1株铜绿假单胞菌对芘的降解特性及代谢途径

多环芳烃（PAHs）因其具有"三致"作用对生态系统产生潜在威胁.微生物降解是多环芳烃降解的主要途径之一,筛选出能高效降解多环芳烃的菌株是微生物修复技术的关键.本文采用富集培养的方法从多环芳烃污染的污泥中分离到1株以芘为唯一碳源的菌株LX2,经形态观察、生理生化和16S rDNA鉴定,LX2属于铜绿假单胞菌（Pseudomonas sp.LX2）.菌株在含芘浓度为50 mg·L^-1的无机盐液体培养基中培养21 d对芘的降解效率达32.1%.经GC-MS分析发现,Pseudomonas sp.LX2降解芘的中间代谢产物主要有4,5-二氢芘、2''-羟基苯丙酮、苯酚、原儿茶酚.基于鉴定的代谢产物得出芘通过"萘"和"邻苯二甲酸"两种不同的途径被铜绿假单胞菌（Pseudomonas sp.LX2）降解.     

3株细菌对土壤中芘和苯并芘的降解及其动力学

研究了3株多环芳烃(PAHs)高效降解菌对土壤中芘和苯并芘(BaP)的降解动态,用Michaelis-Menton和Monod动力学模型对结果进行拟合.结果表明,3株细菌对芘和BaP的降解率有显著性差异.芽孢杆菌(Bacillus sp.SB02)42 d对芘和BaP的降解率均最高.当土壤中芘和BaP的初始浓度为50　mg/kg时,芽孢杆菌(Bacillus sp. SB02)、动胶杆菌(Zoogloea sp. SB09)、黄杆菌(Flavobacterium sp. SB10)42 d对芘的降解率分别为42.69%、32.88%、25.07%, 对BaP的降解率分别为33.04%、25.39％、22.02%.3株细菌对芘和BaP的降解速率也存在显著性差异.芽孢杆菌(Bacillus sp., SB02)最快,1周可降解20.88％芘和12.6%的BaP,动胶杆菌(Zoogloea sp.SB09)次之,黄杆菌(Flavobacterium sp.SB10)降解速率最慢.     

3种氧化剂对焦化场地多环芳烃的修复效果与土著微生物的响应关系

为了探究化学氧化对污染土壤修复过程土著微生物生理生态功能的影响,以焦化场地多环芳烃（PAHs）污染土壤为实验对象,研究了高锰酸钾、过硫酸钠和臭氧这3种氧化剂在不同液固比条件下对PAHs的修复效果和土著微生物的响应关系.结果表明,该焦化场地土壤ΣPAHs含量为679.1 mg·kg^-1,高锰酸钾和过硫酸钠投加量为1%时,液固比为6：1条件下ΣPAHs（16种PAHs）的去除率最高,分别为96.9%和95.7%,而臭氧剂量为72 mg·min^-1、液固比为8：1时ΣPAHs的去除率（82.3%）最高;不同液固比条件下低环PAHs （3~4环）的去除率高于高环PAHs （5~6环）,去除率最高的是菲和二氢苊;而对于高环的苯并[a]芘,仅高锰酸钾对其去除效果较优,去除率达到97.4%;微生物数量分析表明,土壤微生物数量经高锰酸钾处理后骤降,由10⁸ copies·g^-1降至10⁵ copies·g^-1,而过硫酸钠和臭氧处理变化不明显,数量级未发生显著变化;微生物群落结构分析表明,污染原土中Proteobacteria占绝对优势,相对丰度为99.5%,高锰酸钾和过硫酸钠处理后微生物多样性显著增加,多种能够降解PAHs的微生物（如Ralstonia和Acinetobacter等）相对丰度大幅提高;微生物代谢功能路径分析表明,化学氧化处理增加了PAHs降解菌的相对丰度,提高了有机物代谢能力.总体而言,液固比为6：1时高锰酸钾处理会显著改变土著微生物数量,微生物群落结构和PAHs降解微生物相对丰度.     

