Statistically-based Experimental Designs Research Articles

Optimization of bacterioruberin production from Halorubrum ruber and assessment of its antioxidant potential

Haloarchaea produce bacterioruberin, a major C50 carotenoid with antioxidant properties that allow for its potential application in the food, cosmetic, and pharmaceutical industries. This study aimed to optimize culture conditions for total carotenoid, predominantly comprising bacterioruberin, production using Halorubrum ruber MBLA0099. A one-factor-at-a-time and statistically-based experimental design were applied to optimize the culture conditions. Culture in the optimized medium caused an increase in total carotenoid production from 0.496 to 1.966 mg L− 1 Maximal carotenoid productivity was achieved in a 7-L laboratory-scale fermentation and represented a 6.05-fold increase (0.492 mg L–1 d–1). The carotenoid extracts from strain MBLA0099 exhibited a 1.8–10.3-fold higher antioxidant activity in vitro, and allowed for a higher survival rate of Caenorhabditis elegans under oxidative stress conditions. These results demonstrated that Hrr. ruber MBLA0099 has significant potential as a haloarchaon for the commercial production of bacterioruberin.

Statistical Optimization of Microcystis Growth and Microcystin Production in Natural Phytoplankton Community using Microcosm Bioassays

Eutrophication of freshwater lakes and reservoirs causing toxic cyanobacterial blooms has become a global health concern. Commonly used approaches of statistical modelling have not fully captured the complex response of cyanobacterial biomass and mi- crocystin concentration in response to stochastic variation of ambient conditions. This study applied statistically-based experimental design to screen out critical environmental factors, and to investigate the interactive effects of total nitrogen (TN), total phosphorus (TP), temperature (T) and light intensity (L) on Microcystis (MA) growth and microcystin (MC) production in the natural phytoplankton com- munity. A batch of central composite designed nutrient enrichment bioassays (CCD-NEBs) were conducted in an ambient-controlled microcosm based on pneumatic annular flume system. Second-order polynomial CCD regression models predicting MA density and MC concentration were established and validated. MC concentration was found positively related to MA dominance. The interactive effects of TN-TP and TN-L on both MA density and MC concentration were determined to be more significant than other factors. Ranges of the optimum TN/TP ratio for MA growth and MC production under various environmental conditions were defined as 9.5 ~ 10.2 and 9.9 ~ 10.4, respectively. The ranges of nutrient thresholds for MA density of 2.00 × 104 cells mL-1 and MC concentration of 1.0 μg L-1 under different temperature and light conditions were evaluated, respectively. This study revealed that nutrient thresholds for MA blooming events and cyanotoxins safety guideline were fluctuant under lake-dependant conditions of temperature and light intensity, and the optimum TN/TP mass ratios should be alerted for the eutrophication management of blooming formation and microcystin safety.

Statistical optimization of culture medium for improved production of antimicrobial compound by Streptomyces rimosus AG-P1441.

The nutritional requirements for antimicrobial activity of Streptomyces rimosus AG-P1441 were optimized using statistically-based experimental designs at a flask level. Based on a one-factor-at-a-time (OFAT) approach, glucose, corn starch and soybean meal were identified as the carbon and nitrogen sources having a significant effect on antimicrobial productivity. As a result of investigating the effect of glucose concentration, the highest antimicrobial activity was observed at 3% concentration. Response surface methodology (RSM) was then applied to optimize the growth medium components (corn starch, soybean meal, MgCl2 and glutamate). Antimicrobial productivity increased sharply when the medium consisted of 3% glucose, 3.5% corn starch, 2.5% soybean meal, 1.2mM MgCl2 and 5.9mM glutamate. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in antimicrobial activity, evaluated by the paper disc assay, revealed a 29mm inhibition zone diameter against Phytophthora capsici.

Optimization of the photoelectrocatalytic oxidation of landfill leachate using copper and nitrate co-doped TiO2 (Ti) by response surface methodology.

In this paper, a statistically-based experimental design with response surface methodology (RSM) was employed to examine the effects of functional conditions on the photoelectrocatalytic oxidation of landfill leachate using a Cu/N co-doped TiO2 (Ti) electrode. The experimental design method was applied to response surface modeling and the optimization of the operational parameters of the photoelectro-catalytic degradation of landfill leachate using TiO2 as a photo-anode. The variables considered were the initial chemical oxygen demand (COD) concentration, pH and the potential bias. Two dependent parameters were either directly measured or calculated as responses: chemical oxygen demand (COD) removal and total organic carbon (TOC) removal. The results of this investigation reveal that the optimum conditions are an initial pH of 10.0, 4377.98mgL-1 initial COD concentration and 25.0 V of potential bias. The model predictions and the test data were in satisfactory agreement. COD and TOC removals of 67% and 82.5%, respectively, were demonstrated.Under the optimal conditions, GC/MS showed 73 organic micro-pollutants in the raw landfill leachate which included hydrocarbons, aromatic compounds and esters. After the landfill leachate treatment processes, 38 organic micro-pollutants disappeared completely in the photoelectrocatalytic process.

