Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Other Two-Level Factorial Designs <s> A Bacillus subtilis strain isolated from contaminated soil from a refinery has been screened for biosurfactant production in crystal sugar (sucrose) with different nitrogen sources (NaNO3, (NH4)2SO4, urea, and residual brewery yeast). The highest reduction in surface tension was achieved with a 48-h fermentation of crystal sugar and ammonium nitrate. Optimization of carbon/nitrogen ratio (3, 9, and 15) and agitation rate (50, 150, and 250 rpm) for biosurfactant production was carried out using complete factorial design and response surface analysis. The condition of C/N 3 and 250 rpm allowed the maximum increase in surface activity of biosurfactant. A suitable model has been developed, having presented great accordance experimental data. Preliminary characterization of the bioproduct suggested it to be a lipopeptide with some isomers differing from those of a commercial surfactin. <s> BIB001 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Other Two-Level Factorial Designs <s> Abstract In this work, optimizing trace element composition was attempted as a primary strategy to improve surfactin production from Bacillus subtilis ATCC 21332. Statistical experimental design (Taguchi method) was applied for the purpose of identifying optimal trace element composition in the medium. Of the five trace elements examined, Mg 2+ , K + , Mn 2+ , and Fe 2+ were found to be more significant factors affecting surfactin production by the B. subtilis strain. In the absence of Mg 2+ or K + , surfactin yield decreased to 0.4 g/l, which was only 25% of the value obtained from the control run. When Mn 2+ and Fe 2+ were both absent, the production yield also dropped to ca. 0.6 g/l, approximately one-third of the control value. However, when only one of the two metal ions (Fe 2+ or Mn 2+ ) was missing, the B. subtilis ATCC 21332 strain was able to remain over 80% of original surfactin productivity, suggesting that some interactive correlations among the selected metal ions may involve. Taguchi method was thus applied to reveal the interactive effects of Mg 2+ , K + , Mn 2+ , Fe 2+ on surfactin production. The results show that interaction of Mg 2+ and K + reached significant level. By further optimizing Mg 2+ and K + concentrations in the medium, the surfactin production was boosted to 3.34 g/l, which nearly doubled the yield obtained from the original control. <s> BIB002 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Other Two-Level Factorial Designs <s> In order to improve biosurfactant production by Yarrowia lipolytica IMUFRJ 50682, a factorial design was carried out. A full factorial design was used to investigate the effects of nitrogen sources (urea, ammonium sulfate, yeast extract, and peptone) on maximum variation of surface tension (ST) and emulsification index (EI). The best results (67.7% of EI and 20.9 mN of ST) were obtained in a medium composed of 10 g of ammonium sulfate and 0.5 g of yeast extract. Then, the effects of carbon sources (glycerol, hexadecane, olive oil, and glucose) were evaluated. The most favorable medium for biosurfactant production was composed of both glucose (4% w/v) and glycerol (2% w/v), which provided an EI of 81.3% and a ST of 19.5 mN . The experimental design optimization enhanced EI by 110.7% and ST by 108.1% in relation to the standard process. <s> BIB003 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Other Two-Level Factorial Designs <s> Abstract Bacillus subtilis SPB1 is known to produce a highly effective biosurfactant that belongs to the class of lipopeptides. This biosurfactant has shown relevant properties that could be efficiently applied in various domains. However, high production and purification costs limit the use of B. subtilis SPB1 in high-volume applications. The present work aimed to promote an economical production of this lipopeptide biosurfactant. Statistical experimental designs and response surface methodology were employed to optimize the concentrations of agro-industrial residues, inoculum size and humidity for B. subtilis SPB1 biosurfactant production under solid-state fermentation. The best production yield was approximately 30.67 mg of crude lipopeptide biosurfactant per gram of solid material. This yield was obtained using a solid substrate ratio of 1.5, a moisture content of 90% and an inoculum size (OD600) of 0.08. These data support the utilization of a mixture of 6 g of olive leaf residue flour and 4 g of olive cake