质粒pT7降解三氯苯的功能研究

从以1,2,4-三氯苯为唯一碳源的降解菌--施氏假单胞菌(Pseudomonas Stutzeri)T7中提取到一个降解质粒,为研究该质粒的降解功能,将其转化到E.coli JM109中,转化子获得了降解1,2,4-三氯苯的能力.采用色谱/质谱联用(GC/MS)技术检测到转化子E.coli JM109(含质粒pT7)降解1,2,4-三氯苯过程中的部分中间产物分别为2,3,5-三氯-2,4-己二烯二酸、2,5-二氯-2,4-二烯-1,4-内酯-己酸、2,5-二氯-4-氧代-2-烯己二酸;在转化子中分别检测了三氯苯双加氧酶、脱氢酶、邻苯二酚1,2-双加氧酶的酶活性.     

降解1,2,4-三氯苯的硝基还原假单胞菌J5-1的分离鉴定和邻苯二酚1,2-双加氧酶基因的克隆

从受氯苯污染的土壤中分离到1株以1,2,4-三氯苯为唯一碳源生长的细菌,命名为J5-1.根据其生理生化特征和16S rDNA(GenBank Accession No.EF107515)序列相似性分析,将该菌株初步鉴定为硝基还原假单胞菌(Pseudomonas nitroreducens).当1,2,4-三氯苯初始浓度为400 mg/L时,J5-1对其最大降解率接近90%;当1,2,4-三氯苯浓度初始为20 mg/L时,降解效果最好.J5-1对1,2,4-TCB的降解服从一级反应动力学.从J5-1的基因组DNA中克隆到CC120的全长序列.     

低温条件下1,2,4-三氯苯降解菌的筛选及降解特性

在10 ℃的低温条件下, 从吉林石化化工厂排污管和长春第一汽车制造厂污水处理厂的混合污泥中驯化并分离出10株能以1,2,4-三氯苯（1,2,4-TCB）为唯一碳源的降解菌株, 通过筛选得到6株高效1,2,4-TCB降解菌株. 以6株菌的混合菌为研究对象, 考察其对1,2,4-TCB的降解特性. 结果表明, 混合菌在以葡萄糖为辅助碳源, 硫脲为氮源, pH为7.0, 盐度为2.0%, 溶液体积100 mL的条件下对1,2,4-TCB的降解效果最佳, 且添加辅助碳源对1,2,4-TCB的降解有一定的促进作用. 根据菌种的生理生化特征对降解菌进行初步鉴定, 结果表明,6株菌分别属于弧菌属（Vibrio sp.）, 奈瑟氏球菌属（Neisseria sp.）, 螺菌属（Spirillum sp.）, 无色细菌属（Achromobacter sp.）, 醋酸单胞菌属（Acetobacter sp.）和节细菌属（Archrobacter sp.）.      

BHC降解性质粒的研究

分离获得了一株对β-及γ-BHC均具有一定降解效果的Ⅱ_5-A菌株,该菌在好气条件下以BHC为唯一碳源。检测发现。它携带一个质粒。用丝裂霉素C进行的质粒消除试验表明,凡是消除了质粒的菌株,均丧失在以BHC为唯一碳源的无机盐培养基上的生长能力。该质粒经提取和纯化后,通过转化,成功地转移到大肠杆菌(Escherichia coli C600),使该转化子获得降解BHC的能力,并在转化子中检测出与原菌株电泳泳动度相同的质粒。因此,认为Ⅱ_5-A菌株降解BHC的基因是位于它所携带的质粒上。作者暂将此降解性质粒命名为BHC质粒,并编号为pWE1。     

1株氯苯高效降解菌的分离鉴定及降解特性

分离筛选到1株能以氯苯为唯一碳源和能源的菌株LW26,根据菌株的形态、生理生化特征、16S rRNA序列分析以及Biolog鉴定,确定该菌株为戴尔福特菌(Delftia tsuruhatensis),其为新发现的具有氯苯降解能力的菌株,并且该菌株能降解BETX、正己烷和环己烷等常见的有机污染物.实验考察了温度、pH、氯苯初始浓度、Cl-浓度等因素对菌株生长和降解性能的影响.结果表明,该菌株较为适宜的生长和降解条件为:温度25℃、pH 7.0;底物耐受浓度高达500 mg·L~(-1);当Cl-浓度超过0.14 mol·L~(-1)时,菌株生长会受到抑制.利用Haldane模型对实验数据拟合得到菌株LW26的最大比生长速率μmax和最大比降解速率γmax分别为0.42 h~(-1)和2.53 h~(-1).利用GC-MS进行中间产物分析,结果表明菌株LW26降解氯苯的过程中产生邻氯苯酚,结合邻苯二酚双加氧酶活性分析,推测氯苯经历邻位开环、脱氯、氧化等过程,最终矿化为CO2或转化为生物质.     

