Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 g l⁻¹) in the fermentation medium further increased naringinase production (8.9 IU ml⁻¹) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.     

Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein from Escherichia coli by fed-batch fermentation

The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett–Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments.     

Optimization of nutrient components for enhanced phenazine-1-carboxylic acid production by <Emphasis Type="Italic">gacA</Emphasis>-inactivated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18G using response surface method

The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’ optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l⁻¹ of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l⁻¹), glucose (12.7 g l⁻¹), soy peptone (10.9 g l⁻¹), and ethanol (13.8 ml l⁻¹) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l⁻¹, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l⁻¹ PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.     

Application of statistically-based experimental designs for the optimization of nisin production from whey

Statistically-based experimental designs were applied for the optimization of nisin production by Lactococcus lactis in a whey-based medium. Yeast extract, KH₂PO₄, and MgSO₄ were identified to have significant effects on nisin biosynthesis by a Plackett–Burman design. These three significant factors were subsequently optimized using central composite design, and the optimal conditions were determined to be 12.067 g l^–1 for yeast extract, 0.569 g l^–1 for KH₂PO₄, and 0.572 g l^–1 for MgSO₄. The validity of the optimal conditions was verified by a separate experiment.     

The effect of nutrients and environmental conditions on biomass and oil production in Botryococcus braunii Race B strains

The green alga Botryococcus braunii is widely recognized as a source of non-fossil oil. However, limitations in Botryococcus biomass production hamper its commercial exploitation. This study examines the effects of nutrients (nitrogen and iron) and environmental conditions (temperature, light intensity and photoperiod) on biomass and oil production in two B. braunii Race B strains, Kossou-4 and Overjuyo-3. The highest biomass and oil production were obtained at a nitrogen concentration of 750 mg l^?1, iron concentration of 6 mg l^?1, at 25°C and at 135 µmol photons m^?2 s^?1 with a photoperiod of 16 h light:8 h darkness. Culturing the strains in Blue-green (BG11) medium containing optimized nutrients under optimal conditions resulted in an up to ~10.6-fold increase in biomass. In Kossou-4 and Overjuyo-3 strains, biomass increased from 1.647 g 10 l^?1 and 3.137 g 10 l^?1 respectively in normal BG11 medium to 17.390 g 10 l^?1 and 21.721 g 10 l^?1 in optimized BG11 media and growth conditions. This was accompanied by ~8–10.5-fold increase in oil production compared with that in normal BG11 medium. Oil (0.324 g 10 l^?1 and 0.211 g 10 l^?1) was produced in normal BG11 medium in Kossou-4 and Overjuyo-3 strains respectively, compared with 2.642 g 10 l^?1 (Kossou-4) and 2.206 g 10 l^?1 (Overjuyo-3) in modified BG11 media under optimized conditions. Therefore, optimization of nutrients and environmental conditions can increase biomass and oil production in the two strains of B. braunii.     

Application of response surface methodology in medium optimization for spore production of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in solid-state fermentation

Summary Spore production of Coniothyrium minitans was optimized by using response surface methodology (RSM), which is a powerful mathematical approach widely applied in the optimization of fermentation process. In the first step of optimization, with Plackett–Burman design, soluble starch, urea and KH²PO⁴ were found to be the important factors affecting C. minitans spore production significantly. In the second step, a 2³ full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components for the maximum were obtained as follows: soluble starch 0.643 (36.43 g. l⁻¹), urea −0.544 (3.91 g l⁻¹) and KH²PO⁴ 0.049 (1.02 g l⁻¹) with a predicted value of maximum spore production of 9.94 × 10⁹ spores/g IDM. Under the optimal conditions, the practical spore production was 1.04 × 10¹⁰ spores/g IDM. The determination coefficient (R²) was 0.923, which ensure an adequate credibility of the model.     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens

Ruta graveolens in vitro cultures are a potential source of secondary metabolites (furanocoumarins) of significant medical interest. Experiments led in our laboratory showed that micropropagated shoots were richer in furanocoumarins than any other plant material. In order to optimize the molecule production by such cultivation systems, several factors related to the culture medium were studied. Effects of medium composition on biomass growth and furanocoumarin content were analysed and optimal conditions were determined for phosphate (300 mg l⁻¹ of NaH₂PO₄), nitrate (2527 mg l⁻¹ of KNO₃), carbon source (10 g l⁻¹ of sucrose) and phytohormones (2,4-dichlorophenoxyacetic acid (2,4-D) 50 μM and benzylaminopurine (BAP) 50 μM). Ruta shoot growth and furanocoumarin production were compared for optimized and standard culture conditions. Specific medium gave better results in terms of growth (t_D equal to 6.9 days against 8.6 for standard conditions) but no significant differences appeared concerning metabolite concentrations. However, the present study opens the way to scale-up studies with bioreactor cultivation systems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures

