Degradation of 2,4-dinitrophenol by free and immobilized cells of Rhodococcus erythropolis HL PM-1

Degradation of 2,4-dinitrophenol (2,4-DNP) by the cells of Rhodococcus erythropolis HL PM-1 was studied. The enzymes involved in 2,4-DNP degradation were inducible, and their resynthesis took place during the process. Cell immobilization by embedding into agar gels decreased the degrader activity. Maximum rates of 2,4-DNP degradation by free and immobilized cells were 10.0 and 5.4 nmol/min per mg cells, respectively. The concentration dependence of 2,4-DNP degradation was typical of substrate inhibition kinetics. The immobilized cells were used in a model reactor designed for 2,4-DNP biodegradation. Its maximum capacity was 0.45 nmol/min per mg cells at a volumetric flow rate of 20 h-1. The reactor operated for 14 days without losing capacity; its half-lifetime equaled 16 days.     

A Reactor-Type Biosensor Based on Rhodococcus erythropolis HL PM-1 Cells for Detecting 2,4-Dinitrophenol

A model of a reactor-type biosensor based on the Rhodococcus erythropolis HL PM-1 was developed for amperometric detection of 2,4-dinitrophenol (2,4-DNP). The effects of the matrix material (agar and calcium alginate gels, ceramic support, and cellulose powder) on the biosensor signal concentration dependence, detection time, and biosensor stability were studied. In the case of bacterial cells immobilized on cellulose powder, the lower limit of 2,4-DNP detection was 20 M and the time of single analysis, the biosensor recovery included, was 30–50 min. In the continuous detection mode, the biosensor response was maintained at a stable level without biosensor inactivation for ten days. The biosensor can be used as an element of a complex analytical system for detecting nitroaromatic compounds in samples.     

Degradation of 2,4-dinitrophenol by two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2.

Two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2, were isolated from soil and river water by their abilities to utilize 2,4-dinitrophenol (0.5 mM) as the sole source of nitrogen. Although succinate was supplied as a carbon and energy source during selection, both isolates could utilize 2,4-dinitrophenol also as the sole source of carbon. Both strains metabolized 2,4-dinitrophenol under concomitant liberation of stoichiometric amounts of nitrite and 4,6-dinitrohexanoate as a minor dead-end metabolite.     

Degradation of 2,4-dinitrophenol by two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2.

Two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2, were isolated from soil and river water by their abilities to utilize 2,4-dinitrophenol (0.5 mM) as the sole source of nitrogen. Although succinate was supplied as a carbon and energy source during selection, both isolates could utilize 2,4-dinitrophenol also as the sole source of carbon. Both strains metabolized 2,4-dinitrophenol under concomitant liberation of stoichiometric amounts of nitrite and 4,6-dinitrohexanoate as a minor dead-end metabolite.     

Degradation of 2,4-Dinitrophenol by Free and Immobilized Cells of Rhodococcus erythropolis HL PM-1

The degradation of 2,4-dinitrophenol (2,4-DNP) by Rhodococcus erythropolis HL PM-1 was studied. The enzymes involved in 2,4-DNP degradation were inducible, and their resynthesis took place during the process. Cell immobilization by embedding into agar gels decreased the degrader activity. The maximum rates of 2,4-DNP degradation by free and immobilized cells were 10.0 and 5.4 nmol/min per mg cells, respectively. The concentration dependence of 2,4-DNP degradation was typical of substrate inhibition kinetics. The immobilized cells were used in a model reactor designed for 2,4-DNP biodegradation. Its maximum capacity was 0.45 nmol/min per mg cells at a volumetric flow rate of 20 h^–1. The reactor operated for 14 days without losing capacity; its half-life equaled 16 days.     

Degradation of phenol by Rhodococcus erythropolis UPV-1 immobilized on Biolite in a packed-bed reactor

A strain of Rhodococcus erythropolis has been isolated and identified by 16S rRNA sequencing. Cells acclimated to phenol can be adsorbed on the external surface of beads of the ceramic support Biolite where they grow forming a network of large filaments. Exponentially-growing cells were adsorbed faster than their stationary-phase counterparts. Immobilization resulted in a remarkable enhancement of the respiratory activity of cells and a shorter lag phase preceding the active phenol degradation. Under optimum operation conditions, the immobilized cells in a laboratory-scale column reactor packed with support beads were able to degrade completely phenol in defined mineral medium at a maximum rate of 18 kg phenol m(-3) per day. The performance of the bioreactor in long-term continuous operation was characterized by pumping defined mineral medium which contained different concentrations of phenol at different flow-rates. Once phenol biodegradation in defined mineral medium was well established, an industrial wastewater from a resin manufacturing company, which contained both phenol and formaldehyde, was tested. In this case, after wastewater conditioning (i.e. pH, nitrogen source and micronutrient amendments) the immobilized cells were able to remove completely formaldehyde and to partly biodegrade phenols at a rate of 1 kg phenol m(-3) per day.     

