【作者】 孔赟；

【导师】 徐向阳；

【作者基本信息】 浙江大学 ， 环境工程， 2013， 博士

【摘要】 近年来,我国近岸海域、湖库水体污染严重,富营养化程度加剧,赤潮水华频发且影响范围扩大,探索行之有效的抑藻控藻技术尤为重要。微生物控藻技术具有高效、成本低、二次污染风险低等特点,已成为近年来水体富营养化治理的研究热点。论文以我国发生频率高、分布范围广的淡水水华蓝藻铜绿微囊藻为研究对象,从浙江省富营养化水体筛选分离获得具有溶藻特性的微生物,在此基础上开展溶藻菌分子鉴定与生理生化特性、溶藻菌发酵培养基和发酵条件优化、溶藻产物特性与生物急性毒性、溶藻过程作用机理以及环境水体除藻及脱氮性能等研究,取得以下结果：1.从富营养化水体中分离筛选获得4株具有高效抑藻作用的溶藻菌HJC-D1、 ZJG-24、ZJG-215和KY-63,,菌株HJC-D1抑藻效果最佳,结合形态学、生理生化特征及16S rRNA序列分析,将其鉴定为链霉菌属Streptomyces sp. HJC-D1;溶藻菌HJC-D1发酵培养4d后,在铜绿微囊藻初始叶绿素a初始浓度0.2713±0.0192～0.6375±0.0321mg·L-1时,叶绿素a去除率为53.5±4.3%～62.6±3.3%;溶藻菌Streptomyces sp. HJC-D1溶藻活性物质主要存在于胞外无菌滤液中,并具有热稳定性、嗜碱性,可被乙酸乙酯萃取,推测其作用方式为间接抑藻。基于上述研究结果,为优化溶藻菌Streptomyces sp. HJC-D1大规模发酵产溶藻活性物质的培养条件,通过单因素实验筛选,获得溶藻菌Streptomyces sp. HJC-D1发酵培养基的最佳碳氮源为蔗糖和硝酸钾,并采用PB实验设计获得影响溶藻菌抑藻活性的三种显著因素为蔗糖、硝酸钾和初始pH值；在此基础上,应用CCD响应曲面法分析获得溶藻菌Streptomyces sp. HJC-D1的优化发酵培养基为(g·L-1,)：蔗糖,22.73; KNO3,0.96; NaCl,0.5; K2HPO4,0.5; MgSO4,0.5; FeSO4,0.002,初始pH为8.8；在溶藻菌Streptomyces sp. HJC-D1发酵条件优化方面,研究了培养温度、摇床转速、装液量和初始pH值等参数对发酵工艺的影响,确定各因素的最佳条件为：培养温度33.1℃、摇床转速337.5rpm,装液量91.2mL/250mL摇瓶、初始pH11.8,在此条件下培养4d后,藻叶绿素a去除率高达93.7%。2.运用傅里叶红外光谱、三维荧光光谱、凝胶渗透色谱等技术,研究了溶藻菌Streptomyces sp. HJC-D1抑制铜绿微囊藻过程中溶藻产物的组成与特性。结果表明,投加比例为3%(v/v)时溶藻菌Streptomyces sp. HJC-D1发酵液能有效抑制初始叶绿素a含量为0.2835±0.0721mg-L-1铜绿微囊藻生长,培养8d后藻类去除率为46.8±5.1%；在5%和10%投加比例下,藻类去除率可达72.6±5.5%和82.8±1.8%。不同处理组溶藻产物的DOM光谱学特性研究发现,溶藻产物分子量约1000Da,其DOM组分荧光峰特征基本相似,DOM以类腐殖酸物质为主,且随溶藻菌发酵液浓度增加,溶藻产物DOM中类腐殖酸和类富里酸荧光物质的强度总体呈增大趋势。傅里叶红外分析发现,溶藻产物HPI和HPO-A组分红外吸收峰位置与峰形大致相似,但各吸收峰的相对强度有所差异,TPI-A在1031cm-1处的特征峰则是由糖类和多糖类物质的伸缩振动引起；应用发光细菌(Photobacterium phosphoreum)急性毒性试验研究了溶藻产物的生物急性毒性,发现溶藻菌发酵液本身对发光细菌具有一定的低毒性,发酵3-5d时相对发光度为67.6±3.1%～72.4±2.8%；以微囊藻毒素为主要溶藻产物进行毒性试验发现,水体藻毒素浓度低于20μg-L-1时其生物毒性较低。3.通过对功能菌株抑制前后铜绿微囊藻细胞完整性和生理特征、抗氧化酶活、氧化损伤变化的影响研究,初步揭示了溶藻菌Streptomyces sp. HJC-D1对铜绿微囊藻的溶藻机制。结果表明,铜绿微囊藻的生长抑制程度和活藻细胞比例随发酵液浓度和共培养时间变化,共培养8d时3%、5%和10%处理组中活藻细胞含量分别为51.4%、15.7%和2.3%,TEM镜检发现藻细胞壁受到大幅破坏,细胞膜通透性增大,胞内K+和Ca2+离子外渗；在溶藻菌无菌滤液胁迫下,铜绿微囊藻抗氧化酶活发生明显变化,SOD、POD和CAT酶活随发酵液浓度的增加而增加,活性氧ROS水平亦显著提升,其过量增加导致藻细胞氧化损伤、细胞膜发生脂质过氧化,从而破坏细胞质膜,导致藻细胞破裂；综上分析,推测溶藻菌Streptomyces sp. HJC-D1抑制铜绿微囊藻细胞生长的溶藻机理为：溶藻菌破坏藻细胞壁,导致细胞内类囊体、光合片层、磷酸颗粒等细胞器释放并失活,最终引起藻细胞衰亡。4.针对我国环境水体富营养化日趋严重、藻类污染频发等问题,应用分离获得的溶藻菌Streptomyces sp. HJC-D1开展固定化微生物技术强化污染水体生物膜除藻及脱氮性能研究。结果表明,对照组和试验组的水体叶绿素a平均去除率分别为71.7±5.4%和80.9±4.4%,NH4+-N的平均去除率为77.8±2.8%和72.4±3.2%,而高锰酸盐指数(CODMn)平均去除率为25.0±1.5%和18.7±1.4%；不同曝气强度研究发现,曝气/停曝时间比2h：4h、曝气量60L·h-1工况下,系统CODMn和NH4+-N去除率均有所提高,相比对照组NO3--N积累更为明显；水力停留时间(HRT)变化对系统NH4+-N、CODMn等的去除影响不大,但缩短HRT时叶绿素a去除率有所降低；分析填料表面微生物相发现,试验组填料表面有溶藻菌富集,推测对照组除藻主要通过填料对藻类的吸附去除,而试验组则是藻类吸附在填料表面后通过溶藻微生物实现藻类去除。综上,通过对溶藻菌Streptomyces sp. HJC-D1溶藻特性、溶藻作用方式、溶藻产物特性以及微生物固定化技术除藻脱氮等研究,分析认为本研究分离获得的溶藻菌所分泌的胞外活性物质可作为生物除藻剂,可为我国水华控制提供可行的生物修复技术。更多还原

