微生物转化污泥制备苏云金杆菌生物杀虫剂

以城市污水处理厂的污泥为原料,探索了微生物转化污泥制备苏云金杆菌生物杀虫剂的可行性,并与常规培养基的发酵进程进行了对比.主要考察了苏云金杆菌的代谢特征、菌体形态与杀虫晶体蛋白产量.研究表明:无需任何预处理工序,污泥所含营养成分即可基本满足苏云金杆菌生长需求,且增殖较快,24h即可达活菌数与活芽孢数的最大值:9.48×10⁸CFU·mL^-1和8.51×10⁸CFU·mL^-1,比常规培养基提前12h,数量分别提高17%和21%;36h SEM表征显示,污泥中晶体与芽孢大部分游离,且晶体较大,呈规则的菱形,而常规培养基中苏云金杆菌的代谢进程相对滞后,且晶体较小;至发酵终点污泥中杀虫晶体蛋白含量为2.80 mg·mL^-1,亦略高于常规培养基.采用污泥发酵制备苏云金杆菌生物杀虫剂大大降低了生产成本,且发酵性能优良,为污泥处置开辟了崭新途径.     

利用城市污泥生产苏云金杆菌生物农药

用城市污水污泥和经酸水解后的城市污泥作为培养基发酵苏云金杆菌Bacillusthringiensis(B.t),与常规的黄豆粉培养基作比较.检测B.t发酵液的pH值变化,总细胞数及活孢子数以及在发酵48h后,用2龄家蚕作试虫比较3种发酵液的生物毒性.结果表明,用污泥培养的B.t总细胞数及活孢子数略低于黄豆粉培养基生产的B.t,但两者的细菌总数都在同一数量级,差别不显著.用污泥培养B.t,芽孢大量形成109个/mL的时间,比用黄豆粉培养基生产的B.t约提前10h,发酵周期明显缩短.生物毒性实验表明用污泥培养的B.t孢晶混合液比用黄豆粉生产的B.t孢晶混合液毒性高,在相同的稀释倍数下,家蚕的死亡率提高了10%,酸水解污泥毒性略差.     

养猪发酵床中净水芽孢杆菌的分离及其固体发酵研究

为解决养猪场污染物问题,从猪场生物发酵床中分离10株芽孢杆菌,用养猪污水进行培养,筛选出净化污水能力最强的一株,通过鉴定其为蜡状芽孢杆菌,并优化了该菌固体发酵培养基及固体发酵工艺条件。结果表明:此蜡状芽孢杆菌能使养猪污水中COD和氨氮分别降低47.1%和54.4%。优化后的培养基组分和条件为m(豆粕)∶m(稻壳)∶m(麸皮)=2∶9∶9(样品干重比),含水率为50%,KH2PO4含量为1.5%,MgSO4·7H2O含量为0.1%,初始pH为6.5。3%的菌株接种量在发酵温度为30℃的培养基中生长状况最佳,24 h后菌落数达到3.8×108CFU/g。     

Pb2+对城市污水处理厂污泥中苏云金芽孢杆菌发酵的影响

以城市污水处理厂污泥为培养基,研究了添加Pb2+对Bt(Bacillus thuringiensis,苏云金芽孢杆菌)生长和晶体蛋白合成的影响,同时分析了Bt发酵过程中有效态Pb含量(以ρ计)的变化,并采用FTIR(傅立叶红外光谱)和XPS(X射线光电子能谱),初步探讨了Pb2+在Bt表面的作用机制.结果表明:污泥培养基在支持Bt生长代谢和降低重金属有效态含量方面均优于商用培养基,当初始ρ(Pb2+)高达400 mg/L时,与未添加Pb2+相比,污泥培养基中Bt活菌数与晶体蛋白产量约分别下降48%和54%,而商用培养基中约分别下降82%和75%,证实了污泥作为Bt发酵培养基的可行性;Bt对Pb2+有较好的耐受性,当ρ(有效态Pb)在80 mg/L以下时,Bt生长代谢不会受到显著影响.光谱学分析表明,Bt对Pb2+有少量吸附,从而降低Bt发酵过程中Pb的生物有效性,吸附位点主要为羟基,部分C O和S O也参与了吸附反应.     

Effect of Lead lons on Bacillus thuringiensis Fermentation with Sewage as Medium

以城市污水处理厂污泥为培养基,研究了添加Pb2+对Bt(Bacillus thuringiensis,苏云金芽孢杆菌)生长和晶体蛋白合成的影响,同时分析了Bt发酵过程中有效态Pb含量(以ρ 计)的变化,并采用FTIR(傅立叶红外光谱)和XPS(X射线光电子能谱),初步探讨了Pb2+在Bt表面的作用机制.结果表明:污泥培养基在支持Bt生长代谢和降低重金属有效态含量方面均优于商用培养基,当初始p( Pb2+)高达400 mg/L时,与未添加Pb2+相比,污泥培养基中Bt活菌数与晶体蛋白产量约分别下降48％和54％,而商用培养基中约分别下降82％和75％,证实了污泥作为Bt发酵培养基的可行性;Bt对Pb2+有较好的耐受性,当ρ(有效态Pb)在80 mg/L以下时,Bt生长代谢不会受到显著影响.光谱学分析表明,Bt对Pb2+有少量吸附,从而降低Bt发酵过程中Pb的生物有效性,吸附位点主要为羟基,部分C=O和S＝O也参与了吸附反应.     

