Augmented cellulase production by Bacillus subtilis strain MU S1 using different statistical experimental designs.

Optimization of the media components for cellulase production usingTrichoderma reeseiwas carried out. The optimization of cellulase production using pineapple waste as substrate was performed with statistical methodology based on experimental designs. The screening of nutrients and their influence on the cellulase production was studied using a Plackett-Burman design. Avicel, soybean cake flour, KH2PO4, and yeast extract were found to have the positive influence for the production of cellulase. The selected components were optimized using response surface methodology. The optimum concentrations are avicel: 26.5 g/L, soybean cake flour: 22.5 g/L, KH2PO4: 4.5 g/L, and yeast extract: 12.3 g/L. A maximum cellulase activity of 8.61 IU/mL was obtained under the optimized medium in the validation experiment.

L‐Asparaginase is a therapeutic enzyme widely used as an antitumor agent for the treatment of acute lymphoblastic leukemia and lymphosarcoma. Its potential applications as a therapeutic agent and food additive require production at high yields in a low‐cost medium with high purity and stability. Statistical experimental designs were applied to identify and optimize the significant parameters for the fermentative production of L‐asparaginase by Pectobacterium carotovorum Microbial Type Culture Collection (MTCC) 1428. Screening of different carbon sources identified galactose as the best carbon source for the production of L‐asparaginase. Twelve medium components were selected and sixteen experiments were carried out based on the Plackett–Burman (PB) design for identifying the most significant medium components. Yeast extract, galactose, monosodium glutamate, MgSO4.7H2O and CaCl2.2H2O were found to be the most significant components that affected the production of L‐asparaginase, and they were optimized by response surface methodology (RSM) using central composite design. This optimized medium showed maximum production rates of periplasmic and extracellular L‐asparaginase of 3.25 and 0.83 U ml−1, respectively. The periplasmic L‐asparaginase production in the optimized medium was twofold higher than in the basal medium. Unstructured kinetic models were applied and satisfactorily described the cell growth, product formation and substrate utilization profile, thus providing a better understanding and prediction of the fermentation profile. This paper details the application of PB experimental design and RSM for the optimization of L‐asparaginase production from Pe. carotovorum MTCC 1428 in submerged fermentation. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd.

The enhancement of the cellulase activity of Aspergillus nidulans by combinational optimization technique was investigated. The strain isolated from decayed, dry leaf of Ficus caricus was compared for the first time for its ability to produce cellulolytic enzyme in submerged fermentation (SmF). The medium ingredients enhancing the cellulase production were optimized by combinational statistical approach by one factor at a time methodology (OFAT), Plackett Burmann methodology (PB) and response surface methodology (RSM). A four-factor-five-level central composite design (CCD) was employed to determine the maximum activity of cellulase at optimum levels of carboxy methyl cellulose (CMC), ammonium nitrate and potassium dihydrogen phosphate at varying pH values. The optimum fermentation parameters were found to be 1.2 g/l CMC, 0.9 mg/l ammonium nitrate and 0.75 mg/l potassium dihydrogen phosphate at pH 6. The optimization of medium by combinational statistical approach led to the fine tuning of the cellulase production thereby enhancing the cellulase activity from 4.91 U/ml to 39.56 U/ml. The predicted results were in agreement with the actual experimental values. The cellulase activity obtained with this strain may be one of the best obtained in Aspergillus nidulans .

In this paper, we have attempted optimization of production of enzyme carboxymethylcellulase or endoglucanase from the bacterium Bacillus amyloliquefaciens SS35. Optimization has been carried out in two stages using statistical experimental design, viz. medium optimization and optimization of fermentation parameters. For medium optimization, Plackett–Burman design followed by central composite design (CCD) was used, while for optimization of fermentation parameters one-variable-at-a-time method followed by CCD was used. Carbon and nitrogen sources in the medium have been revealed to be the significant factors for enzyme production (carboxymethylcellulose 18.05 g/L; yeast extract 8 g/L and peptone 2 g/L). The inorganic salts have been found to be insignificant components of medium. Optimum fermentation parameters for optimized medium were: initial medium pH 5.65, incubation temperature = 40 °C, shaking speed = 120 rpm, and inoculum size = 6.96 %, v/v. Interestingly, the influence of all four parameters was almost independent with no interlinks. Secondly, the overall effect of all parameters was also low, as indicated by linear, square and interaction regression coefficients that were at least one order of magnitude lower than the intercept in the model equation. These results essentially meant that medium components dominate overall enzyme production process in comparison to fermentation parameters.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-013-0169-6) contains supplementary material, which is available to authorized users.