Host-vector system for phenol-degrading Rhodococcus erythropolis based on Corynebacterium plasmids

Two integrating vectors developed for use in Saccharomyces cerevisiae were successfully employed for cloned gene integration in the yeast Kluyveromyces lactis. A delta-integrating vector carrying the dominant selection marker neo allowed tandem integrations of a CUP1p-lacZ cassette into one or two chromosomal sites. A delta/UB-integrating vector, which contains a reusable selection cassette, enabled multiple rounds of integration and the sequential insertion of stable, dispersed copies of CUP1p-lacZ. Subsequent gene expression was closely correlated with integrated copy number illustrating the promise of this method for metabolic engineering in K. lactis. While both vectors contain an S. cerevisiae delta target sequence, the presence of delta-like elements in K. lactis has not been confirmed. Given the degree of illegitimate recombination in this yeast species, the insertions likely occurred at random locations in the chromosomes.     

Intensification of surfactant synthesis in Rhodococcus erythropolis 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 micromol/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.     

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.     

The hyperpolarizing and depolarizing effects of 2,4-dinitrophenol on ehrlich cells

The ability of glucose to reverse the effects of dinitrophenol on amino acid uptake in Ehrlich cells is a function of pH. At pH 6.0, the presence of glucose does not reverse the inhibitory action of the uncoupler. Nearly complete restoration occurs with glucose at pH 7.4. At pH 8, the presence of glucose may cause a modest increase in amino acid uptake in presence of dinitrophenol. At all pH values, glucose restores ATP and cellular K⁺ to the control levels at the same pH. Although the cytoplasmic pH changes with changes in the external pH, the cell interior is more alkaline than the medium near pH 6.0 and more acid than the medium at pH 7.8 even after 45 min incubation at 37°C. With dinitrophenol and in presence of glucose the difference in pH between the medium and the cell is minimal at both pH 6.0 and 7.8.     

Scaling of the process of biosynthesis of surfactants by Rhodococcus erythropolis 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.     

Biodegradation of phenol in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1 immobilized in an air-stirred reactor with clarifier

Phenol biodegradation by suspended and immobilized cells of Rhodococcus erythropolis UPV-1 was studied in discontinuous and continuous mode under optimum culture conditions. Phenol-acclimated cells were adsorbed on diatomaceous earth, where they grew actively forming a biofilm of short filaments. Immobilization protected cells against phenol and resulted in a remarkable enhancement of their respiratory activity and a shorter lag phase preceding active phenol degradation. Under optimum operation conditions in a laboratory-scale air-stirred reactor, the immobilized cells were able to completely degrade phenol in synthetic wastewater at a volumetric productivity of 11.5 kg phenol m(-3) day(-1). Phenol biodegradation was also tested in two different industrial wastewaters (WW1 and WW2) obtained from local resin manufacturing companies, which contained both phenols and formaldehyde. In this case, after wastewater conditioning (i.e., dilution, pH, nitrogen and phosphorous sources and micronutrient amendments) the immobilized cells were able to completely remove the formaldehyde present in both waters. Moreover, they biodegraded phenols completely at a rate of 0.5 kg phenol m(-3) day(-1) in the case of WW1 and partially (but at concentrations lower than 50 mg l(-1)) at 0.1 and 1.0 kg phenol m(-3) day(-1) in the cases of WW2 and WW1, respectively.     

The hyperpolarizing and depolarizing effects of 2,4-dinitrophenol on Ehrlich cells.

The ability of glucose to reverse the effects of dinitrophenol on amino acid uptake in Ehrlich cells is a function of pH. At pH 6.0, the presence of glucose does not reverse the inhibitory action of the uncoupler. Nearly complete restoration occurs with glucose at pH 7.4. At pH 8, the presence of glucose may cause a modest increase in amino acid uptake in presence of dinitrophenol. At all pH values, glucose restores ATP and cellular K+ to the control levels at the same pH. Although the cytoplasmic pH changes with changes in the external pH, the cell interior is more alkaline than the medium near pH 6.0 and more acid than the medium at pH 7.8 even after 45 min incubation at 37 degrees C. With dinitrophenol and in presence of glucose the difference in pH between the medium and the cell is minimal at both pH 6.0 and 7.8.     

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.     

Alterations in the survival of X-irradiated cells by 2,4-dinitrophenol depending on ATP deprivation.

The dose-survival curve of cultured melanoma cells was changed by post-irradiation treatment with 2,4-dinitrophenol (DNP). The parameters of the curves were Do = 147 R and n = 5 . 6 for untreated cells and Do = 143 R, n = 7 . 9 and Do = 142 R, n = 2 . 0 for the cells treated with 10(-5) M DNP and 5 x 10(-5) M DNP in phosphate-buffered saline, respectively. The content of ATP in the cell decreased to 5% of the control level after treatment with either concentration of DNP. The recovery of ATP content was rapid and complete after 2 hours' incubation in culture medium after the removal of 10(-5) M DNP, but was retarded and incomplete after 4 hours with 5 x 10(-5) M DNP. Thus prolonged ATP deprivation with a high concentration of DNP results in an inhibition of recovery and a reduction in the n-value.     

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).