球形红细菌厌氧降解2,4-二硝基甲苯

【目的】研究不同环境条件对2,4-二硝基甲苯(2,4-DNT)生物降解的影响。【方法】采用光合细菌球形红细菌在温度为30 °C的光照培养箱中厌氧降解2,4-DNT,并用高效液相色谱仪测定其浓度。【结果】去除2,4-DNT的最佳条件是初始浓度40 mg/L、初始pH 7.0和接种量15%。另外,2,4-DNT在菌体延滞期被细胞吸收,然后在指数期作为碳源被降解。2,4-DNT的去除率在72 h达到98.8%。从液相色谱图中观察到有2种中间代谢产物,但在120 h内产物被逐渐降解。2,4-DNT的去除动力学符合一级速率模型。【结论】不同条件下2,4-DNT的去除率表明球形红细菌能有效降解2,4-DNT。     

球形红细菌厌氧降解2,4-二硝基甲苯（英文）

【目的】研究不同环境条件对2,4-二硝基甲苯(2,4-DNT)生物降解的影响。【方法】采用光合细菌球形红细菌在温度为30°C的光照培养箱中厌氧降解2,4-DNT,并用高效液相色谱仪测定其浓度。【结果】去除2,4-DNT的最佳条件是初始浓度40 mg/L、初始p H 7.0和接种量15%。另外,2,4-DNT在菌体延滞期被细胞吸收,然后在指数期作为碳源被降解。2,4-DNT的去除率在72 h达到98.8%。从液相色谱图中观察到有2种中间代谢产物,但在120 h内产物被逐渐降解。2,4-DNT的去除动力学符合一级速率模型。【结论】不同条件下2,4-DNT的去除率表明球形红细菌能有效降解2,4-DNT。     

铁还原细菌Shewanella oneidensis MR-4诱导水合氧化铁形成蓝铁矿的过程

【目的】研究铁还原细菌Shewanella oneidensis MR-4在细胞外诱导形成含铁矿物的矿物相、化学成分和形貌结构等特性及其变化,深化对铁还原细菌细胞外诱导矿化过程的认识。【方法】在以30 mmol/L乳酸钠为电子供体,10 mmol/L水合氧化铁为电子受体,[HCO_3~–]为30 mmol/L,[PO_4~(3–)]为5 mmol/L条件下,30°C恒温下厌氧培养,进行细菌生长和细胞外诱导矿化实验,定期采样测量反应体系的pH、生物量、Fe(Ⅱ)浓度;采用激光拉曼光谱(Raman)、扫描电子显微镜(SEM)、透射电子显微镜(TEM)和X-射线衍射(XRD)等方法对不同时间点的矿化产物进行分析。【结果】MR-4在还原Fe(Ⅲ)的过程中,细胞快速生长,表明MR-4的Fe(Ⅲ)还原和乳酸氧化过程相互耦合,从而进行细胞生长,并在细胞外诱导矿物形成。对不同阶段矿化产物的综合分析表明,反应进行到约8 d时,无定形-弱结晶的水合氧化铁部分地转化为纳米尺寸的磁铁矿晶体颗粒;约16 d时,反应体系中开始出现蓝铁矿晶体颗粒;约20 d后,几乎所有矿物转化为纤维状或者叶片状的蓝铁矿。【结论】铁还原细菌Shewanella oneidensis MR-4细胞外诱导矿化过程受环境条件控制,当以乳酸钠和水合氧化铁分别作为电子供体和受体,相对高的[PO_4~(3–]/[HCO_3~–](1:6)时,水合氧化铁先转化为磁铁矿,最后大量转化为蓝铁矿。本研究为全面认识铁还原细菌的生物诱导矿化过程和评估其参与铁元素地球化学循环提供了新的数据。     