胶质芽孢杆菌(Bacillus mucilaginosus)在餐厨垃圾废水中生长条件优化

为了探讨餐厨垃圾废水用作发酵基质生产液态解钾菌肥的可行性,选用胶质芽孢杆菌（Bacillus mucilaginosus）作为试验菌种,采用正交和单因素方法对相关生长因素进行了优化.结果表明,在餐厨垃圾废水中培养胶质芽孢杆菌经过3 d的调整期后进入对数生长期,6~7 d时活菌数达到最大,Ⅰ类废水活菌数为1.55×1010 CFU/mL,Ⅱ类废水活菌数为6.60×1010 CFU/mL.以Ⅱ类废水为基质进行正交试验确定的较优培养条件为pH=7、温度30℃、摇床转速160 r/min、接种量2.0%（V/V）.废水的pH和盐分对胶质芽孢杆菌的生长代谢影响极为显著:最适初始pH为7（活菌数为3.80×1010 CFU/mL和9.20×1010 CFU/mL）;随着ρ（NaCl）的增加,活菌数先升高后快速降低,最适ρ（NaCl）为4 g/L.Ⅰ类和Ⅱ类废水的最佳接种量分别为1.5%（活菌数为1.60×1010 CFU/mL）和2.0%（活菌数为6.40×1010 CFU/mL）.研究显示,胶质芽孢杆菌在餐厨垃圾废水中经过培养后可达到GB 20287-2006《农用微生物菌剂》中液态菌肥的活菌数（2.0×108 CFU/mL）,经湿热处理后的Ⅱ类废水对胶质芽孢杆菌的生长有明显的促进作用.      

巨大芽孢杆菌在餐厨垃圾湿热处理脱出液中生长条件优化研究

选用巨大芽孢杆菌(Bacillus megatherium)作为实验菌种,以餐厨垃圾湿热处理脱出液为发酵培养基制作解磷液态菌肥。将巨大芽孢杆菌接种于餐厨垃圾湿热处理脱出液中进行培养并确定最佳发酵时间,考察初始p H值、接种量、培养温度、摇床转速、装液量和脱出液与水的混合比例对活菌数的影响。结果表明:巨大芽孢杆菌在餐厨垃圾湿热处理脱出液中最佳发酵时间为36 h,发酵最佳初始p H值、接种量、培养温度、摇床转速、装液量和脱出液与水的混合比例分别为7.5、2%、30℃、210 r/min、50 m L(250 m L锥形瓶)和1∶1。巨大芽孢杆菌在餐厨垃圾湿热处理脱出液中进行培养后可达到农业部规定的液态菌肥的活菌数(2×108cfu/m L)标准。     

莠去津降解菌HB-5的最佳产酶培养基及发酵条件

从农药厂废水中分离到一株降解莠去滓的节杆菌(Arthrobacter sp.)HB-5,以从该菌中提取到的降解酶对莠去津的降解率为指标,进行最佳产酶培养基及发酵条件的优化研究,对其产酶量进行了评价.通过正交试验和均匀试验,对细菌HB-5的发酵培养基进行了优化研究.运用SAS软件进行结果分析,所获优化培养基配方为:蔗糖3.0g·L-1,莠去津0.38g·L-1,K2HPO40.5g·L-1,KH2PO41.2 g·L-1,MgSO4·7H2O 1.2g·L-1,NaCl 0.1g·L-1,微量元素溶液3.8mL·L-1.得到菌株培养的最佳优化条件为:菌株发酵液培养时间为48h.接种量为2%,发酵液初始pH值为9,250mL三角瓶中装液量为80mL经优化后,降解酶对莠去津的降解率(91.64%)比原培养基(40.67%)提高了125%.     

