一株高效降解芘的细菌分离、鉴定及其降解效果

摘要：【目的】获得高效降解高分子量多环芳烃的细菌,并研究其对多环芳烃的降解能力。【方法】利用富集培养和芘升华平板方法,从焦化厂污染土壤中分离多环芳烃降解细菌,对分离菌株通过形态特征、16S rRNA基因和gyrb基因序列相似性分析进行鉴定,并研究该菌对高分子量多环芳烃（HMW-PAHs）的降解效果。【结果】筛选到一株能以芘、苯并蒽、屈、苯并芘、茚并芘、苯并苝、荧恩为碳源和能源生长并降解这些底物的菌株HBS1,该菌株的16S rRNA基因和gyrb基因序列与Gordonia amicalis的相应基因的相似     

多环芳烃降解菌的筛选、鉴定及降解特性

【目的】多环芳烃(PAHs)是一类普遍存在于环境中且具有高毒性的持久性有机污染物,高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。研究拟从供试菌株中筛选多环芳烃高效降解菌,并分析其降解特性,为多环芳烃污染环境的微生物修复提供资源保障和科学依据。【方法】采用平板法从25株供试菌株中筛选出以菲和芘为唯一碳源和能源的高效降解菌,经16S rRNA基因序列进行初步鉴定,通过单因素实验法分析其在液体培养基中的降解特性。【结果】筛选出的3株多环芳烃高效降解菌SL-1、02173和02830经16S rRNA基因序列分析,02173和02830分别与假单胞菌属中的Pseudomonas alcaliphila和Pseudomonas corrugate同源性最近,SL-1为本课题组发表新类群Rhizobium petrolearium的模式菌株;降解实验表明,菌株SL-1 3 d内对单一多环芳烃菲(100 mg/L)和芘(50 mg/L)的降解率分别达到100%和48%,5 d后能够降解74%的芘;而其3 d内对混合PAHs中菲和芘的降解率分别为75.89%和81.98%。菌株02173和02830 3 d内对混合多环芳烃中萘(200 mg/L)、芴(50 mg/L)、菲(100 mg/L)和芘(50 mg/L)的降解率均分别超过97%。【结论】筛选出的3株PAHs降解菌SL-1、02173和02830不仅可以高效降解低分子量PAHs,还对高分子量PAHs具有很好的降解潜力。研究表明,由于共代谢作用低分子量多环芳烃可促进高分子量多环芳烃的降解,而此时低分子量多环芳烃的降解将受到抑制。     

一株可同时降解多种高环PAHs的丝状真菌——宛氏拟青霉(Paecilomyces variotii)

采用液体培养的方法,分离纯化了丝状真菌宛氏拟青霉(Paecilomyces variotii)并研究了其对苯并[a]蒽、苯并[a]芘、苯并[b]荧蒽、苯并[k]荧蒽、茚并[1,2,3-cd]芘的降解效果.结果表明:接种处理30 d,该菌株对混合体系中5种PAHs的降解率为16.1%～24.6%,而对单一体系中的降解率为10.4%～33.3%;同时,对单一与混合体系中PAHs的降解作用存在一定差异,苯并[k]荧蒽和苯并[b]荧蒽在单一体系中降解率增大,而其他种类的则减小.本研究结果为高环多环芳烃共代谢机理研究和多环芳烃复合污染水土环境的生物修复提供了一种新的种质资源.     