培养条件对Shewanella oneidensis MR-1电极生物膜及细胞形貌的影响

【目的】Shewanella oneidensis MR-1是电活性模式微生物,但目前仍缺乏对其细胞及生物膜形貌变化的系统研究,本研究旨在完善对其形貌特征的理解,为支持其作为模式微生物提供有力的基础数据。【方法】选取培养基类型、缓冲液浓度、维生素、微量元素、无机盐、电子穿梭体、电子供体、电子受体等培养条件作为变量,采用恒电位培养法获得生物膜,通过扫描电子显微镜对生物膜形貌进行观察。【结果】低浓度缓冲液中(30 mmol/L和100 mmol/L),其细胞多为短杆状,高浓度缓冲液中(200 mmol/L和300 mmol/L)细胞卷曲伸长;缺乏维生素、微量元素、无机盐则可使生物膜紧贴电极生长,变得致密;而穿梭体和电子受体对于S. oneidensis MR-1极为关键,前者的存在可显著促进生物膜的厚度,后者的缺失可迫使生物膜细胞裂解;此外,通过形貌研究发现,S. oneidensis MR-1可首尾相连形成超过100μm的长线状结构。【结论】可通过改变缓冲液浓度、培养基类型、电子穿梭体和电子供受体等变量,实现Shewanella oneidensis MR-1电极生物膜及细胞形貌的调控。     

金属还原菌希瓦氏菌厌氧呼吸能力及其在环境修复中的研究进展

Shewanella oneidensis MR-1是一种模式金属还原菌,它能够在厌氧条件下,将多种金属化合物和人工合成染料等作为电子受体还原代谢。因此,该菌常常被用于生态修复等研究。厌氧条件下,S.oneidensis MR-1能够将细胞质内或细胞内膜产生的电子通过定位于细胞内膜、细胞膜周质和细胞外膜上的c-血红色素蛋白或还原酶所组成的具有多样性的电子传递系统,最终传递到存在于细菌细胞外环境中的电子受体。通过对多种电子传递过程的介绍,进一步阐明其对污染物修复和纳米材料合成的机理,从而为未来对该类微生物的利用和开发提供更为充分的理论依据。     

一株林可霉素降解菌的筛选鉴定及其降解机制

【背景】抗生素污染越来越引起人们的关注。利用微生物处理抗生素污染被认为是一种环境友好型的方法。【目的】筛选林可霉素高效降解菌并研究其降解机制。【方法】经形态学观察、生理生化鉴定和16S rRNA基因测序分析进行鉴定;通过PCR技术和质谱分析技术对该菌抗性基因和降解产物等进行分析。【结果】从林可霉素菌渣堆肥样本中获得一株高效降解林可霉素的假单胞菌(Pseudomonas RST-1),该菌在林可霉素浓度为3.0 g/L的牛肉膏蛋白胨培养基上培养40 h后,林可霉素降解率高达57.3%。该菌含有intI1、sul1、sul2等抗性基因,降解产物为去甲基林可霉素和2-丙基-N-甲基脯氨酸。【结论】菌株RST-1具有高效降解林可霉素的能力,推测可能的降解机制为去甲基化和酰胺键水解作用,该菌株降解特性及降解机制研究为林可霉素降解工程菌及其高效降解菌剂的研制奠定了基础。     

典型胞外呼吸细菌的胞内电子转移机制研究进展

胞外呼吸在污染物的降解转化和微生物产电过程中具有重要作用。微生物进行胞外呼吸时,其电子受体多以固态形式存在于胞外,氧化产生的电子必须通过电子传递链从胞内经细胞周质转移到外膜。S.oneidensis MR-1与G.Sulfurreducens作为微生物燃料电池中最常用的模式菌株,是现阶段研究最深入和系统的胞外呼吸细菌,其胞内电子传递过程目前研究最为清楚。这两种胞外呼吸细菌的电子传递需多种细胞色素c的参与,S.oneidensis MR-1位于内膜及周质上的细胞色素c-Cym A和MtrA可将电子由内膜上的醌池通过周质到外膜蛋白MtrC和OmcA,MtrC和OmcA接收电子后可直接还原胞外受体,Type Ⅱ secretion system对外膜蛋白中的MtrC和OmcA起到了转运及定位的作用。而在G.sulfurreducens中,电子由MacA传递到PpcA,最终由外膜蛋白OmcB、OmcE、OmcS及OmcZ接受电子,并在Type Ⅳ pili的共同作用下将电子传递到胞外电子受体。本文最后指出目前对Shewanella与Geobacter胞内电子转移研究尚不清楚的地方提出展望。     