T HE CURRENT research deals with optimization of the factors affecting lipase production under submerged culture system. The most efficient isolate R1 was identified depending on cultural and morphological characteristics together with 18S rRNA sequence as Rhizopus oryzae. Using one variable at a time, the maximum lipase activity (171.8 U/mL) was recorded in the presence of 1% fish-frying oil, mixture of peptone and yeast extract at pH 5 with 8% v/v of fungal inoculum after 4 days at 30°C. The screening of the most significant factors using Plackett-Burman design revealed that among ten variables, four, i.e. incubation temperature, inoculum size, incubation period and agitation speed, affected significantly (p-values ranged from 0.003 to 0.049) on the lipase activity. Optimization by using response surface methodology (RSM) through central composite design (CCD) resulted in the highest predicted lipase activity (216.2 U/mL) in which fermentation medium was inoculated with 8% inoculum size and incubated at 28°C under agitation speed of 150 rpm for 4 days.

Paenibacillus sp. 1-49 is a nitrogen-fixing bacterium that has the potential as a fertilizer. However, it grows poorly (OD600≤1) in both mineral medium and rich medium. To achieve high-yield biomass, we optimized fermentation medium for Paenibacillus sp. 1-49. Plackett-Burman Design and Central Composite Design in response surface methodology were used to optimize medium composition. The optimal fermentation medium contained:(per liter) 36.22 g Sucrose, 5.31 g Tryptone, 10.92 g Yeast Extract, 0.51 g MgSO4, 3.5 g NaCl, 0.02 g Na2MoO4 and 0.02 g FeSO4. The maximum OD600 of 10.280±0.009 was obtained in shake flask fermentation, reached 94.6% of the predicted value. We succeeded in using a response surface methodology to optimize the fermentation medium for Paenibacillus sp. 1-49. Our results will be useful for largescale fermentation of this strain and other Paenibacillus spp.

A novel thermophilic lignocellulolytic bacterium, Geobacillus stearothermophilus TP-3, was isolated and characterized from the Tapovan hot spring in India. The cellulase production of TP-3 was optimized using a One-Factor-at-a-Time (OFAT) approach followed by a Plackett-Burman design, leading to a three-fold enhancement in enzyme yield. Phylogenetic analysis based on 16S rRNA sequencing revealed high sequence similarity with Geobacillus sp. H6a. The cellulase enzyme exhibited optimal activity at 50 °C under alkaline conditions (pH 8.0) and retained ~68% of its activity across a broad temperature range (40-70 °C) for up to three hours, demonstrating remarkable thermo-alkali stability. The ANOVA revealed that three factors-glucose, carboxymethyl cellulose (CMC), and yeast extract-significantly affected cellulase production, with yeast extract emerging as the most influential factor. Notably, TP-3 efficiently degraded agronomic residues, including wheat bran and sugarcane molasses, highlighting its potential for sustainable agricultural waste valorization and bioethanol production. The exceptional thermostability and lignocellulolytic potential of G. stearothermophilus TP-3 position it as a promising candidate for industrial bioconversion processes.

This study aimed to optimize the parameters, including medium formulations and culture conditions, for submerged fermentation (SmF) systems using a mutant strain of Trichoderma sp., Mut-4. Optimization was performed using the one-factor-at-a-time (OFAT) method to enhance cellulase activity and productivity. Parameters such as the blending ratio of carbon sources, type of nitrogen source, and initial pH were evaluated for their effects on enzyme activity and productivity. The optimal conditions were determined to be as follows: a 3:1 Avicel-to-cellulose ratio, yeast extract as the nitrogen source, and an initial pH of 5.5. Under these conditions, cellulase production was initiated earlier, and the activity of all cellulase components, along with protein concentration, increased by 1.17- to 1.43-fold at the flask scale and by 1.3- to 2.0-fold at the reactor scale. These results demonstrate the superior activity and productivity of Mut-4 under optimized conditions, highlighting its potential for application in large-scale cellulase production.