酚类及羟基化合物对BaP代谢影响的研究

经叔-丁基-羟基-苯甲醚(BHA)对照、HPLC分析结果表明,天然化合物如从甜瓜蒂(Cucumis melo L. cucurbitaceae)提取的葫芦素B、从徐长卿根(Pycnostelma paniculatum(Bge)K.Shum)提取的牡丹酚、人工合成的对乙氧基苯酚及维生素E中间体2,3,5-三甲基氢醌对BaP在大鼠肝微粒体体外代谢均有不同程度的抑制作用。当其浓度为25μM时,对BaP9,10-二醇及9-OHBaP的生成抑制较对BaP7,8-二醇及BaP4,5-二醇的更强。     

A reactor system combining reductive dechloirnation with cometabolic oxidation for complete degradation of tetrachloroentylene

A laboratory sequential anaerobic aerobic bioreactor system, which consisted of an anaerobic fixed film reactor and two aerobic chemostats, was set up to degrade tetrachloroethylene (PCE) without accumulating highly toxic degradation intermediates. A soil enrichment culture, which could reductively dechlorinate 900μM (ca. 150 mg/L) of PCE stoichiometrically into cis-1,2-dichloroethylene ( cis-DCE), was attached to ceramic media in the anaerobic fixed film reactor. A phenol degrading strain, Alcaligenes sp. R5, which can efficiently degrade cis-DCE by co metabolic oxidation, was used as inoculum for the aerobic chemostats consisted of a transformation reactor and a growth reactor. The anaerobic fixed film bioreactor showed more than 99 % of PCE transformation into cis DCE in the range of influent PCE concentration from 5μM to 35μM at hydraulic retention time of 48h. On the other hand, efficient degradation of the resultant cis-DCE by strain R5 in the following aerobic system could not be achieved due to oxygen limitation. However, 54% of the maximum cis-DCE degradation was obtained when 10 μmol of hydrogen peroxide (H₂O₂) was supplemented to the transformation reactor as an additional oxygen source. Further studies are needed to achieve more efficient co metabolic degradation of cis DCE in the aerobic reactor.     

β-Proteobacteria菌降解甲基叔丁基醚的条件及中间代谢产物研究

采用可利用甲基叔丁基醚(methyl tert-butyl ether,MTBE)为唯一碳源和能源生长的1株β-Proteobacteria菌进行MTBE在密闭系统中的降解试验,确定了该菌降解MTBE的最适条件为:培养液初始pH值7.2,初始细胞浓度10⁷cells/mL,初始MTBE浓度为25 mg/L.考察了密封培养系统内培养液溶解氧对降解效果的影响,结果表明,在培养系统密闭前充入氧气可提高菌体对MTBE的降解速率.以气相色谱-质谱联用法检测到MTBE降解主要中间代谢产物是叔丁基醇、异丙醇、丙酮.在选择离子扫描模式下定量分析,得到降解过程中主要中间代谢产物的浓度变化曲线,据此推断MTBE的降解途径属于“丙酮途径”.     

苯高效降解菌群富集及其降解机理研究

微生物绿色、低耗、原位修复去除污染土壤与地下水中可降解污染物日益受到关注.本研究从江苏某农药厂苯系物污染土壤中以苯为唯一碳源富集获得苯降解菌群.在28℃、pH=8.0、添加100 mg·L^-1酵母粉条件下,该菌群在6 h内完全降解100 mg·L^-1的苯,利用Q-TOF解析出苯酚和邻苯二酚等主要代谢产物,并通过PCR扩增出苯酚单加氧酶基因（GenBank登录号：MW388722）.该菌群30 h内可完全降解30 mg·L^-1的苯、甲苯和乙苯混合物,高通量测序结果显示该菌群主要含有Cupriavidus、Arthrobacter、Dyella、Corynebacterium、Microbacterium等菌属,其中Cupriavidus为优势菌,菌群群落结构稳定且可高效降解苯,具有潜在的应用前景.     

