Enhanced production of amidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket–Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.     

Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574

The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L(-1)) and achieved a critical micelle concentration (CMC) value of 120 mg L(-1).     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria <Emphasis Type="Italic">Mycobacterium neoaurum,Pimelobacter simplex</Emphasis>, and <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.     

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.     

Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO₄, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.     

Enhanced antibiotic production by Streptomyces sindenensis using artificial neural networks coupled with genetic algorithm and Nelder-Mead downhill simplex

Antibiotic production with Streptomyces sindenensis MTCC 8122 was optimized under submerged fermentation conditions by artificial neural network (ANN) coupled with genetic algorithm (GA) and Nelder-Mead downhill simplex (NMDS). Feed forward back-propagation ANN was trained to establish the mathematical relationship among the medium components and length of incubation period for achieving maximum antibiotic yield. The optimization strategy involved growing the culture with varying concentrations of various medium components for different incubation periods. Under non-optimized condition, antibiotic production was found to be 95 microgram/ml, which nearly doubled (176 microgram/ml) with the ANN-GA optimization. ANN-NMDS optimization was found to be more efficacious, and maximum antibiotic production (197 microgram/ml) was obtained by cultivating the cells with (g/l) fructose 2.7602, MgSO4 1.2369, (NH4)2PO4 0.2742, DL-threonine 3.069%, and soyabean meal 1.952%, for 9.8531 days of incubation, which was roughly 12% higher than the yield obtained by ANN coupled with GA under the same conditions.     

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.     

Optimization of fermentation medium for triterpenoid production from <Emphasis Type="Italic">Antrodia camphorata</Emphasis> ATCC 200183 using artificial intelligence-based techniques

In this study, alteration in morphology of submergedly cultured Antrodia camphorata ATCC 200183 including arthroconidia, mycelia, external and internal structures of pellets was investigated. Two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) were built to optimize the inoculum size and medium components for intracellular triterpenoid production from A. camphorata. Root mean squares error, R ², and standard error of prediction given by ANN model were 0.31%, 0.99%, and 0.63%, respectively, while RSM model gave 1.02%, 0.98%, and 2.08%, which indicated that fitness and prediction accuracy of ANN model was higher when compared to RSM model. Furthermore, using genetic algorithm (GA), the input space of ANN model was optimized, and maximum triterpenoid production of 62.84 mg l⁻¹ was obtained at the GA-optimized concentrations of arthroconidia (1.78 × 10⁵ ml⁻¹) and medium components (glucose, 25.25 g l⁻¹; peptone, 4.48 g l⁻¹; and soybean flour, 2.74 g l⁻¹). The triterpenoid production experimentally obtained using the ANN–GA designed medium was 64.79 ± 2.32 mg l⁻¹ which was in agreement with the predicted value. The same optimization process may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoid production from A. camphorata and to improve the production of bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Isolation of biosurfactant producers, optimization and properties of biosurfactant produced by Acinetobacter sp. from petroleum-contaminated soil

Aims: To screen and identify biosurfactant producers from petroleum‐contaminated soil; to use response surface methodology (RSM) for medium optimization to enhance biosurfactant production; and to study the properties of the newly obtained biosurfactant towards pH, temperature and salinity. Methods and Results: We successfully isolated three biosurfactant producers from petroleum‐contaminated soil and identified them through 16S rRNA sequence analysis, which exhibit the highest similarities to Acinetobacter beijerinckii (100%), Kocuria marina (99%) and Kineococcus marinus (99%), respectively. A quadratic response model was constructed through RSM designs, leading to a 57·5% increase of the growth‐associated biosurfactant production by Acinetobacter sp. YC‐X 2 with an optimized medium: beef extract 3·12 g l^?1; peptone 20·87 g l^?1; NaCl 1·04 g l^?1; and n‐hexadecane 1·86 g l^?1. Biosurfactant produced by Acinetobacter sp. YC‐X 2 retained its properties during exposure to a wide range of pH values (5–11), high temperatures (up to 121°C) and high salinities [up to 18% (w/v) Na⁺ and Ca²⁺], which was more sensitive to Ca²⁺ than Na⁺. Conclusions: Two novel biosurfactant producers were isolated from petroleum‐contaminated soil. Biosurfactant from Acinetobacter sp. YC‐X 2 has good properties to a wide range of pH, high temperature and high salinity, and its production was optimized successfully through RSM. Significance and Impact of the Study: The fact, an increasing demand of high‐quality surfactants and the lack of cost‐competitive bioprocesses of biosurfactants for commercial utilization, motivates researchers to develop cost‐effective strategies for biosurfactant production through isolating new biosurfactant producers with special surface‐active properties and optimizing their cultural conditions. Two novel biosurfactant producers in this study will widen our knowledge about this kind of micro‐organism. This work is the first application of RSM designs for cultural optimization of biosurfactant produced by Acinetobacter genus and the first report that biosurfactant may be more sensitive to Ca²⁺ than Na⁺.     

