鞘氨醇单胞菌USTB-05对微囊藻毒素的生物降解

研究了云南滇池水华蓝藻细胞中微囊藻毒素(MCs)的分析和鞘氨醇单胞菌USTB-05在细胞水平和酶水平下对MC-YR、RR和LR的生物降解.结果表明,云南滇池水华蓝藻细胞中MC-YR、RR和LR的含量分别为0.16, 0.96, 0.47mg/g.在初始浓度分别为19.5mg/L MC-YR、79.5mg/L MC-RR和43.6mg/L MC-LR下,鞘氨醇单胞菌USTB-05在2d内可将上述3种MCs全部降解,鞘氨醇单胞菌USTB-05粗酶液可以以更快的速率对MC-YR、MC-RR和MC-LR进行高效酶催化降解,在10h内可以将初始浓度分别为14.8mg/L MC-YR、28.4mg/L MC-RR和19.5mg/L MC-LR全部降解.同时发现了MC-YR降解过程中的2个中间和1个最终代谢产物.     

微囊藻毒素降解菌的分子鉴定和特性研究

在成功筛选出1株能够降解微囊藻毒素(Microcystins,MCs)菌株的基础上,利用16S rDNA的PCR扩增和序列分析对其进行了分子鉴定。由BLAST序列比对及进化树分析可将该菌株定属至鞘氨醇单胞菌USTB-01(Sphingopyxissp.USTB-01),这是我国首次筛选出高效降解MCs的鞘氨醇单胞菌。在鞘氨醇单胞菌USTB-01降解MC-RR和MC-LR活性研究方面发现,温度30℃和pH7.0条件下该菌降解MCs的速率最大,日均降解MC-RR和MC-LR分别达到了23.4mg/L和7.9mg/L,在进一步去除水体中的MCs的基础和应用研究方面都具有非常重要的意义。     

Microcystin-LR biodegradation by Sphingopyxis sp. USTB-05

A promising bacterial strain for biodegrading microcystin-LR (MC-LR) as the sole carbon and nitrogen source was successfully isolated from Lake Dianchi, China. The strain was identified as Sphingopyxis sp. USTB-05, which was the first isolated MCs-biodegrading Sphingopyxis sp. in China. The average biodegradation rate of MC-LR by Sphingopyxis sp. USTB-05 was 28.8 mg·L⁻¹ per day, which was apparently higher than those of other bacteria reported so far. The optimal temperature and pH for both strain USTB-05 growth and MC-LR biodegradation were 30°C and 7.0, respectively. The release of MC-LR from the cyanobacterial cells collected from Lake Guishui and the biodegradation of MC-LR by both strain and cell-free extract (CE) were investigated. The results indicated that MC-LR with the initial concentration of 4.0 mg·L⁻¹ in water was biodegraded by Sphingopyxis sp. USTB-05 within 4 d, while MC-LR with the initial concentration of 28.8 mg·L⁻¹ could be completely removed in 3 h by CE of Sphingopyxis sp. USTB-05 containing 350 mg·L⁻¹ protein. During enzymatic biodegradation of MC-LR, two intermediate metabolites and a dead-end product were observed on an HPLC chromatogram. Moreover, the similar scanning profiles of MC-LR and its metabolic products indicate that the Adda side-chain of MC-LR was kept intact in all products.     

