芘高效降解菌的分离鉴定及其降解特性研究

采用富集培养的方法从多环芳烃污染的土壤中分离到3株能高效降解四环芳烃芘的细菌J1、J2、J3,经形态观察、生理生化和16SrDNA鉴定,J1属于铜绿假单胞菌属(Pseudomonas aeruginosa),J2属于黄杆菌属(Flavobacterium mizutaii),J3属于短短芽孢杆菌属(Brevibacillus parabrevis).3株细菌均能以芘作为碳源生长,在含芘50、100、200、500、1 000 mg/L的无机盐液体培养基中培养7 d后细菌总数达到最高,在含芘200 mg.L-1的无机盐液体培养基中7d的降解效率分别达到53.04%、65.03%、51.02%.3株细菌对培养基具有较广泛的pH适应范围,在芘浓度200 mg/L,pH为4～9的液体条件下,均可生长,且对芘有很好的降解.虽然可以采用芘的二氯甲烷溶液或者N,N-二甲基甲酰胺(DMF)溶液向培养液中添加芘,但是以N,N-二甲基甲酰胺添加的方式芘在水溶液中起到很好的分散作用,更有利于3株细菌对芘的降解.     

耐盐石油烃降解菌的筛选鉴定及其特性研究

为得到高效耐盐石油烃降解菌,从黄河三角洲石油污染盐渍化土壤中分离出39株细菌,经液体培养初筛和土壤培养复筛实验,得到1株高效耐盐石油烃降解菌BM38.通过形态特征、生理生化特征和16S rDNA序列分析,确定该菌为恶臭假单胞菌（Pseudomonas putida）.通过液体培养实验,研究了BM38的耐盐和产生物表面活性剂特性以及对不同烃的利用能力.结果表明,在含0.5%～6.0%NaCl液体培养基中BM38生长良好,属中度耐盐菌.在高盐环境下BM38具有较强的分解石油烃能力,其中在含1.0%NaCl液体培养基中,降解7 d后,原油降解率达到73.5%;在含盐量0.22%和0.61%土壤中添加BM38,降解40 d后,土壤总石油烃降解率达到40%以上.BM38能产生一种生物乳化剂,盐浓度对这种乳化剂的乳化能力影响较大,当NaCl浓度增加到1.0%,乳化值（EI24）开始迅速降低,但在NaCl浓度为2.0%时,EI24仍达到61.0%.BM38能够利用环己烷、甲苯、异辛烷、菲和正十六烷为唯一碳源生长,其中对正构烷烃和芳烃具有较强的利用能力.     

高效石油降解菌的筛选及其生长特性研究

从上海、大连和天津的附近海域海水中分离出的15种石油降解菌中筛选出6种柴油降解率在75%以上的高效石油降解菌D3、T4、R4、T1、D4和R3。经16S rDNA测序鉴定,D4和T4属假单胞菌属,D3和R4属短波单胞菌属;R3属迪茨菌属,T1属盐单胞菌属。分析6种菌株在3种培养基中的生长特性发现,在LB培养基中,D4的适应期较长,需要25 h,而稳定期较短;在葡萄糖培养基中,R4在3 d后进入稳定期,适应期较长,其他5种菌株在2 d后就进入稳定期;在柴油海盐培养基中6种菌株在3 d后生物量出现明显增长,其中R3、R4和D3的生物量高于T1、D4和T4的生物量。对比6种高效石油降解菌株在3种培养基中的生长状况得出其生长的难易顺序是柴油海盐培养基>葡萄糖培养基>LB培养基。     

