Statistical analysis of optimal culture conditions for Gluconacetobacter hansenii cellulose production

AIM: The purpose of this study was to analyse the effects of different culture parameters on Gluconacetobacter hansenii (ATCC 10821) to determine which conditions provided optimum cellulose growth. METHODS AND RESULTS: Five culture factors were investigated: carbon source, addition of ethanol, inoculation ratio, pH and temperature. jmp Software (SAS, Cary, NC, USA) was used to design this experiment using a fractional factorial design. After 22 days of static culture, the cellulose produced by the bacteria was harvested, purified and dried to compare the cellulose yields. The results were analysed by fitting the data to a first-order model with two-factor interactions. CONCLUSIONS: The study confirmed that carbon source, addition of ethanol, and temperature were significant factors in the production of cellulose of this G. hansenii strain. While pH alone does not significantly affect average cellulose production, cellulose yields are affected by pH interaction with the carbon source. Culturing the bacteria on glucose at pH 6.5 produces more cellulose than at pH 5.5, while using mannitol at pH 5.5 produces more cellulose than at pH 6.5. The bacteria produced the most cellulose when cultured on mannitol, at pH 5.5, without ethanol, at 20 degrees C. Inoculation ratio was not found to be a significant factor or involved in any significant two-factor interaction. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings give insight into the conditions necessary to maximize cellulose production from this G. hansenii strain. In addition, this work demonstrates how the fractional factorial design can be used to test a large number of factors using an abbreviated set of experiments. Fitting a statistical model determined the significant factors as well as the significant two-factor interactions.     

Optimization of extracellular fungal peroxidase production by 2 Coprinus species

Optimum culture conditions for the batch production of extracellular peroxidase by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were explored using 2 statistical experimental designs, including 2-level, 7-factor fractional factorial design and 2-factor central composite design. Of the 7 factors examined in the screening study, the concentrations of carbon (glucose) and nitrogen (peptone or casitone) sources showed significant effects on the peroxidase production by Coprinus sp. UAMH 10067. The optimum glucose and peptone concentrations were determined as 2.7% and 0.8% for Coprinus sp. UAMH 10067, and 2.9% and 1.4% for C. cinereus UAMH 4103, respectively. Under the optimized culture condition the maximum peroxidase activity achieved in this study was 34.5 U x mL(-1) for Coprinus sp. UAMH 10067 and 68.0 U x mL(-1) for C. cinereus UAMH 4103, more than 2-fold higher than the results of previous studies.     

Optimization of bacteriocin production by batch fermentation of Lactobacillus plantarum LPCO10

Optimization of bacteriocin production by Lactobacillus plantarum LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 2(3)(5-2) fractional factorial two-level design and tested for their influence on maximal bacteriocin production by L. plantarum LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 3(2)(3-1) fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27 degrees C, salt concentration from 2.3 to 2.5%, and L. plantarum LPCO10 inoculum size ranging from 10(7.3) to 10(7.4) CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 x 10(4) times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from L. plantarum LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from L. plantarum LPCO10 so that bacteriocins can be used as preservatives in canned foods.     

Multifactorial experimental design for optimizing transformation: Electroporation of Streptococcus thermophilus

A multifactorial process was used to optimize transformation of Streptococcus thermophilus by electroporation. Simple experimental designs were applied to study three, four, or five factors in eight experiments. Four qualitative factors, growth and recovering media, and plasmid and bacterial strains, were studied empirically. Eight quantitative factors, including electrical, physiological, and chemical parameters, were studied by fractional factorial designs. Effects of individual parameters as well as interactions between them were investigated and optimized. Optimization was performed for one S. thermophilus strain, ST11, and proved to work for all other tested strains of the same species. Transformation efficiencies of 9 x 10(2) to 6 x 10(5) transformants per microgram DNA were achieved, depending on the strains and vectors used. (c) 1994 John Wiley & Sons, Inc.     

