Production of surfactants by Rhodococcus erythropolis strain EK-1, grown on hydrophilic and hydrophobic substrates

The ability of Rhodococcus erythropolis strain EK-1 to produce surfactants when grown on hydrophilic (ethanol and glucose) and hydrophobic (liquid paraffins and hexadecane) substrates was studied. The strain was found to produce surfactants with emulsifying and surface-active properties. The production of surfactants depended on the composition of the nutritive medium, nature and concentration of the sources of carbon and nitrogen, and duration of cultivation. Chemically, surfactants produced by Rhodococcus erythropolis EK-1 grown on ethanol are a complex of lipids with polysaccharide-proteinaceous substances. The lipids include glycolipids (trehalose mono- and dicorynomycolates) and common lipids (cetyl alcohol, palmitic acid, methyl n-pentadecanoate, triglycerides, and mycolic acids).     

Peculiarities of C<Subscript>2</Subscript> metabolism and intensification of the synthesis of surface-active substances in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 grown in ethanol

Oxidation of ethanol, acetaldehyde, and acetate in Rhodococcus erythropolis EK-1, producer of surface-active substances (SAS), is catalyzed by N,N-dimethyl-4-nitrosoaniline (DMNA)-dependent alcohol dehydrogenase, NAD⁺/NADP⁺-dependent dehydrogenases (optimum pH 9.5), and acetate kinase/acetyl-CoA-synthetase, respectively. The glyoxylate cycle and complete tricarboxylic acid cycle function in the cells of R. erythropolis EK-1 growing on ethanol; the synthesis of phosphoenolpyruvate (PEP) is provided by the two key enzymes of gluconeogenesis, PEP carboxykinase and PEP synthetase. Introduction of citrate (0.1%) and fumarate (0.2%) into the cultivation medium of R. erythropolis EK-1 containing 2% ethanol resulted in the 1.5-and 3.5-fold increase in the activities of isocitrate lyase and PEP synthetase (the key enzymes of the glyoxylate cycle and gluconeogenesis branch of metabolism, respectively) and of lipid synthesis, as evidenced by the 1.5-fold decrease of isocitrate dehydrogenase activity. In the presence of fumarate and citrate, the indices of SAS synthesis by strain R. erythropolis EK-1 grown on ethanol increased by 40–100%.     

Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil

Oil-oxidizing bacteria were isolated from oil-polluted soil and water samples and identified as Acinetobacter calcoaceticus K-4, Nocardia vaceinii K-8, Rhodococcus erythropolis EK-1, and Mycobacterium sp. K-2. It was found that immobilization of the bacteria on an expanded clay aggregate accelerated their growth and consumption of hydrocarbon substrates. It was also found that water polluted with 100 mg/l oil could be purified with Rhodococcus erythropolis EK-1 and Nocardia vaceinii K-8 cells immobilized in this way. The dependence of the degree of water purification on its flow rate, aeration, and availability of nitrogen and phosphorus sources was determined. The efficiency of water purification from oil by immobilized Rhodococcus erythropolis EK-1 cells at high flow rates (of up to 0.68 l/h), low aeration (of 0.1 l/l per min) and an intermittent supply of 0.01% diammonium phosphate reached 99.5–99.8%.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 58–63.Original Russian Text Copyright © 2005 by Pirog, Shevchuk, Voloshina, Gregirchak.     

The prospects of using bacteria of the genus Rhodococcus and microbial surfactants for the degradation of oil pollutants

The possibility of accelerating oil degradation by an enrichment culture of oil-oxidizing microorganisms in the presence of bacteria of the genus Rhodococcus and microbial surfactants was studied. It was shown that the degree of consumption of crude oil (2vol %) after 192 h of enrichment culture growth reached 84%. Inoculation of the active hydrocarbon-oxidizing strain Rhodococcus erythropolis EK-1 and exogenous surfactants produced by Pseudomonas sp. PS-27 increased this degree to 90 and 93–94%, respectively. On the grounds of these results, efficient methods of purification of the environment from oil pollutants can be developed.     

Production of a Bioflocculant by Rhodococcus erythropolis S-1 Grown on Alcohols

Rhodococcus erythropolis strain S-1, which was isolated from soil, produces a bioflocculant. We have found that alcohols are useful carbon sources for its flocculant production. Ethanol was best for flocculant production and culture time. The bioflocculant produced on ethanol medium flocculated a wide range of suspended soils, alkaline and acid.     

