Plackett-Burman Method Research Articles

Acid Pretreatment of Two Phase Olive Mill Waste to Improve Bioavailable Sugars: Conditions Optimization Using Response Surface Methodology

Two phase olive mill waste (TPOMW) is the semisolid waste generated for olive oil production. The organic fraction of this waste includes compounds that serve as a source of carbon and other nutrients for microorganisms, a feature that makes it potentially attractive for bioethanol production. To increase the bioavailability of cellulose and hemicellulose, no soluble carbohydrates, the application of pretreatments is required. This paper presents the results of the optimization of the sulfuric acid pretreatment variables of TPOMW applied to improve sugars bioavailability, using the response surface methodology. First, the Plackett–Burman method was applied to find the relevant variables of the process, using total sugars content as response variable. The results showed that temperature, acid concentration and solid:liquid ratio are significant for this process. Subsequently, these variables were optimized to maximize total sugars content, using the Box–Behnken method, an experimental array based on an incomplete three-level factorial design. The results indicate that the optimum working conditions were 10.10 % w/w for H2SO4 concentration, 95.2 °C for temperature, and 0.17 g dry solid TPOMW per ml of acid solution, obtaining 67 % of sugars content increase.

English

A protease producing Bacillus sp. GA CAS10 was isolated from ascidian Phallusia arabica , Tuticorin, Southeast coast of India. Response surface methodology was employed for the optimization of different nutritional and physical factors for the production of protease. Plackett-Burman method was applied to identify important factors (anchovy waste, KH 2 PO 4 , NaCl and temperature) influencing protease production. Further optimization was done by response surface methodology using central composite design. Under the optimized conditions by central composite design, the protease experimental yield (842.102 U/ml) closely matched the predicted yield by the statistical model (830.307 U/ml) with R 2 = 99.94%. The time course of protease production was increased using the RSM optimized medium (856.29 U/ml) (anchovy waste 20.50 g/l, KH 2 PO 4 3.06 g/l, NaCl 42.91 g/l, temperature 43.36°C, 42 h and pH 9) compared with the un-optimized basal medium (267.33 U/ml). The improvement of protease production by microbial conversion of anchovy waste suggested its potential utilization to generate high value added products using cheap carbon and nitrogen substrates. Keywords: Protease, anchovy waste, statistical optimization, ascidian associated bacteria. African Journal of Biotechnology Vol 13(27) 2741-2749

Improved islet purity by the hypertonic-hypotonic method.

Islet purification is usually performed using the density gradient separation method, but the purity of islets is low because exocrine cells and the embedded islets are hard to remove by using only the density gradient method. The aim of this study was to establish a new islet purification process comprising a hypertonic-hypotonic treatment step followed by a density gradient centrifugation step to improve the purity of islets. The Plackett-Burman method was used to determine which factors had a significant influence on the purity of islets obtained after the hypertonic-hypotonic treatment step. The hypertonic solution concentration and the incubation time were both found to have a significant effect on islet purity. The purity of islets obtained using the modified purification process was significantly higher than that of islets obtained by density gradient alone (97% vs. 87.23%). Importantly, good cell viability and normal insulin secretion ability of islets were maintained following the modified purification. The new purification process allows isolation of islets with improved purity and does not compromise the viability or function of the islets.

Optimization of reaction conditions and methodological investigation on microtox-based fast testing system for traditional Chinese medicine injection