BackgroundCellulose is the major part of lignocellulosic biomass. It can be hydrolyzed into glucose units via specific enzymes called cellulases that have been applied in many commercial fields. There are several studies illustrate the influence of enzymes on apple juice clarification. However, to the best of our knowledge, the effect of microbial cellulase on volatile compounds of apple juice is not well known. The present study aimed to assess the effect of cellulase from a new bacterial isolate on the physicochemical properties of apple juice as well as volatile compounds. The hydrolysis of some polysaccharides (cellulose, hemicellulose, pectin) and polyphenols during apple juice production is necessary to reduce cloud sedimentation or color deterioration and increase the yield of juice. So, enzymes from new microbial isolates serve as processing aids to obtain clear juice with a high yield.ResultsCellulase-producing bacterium was isolated, characterized and molecularly identified as Bacillus licheniformis strain-MA1 with an accession number of ON840115. Optimization of medium parameters was implemented using Plackett–Burman design (PBd) followed by Box-Behnken design (BBd) of response surface methodology (RSM). The PBd revealed the three most important (significant) variables including carboxymethyl cellulose (CMC), corn cob, and peptone that had positive impact on cellulase production. Additionally, using the agricultural residue (corn cob) by the bacterial strain as a carbon source helps in reducing the costs of enzyme production, recycling the by-products, and preserving the environment. The optimized medium using PBd and BBd enhanced cellulase production from B. licheniformis strain-MA1 by 6.8-fold. A remarkable increase was observed in juice yield in enzyme treated-juice sample (88.2 ± 0.15%) in comparison with control juice (75.4 ± 0.09%). The total phenolic contents in cloudy and clarified apple juices were 0.957 ± 0.09 and 0.412 ± 0.03 mg/mL, respectively. Also, DPPH and FRAP assays showed a remarkable increase in antioxidant activity (Low IC50) in the control sample compared to enzyme treatment. Twenty-seven volatile compounds were extracted using headspace solid-phase microextraction-gas and analysis was performed by GC–MS. The identified volatile constituents belonged to several chemical classes: 15 esters; 6 alcohols; 4 aldehydes and 2 acids. The predominant class in apple juice volatile fraction was esters with a sweet and fruity odor.ConclusionThe crude cellulase obtained from the novel bacterial isolate B. licheniformis strain-MA1 was successfully applied as a clarifying agent in apple juice.

In previous study, we identified a novel protein elicitor AMEP412 from Bacillus subtilis BU412, which could trigger plant defense response and induce systemic acquired resistance against diseases. In this paper, the production of the new elicitor was optimised. First, optimal carbon source and nitrogen source were determined as glucose and yeast extract by single factor test. Then, effects of seven different factors on AMEP412 production was studied by Plackett-Burman design. Significant factors (yeast extract, culture temperature and culture time) were selected for further optimisation using central composite design by response surface methodology. The optimum conditions for AMEP412 production were yeast extract 21.72 g/L, culture temperature 30.84 °C and culture time 31.84 h. Under these conditions, the yield of AMEP412 was enhanced from 0.32 to 2.39 mg/mL (7.47-fold), which was quite close to the predicted one. This result significantly raised the yield of AMEP412, which laid solid foundation for its application as a biocontrol agent.

Statistical experimental designs were carried out for enhancing the ethanol production from cashew apple juice after obtaining the biochemical analysis of cashew apple juice. The biochemical analysis of the cashew apple juice was as follows: 79.2 % moisture, 12.5 % total sugars, 4.9 % reducing sugars, 0.16 % protein, 0.2 % fat, 1.91 % tannin and 1.13 % ascorbic acid. Cashew apple juice can be preserved at -18oC for several months. The Plackett -Burman design was used initially to screen seven nutritional parameters which are critical and important for growth of Saccharomyces cerevisiae as well as enhancing ethanol production. Among them three parameters were found to have a significant effect on ethanol production and these three elements were further optimized using central composite design (CCD) by response surface methodology. The optimal values of three variables obtained for maximum production of ethanol was (g/l): ammonium chloride 0.45, magnesium sulfate 0.08 and dipotassium phosphate 0.21 and the predicted ethanol production was 61.34 g/l. With the predicted optimal values, experiments conducted in the laboratory yielded an ethanol production of 59.80 g/l equal to 93.62% of its theoretical yield. Statistical experimental designs such as Plackett-Burman design and CCD are critical and important for rapid screening and determining significant nutritional parameters and then optimizing them for enhancing the ethanol production.