Statistical Optimization of Crude Oil Biodegradation by Marinobacter sp. Isolated from Qeshm Island, Iran

Background: Hydrocarbons degradation is principally achieved by microorganisms in natural environments. The extent of hydrocarbons biodegradation is mainly conditioned by environmental factors and its success depends on the optimal condition for the crude oil degrading isolates. Objectives: The aims of the current study was to isolate and identify crude oil degrading bacterium from surface sediments of Qeshm Island, Iran and to evaluate the efficiency of a statistically-based experimental design for the optimization of crude oil degradation performed by the isolated strain. Materials and Methods: Crude oil degrading bacteria were isolated by serial dilutions of bacterial consortium. In order to optimize crude oil biodegradation by isolated strains, Plackett-Burman experimental design was used to evaluate nine factors affecting crude oil biodegradation in twelve experimental trials. To observe the best yield in crude oil biodegradation, factors that had higher effects were considered for the next stage in the biodegradation optimization process using the Taguchi experimental design. Results: A gram-negative bacterium strain named as the KK1- strain (with 98% homology with Marinobacter litoralis) was isolated from enrichment consortium. Among the various variables screened using the Plackett-Burman experimental design, pH, temperature, salinity and NH 4 Cl were determined as the most significant factors and considered for the next stage of the biodegradation optimization process using the Taguchi experimental design. Theoretically, the optimum degradation conditions were determined to be: pH = 8, temperature = 35˚C, salinity = 30 ppt and NH 4 Cl = 1 g.L -1 . The validity of the predicted optimized condition was tested by conducting experiments considering the predicted criteria. Biodegradation efficiency of 58.32±5.57% was achieved under the suggested conditions, which was significantly higher than that achieved by the primary conditions (35%). Conclusions: Indigenous bacteria from surface sediments of Qeshm Island were found to be able to degrade crude oil. Our results showed that a combination of the Plackett-Burman and the Taguchi experimental designs may be successfully used to find the optimal amounts of factors required for crude oil biodegradation.

Screening of Lipid High Producing Mutant from Rhodotorula Glutinis by Low Ion Implantation and Study on Lipid Extration Technique

The paper studied on parameters of ion implantation into lipid producing strain Rhodotorula glutinis and lipid extration technology. It was found that the strain had a higher positive mutation rate when the output power was 10keV and the dose of N+ implantation was 80×2.6×1013 ions/cm2. Then a high-yield mutant strain D30 was obtained and it’s lipid yield which was 3.10 g/L increased by 33.05% than that of the original. Additionally, statistically-based experimental designs were applied for the optimization of lipid extraction by acid-heating coupling ultrasonic technique. By a Plackett-Burman design, it was found that three factors, treatment time of HCl (p=0.036) , ultrasonic time (p=0.0105) and rate of extracting solution (VCHCl3:VCH3OH) (p=0.0105), had significant effect on lipid extraction. Subsequently, these significant factors were optimized using response surface methodology (RSM), and the optimized parameters of lipid extraction were as follows: 34 min for treatment time of HCl, 7.5 min for ultrasonic treating time, 1.9:1 for rate of extracting solution (VCHCl3:VCH3OH). Finally the fermentation characteristic of high-yield mutation strain D30 was studied, when fermentation time was 10 d, it’s lipid yield increased to 7.81 g/L

Biodegradation of crude petroleum oil and environmental pollutants by Candida tropicalis strain

ABSTRACT A local yeast isolate named A was isolated from polluted area of Abou-Qir gulf (Alexandria, Egypt), identified according to a partial sequence of 18sRNA as Candida tropicalis . The isolate showed a high potency in petroleum oil biodegradation as well some hydrocarbons. Morphological changes in cell diameter of this yeast were recognized upon growing the target cell in sea water medium supplemented with petroleum oil as sole carbon source in comparison to the growth in enriched medium. Statistically-based experimental design was applied to evaluate the significance of factors on petroleum oil biodegradation by this yeast isolate. Eleven culture conditions were examined by implementing Plackett-Burman factorial design where aeration, NH 4 Cl and K 2 HPO 4 had the most positive significance on oil degradation. Key words: Key words : petroleum oil, Candida tropicalis , 18Sr RNA, biodegradation, Plackett-Burman * Author for correspondence: nadiastuttgart@yahoo.com

