Mechanistic Understanding of Gordonia sp. in Biodesulfurization of Organosulfur Compounds.

Although conventional oil refining process like hydrodesulfurization (HDS) is capable of removing sulfur compounds present in crude oil, it cannot desulfurize recalcitrant organosulfur compounds such as dibenzothiophenes (DBTs), benzothiophenes (BTs), etc. Biodesulfurization (BDS) is a process of selective removal of sulfur moieties from DBT or BT by desulfurizing microbes. Therefore, BDS can be used as a complementary and economically feasible technology to achieve deep desulfurization of crude oil without affecting the calorific value. In the recent past, members of biodesulfurizing actinomycete genus Gordonia, isolated from versatile environments like soil, activated sludge, human beings etc. have been greatly exploited in the field of petroleum refining technology. The bacterium Gordonia sp. is slightly acid-fast and has been used for unconventional but potential oil refining processes like BDS in petroleum refineries. Gordonia sp. is unique in a way, that it can desulfurize both aliphatic and aromatic organosulfurs without affecting the calorific value of hydrocarbon molecules. Till date, approximately six different species and nineteen strains of the genus Gordonia have been recognized for BDS activity. Various factors such as enzyme specificity, availability of essential cofactors, feedback inhibition, toxicity of organic pollutants and the oil-water separations limit the desulfurization rate of microbial biocatalyst and influence its commercial applications. The current review selectively highlights the role of this versatile genus in removing sulfur from fossil fuels, mechanisms and future prospects on sustainable environment friendly technologies for crude oil refining.

Analysis of genome sequence and trehalose lipid production peculiarities of the thermotolerant Gordonia strain.

Gordoniae are one of the most promising hydrocarbon-oxidizing actinobacteria. Here we present the genome sequence analysis of thermotolerant strain Gordonia sp. 1D isolated from oil-refinery soil. It is capable of alkane consumption and biosurfactant production at temperatures of up to 50°C. Gordonia sp. 1D demonstrates maximum biosurfactant production when grown on hexadecane, and at 40°C it was slightly higher than at 27°C: 35 and 39 mN/m, respectively. For the first time, it was experimentally confirmed that the carbohydrate component of extracellular biosurfactants produced by strain 1D is trehalose. In addition, genes for the production of trehalose lipid biosurfactants were identified. The genetic determinants for two different pathways for trehalose synthesis were found. The strain carries genes otsA and otsB involved in de novo trehalose biosynthesis. Moreover, the genes treY and treZ responsible for trehalose biosynthesis from maltooligosaccharides and starch or glycogen were identified.

Characterization and genomic analysis of highly efficient thermotolerant oil-degrading bacterium Gordonia sp. 1D.

A thermotolerant bacterial strain 1D isolated from refinery oil-contaminated soil was identified as Gordonia sp. based on the analysis of 16S rRNA and gyrB gene sequences. The strain was found to utilize crude oil, diesel fuel, and a wide spectrum of alkanes at temperatures up to 50 °C. Strain 1D is the first representative of Gordonia amicalis capable of utilizing alkanes of chain length up to С36 at a temperature of 45-50 °C. The degree of crude oil degradation by Gordonia sp. 1D at 45 °C was 38% in liquid medium and 40% in soil (with regard to abiotic loss). There are no examples of so effective hydrocarbon-oxidizing thermotolerant Gordonia in the world literature. The 1D genome analysis revealed the presence of two alkane hydroxylase gene clusters, genes of dibenzothiophene cleavage, and the cleavage of salicylate and gentisate - naphthalene metabolism intermediates. The highly efficient thermotolerant strain Gordonia sp. 1D can be used in remediation of oil-contaminated soils in hot climates.

Substrate specificity of ß-glucosidase from Gordonia terrae for ginsenosides and its application in the production of ginsenosides Rg3, Rg2, and Rh1 from ginseng root extract.

A ß-glucosidase from Gordonia terrae was cloned and expressed in Escherichia coli. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb1 was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg3 from Rb1 by the enzyme were pH 6.5, 30°C, 20 mg/ml enzyme, and 4 mg/ml Rb1. Under these conditions, G. terrae ß-glucosidase completely converted Rb1 and Re to Rg3 and Rg2, respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg1 to Rh1 at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg3, 1.47 mg/ml Rg2, and 1.17 mg/ml Rh1 from Rb1, Re, and Rg1, respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30°C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg2, Rg3, and Rh1 from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg3, Rg2, and Rh1.

