Research Of Removal Typical Environmental Pollutants By Cell Surface Display

Environmental pollution has become increasingly prominent,especially heavy metals and antibiotics.It is found these two pollutants everywhere.Mercury is one of the most toxic metals.It always has two forms in the environment,including inorganic mercury(Hg²⁺)and organic mercury.Hg²⁺usually accounts for a large proportion of the total mercury in the environment.However,organic mercury is the most toxic form of mercury.It is a neurotoxic pollutant,causing irreversible harm to human beings.The common organic mercury is methylmercury（MeHg）.Human are exposed MeHg mainly through fish consumption.In fact,more than 94%of mercury in fish is MeHg.The ingested MeHg is accumulated fastly in tissues of animal body.In addition,antibiotics are an emergent environmental pollutant.China is a large country in the production and consumption of antibiotics.Most of these antibiotics are used in the livestock and poultry.Most of the antibiotics（50%-90%）taken by humans and animals can not be completely absorbed and metabolized,which causes large quantities of antibiotics are released into the environment as their original form.Antibiotics into the environment pose a great threat to human health.Furthermore,antibiotics in livestock manure inhibit methane production during anaerobic digestion.The cell surface display technology was used to remove mercury and to degrade antibiotics in this study.This new microbial remediation technology was preliminarily applied to the treatment of these pollutants.The main research contents are as follows:1.Most of the mercury in phytoplankton is inorganic mercury,which will be continuously enriched and bioaccumulated after entering the food chain.A mercury-binding peptide was displayed on the surfaces of Escherichia coli cells using an ice nucleation protein anchor.The surface-engineered E.coli facilitated selective adsorption of Hg²⁺from a solution containing various metal ions.It was found that the maximum adsorption capacity of engineering strains for Hg²⁺reached 394.9μmol/g stem cells.The Hg²⁺adsorption capacity of the whole-cell was four-fold higher than that of the original bacterial host cell.Approximately 95%of Hg²⁺was removed from solution by these whole-cell sorbents.Using edible Carassius auratus as research model,the engineered strains were fed to C.auratus and the bacteria colonized the intestines.C.auratus with engineered strains showed significantly less（51.1%）accumulation of total mercury compared with that in the group without any engineered bacteria.The engineered bacteria facilitate Hg²⁺excretion in feces.The surface-engineered E.coli effectively protected fish against the toxicity of Hg²⁺in aquatic environments.The toxicity of Hg²⁺in fish was alleviated by an effective surface-engineered strain via the adsorption of Hg²⁺.Furthermore,the surface-engineered E.coli mitigated microbial diversity changes caused by Hg²⁺exposure,which protected the intestinal microbial community.This strategy is a novel approach to controlling Hg²⁺contamination in fish.2.Hg²⁺in environment is usually converted MeHg by certain anaerobic microorganisms.MeHg can bioaccumulate to high concentration levels in natural aquatic food webs.In this work,E.coli strain W-1 was isolated from C.auratus feces.A peptide with seven amino acids was displayed on the cell surfaces of E.coli strain W-1.These engineered strains exhibited high affinity and selectivity toward MeHg.They efficiently removed more than 96%of 12μM methylmercury,and accumulation of methylmercury in the engineered strain was four times higher than that in the wild type.It’s found thattemperature and other metal ions did not affect MeHg adsorption.Transmission electron microscopy confirmed MeHg accumulation on cell membranes.C.auratus was fed by engineered bacteria,which showed a decrease in MeHg concentration in muscles of about 36.3%;whereas an increase in MeHg concentration was observed in the feces（36.7%）in comparison to the control group.The engineered strain in the gut captured MeHg and prevented it’s absorption by muscles,while some bacteria with MeHg were excreted in the feces.This method can reduce the accumulation of MeHg in fish.3.Large quantities of antibiotics are used for livestock resulting in the presence of high levels of antibiotics in manure,inhibiting biogas production during anaerobic digestion.Hence,a novel biocatalyst was developed by displayingβ-lactamase on the cell surface of E.coli.This whole-cell biocatalyst showed high enzyme activity（4.93U/mg dry cells）in degradingβ-lactam antibiotics and higher stability than freeβ-lactamase.The whole-cell biocatalyst retained over 82.7%of the initial enzyme activity after 60 days storage at room temperature,while the activity of freeβ-lactamase declined to 18.0%of its initial activity.Penicillin,cefamezin,and amoxicillin were completely degraded in solution by the biocatalyst within one hour.Pretreatment of swine manure containingβ-lactam antibiotics with it enhanced methane production（93.2%）during anaerobic digestion due to antibiotic degradation.The engineered biocatalyst protected the microbial community from antibiotic inhibition and hence is conducive for the growth of methanogens.This new strategy eliminates the inhibiting effects of antibiotics on manure,increasing the methane production and therefore achieving maximum utilization of renewable energy during anaerobic digestion.4.Erythromycin is one of the macrolide antibiotics.It is widely used in livestock and poultry industry.Erythromycin is one of the antibiotics with high detection rate in the environment.Therefore,erythromycin esterase was displayed on the surface of E.coli cell as whole-cell biocatalyst.The erythromycin esterase activity of whole-cell biocatalyst is 1.0 U/mg dry cells.The optimum temperature and pH of enzyme activity were 30℃and 7 respectively.Whole-cell biocatalyst can completely degrade50 mg/L erythromycin within 24 hours.The stability of the enzyme is very high.After40 days storage at room temperature,the enzyme can degrade 50 mg/L erythromycin completely within 24 hours,and maintain 86%of initial enzyme activity.The engineering strain can be recycled and reused for 7 times,and the enzyme activity reached 80%of the initial activity.In order to reduce erythromycin in the environment,the whole-cell biocatalyst was used in the treatment of wastewater containing erythromycin,and the degradation efficiency reached 100%.