Aims: To study the optimization of submerged culture conditions for exopolysaccharide (EPS) production by Armillaria mellea in shake‐flask cultures and also to evaluate the performance of an optimized culture medium in a 5‐l stirred tank fermenter. Methods and Results: Shake flask cultures for EPS optimal nutritional production contained having the following composition (in g l^?1): glucose 40, yeast extract 3, KH₂PO₄ 4 and MgSO₄ 2 at an optimal temperature of 22°C and an initial of pH 4·0. The optimal culture medium was then cultivated in a 5‐l stirred tank fermenter at 1 vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150 rev min^?1 agitation speed, controlled pH 4·0 and 22°C. In the optimal culture medium, the maximum EPS production in a 5‐l stirred tank fermenter was 588 mg l^?1, c. twice as great as that in the basal medium. The maximum productivity for EPS (Q_p) and product yield (Y_P/S) were 42·02 mg l^?1 d^?1 and 26·89 mg g^?1, respectively. Conclusions: The optimal culture conditions we proposed in this study enhanced the EPS production of A. mellea from submerged cultures. Significance and Impact of the Study: The optimal culturing conditions we have found will be a suitable starting point for a scale‐up of the fermentation process, helping to develop the production of related medicines and health foods from A. mellea.     

Optimization of l‐DOPA production by Brevundimonas sp. SGJ using response surface methodology

l ‐DOPA (3,4‐dihydroxyphenyl‐l ‐alanine) is an extensively used drug for the treatment of Parkinson's disease. In the present study, optimization of nutritional parameters influencing l ‐DOPA production was attempted using the response surface methodology (RSM) from Brevundimonas sp. SGJ. A Plackett–Burman design was used for screening of critical components, while further optimization was carried out using the Box–Behnken design. The optimized levels of factors predicted by the model were pH 5.02, 1.549 g l^?1 tryptone, 4.207 g l^?1 l ‐tyrosine and 0.0369 g l^?1 CuSO₄, which resulted in highest l ‐DOPA yield of 3.359 g l^?1. The optimization of medium using RSM resulted in a 8.355‐fold increase in the yield of l ‐DOPA. The anova showed a significant R² value (0.9667), model F‐value (29.068) and probability (0.001), with insignificant lack of fit. The highest tyrosinase activity observed was 2471 U mg^?1 at the 18th hour of the incubation period with dry cell weight of 0.711 g l^?1. l ‐DOPA production was confirmed by HPTLC, HPLC and GC‐MS analysis. Thus, Brevundimonas sp. SGJ has the potential to be a new source for the production of l ‐DOPA.     

Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg’s medium vitamins, and 30 g l⁻¹ sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g⁻¹) and azadirachtin production (∼44 mg l⁻¹) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l⁻¹ dry weight. To enhance the production of azadirachtin, a Plackett–Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l⁻¹ sucrose, 0.19 g l⁻¹ potassium dihydrogen phosphate, 3.1 g l⁻¹ potassium nitrate, and 0.41 g l⁻¹ magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l⁻¹ and azadirachtin accumulation of 5.2 mg g⁻¹, which was equivalent to an overall azadirachtin production of 73.84 mg l⁻¹, 68% more than that obtained under non-optimized conditions.     

Optimal culture conditions for keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFR‐H49‐1

Aims: To investigate the effect of medium compositions and culture conditions on keratinase production by a novel thermophilic fungus Myceliophthora thermophila (Apinis) Oorschot strain GZUIFR‐H49‐1. Methods and Results: The thermophilic strain GZUIFR‐H49‐1 with keratinolytic ability was characterized and identified as a strain of M. thermophila on the basis of its morphological characters and molecular analysis of ITS1‐5.8S‐ITS2 rDNA sequence. Among the medium compositions tested, the soluble starch (SS), urea, sodium thiosulfate and CaCl₂ were the most effective C‐source, N‐source, S‐source and mineral ion, respectively, by employing the single‐factor experiment. The urea and pH value were the significant factors (P < 0·05) for the keratinase production in this experiment condition using Plackett–Burman factorial design. The conditions of keratinase production were further optimized by Box–Behnken design. Consequently, there was a 6·4‐fold increase (5100 U l^?1) in the keratinase activity than the initial value (800 U l^?1) by this optimal process. Conclusions: This study indicated that the optimization design proved a useful and powerful tool for the development of optimal medium compositions and culture conditions. Myceliophthora thermophila strain GZUIFR‐H49‐1 was a promising fungus strain for keratinase production. Significance and Impact of the Study: This study characterized a novel thermophilic M. thermophila strain GZUIFR‐H49‐1 with potential applications for keratinase production. These conditions of keratinase production obtained by means of optimization design will be accumulated as potential information for exploration and utilization to the new fungus isolate.     