Vibrio fischeri CS234 was used to establish and optimize microtox assay system, laying a foundation for the application of this method in comprehensive acute toxicity test of traditional Chinese medicine (TCM) injections. Firstly, the Plackett-Burman method was carried out to optimize the factors which would affect Vibrio fischeri CS234 luminescence. Secondly, ZnSO4•7H2O was chosen as reference substance to establish its reaction system with quality control samples. The optimal luminescence conditions were achieved as follows: ①At a temperature of (15±1) ℃, Vibrio fischeri CS234 lyophilized powders were balanced for 15 min, then, 1 mL resuscitation fluid was added and blended for 10 min. 100 μL bacteria suspension was taken to measure the initial luminescence intensity, and then 1 mL resuscitation fluid or test sample was immediately added; after reaction for 10 min, corresponding luminescence intensity was measured again. Resuscitation diluent, osmotic pressure regulator and ZnSO4•7H2O stock solution showed no interference to the determination of Vibrio fischeri CS234 luminescence intensity, so this method was of good specificity. The within-and between-batch precisions of quality controls and the lower limit of quantification (LLOQ) samples were <5% and <10% respectively, while the accuracy ranged between 85.8% and 103.2%. The standard curve equation of ZnSO4•7H2O ranged from 3.86 mg•L⁻¹ to 77.22 mg•L⁻¹ (final concentrations) was y=21.78lnx-15.14, R2=0.998; meanwhile, IC₅₀ of ZnSO4•7H2O to Vibrio fischeri CS234 was 19.90 mg•L⁻¹. ZnSO4•7H2O stock solution and its quality controls were continuously investigated for 120 h and 8 h respectively, and their RSD was lower than 2%, indicating stability at room temperature and 4 ℃ storage conditions. Between pH 4.5-8.0, luminescence intensity of Vibrio fischeri CS234 was controlled within ±10%, and such pH value range could meet the testing needs of the vast majority of traditional Chinese medicine injections. The Vibrio fischeri strain CS234 assay system was specific, stable, sensitive, accurate and adaptable after optimization, so it was suitable for the comprehensive acute toxicity assessment of TCM injections.

Co-digestion of oil palm trunk hydrolysate with slaughterhouse wastewater for thermophilic bio-hydrogen production by Thermoanaerobacterium thermosaccharolyticm KKU19

The key factors influencing a co-digestion of the oil palm trunk (OPT) hydrolysate with a slaughterhouse wastewater (SHW) to produce hydrogen by Thermoanaerobacterium thermosaccharolyticum KKU19 were investigated. The OPT hydrolysate was obtained by the hydrolysis of OPT by microwave-H2SO4 method using 1.56% (w/v) H2SO4 and 7.50 min reaction time at 450 W. The Plackett–Burman method was used to screen the key factors that influenced the hydrogen production potential (Ps). Results indicated that initial cell concentration, tCOD/TN (total COD/total nitrogen) ratio and CuSO4 concentration influenced the Ps. These factors were further optimized using response surface methodology (RSM) with central composite design (CCD). A maximum Ps of 2604 ± 86 mL H2/L substrate was achieved at an initial cell concentration of 224 mg dry cell/L, tCOD/TN ratio of 49.87 and CuSO4 concentration of 13.33 mg/L. The main soluble metabolite products were butyric and acetic acids. The Ps obtained when the hydrolysate was supplemented with SHW (2604mL ± 86 mL H2/L substrate) was comparable to the Ps obtained when it was supplemented with yeast extract at the same tCOD/TN (2802 ± 87 mL H2/L substrate). This result suggests that SHW can be used to replace the costly nitrogen source.

Statistical approach to optimize production of biosurfactant by Pseudomonas aeruginosa 2297.

The main objective of this paper is to optimize biosurfactant production by Pseudomonas aeruginosa 2297 with statistical approaches. Biosurfactant production from P. aeruginosa 2297 was carried out with different carbon sources, and maximum yield was achieved with sawdust followed by groundnut husk and glycerol. The produced biosurfactant has showed active emulsification and surface-active properties. From the kinetic growth modeling, the specific growth rate was calculated on sawdust and it was 1.12 day−1. The maximum estimated value of product yield on biomass growth (Y p/x) was 1.02 g/g. The important medium components identified by the Plackett–Burman method were sawdust and glycerol along with culture parameter pH. Box–Behnken response surface methodology was applied to optimize biosurfactant production. The obtained experimental result concludes that Box–Behnken designs are very effective statistical tools to improve biosurfactant production. These results may be useful to develop a high efficient production process of biosurfactant. In addition, this type of kinetic modeling approach may constitute a useful tool to design and scaling-up of bioreactors for the production of biosurfactant.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-014-0203-3) contains supplementary material, which is available to authorized users.

