【作者】 李卓；

【导师】 朱联东；

【作者基本信息】 武汉大学 ， 环境工程， 2023， 硕士

【摘要】 氟喹诺酮类抗生素能够通过生活污水、养殖废水和医疗或制药等工业废水进入自然环境,并导致抗生素抗性基因的产生与传播,对水生生物及人类造成潜在威胁。微藻生物处理技术具备对氟喹诺酮类抗生素的去除潜力,吸引了研究者的广泛关注。然而,目前的研究集中于微藻在抗生素胁迫下的生理响应和抗生素的去除效率等方面,对氟喹诺酮类药物的具体去除机制尚缺乏深入研究。本研究选取小球藻(Chlorella sorokiniana)与栅藻(Scenedesmus dimorphus)为研究对象,以环丙沙星(CIP)为主要处理目标,解析微藻在CIP暴露下的毒性响应及其对CIP的去除机制。通过转录组学,深入分析微藻在分子层面的生理变化,解析CIP的生物降解途径及机制,为微藻处理含抗生素废水的应用提供技术指导。本文的主要研究结论如下:通过考察CIP对C.sorokiniana与S.dimorphus的生长影响,结果表明1～20mg/L的CIP对C.sorokiniana和S.dimorphus生长抑制率分别为1.77%～5.14%和12.19%～22.74%。C.sorokiniana对CIP的耐受性更强,这主要归因于C.sorokiniana受CIP胁迫时,其有效与最大光合速率与对照组差异较小,而S.dimorphus的有效与最大光合速率需要一定时间才能恢复到对照组水平。在20 mg/L的CIP暴露下,C.sorokiniana的叶绿素a、叶绿素b和类胡萝卜素含量与对照组相比分别从2.50、0.66和0.89 mg/g上升至3.20、0.88和1.23 mg/g,而S.dimorphus则从3.28、0.77和2.68 mg/g下降到1.91、0.50和2.07 mg/g。同时,当受到CIP的胁迫时,C.sorokiniana和S.dimorphus抗氧化酶系统通过提高超氧化物歧化酶(SOD)的活性,以减轻细胞所受到的氧化损伤。通过考察C.sorokiniana与S.dimorphus对CIP的去除效能,结果表明两种微藻对CIP均有较好的去除效果,C.sorokiniana与S.dimorphus对20 mg/L的CIP在10天培养期内的去除率分别达89.43%和91.73%。通过去除路径分析,发现光降解与水解对总CIP去除率贡献仅约5%,说明大部分去除依赖生物积累与生物降解。通过三维荧光光谱-平行因子分析(EEM-PARAFAC),结果表明,C.sorokiniana和S.dimorphus释放的溶解性有机物(DOMs)主要是胡敏酸类物质和富里酸类物质,这些物质与CIP的结合和降解相关。基于高效液相色谱串联质谱(HPLC-MS/MS)技术,解析了CIP的光降解和生物降解中间产物。其中,光降解涉及到的降解中间产物有5种,生物降解的中间产物11种,CIP可以通过羟化、脱羧、去甲基化、环裂解等途径进行生物降解。研究了不同浓度CIP暴露下,C.sorokiniana与S.dimorphus在分子水平上的响应机制。相比于不添加CIP,C.sorokiniana在低浓度CIP(5 mg/L)暴露下产生了334个差异基因(DEGs),上调164个,下调170个;在高浓度CIP(20 mg/L)下产生了427个DEGs,上调247个,下调180个。S.dimorphus在低浓度CIP下产生了771个DEGs,上调231个,下调540个;在高浓度CIP下产生了3382个DEGs,上调1124个,下调2258个。通过GO富集分析,发现CIP主要影响C.sorokiniana内与核糖体相关的基因表达,进而调控抗氧化酶和氧化还原酶等蛋白类物质的合成,而S.dimorphus内的差异基因集中于膜的损伤与修复及类囊体功能,主要影响S.dimorphus的光合作用能力。通过对DEGs的功能性分析,发现C.sorokiniana与S.dimorphus胞内与抗氧化相关的酶合成的基因均有上调,对微藻细胞形成了有力的保护能力,与CIP代谢相关的一些氧化还原酶相关的基因表达量也有所上升,可能与CIP在微藻胞内的降解相关。本研究对CIP的降解产物和去除途径进行了深入分析,同时对微藻内部基因表达进行了差异性与富集分析,为微藻去除抗生素的研究提供了新的见解。更多还原