Medium Optimisation for Improved Growth-Rate Inhibition of Microcystis Aeruginosa Exposed to AgBiO<sub>3</sub> Using Response-Surface Methodology

Blooms of the Microcystis aeruginosa in water have been a long-standing problem for water quality and a human-health issue. Public concern for the inhibition of Microcystis aeruginosa has become increasingly urgent. AgBiO3 is an algaecide having good inhibitory effects on the growth of Microcystis aeruginosa with hydroxyl radicals generated by photocatalyst. The inhibition of Microcystis aeruginosa exposed to AgBiO3 was optimised using statistically-based experimental designs. The four variables, namely, ZnSO4, CaCl2, CuSO4 and NaNO3, were screened as significant effects and were further optimised to determine the optimum levels and their interactions. The optimal concentrations of variables for maximum inhibition were 0.24 mg/L ZnSO4, 125.94 mg/L CaCl2, 0.28 mg/L CuSO4 and 600 mg/L NaNO3. The maximum inhibition rate predicted by the model was 61.86%, in near-perfect agreement with the experimental value (60.07%), and significantly better than unoptimised medium (42.79%).

Production of laccase from Pleurotus florida NCIM 1243 using Plackett–Burman Design and Response Surface Methodology

Statistically-based experimental designs were applied to optimize the fermentation for the production of laccase by Pleurotus florida NCIM 1243. Eleven components were screened for their significant effect on laccase production using Plackett-Burman factorial design. Glucose (carbon source), asparagine (nitrogen source), CuSO(4)(inducer) and incubation period were found to have highest positive influence on the laccase production. The combined effect of these factors on laccase production was studied using central composite design of Response surface methodology. The optimal point of variables for maximum laccase production using Response surface methodology are glucose (15.21 g/l), asparagine (6.40 g/l), CuSO(4) (91.78 microM) and incubation period (178.55 h), respectively. The maximum enzyme activity predicted by the model was 5.0 U/ml which was in perfect agreement with the actual experimental value (4.8 U/ml). Further, partially purified laccase from the optimized cultural condition was used for the decolorization of reactive dyes, Reactive Blue 198 and Reactive Red 35.

Cyclodextrin glucosyltransferase production by Bacillus megaterium NCR: evaluation and optimization of culture conditions using factorial design

Statistically-based experimental designs were used to optimize the production of cyclodextrin glucosyltransferase (CGTase) from a local isolate of Bacillus megaterium using shack culture fermentation. Seven cultural conditions were examined for enzyme production and specific activity using Plackett-Burman factorial design. Fermentation time and K(2)HPO(4) level were the crucial for factors improving enzyme production process. The steepest ascent design was adopted-based on the results recorded with Plackett-Burman design. Maximal enzyme estimates (activity 56.1 U/ml, and specific activity 62.7 U/mg protein) were achieved. A verification experiment was carried out to examine model validation of this optimization.

Medium optimization for enhanced production of cytosine-substituted mildiomycin analogue (MIL-C) by Streptoverticillium rimofaciens ZJU 5119.

Cytosine-substituted mildiomycin analogue (MIL-C) was produced effectively by supplementing cytosine into the culture of Streptoverticillium rimofaciens. In order to improve the yield of MIL-C, statistically-based experimental designs were applied to optimize the fermentation medium for S. rimofaciens ZJU 5119. Fifteen culture conditions were examined for their significances on MIL-C production using Plackett-Burman design. The Plackett-Burman design and one-variable-at-a-time design indicated that glucose and rice meal as the complex carbon sources, and peanut cake meal and NH4NO3 as the complex nitrogen sources were beneficial for MIL-C production in S. rimofaciens ZJU 5119. The results of further central composition design (CCD) showed that the optimal concentration of glucose, rice meal and peanut cake meal were 18.7 g/L, 64.8 g/L and 65.1 g/L, respectively. By using this optimal fermentation medium, the MIL-C concentration was increased up to 1336.5 mg/L, an approximate 3.8-fold improvement over the previous concentration (350.0 mg/L) with un-optimized medium. This work will be very helpful to the large-scale production of MIL-C in the future.

