Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Box-Behnken <s> A culture medium formulation was established using a Box-Behnken experimental design aimed at enhancing biosurfactant production and phenanthrene removal by Pseudomonas putida CB-100. The independent variables selected (+, 0, −, levels) were: glucose (9.1, 13.6, and 18.2 g/l), NH4Cl (0.5, 0.75, and 1.0 g/l), and yeast extract (0.005, 0.0075, and 0.01 g/l), with 200 mg/l phenanthrene at 37°C, shaken at 150 rpm, at pH 7, for 5 days. Analyses of results by one-way analysis of variance showed that biosurfactant production was improved by the glucose–ammonium chloride interaction at high values (p < 0.004). Phenanthrene removal was enhanced by the ammonium chloride-yeast extract interaction at low and high values, respectively (p < 0.02). The highest phenanthrene removal (82.4%) was observed in formulation 11, which had a C:N ratio of 5:1 and a C:P ratio of 10:1. In addition, 47.5 mN/m surface tension, 20% emulsion capacity, and 23.5 mg/l biosurfactant production were obtained. With this medium, we followed the kinetic growth of P. putida for 118 h, the culture conditions were the same as those used in the experimental design. At 46 h, we obtained 57.8% phenanthrene removal and 27 mg/l biosurfactant production. The critical micelle concentration of the biosurfactant was 430 mg/l. The biosurfactant produced by P. putida was characterized as a rhamnolipid type. <s> BIB001 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Box-Behnken <s> The biosurfactant production of a marine actinobacterium Brevibacterium aureum MSA13 was optimized using industrial and agro-industrial solid waste residues as substrates in solid state culture (SSC). Based on the optimization experiments, the biosurfactant production by MSA13 was increased to threefold over the original isolate under SSC conditions with pre-treated molasses as substrate and olive oil, acrylamide, FeCl3 and inoculums size as critical control factors. The strain B. aureum MSA13 produced a new lipopeptide biosurfactant with a hydrophobic moiety of octadecanoic acid methyl ester and a peptide part predicted as a short sequence of four amino acids including pro-leu-gly-gly. The biosurfactant produced by the marine actinobacterium MSA13 can be used for the microbially enhanced oil recovery processes in the marine environments. <s> BIB002 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Box-Behnken <s> The strain SmSA, identified as Serratia marcescens and known as a biosurfactant producer, was isolated from hydrocarbon contaminated soil from Veracruz, México. The interactions among the C/N, C/Mg and C/Fe ratios have not been examined for this microorganism. In this work was evaluated the effect of these nutrients at three levels using a mineral medium with glucose as the carbon source. A Box-Behnken experimental design was utilised to maximise biosurfactant production, which was assessed by oil spreading and surface tension tests. The treatment with C/N=5, C/Fe=26,000 and C/Mg=30 showed the best result since the surface tension was reduced to 30 mN m(-1). The multiple regression and response surface analyses indicated that the interaction between C/N and C/Mg had the utmost effect on the reduction of surface tension and biosurfactant production. The conditions of the best treatment were used to scale up biosurfactant production in a 3L bioreactor giving a yield of 4.1 gL(-1) of pure biosurfactant. It was found that the biosurfactant was mainly produced in the exponential phase and decreased the surface tension to 31 mN m(-1). The contact between the biosurfactant with heavy oil (15° API) increased its displacement from 9.3 to 18 cm. <s> BIB003 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Box-Behnken <s> The inoculum age and density can influence considerably the production yield and cost of the fermentation process. Some literature studies report the use of two-stage inocula to enhance metabolite production. In the present study, optimization studies were done in order to define the best inocula conditions supporting a maximum biosurfactant production by Bacillus subtilis SPB1. Hence, by adjusting the levels of the two-stage inocula strategy, lipopeptide production was effectively enhanced to almost 3.4 g/l as estimated gravimetrically. The new defined parameters were as follows; a first inoculum age of 23 h followed by a second inoculum age and size of 4 h and 0.01, respectively. Thereby, we note an improved production as compared to the production yield described under non-optimized inocula conditions reported in our previous work. <s> BIB004 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Box-Behnken <s> Many studies have investigated the effects of pH, temperature, and salinity on the surface-active properties of various surfactants, although in most cases the variables have been studied separately, without considering the effects of any interactions between them. In the present study, a Box-Behnken factorial design was applied to study