双酚A降解菌的分离鉴定及降解基因的定位

从土壤中分离到一株双酚A降解菌W2。该菌可在饱和的双酚A溶液里以其为唯一碳源与能源生长。具有较强的双酚A降解能力。通过16SrDNA序列分析与形态与生理生化分析,该菌株为假单胞菌。通过质粒丢失实验、质粒转化实验证明该基因位于一个19kb左右的质粒上。     

氯苯类化合物的生物降解

经过2个月的驯化,从某染料厂和某毛纺厂活性污泥中分离出能够生长于1,4-二氯苯、1,2,4-三氯苯和六氯苯的4种微生物.通过测定该混合菌降解氯苯类化合物过程中的累积好氧量、微生物生长曲线及降解产物Cl^-的释放,证明在好氧条件下该混合菌能够以1,4二氯苯和1,2,4-三氯苯为唯一碳源和能源,降解产物Cl^-浓度的变化与微生物生长周期有关.通过好氧振荡瓶培养法测得3种氯苯的生物降解顺序为:1,4-二氯苯[356.7μg/(L·d)]>1,2,4-三氯苯[110.4μg/(L·d)]>六氯苯[～6μg/(L·d)],说明氯取代数越多,氯苯类化合物越难被好氧降解.     

菲降解菌株的分离鉴定及特性研究

从某石化公司污水处理厂的活性污泥中分离到1株对菲具有降解活性的菌株AFF,该菌株能够以菲为唯一碳源和能源生长.根据形态特征及16S rRNA基因序列分析,该菌株初步鉴定为Burkholderia sp..在以菲为唯一碳源的培养基中,84 h内菌株对100 mg·L-1菲的降解率可达74.1%.通过液相-质谱联用及紫外可见分光光度法分析,推测该菌株代谢菲的途径为水杨酸途径.测定了降解途径中相关酶的活性,表明该菌株降解酶系中具有邻苯二酚2,3-双加氧酶活性,并证明该酶为诱导酶;确定了粗酶降解邻苯二酚的动力学常数:米氏常数Km为10.93μmol·L-1,最大反应速率Vmax为106.38μmol·min-1,说明酶与邻苯二酚的亲和力较大,邻苯二酚是该酶的良好底物.     

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).      

甲基对硫磷降解菌DLL-E4降解对-硝基苯酚特性

甲基对硫磷降解菌DLL-E4(Pseudomonas putida)能以对-硝基苯酚(PNP)为唯一碳源和氮源生长,还可以利用对-硝基苯酚代谢产生的亚硝酸根为唯一氮源.DLL-E4降解对-硝基苯酚的酶系是诱导产生的,甲基对硫磷(MP)和PNP驯化菌株,能有效诱导DLL-E4对PNP的降解,生长延滞期由原来的6h减少到3h,降解完全的时间由9h缩短为7h和6h.对苯二酚(HQ)诱导,降解完全的时间延长为15h.该诱导酶同时能够降解2-硝基酚.DLL-E4菌株中含有大质粒,在丢失对-硝基苯酚降解性状的突变株M+P-中依然存在.     

Toxic effects of acetochlor, methamidophos and their combination on nifH gene in soil

Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis （DGGE） and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity （both in gene richness and diversity index values） and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patterns showed that nifH gene composition which had been affected by low concentration of acetochlor （50 mg/kg） recovered firstly. Methamidophos reduced nifH gene richness that except at 4 weeks. The medium concentration of methamidophos （150 mg/kg） caused the most apparent changes in nifH gene diversity at the first week while the high concentration of methamidophos （250 mg/kg） produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes （bands） responded differently to the impact of agrochemicals： four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha- and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.     

Arsenic uptake by arbuscular mycorrhizal maize （Zea mays L.） grown in an arsenic-contaminated soil with added phosphorus