Cloning and expression of the first gene for biodegrading microcystin LR by Sphingopyxis sp. USTB-05

Harmful cyanobacterial blooms are a growing environmental problem worldwide in natural waters, the biodegradation is found to be the most efficient method for removing microcystins (MCs) produced by harmful cyanobacteria. Based on the isolation of a promising bacterial strain of Sphingopyxis sp. USTB-05 for biodegrading MCs, we for the first time cloned and expressed a gene USTB-05-A (HM245411) that is responsible for the first step in the biodegradation of microcystin LR (MC-LR) in E. coli DH5αup, with a cloning vector of pGEM-T easy and an expression vector of pGEX-4T-1, respectively. The cell-free extracts (CE) of recombinant E. coli DH5αup containing USTB-05-A had high activity for biodegrading MC-LR. The initial MC-LR concentration of 40 mg/L was completely biodegraded within 1 hr in the presence of CE with a protein concentration of 0.35 mg/mL. Based on an analysis of the liquid chromatogram-mass spectrum (LC-MS), the enzyme encoded by gene USTB-05-A was found to be active in cleaving the target peptide bond between 3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda) and arginine of MC-LR, and converting cyclic MC-LR to linear MC-LR as a first product that is much less toxic than parent MC-LR, which offered direct evidence for the first step on the pathway of MC-LR biodegradation by Sphingopyxis sp. USTB-05.     

Carbon dioxide fixation by Chlorella sp. USTB-01 with a fermentor-helical combined photobioreactor

A promising microalgal strain isolated from fresh water, which can grow both autotrophically on inorganic carbon under lighting and heterotrophically on organic carbon without lighting, was identified as Chlorella sp. USTB-01 with the phylogenetic analysis based on 18S ribosomal ribonucleic acid (rRNA) gene sequences. In the heterotrophic batch culture, more than 20.0 g·L⁻¹ of cell dry weight concentration (DWC) of Chlorella sp. USTB-01 was obtained at day 5, and which was used directly to seed the autotrophic culture. A novel fermentor-helical combined photobioreactor was established and used to cultivate Chlorella sp. USTB-01 for the fixation of carbon dioxide (CO₂). It showed that the autotrophic growth of Chlorella sp. USTB-01 in the combined photobioreactor was more effective than that in the fermentor alone and the maximum DWC of 2.5 g·L⁻¹ was obtained at day 6. The highest CO₂ fixation of 95% appeared on day 1 in the exponential growth phases of Chlorella sp. USTB-01 and 49.8% protein was found in the harvested microalgal cells.     

Effects of nicotinamide and riboflavin on the biodesulfurization activity of dibenzothiophene by Rhodococcus erythropolis USTB-03

Rhodococcus erythropolis USTB-03 is a promising bacterial strain for the biodesulfurization of dibenzothiophene (DBT) via a sulfurspecific pathway in which DBT is converted to 2-hydroxybiphenyl (2HBP) as an end product. The effects of nicotinamide and riboflavin on the sulfur specific activity (SA) of DBT biodesulfurization by R. erythropolis USTB-03 were investigated. Both nicotinamide and riboflavin were found to enhance the expression of SA, which was not previously reported. When R. erythropolis USTB-03 was grown on a medium containing nicotinamide of 10. 0 mmol or riboflavin of 50. 0 μnol, SA was raised from 68. 0 or so to more than 130 mmol 2HBP/(kg dry cells. h). When R. erythropolis USTB-03 was grown in the presence of both nicotinamide of 5. 0 mmol and riboflavin of 25. 0 μmol, SA was further increased to 159. 0 mmol 2HBP/(kg dry cells. h). It is suggested that the biological synthesis of reduced form of flavin mononucleotide (FMNH2), an essential coenzyme for the activities of biodesulfurization enzyme Dsz C and A, might be enhanced by nicotinamide and riboflavin, which was responsible for the increased SA of R. erythropolis USTB-03.     

二苯并噻吩脱硫微生物菌种的筛选与活性

从被石油污染的土壤中筛选出1株能对二苯并噻吩(DBT)进行高效脱硫的微生物菌种,初步鉴定为红串红球菌USTB-03(RhodococcuserythropolisUSTB-03).该菌种可以按特异性脱硫途径(简称4S途径)将DBT转化为2-羟基联苯(2HBP)和亚硫酸作为最终脱硫产物.在葡萄糖、甘油和乙醇分别作为微生物生长的唯一碳源时,葡萄糖是支持该菌生长和提高其脱硫比活性的较好碳源,使培养出的微生物对DBT的脱硫比活性达到了68.63mmol2HBP/(kg·h).该菌株还可以对4,6-二甲基二苯并噻吩(4,6-DMDBT)进行脱硫.     

