Degradation of car engine base oil by Rhodococcus sp. NDKK48 and Gordonia sp. NDKY76A

Two microorganisms (NDKK48 and NDKY76A) that degrade long-chain cyclic alkanes (c-alkanes) were isolated from soil samples. Strains NDKK48 and NDKY76A were identified as Rhodococcus sp. and Gordonia sp., respectively. Both strains used not only normal alkane (n-alkane) but also c-alkane as a sole carbon and energy source, and the strains degraded more than 27% of car engine base oil (1% addition).     

三株降解芘的戈登氏菌鉴定及其降解能力

从沈抚灌区多环芳烃污染土壤中筛选出的芘降解菌D44、D82S和D82Q,经形态观察、生理生化试验和16S rDNA序列分析确定均为戈登氏菌属(Gordonia sp.).3株菌的最适生长pH值均为7,当pH值低于5或高于9时,生长均受到明显抑制.降解试验表明,3株菌能以芘、苯并芘、蒽、萘、菲和荧蒽为唯一碳源和能源生长.经过7 d的培养,3株菌对初始浓度为100 mg.L-1的芘的降解率均在65%以上,对初始浓度为50 mg.L-1的苯并芘的降解率分别为79.6%、91.3%和62.8%.通过PCR检测发现D82Q和D82S含有烷烃单加氧酶基因alkB.     

红球菌DS—3脱除二苯并噻吩中有机硫的性能初探

从孤岛油田分离到一株红球菌(Rhodococcus sp.)DS—3,能专一地切断二苯并噻吩(DBT)中的C－S键,沿4S途径代谢,生成二羟联苯。实验证明,以2％的接种量脱除50μｇ／mL DBT底物中的硫效果最佳。在此条件下,适宜菌株生长和脱硫的碳源为葡萄糖,氯源为硝酸铵,初始PH为8．2,生长温度为30℃,15mmol/L的硫酸根离子能使其丧失脱硫能力。在上述适宜条件下,培养72h后DBT中34．04％的硫被脱除。     

Degradation pathways of cyclic alkanes in Rhodococcus sp. NDKK48

The degradation pathways for cyclic alkanes (c-alkanes) in Rhodococcus sp. NDKK48 were investigated. Strain NDKK48 used dodecylcyclohexane as a sole carbon and energy source, and five metabolites in the dodecylcyclohexane degradation pathway were detected by gas-chromatography/mass spectra. The metabolites were identified as cyclohexanecarboxylic acid, cyclohexylacetic acid, 1-cyclohexene-1-acetic acid, 4-dodecylcyclohexanol, and 4-dodecylcyclohexanone. The strain degrades dodecylcyclohexane via a ring oxidation pathway and an alkyl side chain oxidation pathway. Cyclohexanecarboxylic acid was further oxidized to muconic acid via 1-cyclohexene-1-carboxylic acid and benzoic acid, and the muconic acid was finally used by strain NDKK48 for growth. Methylcyclohexane and cyclohexane were co-oxidized with hexadecane by strain NDKK48. Methylcyclohexane was degraded via a ring oxidation pathway, and the degradation pathway contained part of the Baeyer-Villiger oxidation for ring cleavage. Cyclohexane was also degraded by the same pathway as methylcyclohexane. Thus, strain NDKK48 has two pathways for the complete degradation of c-alkanes.     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

Degradation of benzotrifluoride via the dioxygenase pathway in Rhodococcus sp. 065240

We previously isolated Rhodococcus sp. 065240, which catalyzes the defluorination of benzotrifluoride (BTF). In order to investigate the mechanism of this degradation of BTF, we performed proteomic analysis of cells grown with or without BTF. Three proteins, which resemble dioxygenase pathway enzymes responsible for isopropylbenzene degradation from Rhodococcus erythropolis BD2, were induced by BTF. Genomic PCR and DNA sequence analysis revealed that the Rhodococcus sp. 065240 carries the gene cluster, btf, which is highly homologous to the ipb gene cluster from R. erythropolis BD2. A mutant strain, which could not catalyze BTF defluorination, was isolated from 065240 strain by UV mutagenesis. The mutant strain had one mutation in the btfT gene, which encodes a response regulator of the two component system. The defluorinating ability of the mutant strain was recovered by complementation of btfT. These results suggest that the btf gene cluster is responsible for degradation of BTF.     

