Key Media Components Research Articles

Medium optimization to improve growth and iron uptake by Bacillus tequilensis ASFS1 using fractional factorial designs

Many notable applications have been described for magnetic nanoparticles in delivery of diverse drugs and bioactive compounds into cells, magnetofection for the treatment of cancer, photodynamic therapy, photothermal therapy, and magnetic particle imaging (MPI). In response to the growing demand for magnetic nanoparticles for drug delivery or biomedical imaging applications, more effective and eco-friendly methodologies are required for large-scale biosynthesis of this nanoparticles. The major challenge in the large-scale biomedical application of magnetic nanoparticles lies in its low efficiency and optimization of nanoparticle production can address this issue. In the current study, a prediction model is suggested by the fractional factorial designs. The present study aims to optimize culture media components for improved growth and iron uptake of this strain. The result of optimization for iron uptake by the strain ASFS1 is to increase the production of magnetic nanoparticles by this strain for biomedical applications in the future. In the present study, design of experiment method was used to probe the effects of some key medium components (yeast extract, tryptone, FeSO4, Na2-EDTA, and FeCl3) on Fe content in biomass and dried biomass of strain ASFS1. A 25−1 fractional factorial design showed that Na2-EDTA, FeCl3, yeast extract-tryptone interaction, and FeSO4-Na2-EDTA interaction were the most parameters on Fe content in biomass within the experimented levels (p < 0.05), while yeast extract, FeCl3, and yeast extract-tryptone interaction were the most significant factors within the experimented levels (p < 0.05) to effect on dried biomass of strain ASFS1. The optimum culture media components for the magnetic nanoparticles production by strain ASFS1 was reported to be 7.95 g L−1 of yeast extract, 5 g L−1 of tryptone, 75 μg mL−1 of FeSO4, 192.3 μg mL−1 of Na2-EDTA and 150 μg mL−1 of FeCl3 which was theoretically able to produce Fe content in biomass (158 μg mL−1) and dried biomass (2.59 mg mL−1) based on the obtained for medium optimization. Using these culture media components an experimental maximum Fe content in biomass (139 ± 13 μg mL−1) and dried biomass (2.2 ± 0.2 mg mL−1) was obtained, confirming the efficiency of the used method.

Optimization of media components for enhanced carotenoid production by Paracoccus marcusii RSPO1 and assessment of their cytotoxicity against A549 and vero cells

In this study, we tried to explore the influence of various tricarboxylic acid (TCA) cycle intermediates on carotenoid production and with a focus on enhancing pigment biosynthesis, we conducted two statistical analysis. In case of TCA intermediates influence on pigment production by Paracoccus marcusii RSPO1; fumaric acid, and malic acid were observed as potent enhancers of pigment biosynthesis. Further, to optimize key media components for enhanced carotenoid production, the Plackett–Burman design was employed encompassing carbon, nitrogen sources, TCA cycle intermediates, and metal salts. The selected factors after Plackett Burman were fine-tuned through Response Surface Methodology and the optimal concentrations that have remarkably elevated carotenoid production were starch-2.24 g/l, MgSO4-0.416 g/l, ZnSO4-0.0157 g/l, and fumaric Acid-16 mM. Further, evaluation of pigment cytotoxicity against normal (Vero) and Non-Small Cell Carcinoma (A549) cells was performed. The resultant IC50 values were quantified as 161.3 µg/ml and 7.623 µg/ml for Vero and A549 cells, respectively. Moreover, a reactive oxygen species (ROS) determination study in A549 cells was done which have shown a noteworthy threefold ROS production in A549 cells through fluorescence spectroscopic observation. This implies that the bacterial carotenoids can act as potent pro-oxidants against cancerous cells while being nontoxic toward normal cells.

Promoting cell growth and characterizing partial symbiotic relationships in the co-cultivation of green alga Chlamydomonas reinhardtii and Escherichia coli.