Biodegradation of methyl parathion by Acinetobacter radioresistens USTB-04

Biodegradation of methyl parathion (MP),a widely used organophosphorus pesticide,was investigated using a newly isolated bacterium strain Acinetobacter radioresistens USTB-04.MP at an initial concentration of 1200 mg/L could be totally biodegraded by A.radioresistens USTB-04 as the sole carbon source less than 4 d in the presence of phosphate and urea as phosphorus and nitrogen sources,respectively.Biodegradation of MP was also achieved using cell-free extract of A.radioresistens USTB-04.MP at an initial concentration of 130 mg/L was completely biodegraded in 2 h in the presence of cell-free extract with a protein concentration of 148.0 mg/L,which was increased with the increase of pH from 5.0 to 8.0.Contrary to published reports,no intermediate or final degradation metabolites of MP could be observed.Thus we suggest that the cleavage of C--C bond on the benzene ring other than P-O bond may be the biodegradation pathway of MP by A.radioresistens USTB-04.     

微囊藻毒素-R R 高效降解菌的分离鉴定及降解特性

从蓝藻爆发期的上海市淀山湖表层水体中筛选分离降解微囊藻毒素-RR(MC-RR)的细菌,研究其降解特性。根据分离菌株的细胞形态结构、生理生化特征及其16S rDNA序列分析鉴定降解菌,高效液相色谱法测定该菌株降解MC-RR的能力。分离菌株DHU-38(GenBank序列登录号为HM047515)属荧光假单胞菌(Pseudomonas fluorescens)。微囊藻毒素降解实验结果表明,该菌株能在以MC-RR为唯一碳源、氮源的无机盐培养基中生长,6 d内可将初始质量浓度为20 mg/L的MC-RR降解为6.23 mg/L,降解效率达到69%。菌株DHU-38的最适生长温度是30℃,最适生长pH值为7.0。酵母粉、蛋白胨、葡萄糖等营养物质可以明显促进菌株对MC-RR的降解效率,尤其是加入100 mg/L酵母粉后,6 d降解率达到89.6%。     

筛选菌种酶催化降解微囊藻毒素的特点

从云南滇池底泥中筛选出1株能够高效降解微囊藻毒素(Microcystins,MCs)的纯菌种.该菌无细胞提取液能进行降解MC-RR和MC-LR的酶促反应,并有最终产物的积累.在pH 6～8范围内酶对MC-RR和MC-LR的催化降解活性较高,Cu²⁺、Mn²⁺和Zn²⁺对降解MCs的酶促反应没有明显的促进作用.     

1株高效反硝化聚磷菌的生物学特性研究

采用专性培养基,从稳定运行的A/O/A SBR反应器中分离得到1株高效的反硝化聚磷菌Q-hrb05.菌株Q-hrb05的16S rDNA序列登录GenBank,登录号为GU214826.分析了该菌株的胞外聚合物的成分,探讨了pH、温度和碳源对株菌的生长及脱氮除磷效能的影响.结果表明,菌株Q-hrb05为芽孢杆菌,胞外聚...     

一株嗜盐聚磷菌的筛选及除磷性能初探

从运行稳定的以生活污水为碳源的污泥中富集分离,并筛选出一株嗜盐聚磷菌qdp05,通过对菌株的形态、生理生化特征及16SrDNA序列进行分析后,鉴定该菌株qdp05为肠杆菌属.当碳源为乙酸钠,盐度为2%的条件下,好氧条件下培养48h后,qdp05对磷的最终去除率为87.8%.在厌氧好氧连续培养过程,qdp05表现出明显的...     