Utilization of rice straw for laccase production by <Emphasis Type="Italic">Streptomyces psammoticus</Emphasis> in solid-state fermentation

Laccase production from a novel actinobacterial strain, Streptomyces psammoticus, MTCC 7334 was optimized in solid-state fermentation. The process parameters were initially optimized by the conventional “one factor at a time” approach, and the optimal levels of the factors that had considerable influence on enzyme production were identified by response surface methodology. Rice straw was identified as a suitable substrate for laccase production (17.3 U/g), followed by coffee pulp (15.8 U/g). Other optimized conditions were particle size, 500–1,000 μm (21.2 U/g); initial moisture content, 65% (26.8 U/g); pH of moistening solution, 8.0 (26.9 U/g); incubation temperature, 32°C (27.6 U/g) and inoculum size, 1.5 × 10⁷ CFU (33.8 U/g). Yeast extract served as the best nitrogen source (34.8 U/g). No enhancement in enzyme yield was observed with carbon supplementation. The level of yeast extract, inoculum size and copper sulphate were optimized statistically. Statistical optimization performed using a central composite design resulted in threefold increase in laccase activity (55.4 U/g) as compared to the unoptimized medium (17.3 U/g). The upgrading of fermented rice straw for fodder enhancement is also discussed briefly.     

beta-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant

Several wild strains and mutants of Rhodotorula spp. were screened for growth, carotenoid production and the proportion of -carotene produced in sugarcane molasses. A better producer, Rhodotorula glutinis mutant 32, was optimized for carotenoid production with respect to total reducing sugar (TRS) concentration and pH. In shake flasks, when molasses was used as the sole nutrient medium with 40 g l⁻¹ TRS, at pH 6, the carotenoid yield was 14 mg l⁻¹ and -carotene accounted for 70% of the total carotenoids. In a 14-l stirred tank fermenter, a 20% increase in torulene content was observed in plain molasses medium. However, by addition of yeast extract, this effect was reversed and a 31% increase in -carotene content was observed. Dissolved oxygen (DO) stat fed-batch cultivation of mutant 32 in plain molasses medium yielded 71 and 185 mg l⁻¹ total carotenoids in double- and triple-strength medium, respectively. When supplemented with yeast extract, the yields were 97 and 183 mg l⁻¹ total carotenoid with a 30% increase in -carotene and a simultaneous 40% decrease in torulene proportion. Higher cell mass was also achieved by double- and triple-strength fed-batch fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 327–332. Received 18 September 2000/ Accepted in revised form 02 March 2001     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l⁻¹) and at 72 h (751 mg l⁻¹) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30°C and 100 rpm. It reached 8373 mg l⁻¹ when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

Optimization of culture conditions for Aspergillus sojae expressing an Aspergillus fumigatus α-galactosidase

Using Response Surface Methodology, carbon and nitrogen sources and agitation speed for cultivation of Aspergillus sojae expressing the α-galactosidase gene, aglB of Aspergillus fumigatus IMI 385708 were optimized. Compared to cultivation in modified YpSs medium, cultivation in 250-mL Erlenmeyer flasks agitated at 276 rpm and containing 100 mL of optimized medium consisting of 10.5% molasses (w/v) and 1.3% NH₄NO₃ (w/v), 0.1% K₂HPO₄, and 0.005% MgSO₄·7H₂O achieved a 4-fold increase in α-galactosidase production (10.4 U/mL). These results suggest the feasibility of industrial large scale production of an α-galactosidase known to be valuable in galactomannan modification.     

Enhancement of oxytetracycline production after gamma irradiation-induced mutagenesis of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> CN08 strain

Streptomyces rimosus CN08 isolated from Tunisian soil produced 8.6 mg l⁻¹ of oxytetracycline (OTC) under submerged fermentation (SmF). Attempts were made for enhancing OTC production after irradiation-induced mutagenesis of Streptomyces rimosus CN08 with Co⁶⁰-γ rays. 125 OTC-producing colonies were obtained after screening on kanamycin containing medium. One mutant called Streptomyces rimosus γ-45 whose OTC production increased 19-fold (165 mg l⁻¹) versus wild-type strain was selected. γ-45 mutant was used for OTC production under solid-state fermentation (SSF). Wheat bran (WB) was used as solid substrate and process parameters influencing OTC production were optimized. Solid-state fermentation increased the yield of antibiotic production (257 mg g⁻¹) when compared with submerged fermentation. Ammonium sulphate as additional nitrogen source enhanced OTC level to 298 mg g⁻¹. Interestingly, OTC production by γ-45 mutant was insensitive to phosphate which opens the way to high OTC production even in medium containing phosphate necessary for optimal mycelia growth.     

Optimization of carbon and nitrogen sources in the medium and environmental factors for enhanced production of chitinase by Trichoderma harzianum

Statistical design was used to determine the optimal levels of medium components, the optimal initial pH of the enzyme production medium, the temperature of fermentation, age of the organism in the slant growth and the age of the inoculum for the production of chitinase in shake flask fermentations. The use of high concentrations of chitin and ammonium sulphate and exclusion of peptone and urea from the medium resulted in the production of higher level of the enzyme. The optimal concentrations of the medium components were 12.5 kg/m³ and 4.2 kg/m³ for the chitin and ammonium sulphate respectively. The effect of the addition of peptone and urea to the optimized medium was studied. The optimal values of initial pH and temperature were 5.6 and 28 °C respectively. The optimal age of the slant and the inoculum were found to be 105 h and 43 h respectively. The highest level of chitinase before optimization of the above variables was 0.054 U which was maximized to the level of 0.197 U.