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.     

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.     

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.     

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 ...     

锰过氧化物酶(MnP)对农药毒死蜱降解的初步研究

以广泛使用的有机磷杀虫剂毒死蜱为研究对象,利用白腐菌P.sordida YK-624菌株所产锰过氧化物酶(MnP)对其进行生物降解的初步研究。首先比较确定了MnP降解毒死蜱所需要的降解体系,即对于毒死蜱的降解,需要在有机酸(丙二酸)存在的缓冲体系中进行,此体系不需要表面活性剂Tween 80,但必须含有Mn2+和葡萄糖氧化酶氧化葡萄糖产生的H2O2。随后对主要影响因素的试验结果表明,MnP降解毒死蜱较好的条件是在pH 4.5的丙二酸缓冲体系中含7.5 mmol/L MnSO4、2.5 mmol/L葡萄糖以及40 U葡萄糖氧化酶,温度30℃。此时,400 U/L的MnP可将30 mg/L的毒死蜱经3 d时间降解77.51%。同时,利用GC-MS对毒死蜱酶降解后的产物进行了初步分析,得到1种可能的酶解产物O,O,O’,O’-四乙基二硫代焦磷酸酯,不同于水解和细菌降解途径,值得进行进一步研究。     

降解甲基对硫磷菌株YL8的分离、纯化及降解机理

从农药厂污水处理池的活性污泥中分离到1株能降解甲基对硫磷(MP)和对硝基苯酚(PNP)的菌株YL8,经生理生化试验和16SrRNA同源性分析,初步将YL8归类为节杆菌属(Arthrobactersp.).该菌株能以MP和PNP为唯一碳源生长,在5h内将起始浓度为50mg/L的MP降解近90%;在9h内将50mg/L的PNP完全降解.YL8在降解MP的过程中几乎检测不到PNP的产生.     

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。     

硝基苯污染底质的微生物强化修复研究

采用从污染底质中分离出的可降解硝基苯的恶臭假单胞菌,对硝基苯污染底质的微生物强化修复进行了实验室和现场实验研究.该细菌在未灭菌的河水中可以硝基苯为唯一碳源生长,低温条件下(5℃),对于100g的含有11.8mg/kg硝基苯的污染底质,投加2mL(107cells/mL)菌液可以在4d完全降解底质中的硝基苯,实现对污染底质的强化修复.该过程中无须投加额外的氮、磷及其他的营养盐,说明污染底质中含有足够的细菌生长所需的营养物质.在使用河水和底质的现场实验中,当底质和河水中的硝基苯初始浓度在7～8mg/kg,50～61mg/L之间时,投加硝基苯降解菌可使底质和河水中硝基苯的降解时间缩短了40h以上,河水中的硝基苯先于底质中的硝基苯被细菌所降解.     

一株甲基对硫磷高效降解菌的鉴定及特性研究

从长期施用甲基对硫磷(MP)的污染土壤中分离到一株能以甲基对硫磷为唯一碳源和氮源生长的新型降解菌HS-D38,并利用该菌既降解MP.结果表明,该菌既在降解MP的同时,可对中间产物对硝基苯酚(PNP)进行降解.该菌既能利用苯胺类物质作为唯一的氮源生长,又能利用对苯二酚作为唯一的碳源生长.经SDS或吖啶橙消除质粒后,HS-D38降解MP和PNP的能力丧失. 表明该菌降解酶可能由质粒DNA编码.对该菌16S rDNA 进行PCR扩增、测序,运用BLAST检索分析并构建了系统进化树.结合生理生化鉴定结果,HS-D38被鉴定为铜绿假单胞菌(Pseudomonas aeruginosa).      