微囊藻毒素-RR的臭氧降解研究

采用臭氧氧化的方法对微囊藻毒素-RR(MC-RR)进行降解.结果表明,在O3∶MC-RR(物质量比)为6的条件下,MC-RR的去除率最高可达到83.0%;pH上升、水中NOM含量增加都能显著降低MC-RR的臭氧降解效果.使用HPLC-MS考察了MC-RR的臭氧降解产物,并在此基础上探讨了MC-RR的臭氧氧化降解途径:主要通过Adda途径和Mdha途径来完成对MC-RR的降解和脱毒作用.臭氧氧化的Adda途径是通过对MC-RR上Adda侧链的进攻,断开具有活性的Adda支链,而达到脱毒的目的,其中Adda途径过程中的苯环羟基化作用对整个过程有促进作用;臭氧氧化的Mdha途径是通过对MC-RR肽环上面Mdha和Ala的断键,打开环状肽链,使藻毒素失去活性.在整个过程中Adda途径占主导地位.     

Alga-lysing bioreactor and dominant bacteria strain

Alga-lysing bacteria have been paid much attention to in recent years. In this study, the alga-lysing strain P05 which was isolated from an immobilizing biosystem was immobilized by coke and elastic filler, forming two biological reactors. The removal efficiencies of algae, NH4^＋-N and organic matter using the two reactors were studied. The results showed that strain P05 was an ideal algal-lysing bacteria strain because it was easy to be immobilized by coke and elastic filler which are of cheap, low biodegradability and the simple immobilization procedure. After 7 d filming, the biological film could be formed and the reactors were used to treat the eutrophic water. These two reactors were of stability and high effect with low cost and easy operation. The optimal hydraulic retention time （HRT） of each reactor was 4 h. The algae removal rates were 80.38% and 82.1% （in term of Chl-α） of coke reactor and filler reactor, respectively. And that of NH4^＋-N were 52.3% and 52.7%. The removal rates of CODMn were 39.03% and 39.64%. The strain P05 was identified as Bacillus sp. by PCR amplification of the 16S rRNA gene, BLAST analysis, and comparison with sequences in the GenBank nucleotide database.     

Alga-lysing bioreactor and the dominant bacteria strain

Alga-lysing bacteria have been paid much attention to in recent years. In this study, the alga-lysing strain P05 which was isolated from an immobilizing biosystem was immobilized by coke and elastic filler, forming two biological reactors. The removal efficiencies of algae, NH3-N and organic matter using the two reactors were studied. The results showed that strain P05 was an ideal algal-lysing bacteria strain because it was easy to be immobilized by coke and elastic filler which are of cheap, low biodegradability and the simple immobilization procedure. After 7 d filming, the biological film could be formed and the reactors were used to treat the eutrophic water. These two reactors were of stability and high effect with low cost and easy operation. The optimal hydraulic retention time (HRT) of each reactor was 4 h. The algae removal rates were 80.38% and 82.1% (in term of Chl-a) of coke reactor and filler reactor, respectively. And that of NH3-N were 52.3% and 52.7%. The removal rates of CODMn were 39.03% and 39.64%. The strain P05 was identified as Bacillus sp. by PCR amplification of the 16S rRNA gene, BLAST analysis, and comparison with sequences in the GenBank nucleotide database.     

不同波段紫外光对微囊藻毒素光降解的影响

为优化光降解法去除饮用水中的微囊藻毒素,分别用UVA(l=300～400nm)和UVC(l=253.7nm)2种紫外光源研究了紫外光波长对MC-RR降解效率的影响.结果表明,不同波段的紫外光具有不同的催化效率和降解中间产物.在UVA下照射12h后,MC-RR仍有30%～50%残余,同时产生2种几何异构体4(Z)-Adda-MC-RR和6(Z)-Adda-MC-RR,且二者在整个反应过程中保持恒定的比例.而在UVC下,MC-RR除生成2种异构体外,还生成中间产物[三环-Adda]MC-RR,在0.850mW/cm2光强下照射60min后,这些中间产物随MC-RR一起基本消失.两种情况下,MC-RR浓度随时间的变化均可用二级反应速率方程描述,表观反应速率常数(k)均随着光强度的增加而增大.尽管UVC强度仅为UVA的10%,但MC-RR在UVC下的反应速率常数却比UVA下高了近2个数量级.这表明,利用光降解技术去除饮水中的微囊藻毒素宜选用UVC光源.     

