一株丁草胺降解菌的分离鉴定及其降解特性的研究

利用富集培养技术从长期施用丁草胺的稻田土壤中分离得到能够降解丁草胺的细菌1株, 标记为LYC-1。经形态特征、生理生化特征和16S rRNA序列分析, 将该菌株鉴定为不动杆菌属(Acinetobacter sp.), 菌株LYC-1的最适生长温度为30°C, 最适pH值为7.5。当接种量为5%时, 该菌株在含100 mg/L 的丁草胺无机盐基础培养液中培养7 d后, 可使丁草胺降解达80%以上。     

1株高效菲降解不动杆菌的筛选、鉴定及性能研究

从兰州某化工厂石油废水中分离筛选出1株高效降解菲的细菌F-1并对其菌种进行鉴定,结合紫外分光光度法及气相色谱-质谱联用(GC-MS)对菌株生长特性、不同烃类化合物降解特性及菲降解动力学等进行了研究,利用PCR技术检测了芳香烃代谢相关基因。结果表明,菌株F-1属于约翰逊不动杆菌(Acinetobacter johnsonii),可在终浓度为50～800 mg/L的含菲基础培养基中正常生长。在温度30℃、pH 7. 0、盐度0. 3%(质量分数)、转速180 r/min条件下培养5 d后菲(终浓度为100 mg/L)降解率为43. 57%,降解过程符合一级动力学特征。菌株F-1也能利用联苯、萘、蒽、芘为唯一碳源生长。GC-MS分析显示菌株对C10-C28部分直链烷烃具有较强的降解能力。PCR扩增结果表明,菌株F-1基因组中存在邻苯二酚-1,2-双加氧酶、苯甲酸盐双加氧酶、铁氧化还原蛋白还原酶、乙醇脱氢酶、二羟酸脱水酶、醛缩酶和氧化还原蛋白基因。研究结果为该菌株应用到含菲废水及多环芳烃污染土壤的处理和深度修复研究提供参考。     

假交替单胞菌Pseudoalteromonas sp. AJ5 产k-卡拉胶酶的培养条件优化

[目的]本研究的目的是优化Pseudoalteromonas sp. AJ5菌株的培养条件使之产生高活性的胞外κ-卡拉胶酶.[方法]通过富集培养技术从刺参肠道分离出一株卡拉胶降解菌AJ5,该菌株能利用卡拉胶作为惟一碳源和能源.依据形态学和生理学特征及16S rRNA基因序列分析,将该菌株鉴定为假交替单胞菌属(Pseudoalteromonas).通过单因素试验和正交试验对Pseudoalteromonas sp. AJ5菌株产胞外κ-卡拉胶酶的培养条件进行了优化.[结果]单因素试验结果表明,Pseudoalteromonas sp. AJ5菌株的最佳培养条件为250 mL三角瓶装入75 mL发酵培养基、摇床转速150 r/min、接种量7%、pH8.0.单因素试验和正交试验结果显示该菌株的最佳培养基组成为κ-卡拉胶 1 g/L、牛肉膏2 g/L、 NaCl 20 g/L、K2HPO4·3H2O 1 g/L、 MgSO4·7H2O 0.5 g/L、 MnCl2· 4H2O 0.2 g/L、 FePO4 · 4H2O 0.01 g/L; 培养温度为28℃,培养时间为28 h.[结论]Pseudoalteromonas sp. AJ5菌株分泌胞外κ-卡拉胶酶,在最佳培养条件下,该菌株的κ-卡拉胶酶活力比优化前提高了4倍.     

1株兼有烟碱降解活性解磷菌的分离鉴定

从烟草(Nicotiana tabacum L.)根际土壤中分离到1株兼有烟碱降解活性的解磷菌株,编号为YF24;菌株YF24经过48 h发酵后,培养基上清液中游离态磷含量达到141.6μg/m L,烟碱降解率达到77%,确定YF24具有解磷和烟碱降解的双重活性;对菌株YF24生长特性的研究表明,其最适生长温度是30~35℃,最适p H值范围是5.5~7.0,耐盐性差。通过形态特征观察、生理生化特性测定和16S r DNA序列分析,确定菌株YF24隶属于不动杆菌属Acinetobacter,暂命名为Acinetobacter sp.YF24。菌株YF24可作为烟草专用菌肥开发、诱变育种的良好出发菌株。     