By co-culturing selected microalgae and heterotrophic microorganisms, the growth rate of microalgae can be improved even under atmospheric conditions with a low CO2 concentration. However, the detailed mechanism of improvement of proliferative capacity by co-culture has not been elucidated. In this study, we investigated changes in the proliferative capacity of the green alga Chlamydomonas reinhardtii by co-culturing with Escherichia coli. In the co-culture, the number of C. reinhardtii cells reached 2.22 × 1010 cell/L on day 14 of culture. This was about 1.9 times the number of cells (1.16×1010 cell/L) on day 14 compared to C. reinhardtii cells in monoculture. The starch content per cell in the co-culture of C. reinhardtii and E. coli on the 14th day (2.09 × 10-11 g/cell) was 1.3 times higher than that in the C. reinhardtii monoculture (1.59 × 10-11 g/cell), and the starch content per culture medium improved 2.5 times with co-cultivation. By analyzing the gene transcription profiles and key media components, we clarified that E. coli produced CO2 from the organic carbon in the medium and the organic carbon produced by photosynthesis of C. reinhardtii, and this CO2 likely enhanced the growth of C. reinhardtii. Consequently, E. coli play a key role in promoting the growth of C. reinhardtii as well as the accumulation of starch which is a valuable intermediate for the production of a range of useful chemicals from CO2 . This article is protected by copyright. All rights reserved.

Optimization of culture conditions for biomass and lipid production by oleaginous fungus Penicillium citrinum PKB20 using response surface methodology (RSM)

Penicillium citrinum is a naturally occurring filamentous fungus with immense potential for biomass-based diesel production because they are prolific producers of cellulose-degrading enzymes and also accumulate significant amount of lipids. In this study, four media components, namely, carbon source, nitrogen (organic and inorganic) source and metal salts, and three fermentation parameters viz. Incubation time, temperature and pH were first screened for their effect on the biomass and lipid production by P. citrinum PKB20 using ‘one variable at a time’ (OVAT) approach. OVAT based experiments revealed that glucose ( X G ), yeast extract ( X Y ), ammonium sulphate ( X A ) and magnesium ion ( X M ) had the greatest impact on biomass yield and lipid accumulation when grown under batch cultivation mode at pH 7, temperature 30 °C and incubation time of 6 days. A central composite design based response surface methodology (RSM) was then adopted to model and optimize the ideal concentrations of the media constituents for maximum production of biomass and lipid from oleaginous fungus Penicillium citrinum PKB20.The optimization condition was found to be 10% of X G , 0.75 g/L of X Y , 0.38 g/L of X A and 0.06 g/L of X M and under optimum condition, the biomass and lipid production was 7.9 g/L and 1.73 g/L respectively. The study thus presents the optimum concentrations of key media components and culture conditions for maximum biomass and lipid production by Penicillium citrinum PKB20 under the studied cultivation and harvesting conditions. • Glucose, yeast extract, ammonium sulphate, magnesium significantly affect growth and lipid production by P. citrinum PKB20. • RSM was used to optimize the concentration of media constituents for maximum biomass (7.9 g/L) and lipid (1.73 g/L) production. • Influence of interaction of media components on growth and lipid biosynthesis were analyzed using RSM.

Modeling and Optimizing Culture Medium Mineral Composition for in vitro Propagation of Actinidia arguta.

The design of plant tissue culture media remains a complicated task due to the interactions of many factors. The use of computer-based tools is still very scarce, although they have demonstrated great advantages when used in large dataset analysis. In this study, design of experiments (DOE) and three machine learning (ML) algorithms, artificial neural networks (ANNs), fuzzy logic, and genetic algorithms (GA), were combined to decipher the key minerals and predict the optimal combination of salts for hardy kiwi (Actinidia arguta) in vitro micropropagation. A five-factor experimental design of 33 salt treatments was defined using DOE. Later, the effect of the ionic variations generated by these five factors on three morpho-physiological growth responses – shoot number (SN), shoot length (SL), and leaves area (LA) – and on three quality responses - shoots quality (SQ), basal callus (BC), and hyperhydricity (H) – were modeled and analyzed simultaneously. Neurofuzzy logic models demonstrated that just 11 ions (five macronutrients (N, K, P, Mg, and S) and six micronutrients (Cl, Fe, B, Mo, Na, and I)) out of the 18 tested explained the results obtained. The rules “IF – THEN” allow for easy deduction of the concentration range of each ion that causes a positive effect on growth responses and guarantees healthy shoots. Secondly, using a combination of ANNs-GA, a new optimized medium was designed and the desired values for each response parameter were accurately predicted. Finally, the experimental validation of the model showed that the optimized medium significantly promotes SQ and reduces BC and H compared to standard media generally used in plant tissue culture. This study demonstrated the suitability of computer-based tools for improving plant in vitro micropropagation: (i) DOE to design more efficient experiments, saving time and cost; (ii) ANNs combined with fuzzy logic to understand the cause-effect of several factors on the response parameters; and (iii) ANNs-GA to predict new mineral media formulation, which improve growth response, avoiding morpho-physiological abnormalities. The lack of predictability on some response parameters can be due to other key media components, such as vitamins, PGRs, or organic compounds, particularly glycine, which could modulate the effect of the ions and needs further research for confirmation.