2株降解菲的植物内生细菌筛选及其降解特性

为了获得具有菲降解特性的植物内生细菌,通过选择性富集培养,本研究从多环芳烃污染区植物体内分离得到2株能够降解液体培养基中高浓度菲(200 mg·L-1)的植物内生细菌(菌株P1和P3).经形态观察、生理生化特征鉴定和16S rDNA序列同源性分析,分别将菌株P1和P3鉴定为寡养单胞菌属(Stenotrophomonas sp.)和假单胞菌属(Pseudomonas sp.)的细菌.菌株P1和P3均为好氧生长,28℃、150 r·min-1摇床培养7 d,2株菌对无机盐培养基中菲(100 mg·L-1)的降解率均高于90%.条件实验表明,温度20～30℃,pH 6.0～8.0,盐含量0%～4%,装液量10～30 mL(100 mL三角瓶)2菌株生长良好且对菲降解率高于70%.其最适生长和降解温度为30℃,pH为7.0,盐含量≤4%,装液量≤30 mL.综合比较2株菌对菲的降解特性,P1菌株高温耐受性稍强,而P3菌株对环境pH改变和缺氧的耐受性稍强.     

微生物-化学水解联合作用下烟嘧磺隆的降解

从被烟嘧磺隆污染的人工湿地土壤中分离出1株能够在葡萄糖存在下降解烟嘧磺隆的微生物,通过16S rDNA序列同源相似性分析,初步鉴定该微生物为Klebsiella sp..它能够以烟嘧磺隆为唯一氮源生长,其最适生长条件为:温度35℃,初始pH为中性偏酸.水解试验表明,烟嘧磺隆在中性和碱性条件下比较稳定,在酸性条件下水解较快.生物降解试验发现,当培养液中葡萄糖浓度为5 g.L-1时,在温度35℃、初始pH为7的条件下培养10 d后烟嘧磺隆有99.4%得到降解,同时溶液的pH从7.0降低至4.0;而降低葡萄糖浓度分别为500 mg.L-1和100 mg.L-1时,培养10 d后烟嘧磺隆的降解率仅为11.7%和6.6%,溶液的pH始终在7左右.进一步研究表明烟嘧磺隆的降解是由于微生物代谢葡萄糖产生了酸性环境,pH降低引起了烟嘧磺隆水解,菌种对烟嘧磺隆降解的实质是微生物-化学水解联合作用.     

阿特拉津降解菌株的分离、鉴定及降解特性研究

通过贫营养原水曝气培养法,以SBR方式驯化和稳定运行2个月,分离得到1株能够降解除草剂阿特拉津的降解菌株,命名为L-6.在以阿特拉津为唯一氮源(500 mg.L-1)的无机盐液体培养基中培养96 h后,降解率达到89.2%.扫描电镜观察发现该降解菌株细胞呈长杆状.提取菌株的DNA,进行16S rRNA基因序列分析,并构建系统进化树.结果表明该菌株与GenBank数据库中多株Pseudomonas菌株16S rRNA基因的同源性在99%以上.结合生理生化检测,判断该降解菌为假单胞菌属(Pseudomonas sp.).碳源利用实验发现,降解菌株L-6可以利用葡萄糖、果糖和柠檬酸钠生长,不能利用蔗糖、乳糖和淀粉.环境因素对降解效率的影响实验表明,温度在30℃,初始pH 7～9条件下,降解效率最佳.     

1株低温石油烃降解菌的分类鉴定及降解特性研究

从渤海油船泄漏区域的海底泥中筛选到1株能降解柴油的低温石油烃降解菌T7-2,初步鉴定为红平红球菌(Rhodococcus erythropolis).研究表明,利用工业乙醇无机盐培养基培养液体种子是适宜的,其最适温度和pH分别为15℃和7.8,工业乙醇最适加入量为0.5％,菌体浓度为10⁸ CFU/mL.接种人工海水烃降解培养基后,通过补加氮源 (NH₄)₂SO₄ 2.64　g/L、磷源Na₂HPO₄ 2.5 g/L和酵母粉 0.015 g/L后,15℃振荡培养7　d,降解率可以达到73.2％.该菌降解烷烃的范围很广泛,C12～C36均有不同程度的降解.     

