Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Central Composite Design <s> Abstract In this study, we have investigated the potential of a native bacterial strain isolated from an Iranian oil field for the production of biosurfactant. The bacterium was identified to be Bacillus mycoides by biochemical tests and 16S ribotyping. The biosurfactant, which was produced by this bacterium, was able to reduce the surface tension of media to 34 mN/m. Compositional analysis of the produced biosurfactant has been carried out by thin layer chromatography (TLC) and FT-IR. The biosurfactant produced by the isolate was characterized as lipopeptide derivative. Biosurfactant production was optimized by the combination of central composite design (CCD) and response surface methodology (RSM). The factor selected for optimization of growth conditions were pH, temperature, glucose and salinity concentrations. The empirical model developed through RSM in terms of effective operational factors mentioned above was found to be adequate to describe the biosurfactant production. Through the analysis, glucose and temperature were found to be the most significant factors, whereas pH and salinity had less effect within the ranges investigated. A maximum reduction in surface tension was obtained under the optimal conditions of 16.55 g/l glucose concentration, 39.03 °C, 55.05 g/l total salt concentration and medium pH 7.37. <s> BIB001 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Central Composite Design <s> Aims: To screen and identify biosurfactant producers from petroleum-contaminated soil; to use response surface methodology (RSM) for medium optimization to enhance biosurfactant production; and to study the properties of the newly obtained biosurfactant towards pH, temperature and salinity. ::: Methods and Results: We successfully isolated three biosurfactant producers from petroleum-contaminated soil and identified them through 16S rRNA sequence analysis, which exhibit the highest similarities to Acinetobacter beijerinckii (100%), Kocuria marina (99%) and Kineococcus marinus (99%), respectively. A quadratic response model was constructed through RSM designs, leading to a 57AE5% increase of the growth-associated biosurfactant production ::: by Acinetobacter sp. YC-X 2 with an optimized medium: beef extract ::: 3AE12 g l)1 ; peptone 20AE87 g l)1 ; NaCl 1AE04 g l)1 ; and n-hexadecane 1AE86 g l)1. Biosurfactant produced by Acinetobacter sp. YC-X 2 retained its properties during exposure to a wide range of pH values (5–11), high temperatures (up to 121C) and high salinities [up to 18% (w⁄ v) Na+ and Ca2+], which was more sensitive to Ca2+ than Na+ . ::: Conclusions: Two novel biosurfactant producers were isolated from petroleumcontaminated soil. Biosurfactant from Acinetobacter sp. YC-X 2 has good properties to a wide range of pH, high temperature and high salinity, and its production was optimized successfully through RSM. <s> BIB002 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Central Composite Design <s> The present study aims to evaluate and validate a statistical model for maximizing biosurfactant productivity by Bacillus brevis using response surface methodology. In this respect, twenty bacterial isolates were screened for biosurfactant production using hemolytic activity, oil spreading technique, and emulsification index (E24). The most potent biosurfactant-producing bacterium (B. brevis) was used for construction of the statistical response surface model. The optimum conditions for biosurfactant production by B. brevis were: 33 °C incubation temperature at pH 8 for 10 days incubation period and 8.5 g/L glucose concentration as a sole carbon source. The produced biosurfactant (BS) (73%) exhibited foaming activity, thermal stability in the range 30-80 °C for 30 min., pH stability, from 4 to 9 and antimicrobial activity against (Escherichia coli). The BS gave a good potential application as an emulsifier. <s> BIB003 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Central Composite Design <s> Surfactants are amphipathic compounds containing both hydrophilic and hydrophobic groups, capable to lower the surface or interfacial tension. Considering the advantages of the use of biosurfactants produced by microorganisms, the aim of this paper was to develop and characterize a biosurfactant produced by Streptomyces sp. DPUA1559 isolated from lichens of the Amazon region. The microorganism was cultured in a mineral medium containing 1% residual frying soybean oil as the carbon source. The kinetics of biosurfactant production was accompanied by reducing the surface tension of the culture medium from 60 to values around 27.14 mN/m, and by the emulsification index, which showed the efficiency of the biosurfactant as an emulsifier of hydrophobic compounds. The yield of the isolated biosurfactant was 1.74 g/L, in addition to the excellent capability of reducing the surface tension (25.34 mN/m), as observed from the central composite rotational design when the biosurfactant was produced at pH 8.5 at 28°C. The critical micelle concentration of the biosurfactant was determined as 0.01 g/mL. The biosurfactant showed thermal and pH stability regarding the surface tension reduction, and tolerance under high salt concentrations. The isolated biosurfactant showed no toxicity to the micro-crustacean Artemia salina, and to the seeds of lettuce (Lactuca sativa L.) and cabbage (Brassica oleracea L.). The biochemistry characterization of the biosurfactant showed a single protein band, an acid character and a molecular weight around 14.3 kDa, suggesting its glycoproteic nature. The results are promising for the industrial application of this new biosurfactant. <s> BIB004
RSM that are most commonly used for enhancing Bs production include the CCD and the BBD, although the one-factor strategy has been used. For the CCD each numeric factor is varied over five levels: plus and minus alpha (axial points), plus and minus 1 (factorial points), and the center points. In the BBD design each numeric factor is varied over three levels. Chen et al. BIB002 decided to optimize the production of a Bs from Acinetobacter sp. YC-X 2 that showed stability under high temperatures, salinity, and harsh pH conditions. In order to determine the factors that had a significant effect on Bs production a one-factor RSM was used. Once optimal conditions were obtained, a CCD quadratic response model obtained with experimental data was implemented. Analysis of the data showed a 57.5% increase in Bs production with beef extract 3.12 gL −1 , peptone 20.87 gL −1 , NaCl 1.04 gL −1 , and n-hexadecane 1.86 gL −1 . According to the model, peptone was the most significant factor. Bs production was directly related to cell growth where the highest emulsion values were at high concentrations of peptone and beef extract. Bs production was stimulated at lower levels of hexadecane and NaCl. A RSM (CCD) optimized Bs production by B. brevis. The effect of temperature, pH, incubation period, and glucose concentration were evaluated BIB003 . Emulsion reached 73%. All the factors studied had an influence of Bs production. Long incubation periods, slightly alkaline pH (8) (interaction incubation-period-pH), and intermediate temperatures (33 • C) (interaction incubation period-temperature) favored Bs production. High glucose concentrations and slightly alkaline pH influenced Bs production (interaction glucose-pH). The interaction between glucose concentration and incubation periods was not very significant. The production of a Bs of a glycoproteic nature was enhanced using the CCRD method BIB004 . The Streptomyces sp. DUPA1559 strain was isolated from lichens in the Amazon region in Brazil. These Bs showed promising biochemical properties like low Critical Micelle Concentration (CMC), high thermal and pH stability, salt tolerance, and low toxicity, thus production was optimized. Temperature and pH were the variables analyzed using 12 experiments with four replicates at the central points. Statistical analysis showed that pH had a greater effect than temperature on ST. Under optimized conditions the Bs reduced ST to 25.34 mNm −1 compared to 27.14 mNm −1 . The Bs production yield reported was 1.74 gL −1 after 96 h. Najafi et al. BIB001 also applied a CCD to enhance the Bs production (lipopeptide) from an indigenous strain of Bacillus mycoides. Temperature, pH, salinity, and glucose were the factors evaluated. Although this strain produced Bs over a wide range of temperatures, maximum Bs production was at a higher temperature (39.03 • C), suggesting that the bacteria could be a moderate thermophile. Their analysis showed that low salt concentrations negatively affected Bs production. Glucose also positively affected Bs production. ST was reduced from 61 to 34 mNm −1 .