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。     

USTB-05-B基因在大肠杆菌中的表达与纯化研究

采用生物学技术对高效降解线性微囊藻毒素-LR(microcystin-LR,MC-LR)的USTB-05-B酶进行克隆表达及纯化研究。首先利用分子克隆技术得到重组菌pET30a(+)/USTB-05-B/BL21(DE3)。在诱导剂IPTG浓度为0.25 mmol/L、温度30℃和诱导时间4 h条件下,重组菌能大量表达目的蛋白。然后利用亲和层析柱对重组菌破碎后的无细胞提取液(cell free extracts,CE)进行纯化,当咪唑浓度为100 mmol/L时,洗脱下来的蛋白纯度高。最终得到USTB-05-B蛋白纯度为91.1%,浓度为0.205 mg/m L。纯化后的USTB-05-B酶具有较高的活性,能在1 h内将10.8 mg/L的线性MC-LR降解完全。纯化后的目的蛋白为研究USTB-05降解MCs分子机理奠定基础,为有效提高降解MCs速率提供新材料。     

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.     

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 .     

农田土壤中微囊藻毒素污染特征及风险评价

蓝藻水华释放的微囊藻毒素(MCs)通过灌溉、堆肥沤田等途径进入农田土壤造成污染.采用固相萃取-高效液相色谱串联质谱方法(HPLC-MS/MS)研究了滇池周边35个代表性农田土壤样品中3种典型微囊藻毒素(MC-LR、MC-RR、MC-YR)的含量、分布特征及风险水平.结果表明,MCs检出率为85.7%,总含量为n.d.~7.8μg/kg,平均含量为1.6 μg/kg,其中MC-RR检出率(82.9%)和含量(n.d.~5.3μg/kg)最高. 3种MCs的健康风险和生态风险均在可接受范围内,健康风险以MC-YR最大,生态风险以MC-LR最大.儿童以口腔暴露MCs为主,成人以皮肤暴露MCs为主,儿童暴露MCs的健康风险高于成人.     

一株降解微囊藻毒素菌种的鉴定及其活性研究

从巢湖水体中分离出一株能够降解微囊藻毒素(MCs)的菌株M9,通过生理生化实验以及16S rDNA序列比对和序列分析,表明该菌株16S rDNA的全序列与吉氏库特菌kurthia gibsonii的相似性达99%.研究了pH值、外加碳源、氮源以及金属离子对菌株M9降解MCs活性的效应,发现菌株M9降解MCs的最适pH值为7.0.乙醇可显著提高MCs的降解活性,在48h内对微囊藻毒素RR(MC-RR)和微囊藻毒素LR(MC-LR)的降解率分别达到70.0%和81.4%.以硫酸铵为氮源时该菌株对MC-RR和MC-LR的降解率最高,分别为70.4%和80.9%.金属离子Cu2+、Zn2+和Mn2+对菌株M9降解MCs有明显的促进作用,48h时MC-RR和MC-LR的降解率分别达到了85%和93%以上;而Fe3+和Mg2+对降解过程的促进作用不如Cu2+、Zn2+和Mn2+.     

大孔吸附树脂分离纯化微囊藻毒素

以5%醋酸为提取剂从滇池有毒蓝藻中提取了微囊藻毒素,经磁力搅拌、离心、过滤后得到MCs粗提液. 此粗提液先后2次通过筛选后的大孔吸附树脂D101,用不同浓度的乙醇-水溶液洗脱后,获得纯度为82.6%、82.1%、62.5%的MC-RR、MC-YR和MC-LR.另外,条件实验结果表明,pH值＝3时,D101对MCs的吸附性能最佳.实验建立了一种安全、高效、低成本的分离纯化MCs的方法.     

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.     

基于太湖微囊藻毒素的叶绿素a阈值研究

以探讨叶绿素a(Chl-a)阈值为主要目的于2013年6~10月间每月在太湖采样,通过固相萃取超高效液相色谱串联质谱法对水体中3种微囊藻毒素(MC-LR、MC-RR、MC-YR)进行检测.利用SPSS软件分析各种形态的微囊藻毒素(MCs)与总氮(TN)、总磷(TP)、Chl-a、高锰酸盐指数等富营养指标的相关性,分析了MC-LR、MCs与Chl-a的关系.结果表明,太湖MCs污染较严重,其浓度的空间分布特征为:梅梁湾>贡湖、西部沿岸区>湖心区>胥湖区、南部湖区,并以MC-LR浓度最高;相关性分析表明MC-LR、MC-RR、MC-YR及MCs均与高锰酸盐指数、TN、TP、Chl-a呈极显著正相关(P<0.01).结合饮用水中MC-LR和MCs的标准限值分析得出,太湖Chl-a的阈值是12.26 mg·m-3,与美国北卡罗莱纳州湖泊的Chl-a标准值比较属于安全阈值,具有一定的科学性.     