一株深海热液环境来源的PAHs降解菌TVG9-Ⅶ的系统发育与降解基因

【目的】对一株深海热液环境来源的多环芳烃(PAHs)降解菌进行系统发育分析并对其降解特性和降解机制进行研究。【方法】对16S rRNA基因进行扩增和测序,进行基于16S rRNA基因序列的系统发育分析;利用GC-MS测定其对PAHs的降解率;通过构建基因组Fosmid文库,克隆PAHs降解基因簇;并利用RT-PCR和qPCR研究关键降解酶基因在不同PAHs诱导下的表达情况。【结果】从西南太平洋劳盆地热液沉积物中分离到一株PAHs降解菌株TVG9-Ⅶ,系统发育分析结果表明,该菌株属于新鞘氨醇杆菌属(Novosphingobium),与该属的Novosphingobium indicum H25T系统发育关系最为密切,它们的16S rRNA基因序列相似性高达99.7%。该菌株在21 d内对菲、荧蒽和芘的降解率分别为95.2%,57.3%和69.6%。从Fosmid文库中筛选得到一个负责PAHs降解的上游基因簇,包含了PAHs起始降解双加氧酶大小亚基(pheA1a/b)基因和一个脱氢酶基因;RT-PCR和qPCR实验表明,双加氧酶大亚基基因pheA1a在菲的诱导下上调表达4.2倍,而在萘及高环荧蒽和芘的诱导下无上调。【结论】菌株TVG9-Ⅶ是Novosphingobium属深海热液来源的PAHs降解菌,具有良好的降解特性,特别是对高环PAHs的降解效果较好。     

两株多环芳烃降解菌协同对菲-镉污染的去除特性北大核心

【目的】从污染土壤中分离筛选一株多环芳烃降解菌,并探究其与Pseudomonas aeruginosa B6-2构建的混菌体系对菲-镉复合污染的修复效能,以及微生物代谢特性对不同镉浓度赋存的响应特性,以期为复合污染的生物修复提供优良菌株资源及应用技术参考。【方法】采用富集驯化、筛选纯化方法得到一株多环芳烃降解菌,通过生理生化特征和16S rRNA基因序列分析进行鉴定。利用高效液相色谱法和电感耦合等离子体质谱法评估不同镉浓度赋存下各反应体系对菲和镉的去除效能;通过菌体细胞形态的扫描电镜观测及菌株代谢活性检测,探讨镉胁迫对菲生物降解过程的影响机制。【结果】筛选得到一株具有重金属耐受性和多环芳烃高效降解菌SZ-3,经鉴定为节杆菌属;降解菌协同体系(M)具有良好的菲降解效能和抗镉胁迫优势。镉胁迫浓度为0.5、10 mg/L时,M对菲和镉的去除率分别高于85%、80%;镉胁迫浓度为25、50 mg/L时,2种污染物的去除率均大于65%。扫描电镜分析表明,镉胁迫导致菌体表面粗糙且出现不同程度变形,菌体间黏附性和聚集性提高。反应周期内,邻苯二酚1,2-双加氧酶活性与电子传递体系活性随镉浓度增加而降低,两者变化与菲降解速率变化一致。【结论】Arthrobacter sp.SZ-3是一株PAHs高效降解菌,能与Pseudomonas aeruginosa B6-2协同高效修复菲-镉复合污染,随着初始镉胁迫浓度增加,混菌协同对目标污染物去除的优势显著。     

嗜麦芽寡养单胞菌PX1的降解芘特性及定殖效能

为丰富多环芳烃降解菌菌种库、降低农作物的污染风险,本研究对一株可高效降解多环芳烃(PAHs)的植物内生菌进行筛选鉴定,并初步探究其降解途径以及定殖效能。结果表明: 菌株PX1为嗜麦芽寡养单胞菌。该菌株对多环芳烃的降解具有广谱性,7 d几乎可彻底降解PAH无机盐培养基中的萘,在分别含有50.0 mg·L^-1菲、20.0 mg·L^-1芘、20.0 mg·L^-1荧蒽和10.0 mg·L^-1苯并[a]芘的培养体系中,对菲、芘、荧蒽、苯并[a]芘的降解率分别为72.6%、50.7%、31.9%和12.9%。选取芘作为PAHs模型研究菌株PX1的降解特性。酶活性试验表明,芘可诱导菌株PX1体内邻苯二甲酸双加氧酶、邻苯二酚-1,2-双加氧酶和邻苯二酚-2,3-双加氧酶的活性。在芘降解过程中检测到4,5-环氧化芘、4,5-二羟基芘、龙胆酸/原茶儿酸、水杨酸、顺-己二烯二酸/2-羟粘糠酸半醛、顺-2′-羧基苯丙酮酸、1-羟基-2-萘甲酸、水杨醛等中间产物。浸种定殖试验表明,菌株PX1可高效定殖到空心菜和小麦体内,显著促进空心菜和小麦生长,并能够将空心菜、小麦体内及其生长基质中的芘浓度分别降低29.8%～50.7%、52.4%～67.1%和8.0%～15.3%。表明菌株PX1主要通过“水杨酸途径”和“邻苯二甲酸途径”降解芘,且可以定殖到植物体内,促进植物生长。     