Development of a Defined Medium for Pythium oligandrum Oospore Production

Complex and defined media have been previously proposed for production of oospores of Pythium oligandrum , a fungal mycoparasite of several disease-causing fungi. However, oospore production in synthetic media requires long periods of incubation and yields lower oospore numbers than in complex media. Moreover, although complex media produce high oospore yields, these yields are not reproducible because of the variability in the composition of complex nutrient sources. In the present study, the average composition of molasses reported in the literature was utilized as a base to develop a new defined medium for P. oligandrum oospore production. Forty-two substrates defined in nine stock solutions: carbohydrates, vitamins, sterols, non nitrogenous acids, amino acids, minerals, nucleic acid bases, CaCl 2 and MgSO 4 were tested at two levels (present or absent) in two fractional factorial designs. Each of these nine variables had a significant main effect on oospore production. Furthermore, the effect of each variable, except for vitamins, depended on the level of each other variable, expressed by two-variable interactions. The maximal predicted oospore production was calculated from the polynomial regressions associated with the two fractional factorial designs. Oospore productions were 1.3 and 2 ×10 6 oospores mL -1 from the first and second designs. In order to optimize the oospore production, the two levels of each variable were modified and all variables were experimented in a selection of a complete factorial design (Plackett and Burman design). A fitted first-order polynomial regression equation provided the combination of levels of variables for optimal oospore production. A defined medium, based on this combination of levels, was used for P. oligandrum growth. The optimized oospore production after 7 days growth was 4 ×106 oospores mL -1 as predicted by the polynomial regression. Oospore yields, biomass produced from oospores and oospore freeze-drying tolerance were similar when P. oligandrum was previously grown in molasses or in the new defined medium.     

重组巴斯德毕赤酵母发酵生产内切几丁质酶的培养条件优化

探讨重组巴斯德毕赤酵母发酵生产内切几丁质酶的最适培养条件,以期获得最佳的内切几丁质酶活力。以内切几丁质酶活力为指标,通过部分因子试验设计以及响应面法优化确定重组巴斯德毕赤酵母高产内切几丁质酶的最适培养条件。部分因子试验设计筛选的影响重组巴斯德毕赤酵母高产内切几丁质酶的3个关键因子为甲醇、油酸和吐温-80。响应面法优化的上述3个关键因子的最佳浓度分别为0.71%、0.086%和0.31%。重组巴斯德毕赤酵母发酵生产内切几丁质酶的最适培养条件为：酵母膏1%、酵母氮碱（YNB）1.34%、蛋白胨2%、甲醇0.71%、油酸0.086%、吐温-80 0.31%、PTM₁ 0.8%、pH 6.0。在上述培养条件下,重组巴斯德毕赤酵母产内切几丁质酶的活力高达30.92U/mL。与未优化前相比,酶活力提高了1.44倍。研究结果为内切几丁质酶的产业化生产和应用奠定了良好基础。     

A statistical approach to improve compound screening in cell culture media

The Chinese hamster ovary (CHO) cell line is widely used for the production of recombinant proteins due to its high growing capacity and productivity, as well as other cell lines derived later than CHO. Adapting cell culture media for each specific cell line is a key to exploit these features for cost effective and fast product generation. Media supplementation is generally addressed by means of one‐factor‐at‐a‐time or classical design of experiments approaches but these techniques may not be efficient enough in preliminary screening phases. In this study, a novel strategy consisting in folding over the Plackett–Burman design was used to increase cell growth and trastuzumab production of different CHO cell lines through supplementation with nonanimal recombinant compounds. Synergies between compounds could be detected with a reduced number of experiments by using this methodology in comparison to more conventional fractional factorial designs. In the particular case reported here, the sequential use of this modified Plackett–Burman in combination with a Box‐Behnken design led to a 1.5‐fold increase in cell growth (10 × 10⁶ cells/mL) and a two‐fold in trastuzumab titer (122 mg/L) in suspension batch culture.     

Optimization of Bacteriocin Production by Batch Fermentation of Lactobacillus plantarum LPCO10

Optimization of bacteriocin production by Lactobacillus plantarum LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 2³_5-2 fractional factorial two-level design and tested for their influence on maximal bacteriocin production by L. plantarum LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 3²_3-1 fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27°C, salt concentration from 2.3 to 2.5%, and L. plantarum LPCO10 inoculum size ranging from 10^7.3 to 10^7.4 CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 × 10⁴ times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from L. plantarum LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from L. plantarum LPCO10 so that bacteriocins can be used as preservatives in canned foods.     