Medium Optimization for Spore Production of a Straw-Cellulose Degrading Actinomyces Strain under Solid-State Fermentation Using Response Surface Method

The strains capable of degrading cellulose have attracted much interest because of their applications in straw resource utilization in solid-state fermentation (SSF). However, achieving high spore production in SSF is rarely reported. The production of spores from Streptomyces griseorubens JSD-1 was investigated in shaker-flask cultivation in this study. The optimal carbon, organic nitrogen and inorganic nitrogen sources were sucrose, yeast extract and urea, respectively. Plackett–Burman design (PBD) was adopted to determine the key medium components, and the concentration levels of three components (urea, NaCl, MgSO4·7H2O) were optimized with the steepest ascent path and central composite design (CCD), achieving 1.72 × 109 CFU/g of spore production. Under the optimal conditions (urea 2.718% w/v, NaCl 0.0697% w/v, MgSO4·7H2O 0.06956% w/v), the practical value of spore production was 1.69 × 109 CFU/g. The determination coefficient (R2) was 0.9498, which ensures an adequate credibility of the model.

Optimization of cultivation conditions for efficient production of carotenoid-rich DHA oil by Schizochytrium sp. S31

The high carotenoid content in docosahexaenoic acid (DHA) oil may serve as an avenue to avoid DHA oxidation during storage. To obtain carotenoid-rich DHA oil from Schizochytrium sp. S31, the effects of several key medium components were investigated. Shake-flask experiment revealed that in optimal conditions, glycerol was used as the carbon source with 0.25 g/L of KH2PO4 and a C/N ratio of 75:20. Fed-batch cultivation further showed that a high lipid accumulation (30.81 g/L) with 497.56 μg/g carotenoid and 12.24 g/L DHA was obtained under the optimized conditions. The increased carotenoid content resulted in improved defense against environmental stress, as indicated by the catalase (CAT) and superoxide dismutase (SOD) activities. Further transcriptome analysis demonstrated that the synthesis pathway of β-carotene was enhanced during the lipid turnover stage, which suggested that more carotenoids were produced to reduce oxidative stress. This study presented an efficient strategy for DHA oil production with high carotenoid content.

Optimized medium via statistical approach enhanced threonine production by Pediococcus pentosaceus TL-3 isolated from Malaysian food

BackgroundThreonine is an essential amino acid that is extensively used in livestock industry as feed supplement due to its pronounced effect in improving the growth performance of animals. Application of genetically engineered bacteria for amino acid production has its share of controversies after eosinophils myalgia syndrome outbreak in 1980s. This has urged for continuous search for a food grade producer as a safer alternative for industrial amino acid production. Lactic acid bacteria (LAB) appear as an exceptional candidate owing to their non-pathogenic nature and reputation of Generally Recognized as Safe (GRAS) status. Recently, we have identified a LAB, Pediococcus pentosaceus TL-3, isolated from Malaysian food as a potential threonine producer. Thus, the objective of this study was to enhance the threonine production by P. pentosaceus TL-3 via optimized medium developed by using Plackett–Burman design (PBD) and central composite design (CCD).ResultsMolasses, meat extract, (NH4)2SO4, and MnSO4 were identified as the main medium components for threonine production by P. pentosaceus TL-3. The optimum concentration of molasses, meat extract, (NH4)2SO4 and MnSO4 were found to be 30.79 g/L, 25.30 g/L, 8.59 g/L, and 0.098 g/L respectively based on model obtained in CCD with a predicted net threonine production of 123.07 mg/L. The net threonine production by P. pentosaceus TL-3 in the optimized medium was enhanced approximately 2 folds compared to the control.ConclusionsThis study has revealed the potential of P. pentosaceus TL-3 as a safer alternative to produce threonine. Additionally, the current study has identified the key medium components affecting the production of threonine by P. pentosaceus TL-3, followed by optimization of their concentrations by means of statistical approach. The findings of this study could act as a guideline for the future exploration of amino acid production by LAB.