环境激素对叔辛基酚降解菌的筛选、鉴定及降解特性

为了研究环境激素4-t-OP(对叔辛基酚)的生物降解,从扬州市汤汪生活污水处理厂二沉池污泥中筛选得到1株能以4-t-OP为唯一碳源进行生长的降解菌株,标示为TW30,16S rRNA测试其为不动杆菌属(Acinetobacter sp.),通过摇瓶试验测试其降解活性.结果表明:在40℃、初始pH为6.0、ρ(4-t-OP)为5 mg/L的无机盐培养基中,5 d后降解率可达99.03%;降解过程满足一级反应模型,降解速率常数(k)为0.875 d-1,半衰期(t1/2)为0.8 d.这说明TW30是一株高效的4-t-OP降解菌.此外,培养温度的升高和额外Ca2+、Mn2+的加入可以提高TW30降解4-t-OP的效率,而在5～25 mg/L范围内提高初始ρ(4-t-OP)以及额外加入磷酸盐、NH4+、Mg2+、Fe2+、Na+、Zn2+、Cu2+等无机盐和葡萄糖、CH3COO-等碳源则会降低降解率.      

己烯雌酚降解菌株沙雷氏菌的分离鉴定及其降解特性

利用具有降解己烯雌酚(diethylstilbestrol,DES)特性的功能微生物来降解DES、有望实现环境中DES的有效去除,然而迄今关于降解DES的功能菌株及其降解特性的报道很少.本研究通过选择性富集培养,从某污水处理站活性污泥中分离获得1株具有DES降解特性的细菌(菌株S).经形态观察、生理生化试验及16S rDNA序列同源性分析将菌株S鉴定为沙雷氏菌属(Serratia sp.)细菌.菌株S好氧生长,30℃、150 r·min-1摇床培养7 d后,对无机盐培养基中DES(50 mg·L-1)的降解率达68.3%.通过摇瓶实验,优化了菌株S生长和降解DES的最适环境条件:温度30℃,底物浓度40～60 mg·L-1,pH 7.0,接种量5%,盐添加量0 g·L-1,装液量10 mL(100 mL三角瓶).     

外加碳源对菌株GH10降解BDE-209的影响

通过研究6种外加碳源对菌株GH10降解BDE-209产生的影响,证明葡萄糖是菌GH10降解BDE-209的最佳外加碳源,降解培养基中葡萄糖的最适浓度为2 mg/L。经过5 d的降解培养,在第3天时培养基中BDE-209的残余浓度最低,其浓度从10 mg/L降为1.24 mg/L;与不添加葡萄糖的试验相比,BDE-209的降解率从42.24%提高到87.61%;菌株GH10对BDE-209的降解符合一级反应动力学,降解动力学方程为C=12.064e-0.670 t,半衰期为1.03 d,说明添加葡萄糖能够有效促进菌GH10降解BDE-209。     

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.     

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.     

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.     

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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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.     

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.     

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.     

Alterations of organ histopathology and metallolhionein mRNA expression in silver barb, Puntius gonionotus during subchronic cadmium exposure

Common silver barb,Puntius gonionotus,exposed to the nominal concentration of 0.06 mg/L Cd for 60 d,were assessed for histopathological alterations(gills,liver and kidney),metal accumulation,and metallothionein(MT)mRNA expression.Fish exhibited pathological symptoms such as hypertrophy and hyperplasia of primary and secondary gill lamellae,vacuolization in hepatocytes,and prominent tubular and glomerular damage in the kidney.In addition,kidney accumulated the highest content of cadmium,more than gills and liver.Expression of MT mRNA was increased in both liver and kidney of treated fish.Hepatic MT levels remained high after fish were removed to Cd-free water.In contrast,MT expression in kidney was peaked after 28 d of treatment and drastically dropped when fish were removed to Cd-free water.The high concentrations of Cd in hepatic tissues indicated an accumulation site or permanent damage on this tissue.