Modeling and optimization of fermentative hydrogen production

Biohydrogen is a sustainable energy resource due to its potentially higher efficiency of conversion to usable power, non-polluting nature and high energy density. The purpose of modeling and optimization is to improve, analyze and predict biohydrogen production. Biohydrogen production depends on a number of variables, including pH, temperature, substrate concentration and nutrient availability, among others. Mathematical modeling of several distinct processes such as kinetics of microbial growth and products formation, steady state behavior of organic substrate along with its utilization and inhibition have been presented. Present paper summarizes the experimental design methods used to investigate effects of various factors on fermentative hydrogen production, including one-factor-at-a-time design, full factorial and fractional factorial designs. Each design method is briefly outlined, followed by the introduction of its analysis. In addition, the applications of artificial neural network, genetic algorithm, principal component analysis and optimization process using desirability function have also been highlighted.     

Micronutrient Requirements for Growth and Hydrocarbon Production in the Oil Producing Green Alga Botryococcus braunii (Chlorophyta)

The requirements of micronutrients for biomass and hydrocarbon production in Botryococcus braunii UTEX 572 were studied using response surface methodology. The concentrations of four micronutrients (iron, manganese, molybdenum, and nickel) were manipulated to achieve the best performance of B. braunii in laboratory conditions. The responses of algal biomass and hydrocarbon to the concentration variations of the four micronutrients were estimated by a second order quadratic regression model. Genetic algorithm calculations showed that the optimal level of micronutrients for algal biomass were 0.266 μM iron, 0.707 μM manganese, 0.624 μM molybdenum and 3.38 μM nickel. The maximum hydrocarbon content could be achieved when the culture media contained 10.43 μM iron, 6.53 μM manganese, 0.012 μM molybdenum and 1.73 μM nickel. The validation through an independent test in a photobioreactor suggests that the modified media with optimised concentrations of trace elements can increase algal biomass by 34.5% and hydrocarbon by 27.4%. This study indicates that micronutrients play significant roles in regulating algal growth and hydrocarbon production, and the response surface methodology can be used to optimise the composition of culture medium in algal culture.     

Optimization of Spongiochloris sp. biomass production in the abattoir digestate

In the present study, the abattoir digestate was used as a culture medium for Spongiochloris sp. growth with added mineral components under optimized conditions in batch culture. Firstly, an Hadamard matrix was used to investigate the impact of certain influencing factors on the Spongiochloris sp. growth. Then, a fractional factorial design 2^7-4 was successfully employed to optimize the concentration of different added components to abattoir digestate for increased Spongiochloris sp. biomass production. The major influencing factors were NaHCO₃ and FeSO₄ at a level of 2000 mg/L and 5 mg/L, respectively. A high biomass production of 5.29 × 10⁶ cell/mL and an important content of chlorophyll a of 65.32 mg/L were obtained after 42 days of culture of Spongiochloris sp. on the defined abattoir medium at static conditions.     

Effect of mineral nutrients on cell growth and self-flocculation of Tolypothrix tenuis for the production of a biofertilizer

The influence of mineral nutrients on the growth and self-flocculation of Tolypothrix tenuis was studied. The identification of possible limiting nutrients in the culture medium was performed by the biomass elemental composition approach. A factorial experimental design was used in order to estimate the contribution of macronutrients and micronutrients, as well as their interactions. Iron was identified to be limiting in the culture medium. The micronutrients influenced mainly cellular growth without effects on self-flocculation. Conversely, the self-flocculation capacity of the biomass increased at higher concentrations of macronutrients. The optimization of mineral nutrition of T. tenuis allowed a 73% increase in the final biomass level and 3.5 times higher flocculation rates.     

Response surface designs for experiments in bioprocessing

Many processes in the biological industries are studied using response surface methodology. The use of biological materials, however, means that run-to-run variation is typically much greater than that in many experiments in mechanical or chemical engineering and so the designs used require greater replication. The data analysis which is performed may involve some variable selection, as well as fitting polynomial response surface models. This implies that designs should allow the parameters of the model to be estimated nearly orthogonally. A class of three-level response surface designs is introduced which allows all except the quadratic parameters to be estimated orthogonally, as well as having a number of other useful properties. These subset designs are obtained by using two-level factorial designs in subsets of the factors, with the other factors being held at their middle level. This allows their properties to be easily explored. Replacing some of the two-level designs with fractional replicates broadens the class of useful designs, especially with five or more factors, and sometimes incomplete subsets can be used. It is very simple to include a few two- and four-level factors in these designs by excluding subsets with these factors at the middle level. Subset designs can be easily modified to include factors with five or more levels by allowing a different pair of levels to be used in different subsets.     