Keratinase is a class of proteases that degrade keratin into polypeptides and amino acids by breaking peptide and disulfide bonds in keratinous proteins. Protease is one of the largest industrial enzymes, the global protease market is increasing rapidly every year. In previous studies, a keratinase-producing microbe was isolated from Bleduk Kuwu which was identified as Bacillus haynesii BK1H. However, further study needs to be done to optimize the production by observing microenvironmental factors that influence keratinase production. In this study, screening some microenvironmental factors is reported. Screening factors including carbon sources, type and concentration of metal ions, agitation speed, amount of inoculum, pH, and temperature. Screening for these factors was started with the One Factor at A Time (OFAT) method and followed by the Plackett-Burman Design (PBD) method. In this study, sequential work was done: (1) Regeneration of Bacillus haynesii BK1H Microbes, (2) Preparation of Tyrosine Standard Series Solutions, (3) Screening of Factors Affecting Protease Fermentation of Bacillus haynesii BK1H using the One Factor at A Time (OFAT) method. ), and (4) Maintaining Significantly Influential Factors by Using Plackett-Burman Design (PBD). The results of the OFAT approach showed that the best condition for keratinase production was achieved at rice husk concentration, additional carbon source, of 1%; pH of 7; a temperature of 35°C; the amount of inoculum of 1%; agitation speed of 150 rpm; magnesium sulfate concentration of 0.04 g/mL, and calcium chloride concentration of 0.0005 g/m. Justification of those factors using PBD confirmed that only additional rice husk, magnesium sulfate and calcium chloride concentration, and agitation speed were significantly important toward keratinase production at selected experiment level limits.

The production of dextransucrase from Leuconostoc mesenteroides NRRL B-640 was investigated using statistical approaches. Plackett-Burman design with six variables, viz. sucrose, yeast extract, K(2)HPO(4), peptone, beef extract, and Tween 80, was used to screen the nutrients that significantly affected the dextransucrase production. 2(4)-Central composite design with four selected variables (sucrose, K(2)HPO(4), yeast extract, and beef extract) was used for response surface methodology (RSM) for optimizing the enzyme production. The culture was grown under flask culture with 100 ml optimized medium containing 30 g/l sucrose, 18.5 g/l yeast extract, 15.3 g/l K(2)HPO(4), and 5 g/l beef extract at 25 degrees C and shaking at 200 rpm gave dextransucrase with specific activity of 0.68 U/mg. Whereas the same optimized medium in a 3.0-l bioreactor (1.4 l working volume) gave an experimentally determined value of specific activity of 0.70 U/mg, which was in perfect agreement with the predicted value of 0.65 U/mg by the statistical model.

Production, optimization, partial-purification and pyrolysis kinetic studies of exopolysaccharide from a native brown-rot fungi Fomitopsis meliae AGDP-2

This study aimed to optimize the growth conditions for yeast strain Pichia kluyveri K-1 to maximize cell growth. The effect of several carbon sources (glucose, fructose, maltose, galactose, and glycerol) and their concentrations on the cell growth of P. kluyveri K-1 was examined using the one-factor-at-a-time method. Additionally, the Plackett-Burman design was used to assess the impact of process conditions (temperature and agitation speed) and components of the medium (yeast extract, ZnSO4·7H2O, riboflavin, biotin, MgSO4·7H2O, KH2PO4, MnCl2·4H2O and CaCl2) on the number of viable cells and biomass yield (response variables). The results of the one-factor-at-a-time and Plackett-Burman design studies revealed that temperature, agitation speed, and glucose concentration were the main factors affecting cell growth. The Box-Behnken design and response surface method were used to evaluate the impact of factors, temperature (20-30 °C), agitation speed (40-200 rpm), and glucose concentration (0.5-5% (w/v)), as well as to optimize the process. Based on the experimental results, the predicted models for both response variables were statistically significant. In both predicted models, the agitation speed and glucose concentration had significant impacts on the response variables. The optimum conditions for maximization of a number of viable cells and biomass yield were 21.9 °C, 184 rpm, and 4.8% (w/v) of glucose. Under these optimal conditions, the predicted values of the number of viable cells and biomass yield were 8.68 log CFU/mL and 58.40 g/L, respectively. Validation of the optimal conditions at the experimental level proved the suitability of the proposed models.