Basal Production Medium Research Articles

Optimization of culture parameters for α-glucosidase production from suspension culture of moss Hyophilla nymaniana (Fleish.) Menzel

BackgroundBryophytes, comprising of the second largest group in the plant kingdom, has attracted a great deal of attention in recent years due to its immense potential to produce biopharmaceuticals. But studies conducted to better understand their chemical composition are limited and scattered. In the present investigation, sequential optimization strategy, based on statistical experimental designs, was employed to enhance the production of α-glucosidase enzyme from moss Hyophilla nymaniana (Fleish.) Menzel by using Taguchi methodology. L16 orthogonal array and five physical parameters including sugar, temperature, pH, rpm, nitrogen source were considered as key parameters for enzyme production. The optimal level of each parameter for maximum glucosidase production by the moss was determined. Analysis of variance (ANOVA) was performed to evaluate statistically significant process factors. ResultsBased on statistical analysis (ANOVA), the optimal combinations of the major constituents of media for maximal α-glucosidase production were evaluated as follows: dextrose 2% contributed maximum on α-glucosidase production followed by ammonium nitrate 1.5%, temperature 24 °C, pH 5.6, and RPM 120. Predicted results showed an enhanced glucosidase (53%) than the basal production medium. ConclusionThe present study highlighted that Taguchi design of experiments approach is better than the conventional optimization technique to determine the optimum level of each of the significant parameters that brings maximum enzyme production.

Influence of bioprocess variables on the production of extracellular chitinase under submerged fermentation by Streptomyces pratensis strain KLSL55.

Chitinases are the enzymes which are capable of hydrolyzing chitin to its monomer N-acetyl glucosamine (GlcNac). Present study emphasizes on the impact of critical process variables on the production of chitinase from Streptomyces pratensis strain KLSL55. Initially the isolate was noticed to produce 84.67 IU chitinase in basal production medium. At optimization of bioprocess variables, the physical parameters pH of 8.00, 40 °C of incubation temperature, agitation speed of 160 rpm and 1.25 mL of spore suspension were found optimum for improved production of chitinase. Further, formulated production medium with 1.5% colloidal chitin, 1.25% fructose greatly influenced the chitinase production. At all described optimum conditions with formulated production media, a total of 14.30-fold increment was achieved in the chitinase production with final activity of 1210.67 IU when compared to the initial fermentation conditions in basal production medium.

Bioprocess Development Employing Design of Experiments for Antibiotic Production from Streptomyces parvulus Strain Sankarensis-A10

Optimization of antibiotic production medium and fermentation conditions of Streptomyces parvulus strain sankarensis-A10 was studied using classical and statistical methods. After selection of the basal production medium, the effect of sea water and incubation time on antibiotic production was investigated by classical method. In the first step of optimization, factors that significantly affect antibiotic production such as starch, K2HPO4, FeSO4.7H2O, KNO3 and pH were selected based on Plackett-Burman design. In the second step, three significant factors, starch, KNO3, pH, with a positive effect were optimized with a 33 factorial Box-Behnken design and analysed using response surface analysis. The optimal values for maximum antibiotic production were as follows, starch- 20 g/l, pH- 8.0 and KNO3- 3 g/l. Under optimal conditions, the antibacterial activity was found to be 620 U/ml and 616.66 U/ml against Staphylococcus aureus and Escherichia coli, respectively, which indicated that the activity was 1.4 folds higher in contrast to antibacterial activity prior to optimization. TLC-based partial purification of crude extract and bioassay of the fractions revealed the presence of four active compounds with Rf values of 0.275, 0.2, 0.125 and 0.062. These could probably be novel bioactive compound(s) in the crude extract, which could prove to be a novel source of antibiotics.

Production of thermostable extracellular α-amylase by a moderate thermophilic Bacillus licheniformis isolated from Qinarje Hot Spring (Ardebil prov. of Iran)

