乳白耙齿菌F17好氧降解四溴双酚A的特性及机理研究

[目的] 为了探究乳白耙齿菌F17（Irpex lacteus F17）降解溴代阻燃剂的可能性,研究了该菌好氧降解四溴双酚A （Tetrabromobisphenol A,TBBPA）的特性以及影响降解的因素,并结合降解产物的分析,推测其降解途径。[方法] 采用高效液相色谱法测定TBBPA的浓度,并通过气相色谱-质谱联用仪分析降解过程的中间产物。[结果] I.lacteus F17可以通过共代谢的方式好氧降解TBBPA,最适共代谢基质是葡萄糖。在葡萄糖浓度为8 g/L、菌悬液接种量为5%、pH 5.0的优化条件下,当TBBPA初始浓度为20 mg/L时降解率可达85.5%,脱溴率为14.6%。对降解过程中锰过氧化物酶的研究发现TBBPA的降解率受到该酶活性的影响。通过气相色谱-质谱联用仪检测到7种中间产物。[结论] I.lacteus F17可以有效降解四溴双酚A,其降解机理主要包括脱溴、β-断裂、羟基化、去质子和氧化等过程。     

乳白耙齿菌F17对单一和复合多环芳烃的降解差异解析

[目的] 针对菲、蒽、荧蒽多环芳烃（PAHs）污染物,利用乳白耙齿菌F17,研究单一和复合PAHs污染物的生物降解规律。[方法] 采用气相色谱-质谱法（GC-MS）分析降解过程中PAHs的浓度,并采用准一级反应动力学模型对降解结果进行拟合。[结果] 对于单一PAHs,第15天时菲、蒽、荧蒽的降解率由高到低依次为菲（97.8%） > 蒽（89.3%） > 荧蒽（81.5%）。菲、蒽和荧蒽的降解过程具有准一级反应动力学特征,菲的生物降解速率最快,其次是蒽,荧蒽的降解速率最慢。与单一PAHs的降解相比,在复合PAHs的降解过程中,乳白耙齿菌F17的生长和锰过氧化物酶的合成均表现出不同的特征。此外,水溶性极可能是复合污染物降解的重要控制因子,三者水溶性为：菲 > 荧蒽 > 蒽。因此,在菲或荧蒽加入条件下,微生物能优先降解这些污染物,抑制了污染物蒽的降解;同时,蒽或菲的存在对荧蒽的降解也有抑制作用;然而外源加入水溶性较差的蒽和荧蒽,则对菲的生物降解无显著影响。[结论] 复合PAHs的生物降解主要表现为相互竞争的特点,通过GC-MS分析了PAHs的生物降解途径。     

固定化乳白耙齿菌脱色茜素红的研究

利用海藻酸钙法对乳白耙齿菌进行固定化,检测固定化乳白耙齿菌（固定化菌）对几种染料的脱色能力。同时,考察pH值、染料浓度、金属离子、碳源种类、氮源种类、盐浓度对固定化菌脱色茜素红的影响。结果表明,固定化菌的优化条件为海藻酸钠3%、氯化钙5%、固定化时间6h、接菌量10g/100mL;固定化菌对6种染料均可脱色,其中对茜素红染料的脱色效果最为明显;固定化菌对茜素红的脱色率随染料浓度的增加而下降,当染料浓度高于250mg/L时,其脱色效果明显下降;固定化菌对茜素红脱色的适宜pH为7,适宜碳源为可溶性淀粉、适宜氮源为硝酸铵。另外,固定化菌对茜素红的脱色率随盐浓度的升高,呈下降趋势,当盐浓度高于3%时,脱色率下降明显;固定化菌于生理盐水中保存10d后,脱色率维持在较高水平,达94.20%;固定化菌重复利用5次后,脱色率仍高达88.70%。     

裂褶菌F17对刚果红的脱色及主要降解酶的研究

裂褶菌F17在限氮培养基中对偶氮染料刚果红表现出较高的脱色能力。最佳脱色条件为:温度28℃、初始pH值4.5以及菌体摇瓶培养3d加入染料,染料浓度以100mg/L为宜。诱导因子藜芦醇、吐温80、土豆汁和松木屑的加入明显提高了脱色率,而叠氮化钠和氰化钾的加入对脱色有显著的抑制作用。酶活检测表明,裂褶菌F17在刚果红脱色过程中主要产生MnP,LiP活力很小,未检测到Lac。此外,刚果红的脱色率与累积MnP酶活具有良好的线性关系,相关系数为0.973。推测裂褶菌F17对刚果红脱色的主要降解酶为MnP。     