Biodegradation of malathion by Acinetobacter johnsonii MA19 and optimization of cometabolism substrates

To enhance the removal efficiency of malathion in the wastewater from organophosphate pesticide mill, a bacterium, Acinetobacter johnsonii MA19, that could degrade malathion with cometabolism was isolated from malathion-polluted soil samples using enrichment culture techniques. Four kinds of additional compounds, sodium succinate, sodium acetate, glucose, and fructose were tested to choose a favorite carbon source for the cometabolism of strain MA19. The results showed that sodium succinate and sodium acetate could promote malathion biodegradation and cell growth. The investigation results of effects of sodium succinate concentrations on the malathion biodegradation indicated that the more sodium succinate supplied resulted in quick degradation ofmalathion and fast cells multiplied. Zero-order kinetic model was appropriate to describe the malathion biodegradation when the concentration of sodium succinate was more than 0.5144 g/L. The degradation rate constant (K) reached the maximum value of 3.5837 mg/(L·h) when the mass ratio of sodium succinate to malathion was 128.6 mg/mg. The aquatic toxicity of the malathion was evaluated using the test organism, Limnodrilus hoffmeisteri. The data obtained suggested that the toxicity of malathion could be ignored after 84 h biodegradation. Our result demonstrates the potential for using bacterium A. Johnsonii MA19 for malathion biodegradation and environmental bioremediation when some suitable conventional carbon sources are supplied.     

选择性生物强化处理二元互抑体系中苯胺和硝基苯

采用树脂吸附与生物强化相组合的方法处理含有苯胺和硝基苯的混合废水,对苯胺和硝基苯的降解抑制类型、吸附分离条件、生物强化降解过程与树脂性能变化等进行了研究.结果表明,硝基苯与苯胺均对对方的生物降解产生抑制;当进水中苯胺与硝基苯浓度分别为330与44mg/L时,在pH为4且流速为110mL/h条件下,通过装填有10mL吸附树脂NDA-150(7.2g)的吸附柱,吸附出水中硝基苯浓度低于4mg/L;吸附出水中苯胺的浓度保持不变,可通过生物强化而得到降解;吸附过程中约有597mg的硝基苯被树脂所吸附,其中约有224mg可通过生物强化方法得到脱附降解,系统降解硝基苯的容积负荷为315mg/(L·d);在此过程中树脂吸附能力获得部分恢复,其再生程度受到微生物对硝基苯降解能力的限制;70d的重复性实验证明,树脂性能保持稳定.     

一株Acinetobacter johnsonii的部分反硝化特性及动力学研究

从实验室A²/O小试设备中分离纯化出一株具有高亚硝酸盐氮（NO₂^--N）积累率的反硝化菌株ZY04,经过16SrDNA鉴定和基因比对后,初步鉴定为Acinetobacter johnsonii.使用Logistic模型可以很理想地拟合菌株ZY04的生长特性曲线,得到生长方程常数a=0.6588,b=24.08,k=0.2413.在维持初始基质中硝酸盐氮（NO₃^--N）浓度为100mg/L的条件下,改变碳源乙酸钠的浓度,使碳氮比（TOC/TN）为3.5,4.5,5.5,6.5,研究菌株ZY04部分反硝化性能,发现该菌株在不同碳氮比条件下均能够保持95%以上的NO₃^--N降解率,在碳氮比为3.5和4.5时,17h后NO₂^--N积累率达到70%以上;在碳氮比为5.5和6.5时,NO₂^--N积累率在更快的11h后达到85%以上,碳氮比为5.5时达到最高NO₂^--N积累率91%.使用Aiba,Edwards和Andrews模型对菌株的基质抑制动力学进行拟合,结果表明,3种模型均可以很好的拟合NO₃^--N和乙酸钠对菌株的单基质抑制动力学,在双基质抑制的9种组合中,有6种模型成功拟合了NO₃^--N和乙酸钠对菌株的双基质抑制动力学,得到了相关半饱和参数和基质抑制参数,相关系数（R²）可以达到98%.     