纳米乳化油修复硝酸盐污染地下水过程中的微生物特征模拟实验研究

为探寻纳米乳化油原位修复地下水硝酸盐氮污染过程中微生物堵塞的形成原因,本研究采用市售的反硝化细菌接种微生物,以纳米乳化油为碳源,中砂为介质,分别建立2组反应器进行模拟实验,分析不同反应器中硝氮的降解情况,同时采用MiSeq高通量测序技术表征不同反应器的微生物菌落结构和多样性.结果表明,纳米乳化油作为碳源具有良好的降解效果,添加纳米乳化油的反应器,反应周期内硝酸盐氮的总降解效率为91.76%,而对照反应器的降解效率仅为38.11%.在硝酸盐氮降解过程中,均存在以蛋白质和多糖为主的代谢产物胞外聚合物增加的趋势,且蛋白质的含量均显著高于多糖.反应结束时,实验组和对照组的胞外聚合物累积量分别为384.49 mg和279.45 mg,单位质量硝氮降解产生的胞外聚合物分别为1.79 mg·mg^-1和39.43 mg·mg^-1.高通量测序结果显示,添加纳米乳化油会引起细菌浓度的升高及细菌群落多样性的降低,但具有反硝化作用的微生物相对丰度增加.实验组和对照组反应器中共同的优势菌门为Proteobacteria、Bacteroidetes和Actinobacteria,相对丰度分别为73.35%、6.77%、8.49%及33.46%、47.15%、7.15%,纳米乳化油的添加会刺激Proteobacteria等具有较高反硝化作用的微生物增多,因此,以纳米乳化油作为碳源能够有效提高硝酸盐氮的降解效率,但与此同时纳米乳化油也会刺激微生物的生长及影响微生物群落演变.Sphingamonas、Rhodopseudomonas和Microbacterium菌属相对丰度增加,会引起粘性代谢产物增多,造成多孔介质渗透性下降和生物堵塞.     

零价铁预厌氧协同MFC强化降解吲哚性能与功能菌群分析

通过零价铁（ZVI）预厌氧与微生物燃料电池（MFC）的协同作用,研究其对单一基质碳源吲哚的降解特性、MFC产电性能及群落分析.结果表明：ZVI（4 g·L^-1）的预厌氧可明显促进吲哚（250 mg·L^-1）在MFC体系中的降解;其中吲哚与TOC在96 h内的降解率分别为97.17%和89.50%,且吲哚的降解符合一级反应动力学模型;体系中MFC最大输出电压和功率密度可达600 mV和439.55 mW·m^-2;通过LC-MS分析,吲哚在协同体系中的主要中间代谢产物为靛红和水杨酸.高通量测序结果表明,ZVI预厌氧体系的引入有利于MFC体系中梭菌（Clostridium sensu stricto）、链霉菌（Streptomyces sp.）、热单胞菌（Thermomonas）的富集与吲哚的厌氧降解;同时促进假单胞菌（Pseudomonas）和梭菌等在代谢过程中的电子转移,提高了MFC的产电性能.     

高效石油降解菌修复石油污染土壤与强化机制分析

利用定向驯化高效石油降解菌系对石油污染土壤进行为期120 d原位修复,考察生物强化修复效果、土壤理化性质和酶活性的变化,结合宏基因组测序及生物信息学分析揭示其强化机制.结果表明,与空白对照组(Ctrl)相比,生物修复组(Exp-BT)总石油烃降解率显著提升,增幅达81.23%; 高效石油降解菌生物强化修复期间土壤pH变化稳定,体系氧化能力提高,电导率处于适宜农业活动范围内; 脂肪酶和脱氢酶在修复期间保持较高活性; 另对初始污染土壤样本(B0)、驯化所得高效石油降解菌系样本(GZ)和生物修复后土壤样本(BT)的分析显示,门水平上变形菌门与放线菌门相对丰度增加17.1%,属水平上Nocardioides、Achromobacter、Gordonia和Rhodococcus等丰度明显上升,COG和KEGG物种与功能贡献度分析证明以上菌属对石油烃降解有重要贡献; 修复后土壤中发现高丰度的石油烃相关代谢酶及5个降解基因:alkM、tamA、rubB、ladA 和alkB,分析表明外源石油烃降解菌群的引入增强了微生物相关酶的代谢活性与相应功能基因的表达.     