Optimization of Phenol Degradation Using Pseudomonas aeruginosa (MTCC 7814) by Plackett-Burman Design and Response Surface Methodology

In the present study, statistical screening and optimization of phenol degradation was done by Pseudomonas Aeruginosa (MTCC 7814), which can utilize phenol as a sole carbon and energy source. Nine medium variables Phenol, K2HPO4, K2H2PO4, MgSO4, (NH4)2SO4, MnSO4, FeSO4, NaCl and H3BO3 were screened by Plackett-Burman (PB) method. K2HPO4, (NH4)2SO4, MnSO4 and phenol were significant in PB method. Central composite design (CCD) and Response Surface Methodology (RSM) were applied to optimize the significant variables identified from the PB experiment. Statistical analysis of the experimental results showed optimal values were found to be KH2PO4 0.025 g/L, (NH4)2SO4 0.45 g/L, MnSO4 0.05 g/L and phenol 1 g/L with maximum phenol degradation of 83.86%. Maximum phenol degradation of 81.62% was observed in the validation experiment. This experimental result explained the model was fitted 97.32 % as compare with the result predicted by response surface method. This study indicated the excellent ability of Pseudomonas Aeruginosa to degrade phenol of high concentration.

Entomopathogenic marine actinomycetes as potential and low-cost biocontrol agents against bloodsucking arthropods

A novel approach to control strategies for integrated blood-feeding parasite management is in high demand, including the use of biological control agents. The present study aims to determine the efficacy of optimized crude extract of actinomycetes strain LK1 as biological control agent against the fourth-instar larvae of Anopheles stephensi and Culex tritaeniorhynchus (Diptera: Culicidae) and adults of Haemaphysalis bispinosa, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae), and Hippobosca maculata (Diptera: Hippoboscidae). Antiparasitic activity was optimized using the Plackett-Burman method, and the design was developed using the software Design-Expert version 8.0.7.1. The production of the optimized crude actinomycetes LK1 strain extract was performed using response surface methodology to optimize the process parameters of protease inhibitor activity of marine actinobacteria for the independent variables like pH, temperature, glucose, casein, and NaCl at two levels (-1 and +1). The potential actinomycetes strain was identified as Saccharomonas spp., and the metamodeling surface simulation procedure was followed. It was studied using a computer-generated experimental design, automatic control of simulation experiments, and sequential optimization of the metamodels fitted to a simulation response surface function. The central composite design (CCD) used for the analysis of treatment showed that a second-order polynomial regression model was in good agreement with the experimental results at R (2) = 0.9829 (p < 0.05). The optimized values of the variables for antioxidant production were pH 6.00, glucose 1.3%, casein 0.09%, temperature 31.23 °C, and NaCl 0.10%. The LK1 strain-optimized crude extract was purified using reversed-phase high-pressure liquid chromatography, and the isolated protease inhibitor showed antiparasitic activity. The antiparasitic activity of optimized crude extract of LK1 was tested against larvae of A. stephensi (LC₅₀ = 31.82 ppm; r(2) = 0.818) and C. tritaeniorhynchus (LC₅₀ = 26.62 ppm; r(2) = 0.790) and adults of H. bispinosa (LC₅₀ = 106.58 ppm; r(2) = 0.871), R. (B.) microplus (LC₅₀ = 92.96 ppm; r(2) = 0.913), and H. maculata (LC₅₀ = 84.90 ppm; r(2) = 0.857).