flour with a 10g total weight of the solid substrate. A mixture of two by-products of a traditional olive mill factory was demonstrated to be a suitable substrate for biosurfactant biosynthesis, providing enhanced bacterial growth and leading to a strong improvement in the yield of tensioactive lipopeptide production. <s> BIB004 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Other Two-Level Factorial Designs <s> Abstract The maximisation of the production of metabolites of industrial interest from fermentative processes, such as biosurfactants, requires standardisation of the medium and the cultivation conditions. In this sense, experiments were conducted to maximise the production of the biosurfactant from Candida lipolytica UCP0988 cultivated on 5% animal fat and 2.5% corn steep liquor using a 23 full factorial design. The effects and interactions of the agitation speed (200, 300 and 400 rpm), the variables aeration (0, 1 and 2 vvm) and time of cultivation (48, 96 and 144 h) on the surface tension, yield and biomass were evaluated. The results showed that the variable time of cultivation had positive influence on the production of biosurfactant, while the increase of the variables aeration and agitation showed a negative effect. These results indicate that the biosurfactant has a strong potential to be applied as a clean-up of oil spills at sea and on shorelines. <s> BIB005
These are designs used for 2-21 factors where each factor is varied over two levels. They are useful for estimating the main effects and interactions (Design Expert 7, Stat-Ease, Inc., Minneapolis, MN, USA). A 2 3 factorial design was implemented to improve Bs activity of a surfactin produced by Bacillus subtilis. Sucrose was evaluated as the carbon source and NaNO 3 , (NH 4 ) 2 SO 4 , NH 4 NO 3 , urea, and residual brewery yeast were used as the nitrogen sources. NH 4 NO 3 was the most important nitrogen source for Bs production. The C/N ratio also had an impact on Bs production. Surface tension (ST) was reduced to 29.3 mNm −1 BIB001 . In order to optimize Bs production in B. subtilis SPB1 under solid-state fermentation, making production cost effective, a 2 3 factorial design and a central composite were applied BIB004 . To enhance Bs production, olive leaf residue flour, olive cake flour, inoculum size, and moisture content were analyzed. Bs production reached 30.67 mgg −1 . Statistical designs have also been used for optimizing Bs production of yeast. Fontes et al. BIB003 used a 2 4 complete factorial design and RSM to optimize Yarrowia lipolytica IMUFRJ 50,682 Bs production. The effect of nitrogen sources (urea, (NH 4 ) 2 SO 4 , yeast extract, and peptone) were evaluated first, and then the carbon sources (glycerol, hexadecane, olive oil, and glucose). In terms of the nitrogen sources used, yeast extract and (NH 4 ) 2 SO 4 favored Bs production at low levels. The authors argued that this was probably due to the fact that when nitrogen was in excess, Bs production decreased, since carbon was used for cell growth. Glucose and glycerol were the most significant carbon sources (at intermediate levels), while olive oil and hexadecane did not favor Bs production. An increase in shaking resulted in an increase in Bs production, and the authors suggested that this was due to an increase in oxygenation levels. Optimization by statistical design enhanced ∆EI 24 by 110.7% and ∆ST by 108.1% in relation to the standard process. A 2 3 factorial design (RSM with Contour Curve) was also used for optimizing Bs production in the yeast Candida lipolytica UCP0988. Physicochemical parameters were evaluated. The input variables were agitation, aeration, and time of the process, and the output variables were the ST, yield, and biomass. The results of the analysis revealed that although time had a positive effect on Bs production yield, agitation and aeration had a negative effect with an optimum yield of 7.27 gL −1 BIB005 . Trace element composition was optimized by the Taguchi method for improved surfactin production in B. subtilis BIB002 . This is an orthogonal array design from Taguchi's textbook which explores two level factorial and general factorial designs as alternatives. Mg 2+ , K + , Mn 2+ , and Fe 2+ were found to have the greatest effect on Bs production, since the absence of these ions in the culture medium resulted in a decrease in production yield of 0.4-0.6 gL −1 . The Taguchi approach increased production yields from 1.74 gL −1 of the control experiments to 3.34 gL −1 in the optimized medium.