The Microbial Community Structure And Remediation Efficiency In In-situ Bioremediation Of Nitrobenzene Contaminated Groundwater

As a common, high toxicity environmental organic pollutant, nitrobenzene hasbeen detected frequently in organic contaminated groundwater and has been listed asthe environmental priority pollutant by many countries. Nitrobenzene in groundwaterwould result in the damage of ecological environment, destruction of groundwaterresources, and cause human diseases through drinking water or food chain. Based onthese reasons, it is significant to remediate nitrobenzene-contaminated groundwatereffectively. Among all the remediation technologies for groundwater, in-situbioremediation technology is considered as the most potential and green remediationtechnology. As known to all, nitrobenzene can be biodegrade by the correspondingfunctional microorganisms both in aerobic or anaerobic condition. Therefore, thisresearch firstly investigate the microbial community characteristics in actualnitrobenzene-contaminated groundwater, and then study the biostimulation andbioaugmentation methods to bioremediate nitrobenzene-contaminated groundwater inlaboratory. Also PCR-DGGE and RT-FQ-PCR technology were applied to researchthe varieties of microbial community structure and function in groundwater.First of all, the microbial community characteristics and water chemical factorsof actual nitrobenzene-contaminated groundwater were investigated, and therelationships between microbial community characteristics and water chemical factorswere studied by CCA and correlation analysis. The results showed that a measuredamount of microorganisms including bacteria, actinomycetes, mould, yeast and fungi,were determined in nitrobenzene-contaminated groundwater and large differences ofbiomass existed among the sampling points. The microbial communities maintained agood diversity and the species including Acidovorax sp., Flectobacillus Lacus,Pseudomonas. Corrugata, Rhizobium sp., took large degrees of dominance in groundwater microbial communities. The similarities among all the microbialcommunities from different sampling points were high but some differences were stillexisted. As the key gene to degrade nitrobenzene according to the partial reductivepathway, gene nbzA were detected in all groundwater samples and the relativeabundances of nbzA were especially high in sample S2, S8and S9, which indicatedthat there existed nitrobenzene-degrading microbes in nitrobenzene-contaminatedgroundwater. The results of CCA showed that obvious correlation were maintainedbetween the distributions of microorganisms and TN, NB. Correlation analysisshowed that the abundances of cultured microbes were positively correlated with thevariables of TOC, pH, SO42-and NB, and negatively correlated with the variables of F-,Cl-, NO2-and Cd. The relative abundances of gene nbzA were positively correlatedwith F-, Cl-and NB, and negatively correlated with TOC, pH, NO2-, SO42-and Cd.Secondly, the removal effects of nitrobenzene and the changes of microbialcommunity in groundwater were investigated detailed. The results of screeningbiostimulants showed that lactose-phosphate, peptone, beef extract could enhance thegrowth of indigenous microbes, respectively, and the nitrobenzene in groundwaterwas removed effectively. The results of simulated experiments of in-situbioremediation by biostimulation showed that the removal rate of nitrobenzene ingroundwater could be17.20%under natural condition without adding anybiostimulant,62.05%with lactose phosphate,74.87%with peptone and71.27%withbeef extract. After biostimulation, the microbial dehydrogenase activities, biomassand biodiversities were all increased, microbial community structures changed butstill maintained some homology and the dominant species changed synchronously.The relative abundances of gene nbzA and biomass of nitrobenzene-degradingmicrobes (containing gene nbzA) were improved obviously. Above research resultsdemonstrated that adding lactose-phosphate, peptone and beef extract tonitrobenzene-contaminated groundwater could actually activate the indigenousmicroorganisms, especially nitrobenzene-degrading microbes that had biodegradednitrobenzene in groundwater effectively. The order of biostimulation effects waslactose-phosphate> peptone> beef extract. However, only biostimulation could not remove nitrobenzene completely and a measured amount of nitrobenzene was existedin groundwater.Then, nitrobenzene-degrading bacteria were screened from actualnitrobenzene-contaminated groundwater before the experiments of simulatedbioremediation by bioaugmentation. The identification, degradation characteristics,level of nbzA gene expression and degradation kinetics of nitrobenzene-degradingbacteria was also investigated. The results showed that one strain of nitrobenzene-degrading bacterium named as ZG was screened successfully. The ZG strain wasgram-negative short rod bacterium and could utilize nitrobenzene as the sole carbonsource and nitrogen source. The results of16S rDNA sequence analysis showed thatZG strain was most probable Pseudomonas