一株蒽降解细菌的分离及降解特性研究

【目的】从盐碱土壤中筛选蒽降解菌株并分析其降解特性。【方法】采用极度稀释结果流式细胞检测法筛选分离纯化菌株,通过16S rRNA基因序列分析对菌株进行初步鉴定,采用气质联用仪(GC-MS)分析蒽的降解特性。【结果】从盐碱土壤中筛选出一株高效蒽降解菌株。经过16S rRNA基因序列分析,鉴定该菌株为Demequina salsinemorus BJ1。菌株可以利用蒽作为唯一碳源生长,降解率可达92%。在一定浓度范围内,随着蒽浓度的降低,细菌生长速率变快,降解率升高。添加外加碳源后,细菌生长速率明显变快,而对蒽降解率变低。对萃取中间代谢产物的质谱分析表明,降解蒽的中间代谢产物主要有9,10-anthracenedione (9,10-蒽醌)和Phthalic acid (邻苯二甲酸)等,说明它可能通过邻苯二甲酸途径降解蒽。【结论】筛选得到一株新的耐盐碱蒽降解菌,该菌降解效率高,对修复石油污染的土壤有一定的现实意义。     

一株白囊耙齿菌对刚果红染料的脱色研究及其毒力测试

菌株2-1-1于贵州大学校园樱花树干子实体中分离,通过形态学结构、ITS序列及系统发育树分析,鉴定其为白囊耙齿菌Irpex lacteus,对菌株2-1-1产生的3种木质素降解酶进行测定,发现菌株可分泌漆酶(laccase,Lac)、木质素过氧化物酶(lignin peroxidase,LiP)和锰过氧化物酶(manganese peroxidase, MnP)。利用菌株2-1-1对固体培养基条件下7种染料的脱色能力进行检测,筛选出较易脱色的刚果红染料,同时采用气相色谱-质谱联用(gas chromatography-mass spectroscopy,GC-MS)分析确定经白囊耙齿菌降解的刚果红染料代谢产物,并对其进行脱色条件优化和毒力测验,试验分析表明：菌株2-1-1对7种染料均有脱色,对刚果红脱色效果较佳。GC-MS分析刚果红染料降解产物主要为联苯胺、联苯、苯乙烯、十六烷和3-硝基苯酚。单因素和综合优化脱色条件：染料浓度50mg/L,碳源为果糖、金属离子为锌离子、pH7,该条件下刚果红染料脱色效果最优。优化条件下菌株2-1-1对刚果红染料脱色率达91.03%,相比对照组脱色率提高...     

微生物降解菲的机理研究进展*

针对微生物降解菲的机理研究进展,论述了细菌、真菌在好氧、厌氧条件下代谢菲的产物以及推测的降解途径;在此基础上概括了催化反应的酶系以及编码酶系的基因簇。简要介绍了基因探针的应用,并结合本实验室的初步研究,指出了该领域有待深入探讨的问题。     

复合菌对十溴联苯醚的降解特性研究

【目的】利用6株十溴联苯醚(decabromodiphenyl ether, BDE-209)降解细菌,探究复合菌对BDE-209的降解特性和降解路径,为BDE-209污染环境的生物修复提供科学依据。【方法】利用高效液相色谱法测定BDE-209的浓度,通过液相色谱-质谱联用仪分析鉴定BDE-209降解产物。【结果】短芽孢杆菌属(Achromobacter sp.) M1和无色杆菌属(Achromobacter sp.) M2的组合对BDE-209的降解效果最好,在30 ℃、pH值7.0、接种量15%的条件下,120 h后10 mg/L BDE-209的降解效率可达87.7%。相比于单一菌株,复合菌M(1+2)可以更有效、更快地降解BDE-209。在0.5-10 mg/L范围内,复合菌M(1+2)对BDE-209的降解率随着BDE-209初始浓度的增大而增大。通过液相色谱-质谱联用仪(liquid chromatograph-mass spectrometer, LC-MS)检测到11种BDE-209微生物降解产物,复合菌M(1+2)通过脱溴、羟基化、去质子化、醚键断裂和开环等反应对BDE-209进行降解。【结论】复合菌M(1+2)对BDE-209具有良好的降解能力,研究结果为进一步提高微生物对BDE-209污染环境的生物修复能力提供了良好的微生物资源。     

一株高效菲降解菌的筛选及降解条件研究

从南京某石化厂排污口附近采集土样,以菲为碳源的选择性培养基分离筛选到一株菲高效降解菌F10a,根据形态和生理生化特性初步鉴定为芽孢杆菌属,并对其降解菲的特性及各种影响因素进行了研究.结果表明,F10a在50 mg·L^-1的条件下,28 ℃振荡培养27 h,菲的降解率达到98.12%;静置培养8 h,菲的降解率达到98.7%.pH值分别为、6、8时,F10a对菲具有良好的降解效能;pH值为10时F10a不生长.Zn²⁺与Pb²⁺的存在不影响F10a的降解效能,Cu²⁺可以延缓菲的降解,Cr²⁺对F10a有毒性.F10a在菲浓度为200 mg·L^-1时,28 ℃振荡培养8 h,降解率为99.6%.菲的降解程度与细菌数量的增长呈正相关关系.     