The effects of arbuscular mycorrhizal (AM) fungus (Glomus mosseae) and phosphorus (P) addition (100 mg/kg soil) on arsenic (As) uptake by maize plants (Zea mays L.) from an As-contaminated soil were examined in a glasshouse experiment.Non-mycorrhizal and zero-P addition controls were included.Plant biomass and concentrations and uptake of As,P,and other nutrients,AM colonization,root lengths,and hyphal length densities were determined.The results indicated that addition of P significantly inhibited root colonization and development of extraradical mycelium.Root length and dry weight both increased markedly with mycorrhizal colonization under the zero-P treatments,but shoot and root biomass of AM plants was depressed by P application.AM fungal inoculation decreased shoot As concentrations when no P was added,and shoot and root As concentrations of AM plants increased 2.6 and 1.4 times with P addition,respectively.Shoot and root uptake of P,Mn,Cu,and Zn increased,but shoot Fe uptake decreased by 44.6%,with inoculation, when P was added.P addition reduced shoot P,Fe,Mn,Cu,and Zn uptake of AM plants,but increased root Fe and Mn uptake of the nonmycorrhizal ones.AM colonization therefore appeared to enhance plant tolerance to As in low P soil,and have some potential for the phytostabilization of As-contaminated soil,however,P application may introduce additional environmental risk by increasing soil As mobility.     

Degradation of metabolites accumulated of benzo[a]pyrene by coupling Penicillium Chrysogenum with KMnO4

Several main metabolites of benzo[a]pyrene （BaP） formed by Penicillium chrysogenum, Benzo[a]pyrene-1,6-quinone （BP 1,6- quinone）, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene （BP 7,8-diol）, 3-hydroxybenzo[a]pyrene （3-OHBP）, were identified by high-performance liquid chromatography （HPLC）. The three metabolites were liable to be accumulated and were hardly further metabolized because of their toxicity to microorganisms. However, their further degradation was essential for the complete degradation of BaP. To enhance their degradation, two methods, degradation by coupling Penicillium chrysogenum with KMnO4 and degradation only by Penicillium chrysogenum, were compared; Meanwhile, the parameters of degradation in the superior method were optimized. The results showed that （1） the method of coupling Penicillium chrysogenum with KMnO4 was better and was the first method to be used in the degradation of BaP and its metabolites; （2） the metabolite, BP 1,6-quinone was the most liable to be accumulated in pure cultures; （3） the effect of degradation was the best when the concentration of KMnO4 in the cultures was 0.01% （w/v）, concentration of the three compounds was 5 mg/L and pH was 6.2. Based on the experimental results, a novel concept with regard to the bioremediation of BaP-contaminated environment was discussed, considering the influence on environmental toxicity of the accumulated metabolites.     

Impact of atrazine and nitrogen fertilizers on the sorption of chlorotoluron in soil and model sorbents

Sorption of chlorotoluron in ammonium sulfate, urea and atrazine multi-solutes system was investigated by batch experiments. The results showed application of nitrogen fertilizers to the soil could affect the behavior of chlorotoluron. At the same concentration of N, sorption of chlorotoluron decreased as the concentration of atrazine increased on the day 0 and 6 in soil, respectively. The sorption of chlorotoluron increased from 0 to 6 d when soils were preincubated with deionized water, ammonium sulfate and urea solution for 6 d. That indicated incubation time was one of the most important factors for the sorption of chlorotoluron in nitrogen fertilizers treatments. The individual sorption isotherms of chlorotoluron in rubbery polymer and silica were strictly linear in single solute system, but there were competition sorption between pesticides or between pesticides and nitrogen fertilizers. That indicated the sorption taken place by concurrent solid-phase dissolution mechanism and sorption on the interface of water-organic matter or water-mineral matter.     

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.     

Removal of heavy metals from sewage sludge by low costing chemical method and recycling in agriculture

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Single and joint effects of pesticides and mercury on soil urease

The influence of two pesticides including chlorimuron-ethyl and furadan and mercury （Hg） on urease activity in 4 soils （meadow burozem and phaeozem） was investigated. The soils were exposed to various concentrations of the two pesticides and Hg individually and simultaneously. Results showed that there was a close relationship between urease activity and organic matter content in soil. Chlorimuron-ethyl and furadan could both activate urease in the 4 soils. The maximum increment of urease activity by chlorimuronethyl was up to 14%-18%. There was almost an equal increase （up to 13%-21%） in the urease activity by furadan. On the contrary, Hg markedly inhibited soil urease activity. A logarithmic equation was used to describe the relationship （P〈0.05） between the concentration of Hg and the activity of soil urease in the 4 tested soils. Semi-effect dose （ED50） values by the stress of Hg based on the inhibition of soil urease in the 4 soils were 88, 5.5, 24 and 20 mg/kg, respectively, according to the calculation of the corresponding equations. The interactive effect of chlorimuron-ethyl or furadan with metal Hg on soil urease was mainly synergic at the highest tested concentrations.     