【Abstract】 In recent years, with the rapid development of coastal area economy and aquaculture industry, water environment pollution is increasingly serious. Red tide/harmful algal blooms (HABs) have been occured frequently in various coastal area, lakes and reservoirs, and the affected regions increased gradually. Therefore, it is extremely urgent to explore the effective, economical and environment-friendly eutrophication control methods. For controlling cyanobacteria over-growing, biological control methods have been recently explored. Antialgal microbial technology has been regarded as a novel and safe method for eutrophication remediation because of its low cost, low secondary pollution risk characteristics and high efficiency. Based on the advantages of antialgal microbial technology, the antialgal bacteria with highly effective inhibiting activity were isolated from eutrophication waters in Zhejiang province using Microcystis aeruginosa as a model species. The molecular identification, physiological and biochemical characteristics, the optimization of the culture medium and fermentation conditions of antialgal bacterium strain HJC-D1, the characteristics and acute toxicity of antialgal products, antialgal process and mechanism, and algae and nitrogen removal performance of biofilm process with the addition of strain HJC-D1from micro-polluted source water were investigated in this paper, the main results are as follows:1. Four antialgal bacteria (HJC-D1, ZJG-24, ZJG-215and KY-63) which exhibited antialgal activity against a representative bloom-forming cyanobacterium M. aeruginosa were isolated from eutrophication waters, and the antialgal effect of strain HJC-D1was the best. Based on its morphology, physiological and biochemical characteristics and16S rRNA sequence, the isolated strain was identified as Streptomyces sp. The best antialgal effect was obtained as the strain HJC-D1fermented for4d, and the M. aeruginosa could be inhibited by the isolated strain and the Chi a removal efficiency was53.5±4.3%～62.6±3.3%at the initial Chi a concentration of0.2713～0.6375mg L-1.The antialgal active substances mainly existed in extracellular aseptic filtrate, with thermal stability, basophilic and could be extracted by ethyl acetate. In such a way it was speculated that the antialgal way of Streptomyces sp. HJC-D1was indirect effect. Based on the above research results, in order to improve the antialgal effect and raise the production of antialgal active substances, a sequential optimization of the culture medium based on statistical design was employed. Sucrose and KNO3were selected as the most suitable carbon and nitrogen sources based on the one-factor-at-a-time method. Sucrose, KNO3and initial pH were found to be the major impact factors for further optimization obtained from the Plackett-Burman design. The optimum levels for these key variables were analysed by central composite design, and the optimum condition of the culture medium was obtained as following (g·L-1):sucrose,22.73; KNO3,0.96; NaCl,0.5; K2HPO4,0.5, MgSO4,0.5; FeSO4,0.002, and with initial pH of8.8; On the basis of the optimised culture medium, the influence of fermentation conditions such as temperature, initial pH, volume and agitation rate were carried out, and the best level of operating parameters was33.1℃for temperature,11.8for initial pH,91.2mL for volume and337.5rpm for agitation