烟草悬浮细胞抗氧化系统对微囊藻毒素-RR的响应

采用0.1mg/L的微囊藻毒素-RR(MC-RR)处理烟草BY-2悬浮细胞,测定了细胞活力、细胞内活性氧(ROS)及抗氧化系统的变化.结果表明,处理144h后,BY-2的细胞活力与对照相比无显著差异,但是处理细胞的ROS含量随着MC-RR处理时间的延长而迅速上升,96h后,细胞的ROS含量与对照差异显著;毒素处理细胞的过氧化物酶(POD)和谷胱甘肽过氧化物酶(GPX)活性明显升高,分别在处理48h和72h后与对照差异显著;细胞中还原型谷胱甘肽(GSH)含量有一个先下降后升高的过程,96h后,MC-RR处理的细胞中GSH含量显著高于对照;然而,0.1mg/L的MC-RR处理144h后,过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性与对照相比没有显著差异.恢复处理168h后,抗氧化系统各个指标均恢复到对照水平.     

降解微囊藻毒素菌种的筛选和活性研究

研究了滇池底泥和表层水体中的微生物菌群降解微囊藻毒素(Microcystins, MCs)的能力差异,发现底泥中的微生物菌群对MCs有更强的生物降解能力.采用从滇池水华蓝藻细胞中提取提纯的微囊藻毒素作为微生物生长的唯一碳源和氮源,先后经过液体和固体培养基培养后,通过挑取单克隆菌落,分别从底泥中筛选出了能够降解MC-RR和LR的5种不同微生物菌种.其中筛选的菌种D降解MCs的能力最强,在3 d内可将初始浓度分别为60.1 mg·L^-1和38.7 mg·L^-1的MC-RR和LR全部降解,日均降解MC-RR和LR的速率分别高达20.0 mg·L^-1和12.9 mg·L^-1.     

微囊藻毒素的提取和提纯研究

对采自云南滇池水华蓝藻细胞中的微囊藻毒素 (Microcystins,MCs)的提取与提纯方法进行了研究 .结果表明 ,在藻细胞干重浓度为 2 0g·L-1下 ,与不同甲醇浓度提取液相比 ,4 0 %甲醇溶液可以最有效地从藻细胞中提取出MC RR和MC LR .将MCs提取液过Waters固相萃取小柱后 ,用 70 %甲醇溶液洗脱吸附于柱上的MCs ,可以分别获得 7 3%和 3 5 %纯度的MC RR和MC LR .通过观察洗脱液的颜色变化 ,收集蓝绿色和橘黄色后面流出的基本无色的洗脱液 ,可以获得纯度为 2 8 6 %和12 9%的MC RR和MC LR .     

太湖水中微囊藻毒素的测定及其分布特征

为了调查太湖水体中微囊藻毒素的污染程度,运用固相萃取-高效液相色谱技术测定了微囊藻毒素(MC-RR,MC-LR)的含量水平。该方法线性范围0.2～5 mg/L,相关系数大于0.99,2种藻毒素的最低检测限分别为0.05μg/L(MC-RR)和0.048μg/L(MC-LR)。结果表明,夏季太湖水体中MCs总体含量高于冬季;微囊藻毒素MC-LR的含量大于MC-RR。总体上看来,太湖北部(梅梁湾)水域中藻毒素的污染比其它区域水体严重。