distribution,the population densities as well as 10?m particulate matter(PM 10)concentrations were also used as parameters and the spatial distribution of b-Hg and b-PHE were illustrated.The result demonstrated that biosensors,which are time-and cost-efficient,could offer a probable way to test soil samples from contaminated areas or provide a rapid screening tool for environmental risk assessment.It was found that the spatial distribution of b-Hg,instead of b-PHE in YRD,were significantly related to the population density.PM10 distribution seems to have little effect on b-PHE,while The distribution and concentration of b-Hg in YRD soil were significantly affected by PM10.From west to East,b-Hg showed a significant decreasement with PM 10.Results of this study for the first time verified the feasibility of bacteria reportor method for a wide range environmental investigation,on the other hand,large scale geographical distribution characteristics of bioavailable Hg/PHE were also found.This provided an important reference for typical environmental pollutants bioavailability evaluations in key areas..Finally,Based on the typical red soil from Jiangxi,single and combined pollution treatments of cultivated rice,cole,peanut,and uncultivated red soil were designed.At the same time,the Hg treatments had also been designed to simulate different conditions of sewage irrigation with the fixed and continuous dose pollution experiment.Based on the preliminary construction of the whole cell bacterial sensors,combined with soil enzyme activity analysis,soil bacterial DGGE results,bioavailabilities and ecological toxicities of Hg/PHE pollutants were comprehensively evaluated after the red soil was incubated a certain period of time.After analysis different crops on the soil diversity index by RDA or CCA,bioavailability and pollution time of pollutants were demonstrated to be negatively correlated,that is consistent with the expectation.In addition,no significant differences of microbial populations in fixed dose mercury or phenanthrene soil treatments were observed,while continuous dose of Hg in paddy soil showed negatively correlation with b-Hg.This demonstrated that the red soil,which cultivated with rice,could alleviate continuous dose of mercury pollution,and this alleviation effcect was significantly better than that of the peanut or cole plantcd soil.The research objectively illustrated the relief characteristics of crop types on Hg and PHE toxicities;This was the first time to comprehensively analyze the bioavailability of pollutants and soil microbe by different ways;A new system for evaluating the soil quality was established based on the bioavailability of pollutants;The general rule of paddy soil on the relief of Hg and PHE pollution was found on the level of bacterial diversity.All these findings have certain guiding significance to the quality improvement of the polluted farmland.In conclusion,this research exploit specific bacterial luminescence biosensors,with mercury and phenanthrene as typical heavy metals and polycyclic aromatic hydrocarbons representatives,established methods and evaluated bioavailabilities of the single and combined pollution in soil.Real environmental samples determination and relationship analysis between bioavailable pollutants and soil bacterial community changes had also been conducted.This research expatiated characteristics of bioavailable Hg/PHE in soil from many aspects,and establish a reliable theoretical and practical basis for evaluation EMWCBs application in single or combined contaminated soil. |
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