Gordonia phosphorivorans sp. nov., isolated from a wastewater bioreactor with phosphorus removal.

Two Gram-stain-positive, non-endospore-forming actinobacteria (Ca8(T)and Ca14) were isolated from a bioreactor with extensive phosphorus removal. Based on 16S rRNA gene sequence similarity comparisons, strains Ca8(T) and Ca14 were shown to belong to the genus Gordonia and were most closely related to Gordonia hirsuta DSM 44140(T) (98.0 % sequence similarity) and Gordonia hydrophobica DSM 44015(T) (97.2 %). In comparison with the sequences of the type strains of all other species of the genus Gordonia tested, similarities were below 97 %. The quinone systems of the strains were determined to consist predominantly of MK-9H(2). The polar lipid profile for both organisms consisted of diphosphatidylglycerol, phosphatidylglycerol, phospatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Whole-organism hydrolysates contained meso-diaminopimelic acid as the diamino acid of the peptidoglycan; mycolic acids were detected as well. These chemotaxonomic traits and the major fatty acids, which were C(16 : 1)cis-9, C(16 : 0) and C(18 : 1) and tuberculostearic acid strongly supported the grouping of strains Ca8(T) and Ca14 into the genus Gordonia. The two strains showed a DNA-DNA similarity of 96 %. DNA-DNA hybridizations of strain Ca8(T) with G. hirsuta DSM 44140(T) and G. hydrophobica DSM 44015(T) resulted in values of 26.3 and 25.0 %, respectively. These results and those of the physiological and biochemical tests allowed a clear phenotypic differentiation of strains Ca8(T) and Ca14 from the most closely related species of the genus Gordonia. It is concluded that strains Ca8(T) and Ca14 represent a novel species, for which the name Gordonia phosphorivorans sp. nov. is proposed, with the type strain Ca8(T) (= DSM 45630(T) = CCUG 61533(T) = CCM 7957(T) = LMG 26648(T)).

Biodesulfurization of dibenzothiophene by Gordonia sp. AHV-01 and optimization by using of response surface design procedure.

A novel desulfurizing bacterium has been isolated from oil-contaminated soils in Khuzestan. The ability for dibenzothiophene desulfurization and its biochemical pathway were investigated. The bacterium was identified as Gordonia sp. AHV-01 (Genbank Accession No HQ607780) by 16S rRNA gene sequencing. HPLC results and Gibb's assay were shown that dibenzothiophene desulfurized via 4S-pathway Maximum growth (0.426 g dry cells/L) and produced 2-hydroxybiphenyl (63.1 microM) were observed at 120 h of cultivation. By using of response surface design procedure the optimization of pH, temperature and rotary shaker round on the desulfurization reaction of isolate AHV-01 were performed. The optimum conditions were determined at pH of 7.0, temperature of 30 degrees C and rotary shaker round of 180 rpm. At these conditions, the dibenzothiophene desulfurization activity was increased and maximum 2-hydroxybiphenyl production was detected 70.29 microM at 96 h. According to these results, Isolate AHV-01 was capable to desulfurize dibenzothiophene via 4S-pathway and likely it can be useful to reduce organic sulfur contents of crude oil.

Molecular detection and phylogenetic characterization of Gordonia species in heavily oil-contaminated soils.

This study was undertaken to assess genetic diversity among Gordonia species present in heavily oil-contaminated sites using both a culture-dependent and a culture-independent (PCR-denaturing gradient gel electrophoresis (DGGE)) approach. Soil samples for this purpose were collected from 8 different heavily (crude) oil-contaminated industrial park sites located around Kaohsiung County, Taiwan. Using Gordonia-specific PCR-DGGE, a significant increase in Gordonia species diversity was noted in 1% heavily oil-enriched soil. A total of 67 strains were scored and identified as Gordonia after genus-specific PCR amplification and sequencing. BOX-PCR fingerprinting of culturable Gordonia showed wide strain diversity. A total of 33 different strains were identified from most of the sampling sites. Based on gyrB gene sequence analysis, all Gordonia strains could be segregated into five major clusters. Gordonia amicalis was the predominant species in all oil-amended soil samples. Isolates sharing <98.5% gyrB gene sequence similarities with Gordonia type strains represent indigenous novel Gordonia species. Variations in phenotypic characteristics further confirm the presence of a wide range of species and strain diversity among Gordonia isolates. Based on the genotypic and phenotypic details obtained here, we conclude that heavily oil-contaminated soil supports diverse indigenous Gordonia strains.