Factorial design and response surface optimization of crude violacein for Chromobacterium violaceum production

Initially an eleven variable, sixteen assay 2^15–11 fractional factorial design, was used to determine the key factors in the production of violacein produced by Chromobacterium violaceum, CCT 3496. Subsequently five and three factor central composite designs were executed to determine response surfaces with the aim of optimizing cellular mass and crude violacein production. The 7.5 g l^–1 dry cell mass and 0.17 g l^–1 crude violacein productions obtained with our initial culture medium were increased to 21 g l^–1 and 0.43 g l^–1, respectively, for a medium investigated in the three factor design.     

Reduction in laboratory animal use by factorial design

Factorial experimental designs (FEDs) can be used to study the effects of controllable variables, such as an experimental treatment, sex, strain, age, diet and prior treatment of animals, on some defined response. Such designs have been widely used in optimising manufacturing processes, but have rarely been used in optimising animal experiments in drug discovery. FEDs generally provide more information than the alternative "one-variable-at-a-time" approach, because each animal contributes information on the effect of every factor, and because such designs can highlight any interactions among the variables. Although FEDs can have any number of factors and levels of each factor, where many factors are to be explored, it is common to do an initial experiment using two levels of each factor, and in some cases fractional factorial designs can be used to reduce the total number of treatment combinations to manageable levels. These designs have been used successfully at AstraZeneca in the optimisation of in vivo drug screening experiments, where their use has effectively reduced the numbers of animals used in some routine screens.     

Use of a Fractional Factorial Design to Evaluate Interactions of Environmental Factors Affecting Biodegradation Rates

For investigation of main and interactive effects of six experimentally controlled environmental factors on phenol biodegradation in a shake-flask system, a largely neglected statistical procedure was applied. A major benefit resulting from the application of the orthogonal, fractional factorial design is that the number of experiments necessary to evaluate multifactor interactions is limited. In our investigation, the required number of experiments was reduced to 81 from the 324 necessary with conventional factorial designs; information was sacrificed for only 3 of 15 possible two-factor interactions. Six experimentally controlled factors were investigated at two or three treatment levels each; the six factors were (1) amount of phenol substrate, (2) amount of bacterial inoculum, (3) filtration of inoculum, (4) type of basal salts medium, (5) initial pH of basal salts medium, and (6) flask closure. Significant main effects were found for factors 1, 2, and 4; whereas significant interactive effects were found only for factor 2 with factor 3 and for factor 2 with factor 5. Our results suggest that the application of these statistical designs will greatly reduce the number of experiments necessary to evaluate multifactor effects on degradation rates during optimization of both hazard screening systems and waste treatment systems.     

Identification of in vitro folding conditions for procathepsin S and cathepsin S using fractional factorial screens

Human procathepsin S and cathepsin S were expressed as inclusion bodies in Escherichia coli. Following solubilization of the inclusion body proteins, fractional factorial protein folding screens were used to identify folding conditions for procathepsin S and cathepsin S. A primary folding screen, including eight factors each at two levels, identified pH and arginine as the main factors affecting procathepsin S folding. In a second simple screen, the yields were further improved. The in vitro folding of mature cathepsin S has never been reported previously. In this study we used a series of fractional factorial screens to identify conditions that enabled the active enzyme to be generated without the prodomain although the yields were much lower than achieved with procathepsin S. Our data show the power of fractional factorial screens to rapidly identify folding conditions even for a protein that does not easily fold into its active conformation.     

A Stochastic Algorithm for Selecting of Defining Contrasts in Two-Level Experiments

Franklin and Bailey (1977) provided an algorithm for construction of fractional factorial designs for estimating a user specified set of factorial effects. Their algorithm is based on a backtrack procedure. This is computer intensive when the number of factors is not small. We propose a stochastic search method called SEF (sequential elimination of factors) algorithm. The SEF algorithm is a simple modification of the exhaustive approach of the Franklin-Bailey algorithm since defining contrasts for the design of interest are chosen stochastically rather than choosing them in a systematic and exhaustive manner. Our experience shows the probability of success of obtaining a required design to be sufficiently large to make this a practical approach. The success probability may be expected to be rather small if the required design is close to a saturated design. We suggest the use of this stochastic alternative particularly when the number of factors is large. This can offer substantial savings in computing time relative to an exhaustive approach. Moreover, if the SEF algorithm fails to produce a design even after several attempts, one can always revert back to the Franklin-Bailey approach.     

Cyclodextrin glucosyltransferase production by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> 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 K₂HPO₄ 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.     

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS-22.

Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.