Scaling of the process of biosynthesis of surfactants by <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 on hexadecane

Peculiarities of synthesis of surface-active substances (SAS) are studied at periodical cultivation of Rhodococcus erythropolis EK-1 in the AK-210 fermenter on medium containing n-hexadecane. Maximum indicators of SAS synthesis (concentration of extra cellular SAS is 7.2 g/l; factor of emulsification of the cultural liquid 50%; SAS yield from the substrate 50%) have been observed at 60–70% concentration of dissolved oxygen from the saturation level with aerial oxygen (pH 8.0) fractional supply of the substrate by portions each being 0.3–0.4% every 5–6 h to a final volume concentration of 2.4% and with the use of 10% inoculate grown until mid-exponential phase on the medium with 1.0 vol % of n-hexadecane. Implementation of the process of SAS biosynthesis with the fermentation equipment provided the possibility to increase almost two-fold the amount of the synthesized SAS and reduce 3.5-fold the time of cultivation of the producer strain compared with the growth in flasks at shake-flask propagator.     

Intensification of surfactant synthesis in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP⁺-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.     

Surfactant production by the Rhodococcus erythropolis sH-5 bacterium grown on various carbon sources

It has been shown that the Rhodococcus erythropolis sH-5 strain can produce surfactants associated and not associated with the cell wall. Their content depends on medium composition, the nature of the carbon source, and oxygen supply. The highest biosurfactant (bioSF) yield is achieved by growing R. erythropolis sH-5 in medium with 2% kerosene at neutral pH. It has been found that the bioSF yield and emulsification index for various hydrocarbons depend on the kind of the nitrogen source used by the bacterium, increasing with replacement of KNO₃ by NaNO₃. The yields of biomass and bioSF in R. erythropolis depend on growth temperatures (max at 30°C) but not on water quality (bidistillate, catholyte, or anolyte). It has been found that sH-5 produces more cell-associated bioSF than extracellular species.     

Anthranilic Acid Production from Aniline by Rhodococcus erythropolisAN-13

When Rhodococcus erythropolis AN-13 grew on aniline, a fluorescent substance accumulated in the cultural fluid. It was obtained as crystals and identified as anthranilic acid (AnA). An A was also produced from aniline following incubation with resting cells of the bacterium grown on aniline. Heated cells lost the activity to produce it, and aniline was essential for its production. The production of AnA was promoted by sodium bicarbonate; when [¹⁴C]sodium bicarbonate was added to the incubation mixture, [¹⁴C]AnA was formed. The optimal pH for AnA production by the resting cells was 7.0 to 7.5. These results suggest that microbial activities of R. erythropolis AN-13 catalyzed the formation of AnA from aniline.     

<Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis,purification and concentration

Background  

The suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114.     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Cloning of a rhodococcal promoter using a transposon for dibenzothiophene biodesulfurization

The expression of biodesulfurization genes (dsz) in Rhodococcus erythropolis strain KA2-5-1 is repressed by sulfate which is the product of biodesulfurization. The application of a sulfate non-repressible promoter could be effective in enhancing biodesulfurization. A promoter-probe transposon was constructed using the promoterless, red-shifted green fluorescence protein gene (rsgfp). A 340 bp putative promoter element, designated kap1, was isolated from a strain KA2-5-1 recombinant that had shown high fluorescence intensity. The activity of kap1 was not affected by 1 mM sulfate. It gave about a 2-fold greater activity than the 16S ribosomal RNA promoter in R. erythropolis strain KA2-5-1 and is therefore useful for expressing desulfurization genes in rhodococcal strains.     

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.     

The prospects of using bacteria of the genus Rhodococcus and microbial surfactants for the degradation of oil pollutants

The possibility of accelerating oil degradation by an enrichment culture of oil-oxidizing microorganisms in the presence of bacteria of the genus Rhodococcus and microbial surfactants was studied. It was shown that the degree of consumption of crude oil (2% v/v) after 192 h of enrichment culture growth reached 84%. Inoculation of the active hydrocarbon-oxidizing strain Rhodococcus erythropolis EK-1 and exogenous surfactants produced by Pseudomonas sp. PS-27 increased this degree to 90% and 93-94%, respectively. On the grounds of these results, efficient methods of purification of the environment from oil pollutants can be developed.     