陕北地区石油污染土壤中不动杆菌属的筛选、鉴定及降解性能

从陕北地区石油污染土壤中分离鉴定得到两株不动杆菌属(Acinetobacter sp.)的高效石油降解菌A.sp 1和A.sp 2,分别从盐浓度、pH值、氮源、磷源和接种量等因素进行研究以确定其最佳石油降解条件,并进一步通过GC-MS(Gas ChromatographyMass Spectrometer)方法分析其在最佳条件下对原油组分的不同降解性能。结果显示:A.sp 1在盐浓度为1%、pH值为6—7、磷源为KH2PO4和K2HPO4、氮源为尿素和接种量为4%的条件下,最高降解率可达到60%。A.sp 2在盐浓度为1%、pH值为7—9、磷源为KH2PO4和K2HPO4、氮源为硝酸铵和接种量为8%的条件下,最高降解率可达到67%。GC-MS分析结果表明,菌株A.sp 1对石油烃类C21—C25有明显的降解效果,菌株A.sp 2对石油烃类C20—C30的降解效果较好。     

盐碱土壤PAHs 降解菌的筛选鉴定及其降解特性

采用富集培养的方法,从天津大港油田PAHs污染盐碱化土壤中分离出一株能以菲、芘为唯一碳源和能源的优势菌TJB5。经形态观察和16S rDNA序列分析结果表明,该菌株为成团泛菌(Pantoea agglomerans)。采用液体培养的方法,研究了pH、盐度、菲芘的初始浓度对TJB5菌株降解菲芘效果的影响,确定了最佳降解条件。结果表明,该菌对菲、芘的降解具有较广泛的pH、盐度范围和良好的降解效果。在菲、芘浓度分别为50 mg/L、pH 6.8-9.5、盐度2%-3%、温度30°C条件下,接种15 d后菲降解率在93.3%以上,芘降解率在20%以上。     

一株高分子量多环芳烃降解菌的筛选、鉴定及降解特性研究

采用富集培养方法从多环芳烃污染土壤中筛选分离得到1株能以苯并[a]芘(B[a]P)为唯一碳源和能源生长的菌株.形态特征观察和16S rDNA序列分析结果表明,该菌株为副球菌属(Paracoccus sp.),编号为HPD-2.HPD-2在3.0 mg/L的B[a]P液体培养基中生长较慢,培养5 d后B[a]P的降解率为89.7%.同时,该菌株对四环的芘和荧蒽也具有较好的降解能力,培养7 d后芘和荧蒽的降解率分别达到47.2%和84.5%.可见,该菌株对高分子量PAHs具有很好的降解潜力.     

中度嗜盐菌Martelella sp. AD-3 降解菲过程中水杨酸-5-羟化酶的酶学性质

[目的]研究中度嗜盐菌Martelella sp.AD-3在降解菲过程中水杨酸-5-羟化酶的活性与菲降解效率的关系及其酶学性质.[方法]通过HPLC分析菲的降解效率和AD-3菌粗酶液催化水杨酸的产物,根据NADH在340 nm处的吸光度变化计算水杨酸-5-羟化酶的活性.[结果]水杨酸-5-羟化酶是一种诱导酶,在AD-3菌的对数生长期和稳定初期时活性较高,酶活力大小与该菌对菲的降解速率基本一致.在菲浓度为200 mg/L、生长盐度为3％、pH为9.0的培养条件下,AD-3菌株表达的水杨酸-5-羟化酶的活力最高,为132.8 nmol/(min·mg).水杨酸-5-羟化酶催化水杨酸降解时的最适温度、pH和盐度分别为30℃、7.5和3％,酶的最大反应速率为200 nmol/(min· mg)、米氏常数Km为8.7μmol/L.[结论]AD-3菌在降解菲的过程中表达水杨酸-5-羟化酶,该酶的活性与菲降解速率具有相关性.     