节杆菌(Arthrobacter sp.)降解阿魏酸的效能

[背景]蓄积在土壤中的阿魏酸类化感自毒物质对农作物生长产生危害,利用有益微生物分解该类物质是一项有效的治理措施.[目的]从自然界土壤分离获得能高效降解阿魏酸的菌株,并评估典型环境因子对降解效能的影响,以期为该菌在阿魏酸类自毒物质降解领域中的应用提供理论依据.[方法]采用一次性投加高浓度化合物的驯化方法分离筛选得到能有效...     

红球菌HX-2所产胞外多糖的特性和对Cu2+的吸附

【背景】目前,微生物所产胞外多糖(exopolysaccharide,EPS)的理化性质及其在重金属吸附中的应用受到了广泛关注。【目的】研究红球菌HX-2所产胞外多糖的理化性质,并探究其对重金属的吸附情况。【方法】使用离子交换和凝胶色谱分离法对胞外多糖粗品进行纯化;利用苯酚硫酸法测胞外多糖中糖含量;用Bradford试剂盒检测胞外多糖中蛋白含量;使用甲醇萃取法检测胞外多糖中脂质含量;用高效液相色谱(high performance liquid chromatography,HPLC)法分析胞外多糖中单糖组成;用扫描电镜(scanningelectronmicroscopy,SEM)法观察多糖表面形态;通过等温吸附模型和动力学模型探究胞外多糖对重金属的吸附效果。【结果】测得胞外多糖主要成分EPS-G-1中总糖含量为78.43%,蛋白含量为8.31%,脂质含量为8.22%;纯化后胞外多糖中单糖组成为葡萄糖、甘露糖、半乳糖、葡萄糖醛酸和岩藻糖,质量比为27.31:26.67:24.83:15.85:4.80;通过等温吸附模型拟合得到HX-2所产胞外多糖对Cu~(2+)的最大吸附量为144.93 mg/g。【结论】红球菌HX-2所产胞外多糖对水体中Cu~(2+)具有良好的吸附作用,可用于工业废水中重金属离子的处理。     

Desulphurisation of benzothiophene and dibenzothiophene by actinomycete organisms belonging to the genus Rhodococcus, and related taxa

Desulphurising enzymes remove the sulphur moiety from an organosulphur molecule leaving the carbon skeleton intact. Two kinds of desulphurisation reaction are recognised. The dibenzothiophene (DBT)-specific pathway desulphurises DBT to inorganic sulphite and 2- hydroxybiphenyl (HBP), and the benzothiophene (BTH)-specific pathway desulphurises BTH to 2-(2-hydroxyphenyl)ethan 1-al (HPEal) and probably inorganic sulphite. The DBT-desulphurisation pathway was originally identified in Rhodococcus erythropolis strain IGTS8 (ATCC 53968), and the BTH-desulphurisation pathway in Gordonia sp. strain 213E (NCIMB 40816). These organisms do not further metabolise the organic product of desulphurisation.In this article current knowledge of the biochemistry and genetics of the desulphurisation enzymes is reviewed. The need for separate, DBT- and BTH-specific desulphurisation routes is rationalised in terms of the chemical differences between the two compounds. The desulphurisation pathway is compared with other microbial DBT- degrading enzyme systems. Finally some comments are made concerning the application of desulphurisation enzymes for fuel desulphurisation and on the relevance of these enzymes to the ecology of the mycolata (sensu Chun et al, 1996).     