鼠李糖脂对微生物降解正十六烷以及细胞表面性质的影响

使用大庆油田石油污染土壤中分离出的优势菌种（蜡状芽孢杆菌DQ01和芽孢杆菌DQ02）,在实验室可控条件下,研究了生物表面活性剂鼠李糖脂对微生物降解正十六烷以及降解菌生长、菌体表面疏水性和外表形态的影响.结果表明,鼠李糖脂可以提高正十六烷的降解率,48 h时蜡状芽孢杆菌DQ01和芽孢杆菌DQ02对正十六烷的降解率比未加鼠李糖脂的体系分别增加了8.1％和11.6％. 正十六烷培养基中的鼠李糖脂能引起菌体表面疏水性的明显增大,且低浓度鼠李糖脂的这种能力明显优于高浓度鼠李糖脂,蜡状芽孢杆菌DQ01和芽孢杆菌DQ02分别在加入0.4 mmol/L和0.2 mmol/L鼠李糖脂时疏水性最大. 尤其是芽孢杆菌DQ02在对数生长后期BATH（bacterial adherence to hydrocarbon）达到44％,远大于未加鼠李糖脂时的BATH. 但在葡萄糖培养基中鼠李糖脂并没有明显影响菌种的生物量和BATH. 另外,加入鼠李糖脂后, 2株菌的培养基接触角比未加鼠李糖脂的培养基接触角均减小了约一半. 未加入鼠李糖脂的菌体细胞表面比较光滑,且菌体之间独立生长,而加入鼠李糖脂后影响了降解菌的外表形态,菌体细胞表面略显粗糙且相互粘连,有助于细胞与疏水性有机物的接触.     

硝基苯好氧降解的共基质及生物协同作用

通过研究多菌种多基质条件下硝基苯的降解以便寻找提高实际废水处理效率的途径,从不同菌源中筛选与驯化获得硝基苯的高效降解菌枯草芽孢杆菌、门多萨假单孢菌、肺炎克雷伯氏菌及人苍白杆菌.研究这些菌种好氧降解硝基苯过程中的生物协同作用及其基质效应,构建实际废水处理的生物降解体系．结果表明,枯草芽孢杆菌和人苍白杆菌之间存在协同降解作用．加入人苍白杆菌后可使质量浓度为232．0mg/L硝基苯被完全降解的时间从145h缩短为85h;由枯草芽孢杆菌、门多萨假单孢菌和肺炎克雷伯氏菌组成的混合降解实验发现,枯草芽孢杆菌和门多萨假单孢菌之间存在一定程度的相互抑制作用;当加入肺炎克雷伯氏菌后抑制作用消失,降解速度加快.复合菌的作用明显优于各菌单独作用或任何两种菌之间的配合效果.经氯霉素制药厂含硝基苯实际废水的检验,混合菌在降解硝基苯的同时也使COD成分得到有效去除．     

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.     

Effects of two sludge application on fractionation and phytotoxicity of zinc and copper in the soil

The potential harm of heavy metals is a primary concern in application of sludge to the agricultural land. A pot experiment was conducted to evaluate the effect of two sludges on fractionation of Zn and Cu in soil and their phytotoxicity to pakchoi. The loamy soil was mixed with 0%, 20%, 40%, 60% and 80% (by weight) of digested sewage sludge (SS) and composted sludge (SC). The additions of both sludges caused a significant raise in all fractions, resulting in that exchangeable (EXCH) and organic bound (OM) became predominance of Zn and organic bound Cu occupied the largest portion. There was more available amount of Zn and Cu in SS treatments than SC treatments. During the pot experiment, the concentration of Zn in EXCH, carbonate (CAR) and OM and Cu in EXCH and OM fractions decreased in all treatments, so their bioavailability reduced. Germination rate and plant biomass decreased when the addition rate was high and the best yield appeared in 20% mixtures at the harvest of pakchoi. The two sludges increased tissue contents of Zn and Cu especially in the SS treatments. Zn in pakchoi was not only in relationship to ΔEXCH and ΔCAR forms but also in ΔOM forms in the sludge-soil mixtures. Tissue content of Cu in pakchoi grown on SC-soils could not be predicted by ΔEXCH. These correlation rates between Zn and Cu accumulation in pakchoi and variation of different fractions increased with time, which might indicate that sludges represented stronger impacts on the plant in long-term land application.     

Intraspecific differences in effects of co-contamination of cadmium and

A hydroponic experiment was carried out to study intraspecific differences in the effects of different concentrations of cadmium (Cd)(0-10 mg/L) and arsenate (As(V)) (0-8 mg/L) on the growth parameters and accumulation of Cd and As in six wheat varieties Jing-9428, Duokang-1, Jingdong-11, Jing-411, Jingdong-8 and Zhongmai-8. The endpoints of wheat seedlings, including seed germination,biomass, root length and shoot height, decreased with increasing the Cd and As concentrations. Significant differences in seed germination, biomass, root length, shoot height and the accumulation of Cd and As were observed between the treatments and among the varieties (p ＜ 0.05). The lethal dosage 50% were about 20, 80, 60, 60, 80 and 20 mg As/L for Jing-9428, Duokang-1, Jingdong-11,Jing-411, Jingdong-8 and Zhongmai-8, respectively, and the corresponding values for Cd were about 30, 80, 20, 40, 60 and 10 mg Cd/L, respectively. Among the six varieties, Duokang-1 was found to be the most resistant to Cd and As toxicity, and Zhongmai-8 was the most sensitive to Cd and As co-contamination. The resistance of the six varieties was found dependant on the seedling uptake of Cd and As. Duokang-1 was the most suitable for cultivation in Cd and As co-contaminated soils.     

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.