海洋石油降解菌AH07的分离鉴定及其石油降解性能分析

以原油为唯一碳源,从大连新港石油污染区域海底沉积物中分离获得1株石油高效降解菌AH07。通过形态学观察、生理生化特征检验及16S rDNA序列分析,确定菌株AH07为人苍白杆菌(Ochrobactrum anthropi),GenBank序列登录号为KT831449。通过单因素试验确定了菌株AH07的最优生长条件,即培养温度为30℃,培养基pH 7.0。为了进一步了解并提高菌株AH07的降解性能,选用5种氮源考察不同氮源对菌株石油降解性能的影响。结果表明,玉米粉为最佳有机氮源,NH_4NO_3为最佳无机氮源;28℃、150 r/min振荡培养10 d,菌株AH07对原油的降解率分别为58.25%和31.98%。     

P-Postlabeling high-performance liquid chromatographic improvements to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene, and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene

Single compounds can generate complex DNA adduct patterns by reactions through different pathways, with different target nucleotides and through different configurations of the products. DNA adduct analysis by ³²P-HPLC was improved by adding an isocratic plateau in an otherwise linear gradient, thereby enhancing resolution of predictable retention time intervals. This enhanced ³²P-HPLC technique was used to analyze and at least partly resolve 14 out of 16 available benzo[c]phenanthrene deoxyadenosine and deoxyguanosine adduct standards, 8 out of 8 available benzo[a]pyrene deoxyadenosine and deoxyguanosine adduct standards, and 51 peaks from 7,12-dimethylbenz[a]anthracene-calf thymus DNA reaction products. The same type of gradient modifications could be used to enhance resolution in analyses of other complex DNA adduct mixtures, e.g., in vivo in humans.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Transformation of 2,4,6-trinitrotoluene by white rot fungus Irpex lacteus

Up to 200 mg 2,4,6-trinitrotoluene (TNT) l^–1 was removed within 12 h after adding it to a 5-day old culture of Irpex lacteus. The initial formation of hydroxylamino-dinitrotoluenes (2- and 4-OHAmDNT) from TNT was detected, followed by their successive transformation to aminodinitrotoluenes (2- and 4-AmDNT). Transformation of TNT to AmDNT via OHAmDNT was fast, but the next step was slow and seemed to be a rate-limiting step in TNT degradation. OHAmDNT isomers were also rapidly transformed by an in vitro enzymatic system. Both the mycelium and extracellular enzymes of I. lacteus were required for the TNT degradation.     

一株尼古丁降解菌株Arthrobacter sp.D4的分离鉴定及其降解代谢途径

【背景】烟草在生产和加工中会产生高浓度的尼古丁废弃物,对环境造成较大的污染。【目的】筛选降解尼古丁的微生物菌种并解析其降解尼古丁的代谢途径,理解微生物如何降解尼古丁。【方法】用常规分离筛选方法、结合形态学观察和分子鉴定手段分离和鉴定菌株类别,进而利用单因素试验方法,通过设置不同的尼古丁浓度、温度和pH确定菌株降解尼古丁的最适发酵条件和降解率,利用气相色谱-质谱联用技术检测菌株在尼古丁降解过程中的主要代谢产物。【结果】获得一株以尼古丁为唯一碳源和氮源的节杆菌属(Arthrobacter)菌株,编号为D4;该菌株降解尼古丁的最适温度和pH分别为30.0℃和7.0;在1 g/L的尼古丁浓度下具备较快的尼古丁降解速率,培养18 h时尼古丁降解率可达到90%以上;尼古丁浓度≥4 g/L时菌株生长受到明显抑制;与目前报道的节杆菌属降解途径不同,该菌株降解尼古丁过程中产生了新的终产物N-甲基吡咯烷酮、可替宁及中间产物麦斯明。【结论】本研究分离鉴定到一株具有较快尼古丁降解速率的节杆菌,该菌株很可能存在新的尼古丁降解途径。     