红球菌SDUZAWQ对有机硫和无机硫的耐受性研究及脱硫基因的克隆

以筛选得到的红球菌SDUZAWQ为对象,研究其在不同浓度的有机硫化合物二苯并噻吩(DBT)存在下的脱硫能力,以及在0.2mmolLDBT和不同浓度Na2SO4同时存在下的脱硫情况。当DBT浓度高达6mmolL时,菌株仍能生长,而且检测出产物2-羟基联苯(2-HBP)的存在,说明该菌株具有耐受较高浓度DBT的能力。当DBT和Na2SO4同时存在时,DBT为菌株SDUZAWQ所利用,并且也检测出2-HBP,并非如文献所报道的红球菌在无机硫存在下不代谢DBT,表明该菌株能够耐受一定浓度的无机硫酸盐。对相关脱硫基因的克隆和测序结果显示,完整脱硫基因dszABC、其上游调控序列和dszD的序列与模式菌株RhodococcuserythropolisIGTS8的同源性分别是99%、100%和100%。     

Gordonia sp. LAM0048的分离鉴定及其降解正十六烷的研究

以正十六烷为唯一碳源,从长期受石油污染的土壤中筛选到一株高效降解正十六烷的菌株LAM0048。通过形态学观察、生理生化试验、细胞化学组分分析、16S rRNA基因序列分析、细胞脂肪酸和极性脂试验,确定其属于棒杆菌亚目(Corynebacterineae)、诺卡菌科(Nocardiaceae)、戈登氏属(Gordonia),且可能为戈登氏属的一株新种。采用单因素实验对菌株LAM0048在无机盐培养基中降解正十六烷的降解率进行初步探讨,发现该菌株能在以正十六烷为唯一碳源的培养基中生长,菌株LAM0048能够在36 h内完全降解0.05%(V/V)的正十六烷,当烷烃浓度达到1.0%(V/V)时,降解率达46.4%。结果表明LAM0048是一株具有耐受高浓度烷烃的石油降解菌,在石油污染环境的微生物修复中具有一定的应用潜力。     

Characterization and functional analysis of a novel gene cluster involved in biphenyl degradation in Rhodococcus sp. strain R04

AIMS: Isolation of the genes relative to PCB biodegradation and identification of the bph gene function in Rhodococcus sp. R04. METHODS AND RESULTS: A 8.7-kb fragment carrying the biphenyl catabolic genes bphABCD was isolated from the gene library in Rhodococcus sp. R04. Based on the deduced amino acid sequence homology, seven bph genes, bphA1A2A3A4, bphB, bphC and bphD, were thought to be responsible for the initial four steps of biphenyl degradation. In Escherichia coli, BphA exhibited poor activity for biphenyl transformation, and BphB, BphC and BphD were found to be catalytically active towards 2,3-dihydro-2,3-dihydroxybiphenyl, 2,3-dihydroxybiphenyl and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate, respectively (activities of 50, 8.1 and 2.4 micromol l(-1) min(-1) mg(-1)). SDS-PAGE analysis indicated that the sizes of bphA1A2A3A4, bphB, bphC and bphD gene products were 49, 19, 14, 47, 32, 30 and 31 kDa, respectively. After disruption of bph genes, the bphA1 mutants lost the ability to grow on biphenyl, the bphB and bphD mutants were able to transform a little of biphenyl, but hardly grew on biphenyl. CONCLUSION: The cloned bph genes indeed play an important role in the biphenyl catabolism in this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This bph gene organization in Rhodococcus sp. R04 differs from that of other biphenyl degraders reported previously, indicating it is a novel type of bph gene cluster. Analysis of the phylogenetic tree suggested that BphA1 and BphA2 in Rhodococcus sp. R04 had a different evolutionary relationship with those in the other PCB degraders.     

Degradation of benzene by a Rhodococcus sp. using immobilized cell systems

The continuous degradation of benzene by a Rhodococcus sp. using free and immobilized cell systems was compared. Cell entrapment in calcium and strontium alginate beads and adhesion on support materials such as glass beads were found to be unsatisfactory. Degradation of benzene by cells immobilized in either ceramic or cellulose carriers proved to be more efficient than its non-immobilized counterpart. A retention time of 36 h was required to effect a 97% degradation of benzene using suspended free cells while cells immobilized on cellulose or ceramic carriers effected 97% degradation at 24 and 18 h, respectively. Recycling of the ceramic carriers was also possible and resulted in an even shorter retention time of 12h to effect a 97% degradation of benzene. Cell adhesion on the support materials was confirmed by scanning electron microscopy.     