多环芳烃降解菌X20的鉴定及降解特性

从多环芳烃降解高效的混合菌群中分离筛选到1株多环芳烃降解菌X20,经形态观察和16SrRNA序列分析,属于假单胞菌(Pseudomonas sp.)。采用室内摇瓶培养的方法,研究了该菌在不同环境条件下对菲和芘的降解。结果表明:弱碱环境有利于菌株X20对菲和芘的降解,最适pH为8.0;葡萄糖对菲芘降解率的影响呈抛物线变化,当葡萄糖浓度为0.2%时,X20对菲和芘的降解达到最高;X20对菲和芘的降解率随其初始浓度的上升而降低,菲和芘在初始浓度为10、20和40mg.L-1时的7d降解率分别为56.3%、39.25%、29.75%和41.8%、29.55%、23.50%,芘对X20降解的抑制强度高于菲。本研究结果将为构建高效的多环芳烃降解菌群,提高多环芳烃原位污染土壤的生物修复效果奠定基础。     

糙皮侧耳和鞘脂菌NS7对多环芳烃污染土壤的生物强化与协同作用

[背景] 真菌和细菌被认为在多环芳烃污染土壤生物修复过程中发挥协同作用,目前在真实土壤体系中开展真菌-细菌协同降解研究较少。[目的] 研究真菌和细菌对不同种类多环芳烃降解的差异及对蒽和苯并[a]蒽的生物强化与协同作用。[方法] 选用多环芳烃降解真菌和细菌各一株,在液体纯培养体系下分析它们对不同种类多环芳烃降解的差异,在土壤体系中采用放射性同位素示踪技术研究2种微生物对蒽和苯并[a]蒽的生物强化与协同作用。[结果] 供试细菌鞘脂菌NS7能够很好地降解低环种类多环芳烃,以蒽作为唯一碳源时可以将其完全降解,在复合污染条件下对菲、蒽、荧蒽、芘等降解效果突出（>90%）,对苯并[a]芘降解效果较差（9.76%）。相比而言,供试真菌糙皮侧耳菌对苯并[a]芘具有更好的降解效果（21.18%）,对低环多环芳烃降解效果明显不如降解菌NS7。在自然土壤中,蒽和苯并[a]蒽具有明显不同的矿化效率,分别为18.61%和4.28%,在蒽污染土壤中加入鞘脂菌NS7并未显著提高蒽的矿化率（P>0.05）,相比而言,苯并[a]蒽污染土壤中加入糙皮侧耳显著提高了污染物矿化效率（2.24倍）,表明真菌和细菌在土壤环境中的定殖存活能力可能影响了生物强化效果。采用灭菌土壤排除土著微生物的竞争排斥作用,研究了真菌菌丝对生物强化降解的影响,发现在蒽污染土壤中,真菌菌丝的迁移作用显著提高了细菌鞘脂菌NS7对污染物的矿化率,从1.75%提高到5.91%;而在苯并[a]蒽灭菌污染土壤中,接种糙皮侧耳却没有发现苯并[a]蒽矿化率提高的现象,表明自然土壤中真菌强化降解苯并[a]蒽的作用可能是源于真菌菌丝促进污染物和土著降解菌的接触,而非直接来自真菌本身。[结论] 细菌能够很好地降解低环种类多环芳烃,而真菌对高环种类多环芳烃降解效果较好。真菌可能通过菌丝促进土著微生物在土壤中的迁移,增大多环芳烃和土著降解菌的接触,从而促进了多环芳烃降解。研究加深了对多环芳烃污染土壤生物强化修复的认识,对发展基于真菌-细菌协同作用的生物强化与调控技术提供理论指导。     