一株不动杆菌降解石油烃的特性及关键烷烃降解基因分析

【背景】Acinetobacter sp. Tust-DM21 (GenBank登录号KX390866)是本实验室前期从渤海湾海洋石油勘探船废油收集区采集的水油混合样中分离出的一株高效石油降解菌,其对短、中、长链烷烃均表现出很强的降解能力,有较好的应用前景。【目的】从应用层面探究其最佳降解条件,同时从生物信息层面探究其降解基因的作用。【方法】将其在不同温度、pH下培养144h,通过GC-MS内标法测定石油烃各组分的变化情况,计算出其最佳降解条件;同时,通过生物信息学手段确定基因组中的降解基因,每个基因分别选择7个同源基因,对它们的蛋白序列进行比较;最后对2个降解基因在0-144 h的表达情况进行了Real-time PCR分析。【结果】Acinetobacter sp. Tust-DM21最佳降解条件为35°C、pH 8.5,该条件下对石油降解率可达97.5%,其中,对长链烷烃降解率达98.5%,对环烃为81%,对芳香烃为87%;同时,研究发现基因组中含有常见烷烃降解基因alk B(GenBank登录号MH368539)和长链烷烃降解基因alm A (GenBank登录号MH357335),2个降解基因的蛋白经比较均与其同源蛋白表现出一定的相似性,同属菌的相似性最高;通过Real-timePCR发现这2个基因在0-144 h的相对表达量随时间逐步提高。【结论】Acinetobacter sp. Tust-DM21在最佳降解条件下对石油各组分都显示出了优良的降解能力,特别对长链烷烃的降解能力尤为突出;将2个降解基因的相对表达量结合该时间段的生长趋势,证明了菌株Acinetobacter sp. Tust-DM21的生长和降解与alk B和alm A基因的上调表达存在关联。     

盐渍化土壤翅碱蓬根际促生细菌的筛选及对多环芳烃的降解特性

为了强化多环芳烃污染盐渍化土壤的原位植物-微生物修复的应用,获得促进植物降解多环芳烃的高效菌种资源,本研究采用富集培养的方法,从多环芳烃污染的大港油田翅碱蓬根际分离到1株能以菲、芘为唯一碳源同时分泌1-氨基环丙烷~(-1)-脱氨酶的优势菌株B~(-1),通过形态观察和16S rRNA序列分析鉴定该菌株,并对其潜在的促生能力和菲、芘的降解特性进行分析。16S rRNA序列分析结果表明,该菌株为动性球菌属(Planococcus sp.),可产生3-吲哚乙酸,且具有溶磷能力。同时对菲、芘的降解具有较广泛的p H值和盐浓度范围,在菲和芘浓度均为50 mg·L~(-1),p H 8.0,盐度2%时,7 d对菲、芘的降解率为66.6%和52.0%。表面活性剂烷基糖苷的添加使菲、芘降解效率分别提高至94.2%和78.8%。     

Identification of metabolites from phenanthrene oxidation by phenoloxidases and dioxygenases of Polyporus sp. S133

Phenanthrene degradation by Polyporus sp. S133, a new phenanthrene-degrading strain, was investigated in this work. The analysis of degradation was performed by calculation of the remaining phenanthrene by gas chromatography-mass spectrometry. When cells were grown in phenanthrene culture after 92 h, all but 200 and 250 mg/l of the phenanthrene had been degraded. New metabolic pathways of phenanthrene and a better understanding of the phenoloxidases and dioxygenase mechanism involved in degradation of phenanthrene were explored in this research. The mechanism of degradation was determined through identification of the several metabolites; 9,10-phenanthrenequinone, 2,2'-diphenic acid, salicylic acid, and catechol. 9,10-Oxidation and ring cleavage to give 9,10-phenanthrenequinone is the major fate of phenanthrene in ligninolytic Polyporus sp. S133. The identification of 2,2'-diphenic acid in culture extracts indicates that phenanthrene was initially attacked through dioxigenation at C9 and C10 to give cis-9,10-dihydrodiol. Dehydrogenation of phenanthrene-cis-9,10-dihydrodiol to produce the corresponding diol, followed by ortho-cleavage of the oxygenated ring, produced 2,2'-diphenic acid. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase, and 2,3-dioxygenase) produced by Polyporus sp. S133 was detected during the incubation. The highest level of activity was shown at 92 h of culture.     