Enhanced production of exocellular glucansucrase from Leuconostoc dextranicum NRRL B-1146 using response surface method

Statistically-based experimental designs were applied to optimize the fermentation for the production of glucosyltransferase by Leuconostoc dextranicum NRRL B-1146. Eleven medium components were examined for their significance on enzyme production using Plackett–Burman factorial design. Tween 80, sucrose and K 2HPO 4 significantly improved the enzyme production process. The combined effect of these nutrients on glucansucrase production were studied using a 2 2 full-factorial central composite design, a second-order polynomial was established to identify the relationship between the enzyme output and the three medium components. The optimal concentration of variables for maximum glucansucrase production were Tween 80 (0.55%, v/v); sucrose (5.6%, w/v) and K 2HPO 4 (1%, w/v). The maximum enzyme activity by predicted model was 6.53 U/ml that was in perfect agreement with the actual experimental value (6.40 U/ml).

Application of statistically-based experimental designs in medium optimization for spore production of Bacillus subtilis from distillery effluent

Spore production of Bacillus subtilis from distillery effluent was optimized using statistically-based experimental designs. The two-level Plackett–Burman design was applied to choose the nutrient supplements significantly influencing spore production. Among the seven variables we tested, the most significant variables influencing spore production were statistically elucidated for optimization, and included (NH4)2SO4, corn flour and MgSO4. The optimum concentration of each significant variable was then predicted using Box–Behnken design. A second-order polynomial was determined by the multiple regression analysis of this experimental data. The optimum values for the critical nutrient supplements for the maximum were obtained as followed: (NH4)2SO4, 4.54%; corn flour, 1.2%; MgSO4, 0.56% with the corresponding value of maximum spore production of 7.24 × 108 spores/ml. A verification experiment performed under the optimum conditions resulted in 6.95 × 108 spores/ml. The determination coefficient (R2) was 0.98, which ensure an adequate credibility of the model.

Efficiency of Penicillium chrysogenum PTCC 5037 in reducing low concentration of chromium hexavalent in a chromium electroplating plant wastewater

The effectiveness of Penicillium chrysogenum was evaluated for reducing Cr(VI) from the wastewater of a chromium electroplating plant. Statistically-based experimental designs were applied to optimize the condition for reducing Cr(VI) to Cr(III). By applying Plackett–Burman factorial design and central composite design as the optimization step, attempts were made to identify optimal values of the three factors that bringing about maximum microorganism activity and therefore maximum hexavalent chromium(VI) bioreduction. It was found that each gram of P. chrysogenum of dry biomass condition could reduce 66 mg of Cr(VI) to Cr(III) in the wastewater of the chromium electroplating plant.

Optimization of carotenoid production by Rhodotorula graminis DBVPG 7021 as a function of trace element concentration by means of response surface analysis

As the final step of a study aiming at the optimization of culture conditions for the production of carotenoids by red yeasts, a statistically-based experimental design has been applied to assess the influence of selected trace elements on carotenogenesis in Rhodotorula graminis DBVPG 7021. In particular, a central composite design scheme has been used to evaluate the influence of Fe 3+, Co 2+, Mn 2+, Al 2+ and Zn 2+ (within the range 0–50 ppm) on various responses, namely biomass (B), total carotenoid production (TC) and percentage of specific carotenoids (β-CAR, β-carotene; γ-CAR, γ-carotene; TN, torulene; TD, torularhodin) on total carotenoids. Second-order polynomial models were calculated and reduced equations were designed by neglecting non-significant ( P < 0.01) regression coefficients. Reduced equations were used to calculate the optimal concentration of trace elements in view of maximising the level of B, TC, β-CAR, γ-CAR, TN and TD. After optimization, average final values total carotenoids (TC = 803.2 μg/g DW) was about 370% of value observed as central point of the central composite design scheme. Under the same condition, average final values of other responses were: B = 5.40 g/L; β-CAR = 50.3%; γ-CAR = 15.4%; TN = 22.7%; TD = 11.6%. All above experimental data are in good agreement with calculated ones, thus confirming the reliability of the proposed empirical model in describing carotenoid production by R. graminis as a function of trace element concentrations.

Application of statistically-based experimental designs for the optimization of nisin production from whey.

Statistically-based experimental designs were applied for the optimization of nisin production by Lactococcus lactis in a whey-based medium. Yeast extract, KH2PO4, and MgSO4 were identified to have significant effects on nisin biosynthesis by a Plackett-Burman design. These three significant factors were subsequently optimized using central composite design, and the optimal conditions were determined to be 12.067 g l(-1) for yeast extract, 0.569 g l(-1) for KH2PO4, and 0.572 g l(-1) for MgSO4. The validity of the optimal conditions was verified by a separate experiment.