Bioremediation potential of a tropical soil contaminated with a mixture of crude oil and production water.

A typical tropical soil from the northeast of Brazil, where an important terrestrial oil field is located, was accidentally contaminated with a mixture of oil and saline production water. To study the bioremediation potential in this area, molecular methods based on PCR-DGGE were used to determine the diversity of the bacterial communities in bulk and in contaminated soils. Bacterial fingerprints revealed that the bacterial communities were affected by the presence of the mixture of oil and production water, and different profiles were observed when the contaminated soils were compared with the control. Halotolerant strains capable of degrading crude oil were also isolated from enrichment cultures obtained from the contaminated soil samples. Twenty-two strains showing these features were characterized genetically by amplified ribosomal DNA restriction analysis (ARDRA) and phenotypically by their colonial morphology and tolerance to high NaCl concentrations. Fifteen ARDRA groups were formed. Selected strains were analyzed by 16S rDNA sequencing, and Actinobacteria was identified as the main group found. Strains were also tested for their growth capability in the presence of different oil derivatives (hexane, dodecane, hexadecane, diesel, gasoline, toluene, naphthalene, o-xylene, and p-xylene) and different degradation profiles were observed. PCR products were obtained from 12 of the 15 ARDRA representatives when they were screened for the presence of the alkane hydroxylase gene (alkB). Members of the genera Rhodococcus and Gordonia were identified as predominant in the soil studied. These genera are usually implicated in oil degradation processes and, as such, the potential for bioremediation in this area can be considered as feasible.

[Isolation and identification of a PAHs-degrading strain Gordonia sp. He4 and its dynamics during bioremediation of phenanthrene polluted soil].

A bacterial strain, He4, capable of degrading n-hexadecane and other polycyclic aromatic compounds was isolated from petroleum polluted soil. This strain was identified as Gordonia sp. He4 according to its morphology, physiological, biochemical properties and the analysis of its 16S rRNA gene sequence. Based on its 16S rRNA gene sequence, specific primers were designed and a competitor template was amplified by PCR. The dynamics of strain He4 in phenanthrene polluted soil was analyzed by colony forming unit (CFU) method and QC-PCR method. The results showed that partial of He4 become non-culturable and un-detectable by CFU method. But by using QC-PCR, the population density of strain He4 could be measured accurately.

Proposal of a method for the genetic transformation of Gordonia jacobaea.

AIMS: Gordonia jacobaea is a recently isolated bacterial species with potential industrial application on account of its ability to store large quantities of trans-canthaxanthin. Its genetic manipulation is, however, difficult and cumbersome owing to the presence of mycolic acids in the cell wall and, especially, because of current lack of knowledge about its basic genetics. The present work describes a method for the genetic transformation of G. jacobaea. METHODS AND RESULTS: Gordonia jacobaea was grown in media supplemented with different glycine, penicillin G and isoniazid concentrations. The temperature, carbon source, growth phase and ultrasounds were analyzed for improving the method efficiency. The cells were finally transformed by electroporation. Finally, the method was applied to Brevibacteriumlactofermentum and Gordonia bronchialis. CONCLUSIONS: The growth of G. jacobaea in the presence of glycine and isoniazid is essential for obtaining electrocompetents cells. The temperature, growth phase and ultrasounds appeared as the main factors for increasing the transformation efficiency. The use of shuttle plasmids became necessary. The method described can be used with other Corynebacteria species. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the importance of the CNM group (Corynebacteria, Nocardia and Mycobacteria genera) in different areas such as industry, bioremediation improve the knowledge of their molecular mechanisms are becoming essential. The method described here improves the genetic manipulation of this group of bacteria.