Degradation of phenanthrene on plant leaves by phyllosphere bacteria

The activity of phyllosphere bacteria in the degradation of phenanthrene was investigated as a mechanism for the removal of atmospheric phenanthrene after its deposition on plant leaves. Initially, leaf samples of six plant species were collected from two roadsides in Bangkok to determine the presence of phenanthrene-degrading bacteria. The numbers of phenanthrene-degrading phyllosphere bacteria were varied and ranged from 3.5 x 10(4) to 1.95 x 10(7) CFU/g, in which the highest number was found from Ixora sp. Further studies were carried out in the laboratory by spraying phenanthrene on Ixora sp. leaves and then monitoring the amount of deposited phenanthrene and number of phenanthrene-degrading bacteria after incubation. The results showed that the amount of phenanthrene was significantly reduced on leaves containing phenanthrene-degrading bacteria. These were detected along with a rapid increase in the number of bacteria on leaves. The results indicated that many phyllosphere bacteria could utilize phenanthrene to support their growth and thereby reduce the amount of deposited phenanthrene on leaf surfaces. Several phenanthrene-degrading bacteria were later isolated from the leaves and identified with a high 16S rDNA sequence similarity to the genera Pseudomonas, Microbacterium, Rhizobium, and Deinococcus.     

Adaptation of a <Emphasis Type="Italic">Mycobacterium</Emphasis> strain to phenanthrene degradation in a biphasic culture system: influence on interfacial area and droplet size

A phenanthrene-degrading Mycobacterium sp. strain 6PY1 was grown in an aqueous/organic biphasic culture system with phenanthrene as sole carbon source. Its capacity of degradation was studied during sequential inoculum enrichments, reaching complete phenanthrene degradation at a maximim rate of 7 mg l⁻¹ h⁻¹. Water–oil emulsions and biofilm formation were observed in biphasic cultures after four successive enrichments. The factors influencing interfacial area in the emulsions were: the initial phenanthrene concentration, the initial inoculum size, and the silicone oil volume fraction. The results showed that the interfacial area was mainly dependent on the silicone oil/mineral salts medium ratio and the inoculum size.     

Mineralization of phenanthrene by a Mycobacterium sp.

A Mycobacterium sp., designated strain BG1, able to utilize the polycyclic aromatic hydrocarbon phenanthrene as the sole carbon and energy source was isolated from estuarine sediment following enrichment with the hydrocarbon. Unlike other phenanthrene degraders, this bacterium degraded phenanthrene via 1-hydroxy-2-naphthoic acid without accumulating this or other aromatic intermediates, as shown by high-performance liquid chromatography. Degradation proceeded via meta cleavage of protocatechuic acid. Different nonionic surfactants (Tween compounds) solubilized the phenanthrene to different degrees and enhanced phenanthrene utilization. The order of enhancement, however, did not correlate perfectly with increased solubility, suggesting physiological as well as physicochemical effects of the surfactants. Plasmids of approximately 21, 58, and 77 megadaltons were detected in cells grown with phenanthrene but not in those which, after growth on nutrient media, lost the phenanthrene-degrading phenotype. Given that plasmid-mediated degradations of aromatic hydrocarbons generally occur via meta cleavages, it is of interest that the addition of pyruvate, a product of meta cleavage, supported rapid mineralization of phenanthrene in broth culture; succinate, a product of ortho cleavage, supported growth but completely repressed the utilization of phenanthrene. The involvement of plasmids may have given rise to the unusual degradation pattern that was observed.     

A novel phenanthrene dioxygenase from Nocardioides sp. Strain KP7: expression in Escherichia coli

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the alpha and beta subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8. 3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the alpha and beta subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.     

Establishment of plasmid vector and allelic exchange mutagenesis systems in a mycobacterial strain that is able to degrade polycyclic aromatic hydrocarbon

Plasmid vector and allelic exchange mutagenesis systems were established for the genetic analysis of a phenanthrene-degrading mycobacterial strain, Mycobacterium sp. EPa45. Successful application of these systems revealed the necessity of the EPa45 phdI gene for the degradation of 1-hydroxy-2-naphthoate, which has been proposed to be an intermediate product in the degradation pathway of phenanthrene.     

Phenanthrene-degrading phenotype of Alcaligenes faecalis AFK2.

A phenanthrene-degrading bacterium that assimilated a wide range of organic compounds was isolated from a soil sample and identified as Alcaligenes faecalis strain AFK2. The strain degraded phenanthrene through protocatechuate, but did not utilize naphthalene. The phenanthrene-degrading phenotype (Phn+) of AFK2 disappeared after 20 successive subcultures in a mineral salts medium containing o-phthalate or after subculture in nutrient broth containing mitomycin C. The results suggested that the Phn+ phenotype of this strain might be encoded by extrachromosomal genes.     