rate. Additionly, the maximizing removal efficiency of M. aeruginosa under the optimized conditions in flask cultures was93.7%.2. Using fourier transform infrared spectroscopy (FTIR),3-D fluorescence spectroscopy (EEMs) and gel permeation chromatography (GPC), the composition and characteristics of dissolved organic matter (DOM) from antialgal products were studied. The results indicated that the growth of M. aeruginosa could be effectively inhibited as the addition scale of HJC-D1fermentation cultrue was above3%(v/v) at a initial Chl a concentration of0.2835±0.0721mg·L-1, and the removal efficiency was72.6±5.5%and82.8±1.8%with the addition dosage of5%and10%, respectively. The main fluorescent substance in DOM was humic-like acid, and the molecular weight of antialgal products was around1000Da. The antialgal products were divided using resin adsorbents into hydrophilic fraction (HPI), hydrophobic acid (HPO-A), transphilic acid (TPI-A), hydrophobic neutral and transphilic neutral, and the results indicated that HPI component was the most abundant DOM fraction in the antialgal products, and the HPI concentration was increased with the increasing of fermentation liquid concentration. The fluorescence peak location and intensity showed that the protein-, fulvic-and humic-like substances were dominant in HPI, HPO-A and TPI-A fractions, and intensities of the relevant fluorescence peaks were stronger in experimental groups than those in control groups; it was also found that the number averaged molecular weight of DOM fractions ranged from245to1452g mol-1, and thereinto organic acids such as HPO-A and TPI-A exhibited lower molecular weights, and the adsorption band at1031cm-1for TPI-A corresponded to carbohydrates and polysaccharide-like substances. The acute toxicity of Streptomyces sp. HJC-D1fermentation broth and antialgal products was studied with Photobacterium phosphoreum, the results indicated that the fermentation broth of antialgal bacterium had certain biotoxicity via photobacteria tests, and the relative luminosity was67.6±3.1%～72.4±2.8%after3-5d fermentation. The toxicity tests with Microcystin-LR as the main antialgal product showed that there’s no inhibited effect when the Microcystin-LR concentration was lower than20μg·L-1.3. The cell integrity and physiological characteristics of M. aeruginosa, antioxidant enzymes and oxidative damage change were investigated to illustrate the antialgal mechanism of M. aeruginosa by strain Streptomyces sp. HJC-D1. Results showed that the growth of M. aeruginosa was significantly inhibited, and the percentage of live cells depended on the fermentation liquid concentration and exposure time. The percentage of live algal cells in the3%,5%and10%treatment groups were51.4%,15.7%and2.3%after8d incubation, respectively. Furthermore, TEM observation suggested the cell wall of M. aeruginosa was damaged dramatically during the antialgal process, and the permeability of cell membrane was increased with the release of K+and Ca2+; Under the stress