Background and Purpose: Amylases are most important industrial enzymes that account for about 30% of the world’s food, feed, fermentation, textile, detergent and cellulosic industries. This study aimed at optimum production of thermostable α-amylase via moderate thermophilic bacterium (Bacillus licheniformis) which was recently isolated from Qinarje Hot spring.Material and Methods: Initially, ability of bacterium for amylase activity was determined by starch hydrolysis test using Gram’s iodine staining. Then bacterial growth pattern and amylase production curves in basal production medium were graphically determined at different time intervals. Finally, effect of different temperature, pH, carbon source, nitrogen source, minerals and inoculum size were studied on bacterial growth and amylase production using turbidimetric and DNS method, respectively.Results: Optimum enzyme production achieved after 84 hours of inoculation from cultures growing at 40 ˚C and pH 9.0 in a medium containing 0.03% (w/v) of CaCl2, compared to the basal medium, results showed that the best enzyme production happened with inoculum size of 4% (v/v). The addition of 1% (w/v) rice husk (as a Carbon source) enhanced enzyme productivity up to 160% and substitution of the peptone and yeast extract with 1% (w/v) of tryptone (as a Nitrogen source) increased the α-amylase production up to 160%.Conclusion: Our findings show that B. licheniformis-AZ2 strain has an ability to produce the thermostable α-amylase which is suitable in starch processing and food industries. To be commercialized, further investigation is required for enhancement of the enzyme production.Keywords: Bacillus licheniformis; Optimization; Basal medium; Agricultural by-products.

Characterization of saltern based Streptomyces sp. and statistical media optimization for its improved antibacterial activity.

A moderately halotolerant Streptomyces strain, designated JAJ13 was characterized and a culture medium was statistically optimized to improve its antibacterial activity. Based on the phenotypic and molecular characteristics, strain JAJ13 was identified as a moderately halotolerant Streptomyces sp. JAJ13. Novelty of the strain JAJ13 in production of antibacterial compound was assessed by sequence analysis of KSα gene and LC-MS analysis of the active compound. Optimization of the culture medium for antibacterial compound production by the strain JAJ13 was performed with statistical methodology based on experimental designs. Initially, a starch based basal production medium was selected out of eight different production media screened for antibacterial compound production by Streptomyces sp. JAJ13. Plackett-Burman design was employed to screen the influential media components affecting the antibacterial compound production. Subsequently, statistical optimization of selected medium components was performed by employing the response surface methodology (RSM) with Box-Behnken design. The optimum initial level of CuSO4.5H2O, (NH4)2SO4 and K2HPO4 for the highest antibacterial activity was determined to be at 4.45 mg, 1.96 g, and 1.15 g in 1 L of distilled H2O, respectively. PBD and RSM guided design of experiments resulted in a maximum antibacterial activity of 23.37 ± 2.08 mm, which is a 78.8% increase in comparison with that obtained in the unoptimized medium. This study points the success of statistical model in developing an optimized production media for enhanced antibacterial compound production by Streptomyces sp. JAJ13.

Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6T

Levan is a bioactive fructan polymer that is mainly associated with high-value applications where exceptionally high purity requirements call for well-defined cultivation conditions. In this study, microbial levan production by the halophilic extremophile Halomonas smyrnensis AAD6(T) was investigated systematically. For this, different feeding strategies in fed-batch cultures were employed and fermentation profiles of both shaking and bioreactor cultures were analyzed. Initial carbon and nitrogen source concentrations, production pH, NaCl and nitrogen pulses, nitrogen and phosphorous limitations, trace elements and thiamine contents of the basal production medium were found to affect the levan yields at different extends. Boric acid was found to be the most effective stimulator of levan production by increasing the sucrose utilization three-fold and levan production up to five-fold. This significant improvement implied the important role of quorum sensing phenomenon and its regulatory impact on levan production mechanism. Levan produced by bioreactor cultures under conditions optimized within this study was found to retain its chemical structure. Moreover, its biocompatibility was assessed for a broad concentration range. Hence H.smyrnensis AAD6(T) has been firmly established as an industrially important resource microorganism for high-quality levan production.

Optimization of Process Parameters by Statistical Experimental Designs for the Production of Naringinase Enzyme by Marine Fungi

Naringinase has attracted a great deal of attention in recent years due to its hydrolytic activities which include the production of rhamnose and prunin and debittering of citrus fruit juices. Screening of fifteen marine-derived fungi, locally isolated from Ismalia, Egypt, for naringinase enzyme production, indicated thatAspergillus nigerwas the most promising. In solid state fermentation (SSF) of the agroindustrial waste, orange rind was used as a substrate containing naringin. Sequential optimization strategy, based on statistical experimental designs, was employed to enhance the production of the debittering naringinase enzyme. Effects of 19 variables were examined for their significance on naringinase production using Plackett-Burman factorial design. Significant parameters were further investigated using Taguchi’s (L1645) orthogonal array design. Based on statistical analysis (ANOVA), the optimal combinations of the major constituents of media for maximal naringinase production were evaluated as follows: 15 g orange rind waste, 30 mL moisture content, 1% grape fruit, 1% NaNO3, 0.5% KH2PO4, 5 mM MgSO4, 5 mM FeSO4, and the initial pH 7.5. The activity obtained was more than 3.14-fold the basal production medium.

Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium

In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L(-1) starch, 30 g L(-1) soya bean and 9 g L(-1) sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.

Cholesterol oxidase production (COD) by a new isolate characterized as Streptomyces sp. was studied in different production media and fermentation conditions. Individual supplementation of 1 % maltose, lactose, sucrose, peptone, soybean meal and yeast extract enhanced COD production by 80-110 % in comparison to the basal production medium (2.4 U/ml). Supplementation of 0.05 % cholesterol (inducer) enhanced COD production by 150 %. COD was purified 14.3-fold and its molecular weight was found to be 62 kDa. Vmax (21.93 μM/min mg) and substrate affinity Km (101.3 μM) suggested high affinity of the COD for cholesterol. In presence of Ba(2+) and Hg(2+) the enzyme activity was inhibited while Cu(2+) enhanced the activity nearly threefold. Relative activity of the enzyme was found maximum in triton X-100 whereas sodium dodecyl sulfate inactivated the enzyme. The enzyme activity was also inhibited by the thiol-reducing reagents like Dithiothreitol and β-mercaptoethanol. The COD showed moderate stability towards all organic solvents except acetone, benzene and chloroform. The activity increased in presence of isopropanol and ethanol. The enzyme was most active at pH 7 and 37 °C temperature. This organism is not reported to produce COD.

English

  Bacillus sp. SW1-2 producing poly glutamic acid (PGA), locally isolated from Eastern province in Saudi Arabia, was characterized and identified based on 16S rRNA gene sequencing. Phylogenetic analysis revealed its closeness to Bacillus megaterium. The homopolymer consists mainly of glutamic as indicated in the analysis of amino acid. Preliminary optimization of PGA production, through a series of one-variable-at-a-time (OVAT) experiments, revealed maximum PGA production of 10.5 (g/L). Statistically based experiments were applied to optimize culture conditions for production of PGA. Effect of 15 variables were examined for their significance on PGA production using Plackett-Burman factorial design. Among those variables; Na-citrate, (NH4)2SO4, glutamic acid and CaCl2.2H2O were most significant variables that encouraged PGA production, while those negatively affected were; glycerol, glucose and phosphate buffer. Significant parameters were further investigated using Box-Behnken design to define the optimal medium composition. Based on statistical analysis, maximal PGA production on optimized medium was 36.5 (g/L) which was more than 4-folds the basal production medium. Verification experiment was carried out to examine model validation and revealed more than 95% validity.   Key words: Poly glutamic acid, Bacillus megaterium, statistical experimental design, optimization, one-variable-at-a-time (OVAT).

Studies on optimum fermentation conditions for actinomycin-D production by two new strains of Streptomyces spp.

Two biologically active microbial strains were isolated from soil samples collected from different geographical areas of India. These strains were characterized as Streptomyces halstedii MTCC 6817 and Streptomyces anulatus MTCC 6818. Active compounds were extracted from the purified fermented broth and one of them was chemically characterized as actinomycin-D. Though these two strains produced the same antibiotic, their nutritional requirements vary substantially. For S. halstedii, a medium containing 1.5% fructose, 0.05% N-acetylglucosamine, and basal production medium, gave the maximum titre, 754% higher, as compared to production medium alone. In the case of Streptomyces anulatus, maximum antibiotic production was attained with a production medium containing 1.5% galactose and 0.05% L-asparagine monohydrate.

Improved production of Pseudomonas aeruginosa uricase by optimization of process parameters through statistical experimental designs

Sequential optimization strategy, based on statistical experimental designs, was employed to enhance the production of uricase by Pseudomonas aeruginosa local isolate. Glucose supplementation to the basal production medium inhibits uricase production, perhaps by catabolic repression. For screening of bioprocess parameters significantly influencing uricase activity, the two-level Plackett–Burman design was used. Among fifteen variables tested; pH, CuSO 4 and FeSO 4 were selected based on their high significant effect on uricase activity. A near optimum medium formulation was obtained using this method with increased uricase yield by 15-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box–Behnken design was employed. A polynomial model was created to correlate the relationship between the three variables and uricase activity. The optimal combination of the major constituents of media for uricase production evaluated from the non-linear optimization algorithm of EXCEL-Solver was as follows: pH, 5.5; CuSO 4, 10 −3 M; and FeSO 4, 10 −2 M. The predicted optimum uricase activity was 7.1 U/ml/min, which is 16.5 times than the basal medium.