| Arsenic(As)is a toxic metalloid that is widely distributed in the natural environments.In the natural environment,arsenic has many species,and different species of arsenic have different toxicities.Therefore,the conversion of arsenic species a more toxic species to a less toxic species is also one of the important mechanisms of arsenic detoxification.C.salina is a single-cell,eukaryotic green algae that is widely distributed and abundant in the marine environment.In natural water bodies,microalgae rarely exist alone,and generally coexist with other microorganisms such as microalgae,bacteria and fungi to form a symbiotic system of bacteria and algae.The microalgaehas broad application prospects in water pollution restoration.In previous studies,it was found that the remediation of heavy metal(Cd?Cu)pollution by microalgae was affected by many factors,such as symbiotic bacteria species,light conditions,pH value and medium composition.However,little research has been done for the remediation of arsenic pollution by microalgae.The results of symbiotic bacteria on the metabolism and transformation of microalgae are different.Therefore,the mechanism of symbiotic bacteria on arsenic metabolism of microalgae remains to be further studied.This study used C.salina as a test material and a symbiotic bacterium was isolated from C.salina culture medium,which was identified as Agrobacterium tumefaciens WB-1.The axenic C.salina was obtained by adding antibiotic combination.In order to explore the causes of As(III)metabolism and transformation of C.salina,set up the following parts of the experiments separately:(1)Treatment of non-axenic and axenic C.salina and symbiotic bacteria with different concentrations of As(?);(2)Treatment of non-axenic and axenic C.salina and symbiotic bacteria with As(?)at different time;(3)Treatment of non-axenic and axenic C.salina and symbiotic bacteria with different concentrations of As(III)under shading culture conditions;(4)Treatment of without EPS of non-axenic and axenic C.salina with different concentrations of As(?)The experiments results are as follows:1.Speciation transformation of As(?)by the symbiotic bacteriumThe symbiotic bacterium were treated with different concentrations of As(?)(75,150,300,750 ?g·L-1),and for time period experiments the symbiotic bacteria were treated with 300 ?g·L-1 As(?).The As speciation in the culture medium were analyzed and the rates of oxidation and removal of As(?)by symbiotic bacterium were calculated.The results show that,the As(?)was the main arsenic specie in the symbiotic bacterium(A.tumefaciens WB-1)medium,the oxidation rate of As(?)was 4.51%?30.6%and the removal rate was 1.86%?16.2%.No methyl arsenic was detected in the culture medium.At 24?48h,the bacterium reduced As(?)in the culture solution to As(?).The results showed that the bacterium had the ability of As(?)oxidation,As(V)reduction and no methylation.2.Effect of As(?)treatment concentration on the arsenic metabolism of non-axenic and axenic C.salinaA series of As(?)treatments(0,75,150,300,750 ?g·L-1)was applied to the non-axenic and axenic C.salina.After 7 days of exposure,the absorption,adsorption and accumulation of As(?)by C.salina were determined.The arsenic speciation in the culture and algal cells were analyzed and the rates of oxidation and removal of As(?)by non-axenic and axenic C.salina were calculated.The results show that:compared with the axenic C.salina,the non-axenic C.salina grew faster with higher tolerance of As(?).Moreover,in the presence of bacterium,As(?)accumulation by C.salina from culture solution varied from 103 to 448 mg·kg-1,the oxidation and removal rate of As(?)were 78.9%?96.9%and 18.9%?55.2%,respectively.These values were significantly higher than those of the axenic C.salina,which varied from 11.7 to 91.4 mg·kg-1,14.5%?26.4%and 12.8%?29.2%,respectively.In addition,a small amounts of As(?)and methylated As were detected in the cells of the non-axenic C.salina,while no methylated As was present in the axenic C.salina cells.The results showed that the symbiotic bacterium significantly promoted the growth of C.salina and significantly affected the metabolism and speciation transformation of arsenic by C.salina.3.Effects of As(?)exposure time on the arsenic metabolism of non-axenic and axenic C.salinaOn the basis of the above experiments,300 ?g·L-1 As(?)treated non-axenic and axenic C.salina were set at different time periods(0 h,6 h,12 h,1 d,2 d,3 d,4 d,5 d,6 d,7 d).At different times to determine the absorption,adsorption and accumulation of As(?)by non-axenic and axenic C.salina,and the arsenic speciation in the culture and algal cells were analyzed and the rates of oxidation and