红球菌Rhodococcus sp.DS-3 dszABC基因和dszD基因在大肠杆菌中的共表达

二苯并噻吩(DBT)及其衍生物微生物脱硫的4S途径需要4个酶(DszA,DszB,DszC and DszD)参与催化。其中DBT单加氧酶(DszC or DBT-MO)和DBT-砜单加氧酶(DszA or DBTO2-MO)都是黄素依赖型氧化酶,它们的催化反应需要菌体中还原型的黄素单核苷酸(FMNH2),FMNH2由辅酶黄素还原酶(DszD)再生。因此,共表达DszA,DszB,DszC和DszD可以提高整个脱硫途径的速率。构建了两个不相容性表达载体pBADD和paN2并在大肠杆菌中实现了4个脱硫酶基因的共表达。DszA,DszB,DszC和DszD的可溶性蛋白表达量分别占菌体总蛋白质的7.6%,3.5%,3.1%和18%。共表达时的脱硫活性是单独用paN2表达时的5.4倍,并对工程菌休止细胞脱除模拟柴油中DBT的活性进行了研究。     

Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp. AS2C isolated from the consortium

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)-1,3-dichloropropene) was enriched from an enhanced soil. This mixedculture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presenceof a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)-1,3-D, respectively. (E)-1,3-D was degraded faster than (Z)-1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)-1,3-D to (Z)-1,3-D orthe (Z) form to the (E) form did not occur.     

Cloning and expressing DBT (dibenzothiophene) monooxygenase gene (dszC) from Rhodococcus sp. DS-3 in Escherichia coli

Dibenzothiophene (DBT) monooxygenase (DszC)catalysis,the first and also the key step in the microbial DBT desulfurization,is the conversion of DBT to DBT sulfone (DBTO2).In this study,dszC of a DBT-desulfiaizing bacterium Rhodococcus sp.DS-3 was cloned by PCR.The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank.The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E.coli BL21 strain.The expression amount of DszC was about 20% of total supernatant at low temperature.The soluble DszC in the supematant was purified by Ni2+ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity.Only one band was detected by Western-blotting,which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3,paC5) and Rhodococcus sp.DS-3.The activity of purified DszC was 0.36 U.DszC can utilize the organic compound such as DBT and methyl-DBT,hut not DBT derivates such as DBF,which has no sulfur or inorganic sulfur.     

一株红球菌(Rhodococcus sp.)二噁英降解质粒的稳定性与接合转移特性

【目的】考察一株红球菌Rhodococcus sp.strain p52中的二噁英降解质粒pDF01(170 kb)和pDF02(242 kb)的稳定性和接合转移特性。【方法】在无选择压力的条件下对菌株p52进行连续传代培养,考察质粒pDF01、pDF02的丢失;以菌株p52为供体菌,以不同种属的菌株作受体菌,通过平板接合实验探讨质粒pDF01、pDF02接合转移的受体菌范围以及接合转移频率,利用菌落杂交、Southern杂交对质粒转移结果进行确认,利用降解实验测试转移质粒降解基因的表达。【结果】质粒pDF01和pDF02在红球菌p52中均具有较高的稳定性,在LB培养基上连续传代少于47次时pDF02可保持,连续传代少于65次时pDF01可保持。质粒pDF01和pDF02具备在同属和属间接合转移的能力,可向受体菌——紫红红球菌(Rhodococcus rhodochrous)、红串红球菌(Rhodococcus erythropolis)、大地两面神菌(Terrabacter tumescens)和节杆菌(Arthrobacter sp.)转移,其中以节杆菌作受体菌时质粒pDF01和pDF02接合转移频率最高,达到3.5×10~(-6)(接合子/受体菌);对节杆菌接合子质粒进行Southern杂交进一步确认了质粒pDF01、pDF02的存在。另外获得质粒pDF01、pDF02后的节杆菌接合子可以对二苯并呋喃高效利用,且降解能力与红球菌供体菌株p52相当。【结论】红球菌菌株p52可通过降解质粒转移强化生物修复过程,在去除环境中二噁英污染中具有良好的应用前景。     