Proteomic Effects of Magnesium Stress on Biofilm Associated Proteins Isolated from Cellulolytic Bacillus licheniformis YNP5-TSU

Optimization of cellulase activity is vital for synthesizing the end-products of second generation biofuel production. The slightest change in fermentation parameters can reduce the secretion of necessary enzymes to degrade cellulosic biomass. Determining the ecological effects of certain key media components is essential to understand how bacterial species will respond in a fluid environment. For our experiment a cellulosic media was designed to enhance the industrially important thermophile, Bacillus licheniformis YNP5-TSU. After several attempts to simplify the carboxymethylcellulose (CMC) media composition, impaired biofilm maturation and cellulase activity was noticed. This negative artifact occurred only when magnesium sulphate was removed from media. To analyze the shift in gene expression caused by magnesium stress, biofilm associated proteins were extracted from both control (4.0 mM MgSO4) and magnesium depleted (0.0 mM MgSO4) media at 24 hr and 48 hr incubation periods. These proteins were quantified through isobaric labeling and raw data generated from nanoLC-MS/MS identified over 2,000 proteins from the Bacillus licheniformis YNP5-TSU proteome (NCBI accession number ME). After statistical normalization and false discovery rate were calculated, a total of 161 proteins from magnesium depleted media and 238 proteins from control media were deemed statistically relevant. A closer look through STRING interconnected webs, data mining, and NCBI annotations revealed several up/down regulated proteins that had linkage to biofilm formation and cellulase secretion. In this study we are able to provide significant evidence that; (1) biofilm maturation and cellulase production are highly correlated and (2), their optimization is dependent on the expression of several key proteins.

Statistical Optimization of Medium Components by Response Surface Methodology to Enhance Menaquinone-7 (Vitamin K₂) Production by Bacillus subtilis.

Optimization of the culture medium to maximize menaquinone-7 (MK-7) production by Bacillus subtilis strain KCTC 12392BP in static culture was carried out using statistical experimental methods, including one factor at a time, fractional factorial design, and response surface methodology (RSM). Maltose (carbon source), tryptone (nitrogen source), and glycerol (activator) were identified as the key medium components for MK-7 synthesis by the fractional factorial design, and were selected for statistical optimization by RSM. The statistical analysis indicated that, in the range that was studied, maltose, tryptone, and glycerol were all critical factors having profound effects on the production of MK-7, with their coefficients for linear and quadratic all significant at the p < 0.05 level. The established model was efficient and feasible, with a determination coefficient (R²) of 0.9419. The predicted concentrations of maltose, tryptone, and glycerol in the optimal medium were determined as 36.78, 62.76, and 58.90 g/l, respectively. In this optimized medium, the maximum yield of MK-7 reached a remarkably high level of 71.95 ± 1.00 μg/ml after 9 days of static fermentation, which further verified the practicability of this optimized strategy.

Optimization of the Key Medium Components and Culture Conditions for Efficient Cultivation of G. sulfurreducens Strain PCA ATCC 51573 Using Response Surface Methodology