Potential of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> as a bioremediation organism

Summary The capability of Rhodococcus erythropolis CCM 2595(ATCC 11048) to utilize phenol, pyrocatechol, resorcinol, p-nitrophenol, p-chlorophenol, hydroquinone and hydroxybenzoate, respectively, or as respective binary mixtures with phenol, was described. This capability was found to depend on the substrate and its initial concentration. Some monoaromatic compounds had a suppressive effect on the strain’s ability to utilize phenol in a binary mixture and easily utilizable monoaromatics were strong inducers of the phenol 2-monooxygenase (EC 1.14.13.7). The capacity of R. erythropolis to colonize a synthetic zeolite was demonstrated and the enhancement of phenol tolerance of biofilms utilizing phenol was observed. The effect of humic acids on phenol killing was described and discussed as well. To allow use of recombinant DNA technology for strain improvement, methods of genetic transfer (transformation and conjugation) in R. erythropolis were established.     

Identification and structural characterisation of novel trehalose dinocardiomycolates from n-alkane-grown Rhodococcus opacus 1CP

Rhodococcus opacus 1CP, a potent degrader of (chloro-) aromatic compounds was found to utilise C₁₀–C₁₆ n-alkanes as sole carbon sources. Highest conversion rates were observed with n-tetradecane and n-hexadecane, whereas the utilisation of n-dodecane and n-decane was considerably slower. Thin-layer chromatography of organic extracts of n-alkane-grown 1CP cultures indicated the growth-associated formation of a glycolipid which was characterised as a trehalose dimycolate by ¹H-NMR spectroscopy and mass spectrometry. Total chain lengths between 48 and 54 carbons classify the fatty acid residues as nocardiomycolic acids. The presence of two double bonds in each mycolic acid is another feature that distinguishes the corresponding trehalose dinocardiomycolates from trehalose dicorynomycolates reported for Rhodococcus erythropolis DSM43215 and Rhodococcus ruber IEGM231. R. opacus 1CP was not found, even under nitrogen limitation, to produce anionic trehalose tetraesters which have previously been reported for R. erythropolis DSM43215.     

Production of 3,4-dihydroxyphthalate from phthalate by a membrane-bound two-enzyme system from Rhodococcus erythropolis

 Gram-positive Rhodococcus erythropolis strain S1 formed enzymes for the degradation of phthalate when grown in a phthalate-containing minimal medium. The membrane fraction prepared from phthalate-grown cells by ultrasonication converted phthalate to protocatechuate as the final product. Using two membrane-bound enzymes, phthalate 3,4-dioxygenase (PO) and 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH), prepared by solubilization of the membrane fraction, 3,4-dihydroxyphthalate was selectively obtained from phthalata. Fe²⁺ and Mn²⁺ stimulated the formation of 3,4-dihydroxyphthalate by the membrane-bound PO and PH system. Received: 27 April 1994/Received last revision: 19 August 1994/Accepted: 12 September 1994     

Desulfurization of light gas oil by a novel recombinant strain from Pseudomonas aeruginosa

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa NCIMB 9571 by using a transposon vector. Resting cells of the recombinant strain, PAR41, desulfurized 63 mg sulfur l^–1 of light gas oil (LGO) containing 360 mg S l^–1. The desulfurization activity for LGO by the resting cells of strain PAR41 grown with n-tetradecane (50% v/v) was much higher (1018-fold) than in glucose-grown cells. P. aeruginosa NCIMB 9571 is able to take up water-insoluble compounds from an oil phase which is enhanced by n-alkane.     

Identification of Aniline-assimilating Bacteria

Twenty bacterial strains, grown on aniline as a sole source of carbon and nitrogen, and isolated from soil, were identified by morphological and biochemical tests, and cell wall analyses. Eight isolates were Rhodococcus erythropolis (Gray and Thornton) Goodfellow and Alderson, five were Pseudomonas maltophilia Hugh and Ryschenkow and seven were DAB (diaminobutyric acid)-type coryneform bacteria. The Rhodococcus spp. maintained the ability to assimilate aniline, but the pseudomonads and coryneform bacteria readily lost the ability, when grown in the absence of aniline.     

Selection of trichloroethene (TCE) degrading bacteria that resist inactivation by TCE

Two isoprene (2-methyl-1,3-butadiene) utilizing bacteria, Alcaligenes denitrificans ssp. xylosoxidans JE 75 and Rhodococcus erythropolis JE 77, were identified as highly efficient cooxidizers of TCE, cis- and transdichloroethene, 1,1-dichloroethene and vinylchloride. Isoprene grown cells eliminate chloride from TCE in stoichiometric amounts and tolerate high concentrations of TCE.