Repeated inoculation as a strategy for the remediation of low concentrations of phenanthrene in soil

Phenanthrene, a polycyclic aromatic hydrocarbon, becomes increasingly unavailable to microorganisms for degradation as it ages in soil. Consequently, many bioaugmentation efforts to remediate polycyclic aromatic hydrocarbons in soil have failed. We studied theeffect of repeatedly inoculating a soil with a phenanthrene-degrading Arthrobacter sp. on the mineralization kinetics of low concentrations of phenanthrene. After the first inoculation, the initial mineralization rate of 50 ng/g phenanthrene declined in a biphasicexponential pattern. By three hundred hours after inoculation, there was no difference in mineralization rates between the inoculated and uninoculated treatments even though a large fraction of the phenanthrene had not yet been mineralized. A second and third inoculation significantly increased the mineralization rate, suggesting that, though themineralization rate declined, phenanthrene remained bioavailable. Restirring the soil, without inoculation, did not produce similar increases in mineralization rates, suggesting absence of contact between cells and phenanthrene on a larger spatial scale (>mm) is not the cause of the mineralization decline. Bacteria inoculated into soil 280 hours beforethe phenanthrene was added could not maintain phenanthrene degradation activity. We suggest sorption lowered bioavailability of phenanthrene below an induction threshold concentration for metabolic activity of phenanthrene-degrading bacteria.     

Staphylococcus sp. KW-07 contains nahH gene encoding catechol 2,3-dioxygenase for phenanthrene degradation and a test in soil microcosm

We isolated three species of phenanthrene-degrading bacteria from oil-contaminated soils and marine sediment, and assessed the potential use of these bacteria for bioremediation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs). Based on 16S rDNA sequences, these bacteria were Staphylococcus sp. KW-07 and Pseudomonas sp. CH-11 from soil, and Ochrobactrum sp. CH-19 from the marine sediment. By PCR amplification, catechol 2,3-dioxygenase genes (nahH genes) mediating PAH degradation in the chromosome of Staphylococcus sp. KW-07 and Ochrobactrum sp. CH-19, and in plasmid DNA of Pseudomonas sp. CH-11 were detected. All isolates had a similar optimal growth temperature (25 °C) and optimal growth pH (7.0) in a minimal salt medium (MSM) with 0.1% (w/v) phenanthrene as the sole source of carbon and energy. Pseudomonas sp. CH-11 and Staphylococcus sp. KW-07 degraded 90% of added phenanthrene in 3 days and Ochrobactrum sp. CH-19 degraded 90% of the phenanthrene in 7 days under laboratory batch culture conditions. However, Staphylococcus sp. KW-07 was the most effective among the three strains in degradation of phenanthrene in soil. After inoculation of 1 × 10¹¹ cells of Staphylococcus sp. KW-07, over 90% degradation of 0.1% phenanthrene (0.1 g/100 g soil) was achieved after 1 month at 25 °C. The results collectively suggest that the Staphylococcus sp. KW-07 strain isolated may be useful in bioremediation of PAH-contaminated soils.     

Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp. strain A4

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene and acenaphthylene as sole carbon and energy sources, but it is unable to grow on other polycyclic aromatic hydrocarbons (PAHs). The genes encoding terminal oxygenase components of ring-hydroxylating dioxygenase (arhA1 and arhA2) were isolated from this strain by means of the ability to oxidize indole to indigo of the Escherichia coli clone containing electron transport proteins from phenanthrene-degrading Sphingobium sp. strain P2. The translated products of arhA1 and arhA2 exhibited moderate sequence identity (less than 56%) to large and small subunits of dioxygenase of other ring-hydroxylating dioxygenases. Biotransformation with recombinant E. coli clone revealed the broad substrate specificity of this oxygenase toward several PAHs including acenaphthene, acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene. Southern hybridization analysis revealed the presence of a putative arhA1 homologue on a locus different from that of the arhA1 gene. Insertion inactivation of the arhA1 gene in strain A4 suggested that the gene but not the putative homologue one was involved in the degradation of acenaphthene and acenaphthylene in this strain.