removal of As(?)by non-axenic and axenic C.salina were calculated.The results showed that there was no significant difference in the biomass of the non-axenic and axenic C.salina at 0?24 h,but the biomass of the non-axenic C.salina was significantly higher than that of the axenic C.salina after 24 h.At the same time,the accumulation of non-axenic C.salina was significantly higher than that of axenic C.salina.At 0-3 d,the accumulation of non-axenic C.salina was mainly absorption,accounting for the accumulation 74.4%-94.7%,and there is a rapid adsorption process in the early 0?6 h treatment of axenic C.salina,mainly by adsorption.The oxidation rate of As(?)by the non-axenic and axenic C.salina reached the maximum in the logarithmic growth phase,and the oxidation rate of the non-axenic C.salina was significantly higher than that of the axenic C.salina,which was 97.6%and 57.4%.With the increase of exposure time,the removal rate of non-axenic C.salina was 53.7%at the 6 d,while the removal rate of As(?)by axenic C.salina was 32.7%,which was significantly lower than that of non-axenic C.salina.These results suggest that time interval have has a certain effect on the metabolism and transformation of As(?),and the symbiotic bacterium have significancontributuion for metabolism and stransformation of As(?)by C.salina.4.Effects of shading conditions on the arsenic metabolism in non-axenic and axenic C.salinaOn the basis of previous research,in shading conditions a series of As(?)concentrations(0,75,150,300,750 ?g· L-1)were exposured to the non-axenic and axenic C.salina.After 7 days of exposure,the absorption,adsorption and accumulation of As(?)by C.salina were determined.The arsenic speciation in the medium and algal cells were analyzed and the rates of oxidation and removal of As(?)by non-axenic and axenic C.salina were calculated.Our results showed that the growth rate of non-axenic and axenic C.salina under shading culture conditions was significantlydensity)value of axenic C.salina in the shading culture conditions showed a downward trend,indicating lower than that of C.salina was cultured under light conditions,and the OD(optical that the light conditions are favorable for the growth of C.salina.Under shading culture conditions,the arsenic accumulation and removal ratio of non-axenic C.salina and axenic C.salina,both of which were significantly lower than the accumulation and removal rate of the non-axenic and axenic C.salina under light culture conditions.Indicating that light culture conditions had significant effects on the arsenic metabolism in C.salina.Under shading culture conditions,the oxidation rate of As(?)was 88.8%?99.2%by non-axenic C.salina,and the As(?)oxidation rate of axenic C.salina was 9.46%-26.0%,there was no significant difference in the arsenic oxidation rate in C.salina,indicating that the light culture conditions did not affect the oxidation of arsenic.5.Effect of EPS on the As(?)metabolism of non-axenic and axenic C.salinaOn the basis of previous research,a series of As(?)treatments(0,75,150,300,750?g·L-1)was applied to the non-axenic and axenic C.salina without EPS.After 7 days,the absorption,adsorption and accumulation of As(?)by C.salina were determined.The arsenic speciation in the culture and algal cells were analyzed and the rates of oxidation and removal of As(?)by non-axenic and axenic C.salina were calculated.The results showed that the accumulation of non-axenic C.salina was 85.3?170 mg·kg-1 when treated with low concentration of As(?)(75,150 ?g·L-1),which was significantly higher than that of axenic C.salina accumulation(11.1?18.7 mg·kg-1),indicates that the symbiotic bacteria can promote the enrichment of arsenic by non-axenic C.salina without EPS when the concentration of As(?)is low.After removal of EPS,the oxidation rate of As(?)in the non-axenic and axenic C.salina was 88.6%-99.2%and 11.4%?22.4%,compared with the C.salina with EPS,there was no significant difference,the oxidation of As(?)by C.salina is the result of intracellular and extracellular interactions of microalgae,and is mainly caused by intracellular oxidation.In conclusion,this study found that non-axenic C.salina has a strong ability to oxidize As(?).Through a series of experiments,the oxygen produced by photosynthesis and the effect of EPS produced by microalgae on oxidation of As(?)were eliminated.It was confirmed that the oxidation of As(?)by C.salina was the biological oxidation of microalgae.However,how these symbiotic microorganisms promote the oxidation of As(?)by microalgae and what signals between C.salina and symbiotic bacteria are unknown and need to be further explored. |
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