好氧条件下Sphingomonassp.XJ1降解DBP途径的研究

在三角瓶中采用Sphingomonas sp.XJ1对邻苯二甲酸丁酯(DBP)进行好氧降解,以考察DBP的降解途径。分别对降解16h、32h和40h的DBP样品进行代谢产物分析,可判定保留时间为4.79min和5.11min所对应的代谢产物分别为原儿茶酸和邻苯二甲酸。由此可知,菌株Sphingomonas sp.XJ1对DBP的降解遵循DBP好氧生物降解途径的一般途径。即在菌株XJ1的作用下,DBP首先水解为MBP,继而水解为PA,经由PCA最终完全降解为CO2和H2O。     

DBP降解菌株XJ1的分离鉴定及其降解特性

目的：从湘江底泥筛选分离出能够高效降解邻苯二甲酸二丁酯（DBP）的菌株,对其进行鉴定和降解特性研究。方法：采用DBP为唯一碳源和能源的无机盐培养基,通过富集培养、平板划线分离得到一株优势菌,编号为XJ1。采用形态学、生理生化、（G＋C）mol%和16SrDNA序列分析进行鉴定。采用高效液相色谱测定菌株XJ1在摇瓶中对DBP的降解能力,并进行DBP代谢产物的分析和底物广谱性测试。结果：鉴定该菌株为Sphingomonsasp.。摇瓶实验结果表明：最佳降解条件为温度35℃,初始pH为7.0,转速150r/min;在最佳的降解条件下,40h之内DBP可完全降解。HPLC-UV检测出的中间代谢产物为邻苯二甲酸单丁酯（MBP）和邻苯二甲酸（PA）。底物广谱性实验表明菌株XJ1能够利用邻苯二甲酸二甲酯、邻苯二甲酸二乙酯等邻苯二甲酸酯类化合物。结论：菌株XJ1对DBP具有高效的降解能力,在处理含有邻苯二甲酸酯类化合物污染的生物修复方面具有独特的应用潜力。     

一株黄色杆菌的分离鉴定及对邻苯二甲酸酯的降解研究

邻苯二甲酸酯(PAEs)是环境中普遍存在的有机污染物,具有环境雌激素效应,对人体健康和生态安全造成严重威胁。菌株YC-JY1分离自受石油长期污染的土壤中,可以利用邻苯二甲酸二丁酯(DBP)作为唯一的碳源进行生长;经16S rDNA鉴定,确定其属于黄色杆菌属(Xanthobacter sp.)。菌株YC-JY1降解DBP的最适条件为30℃,pH 7.0,无NaCl添加;在此条件下,100mg/L DBP在5 d内能被完全降解。随着DBP浓度的升高,菌株YC-JY1在5 d内对200 mg/L-400 mg/L DBP的降解率在94%以上;通过底物谱实验发现,菌株YC-JY1对其它邻苯二甲酸酯具有广泛的利用能力,其中邻苯二甲酸二戊酯(DPeP),邻苯二甲酸二己酯(DHP)的降解率均在90%以上;通过高效液相色谱-质谱联用确定DBP中间代谢产物为邻苯二甲酸单丁酯(MBP),邻苯二甲酸(PA)。由此推断,在菌株YC-JY1的作用下,DBP的起始代谢途径为DBP首先水解为MBP,继而水解为PA。     

一株邻苯二甲酸二丁酯降解菌的筛选及其降解特性

【目的】从自然环境中筛选邻苯二甲酸二丁酯(Dibutyl phthalate,DBP)降解能力较强的微生物,并研究其降解特性和代谢途径。【方法】从杭州市河道污水出口的淤泥中筛选到DBP降解菌ZJUTW,对其进行形态、生理生化特征、16SrRNA基因序列分析,考察该菌株对DBP的降解特性,并用GC-MS分析降解中间产物。【结果】该菌株经鉴定为Arthrobacter sp.,降解DBP的最适温度和最适pH值分别为30°C和7.0-8.0,可降解多种邻苯二甲酸酯类化合物;当DBP浓度为800 mg/L时,半衰期为10.47 h;菌株的休止细胞(OD_(600)=1.2)可在20 h内将1 200 mg/L的DBP完全降解。利用GC-MS进行中间产物分析,该菌株可通过酯交换方式起始DBP的降解。【结论】Arthrobacter sp.ZJUTW对DBP有较强的降解能力和较高的耐受性,具有潜在的应用前景。     

