活性污泥中一氧化氮还原酶的提取和测定优化

通过正交实验,研究了超声破碎法和低温高压破碎法提取一氧化氮还原酶（nitric oxide reductase,nor）的活性,分析了超声强度和次数,破碎压力和次数,裂解液的添加量等因素对于胞内可溶性蛋白和核酸（DNA）的释放与提取nor活性的影响.根据活性污泥中酶的催化特性以及其他实验条件,完善了nor催化活性的测定方法.结果表明超声法提取nor的参数应设定为,超声次数为100次,超声强度为500W,裂解液的添加量为0.1mL.低温高压破碎法提取nor的参数应设定为破碎次数为4次,破碎压力为50MPa,裂解液的添加量为0.1mL.低温高压破碎法下的胞内可溶性蛋白和DNA的释放量与nor的最大催化活性要高于超声破碎法.此外,在nor催化活性的测定方法中,nor活性测定终点时间应为15min.     

一株异养硝化菌胞内羟胺氧化酶提取条件的优化

采用超声波细胞破碎法从一株异养硝化菌胞内提取硝化过程的关键酶羟胺氧化酶(HAO)。利用响应面分析法对影响超声波破碎效果的工作条件进行优化,然后采用硫酸铵分级沉淀法对HAO进行部分纯化,得到目标酶HAO提取的最佳超声波工作条件为:超声功率310 W、总辐射时间400 s、单次辐射时间5 s、菌液浓度OD600=1.930,硫酸铵分级沉淀的最佳饱和度为50%~65%。在最佳工作条件下所得粗酶液中HAO活性为28 m U/mg,粗酶液经过2次硫酸铵分级沉淀处理后,HAO的纯化倍数达1.907,比活力达到53 m U/mg。     

活性污泥中超氧化物歧化酶(SOD)活性测定影响因素

研究以污水生物处理中的活性污泥为对象,采用氮蓝四唑光还原法测定超氧化物歧化酶(SOD)活性,从样品制备和保存方面探讨了是否添加溶菌酶液、细胞破碎时间、酶液保存时间与保存温度等因素对SOD活性测定的影响。研究表明,溶菌酶液的加入提高了细胞破碎效果;细胞破碎的条件为99次(工作3 s,停3 s),时间为40 min时破碎效果最好;酶液保存时间在4 h内最佳;-20℃保存比4℃保存对酶活性影响小。最佳检测条件为:取样量为1 mL时,反应温度为30℃,反应时间为20 min时,SOD酶活性最大。     

活性污泥系统中酶研究进展

细菌和原生动物在污水处理中的作用与各自的酶活性密切相关。细菌对有机质进行分解,而原生动物对污水及细菌进行澄清,活性污泥酶活性高则出水水质较好。污水中毒性物质(如重金属)对活性污泥酶活性有抑制作用,原因是重金属离子能和酶中的巯基(—SH)结合。     

进水碳氮比对脱氮污泥羟胺氧化酶活性及N_2O产生的影响

在以A/O方式运行的SBR工艺中,研究了3种不同进水碳氮比下硝化与反硝化过程中污泥羟胺氧化酶(HAO)活性变化、N2O的产生/释放规律及两者之间的关联性.结果表明,当C/N=3.5与C/N=9.5时,HAO平均酶活性分别为(283.77±19.64),(348.87±17.94)U/g MLSS,而C/N=6.5条件下的平均酶活性仅为(246.45±23.30)U/g MLSS,总体上3个条件下缺氧阶段HAO活性均较好氧阶段高;反应过程中HAO的活性变化趋势基本与气态N_2O释放速率、溶解态N_2O及亚硝氮的浓度变化趋势成正相关,在C/N=9.5下好氧段HAO活性与后三者呈现完全一致的变化规律.N_2O主要产生于好氧阶段进行的硝化过程,尤其是羟胺氧化是N_2O产生的主要环节;碳源相对不充分的条件下(如C/N=3.5),缺氧段N_2O的释放与HAO活性关系密切;碳源相对较充分的条件下,缺氧段N_2O的产生与HAO酶活性无明显关联.推测可能是因为缺乏电子受体NO_2~-而导致HAO酶未参与反应;在N_2O产生较多的条件下,HAO活性相对也较高.     