Extensive desulfurization of diesel by <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The resting cells of a new isolate of Rhodococcus erythropolis FSD-2 were used to desulfurize diesel fuels. About 97% of the total sulfur content in the hydrodesulfurized diesel was removed by the two consecutive biodesulfurization (BDS) processes with the majority (∼94%) being removed in the first treatment, resulting in diesel with a sulfur content of 5.7 μg ml⁻¹.     

Design of experiments and artificial neural network linked genetic algorithm for modeling and optimization of L-asparaginase production by <Emphasis Type="Italic">Aspergillus terreus</Emphasis> MTCC 1782

The sequential optimization strategy for design of an experimental and artificial neural network (ANN) linked genetic algorithm (GA) were applied to evaluate and optimize media component for L-asparaginase production by Aspergillus terreus MTCC 1782 in submerged fermentation. The significant media components identified by Plackett-Burman design (PBD) were fitted into a second order polynomial model (R² = 0.910) and optimized for maximum L-asparaginase production using a five-level central composite design (CCD). A nonlinear model describing the effect of variables on L-asparaginase production was developed (R² = 0.995) and optimized by a back propagation NN linked GA. Ground nut oil cake (GNOC) flour 3.99% (w/v), sodium nitrate (NaNO₃) 1.04%, L-asparagine 1.84%, and sucrose 0.64% were found to be the optimum concentration with a maximum predicted L-asparaginase activity of 36.64 IU/mL using a back propagation NN linked GA. The experimental activity of 36.97 IU/mL obtained using the optimum concentration of media components is close to the predicted L-asparaginase activity of the ANN linked GA.     

Enhanced decolourization of Direct Red-80 dye by the white rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> employing sequential design of experiments

Decolourization of Direct Red 80 (DR-80) by the white rot fungus Phanerochaete chrysosporium MTCC 787 was investigated employing sequential design of experiments. Media components for growing the white rot fungus were first screened using Plackett-Burman design and then optimized using response surface methodology (RSM), which resulted in enhancement in the efficiency of dye removal by the fungus. For determining the effect of media constituents on the dye removal, both percent dye decolourization and specific dye removal due to maximum enzyme activity were chosen as the responses from the experiments, and the media constituents glucose, veratryl alcohol, KH₂PO₄, CaCl₂ and MgSO₄ were screened to be the most effective with P values less than 0.05. Central composite design (CCD) followed by RSM in the optimization study revealed the following optimum combinations of the screened media constituents: glucose, 11.9 g l⁻¹; veratryl alcohol, 12.03 mM; KH₂PO₄, 23.08 g l⁻¹; CaCl₂, 2.4 g l⁻¹; MgSO₄, 10.47 g l⁻¹. At the optimum settings of the media constituents, complete dye decolourization (100% removal efficiency) and a maximum specific dye removal due to lignin peroxidase enzyme of 0.24 mg U⁻¹ by the white rot fungus were observed.     

Influence of the glycolipid-producing bacterium Rhodococcus erythropolis on the degradation of a hydrocarbon mixture by an original soil population

Summary The purpose of this investigation was to show whether or no employing a starter culture of the bacterium Rhodococcus erythropolis that produces 6,6-trehalosedicorynomycolates could replace the addition of purified biosurfactant known to accelerate hydrocarbon degradation by an original soil population in a stirred reactor. The rate of degradation, degree of elimination of hydrocarbons, mineralization and degree of oxidation were determined in order to assess the extent of degradation. In comparison with degradation by soil microorganisms only an acceleration of utilization of the hydrocarbons was observed in cultivations of growing cells of R. erythropolis, although the effect of purified trehalosedicorynomycolates is not reached. Except for a higher degree of oxidation a sufficient amount of trehalosedicorynomycolates bound to autoclaved biomass of R. erythropolis does not have any effect.     

The Role of Low-Molecular-Weight Nitrogen Compounds in the Osmotolerance of Rhodococcus erythropolis and Arthrobacter globiformis

Investigations showed that Rhodococcus erythropolis E-15 and Arthrobacter globiformis 2F cells respond to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid (80% of the intracellular free amino acid pool). The osmoprotective role of glutamic acid follows from its beneficial effect on the growth of bacteria in high-salinity media. It was found that the addition of this amino acid to the growth medium at a concentration of 2 mM shortened the lag phase and increased the growth rate and biomass yield of either of the two bacteria. The addition of another osmoprotectant, trehalose, to the high-salinity growth medium of R. erythropolis E-15 at the same concentration (2 mM), restored the growth parameters of this bacterium to the control values.     