微生物降解对氯苯胺的一条新代谢途径

迄今为止的研究报道表明,对氯苯胺的生物降解只能以邻位途径或修饰邻位途径进行。采用HPLC、液相色谱质谱联用技术(LC/MS)对Diaphorobacter PCA039菌株降解对氯苯胺的中间代谢产物进行了分析和鉴定,结果表明,对氯苯胺经PCA039菌株的降解形成了氯代邻苯二酚,5-氯-4草酰巴豆酸,5-氯-2-氧戊烯酸,5-氯-2-氧-4-羟戊酸,氯代乙酸等中间代谢产物,这些都是典型的间位代谢途径(meta-pathway)的中间物质,说明Diaphorobacter PCA039菌株以间位裂解途径对对氯苯胺进行降解。这对于对氯代胺的生物降解代谢研究、代谢机理及其遗传表达调控研究具有意义。     

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。     

一株敌草隆降解菌的分离及其应用潜能探索

旨在获得可高效降解敌草隆的土壤微生物,系统阐明菌株降解特性、敌草隆代谢途径并探索菌株的实际应用潜能。通过唯一碳源法进行富集、驯化和分离获得可降解敌草隆的土壤微生物,通过单因素试验研究菌株的环境的适应性,通过代谢中间产物的质谱分析推测菌株降解敌草隆的代谢途径,通过模拟原位土壤修复探索菌株的实际应用潜能。从长期使用敌草隆的棉田分离获得一株可高效降解敌草隆的细菌LX-C-06。形态学、生理生化特征与16S rRNA基因分析表明菌株LX-C-06为木糖氧化无色杆菌(Achromobacter xylosoxidans)。单因素试验结果表明菌株LX-C-06降解敌草隆的最适温度和pH分别为30℃和pH 7.0;菌株LX-C-06对盐离子浓度具有较高耐受能力,当NaCl浓度为1%、3%和5%时,120 h内对50 mg/L敌草隆降解率分别为100%、100%和87.8%;菌株LX-C-06对较高浓度敌草隆也表现出良好的降解能力,当敌草隆浓度≤400 mg/L时,菌株LX-C-06在120 h对敌草隆的降解率均为100%,当敌草隆浓度升高至600 mg/L时,菌株LX-C-06在120 h对敌草隆的降解率为92.1%。基于代谢中间产物分析推测菌株LX-C-06通过酯键水解将敌草隆转化为3,4-二氯苯胺,并进一步通过脱氨、羟基化、开环等,最终生成琥珀酸被生物体所利用。菌株LX-C-06在30 d内对土壤中50 mg/kg的敌草隆最高降解效率为90.7%。敌草隆降解菌LX-C-06为木糖氧化无色杆菌,该菌对环境温度、pH、盐离子浓度均具有较好的耐受能力,通过酯键水解、脱氨、羟基化、开环等步骤实现敌草隆的转化与利用,菌株同时表现出良好的应用潜能。     

一株高效DEHP降解菌的分离、鉴定及其降解特性

【目的】分离得到高效的邻苯二甲酸二乙基己基酯(DEHP)降解菌。【方法】采用富集培养法筛选分离菌株,并对菌株进行驯化;通过PCR扩增得到其16S rRNA和gyrB基因序列,进行同源序列分析及分子系统发育树的构建,同时结合形态学观察和生理生化实验对菌株进行初步鉴定;采用高效液相色谱(HPLC)分析菌株对DEHP的降解特性。【结果】分离得到一株能以DEHP为唯一碳源和能源生长的菌株,命名为HS-NH1,初步鉴定其为戈登氏菌(Gordoniasp.)。菌株HS-NH1最适的生长和降解条件为30°C、pH 7.0,在此条件下,该菌株60 h内能够将浓度为500 mg/L的DEHP降解90%以上。高效液相色谱(HPLC)分析表明,菌株HS-NH1在降解DEHP过程中产生了一种重要的中间代谢产物——邻苯二甲酸。底物广谱性试验证明,菌株HS-NH1能够有效地利用多种常见的邻苯二甲酸酯(PAEs)与芳香族衍生物。【结论】筛选得到了一株DEHP降解菌Gordonia sp.HS-NH1,该菌降解效率高,具有良好的底物广谱性,在邻苯二甲酸酯类化合物的污染治理中将会有一定的应用潜力。     

一株柴油脱硫菌的分离与鉴定

由最终产物为邻苯基苯酚（2-HBP）的二苯并噻吩（DBT）的4-S代谢途径出发,从被高硫原油污染的土样中分离,纯化得到一株能高效降解DBT的菌株,通过形态学,生理生化试验及16SrDNA基因测序,归类为Mycobacteriumsp．对细菌的培养条件进行研究,初步确定较为适宜的培养条件：温度为40℃,pH值为7．0,转速为200r／min．在此培养条件下,利用该菌株处理含有5mmol／LDBT的正十二烷模拟相,24h以后,DBT减少到3．36mmol／L,平均比脱硫率为8．34mmol DBTh^-1kg^-1 DCW（干细胞重）。     