Microbial electrolysis cell (MEC) is a promising method for hydrogen production by microorganisms. The culture conditions were optimized for Geobacter sulfurreducens PCA ATCC 51573 using response surface methodology involving central composite design (CCD). Effects of sodium bicarbonate, sodium acetate, sodium fumarate, incubation temperature, and initial pH value of culture on cell growth of G. sulfurreducens PCA ATCC 51573 were studied. Experimental data obtained from CCD were utilized to generate a second-order polynomial regression model and to evaluate the effect of the variables studied on the optical density (OD) as the main response of bacterial growth of the strain PCA ATCC 51573. The statistical analysis showed that sodium bicarbonate, sodium acetate, and sodium fumarate had significant effect on OD value (P < 0.01). Similar results revealed sodium bicarbonate had significant interaction with sodium acetate and sodium fumarate (P < 0.05). On the other hand, quadratic term of temperature and initial pH value had a significant effect on OD value at 99% probability level (P < 0.01). Furthermore, interaction between culture temperature and initial pH value significantly affected OD value (P < 0.01). The optimum conditions suggested by the model for obtaining high OD value were 3.11 g/L NaHCO3, 4.10 g/L NaC2H3O2, 16.24 g/L Na2C4H2O4, 28.31 °C incubation temperature, and 6.80 pH value. The statistical model predicted that an OD value of 0.88 could be attained under optimum conditions. Result of verification experiment showed OD value of 0.87 was obtained which confirmed the adequacy of the model for the determining optimum conditions.

Medium optimization for pyrroloquinoline quinone (PQQ) production by Methylobacillus sp. zju323 using response surface methodology and artificial neural network–genetic algorithm

ABSTRACTMethylobacillus sp. zju323 was adopted to improve the biosynthesis of pyrroloquinoline quinone (PQQ) by systematic optimization of the fermentation medium. The Plackett–Burman design was implemented to screen for the key medium components for the PQQ production. CoCl2 · 6H2O, ρ-amino benzoic acid, and MgSO4 · 7H2O were found capable of enhancing the PQQ production most significantly. A five-level three-factor central composite design was used to investigate the direct and interactive effects of these variables. Both response surface methodology (RSM) and artificial neural network–genetic algorithm (ANN–GA) were used to predict the PQQ production and to optimize the medium composition. The results showed that the medium optimized by ANN–GA was better than that by RSM in maximizing PQQ production and the experimental PQQ concentration in the ANN–GA-optimized medium was improved by 44.3% compared with that in the unoptimized medium. Further study showed that this ANN–GA-optimized medium was also effective in improving PQQ production by fed-batch mode, reaching the highest PQQ accumulation of 232.0 mg/L, which was about 47.6% increase relative to that in the original medium. The present work provided an optimized medium and developed a fed-batch strategy which might be potentially applicable in industrial PQQ production.

Augmentation of ethanol production through statistically designed growth and fermentation medium using novel thermotolerant yeast isolates

Overproduction of metabolites, high product yield and process economics are greatly influenced by the media composition used for growth and fermentation. The main purpose of this study is to enhance the ethanol production through statistical tool of response surface methodology (RSM) by optimizing media components for the growth and fermentation of thermotolerant isolates Kluyveromyces marxianus NIRE-K1 and NIRE-K3. Five different salts were used in the Face-centered Central Composite Design (FCCD), with the responses of biomass formation and ethanol production for growth and fermentation, respectively. Yeast extract and K2HPO4 were found to be the key media components for the growth and fermentation which is revealed from their interaction in both the yeast isolates. Further studies on batch fermentation kinetics using the optimized values of the medium composition for K. marxianus NIRE-K1 and NIRE-K3 resulted in final ethanol concentration of 17.73 (86.27% of theoretical ethanol yield) and 19.01 g l−1 (94.12% of theoretical ethanol yield), respectively. An increase in the ethanol yield and productivity by 11.36, 10.42% and 2.0, 2.7% was revealed in NIRE-K1 and NIRE-K3, respectively, as compared to our previous study.

Animal Cell Expression Systems.