L-asparaginase production by Pseudomonas aeruginosa in solid-state culture: evaluation and optimization of culture conditions using factorial designs

Statistically-based experimental designs were applied to optimize a solid-state fermentation for the production of l-asparaginase by Pseudomonas aeruginosa 50071. Fifteen culture conditions were examined for their significance on enzyme production and specific activity using Plackett–Burman factorial design. pH, casein hydrolysate and corn steep liquor were the most significant factors improving enzyme production process. The second optimization step was to identify optimal values of the three factors that bringing about maximum l-asparaginase activity, using Box–Behnken design. Maximal enzyme activity (142.8 IU) has been detected under the following conditions, pH 7.9; casein hydrolysate, 3.11%; and corn steep liquor, 3.68% which is more than five folds the activity in basal medium. A verification experiment was carried out to examine model validation and revealed more than 99% validity.

The machinability and tribological characteristics of aluminum alloys with improved elevated temperature properties using rapidly solidified powder

In this investigation the tribological characteristics of rapidly solidified Al–8Fe–4Ce with improved elevated temperature properties were studied. Such characteristics were compared with cast aluminum–silicon alloy and cast zinc–aluminum alloy. These materials included Al–13Si, Zn–35Al, Zn–35Al–Si, Zn–35Al–3.75Si and Zn–35Al–5.8Si. The wear rates of all materials were tested on a crossed-cylinders wear machine against 440C stainless steel counterface lapped by random abrasion using diamond paste to the desired average surface roughness. The effects of sliding distance on both the worn volume and the coefficient of friction were examined. The aluminum–iron–cerium alloy (Al–8Fe–4Ce) showed the lowest wear rate. The experiments were then extended on this material to examine the effect of varying the applied load and sliding speed on its wear rate. It was found that increasing the applied load increased the wear rate while it was slightly sensitive to the change in sliding speed. As the wear results showed that the Al–8Fe–4Ce alloy has the lowest wear rates, its machinability during turning operation was studied. Statistically-based experimental design (response surface methodology) using central composite second-order rotatable design technique was used to improve the experimentation design without loss of accuracy of the results. The interaction of cutting parameters (cutting speed, feed rate and depth of cut) was examined and their effect on the average surface roughness was reported. It was found that employing a combination of high cutting speed and small depth of cut with small feed rate causes a significant reduction in Ra. The data were represented in three-dimensional and contour graphs for selecting the appropriate machining conditions required to achieve desired values of surface roughness.

Application of statistically-based experimental designs for the optimization of eicosapentaenoic acid production by the diatom Nitzschia laevis.

Statistically based experimental designs were applied to the optimization of medium components and environmental factors for eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis in heterotrophic conditions. First, the Plackett-Burman design was used to evaluate the effects of variables including medium components and environmental factors on cell growth and EPA production. Among these variables, NaCl, CaCl(2), PI metal solution, pH, and temperature were identified to have the significant effects (with confidence level > 90 %). Subsequently, the concentrations of NaCl, CaCl(2), PI metal solution as well as the values of pH and temperature were optimized using central composite design. The cell growth and EPA production were found to respectively correlate to NaCl, CaCl(2), pH, and temperature that could be represented by second-order polynomial models. The optimal values of the four parameters were determined by response surface and numerical analyses as 8 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 19.8 degrees C for cell dry weight (DW), and 14 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 18 degrees C for EPA production, respectively. The subsequent verification experiments confirmed the validity of the models. This optimization strategy led to a DW of 9 g/L, an EPA yield of 280 mg/L and an EPA productivity of 28 mg/L/d, respectively, which were considerably higher than those obtained in the previous studies.

Medium optimization for spore production of coniothyrium minitans using statistically-based experimental designs

Statistically-based experimental designs were used to optimize a chemically defined solid medium for the spore production of Coniothyrium minitans. In the first optimization step the influence of starch, urea, phosphate, magnesium, calcium, thiamin and trace elements on spore production was evaluated using a fractional factorial design. Starch and trace elements influenced spore production positively while urea affected spore production negatively. The other components had no significant influence on spore production. In the second and third steps the concentrations of starch, urea and trace elements were further optimized using central composite designs and response surface analysis. This optimization strategy allowed the spore production to be increased by a factor 7 from 4 x 10(9) to almost 3 x 10(10) spores per Petri dish of 9 cm diameter. Copyright 1999 John Wiley & Sons, Inc.