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.     

MnO_2催化KMnO_4氧化降解酚类化合物

研究了MnO2强化KMnO4氧化降解酚类化合物的效能与机制.在假一级动力学实验条件下(KMnO4初始浓度是目标有机物初始浓度的10倍),考察了KMnO4对酚类化合物(2-氯酚和4-氯酚)的氧化降解规律.发现在KMnO4氧化降解酚类化合物过程中存在着明显的自催化现象,即原位产生的胶体MnO2可以促进KMnO4对有机物的氧化降解.实验进一步考察了MnO2浓度、粒径大小和溶液pH对MnO2催化KMnO4氧化降解酚类化合物的影响.结果表明,外加胶体MnO2和颗粒MnO2都可以催化KMnO4氧化降解酚类化合物,而且假一级动力学常数(K)随着MnO2浓度(30～180μmol·L-1)的增加呈线性增加;与胶体MnO2相比,颗粒MnO2的催化能力较弱;随着溶液pH的增加,MnO2催化能力逐渐减弱.实验中还发现外加MnO2能够催化KMnO4氧化降解2-硝基酚(单独MnO2和KMnO4均不能将其氧化),但对于二甲基亚砜(其不具有与金属离子络合配位的能力)则没有催化作用.由此推测MnO2催化KMnO4氧化降解有机物的作用机制可能为表面吸附络合催化,即吸附在MnO2表面形成的络合物比存在于溶液中的有机物本身更易被KMnO4氧化.     

芬顿试剂、高锰酸钾对餐饮业废水的预氧化效果研究

以芬顿试剂、高锰酸钾为氧化剂氧化降解餐饮业废水,通过测定COD、BOD5变化来比较氧化效果。在单因素实验的基础上,采用正交实验研究。芬顿试剂的最佳氧化条件是：FeSO4·7H2O投加量为3mmol／L,pH=3,H2O2／Fe^2＋比为3：1,反应时间为120min。高锰酸钾的最佳氧化条件为投加量10mL／L,pH=2,反应时间为60min。研究表明：与高锰酸钾处理的效果相比,采用芬顿试剂,COD去除率可达80％,处理后废水的可生化性大大提高,为进一步的生化处理创造了良好的条件。     

高锰酸钾氧化去除砂壤土中三氯乙烯的试验研究

以环境中常见的污染物三氯乙烯(TCE)为研究对象,利用高锰酸钾(KMnO4)对工业场地土壤中的TCE进行处理,探讨了不同氧化条件、污染物初始浓度、氧化次数等对去除效果的影响.结果表明,采用正交试验获得优化操作条件为:KMnO4浓度125mg/L,pH7,反应时间30min.在此条件下,100mg/kg的TCE去除率达到93.7%.TCE的去除率随污染物浓度的增加而减小,对于污染程度高的土壤,2次处理能有效提高去除率.土柱实验结果表明经过KMnO4溶液淋洗12d后,初始浓度为50mg/kg的TCE氧化率均达到88.1%以上,降低淋洗流速可提高TCE的去除率.     

Relation of hepatic EROD activity and CYP1A level in Sebastiscus marmoratus exposed to benzo[a]pyrene

