Studies On Toxicity Response And Removal Mechanism Of Nonylphenol By A Strain Of Dictyosphaerium Sp. And Sediment Microorganisms

Nonylphenol(NP)is a kind of refractory organic pollutant that has been frequently detected in different environments,especially in aquatic environment,where its main existing forms are dissolved in water and adsorbed on sediment.Due to its unique lipophilic,bioaccumulative and endocrine disrupting properties,the large amount and long-term existence of NP in aquatic environment is bound to pose a potential threat to the organisms and ecosystems that exposed to it.Therefore,it is of great significance to investigate the toxic response of aquatic organisms to NP deeply and enhance the removal of NP efficiently.However,although algae are widely distributed in water bodies and microbes are the important component of sediment,studies about the toxicity response of algae and sedimentary microorganisms to NP and their NP removal ability are still not sufficient.Hence,in this study,algae Dictyosphaerium sp.and the sediment indigenous microorganisms were regarded as the research object to investigate their toxicity response and removal ability of NP,respectively.Meanwhile,a series of NP exposure and exogenous substances addition(NaHCO₃and Fe₂O₃nanoparticles)culture experiments were carried out,and growth and metabolism monitoring,high performance liquid chromatography,transcriptome sequencing and high throughput sequencing were used.The aim of this study was to better understand the biological toxicity of NP,deepen our cognition of the ecological risk of NP and provide theoretical reference for risk assessment and pollution control of NP in aquatic ecosystems.The main research results were as follows:(1)Toxicity mechanism of NP on algae Dictyosphaerium sp.NP showed a typical dose-dependent inhibition effects on the growth of Dictyosphaerium sp.in the concentration of 0.5–8 mg/L.It didn't inhibit algal growth at 0.5 mg/L,but when the concentration?1 mg/L,severely inhibition on cell growth was observed.However,algae showed high tolerance to NP and its growth could recover in 5 days even under 8 mg/L NP treatment.Oxidative stress is the main mechanism of NP toxicity in algae and the degree of oxidative damage to algal cell was also closely related to the concentration of NP,as the ultrastructure of algae was not affected by low concentration of NP,whereas the high NP concentrations change cell shape,thylakoid density,pyrenoid volume as well as the abundance of functional groups on cell surface.Meanwhile,a large number of genes were differentially expressed when algae exposed to 4 mg/L NP for 5 days.Among them,genes involved in ribosome,photosynthesis,and antioxidant pathways were especially sensitive to NP stress.(2)Removal ability of NP by algae Dictyosphaerium sp.The removal ability of NP(0.5,1,2,4,8 mg/L)from water by algae Dictyosphaerium sp.was analyzed through a 5-day exposure period.Results showed that more than 50%NP was removed within 24 h in spite of its initial concentration and the removal rate increased slowly with the extension of culture time.At the end of experiment,low concentration of NP(0.5 mg/L)could be completely removed by algae;Moreover,even though the NP removal rate decreased with the increase of the initial NP concentration,the 5-day removal rate of 8 mg/L NP still higher than 70%,indicating that the algae had a good removal ability for high concentration of NP.Further analysis showed that NP in the solution was mainly removed by algal bioaccumulation,including the adsorption of cell wall and the absorption of algal cell,and the content of NP bioaccumulated in algae was positively correlated with the initial concentration of NP.What's more,the adsorption of NP is an extremely rapid process and the NP adsorbed on cell wall would then be absorbed into the cells,which accounted for the decreased content of adsorbed NP and the increased content of absorbed NP with the prolongation of culture time.Additionally,NP removed by biodegradation only occupied a small part of the total amount of removed NP,indicating that the degradation of NP by algae Dictyosphaerium sp.was a slow process.(3)Effects of extracellular polymeric substances on NP toxicity response and NP removal of algaeIn this study,the excretion of EPS from algae Dictyosphaerium sp.in exposed to high concentration of NP in 5 days was detected,and effects of EPS on physicochemical properties of algal cell surface,growth and NP removal ability were also investigated.Results showed that algae could enhance the production of EPS in exposed to 4 and 8 mg/L NP and the higher the concentration of NP,the more content of EPS were secreted.Specifically,the content of S-EPS was significantly higher than that of B-EPS in the EPS secreted by Dictyosphaerium sp.,and the polysaccharide content in S-EPS,the aromatic protein content in B-EPS were increased extremely significantly in algal response to NP toxicity.EPS formed a physical barrier around the algal cells,which can significantly change the morphology,hydrophobicity and the abundance of active functional groups such as-OH,-NH₂,and-COOH on the cell surface.When exposed to NP,the existence of EPS around the algal cell wall reduced the connection possibility between NP and cells.Therefore,more NP would be blocked outside the algae and the decrease contents of NP in the cells would alleviate the toxicity of NP to algae directly,which enhance the algal tolerance and removal ability of NP.(4)Effects of NaHCO₃addition on NP toxicity response and NP removal of algae Dictyosphaerium sp.In NaHCO₃concentration ranged from 0–0.8 g/L,the cell concentration and specific growth rate of algae Dictyosphaerium sp.enhanced with the increased concentration of NaHCO₃,and the complex structure of pyrenoid-thylakoid could be formed in algae to ensure efficient photosynthesis when the concentration of NaHCO₃?0.4 g/L.Moreover,when algae were exposed to 1 and 4 mg/L NP,compared to the no NaHCO₃addition treatment,the addition of 0.8 g/L NaHCO₃ could significantly reduce the toxic inhibition effects of NP on algae,which contributed to a higher biomass,specific growth rate,photosynthesis pigment content and EPS excretion level,and the degree of oxidative damage to algal substructure caused by NP were also reduced obviously.Meanwhile,the number of differentially expressed genes induced by NP stress were significantly decreased after NaHCO₃addition.And except for the photosynthetic metabolic pathway,genes involved in nitrogen metabolism and carbon fixation in photosynthetic organism pathways were all significantly down-regulated.Most importantly,the addition of NaHCO₃alleviated the toxicity of NP and enhance the NP removal ability of a single algal cell,which significantly increased the total removal rate of NP eventually.(5)Effects of nFe₂O₃addition on microbial response to NP and the removal of NP from sedimentnFe₂O₃was added to the NP contaminated sediment for a 42-day culture experiment,and the response of microbes and the removal of NP from sediment were measured.Results showed that NP contamination led to an enhancement in the secretion of EPS by sedimentary microbes,whereas the addition of nFe₂O₃significantly reduced the content of EPS,especially the C-EPS.Moreover,although protein content was higher than polysaccharides content both in C-EPS and B-EPS,their contents were both decreased more significantly in C-EPS than in B-EPS,indicating that the secretion level of microbial C-EPS was more sensitive to NP stress.Furthermore,the spectral analysis of C-EPS indicated that contents of soluble microbial by-product-like and protein-like substances as well as the hydrophobicity,aromaticity and humification of C-EPS were significantly decreased in NP-nFe₂O₃co-treated sediment.Additionally,the high-throughput sequencing of 16S rRNA showed that the addition of nFe₂O₃alleviated NP toxicity to sedimentary microorganisms and the proportion of bacterium involved in the degradation of organic pollutants such as that in the phyla Acidobacteria,Firmicutes,and Chloroflexi were all increased,which strengthened the removal of NP from sediment and reduced the ecological risk of NP in aquatic environment directly.