一株十溴联苯醚高效好氧降解菌的筛选、鉴定及降解特性

【目的】从广东贵屿镇电子垃圾拆解地采集的沉积物样品中分离十溴联苯醚(BDE-209)高效好氧降解菌,并考察其对BDE-209的降解特性。【方法】通过生理生化实验和16S rRNA测序鉴定菌种,正交实验优化降解条件,并分析不同降解体系及影响因素对菌降解BDE-209的影响。【结果】鉴定结果显示,该BDE-209好氧降解菌为短短芽孢杆菌(Brevibacillus brevis)。B.brevis对1 mg/L BDE-209 5 d的降解率可达54.38%。正交实验结果表明,B.brevis降解BDE-209的最优条件为:pH 7,投菌量3 g/L,温度30°C。降解特性研究结果显示B.brevis对BDE-209降解的最佳菌龄为36 h,最佳氮源为(NH4)2SO4,B.brevis对Cu2+、Cd2+有较好的耐受性,但Cu2+和Cd2+的存在会影响其对BDE-209的降解。当Cu2+浓度在1 5 mg/L,Cd2+浓度在0.3 0.5 mg/L范围内时,B.brevis对BDE-209降解均可达50%以上。【结论】B.brevis对BDE-209有很好的降解效率,研究结果对BDE-209的好氧微生物降解及环境中BDE-209的生物修复具有较好的科学意义和应用价值。     

白腐菌SQ01锰过氧化物酶对联苯中间代谢物的转化北大核心CSCD

【目的】在对白腐菌栓菌(Trametes sp.)SQ01锰过氧化物酶(MnP)纯化的基础上,通过MnP对HOPDAs的转化实验,了解白腐菌MnP对2-羟基-6-氧-6-苯基-2,4-己二烯酸(HOPDA)及其衍生物的作用,揭示MnP新的催化特性。【方法】利用紫外可见光谱法分析锰过氧化物酶对10种不同取代基的HOPDAs转化情况,并对锰过氧化物酶的稳态动力学参数进行了测定;红外光谱法分析了HOPDA及其产物的分子结构。【结果】锰过氧化物酶可以转化HOPDA及其卤代HOPDAs,特别是锰过氧化物酶可以催化3,8,11-3Cl HOPDA,而这一物质几乎不能被联苯水解酶(2-羟基-6-氧-6-苯基-2,4-己二烯酸水解酶)和红球菌(Rhodococcus sp.)R04转化。稳态动力学分析表明,在5种HOPDAs中,HOPDA是锰过氧化物酶的最适底物,3,10-2F HOPDA的转化效率(k_(cat)/K_m)是最高的。紫外可见光谱分析表明,锰过氧化物酶在转化HOPDA及其衍生物时最大吸收峰在可见光区均会发生蓝移。红外分析表明,锰过氧化物酶可以使HOPDA的共轭双烯转化为单烯,C_β上的羟基消失。【结论】锰过氧化物酶能够有效降解HOPDA及其衍生物,这为联苯及其中间代谢物的顺利降解提供了新的策略。     

厌氧微生物菌群XH-1对2,4,6-三氯苯酚的降解特性

有机污染物2,4,6-三氯苯酚(2,4,6-TCP)普遍存在于地下水和河流底泥等厌氧环境中。为了探究厌氧微生物菌群XH-1对2,4,6-TCP的降解能力,本研究以2,4,6-TCP为底物,接种XH-1建立微宇宙培养体系,并以中间产物4-氯苯酚(4-CP)和苯酚为底物分别进行分段富集培养,利用高效液相色谱分析底物的降解转化,同时基于16S rRNA基因高通量测序分析微生物群落结构变化。结果表明: 2,4,6-TCP(122 μmol·L^-1)以0.15 μmol·d^-1的速率在80 d内被完全降解转化,降解中间产物分别为2,4-二氯苯酚(2,4-DCP)、4-氯苯酚和苯酚,所有中间产物最终在325 d被完全降解。高通量测序结果表明,脱卤杆菌和脱卤球菌可能驱动2,4,6-TCP还原脱氯,其中,脱卤球菌可能在4-CP的脱氯转化中发挥重要作用,并与丁酸互营菌和产甲烷菌联合作用彻底降解2,4,6-TCP。     

