Wa Gao

Statistical Optimization for Production of Carboxymethylcellulase of Bacillus amyloliquefaciens DL-3 by a Recombinant Escherichia coli JM109/DL-3 from Rice Bran Using Response Surface Method

The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran, tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498 rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before optimization.

Optimization of salts in medium for production of carboxymethylcellulase by a psychrophilic marine bacterium, Psychrobacter aquimaris LBH-10 using two statistical methods

The concentration of four mineral salts in the medium for the production of carboxymethylcellulase (CMCase) by Psychrobacter aquimaris LBH-10 were optimized using orthogonal array method (OAM) and response surface method (RSM) and their results from two statistical methods were compared. The analysis of variance (ANOVA) of data from central composite design (CCD) based on OAM indicated that potassium phosphate gave the highest percentage participation for cell growth as well as production of CMCase. However, their relative participations of four salts for cell growth were different from those for production of CMCase. The ANOVA of results from RSM indicated that highly significant factors (“probe>F” less than 0.0001) for cell growth were K2HPO4 and (NH4)2SO4, whereas those for production of CMCase were K2HPO4, NaCl, and MgSO4·7H2O. The optimal concentration of K2HPO4, NaCl, MgSO4·7H2O, and (NH4)2SO4 for cell growth extracted by Design Expert Software based on RSM were 7.10, 0.84, 0.24, and 0.33 g/L, respectively, whereas those for production of CMCase were 3.00, 0.52, 0.34, and 0.45 g/L. The optimal concentrations of salts for cell growth and production of CMCase using RSM basically coincided with those using OAM as well as those from ‘one-factor-at-a-time’ method. The production of CMCase by P. aquimaris LBH-10 with optimized concentrations of salts was 273.0 U/mL, which was enhanced by 1.27 times higher than the previous report.

Statistical Optimization for Production of Carboxymethylcellulase from Rice Hulls by a Newly Isolated Marine Microorganism Bacillus licheniformis LBH-52 Using Response Surface Method

A microorganism utilizing rice hulls as a substrate for the production of carboxymethylcellulase (CMCase) was isolated from seawater and identified as Bacillus lincheniformis by analyses of its 16S rDNA sequences. The optimal carbon and nitrogen sources for production of CMCase were found to be rice hulls and ammonium nitrate. The optimal conditions for cell growth and the production of CMCase by B. lincheniformis LBH-52 were investigated using the response surface method (RSM). The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that a highly significant factor (“probe＞F” less than 0.0001) for cell growth was rice hulls, whereas those for production of CMCase were rice hulls and initial pH of the medium. The optimal conditions of rice hulls, ammonium nitrate, initial pH, and temperature for cell growth extracted by Design Expert Software were 48.7 g/l, 1.8 g/l, 6.6, and 35.7℃, respectively, whereas those for the production of CMCase were 43.2 g/l, 1.1 g/l, 6.8, and 35.7℃. The maximal production of CMCase by B. lincheniformis LBH-52 from rice hulls under optimized conditions was 79.6 U/㎖ in a 7 l bioreactor. In this study, rice hulls and ammonium nitrate were developed to be substrates for the production of CMCase by a newly isolated marine microorganism, and the time for production of CMCase was reduced to 3 days using a bacterial strain with submerged fermentation.

Characterization of Acidic Carboxymethylcellulase Produced by a Marine Microorganism, Psychrobacter equimeris LBH-10

A microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Psychrobacter aquimaris by analysis of 16S rDNA sequences, and named P. aquimari LBH-10. This strain produced an acidic carboxymethylcellulase (CMCase), which hydrolyzed carboxymethylcellulose (CMC), cellobiose, curdlan, filter paper, p-nitrophenyl-β-D-glucopyranoside (pNPG), pullulan, and xylan, but there was no detectable activity on avicel and cellulose. The optimal temperature for CMCase produced by P. aquimari LBH-10 was 50℃ and more than 90% of its original activity was maintained at broad temperatures ranging from 20 to 50℃ after 24 hr. The optimal pH of the CMCase was 3.5, and more than 70% of its original activity was maintained under acidic conditions between pH 2.5 and 7.0 at 50℃ after 24 hr. The optimal pH of CMCase produced by P. aquimaris LBH-10 seems to be lower than those produced by any other bacterial and fungal strain. CoCl₂, EDTA, and PbCl₂ at a concentration of 0.1 M enhanced CMCase-produced P. aquimaris LBH-10, whereas HgCl₂, KCl, MnCl₂, NiCl₂, and SrCl₂ inhibited it.

