Degradation Of Benzo[a] Pyrene In Ocean By Chlorella Vulgaris With Heterotrophic Ability

Benzo[a]pyrene(BaP)is a kind of typical HMW PAHs with stable properties,and the benzene rings are not easy to be destroyed.BaP in the ocean mainly comes from human activities.It can exist widely in seawater,marine sediments,and marine organisms.At present,the remediation of BaP pollution mainly focuses on microorganisms.More studies on BaP degradation by bacteria and fungi and the mechanism of BaP degradation by bacteria and fungi have been relatively clear.However,there are few studies on the degradation of BaP by microalgae,mainly focusing on the catalytic degradation of BaP by microalgae under light conditions,and the mechanism of degradation of BaP by microalgae needs to be further researched.There is no systematic study on the degradation of BaP as the only carbon source by heterotrophic microalgae.Therefore,a marine microalgae Chlorella vulgaris LH-1 was used in this study.To explore the degradation of BaP in two states(mixotrophic and heterotrophic),analyze the metabolites of BaP,and speculate the degradation mechanism of BaP by C.vulgaris combined with RNA-seq,to provide theoretical and technical support for bioremediation of BaP pollution in the ocean.The main research results are as follows:(1)In this study,we evaluate the biodegradation of BaP by C.vulgaris.The results show that C.vulgaris LH-1 has the ability to use BaP as the sole carbon source and energy source.In the first two days,the removal effect of BaP was obvious,which was mainly contributed by bioadsorption.After 2 days,the biodegradation effect of C.vulgaris began to show.The degradation rates of BaP(0.5、1、2 mg/L)of C.vulgaris LH-1 under mixotrophic and heterotrophic conditions were 62.56～74.13%and 52.07～71.67%after 11 days of culture.The removal efficiency of BaP is different under the two conditions.The removal rate,the bioaccumulation rate,and the biodegradation rate of BaP in mixotrophic was higher than that in heterotrophic.The bioadsorption rate of BaP in heterotrophic was higher than that in mixotrophic.The model of BaP removal in two states was in accordance with the secondorder kinetics.The effect of environmental factors on the removal of BaP by C.vulgaris was investigated.It was found that the removal ratio of BaP increased first and then decreased with the increase of pH,and the most suitable pH for degradation was around 8.However,with the change of salinity,the BaP removal rate of both mixotrophic and heterotrophic groups had little fluctuation.The removal ratio of BaP by C.vulgaris increased first and then decreased with the increase of temperature.The temperature range of the best suitable for BaP removal was 20-25℃.(2)To study the toxicity of BaP to C.vulgaris,Transmission Electron Microscopy(TEM)was used to observe the cells of mixotrophic and heterotrophic C.vulgaris exposed to BaP,the effect of BaP on the cell structure of C.vulgaris was studied.TEM results showed that the surface of C.vulgaris exposed to BaP was wrinkled and vacuolized,the internal organelles and cytoplasm were affected,and the damage of heterotrophic C.vulgaris was much higher than that of mixotrophic.Under the condition of mixotrophic culture,low concentration of BaP(<5 mg/L)had little effect on the growth of C.vulgaris.Increasing the concentration of BaP had a significant inhibitory effect on the growth of C.vulgaris,and the inhibition ratio gradually increased with the BaP concentration.The contents of Total Chlorophyll and Carotenoid increased with the increase of culture time but it was significantly lower than that of the control group without BaP.Under the condition of heterotrophic culture,a low concentration of BaP(<20 mg/L)could promote the growth of C.vulgaris,and over a certain concentration(20 mg/L),BaP could significantly inhibit the growth of heterotrophic C.vulgaris.The content of Total Chlorophyll and Carotenoid decreased gradually with the increase of culture time,but higher than that of the control group without BaP.(3)To explore the mechanism of BaP degradation by C.vulgaris.The differential express genes(DEGs)of mixotrophic and heterotrophic C.vulgaris were analyzed by RNAseq.By analyzing the DEGs of C.vulgaris under the two conditions,it was found that the number of significant DEGs in mixotrophic was less than that in heterotrophic.Most of the DEGs enrich in GO terms were related to protein synthesis.The activity of phenol 2monooxygenase,protocatechuate 3,4-dioxygenase,catechol 1,2-dioxygenase in mixotrophic C.vulgaris were up-regulated.The unique KEGG metabolic pathway-styrene degradation and benzoate degradation were up-regulated of heterotrophic C.vulgaris.Combined with the mass spectrum analysis,it is speculated that the C.vulgaris could degrade BaP by dioxygenase and monooxygenase simultaneously,and the mixotrophic C.vulgaris could degrade BaP by phthalic acid pathway,while the heterotrophic C.vulgaris could degrade BaP by benzoic acid pathway.