深渊来源Citricoccus sp.strain NyZ702的分离培养及其降解4-羟基苯甲酸的特性

【背景】深渊沉积物中存在丰富的微生物细胞和活跃的微生物碳周转,因此,分离培养微生物资源对于认识深渊中的物质循环、能量代谢具有重要意义。芳香化合物在环境中广泛存在,基于组学分析揭示了深渊中具有潜在的芳香化合物代谢菌株,然而深渊来源的芳香化合物降解微生物纯培养和相关的代谢机理研究仍然缺乏。【目的】从马里亚纳海沟沉积物样本中分离培养具有降解芳香化合物能力的微生物,对其代谢途径、中间产物和降解酶活力进行初步鉴定。【方法】以4-羟基苯甲酸为唯一碳源对马里亚纳海沟沉积物样本中的降解菌株进行分离培养,结合形态观察、16S rRNA基因扩增与序列分析对菌株进行鉴定,通过底物生长实验验证其降解能力,通过高效液相色谱和超高效液相色谱-飞行时间质谱联用仪初步鉴定全细胞生物转化中间产物,利用紫外分光光度计测定其粗酶液催化4-羟基苯甲酸的活力,进而推测菌株降解4-羟基苯甲酸的代谢途径。【结果】从深渊沉积物中分离培养获得一株好氧细菌,16SrRNA基因序列分析显示该菌株隶属于柠檬球菌属(Citricoccus),命名为Citricoccus sp. strain NyZ702。该菌株在LB固体培养基上经30°C培养4 d后呈柠檬黄色、不透明、表面光滑、边缘整齐、凸出于培养基表面、直径约为1-2 mm的圆形菌落。扫描电镜表明菌体呈球形,直径为0.4-0.6μm,无鞭毛结构。该菌株为耐盐菌,最适生长盐浓度范围为2%-8%(质量体积分数)。该菌株可利用4-羟基苯甲酸为唯一碳源进行生长,可转化4-羟基苯甲酸至中间产物原儿茶酸,推测该菌株通过原儿茶酸途径降解4-羟基苯甲酸。菌株NyZ702的粗酶液具有4-羟基苯甲酸单加氧酶活力,对4-羟基苯甲酸的催化反应需要还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)作为辅因子。【结论】从深渊沉积物样本分离得到一株4-羟基苯甲酸降解菌Citricoccus sp. strain NyZ702,该菌株以原儿茶酸为中间代谢产物降解4-羟基苯甲酸,丰富了深渊来源的微生物菌种资源,为深渊中的芳香化合物降解研究提供了一定的理论基础。     

Co-Metabolic Biodegradation of DBP by Paenibacillus sp. S-3 and H-2

Two di-n-butyl phthalate (DBP)-degrading strains, designated as S-3 and H-2, were isolated from DBP-polluted soil and both identified as Paenibacillus sp. When DBP was provided as the sole carbon source, about 45.5 and 71.7 % of DBP (100 mg/L) were degraded by strain S-3 and H-2, respectively, after incubation for 48 h. However, DBP (100 mg/L) was degraded completely by co-culture of strain S-3 and H-2 after incubation for 60 h. Four phthalic acid (PA) esters could be utilized by co-metabolism in the study and the degradation rates followed the order of dimethyl phthalate > diethyl phthalate > DBP > dioctyl phthalate. The metabolic pathway of DBP was elucidated based on the results of metabolites identification and enzyme assays. For strain S-3, DBP was degraded into butyl hydrogen phthalate which was degraded to PA by carboxyesterase further. But PA could be not hydrolyzed further because strain S-3 lacked 3,4-phthalate dioxygenase. Different with S-3, strain H-2 could hydrolyze PA into 3,4-dihydroxy-PA by 3,4-phthalate dioxygenase. Then 3,4-dihydroxy-PA was converted to protocatechuate and benzoic acid. Finally, the aromatic ring was cleavage and mineralized to CO₂ and H₂O. Above all, co-metabolism could increase the activity of 3,4-phthalate dioxygenase and accelerated the degradation of DBP. This study highlights an important potential use of co-metabolic biodegradation for the in situ bioremediation of DBP and its metabolites-contaminated environment.     