Pilot-scale Optimization of Parameters Related to Dissolved Oxygen for Mass Production of Pullulan by Aureobasidium pullulans HP-2001

Parameters related to dissolved oxygen for the production of pullulan by Aureobasidium pullulans HP-2001 were optimized in 7 l and 100 l bioreactors. The optimal concentrations of glucose and yeast extract for the production of pullulan were 50.0 and 2.5 g/l, respectively, and its conversion rate from glucose was 37% at a flask scale. The optimal initial pH of the medium and temperature for cell growth were 7.5 and 30℃, whereas those for the production of pullulan were 6.0 and 25℃. The optimal agitation speed and aeration rate for cell growth were 600 rpm and 2.0 vvm in a 7 l bioreactor, whereas those for the production of pullulan were 500 rpm and 1.0 vvm. The production of pullulan with an optimized agitation speed of 500 rpm and aeration rate of 1.0 vvm was 18.13 g/l in a 7 l bioreactor. Maximal cell growth occurred without inner pressure, whereas the optimal inner pressure for the production of pullulan was 0.4 ㎏f/㎠ in a 100 l bioreactor. The production of pullulan under optimized conditions in this study was 22.89 g/l in a 100 l bioreactor, which was 1.38 times higher than that without inner pressure.

Enhanced production of heteropolysaccharide-7 by Beijerinckia indica HS-2001 in pilot-scaled bioreactor under optimized conditions involved in dissolved oxygen using sucrose-based medium

Heteropolysaccharide-7 (PS-7) is a possible alternative to xanthan or gellan used as food additives due to its properties and potential applications. Sucrose was developed as a carbon source for production of PS-7 by Beijerinckia indica HS-2001 to overcome catabolite repression against glucose. The inoculum size was established to be 5.0% (v/v) for the pilot-scaled production of PS-7 by B. indica HS-2001 based on productivity and economic aspect. The optimal agitation speed and aeration rate for cell growth of B. indica HS-2001 were found to be 495 rpm and 1.8 vvm using response surface methodology (RSM), whereas those for production of PS-7 were 440 rpm and 1.2 vvm. The optimal inner pressure for cell growth of B. indica HS-2001 in a 100 L bioreactor was 0.02 MPa, whereas that for production of PS-7 was 0.04 MPa. The production of PS-7 by B. indica HS-2001 from 30.0 g/L sucrose under an optimized inner pressure in a 100 L bioreactor was 10.20 g/L, which was 1.32 times higher than that without inner pressure. The maximal production of PS-7 under optimal conditions involved in the dissolved oxygen using the sucrose-based medium developed in this study was 1.55 times higher than that before optimization.

Enhanced production of carboxymethylcellulase of Bacillus subtilis subsp. subtilis A-53 by a recombinant Escherichia coli JM109/A-53 with pH and temperature shifts

Effect of shifts in pH of the medium and temperature on cell growth and production of carboxymethylcellulase (CMCase) by a recombinant Escherichia coli JM109/A-53 was investigated. Lower initial pH (pH 7.0) of the medium and higher temperature (40 °C) stimulated cell growth of E. coli JM109/A-53, whereas higher initial pH (pH 8.0) and lower temperature (35 °C) enhanced production of CMCase. The maximal production of CMCase was obtained when initial pH of 7.0 and temperature of 40 °C were simultaneously changed to 8.0 and 35 °C after 36 h. The production of CMCase with simple shifts in pH and temperature was 783.1 U/mL, which was 2.13 times higher than that under optimal conditions for cell growth. This indicated that a simple process with shifts in pH and temperature could be economically applied for enhanced production of CMCase by E. coli JM109/A-53 with industrial scales.