高粱SbSBP17基因的克隆及表达分析

SBP box(Squamosa promoter Binding Protein box)蛋白是绿色植物中特有的一类转录因子,其功能涉及植物叶片发育、胚胎发生、间隔期长度、营养到生殖生长的更替、育性维持等生长发育的重要过程。该研究以高粱材料BTx623花序总RNA为模板进行RT PCR,克隆了高粱SbSBP17基因,并对其进行了系统进化及半定量PCR表达分析,为进一步研究SbSBP17基因的生物学功能奠定基础。结果表明：SbSBP17含有2个内含子和3个外显子,编码1个444氨基酸的蛋白质,在其195~269 aa区域含有一个典型的SBP box结构域;系统进化分析表明,18个SBP17蛋白可分为三组,第Ⅰ和Ⅱ组只有单子叶植物基因,而第Ⅲ组只有双子叶植物基因,SbSBP17与玉米PWZ17260.1亲缘关系最近;启动子顺式作用元件分析显示,SbSBP17启动子含有细胞周期调控元件MSA like、分生组织发育调控相关元件CAT box和组织特异性表达元件RY element等。基因表达分析显示,SbSBP17是花序特异性表达基因,随着花序发育(长度增加),SbSBP17基因表达量逐渐增高,当花序长至10 cm时基因表达量达到峰值,约是花序长度最小点(≤2 cm)时的40倍,随后表达量逐渐降低,并于花序长度20 cm时显著降低50%以上。研究推测,随着高粱花序进一步发育,SbSBP17基因的表达量可能逐渐降低,最终消失。     

Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505

Catabolic pathways for utilization of naphthalene (NAP), anthracene (ANT), phenanthrene (PHE), and fluoranthene (FLA) by Sphingomonas paucimobilis EPA505 were identified. Accumulation of catabolic intermediates was investigated with three classes of Tn5 mutants with the following polycyclic aromatic hydrocarbon (PAH)-negative phenotypes; (class I NAP(-) PHE(-) FLA(-), class II NAP(-) PHE(-), and class III FLA(-)). Class I mutant 200pbhA had a Tn5 insertion within a meta ring fission dioxygenase (pbhA), and a ferredoxin subunit gene (pbhB) resided directly downstream. Mutant 200pbhA and other class I mutants lost the ability to catalyze the initial dihydroxylation step and did not transform NAP, ANT, PHE, or FLA. Class I mutant 401 accumulated salicylic acid, 2-hydroxy-3-naphthoic acid, 1-hydroxy-2-naphthoic acid, and hydroxyacenaphthoic acid during incubation with NAP, ANT, PHE, or FLA, respectively. Class II mutant 132pbhC contained the Tn5 insertion in an aldolase hydratase (pbhC) and accumulated what appeared to be meta ring fission products: trans-o-hydroxybenzylidene pyruvate, trans-o-hydroxynaphylidene pyruvate, and trans-o-hydroxynaphthyl-oxobutenoic acid when incubated with NAP, ANT, and PHE, respectively. When mutant 132pbhC was incubated with 1-hydroxy-2-naphthoic acid, it accumulated trans-o-hydroxybenzylidene pyruvate. Class III mutant 104ppdk had a Tn5 insertion in a pyruvate phosphate dikinase gene that affected expression of a FLA-specific gene and accumulated a proposed meta ring fission product; trans-o-hydroxyacenaphyl-oxobutenoic acid during incubation with FLA. Trans-o-hydroxyacenaphyl-oxobutenoic acid was degraded to acenaphthenone that accumulated with class III mutant 611. Acenaphthenone was oxidized via incorporation of one molecule of dioxygen by another oxygenase. 2,3-Dihydroxybenzoic acid was the final FLA-derived catabolic intermediate detected. Analysis of PAH utilization mutants revealed that there are convergent and divergent points involved in NAP, ANT, PHE, and FLA utilization by S. paucimobilis EPA505.     

白刺沙堆退化与土壤水分的关系

近几十年来,我国西北干旱区白刺沙堆退化严重,导致固定沙丘活化,流沙掩埋绿洲,造成了严重危害。如何尽可能长期保持白刺沙堆的稳定、防止白刺沙堆活化成为绿洲保护和沙漠化防治急需解决的问题。在多年野外观察的基础上,提出了"土壤水分收支不平衡所导致的土壤水分减少是白刺沙堆退化的主要原因"的研究假设。但是,由于缺少长期的野外观测试验,这个假设一直未被很好地证明。为了证明这个假设,在甘肃民勤的绿洲外围选择了雏形、发育、稳定和死亡四个退化阶段的白刺沙堆,于2008年1月至2012年6月利用中子水分仪和土壤烘干称重法对土壤水分进行了长期观测。结果表明:各样地的土壤含水量均呈现出2008年最大,2009年和2011年次之,2010年最小的趋势。年内变化是春季土壤含水量最低,夏季逐渐增加,随后逐渐减小。在不同发育阶段,雏形阶段的土壤含水量最大,且降水容易下渗。稳定和死亡阶段的白刺沙堆土壤含水量很低,降水难以下渗,只有大的降水事件发生时,水分才可以下渗。因此,稳定和死亡阶段白刺沙堆的土壤水分经常在植物的凋萎点之下,是造成白刺沙堆退化重要原因。证明了"土壤水分减少是白刺沙堆退化的原因"的研究假设。研究结果对今后的植物固沙实践活动会有积极的参考意义。