Pesticide Health Risk Mapping and Sensitivity Analysis of Parameters in Groundwater Vulnerability Assessment

AbstractThe extensive use of pesticides for increasing the agricultural production is affecting the quality of groundwater. The objectives of this article are to (i) develop pesticide relative leaching ranks for well sites, (ii) develop maps for human health risks due to pesticide applications, and (iii) identify the most significant parameters in pesticide simulations for groundwater vulnerability assessment. The methods include (i) development of acifluorfen relative leaching ranks for 25 well sites using ArcPRZM‐3, (ii) development of health risk maps using model simulated maximum dissolved bentazon concentrations on the basis of USA drinking water quality guidelines, (iii) sensitivity analysis for 14 ArcPRZM‐3 input parameters using the Plackett–Burman method. ArcPRZM‐3 is a user‐friendly system for spatial modeling of pesticide leaching from surface to groundwater. Thirteen acifluorfen relative leaching potential ranks were developed in which the pesticide leaching decrease from 1 to 13. The model predicted ranks for well 34 and well 9 were 2nd and 3rd, respectively, and acifluorfen was detected in both wells during the physical monitoring. The percentages of high health risks in the agricultural areas were 48.38 and 72.72% for Randolph and Independence Counties, respectively. The most significant parameters were thickness of horizon compartment, runoff curve number of antecedent moisture condition II for cropping, soil bulk density, and total application of pesticide. The irrigation, soil permeability, and numerical dispersion could impact the pesticide leaching in soils toward groundwater. The ArcPRZM‐3 system could be efficiently applied for spatial modeling and mapping of pesticide concentrations for groundwater vulnerability assessment on a large scale.

Optimization of the production of a maltooligosaccharides producing amylase from the alkalophilic Streptomyces lonarensis strain NCL 716 using SVR Modeling

AbstractThe Streptomyces lonarensis strain NCL 716 hydrolyses starch to produce a mixture of maltotriose (G3) and maltotetraose (G4) along with maltose (G2). The objective of the present work was to determine an optimum cost effective media composition for the production of α‐amylase from this strain. The most influential factor was found to be starch while the least influential factor found was peptone by Plackett–Burman method. Peptone amount was kept constant throughout the fermentation. Peptone, which is one of the expensive media components was used at a concentration of 1 g/L, which made the optimum media composition cost effective. A support vector regression‐based process model was developed for approximating the non‐linear relationship between the fermentation operating variables and the α‐amylase yield. Multicanonical Jump Walk Annealing, a stochastic optimization technique is used to obtain optimal operating variables to maximize amylase yield. The maximum amylase activity thus obtained was in good agreement with the experimental values at the optimized levels. The optimum media composition obtained by this method was: yeast extract: 4.53 g/L, starch: 20.246 g/L, K2HPO4: 0.0827%, MgSO4: 0.15%, peptone: 1 g/L. A maximum enzyme activity of 297 U/mL, which was achieved using the above approaches compares well with the activity of reported amylases producing maltooligosaccharides.

A NEWLY ANTI-Streptococcus suis BACTERIOCIN PRODUCING STRAIN FROM UNWEANED PIGLETS FECAL MATTER: ISOLATION, PRELIMINARY IDENTIFICATION, AND OPTIMIZATION OF MEDIUM COMPOSITION FOR ENHANCED BACTERIOCIN PRODUCTION

A newly isolated anti-Streptococcus suis bacteriocin-producing strain LPL1-5 was obtained from healthy unweaned piglets' fecal matter, and was designated as Lactobacillus pentosus LPL1-5 based on morphology, biochemical properties, and 16S rDNA sequencing analysis. The medium composition for enhanced bacteriocin production by L. pentosus LPL1-5 was optimized by statistical methodology. Yeast extract, K2HPO4 · 3H2O, and MnSO4 · H2O were identified as significant components influencing pentocin LPL1-5 production using the Plackett–Burman method. Response surface methodology was applied for further optimization. The concentrations of medium components for enhanced pentocin LPL1-5 production were as follows (g/L): lactose 20.00, tryptone 10.00, beef extract 10.00, yeast extract 14.00, MnSO4 · H2O 0.84, K2HPO4 · 3H2O 4.92, triammonium citrate 2.00, Na-acetate 5.00, MgSO4 · 7H2O 0.58, Tween 80 1.00. Under the optimized condition, a value of 3154.65 ± 27.93 IU/mL bacteriocin activity was achieved, which was 4.2-fold that of the original medium.