Sulfur-selective desulfurization of dibenzothiophene and diesel oil by newly isolated Rhodococcus sp. strains

New desulfurizing bacteria able to convert dibenzothiophene into 2-hydroxybiphenyl and sulfate were isolated from contaminated soils collected in Mexican refineries. Random amplified polymorphic DNA analysis showed they were different from previously reported Rhodococcus erythropolis desulfurizing strains. According to 16S rRNA gene sequencing and fatty acid analyses, these new isolates belonged to the genus Rhodococcus. These strains could desulfurize 4,6-dimethyldibenzothiophene which is one of the most difficult dibenzothiophene derivatives to remove by hydrodesulfurization. A deeply hydrodesulfurized diesel oil containing significant amounts of 4,6-dimethyldibenzothiophene was treated with Rhodococcus sp. IMP-S02 cells. Up to 60% of the total sulfur was removed and all the 4,6-dimethyldibenzothiophene disappeared as a result of this treatment.     

Isolation and identification of berberine and berberrubine metabolites by berberine-utilizing bacterium Rhodococcus sp. strain BD7100

Based on the finding of a novel berberine (BBR)-utilizing bacterium, Rhodococcus sp. strain BD7100, we investigated the degradation of BBR and its analog berberrubine (BRU). Resting cells of BD7100 demethylenated BBR and BRU, yielding benzeneacetic acid analogs. Isolation of benzeneacetic acid analogs suggested that BD7100 degraded the isoquinoline ring of the protoberberine skeleton. This work represents the first report of cleavage of protoberberine skeleton by a microorganism.     

A novel lipopeptide produced by a Pacific Ocean deep-sea bacterium, Rhodococcus sp. TW53

Aims: Our goal was to find a novel, biosurfactant‐producing bacterium from Pacific Ocean deep‐sea sediments. Methods and Results: An oil‐degrading biosurfactant‐producing bacterium TW53 was obtained from deep‐sea sediment, and was identified through 16S rDNA analysis as belonging to the genus Rhodococcus. It lowered the surface tension of its culture to 34·4 mN m^?1. Thin layer chromatography (TLC) showed that the crude biosurfactants of TW53 were composed of lipopeptides and free fatty acids (FA). The lipopeptides were purified with column chromatography and then hydrolysed with 6 mol l^?1 HCl. Gas chromatography‐mass spectrometry analysis showed that the hydrolyte in the hydrophobic fraction contained five kinds of FA with chain lengths of C₁₄–C₁₉, and C₁₆H₃₂O₂ was a major component making up 59·18% of the total. However, 3‐hydroxyl FA was not found, although it is usually found in lipopeptides. Silica gel TLC revealed that the hydrolyte in the hydrophilic fraction was composed of five kinds of amino acids; consistently, ESI‐Q‐TOF‐MS analysis confirmed the composition results and provided their sequence tentatively as Ala‐Ile‐Asp‐Met‐Pro. Furthermore, the yield and CMC (critical micelle concentrations) of purified lipopeptides were examined. The purified product reduced the surface tension of water to 30·7 mN m^?1 with a CMC value of 23·7 mg l^?1. These results suggest that Rhodococcus sp. TW53 produces a novel lipopeptide that we have named rhodofactin. Conclusion: The deep‐sea isolate Rhodococcus sp. TW53 was the first reported lipopeptide‐producing bacterium of this genus. The lipopeptides had novel chemical compositions. Significance and Impact of the Study: Rhodococcus sp. TW53 has potential in the exploration of new biosurfactants and could be used in bioremediation of marine oil pollution.