the effects of pH, temperature, and salinity on the surface-active properties of a biosurfactant produced by Lactobacillus pentosus. The data obtained enabled development of a second-order model describing the interrelationships between operational and experimental variables, by equations including linear, interaction, and quadratic terms. The variable that had the greatest effect on the surface-active properties of the biosurfactant was pH. Moreover, at pH 3-5.5, decreases in salinity and temperature acted synergistically, reducing the surface tension of the biosurfactant; at pH 8, the same effect was observed with increasing salinity and temperature. <s> BIB005 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Box-Behnken <s> Great emphasis has recently been given to the environmental impacts caused by chemical surfactants due to their toxicity and difficulty in being degraded in the environment [1]. Increasing environmental concerns, the advance in biotechnology and the emergence of more stringent laws have led to biosurfactants being a potential alternative to the chemical surfac‐ tants available on the market [2, 3]. Although biosurfactants have promising use in bioreme‐ diation processes, their industrial scale production is currently difficult due to high rawmaterial costs, high processing costs and low manufacturing output [3]. As a result, the current research challenges are to increase the yield and to reduce the cost of raw materials [4]. <s> BIB006
In this design each numeric factor is varied over three levels. If categorical factors are added, the Box-Behnken design will be duplicated for every combination of the categorical factor levels. These designs have fewer runs than three-level factorials (Design Expert 7, Stat-Ease, Inc., Minneapolis, MN, USA). A Box-Behnken 3 3 factorial design was used to optimize Bs production in Serratia marcescens. The C/N, C/Fe 2+ , and C/Mg 2+ ratios at three different levels were evaluated. For the interaction between C/Fe 2+ and C/N, the greatest reduction in ST was when both ratios were highest. In terms of the interaction between C/Fe 2+ and C/mg 2+ , the same pattern was observed. With the use of this design, ST was reduced to 31 mNm −1 with a yield of 4.1 gL −1 after 48 h BIB003 . Martínez-Toledo and Rodríguez Vázquez BIB001 employed a BBD evaluating the effect of glucose, NH BIB006 Cl and yeast extract on phenanthrene removal by Pseudomonas putida CB-100. Rhamnolipid production was also measured, and their results showed that high concentrations of glucose and NH BIB006 Cl favored Bs synthesis, reaching 50 mgL −1 after 10 h. A B. subtilis strain lipopeptide production was optimized to 3.4 gL −1 using a BBD BIB004 . Primary inoculum age, secondary seed culture age, and size were the factors evaluated. These authors emphasized that although most studies for improvement of Bs production focuses on nutritional factors the physical parameters also have a great importance for increasing the cost-effectiveness of biotechnological processes. Their analysis showed that all the factors and the interaction between the factors were significant, and that increasing the secondary seed culture negatively affected Bs production. A lower initial cell density also increased Bs productivity. This was explained by the fact that in the case of sporulating bacteria like B. subtilis, less and younger biomass would lengthen the exponential phase where the Bs can be produced; since once the stationary phase is reached sporulation processes are induced BIB004 . Brevifactin (lipopeptide) production by Brevibacterium areum MSA13 in a solid-state culture was optimized by RSM. After a number of factors affecting Bs production were initially traditionally screened (one-at-a-time), olive oil, FeCl 3 , inoculum size, and acrylamide were selected for optimization. Different industrial wastes were also chosen for the base formulation of the media culture including oil seed cake, wheat bran, tannery treated sludge, tannery pre-treated sludge, treated molasses, and pre-treated molasses. The ANOVA analysis after optimization showed that inoculum size, and acrylamide (nitrogen source) were the most significant factors, followed by olive oil and FeCl 3 BIB002 . Vecino Bello et al. BIB005 obtained data that enabled them to develop a second-order model describing the interrelationship between operational and experimental variables by the use of different equations (linear, interaction, and quadratic terms). They implemented Box-Behnken factorial design to evaluate the effects of pH, temperature, and salinity on Bs produced by Lactobacillus pentosus. The pH had the most prominent effect on ST reduction and emulsion stability. However, their analysis showed a synergistic effect in reducing ST between pH (3-5.5) and low salt concentrations and low temperature. Interestingly the opposite synergistic effect was observed at pH 8. After optimization ST was reduced to 53.8 from 69.3 nM/m. Emulsion volume reached 45.93% and emulsion stability was optimized to 100%.