Development of optimal medium for production of commercially important monoclonal antibody 520C9 by hybridoma cell

Hybridoma HB-8696 produces monoclonal antibody (mAb) 520C9 (mouse IgG(1)), which recognizes breast cancer oncoprotein c-erbB2. The objective of this study was to optimize the medium recipe of HB 8696 cell for production of mAb 520C9. The optimization consisted of two steps: (1) screening of significant nutrients to make subsequent experiments more efficient with less runs and (2) locating their optimal concentrations. 29 variables including essential and non-essential amino acids, glucose, serum and 6 salts, namely NaCl, KCl, CaCl(2), NaH(2)PO(4), MgSO(4) and Na-pyruvate were chosen in screening phase. The Plackett-Burman method was used to screen the variables influencing mAb production. Seven factors namely glucose, serum, asparagine, threonine, serine, NaCl and NaH(2)PO(4) were identified to have a positive influencing role on mAb production with a confidence level >90% (p<0.1). Finally, Response surface methodology revealed the optimal level of the variables. The mAb production and average specific mAb production rate were enhanced by 111.05 and 105%, respectively, compared to control medium.

Modeling and optimization of membrane preparation conditions of the alginate‐based microcapsules with response surface methodology

Microencapsulation has been a promising approach for drug delivery, cell implantation, cell-based gene therapy and large-scale cell culture. To make use of microcapsules more effectively, it is important to accurately construct the microcapsule membranes with desired properties including a certain thickness, strength, and so forth. To date single factor experiments have been widely used, however, they are time-consuming to obtain the desired membrane preparation conditions. Response surface methodology (RSM) is a mathematical and statistical technique for building empirical models that gained importance for optimizing reacting conditions. In this study, three signifficant effect factors that affect alginate-based microcapsule membrane properties, including membrane thickness, swelling degree, and mechanical stability, were determined with Plackett-Burman method, and then three empirical models were built to optimize the preparation conditions of the microcapsule membranes according to the responses of these three signifficant effect factors respectively with RSM. These models can be used to predict the characteristics of microcapsules under different membrane preparation conditions, which provide a guide for optimizing the microencapsulation technology.

Optimization of grinding parameters in the production of colorant paste

The tinting strength of a final product together with powerdraw of the mill and grinding time are optimized by adjusting the grinding conditions. The following three-step optimization procedure is described in the paper: in order to determine the most significant grinding variables, the first set of experiments was designed and carried out using the Plackett–Burman method. In the second step, a set of experiments with the selected variables was designed using the Box–Behnken method. The models of tinting strength and grinding power were then identified. In the third step, multicriteria optimization was performed. The paper presents and discusses the optimization results. In particular, the sets of Pareto-optimal solutions are provided for different levels of specific energy. The optimization results of tinting strength and grinding time clearly demonstrate that a low rotor speed combined with a low specific energy can sometimes provide better tinting strength compared with the high rotor speed strategy.

Optimization of protease production by Streptomyces sp. A6 using statistical approach for reclamation of shellfish waste

Protease producing Streptomyces sp. A6 was isolated from intertidal zone of the coast of Diu (Gujarat, India). Plackett–Burman method was applied to identify important factors (shrimp waste, FeCl3, ZnSO4 and pH) influencing protease production by Streptomyces sp. A6. Further optimization was done by response surface methodology using central composite design. The concentrations of medium components for higher protease production as optimized using the above approach were (g l−1): Shrimp waste, 14; FeCl3, 0.035; ZnSO4, 0.065 and pH, 8.0. This statistical optimization approach led to production of 129.02 ± 2.03 U ml−1 of protease which was 4.96 fold higher compared to that obtained using the unoptimized medium. The protease production was scaled to 3 l in a 5-l bench fermenter using optimized medium which further increased the production by 63.4%. Deproteinization and chitin recovery obtained at the end of fermentation was 85.12 ± 4.7 and 70.58 ± 1.33%, respectively. The present study is the first report on statistical optimization of medium components for production of protease by Streptomyces species using cheaper raw material such as shrimp waste. The study also explored the possibility Streptomyces sp. A6 for reclamation of shrimp wastes.