芘降解菌群驯化过程中的演变

【目的】筛选、驯化获得芘的高效降解菌群,为利用其对多环芳烃污染土壤进行生物修复奠定理论基础。【方法】利用芘作为底物在矿物盐培养基(MSM)中富集驯化,得到了一个混合菌群PYR,利用分光光度计和HPLC测定混合菌群的生长与降解的关系,并对混合菌群降解多环芳烃底物的广谱性和降解芘性能的稳定性进行了测定;通过冷冻干燥的方法对菌群进行了保藏,通过HPLC的方法对保藏后复壮的菌群的降解性能进行了测定;通过培养和非培养方法对菌群的多样性进行了调查,通过构建16S rRNA基因文库的方法,分析焦化厂原始土壤中菌群组成和混合菌群转接3次(PYR-3)、6次(PYR-6)和9次(PYR-9)后的组成变化。【结果】该混合菌群可以利用芘作为唯一碳源和能源生长,12 d对芘的降解率为89%,对于菲(86%)及荧蒽(49%)也具有较高的降解率,但不能降解萘和茚并芘,并且该菌群的降解活性经过多次转接和冷冻干燥保藏保持稳定,从混合菌群中分离得到了9株菌,这9株菌分布在无色杆菌属(Achromobacter),芽胞杆菌属(Bacillus),节杆菌属(Arthrobacter),微小杆菌属(Exiguobacterium)和类土地杆菌属(Parapedobacter)。系统进化分析表明变形菌门是土壤原样(100%)及以芘为底物富集的混合菌群中的主要类群(PYR-3,83%),与原土壤样品相似,PYR-3中γ-Proteobacteria(占变形菌门的77%)中假单胞菌属的菌依然占主导地位,但由于菌群的多样性增加,假单胞菌属所占比例减少。随着富集代数的增加,菌群的多样性进一步增加,γ-Proteobacteria的比例在混合菌群中的比例下降(PYR-6中占变形菌门的33%,PYR-9中占变形菌门的18%),而β-Proteobacteria在混合菌群中的比例上升(PYR-3中占变形菌门的13%,PYR-6中占变形菌门的36%,PYR-9中占变形菌门的55%)。【结论】混合菌群具有很强的芘的降解性能,并且随着传代次数的增加,菌群的组成趋于稳定。     

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。     

Phenanthrene stimulates the degradation of pyrene and fluoranthene by <Emphasis Type="Italic">Burkholderia</Emphasis> sp. VUN10013

Pyrene and fluoranthene, when supplied as the sole carbon source, were not degraded by Burkholderia sp. VUN10013. However, when added in a mixture with phenanthrene, both pyrene and fluoranthene were degraded in liquid broth and soil. The amounts of pyrene and fluoranthene in liquid media (initial concentrations of 50 mg l⁻¹ each) decreased to 42.1% and 41.1%, respectively, after 21 days. The amounts of pyrene and fluoranthene in soil (initial concentrations of 75 mg kg⁻¹ dry soil each) decreased to 25.8% and 12.1%, respectively, after 60 days. None of the high molecular weight (HMW) polycylic aromatic hydrocarbons (PAHs) tested adversely affected phenanthrene degradation by this bacterial strain and the amount of phenanthrene decreased rapidly within 3 and 15 days of incubation in liquid broth and soil, respectively. Anthracene also stimulated the degradation of pyrene or fluoranthene by Burkholderia sp. VUN10013, but to a lesser extent than phenanthrene. The extent of anthracene degradation decreased in the presence of these HMW PAHs.     