The glycan profile of therapeutic recombinant proteins such as monoclonal antibodies is a critical quality attribute, which affects the efficacy of the final product. The cellular glycosylation process during protein expression is dependent upon a number of factors such as the availability of substrates in the media, the intracellular content of nucleotide sugars, and the enzyme repertoire of the host cells. In order to control the variability of glycosylation it is important to understand the critical process parameters and their acceptable range of values to enable reproducible production of proteins with a predetermined glycan profile providing the desired biological function or therapeutic effect. The depletion of critical nutrients such as glucose or galactose, which may occur toward the end of a culture process, can lead to truncated glycans. Terminal galactosylation and sialyation are particularly variable but may be controlled by the presence of some key media components. Ammonia accumulation, pH, and dissolved oxygen levels are also known to be key bioprocess parameters that affect the glycosylation of recombinant proteins. Specific enzyme inhibitors can be added to the media to drive the formation of selected and predetermined glycan profiles. Various attempts have been made to predict the glycan profiles of cellular expressed proteins and have led to metabolic models based upon knowledge of metabolic flux and the kinetics of individual glycosylation reactions.In contrast to single recombinant proteins, the glycan profiles of viral vaccines are far more complex and difficult to predict. The example of influenzaA virus shows that hemagglutinin, the major antigenic determinant, has three to nine N-glycans, which may influence the antigenicity and efficacy of the vaccine. Glycosylation of the influenzaA virus has been largely unmonitored in the past as production has been from eggs, where glycan profiles of antigens are difficult if not impossible to control. Over the past decade, however, there have been various commercial influenza vaccines made available from cell technology using animal host cells. Analysis of glycosylation control shows that the type of host cell has the greatest influence on the final analyzed glycan profile. Other factors such as the virus strain, the cultivation system, or various process parameters have been shown to have only a minor effect on the glycosylation pattern. We predict that the analysis of glycan profiles in viral vaccines will become increasingly important in the development and consistent manufacturing of safe and potent vaccines. Graphical Abstract.

Enhanced production of thermostable amidase from Geobacillus subterraneus RL-2a MTCC 11502 via optimization of physicochemical parameters using Taguchi DOE methodology.

The specific effect of chemical and physical factors on amidase production from Geobacillus subterraneus RL-2a was investigated using design of experiments (DOE) methodology. The one-factor-at-a-time (OFAT) method was used to study the effects of carbon and nitrogen sources on amidase production. Subsequently, optimal levels of physical parameters and key media components, namely temperature, pH, sucrose, K2HPO4, NaCl, yeast, CaCl2·2H2O and MgSO4·7H2O, were determined using the Taguchi orthogonal array (OA) experimental design (DOE) methodology. Taguchi method based on three levels with a OA layout of L18 (21 × 37) with eight most influential factors on amidase synthesis for the proposed experimental design. Analysis of variance was performed on the obtained results and optimum condition suggested by statistical calculations was tested in a verification test. An increase of 169.56 % in amidase production compared to the unoptimized conditions was observed and the conversion of isonicotinamide was significantly improved after performing optimization techniques, including OFAT and Taguchi method. The result indicated that Taguchi method was effective in optimizing the culture conditions of amidase production.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0390-1) contains supplementary material, which is available to authorized users.

Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production.

Bacillus subtilis spores have important biotechnological applications; however, achieving both, high spore cell densities and sporulation efficiencies in fermentation, is poorly reported. In this study, medium components and culture conditions were optimized with different statistical methods to increase spore production of the plant growth promoting rhizobacteria B. subtilis EA-CB0575. Key medium components were determined with Plackett-Burman (PB) design, and the optimum concentration levels of two components (glucose, MgSO4·7H2O) were optimized with a full factorial and central composite design, achieving 1.37×10(9) CFU/mL of spore cell density and 93.5% of sporulation efficiency in shake flask. The optimized medium was used to determine the effect of culture conditions on spore production at bioreactor level, finding that maintaining pH control did not affect significantly spore production, while the interaction of agitation and aeration rates had a significant effect on spore cell density. The overall optimization generated a 17.2-fold increase in spore cell density (8.78×10(9)CFU/mL) and 1.9-fold increase in sporulation efficiency (94.2%) compared to that of PB design. These results indicate the potential of B. subtilis EA-CB0575 to produce both, high spore cell densities and sporulation efficiencies, with very low nutrient requirements and short incubation period which can represent savings of process production.

Development and manufacturability assessment of chemically-defined medium for the production of protein therapeutics in CHO cells.

Advantages of using internally developed chemically-defined (CD) media for cell culture-based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in-house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large-scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large-scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb-expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors.