of aseptic filtrate of Streptomyces sp. HJC-D1, the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly changed, which was increased with the increasing of exposure concentration and the extending of exposure time, and the significant increase in the lipid-peroxidation product malondialdehyde (MDA) triggered the synthesis of reactive oxygen species (ROS) to disrupt the subcellular structure of M. aeruginosa, and organelles were released which resulted in the death of algae cell finally. Streptomyces sp. HJC-D1therefore not only affected antioxidant enzyme activities and ROS level, but also destroyed the subcellular structure of M. aeruginosa. According to the above results, it was speculated that the antialgal mechanism was as following:the algal cell wall was destroyed by antialgal active substances that secreted by antialgal bacterium, then cell organelle such as thylakoid, photosynthetic lamellar and phosphate particles were released that leading to inactivation, and resulted in the death of algae cells finally.4. Facing increasingly serious problem of micro-polluted water by harmful algae blooms in our country, strain Streptomyces sp. HJC-D1, which was capable of inhibiting M. aeruginosa, was immobilized to AquaMats to evaluate the removal performance of algae and denitrification by biofilm reactor. The results showed that the average removal efficiency of chlorophyll α, NH4+-N and CODMn was71.7±5.4%,77.8±2.8%and25.0±1.5%for the control reactor R1, and80.9±4.4%,72.4±3.2%and18.7±1.4%for the experimental reactor R2, respectively. On the condition of aerobic to anoxic ratio of2h:4h and aeration intensity of60L-h"1, the removal efficiencies of CODMn and NH4+-N were improved in the control group, while the Chi a removal efficiency was not changed significantly, and the accumulation of NO3--N for the control group was much more significantly; The change of the hydraulic retention time (HRT) had little impact on the removal performance of pollutants such as NH4+-N and CODMn, while the Chi a removal efficiency was decreased with HRT; Microscopic observation revealed that the surface of AquaMats in experimental reactor was surrounded with antialgal bacterium. It was presumed that the removal mechanism of algae for the control group mainly through the adsorption of AquaMats, while the algae was absorbed by AquaMats and then degraded by antialgal strain Streptomyces sp. HJC-D1for the experimental reactor.In conclusion, according to the studies of the antialgal characteristics, the antialgal mode of Streptomyces sp. HJC-D1, the characteristics of antialgal degradation products and the enhanced performance of immobilized microorganism technology for algae remonal and denitrification effect with micro-polluted surface watere, it was noteworthy that antialgal substances produced by Streptomyces sp. HJC-D1had the potential to be developed as a commercial biological control agent and it might provide a feasible bioremediation technology for the control of algal blooms in our country.更多还原