Identification of Nitroso Compounds from Biotransformation of 2,4-Dinitrotoluene

The intermediates of microbial transformation of 2,4-dinitrotoluene by a mixed bacterial culture derived from activated sludge were identified as 2-amino-4-nitrotoluene, 4-amino-2-nitrotoluene, 2-nitroso-4-nitrotoluene, and 4-nitroso-2-nitrotoluene. The biotransformation of 2,4-dinitrotoluene occurred only under anaerobic conditions with an exogenous carbon source. The two nitroso compounds were unstable and could be observed only at the early stage of 2,4-dinitrotoluene anaerobic biotransformation.     

TNT and nitroaromatic compounds are chemoattractants for Burkholderia cepacia R34 and Burkholderia sp. strain DNT

Nitroaromatic compounds are toxic and potential carcinogens. In this study, a drop assay was used to detect chemotaxis toward nitroaromatic compounds for wild-type Burkholderia cepacia R34, wild-type Burkholderia sp. strain DNT, and a 2,4-dinitrotoluene (2,4-DNT) dioxygenase mutant strain (S5). The three strains are chemotactic toward 2,4,6-trinitrotoluene (TNT), 2,3-DNT, 2,4-DNT, 2,5-DNT, 2-nitrotoluene (NT), 4NT, and 4-methyl-5-nitrocatechol (4M5NC), but not toward 2,6-DNT. Of these, only 2,4-DNT is a carbon and energy source for B. cepacia R34 and Burkholderia sp. strain DNT, and 4M5NC is an intermediate in the 2,4-DNT degradation pathway. It was determined that the 2,4-DNT dioxygenase genes are not required for the chemotaxis for these nitroaromatic compounds because the DNT DDO mutant S5 has a chemotactic response toward 2,4-DNT although 2,4-DNT is not metabolized by S5; hence, 2,4-DNT itself is the chemoattractant. This is the first report of chemotaxis toward TNT, 2,3-DNT, 2,4-DNT, 2,5-DNT, 2NT, 4NT, and 4M5NC.     

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<2,2′-dimethyl-5,5′-dinitroazoxybenzene (2,2′-DM-5,5′-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB), and aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<4HA2NT4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2,6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2′-dimethyl-3,3′-dinitroazoxybenzene (2,2′-DM-3,3′-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.     

Oxidation of aminonitrotoluenes by 2,4-DNT dioxygenase of Burkholderia sp. strain DNT

Aminonitrotoluenes form rapidly from the reduction of dinitrotoluenes (DNTs) which are priority pollutants and animal carcinogens. For example, 4-amino-2-nitrotoluene (4A2NT) and 2A4NT accumulate from the reduction of 2,4-DNT during its aerobic biodegradation. Here, we show that 2,4-DNT dioxygenase (DDO) from Burkholderia sp. strain DNT oxidizes the aminonitrotoluenes 2A3NT, 2A6NT, 4A3NT, and 5A2NT to 2-amino-3-nitrobenzylalcohol, 2-amino-4-nitro-m-cresol and 3-amino-5-nitro-p-cresol, 4-amino-3-nitrobenzylalcohol and aminonitrocresol, and 2-amino-5-nitro-o-cresol, respectively. 2A5NT and 3A4NT are oxidized to aminonitrocresols and/or aminonitrobenzylalcohols, and 4A2NT is oxidized to aminonitrocresol. Only 2A4NT, a reduced compound derived from 2,4-DNT, was not oxidized by DDO or its three variants. The alpha subunit mutation I204Y resulted in two to fourfold faster oxidization of the aminonitrotoluenes. Though these enzymes are dioxygenases, they acted like monooxygenases by adding a single hydroxyl group, which did not result in the release of nitrite.     

Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain

Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored.     