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.     

A hybrid of back propagation neural network and genetic algorithm for optimization of collagen extraction from Malaysian cultured catfish (Hybrid Clarias sp.)

Fractional factorial design (FFD) was applied to evaluate the effects of various process parameters in influencing the extraction efficiency of pepsin soluble collagen (PSC) from muscles of cultured catfish (Clarias gariepinus×C. macrocephalus). Result of the first order factorial design showed that acetic acid concentration, acid extraction time, acetic acid to muscles ratio, and stirring speed posed significant effect (P<0.05) on the yield of PSC obtained at the end of the extraction process. Two different artificial intelligence techniques namely artificial neural network (ANN) and genetic algorithm (GA) were then integrated for optimizing the extraction conditions to obtain the highest yield of PSC. The ANN was trained using the back propagation algorithm. A model was successfully generated with R ² value of 0.9527 and MSE value of 0.1672 for unseen data set, implying a good generalization of the network. Input parameters of the established ANN model were subsequently optimized using GA. The hybrid of ANN-GA model predicted a maximum extraction yield of PSC at 238.25 mg/g under the following conditions: an acetic acid concentration of 0.70 M, the acetic acid to muscles ratio of 25.78 mL/g, and the stirring speed of 432.50 rpm. Verification of the optimization showed the percentage error differences between the experimental and predicted values were less than 5%, indicating excellent modeling, predicting ability and optimization by the ANN-GA model.     

Media optimization for the production of beta-carotene by Blakeslea trispora: a statistical approach

Blakeslea trispora (+) MTCC, Blakeslea trispora NRRL 2895 (+), Blakeslea trispora NRRL 2896 (-) as well as intraspecific mating of both the strain types have been studied for optimum production of beta-carotene. Intraspecific mating of both the strain types increased the yield of beta-carotene to a considerable level (98+/-2mg/l) as compared to wild strains. Effect of different media components such as carbon, nitrogen, and sulphates, and that of process variables such as pH and inoculum size on beta-carotene production by Blakeslea trispora in shake flask culture was investigated. One factor at-a-time method was employed for the optimization of media components. Response surface methodology (RSM) was further used to determine the optimum values of process variables for maximum beta-carotene production. The fit of the quadratic model was found to be significant. A significant increase in beta-carotene production (139+/-1mg/l) was achieved using RSM.     

Cell adaptation to solvent, substrate and product: a successful strategy to overcome product inhibition in a bioconversion system

Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (−)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to non-adapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h⁻¹ (ml organic phase)⁻¹] and high yield (0.96 g carvone g carveol⁻¹).     

Enhancement of solubilization and biodegradation of petroleum by biosurfactant from Rhodococcus erythropolis HX-2

Abstract

The demand to repair areas contaminated with hydrocarbon products has led to the development of new technologies for the treatment of contaminants in an unconventional method, that is, no physical or chemical methods are used. Biosurfactants are amphiphilic biomolecules produced by microorganisms that can be used in environments contaminated by petroleum products due to their unexceptionable tensile properties. Petroleum degrading strain Rhodococcus erythropolis HX-2 was found to be an effective producer of biosurfactants. The resulting biosurfactant (named NK) exhibits high physicochemical properties in terms of surface activity. It is capable of reducing surface tension from 54.99 to 28.89?mN/m and critical micelle concentration (CMC) is 100?mg/L. NK was found to be a substitute for chemically synthesized surfactants because of its higher solubilization efficiency for petroleum and polycyclic aromatic hydrocarbons, superior to SDS, Tween 80, Triton X-100 and Rhamnolipid (a wide used biosurfactant). In addition, it exhibits favorable emulsion stability over a wide range of pH (3–10), temperature (20–100?°C) and salinity ranges (5–20?g/L). It was found that the addition of biosurfactant can improve the efficiency of petroleum degradation, therefore it has potential applications in bioremediation.

• Highlights

• Rhodococcus erythropolis HX-2 is an effective petroleum degrading strain.

• HX-2 is a potential source of biosurfactant production.

• The biosurfactant NK reduces surface tension and exhibits high emulsification activity.

• The biosurfactant NK is effective over a wide range of temperatures, pH and salinity.

• The biosurfactant NK shows high solubilization efficiency for petroleum as well as polycyclic aromatic hydrocarbons.