Factorial design of experiments in the determination of adsorption equilibrium constants for basic methylene blue using biopolymer

Equilibrium conditions in the adsorption of a basic dye on Chitosan were studied. The Factorial Design methods and Analysis of Variance have been applied in the experimental determination of adsorption equilibrium constants. Factorial design with three levels of temperature (30v°C, 45v°C, 60v°C), pH (6.7, 8.1, 9.5), particle size (0.177 mm, 0.914 mm, 1.651 mm) was used in identification of significant effects and interactions in the calculation of the equilibrium constants. The dye adsorption capacity of chitosan was found to increase by decreasing the particle size and increasing temperature and pH. The methodology identifies the principal experimental variables, which have the greatest effect on the adsorption process.     

Studies on Box-Behnken design experiments: cellulose acetate-polyurethane ultrafiltration membranes for BSA separation

Ultrafiltration membranes were prepared from mixtures of cellulose acetate-polyurethane blend membranes. During the last 1 or 2 decades, the concentration purification and separation of Albumin by ultrafiltration through semipermeable membranes have been put into practice and hence membrane separation is considered as the unit operation. The blend solution was prepared from cellulose acetate and polyurethane in polar solvent in presence of polyvinylpyrrolidone as additive. The performance of modified blend membranes applied for Bovine Serum Albumin (BSA) separation by ultrafiltration technique using Box-Behnken design with three variables: additive, time and pressure. Three different levels was studied to identify a significant correlation between the effect of these variables on the amount of separation of BSA. The methodology identifies the principal experimental variables, which have the greatest effect on the separation process. The experimental values are in good agreement with predicted values, the correlation coefficient was found to be 0.9871.     

Statistical optimization of culture conditions for bacterial cellulose production using Box-Behnken design

Culture conditions in a jar fermentor for bacterial cellulose (BC) production from A. xylinum BPR2001 were optimized by statistical analysis using Box-Behnken design. Response surface methodology was used to predict the levels of the factors, fructose (X1), corn steep liquor (CSL) (X2), dissolved oxygen (DO) (X3), and agar concentration (X4). Total 27 experimental runs by combination of each factor were carried out in a 10-L jar fermentor, and a three-dimensional response surface was generated to determine the effect of the factors and to find out the optimum concentration of each factor for maximum BC production and BC yield. The fructose and agar concentration highly influenced the BC production and BC yield. However, the optimum conditions according to changes in CSL and DO concentrations were predicted at almost central values of tested ranges. The predicted results showed that BC production was 14.3 g/L under the condition of 4.99% fructose, 2.85% CSL, 28.33% DO, and 0.38% agar concentration. On the other hand, BC yield was predicted in 0.34 g/g under the condition of 3.63% fructose, 2.90% CSL, 31.14% DO, and 0.42% agar concentration. Under optimized culture conditions, improvement of BC production and BC yield were experimentally confirmed, which increased 76% and 57%, respectively, compared to BC production and BC yield before optimizing the culture conditions.     

Optimizing performance of a microbial carbon-capture cell using Box-Behnken design

Performance of a microbial carbon-capture cell (MCC), in which a microbial fuel cell and a photo-bioreactor gets mutually benefitted, is reliant on certain critical factors. In present investigation, Box-Behnken design is adopted to statistically evaluate the effect of influential parameters, namely nitrate concentration in catholyte, photoperiod and concentration of lipid extracted algae (LEA) of mixed culture microalgae in the anodic inoculum, on the performance of MCC. As per validation experiment, based on statistical model prediction, the ideal performance (higher power density and algal productivity) was achieved at LEA concentration, nitrate concentration and photoperiod in a range of 5.3–5.7 g/L, 44−50 mg/L and 10.00–10.67 h, respectively. Furthermore, the accuracy of model predictions was successfully validated by operating a separate MCC, which gave a power density and algal productivity (7.12 ± 0.09 W/m³ and 0.83 ± 0.02 g/L.day) close to the predicted output (7.05 ± 0.13 W/m³ and 0.838 ± 0.019 g/L.day). Box-Behnken design aided in optimizing the influential factors so as to make MCC economical and efficient for scale-up.     