Microbial biodegradation of microcystin-RR by bacterium Sphingopyxis sp. USTB-05

A strain,USTB-05,isolated from Lake Dianchi,China,degraded the cyanobacterial toxin microcystin-RR(MC-RR) at the rate of 16.7 mg/L per day.Analysis of 16S rDNA sequence showed that the strain was Sphingopyxis sp.Enzymatic degradation pathways for MC-RR by Sphingopyxis sp.USTB-05 were identified.Adda-Arg peptide bond of MC-RR was cleaved and then a hydrogen and a hydroxyl were combined onto the NH 2 group of Adda and the carboxyl group of arginine to form a linear molecule as intermediate product within the ...     

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.     

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.     

稻草固体介质中白腐菌对喹啉的降解研究

研究了稻草固体介质中侧耳属白腐菌BP对喹啉的降解。实验结果表明,浓度分别为250mg/L和150mg/L喹啉的降解率能达到93.47%和97.40%。白腐菌BP可以有效对喹啉进行降解。漆酶和锰过氧化物酶在降解过程中起着较重要的作用,但酶活的高低与降解率大小并不完全成正比。高效液相色谱分析表明降解过程中没有中间代谢物的积累,喹啉能得到彻底矿化。     

Biodegradation of high concentration phenol containing heavy metal ions by functional biofilm in bioelectro-reactor

Functional microorganisms to high concentration phenol containing Cr^6＋ and Pb^2＋ were cultured and biofilm was formed on polypropylene packings in bioelectro-reactor. It was found that the biodegradation capability of such biofilm to phenol changed with the applied voltage. Under the optimal electric field conditions （voltage of 3.0 V, electric field of strength 17.7 V/m and current density of 1.98 A/m2）, biodegradation efficiency of phenol aof concentration of 1200 mg/L increased 33% compared to the instance without applying electric field. However, voltage had inverse effect on biodegradation, as microorganisms were killed under strong electric field. Voltage had little effect on heavy ions elimination. Higher absorption rate of Cr^6＋ and Pb^2＋ was observed when changing pH fi＇om acidic to neutral. The experiment results indicated that, after treatment, 10 L phenol of 2400 mg/L was biodegraded completely within 55 h and concentrations of Cr^6＋ and Pb^2＋ dropped to less than 1 mg/L within 12 h and 6 h, fi＇om initial values of 50 mg/L and 30 mg/L, respectively.     

曝气生物填料塔降解低浓度对氯苯酚废水研究

针对珠江三角洲地区化工厂排出的低浓度对氯苯酚废水,研究了曝气生物填料塔对对氯苯酚的降解,并对降解效果的影响因素进行了分析。结果表明:初始浓度、溶解氧含量、停留时间和葡萄糖浓度等因素对降解效果影响显著;初始浓度<31mg/L,经过80h,对氯苯酚废水都可降至0.894mg/L以下,满足国家污水综合排放标准(1mg/L);对氯苯酚废水的适宜降解条件为:溶解氧质量含量为5～6.5mg/L、水力停留时间为5h、葡萄糖浓度为800mg/L。     

活性炭固定耐冷菌对硝基苯的降解特性

研究了用颗粒活性炭固定耐冷菌(Pseudomonas putida)对硝基苯降解的强化作用.结果表明,细菌被活性炭固定后,对硝基苯的降解能力提高.降解浓度为200mg/L的硝基苯所用时间由固定前的34h左右缩短为29.5h.浓度为400mg/L的硝基苯,对细菌有强烈抑制作用,细菌无法生长,但固定化细菌可直接降解浓度为400mg/L的硝基苯,且降解浓度为600mg/L的硝基苯只需46h.固定化细菌可连续4次降解浓度为400mg/L以下的硝基苯,但随着次数增多,降解速率减慢.用SEM扫描200mg/L硝基苯降解末期的生物活性炭的结果表明,Pseudomonas putida菌是降解过程中唯一的细菌.