Degradation of 2,4,6-trinitrotoluene (TNT) by immobilized microorganism-biological filter

The combined process of immobilized microorganism-biological filter was used to degrade TNT in an aqueous solution. The results showed that the process could effectively degrade TNT, which was not detected in the effluent of the system. GC/MS analysis identified 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT), 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT), 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) and 2,4-diamino-6-nitrotoluene (2,6-DA-4-NT) as the main anaerobic degradation products. In addition, the Haldane model successfully described the anaerobic degradation of TNT with high correlation coefficients (R² = 0.9803). As the electron donor, ethanol played a major role in the TNT biodegradation. More than twice the theoretical requirement of ethanol was necessary to achieve a high TNT degradation rate (above 97.5%). Moreover, Environment Scan Electron Microscope (ESEM) analysis revealed that a large number of globular microorganisms were successfully immobilized on the surface of the carrier. Further analysis by Polymerase Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis (DGGE) demonstrated that the special bacterial for TNT degradation may have generated during the domestication with TNT for 150 days. The dominant species for TNT degradation were identified by comparing gene sequences with Genebank.     

Bioremediation Potential of Rhodococcus pyridinivorans NT2 in Nitrotoluene-Contaminated Soils: The Effectiveness of Natural Attenuation,Biostimulation and Bioaugmentation Approaches

This work evaluated the effect of bioremediation treatments including natural attenuation, bioaugmentation, biostimulation as well as combined biostimulation and bioaugmentation on degradation of 4-nitrotoluene (4-NT), 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) in soil microcosms. Bioaugmentation with a previously isolated NTs-degrading bacterium, Rhodococcus pyridinivorans NT2, showed an 86–88% decrease in 4-NT, 2,4-DNT or 2,6-DNT after 60 days. Irrespective of the substrate types, least degradation (6–6.5%) was observed in abiotic control. The addition of β-cyclodextrin or rhamnolipid significantly improved NTs degradation efficiency in soil (18.5–74%) than natural attenuation (22–25%). Exogenous addition of preselected bacterial isolate NT2 along with β-cyclodextrin/rhamnolipid resulted in the greatest number (1.8× and 2.5× high) of total heterotrophic aerobic bacteria and NT degraders, respectively, compared to natural attenuation. Irrespective of the treatment types, the population of NT degraders increased steadily in the first 5 weeks of incubation followed by a plateau within the next few weeks. The treatment BABS2 (Soil + rhamnolipid + NT2) yielded highest microbial-C and -N and dehydrogenase activity, consistent with results of NTs degradation and microbial counts in combined bioaugmentation and biostimulation. Thus the results of this study suggest that bioaugmentation by R. pyridinivorans NT2 may be a promising bioremediation strategy for nitroaromatics-contaminated soils.     

Biotransformation of 2,4,6-trinitrotoluene (TNT) by the fungus Fusarium oxysporum

The fungus Fusarium oxysporum was isolated and identified from the aquatic plant M. aquaticum. The capability of this fungus to transform 2,4,6-trinitrotoluene (TNT) in liquid cultures was investigated TNT was added to shake flask cultures and transformed into 2-amino-4,6-dinitrotoluene (2-A-DNT), 4-amino-2,6-dinitrotoluene (4-A-DNT), and 2,4-diamino-6-nitrotoluene (2,4-DAT) via 2- and 4-hydroxylamino-dinitrotoluene derivatives, which could be detected as intermediate metabolites. Transformation of TNT, 2-A-DNT, and 4-A-DNT was observed by whole cultures and with isolated mycelium. Cell-free protein extracts from the extracellular, soluble, and membrane-bound fractions were prepared from this fungus and tested for TNT-reducing activity. The concentrated extracellular culture medium was unable to transform TNT; however, low levels of TNT transformation were observed by the membrane fraction in the presence of nicotinamide adenine dinucleotide phosphate in an argon atmosphere. A concentrated extract of soluble enzymes also transformed TNT, but to a lesser extent. When TNT toxicity was studied with this fungus, a 50% decrease in the growth of F. oxysporum mycelium was observed when exposed to 20 mg/L TNT.     

Rapid separation of reduction products of 2,4,6-trinitrotoluene using TLC

Silica gel TLC methods were developed for the separation of 2,4,6-trinitrotoluene (TNT) in mixtures with possible reduction products. The methods employed repeated elutions with simple binary or ternary solvent systems in either one or two dimensional modes. The resolved analytes include TNT, selected amino derivatives (2-amino-4,6-di-nitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene) and known hydroxylamino derivatives (2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene and 2,4-dihydroxylamino-6-nitrotoluene).