Photocatalytic decolorization of congo red over ZnO powder using Box–Behnken design of experiments

Photocatalytic decolorization of congo red under sunlight illumination has been examined using ZnO catalyst. Batch experiments were conducted and Box-Behnken design has been employed to study the effect of different variables on the photodecolorization. Four variables such as dye concentration (0.05, 0.075, 0.10 g/l), weight of catalyst ZnO (0.025, 0.14, 0.25 g/l), pH (4, 7, 10) and time (1, 2, 3 (h)) were used to identify the significant effects and interactions in the batch studies. A second order polynomial regression model has been developed using the experimental data. It was found that the photodecolorization potential of ZnO was strongly affected by the variations in dye concentration, weight of catalyst ZnO, pH and time. The experimental values were in good agreement with the predicted values and the correlation coefficient was found to be 0.9982. Optimum conditions of the variables for the maximum photodecolorization are dye concentration (0.05 g/l) ZnO (0.16 g/l), pH (7.0) and time (2.0) (h). The maximum percentage of photodecolorization was observed to be 97%.     

The use of Box-Behnken design of experiments to study in vitro salt tolerance by Pisolithus tinctorius

The ectomycorrhizal fungus Pisolithus tinctorius was grown in vitro with different concentrations and combinations of three different sodium salts viz., sodium chloride, sodium sulphate and trisodium citrate (NaCl, Na₂SO4 and Na₃C₆H₅O₇) for 30 days. At the end of the experiment the mycelial biomass and protein content of the fungus was evaluated. Based on the salts tested the combination of NaCl and Na₂SO₄ in optimum concentrations actually promoted the growth of P. tinctorius. Box-Behnken design with three variables like NaCl, Na₂SO₄ and Na₃C₆H₅O₇ was used to identify a significant correlation between the effect of these variables on mycelial biomass production. The experimental values are in good agreement with predicted values, the correlation coefficient being 0.9880.     

Guidelines for the design and statistical analysis of experiments using laboratory animals

For ethical and economic reasons, it is important to design animal experiments well, to analyze the data correctly, and to use the minimum number of animals necessary to achieve the scientific objectives---but not so few as to miss biologically important effects or require unnecessary repetition of experiments. Investigators are urged to consult a statistician at the design stage and are reminded that no experiment should ever be started without a clear idea of how the resulting data are to be analyzed. These guidelines are provided to help biomedical research workers perform their experiments efficiently and analyze their results so that they can extract all useful information from the resulting data. Among the topics discussed are the varying purposes of experiments (e.g., exploratory vs. confirmatory); the experimental unit; the necessity of recording full experimental details (e.g., species, sex, age, microbiological status, strain and source of animals, and husbandry conditions); assigning experimental units to treatments using randomization; other aspects of the experiment (e.g., timing of measurements); using formal experimental designs (e.g., completely randomized and randomized block); estimating the size of the experiment using power and sample size calculations; screening raw data for obvious errors; using the t-test or analysis of variance for parametric analysis; and effective design of graphical data.     

Box-Behnken design in the development of optimized complex medium for phenol degradation using Pseudomonas putida (NICM 2174)

The biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different operating conditions. Box-Behnken design has been employed to study the effect of different experimental variables. Four variables of maltose (0.25, 0.5, 0.75?g/l), phosphate (3, 12.5, 22?g/l), pH (7, 8, 9) and temperature (30?°C, 32?°C, 34?°C) were used to identify the significant effects and interactions in the batch studies. A second order polynomial regression model, has been developed using the experimental data. It was found that the degrading potential of Pseudomonas putida (NICM 2174) was strongly affected by the variations in maltose, phosphate, pH and temperature. The experimental values were in good agreement with the predicted values, the correlation coefficient was found to be 0.9980. Optimum conditions of the variables for the growth of Pseudomonas putida (NICM2174) and for maximum biodegradation of phenol are maltose (0.052?g/l), phosphate (8.97?g/l), pH (7.9) and temperature (31.5?°C).     

Cell encapsulation using biopolymer gels for regenerative medicine

There has been a consistent increase in the mean life expectancy of the population of the developed world over the past century. Healthy life expectancy, however, has not increased concurrently. As a result we are living a larger proportion of our lives in poor health and there is a growing demand for the replacement of diseased and damaged tissues. While traditionally tissue grafts have functioned well for this purpose, the demand for tissue grafts now exceeds the supply. For this reason, research in regenerative medicine is rapidly expanding to cope with this new demand. There is now a trend towards supplying cells with a material in order to expedite the tissue healing process. Hydrogel encapsulation provides cells with a three dimensional environment similar to that experienced in vivo and therefore may allow the maintenance of normal cellular function in order to produce tissues similar to those found in the body. In this review we discuss biopolymeric gels that have been used for the encapsulation of mammalian cells for tissue engineering applications as well as a brief overview of cell encapsulation for therapeutic protein production. This review focuses on agarose, alginate, collagen, fibrin, hyaluronic acid and gelatin since they are widely used for cell encapsulation. The literature on the regeneration of cartilage, bone, ligament, tendon, skin, blood vessels and neural tissues using these materials has been summarised.     

Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments

The need for successful ex-vivo expansion and directed differentiation of haematopoietic stem cells (HSCs) for therapeutic applications has increased over the past decade. Haematopoietic cell cultures are complex and full characterisation of the process environment has yet to be achieved. The complexity and transient nature of HSC cultures make the identification, maintenance and control of optimal operating conditions challenging. Application of real-time, on-line monitoring techniques and process control strategies enhances the ability to operate bioprocesses of desired reproducibility and high product quality. In this review, we discussed the methods by which in vitro culture information necessary for bioprocess control may be obtained, including process considerations, monitoring and analytical tools, and design of experiments (DOE). The successful application of these tools may result in time- and cost-effective cultures for directed differentiation and expansion of haematopoietic components intended for clinical use.     

Performance optimization of polysulfone ultrafiltration membranes for riboflavin separation using design experiments

Polysulfone membranes containing different concentrations of polyethylene glycol (M.Wt: 600 Da) as additive was prepared using N,N-dimethyl formamide as solvent. The parameters chosen for the study of separation of vitamin B₂ were concentrations of additive and feed and solvent evaporation time. Box-Behnken design methods and analysis of variance have been applied to the experimental separation of vitamin B₂ by polysulfone membranes. Box-Behnken design with three levels of additive concentration (2.5, 6.25 and 10 wt%), feed concentration (0.002, 0.005 and 0.008%) and solvent evaporation time (10, 20 and 30 s) is used for the identification of significant effects and interaction for the batch studied. Second order polynomial regression model which was used for analysis of the experiment made significant effect. The experimental values are in good correlation with predicted values, and the correlation coefficient was found to be 0.9194.     

Determining the most important cellular characteristics for fracture healing using design of experiments methods

Computational models are employed as tools to investigate possible mechanoregulation pathways for tissue differentiation and bone healing. However, current models do not account for the uncertainty in input parameters, and often include assumptions about parameter values that are not yet established. The objective of this study was to determine the most important cellular characteristics of a mechanoregulatory model describing both cell phenotype-specific and mechanobiological processes that are active during bone healing using a statistical approach. The computational model included an adaptive two-dimensional finite element model of a fractured long bone. Three different outcome criteria were quantified: (1) ability to predict sequential healing events, (2) amount of bone formation at early, mid and late stages of healing and (3) the total time until complete healing. For the statistical analysis, first a resolution IV fractional factorial design (L₆₄) was used to identify the most significant factors. Thereafter, a three-level Taguchi orthogonal array (L₂₇) was employed to study the curvature (non-linearity) of the 10 identified most important parameters. The results show that the ability of the model to predict the sequences of normal fracture healing was predominantly influenced by the rate of matrix production of bone, followed by cartilage degradation (replacement). The amount of bone formation at early stages was solely dependent on matrix production of bone and the proliferation rate of osteoblasts. However, the amount of bone formation at mid and late phases had the rate of matrix production of cartilage as the most influential parameter. The time to complete healing was primarily dependent on the rate of cartilage degradation during endochondral ossification, followed by the rate of cartilage formation. The analyses of the curvature revealed a linear response for parameters related to bone, where higher rates of formation were more beneficial to healing. In contrast, parameters related to fibrous tissue and cartilage showed optimum levels. Some fibrous connective tissue- and cartilage formation was beneficial to bone healing, but too much of either tissue delayed bone formation. The identified significant parameters and processes are further confirmed by in vivo animal experiments in the literature. This study illustrates the potential of design of experiments methods for evaluating computational mechanobiological model parameters and suggests that further experiments should preferably focus at establishing values of parameters related to cartilage formation and degradation.     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Effect of permeability on aqueous biopolymer interfaces in spinning drop experiments

In this paper we show that interfaces in aqueous phase-separated biopolymer mixtures are permeable for all components present in the system. In spinning drop experiments, droplets of the low-density phase decreased up to 90% in volume over a time span of days to weeks, when inserted in a matrix of the high-density phase. We propose an expression for this change of volume in time in terms of diffusion coefficients of the components. From the magnitude of these coefficients, we conclude that the transfer of gelatin from inside the droplet to the outer phase was the rate-determining step in this process. Since the interfaces are permeable to all components, the properties of the system change in time. Therefore, the spinning drop technique is not an accurate method for the measurement of the equilibrium interfacial tension of these aqueous phase-separated systems.     