Enhanced production of cellobiase by marine bacterium Cellulophaga lytica LBH-14 from rice bran under optimized conditions involved in dissolved oxygen

The optimal conditions for production of cellobiase by C. lytica LBH-14 at flask scale had been previously reported. In this study, parameters involved in dissolved oxygen in 7 and 100 L bioreactors were optimized for pilot-scale production of cellobiase. The optimal agitation speed and aeration rate for cell growth of C. lytica LBH-14 were 400 rpm and 1.11 vvm in a 7 L bioreactor, whereas those for production of cellobiase were 330 rpm and 0.70 vvm. The analysis of variance (ANOVA) implied that significant factor for cell growth was the aeration rate, whereas those for production of cellobiase were the aeration rate as well as the agitation speed. The optimal inner pressures for cell growth and production of cellobiase by C. lytica LBH-14 in a 100 L bioreactor were 0.00 and 0.06 MPa, respectively. The maximal production of cellobiase in a 100 L bioreactor under optimized conditions using rice bran was 140.1 U/mL, which was 1.52 times higher than that in a flask scale.

Enhanced Production of carboxymethylcellulase by a marine bacterium, Bacillus velezensis A-68, by using rice hulls in pilot-scale bioreactor under optimized conditions for dissolved oxygen

The optimal conditions for the production of carboxymethylcellulase (CMCase) by Bacillus velezensis A-68 at a flask scale have been previously reported. In this study, the parameters involved in dissolved oxygen in 7 and 100 L bioreactors were optimized for the pilot-scale production of CMCase. The optimal agitation speed and aeration rate for cell growth of B. velezensis A-68 were 323 rpm and 1.46 vvm in a 7 L bioreactor, whereas those for the production of CMCase were 380 rpm and 0.54 vvm, respectively. The analysis of variance (ANOVA) implied that the highly significant factor for cell growth was the aeration rate, whereas that for the production of CMCase was the agitation speed. The optimal inner pressures for cell growth and the production of CMCase by B. velezensis A-68 in a 100 L bioreactor were 0.00 and 0.04 MPa, respectively. The maximal production of CMCase in a 100 L bioreactor under optimized conditions using rice hulls was 108.1 U/ml, which was 1.8 times higher than that at a flask scale under previously optimized conditions.

Enhanced Production of Gellan by Sphingomonas paucibilis NK-2000 with Shifts in Agitation Speed and Aeration Rate after Glucose Feeding into the Medium

Optimal agitation speed and aeration rate for the production of gellan by Sphingomnas paucibilis NK2000 in a 7 l bioreactor were found to be 400 rpm and 1.0 vvm. The best time for glucose feeding into the medium for enhanced production of gellan by S. paucibilis NK2000 was 36 hr after cultivation. The concentrations of gellan produced by S. paucibilis NK2000 from 1) 20.0 g/l glucose without additional feeding, 2) 20.0 g/l glucose with feeding of 200.0 g/l glucose at 36 hr, in which the final concentration in the medium was 10.0 g/l, 3) 20 g/l glucose with feeding of 200.0 g/l glucose and a shift in an agitation speed from 400 to 600 rpm, 4) 20.0 g/l glucose with feeding of 200.0 g/l glucose at 36 hr and shifts in an agitation speed from 400 to 600 rpm and an aeration rate from 1.0 to 1.5 vvm, 5) and 20.0 g/l glucose with feeding of 200.0 g/l glucose at 36 hr and shifts in an agitation speed from 400 to 600 rpm and an aeration rate from 1.0 to 2.0 vvm, were 5.19, 5.74, 6.73, 7.93, and 9.40 g/l, respectively, and their conversion rates from glucose were 26.0, 19.1, 22.4, 26.4, and 31.3%, respectively. Compared to those developed using a normal process, production of gellan by S. paucibilis NK2000 from 20.0 g/l glucose was 1.81 times higher, and and its conversion rate was 1.20 times higher when the optimized process developed in this study was used.