接种功能内生细菌Diaphorobacter sp.Phe15减少蔬菜亚细胞菲积累的体外试验研究

[目的]土壤中的多环芳烃(polycyclic aromatic hydrocarbons, PAHs)可被蔬菜根系吸收并在可食部分积累进而通过食物链威胁人群健康。接种功能内生细菌能有效减低蔬菜中PAHs的积累,而关于其对蔬菜亚细胞组分中PAHs积累的影响却鲜有报道。[方法]采用体外实验,研究了接种具有菲降解功能的菌株Diaphorobacter sp. Phe15对空心菜茎叶亚细胞组分中菲积累的影响及PAHs代谢相关酶活性的响应。[结果]接种Phe15可以可加速空心菜茎叶亚细胞中菲的降解,显著削减空心菜亚细胞组分中菲的含量,接菌后空心菜亚细胞组分中菲降解率达90%以上。此外,接种功能菌Phe15可以影响空心菜亚细胞组分中PAHs代谢相关酶系的活性,空心菜亚细胞水平POD、PPO、C230活性整体得到提高,且酶系活性与空心菜体内菲积累呈负相关关系。[结论]接种具有菲降解功能的菌株Phe15增加了空心菜亚细胞水平PAHs代谢相关酶系活性,进而降低空心菜体内菲的积累,研究结果为利用功能内生细菌削减蔬菜中多环芳烃污染提供了一定的参考和理论依据。     

Biodegradation of polycyclic aromatic hydrocarbons by an acidophilic Stenotrophomonas maltophilia strain AJH1 isolated from a mineral mining site in Saudi Arabia

The present study aims at analyzing the degradation of polycyclic aromatic hydrocarbons (PAHs) at acidic conditions (pH = 2) by acidophilic Stenotrophomonas maltophilia strain AJH1 (KU664513). The strain AJH1 was obtained from an enrichment culture obtained from soil samples of mining area in the presence of PAH as sole sources of carbon and energy. Strain AJH1was able to degrade low (anthracene, phenanthrene, naphthalene, fluorene) and high (pyrene, benzo(e)pyrene and benzo(k)fluoranthene) molecular weight PAHs in acidophilic mineral salt medium at pH 2, with removal rates of up to 95% (LMW PAH) and 80% (HMW PAH), respectively. In addition, strain AJH1 treated petroleum wastewater with 89 ± 1.1% COD removal under acidic condition (pH 2) in a continuously stirred reactor. Acidophilic S. maltophilia strain AJH1, hence holds the promise as an effective degrader for biological treatment of PAHs contaminated wastewater at acidic pH.

     

Production of metabolites in the biodegradation of phenanthrene, fluoranthene and pyrene by the mixed culture of Mycobacterium sp. and Sphingomonas sp

The effects of the mixed culture of Mycobacterium sp. strain A1-PYR and Sphingomonas sp. strain PheB4 on the degradation characteristics of single polycyclic aromatic hydrocarbon were investigated. In the mixed bacterial culture, phenanthrene, fluoranthene and pyrene were degraded by 100% at Day 3, 71.2% and 50% at Day 7, respectively. Compared to their respective pure cultures, the degradation of phenanthrene and fluoranthene decreased, but that of pyrene increased significantly. Based on GC-MS analysis, eight and six new metabolites were produced from the biodegradation of phenanthrene and fluoranthene, respectively, while only two new metabolites were formed from pyrene. To our knowledge, this is the first report that the mixed bacterial culture could increase the diversity of metabolites from PAH, but the diverse metabolite pattern was not necessarily beneficial to the degradation of the recalcitrant PAH. The enhancement on pyrene degradation was possibly attributed to the rapid growth of strain PheB4.     

Degrading ability of oligocyclic aromates by <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> selected <Emphasis Type="Italic">via</Emphasis> screening of white rot fungi

Seventy-nine white rot strains were screened to determine if they had the potential for use in the degradation of oligocyclic aromates (PAHs) by measuring their dye-decoloration rate. Fourteen strains that were selected based on their dye-decoloration rate were then evaluated for the ability to tolerate various levels of PAHs spiked in agar medium. The ability of white rot fungi to degrade 3- or 4-ring PAHs (anthracene, phenanthrene, fluoranthene, pyrene) was determined. Two strains of Phanerochaete sordida (KUC8369, KUC8370) were possible PAHs degraders, degrading a significantly greater amount of phenanthrene and fluoranthene than the culture collection strain P. chrysosporium (a known PAHs degrader). The production of manganese peroxidase, the only extracellular ligninolytic enzyme detected during the cultivation, was evaluated.     

Identification of a Novel Metabolite in the Degradation of Pyrene by Mycobacterium sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.