【Abstract】 Fluoroquinolone antibiotics(FQs)could enter the environment through domestic wastewater,livestock effluents and idustrial wastewater such as medical or pharmaceutical wastewater,leading to the occurrence and spread of antibiotic resistance genes,posing a threat to aquatic organisms and humans.Microalgae biotreatment technology,as a potential way to remove FQs,has received increasing attention from researchers.However,current studies focus more on the physiological response of microalgae under antibiotic stress and the removal efficiency of antibiotics,but the specific removal mechanisms of FQs have been less studied.Therefore,in this study,Chlorella sorokiniana and Scenedesmus dimorphus were selected to investigate the toxicity of CIP exposure and the removal mechanism of CIP.The physiological changes of microalgae were analyzed at the molecular level through transcriptomics,and the biodegradation mechanism and pathway of CIP were also speculated in this study.This study provided guides for application of microalgae-based wastewater treatment.The main conclusions of this study were as follows:The effect of CIP on the growth of C.sorokiniana and S.dimorphus was analyzed.The growth inhibition rates of 1～20 mg/L CIP to C.sorokiniana and S.dimorphus were 1.77%～5.14% and 12.19%～22.74%,respectively.It was found that C.sorokiniana was more resistant to CIP while both kept normal growth.This might be attributed to the fact that the effective and maximum photosynthetic effeciencies of C.sorokiniana kept constant compared to the control group when exposed to CIP,whereas the effective and maximum photosynthetic effeciencies of S.dimorphus took some time to recover.The contents of chlorophyll a,b and carotenoid contents of C.sorokiniana exposed to 20 mg/L CIP increased from 2.50,0.66 and 0.89 mg/g to 3.20,0.88 and 1.23 mg/g,respectively,compared to the control,while decreased from 3.28,0.77 and 2.68 mg/g to 1.91,0.50 and 2.07 mg/g in S.dimorphus.Meanwhile,when subjected to CIP stress,the oxidative damage caused by CIPexposure was mitigated by the antioxidant systems of C.sorokiniana and S.dimorphus by increasing the activity of superoxide dismutase(SOD).This study evaluated the removal efficiency of CIP by C.sorokiniana and S.dimorphus 89.43% and 91.73% of CIP were removed by C.sorokiniana and S.dimorphus during 10 days at the concentration of 20 mg/L.The removal pathway analysis revealed that photodegradation and hydrolysis contributed only about 5% to the total CIP removal,indicating that most of the removal relied on bioaccumulation and biodegradation.The results of three-dimensional fluorescence spectroscopy-parallel factor analysis(EEM-PARAFAC)showed that dissolved organic matter(DOMs)released by C.sorokiniana and S.dimorphus were mainly huminic acid-like substances and fulvic acid-like substances,which were associated with the binding and degradation of CIP.The photodegradation and biodegradation intermediates of CIP were determined based on high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)technique.Among them,five degradation intermediates were involved in photodegradation and 11 intermediates in biodegradation.CIP was bio-degraded through hydroxylation,decarboxylation,demethylation,and ring cleavage.The molecular response mechanisms of C.sorokiniana and S.dimorphus under different concentrations of CIP exposure were investigated.Compared to the control group,334 differentially expressed genes(DEGs)in which 164 up-regulated and 170down-regulated were detected in experimental groups of C.sorokiniana with 5 mg/L CIP addition,and 427 DEGs in which 247 up-regulated and 180 down-regulated were detected in experimental groups of C.sorokiniana with 20 mg/L CIP addition.In the meantime,771 DEGs in which 231 up-regulated and 540 down-regulated were detected in experimental groups of S.dimorphus with 5 mg/L CIP addition,and 3382 DEGs in which 1124 up-regulated and 2258 down-regulated were detected in experimental groups of S.dimorphu with 20 mg/L CIP addition.The GO enrichment analysis revealed that the expression of ribosome-related genes was affected in C.sorokiniana,which in turn regulated the synthesis of protein-like substances such as antioxidant-related enzymes and oxidoreductases of organic compounds.And the differences within S.dimorphus concentrated on membrane damage and repair and vesicle-like functions,affecting the photosynthesis of S.dimorphus.Functional analysis of DEGs revealed that genes for the synthesis of antioxidant-related enzymes were up-regulated in both C.sorokiniana and S.dimorphus,forming a strong protective capacity for microalgal cells.The expression of some oxidoreductase-related genes associated with CIP metabolism was also increased,which may be related to the bio-degradation process in microalgae.This study provided information of the degradation intermediates and removal pathways of CIP,as well as differential and enrichment analysis of gene expression within microalgae,providing new insights into the removal of antibiotics by microalgae.更多还原