This study was designed to investigate the in vivo effects of benzo[a]pyrene (BaP) on hepatic ethoxyresorufin-O-deethylase (EROD) activity and its correlation with cytochrome P450 1A (CYP1A) protein levels in Sebastiscus marmoratus, which were exposed through a water column to BaP (10, 100, 1000 ng/L, respectively) or were treated with intraperitoneal injections of BaP (0.5, 1, 5, 10 mg/kg, respectively) every 7 d. The results showed that after 25 d of waterborne exposure to 1000 ng/L BaP, fish hepatic CYP1A levels and EROD activity were significantly induced. In contrast, EROD activity was not altered 7 d after second ip injections, whereas, CYP1A protein levels were increased. Dose-dependent increase of biliary BaP metabolites demonstrated that the catalytic activity of CYP1A was induced by treatment with BaP. The lowest observable effect concentration with regard to biliary BaP metabolites (100 ng/L) was much lower than that with reference to EROD activity (1000 ng/L). The results suggest that biliary polycyclic aromatic hydrocarbon (PAH) metabolites were shown to better reflect the contamination gradients of PAHs than EROD activity. It appeared to be necessary to measure CYP1A protein levels to complement the EROD activity in relevant toxicological assessments.     

Relation of hepatic EROD activity and cytochrome P4501A level in Sebastiscus marmoratus exposed to benzo[a]pyrene

This study was designed to investigate the in vivo effects of benzo[a]pyrene （BaP） on hepatic ethoxyresorufin-O-deethylase （EROD） activity and its correlation with cytochrome P4501A （CYP1A） protein levels in Sebastiscus marmoratus, which were exposed through a water column to BaP （10, 100, 1000 ng/L, respectively） or were treated with intraperitoneal injections of BaP （0.5, 1, 5, 10 mg/kg, respectively） every 7 d. The results showed that after 25 d of waterborne exposure to 1000 ng/L BaP, fish hepatic CYP1A levels and EROD activity were significantly induced. In contrast, EROD activity was not altered 7 d after second intraperitoneal injections, whereas, CYP1A protein levels were increased. Dose-dependent increase of biliary BaP metabolites demonstrated that the catalytic activity of CYP1A was induced by treatment with BaP. The lowest observable effect concentration with regard to biliary BaP metabolites （100 ng/L） was much lower than that with reference to EROD activity （1000 ng/L）. The results suggest that biliary polycyclic aromatic hydrocarbon （PAH） metabolites were shown to better reflect the contamination gradients of PAHs than EROD activity. It appeared to be necessary to measure CYP1A protein levels to complement the EROD activity in relevant toxicological assessments.     

Biodegradation of benzo[a]pyrene in soil by Mucor sp. SF06 and Bacillus sp. SB02 co-immobilized on vermiculite

Two indigenous microorganisms, Bacillus sp. SB02 and Mucor sp. SF06, capable of degrading polycyclic aromatic hydrocarbons （PAHs） were co-immobilized on vermiculite by physical adsorption and used to degrade benzo[a] pyrene （BaP）. The characteristics of BaP degradation by both free and co-immobilized microorganism were then investigated and compared. The removal rate using the immobilized bacterial-fungal mixed consortium was higher than that of the freely mobile mixed consortium. 95.3% of BaP was degraded using the co-immobilized system within 42 d, which was remarkably higher than the removal rate of that by the free strains. The optimal amount of inoculated co-immobilized system for BaP degradation was 2%. The immobilized bacterial-fungal mixed consortium also showed better water stability than the free strains. Kinetics of BaP biodegradation by co-immobilized SF06 and SB02 were also studied. The results demonstrated that BaP degradation could be well described by a zero-order reaction rate equation when the initial BaP concentration was in the range of 10--200 mg/kg. The scanning electronic microscope （SEM） analysis showed that the co-immobilized microstructure was suitable for the growth of SF06 and SB02. The mass transmission process of co-immobilized system in soil is discussed. The results demonstrate the potential for employing the bacterial-fungal mixed consortium, co-immobilized on vermiculite, for in situ bioremediation of BaP.     