putida. In addition, ZG strain containedgene nbzA encoded nitroreductase and biodegraded nitrobenzene according to thepartial reductive pathway. ZG strain showed a relatively strong bearing capabilityagainst environmental variety. When temperature was maintained in the range of10³0℃, pH4⁹, initial concentration of nitrobenzene less than600mg/L, ZG straincould biodegrade nitrobenzene effectively. The best condition for ZG strain degradingnitrobenzene was temperature20℃, pH7, initial concentration of nitrobenzene lessthan300mg/L,then the removal rate of nitrobenzene could be more than99%. Therelative expression of gene nbzA changed with the variety of environmentalconditions (temperature and pH) and the variation trends were same. The relativeexpression of gene nbzA and removal effects of nitrobenzene was influenced by ironand manganese significantly. When the concentration of Fe²⁺was less than5mg/L,there was nearly no influence on the removal effects of nitrobenzene and the relativeexpression of gene nbzA. If the concentration of Fe²⁺was more than25mg/L, theretook the opposite results. When the concentration of Mn²⁺was rising from0mg/L to15mg/L, the removal effects of nitrobenzene and the relative expression of gene nbzAalso increased continuously, which indicated that Mn²⁺in the range of0¹5mg/Lwould enhance the relative expression of gene nbzA of ZG strain. In the actualnitrobenzene-contaminated groundwater, the relative expression of gene nbzA of ZGstrain was higher that in mineral medium, which suggested that there would be no inhibiting factor for the nbzA gene expression. On the contrary, there may be somepromoting factors. The results of degrading-kinetics showed that it corresponded tothe first-order kinetics for ZG strain to degrade nitrobenzene and the half-life was ln(1/2)/K, which indicated that nitrobenzene concentration is higher then the removaleffect is better.Finally, laboratory-scale study on in-situ bioremediation of nitrobenzene-contaminated groundwater by bioaugmentation was carried out and the remediationeffects and microbial communities were investigated detailed. Two modes ofbioaugmentation including adding ZG strain to groundwater solely and combinedaeration and adding ZG strain, were operated respectively. The results showed that theremoval rate of nitrobenzene in groundwater could reach66.31%and the residualnitrobenzene was about10.39mg/L after bioremediation with the mode of adding ZGsolely. While the removal rate of nitrobenzene could reach89.92%and the residualnitrobenzene was below4.16mg/L after bioremediation with the mode of combinedaeration and adding ZG strain. After adding ZG strain, the concentrations of dissolvedoxygen in groundwater were0.85mg/L less on average than that before adding ZGstrain, which meant that ZG strain consumed dissolved oxygen which was the keyfactor for in-situ bioremediation with bioaugmentation. ZG strain could migrate torandom direction, especially along the groundwater flow direction, the horizontalmigration rate could reach0.08m/d. The final concentration of ZG strain ingroundwater was about1.0×10⁵¹.8×10⁵cfu/mL because of groundwater scouringand limited microbial environmental nutrition condition. Compared with the naturalgroundwater, the microbial dehydrogenase activity in groundwater was increased by76.25%with single aeration,82.50%with single adding ZG strain,138.75%³56.25%with combined aeration and adding ZG strain. PCR-DGGE results showedthat the microbial community structure had been changed significantly by singleaeration, and the similarity of microbial community structure between before and aftersingle aeration was only42.6%. After single aeration, in indigenous ZG strain andother microbial species were activated and became the dominant species. There waslittle changes for groundwater microbial communities structure with adding ZG strain along, and the similarity of microbial community structure between before and aftersingle adding ZG strain could reach up to61%. After remediation by combinedaeration and adding ZG strain, the microbial distribution in whole simulatedgroundwater system was similar and a measured abundance of ZG strain was detected,which also indicated that ZG strain actually had migrated and kept certain abundancein groundwater. What's more, there was no obvious promoting or inhabiting effect tothe other species in groundwater with adding large number of ZG strain. TheRT-FQ-PCR results showed that both aeration and adding ZG strain could improve therelative abundance of gene nbzA and its relative expressions. Moreover, theconcentration of nitrobenzene may be the key factor to affect the relative abundanceand expression of gene nbzA.In conclusion, in-situ bioremediation technology including biostimulation andbioaugmentation was an economic, practical, efficient and green remediationtechnology for nitrobenzene-contaminated groundwater. In the actual remediationprocess, according to the actual situation of contaminated site, it could choosebioaugmentation method directly or choose the sequencing batch craft of combinedbiostimulation and bioaugmentation.