Optimization of biohydrogen production from sweet sorghum syrup using statistical methods

This study employed statistically based experimental designs to optimize fermentation conditions for hydrogen production from sweet sorghum syrup by anaerobic mixed cultures. Initial screening of important factors influencing hydrogen production, i.e., total sugar, initial pH, nutrient solution, iron (II) sulphate (FeSO 4), peptone and sodium bicarbonate was conducted by the Plackett–Burman method. Results indicated that only FeSO 4 had statistically significant ( P ≤ 0.005) influences on specific hydrogen production ( P s) while total sugar and initial pH had an interdependent effect on P s. Optimal conditions for the maximal P s were 25 g/L total sugar, 4.75 initial pH and 1.45 g/L FeSO 4 in which P s of 6897 mL H 2/L was estimated. Estimated optimum conditions revealed only 0.04% difference from the actual P s of 6864 mL H 2/L which suggested that the optimal conditions obtained can be practically applied to produce hydrogen from sweet sorghum syrup with the least error.

Optimization of medium constituents for improved chitinase production byPaenibacillussp. D1 using statistical approach

Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Urea, K(2)HPO(4), chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett-Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l(-1)): urea, 0.33; K(2)HPO(4), 1.17; MgSO(4), 0.3; yeast extract, 0.65 and chitin, 3.75. This statistical optimization approach led to the production of 93.2 +/- 0.58 U ml(-1) of chitinase. The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K(2)HPO(4), chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2.56-fold increase in chitinase production. The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.

Statistical optimization of medium components for extracellular protease production by an extreme haloarchaeon,Halobacteriumsp. SP1(1)

Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach. The significant factors influencing the protease production as screened by Plackett-Burman method were identified as soybean flour and FeCl(3). Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l(-1)): NaCl, 250; KCl, 2; MgSO(4), 10; tri-Na-citrate, 1.5; soybean flour, 10 and FeCl(3), 0.16. This statistical optimization approach led to production of 69.44 +/- 0.811 U ml(-1) of protease. Soybean flour and FeCl(3) were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3.9-fold increase in extracellular protease production. The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.

Statistical screenings of medium components for the production of chitinase by the marine isolate Pantoea dispersa

Effect of 19 different medium components on chitinase production by marine isolate Pantoea dispersa was studied by Plackett–Burman design. Each component was screened on the basis of ‘ p’ (probability) value, which was at 95% or above of confidence level. In the initial chitinase production medium containing five medium components chitinase production of 108 units/ml was obtained. In the Plackett–Burman method, significant factors influencing the chitinase production were screened. 4.21-fold increase in chitinase production was observed in 22nd medium of Plackett–Burman experimental design. Increase in production of endochitinase (3.95-fold) and chitobiase (2.31-fold) was observed.

Production of Alcaligenes xylosoxydans EMS33 in a Bench-scale Fermenter

Optimization of medium composition and pH for chitinase production by the Alcaligenes xylosoxydans mutant EMS33 was carried out in the present study and the optimized medium composition and conditions were evaluated in a fermenter. The medium components screened initially using Plackett–Burman design were (NH4)2SO4, MgSO4 7H2O, KH2PO4, yeast extract, Tween 20 and chitin in shake flask experiments. The significant medium components identified by the Plackett–Burman method were MgSO4 7H2O, Tween 20 and chitin. Central composite response surface methodology was applied to further optimize chitinase production. The optimized values of MgSO4 7H2O, Tween 20, chitin and pH were found to be 0.6 g/l, 0.05 g/l, 11.5 g/l and 8.0, respectively. Chitinase and biomass production of Alcaligenes xylosoxydans EMS33, was studied in a 2-l fermenter containing (g/l): chitin, 11.5; yeast extract, 0.5; (NH4)2SO4, 1; MgSO4 7H2O, 0.6; KH2PO4, 1.36 and Tween 20, 0.05. The highest chitinase production was 54 units/ml at 60 h and pH 8.0 when the dissolved O2 concentration was 60%, whereas the highest biomass production was achieved at 36 h and pH 7.5 without any dissolved O2 control.