Metal and dye removal using fungal consortium from mixed waste stream: Optimization and validation

Response surface methodology (RSM) involving two-level-three factors (23) full factorial design of experiment (DOE) was employed to optimize the concentrations of three media components (yeast extract, urea and ammonium nitrate) for a fungal consortium comprising Aspergillus lentulus, Aspergillus terreus and Rhizopus oryzae. The interaction between three variables was studied and modelled for four responses: chromium, copper, dyes (mixture of Acid Blue 161 and Pigment Orange 34) removal and biomass production. The results showed that yeast extract had a significant effect on Cu(II) removal (87.6mgL−1) and biomass production while dye removal was significantly affected by the combination of nutrients. It increased from 80.28% to 97.26% as the amount of urea and ammonium nitrate was increased from 0.5gL−1 to 0.75gL−1. A 50% reduction in the nutrient cost incurred for multiple pollutant removal was achieved by RSM based optimization. The results were validated by treating different industrial effluents supplemented with key media components. The utility of fungal consortium in simultaneous removal of dyes and metals from complex synthetic solution as well as industrial effluents has been demonstrated.

RESPONSE SURFACE OPTIMIZATION OF BIOSURFACTANT PRODUCED BY Pseudomonas aeruginosa MA01 ISOLATED FROM SPOILED APPLES

A potent biosurfactant-producing bacterial strain isolated from spoiled apples was identified by 16S rRNA as Pseudomonas aeruginosa MA01. Compositional analysis revealed that the extracted biosurfactant was composed of high percentages of lipid (66%, w/w) and carbohydrate (32%, w/w). The surface tension of pure water decreased gradually with increasing biosurfactant concentration to 32.5 mN m−1 with critical micelle concentration (CMC) value of 10.1 mg L−1. The Fourier transform infrared spectrum of extracted biosurfactant confirmed the glycolipid nature of this natural product. Response surface methodology (RSM) was employed to optimize the biosynthesis medium for the production of MA01 biosurfactant. Nineteen carbon sources and 11 nitrogen sources were examined, with soybean oil and sodium nitrate being the most effective carbon and nitrogen sources on biosurfactant production, respectively. Among the organic nitrogen sources examined, yeast extract was necessary as a complementary nitrogen source for high production yield. Biosurfactant production at the optimum value of fermentation processing factor (15.68 g/L) was 29.5% higher than the biosurfactant concentration obtained before the RSM optimization (12.1 g/L). A central composite design algorithm was used to optimize the levels of key medium components, and it was concluded that two stages of optimization using RSM could increase biosurfactant production by 1.46 times, as compared to the values obtained before optimization.

Metabolic analysis of antibody producing CHO cells in fed‐batch production

Chinese hamster ovary (CHO) cells are commonly used for industrial production of recombinant proteins in fed batch or alternative production systems. Cells progress through multiple metabolic stages during fed-batch antibody (mAb) production, including an exponential growth phase accompanied by lactate production, a low growth, or stationary phase when specific mAb production increases, and a decline when cell viability declines. Although media composition and cell lineage have been shown to impact growth and productivity, little is known about the metabolic changes at a molecular level. Better understanding of cellular metabolism will aid in identifying targets for genetic and metabolic engineering to optimize bioprocess and cell engineering. We studied a high expressing recombinant CHO cell line, designated high performer (HP), in fed-batch productions using stable isotope tracers and biochemical methods to determine changes in central metabolism that accompany growth and mAb production. We also compared and contrasted results from HP to a high lactate producing cell line that exhibits poor growth and productivity, designated low performer (LP), to determine intrinsic metabolic profiles linked to their respective phenotypes. Our results reveal alternative metabolic and regulatory pathways for lactate and TCA metabolite production to those reported in the literature. The distribution of key media components into glycolysis, TCA cycle, lactate production, and biosynthetic pathways was shown to shift dramatically between exponential growth and stationary (production) phases. We determined that glutamine is both utilized more efficiently than glucose for anaplerotic replenishment and contributes more significantly to lactate production during the exponential phase. Cells shifted to glucose utilization in the TCA cycle as growth rate decreased. The magnitude of this metabolic switch is important for attaining high viable cell mass and antibody titers. We also found that phosphoenolpyruvate carboxykinase (PEPCK1) and pyruvate kinase (PK) are subject to differential regulation during exponential and stationary phases. The concomitant shifts in enzyme expression and metabolite utilization profiles shed light on the regulatory links between cell metabolism, media metabolites, and cell growth.