Isolation and preliminary characterization of a 3-chlorobenzoate degrading bacteria

A study was conducted to compare the diversity of 2-, 3-, and 4-chlorobenzoate degraders in two pristine soils and one contaminated sewage sludge. These samples contained strikingly different populations of mono-chlorobenzoate degraders. Although fewer cultures were isolated in the uncontaminated soils than contaminated one, the ability of microbial populations to mineralize chlorobenzoate was widespread. The 3- and 4-chlorobenzoate degraders were more diverse than the 2-chlorobenzoate degraders. One of the strains isolated from the sewage sludge was obtained. Based on its phenotype, chemotaxonomic properties and 16S rRNA gene, the organism S-7 was classified as Rhodococcus erythropolis. The strain can grow at temperature from 4 to 37℃. It can utilize several （halo）aromatic compounds. Moreover, strain S-7 can grow and use 3-chlorobenzoate as sole carbon source in a temperatures range of 10-30℃ with stoichiometric release of chloride ions. The psychrotolerant ability was significant for bioremediation in low temperature regions. Catechol and chlorocatechol 1,2-dioxygenase activities were present in cell free extracts of the strain, but no （chloro）catechol 2,3- dioxygenase activities was detected. Spectral conversion assays with extracts from R. erythropolis S-7 showed accumulation of a compound with a similar UV spectrum as chloro-cis,cis-muconate from 3-chlorobenzoate. On the basis of these results, we proposed that S-7 degraded 3-chlorobenzoate through the modified ortho-cleave pathway.     

Organochlorine pesticides in soils under different land usage in the Taihu Lake region, China

A field study was conducted in the Taihu Lake region, China in 2004 to reveal the organochlorine pesticide concentrations in soils after the ban of these substances in the year 1983. Thirteen organochlorine pesticides (OCPs) were analyzed in soils from paddy field, tree land and fallow land. Total organochlorine pesticide residues were higher in agricultural soils than in uncultivated fallow land soils. Among all the pesticides, ΣDDX (DDD, DDE and DDT) had the highest concentration for all the soil samples, ranging from 3.10 ng/g to 166.55 ng/g with a mean value of 57.04 ng/g and followed by ΣHCH, ranging from 0.73 ng/g to 60.97 ng/g with a mean value of 24.06 ng/g. Dieldrin, endrin, HCB and α-endosulfan were also found in soils with less than 15 ng/g. Ratios of p,p'-(DDD DDE)/DDT in soils under three land usages were: paddy field ＞ tree land ＞ fallow land, indicating that land usage inlfuenced the degradation of DDT in soils. Ratios of p,p'-(DDD DDE)/DDT ＞1, showing aged residues of DDTs in soils of the Taihu Lake region. The results were discussed with data from a former study that showed very low actual concentrations of HCH and DDT in soils in the Taihu Lake region, but according to the chemical half-lives and their concentrations in soils in 1980s, the concentration of DDT in soils seemed to be underestimated. In any case our data show that the ban on the use of HCH and DDT resulted in a tremendous reduction of these pesticide residues in soils, but there are still high amounts of pesticide residues in soils, which need more remediation processes.     

Sorption of 1-naphthol by plant cuticular fractions

The contribution of aliphatic-rich plant biopolymer to sorption of hydrophobic organic compounds is significantly important because of their preservation and accumulation in the soil environment,but sorption mechanism is still not fully understood.In this study, sorption of 1-naphthol by plant cuticular fractions was examined to better understand the contributions of respective fraction.Toward this end,cuticular materials were isolated from the fruits of tomato by chemical method.The tomato cuticle sheet consisted of waxes (6.5 wt%),cuticular monomer (69.5 wt%),and polysaccharide (24.0 wt%).Isotherms of l-naphthol to the cuticular fractions were nonlinear (N value (0.82-0.90)) at the whole tested concentration ranges.The KodKow ratios for bulk cuticle (TC1),dewaxed cuticle (TC2),cutin (TC4),and desugared cuticle (TC5) were larger than unity,suggested that tomato bulk cuticle and cutin are much powerful solption medium.Sorption capability of cutin (TC4) was 2.4 times higher than the nonsaponifiable fraction (TC3).The 1-naphthol interactions with tomato cuticular materials were governed by both hydrophobic-type interactions and polar (H-bonding) interactions. Removal of the wax and polysaccharide materials from the bulk tomato cuticle caused a significant increase in the sorption ability of the cuticular material.There was a linear negative trend between K_(oc) values and the amount of polysaccharides or fraction's polarities ((N O)/C);while a linear positive relationship between K_(oc) values and the content of cutin monomer (linear R~2=0.993) was observed for present in the cuticular fractions.Predominant sorbent of the hydrophobic organic compounds (HOCs) in the plant cuticular fraction was the cutin monomer,contributing to 91.7% of the total sorption of tomato bulk cuticle.