Optimization of medium composition for improving biomass production of Lactobacillus plantarum Pi06 using the Taguchi array design and the Box-Behnken method

An immune-enhancing strain, Lactobacillus plantarum Pi06, isolated from a healthy infant was used for biomass production following optimization of the medium in shake-flask culture. Preliminary studies showed that commercial MRS medium and cultivation under static conditions generated higher biomass production than four other tested media with or without a shaking condition. The selected medium composition, consisting of glucose, yeast extract, soy peptone, ammonium citrate, and corn steep liquor, was further optimized using a systematic method that integrated the Taguchi array design and the Box-Behnken method. The response effects of these factors were first investigated using Taguchi design under an L ₁₆ (4⁵) array. The suggested medium composition, derived from Statistica 7.1 using the Taguchi design, was applied to cultivate cells and a biomass of 7.16 g dry cell weight (DCW)/L was obtained. Response surface methodology based on the Box-Behnken method for the three response variables of glucose, yeast extract, and corn steep liquor was then used to further increase the biomass level to 8.94 g DCW/L. The resulting optimum medium consisted of 35 g/L glucose, 35 g/L yeast extract, and 40 mL/L corn steep liquor. Compared with the initial medium, the biomass yield was improved from 4.31 to 8.94 g DCW/L, an enhancement of approximately 107%.     

Optimization of the media ingredients for cutinase production from Colleotrichum lindemuthianum using mixture design experiments

Optimal concentrations of yeast extract, glucose and potassium phosphate in the fermentation medium have been identified for the maximum cutinase production from the fungi Colleotrichum lindemuthianum. A simplex lattice experimental design for mixtures was used to identify concentration ranges that yield maximum production. Three sets of experiments were performed all involving three ingredients. The sets of experiments differ in the number of concentration levels considered. In the first set we consider four concentration levels (i.e., 0%, 33%, 67%, 100%), and in the second and third sets we consider five and six levels, respectively. Results showed that the interactions among the nutrient ingredients are best captured when five- and six-level experiments are carried out. An algorithm has been proposed in this study to design the optimal medium. Various models were also developed to predict the enzyme production, and it is concluded that the cubic model obtained using six-level experimental data gives the best model. The study also highlighted the synergistic interaction between yeast extract and glucose toward cutinase production.     

On the design of genome mapping experiments using short synthetic oligonucleotides.

The DNA of an organism can be digested into smaller fragments, stored individually as clones in phage, for example, to create a clone library, and retrieved later, when needed. The original ordering of fragments is lost in the process of creating the library. Hence, it is important to be able to place clones in order according to their position along chromosome(s), and this process is referred to as "in vitro reconstruction" or "contig mapping" of an organismal genome. Clones in the phage library can be assigned binary call numbers by scoring each clone for hybridization (0 or 1) with a battery of short manufactured DNA sequences called synthetic oligonucleotides or with restriction enzyme digests of each clone. Those clones with similar call numbers are placed close together in the ordered library. We address the design question of how many clones and probes to use to carry out in vitro reconstruction of an organism's chromosomes. This physical mapping problem is placed in the context of coverage problems in geometrical probability. Various statistics are developed to summarize how an ordered library covers a chromosome, the extent of clone overlap, and the similarity between clone call numbers. Several tests for whether clones overlap are given, together with their power properties. A simulation study is used to determine how robust some of the tests for clone overlap are to model violations. Tables are presented for researchers to choose the number of clones and probes on the basis of both power and technical considerations surrounding the hybridization experiments.     

Design of experiments applications in bioprocessing: Chromatography process development using split design of experiments

Development of a chromatographic step in a time and resource efficient manner remains a serious bottleneck in protein purification. Chromatographic performance typically depends on raw material attributes, feed material attributes, process factors, and their interactions. Design of experiments (DOE) based process development is often chosen for this purpose. A challenge is, however, in performing a DOE with such a large number of process factors. A split DOE approach based on process knowledge in order to reduce the number of experiments is proposed. The first DOE targets optimizing factors that are likely to significantly impact the process and their effect on process performance is unknown. The second DOE aims to fine-tune another set of interacting process factors, impact of whom on process performance is known from process understanding. Furthermore, modeling of a large set of output response variables has been achieved by fitting the output responses to an empirical equation and then using the parametric constants of the equation as output response variables for regression modeling. Two case studies involving hydrophobic interaction chromatography for removal of aggregates and cation exchange chromatography for separation of charge variants and aggregates have been utilized to illustrate the proposed approach. Proposed methodology reduced total number of experiments by 25% and 72% compared to a single DOE based on central composite design and full factorial design, respectively. The proposed approach is likely to result in a significant reduction in resources required as well as time taken during process development. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2730, 2019