河口底泥来源真菌Trichoderma asperellum对壬基酚的降解路径

从壬基酚(Nonylphenol,NP)污染严重的李村河口底泥中分离纯化出可实验室培养的真菌,以高浓度NP为环境选择压力筛选出了一株目标菌株,18S rDNA确定其归属为棘孢木霉(Trichoderma asperellum)。实验室内研究了该菌株对NP的生物降解过程,LC/MS分析其代谢产物,据此提出了NP可能的生物降解路径。菌株的生长曲线表明NP能促进其生长,该菌株3 d对5 mg/L NP的降解率为71.4%,7 d的降解率达到87.2%,14 d则达到了92.2%。LC/MS分析确定了NP四种代谢产物,分别是2-甲基-1-苯基丁醇、3,5-二羟基苯甲酸、苯二酚和苯甲醚(或苄醇)。提出了两种NP可能的生物降解路径,Ⅰ是最终转化成苯二酚,Ⅱ是最终转化成苯甲醚或苄醇。     

过硫酸钠对我国典型土壤中多环芳烃氧化降解效果的影响

过硫酸钠是污染土壤化学氧化修复技术中应用较为广泛的氧化剂.为研究过硫酸钠对不同土壤中PAHs（polycyclic aromatic hydrocarbons,多环芳烃）的修复效果,以我国多种典型土壤（黑土、潮土、黄土、紫色土、褐土、砖红壤）为试验样本,以萘、菲、蒽、芘、苯并[a]芘5种PAHs为目标污染物,分析活化过硫酸钠对人为老化的降解率;此外,通过对氧化前后土壤pH、w（有机碳）等土壤性质变化的比较和分析,探讨氧化修复过程对土壤性质的影响.结果表明:当活化过硫酸钠用量为0.8 mmol/g、温度为25℃时,PAHs污染土壤中萘、菲、蒽、芘、苯并[a]芘的降解率最高,分别为87.82%、79.68%、87.93%、83.40%、94.31%.随着温度的升高,PAHs降解率逐渐升高,当温度达到25℃时,PAHs的降解率（85.69%）达到最高,随后随着温度的继续升高,总PAHs的降解率没有明显增加;随着pH的升高,PAHs的降解率逐渐升高,当pH达到6~7时,PAHs降解率维持在一个较高水平;随后随着pH的继续升高,总PAHs的降解率逐渐降低.随着温度以及pH的变化,5种PAHs的降解率与总PAHs的降解率变化趋势一致. w（有机碳）越低,PAHs环数越高,PAHs降解率越高;高环（5~6环）、中环（4环）、低环（2~3环）PAHs降解率与总PAHs降解率变化趋势一致.此外,过硫酸钠氧化修复后土壤结构遭到一定程度的破坏,土壤的pH、w（有机碳）和土壤肥力会有不同程度的下降,对土壤的再次利用有较大影响.研究显示,过硫酸钠可有效氧化降解不同性质土壤中PAHs,在氧化修复PAHs污染土壤方面具有较好的应用前景.      

假单胞菌N7的萘降解特性及其降解途径研究

应用HPLC和UV分析技术,以萘为代表性多环芳烃污染物,研究了假单胞菌N7对水中萘的降解特性.结果表明,营养盐、微量元素的添加可使萘的降解率提高23.65%;溶解氧高于4.3 mg/L时萘降解率达95.66%并趋于稳定;随萘浓度增加降解率逐渐下降;在中性和弱碱性环境下,降解效果较好,萘降解率均在82.88%以上.在30℃、转速为165 r/min的摇床中处理pH7.5、萘浓度为100 mg/L的水样72 h,其最大降解率为95.66%.通过检测菌株N7处理含不同底物水样时其吸光度、pH和底物的变化情况,证实菌株N7亦能降解甲苯、二甲苯、苯酚、2,4-二硝基苯酚、苯甲酸、1-萘酚和水杨酸,并以其为唯一的碳源和能源生长繁殖,表明该菌株能适应环境中芳烃类物质种类的变化,具有很好的降解多样性.经UV-Vis和GC-MS分析各降解阶段的中间产物,初步确定了该菌对萘的降解途径:一条是邻苯二甲酸途径;另一条是水杨酸途径,萘先被氧化为1,2-二羟基萘,再开环生成水杨酸、邻苯二酚和2-羟基粘康酸半醛,最终进入三羧酸循环(TCA).