Anaerobic benzene biodegradation by a pure bacterial culture of Bacillus cereus under nitrate reducing conditions

A pure culture using benzene as sole carbon and energy sources was isolated by screening procedure from gasoline contaminated soil. The analysis of the 16S rDNA gene sequence, morphological and physiological characteristics showed that the isolated strain was a member of genus Bacillus cereus. The biodegradation performance of benzene by B. cereus was evaluated, and the results showed that benzene could be e ciently biodegraded when the initial benzene concentration was below 150 mg/L. The metabolites of anaerobic nitrate-dependent benzene oxidation by strain B. cereus were identified as phenol and benzoate. The results of substrate interaction between binary combinations for benzene, phenol and benzoate showed that the simultaneous presence of benzene stimulated the degradation of benzoate, whereas the addition of benzene inhibited the degradation of phenol. Benzene degradation by B. cereus was enhanced by the addition of phenol and benzoate, the enhanced e ects were more pronounced at higher concentration. To our knowledge, this is the first report that the isolated bacterial culture of B. cereus can e ciently degraded benzene under nitrate reducing conditions.     

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。     

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.     

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.     

醋酸盐类城市道路抑尘剂的生态毒性及生物降解特性

醋酸盐类抑尘剂因其具有良好的抑尘效果和生态友好性,被越来越多地用于城市道路抑尘.为了揭示醋酸盐类抑尘剂的水生态效应,选取水环境中占据不同生态位的4种水生生物(发光细菌、羊角月牙藻、大型溞和青鳉鱼)进行急性毒性测试.结果表明:4种水生生物对醋酸盐类抑尘剂的敏感程度依次为羊角月牙藻>大型溞>青鳉鱼>发光细菌.确定了抑尘剂与大型溞、发光细菌、青鳉鱼的剂量-效应关系,抑尘剂对三者的EC₅₀/LC₅₀(半数效应浓度/半致死浓度)分别为1.29、37.6、32.7 g/L.抑尘剂对羊角月牙藻的急性毒性及其质量浓度之间没有呈现出明确的数量关系,但是可推断EC₅₀为mg/L量级.以此为基础,推断醋酸盐类抑尘剂的预测无效应浓度范围为21.8～177 μg/L.不同温度下抑尘剂在水中的生物降解速率结果显示,在5和25 ℃时,醋酸盐类抑尘剂均易被降解,主要是微生物和藻类的共同降解作用所致.研究显示,醋酸盐类抑尘剂易于生物降解且生态毒性较低,但在使用时应注意区域总量控制,以防止对水生生物造成潜在威胁.      

染料活性艳红X-3B的微生物脱色研究

实验研究了以葡萄糖为外加碳源、由酵母膏提供氮源时不同的影响条件下紫色非硫光合细菌的混合菌种对染料活性艳红X-3B的脱色效果。结果表明,随着葡萄糖浓度的增大,脱色率反而下降;酵母膏的浓度对脱色效果影响很大,它在脱色过程中起到氮平衡的作用;当葡萄糖和酵母膏的浓度分别为0.125%和0.3%时,X-3B的脱色效果最佳。所选实验条件下,染料浓度在500mg/L以下X-3B脱色效果很好,对菌种没有明显的抑制作用;染料浓度在500～1000mg/L之间变化时,虽然对菌种有抑制作用,染料最终仍能完全脱色。温度在30℃时,pH在3～10范围内染料均有较好的脱色效果。     

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.     

Biofiltration of the antibacterial drug sulfamethazine by the species Chenopodium quinoa and its further biodegradation through anaerobic digestion

The biofiltering capacity, distribution patterns and degradation of the antimicrobial sulfamethazine(SMT) by halophyte Chenopodium quinoa under hydroponic conditions and its further biodegradation through anaerobic digestion were evaluated. C. quinoa was cultivated for a complete life cycle under different concentrations of SMT(0, 2 and 5 mg/L) and sodium chloride(0 and 15 g/L). C. quinoa is able to uptake and partially degrade SMT. The higher the SMT concentration in the culture medium, the higher the SMT content in the plant tissue. SMT has different distribution patterns within the plant organs, and no SMT is found in the seeds.Dry crop residues containing SMT have a great potential to produce methane through anaerobic digestion and, in addition, SMT is further biodegraded. The highest specific methane yields are obtained using crop residues of the plants cultivated in the presence of salt and SMT with concentrations between 0 and 2 mg/L.