废水及活性污泥中的酶活性

本文研究的目的是评价在废水及活性污泥中确定异养微生物活性及微生物密度,选择酶活性测定的可能性,在废水中,酯酶及脱氢酶的活性与四种(?)过异养菌菌落测定酶活性的方法(α糖甘(?)酶、丙氨酸氨肽酶.酯酶及脱氯酶)的图示,可用于确定中试装置的活性污泥和厌氧水解作用污泥的特性 在两种污泥中,酶活性模型明显有差异,表明其微生物种群不同或生理性质存在差别,四个生产规模污水处理活性污泥中,活性污泥酶活性模型受到进水成分影响较大。     

褐煤降解真菌—青霉菌P6的胞外酶研究

具有强降解褐煤能力的青霉菌株P6在液体培养条件下,可产生较强的过氧化物酶(包括木素过氧化物酶和锰过氧化物酶)活性,并具有酯酶活性,不产生内葡聚糖酶和木聚糖酶.粗酶液依次经80%饱和度的硫酸铵盐析、DEAE-纤维素离子交换树脂层析、Sephadex G-100凝胶过滤,得到一个具有藜芦醇氧化活性的的木素过氧化物酶洗脱峰,藜芦醇氧化活性达5760U/L,比活力达7.52U/mg,纯化倍数150.4倍.     

厌氧氨氧化活性污泥DNA高效提取方法探究

为了揭示自动和手动抽提方法所得DNA的差异,分析该差异所造成的本质原因,探求提高厌氧氨氧化(Anammox)活性污泥DNA抽提效率的方法;通过不同人员自动和手动抽提Anammox活性污泥,比较DNA得率与质量,发现手动抽提DNA浓度显著高于自动抽提(P0.05)、DNA质量与自动提取结果无显著差异(A260/280值在1.8~2.0之间);比较不同人员抽提效率,结果表明自动抽提DNA浓度和质量稳定性高于手动抽提。通过调整自动抽提裂解液、洗脱液、污泥用量优化自动提取过程,发现350 mg活性污泥(13 000r/min离心3 min)、900μL裂解液、100μL洗脱液提取的DNA效率最高,是自动提取的最优使用量,对DNA抽提效率的影响由大到小依次为洗脱液、污泥用量、裂解液。综上,手动抽提用于杂质含量少、微生物丰度低的样品,误差较小,自动抽提用于杂质繁杂、微生物富集较好的样品,抽提稳定性较高。     

外源Cu、Zn对猪粪与玉米秸秆混合物料产甲烷特性影响机理分析

为了探讨重金属Cu、Zn对有机废弃物产甲烷的影响机制,研究了在55℃条件下添加外源Cu、Zn对猪粪和玉米秸秆质量比为2∶1混合物料厌氧发酵过程产甲烷特性的影响.结果表明:当厌氧发酵液的NH_4~+-N含量低于706.7 mg·L-1时对厌氧发酵未产生明显的抑制作用.厌氧发酵液中的VFA下降幅度与累积甲烷产生量呈正相关.随着混合物料中Cu、Zn浓度的增加,累积产甲烷量总体上均呈现降低趋势(除Zn100处理外),当Cu413.7 mg·kg-1或Zn500.6 mg·kg-1时,对厌氧发酵的影响不明显,当Cu513.7 mg·kg-1、Zn600.6 mg·kg-1时,对厌氧发酵产生显著的抑制作用,且Cu对厌氧发酵的抑制作用强于Zn.不同浓度Cu、Zn与发酵料液中纤维素酶、蔗糖酶和脲酶活性均呈显著负相关(p≤0.05),Cu处理发酵料液中纤维素酶、蔗糖酶和脲酶活性与累积产甲烷量均呈显著正相关(p≤0.05);Zn处理的纤维素酶活性和蔗糖酶活性与其呈显著正相关(p≤0.05),但脲酶活性与其相关性不显著.表明Cu、Zn可能通过影响酶活性而对厌氧发酵过程和累积甲烷产生量产生影响.     

影响活性污泥脱氮效率的因素

活性污泥对氮的去除主要通过硝化和反硝化作用来进行.温度、pH、溶解氧浓度、污泥龄、毒性物质、污水性质(有机物含量、氮浓度)都会对系统脱氮能力产生影响。一般较高的PH、延长污泥龄、较低的溶解氧浓度和较低的有机碳浓度均能提高系统的硝化能力。反硝化/硝化系统具有投资省、脱氮效率高等优点.除甲醇外,其它一些工业废弃物也可作为促进反硝化作用的碳源.由于厌氧环境有利于反硝化作用,所以厌氧/好氧(A/O)法和间歇式活性污泥法(SBR)具有极高的脱氮效率.     

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.