红树林细菌Rhodococcus ruber 1K降解邻苯二甲酸二丁酯的研究

1引言邻苯二甲酸酯类化合物( PAEs)是世界上生产量大、应用面广的人工合成化合物.1933年Waldo Semon首先将邻苯二甲酸二丁酯用于PVC生产,得到了大幅度改性后的商品化树脂.从此,邻苯二甲酸酯作为增塑剂得到广泛应用.由于作为增塑剂的邻苯二甲酸酯与PVC聚合物及其他材料并非通过共价键结合,在产品的使用过程中及处置后很容易被释放到环境中[2,16].塑料制品的全球性大量应用导致邻苯二甲酸酯在环境中普遍存在[6,7].城市污泥中可以普遍检测到PAEs[6,9,13~15,17,20],由于城市污泥的农用导致在农作物或者农产品中也可以检测到PAEs的存在[3~…     

Isolation and characterization of Sphingomonas sp. GTIN11 capable of carbazole metabolism in petroleum

A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogen-containing heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37 degrees C was determined to be 8.0 micromol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.     

Aerobic degradation of diethyl phthalate by Sphingomonas sp

An aerobic diethyl phthalate (DEP) degrading bacterium, DEP-AD1, was isolated from activated sludge. Based on its 16S rDNA sequence, this isolate was identified belonging to Sphingomonas genus with 99% similarity to Sphingomonas sp. strain C28242 and 98% similarity to S. capsulate. The specific degradation rate of DEP was concentration dependent with a maximum of 14 mg-DEP/(Lh). Results of degradation tests showed that DEP-AD1 could also degrade monoethyl phthalate (MEP), dimethyl phthalate (DMP), dibutyl phthalate (DBP), and diethylhexyl phthalate (DEHP), but not phthalate and benzoate.     

Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.     

Isolation and characterization of 3-N-trimethylamino-1-propanol-degrading Rhodococcus sp. strain A2

The aerobic degradation of 3- N -trimethylamino-1-propanol (homocholine) as a sole source of carbon and nitrogen has been found for a Rhodococcus sp. bacterium isolated from soil. The isolate was identified as Rhodococcus sp. strain A2 based on its phenotypic features, physiological and biochemical characteristics, and results of phylogenetic analysis. The washed cells of strain A2 completely degraded homocholine within 6 h, with concomitant formation of several metabolites. Analysis of the metabolites using capillary electrophoresis, fast atom bombardment–MS, and GC–MS showed that trimethylamine was the major metabolite, in addition to β-alanine betaine (β-AB) and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strain is through consequent oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH). Thereafter, the cleavage of β-AB C–N bonds yielded trimethylamine and alkyl chain.     

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.     

Effect of rhamnolipids on degradation of anthracene by two newly isolated strains, Sphingomonas sp. 12A and Pseudomonas sp. 12B

Anthracene is a PAH that is not readily degraded, plus its degradation mechanism is still not clear. Thus, two strains of bacteria-degrading bacteria were isolated from longterm petroleum-polluted soil and identified as Sphingomonas sp. 12A and Pseudomonas sp. 12B by a 16S rRNA sequence analysis. To further enhance the anthracene-degrading ability of the two strains, the biosurfactants produced by Pseudomonas aeruginosa W3 were used, which were characterized as rhamnolipids. It was found that these rhamnolipids dramatically increased the solubility of anthracene, and a reverse-phase HPLC assay showed that the anthracene degradation percentage after 18 days with Pseudomonas sp. 12B was significantly enhanced from 34% to 52%. Interestingly, their effect on the degradation by Sphingomonas sp. 12A was much less, from 35% to 39%. Further study revealed that Sphingomonas sp. 12A also degraded the rhamnolipids, which may have hampered the effect of the rhamnolipids on the anthracene degradation.     

Isolation and characterization of dibenzofuran-degrading bacteria

Two bacterial strains capable of utilizing dibenzofuran (DF) as a sole carbon source were isolated from soil samples of reclaimed land. The strains designated HL1 and HL7 were identified as Klebsiella sp. and Sphingomonas sp., respectively, on the basis of biochemical characteristics and the sequences of the 16S ribosomal DNA. Sphingomonas sp. strain HL7 degraded non-, mono- and also dichlorinated DF and dibenzo-p-dioxin (DD). Klebsiella sp. strain HL1 was able to degrade non- and monochlorinated DFs and DDs, but not dichlorinated ones. The metabolites formed from DF by strains HL1 and HL7 were similar to those by dioxin-degrading bacteria Sphingomonas sp. strain RW1 except for salicylic acid and catechol. Strain HL7 had a gene homologous to that encoding the dioxin dioxygenase alpha-subunit (dxnA1) gene of Sphingomonas sp. strain RW1. However, Southern hybridization analysis showed that the size of an EcoRV-digested genomic fragment involving the dioxin dioxygenase gene of strain HL7 was smaller than that of strain RW1, and that strain HL1 did not have the homologous gene. Strains HL1 and HL7 provided useful information regarding the dioxygenase genes.