Media Optimization Studies Research Articles

Media optimization for the production of alkaline protease by Bacillus cereus PW3A using response surface methodology

Media optimization studies and production of adenosylcobalamin (Vitamin B12) by environment friendly organism Rhizobium sppNeha Nohwar, Rahul V. Khandare, Neetin S. Desai

Optimization of Media Composition for Maximum Growth of Probiotic Lactobacillus fermentum NBC-08 Using Response Surface Methodology

In this study, it was aimed to determine the medium where Lactobacillus fermentum microorganisms with probiotic properties grow most efficiently by using Response Surface Method (RSM). Studies have been conducted on a 500 ml erlenmeyer scale, the medium optimization of the Lactobacillus fermentum strain was carried out according to the Central Composite Design (CCD) included in RSM. The effects of glucose, yeast extract, inorganic salts, and Tween 80 were investigated on the growth rate of the L. fermentum NBC-08 strain. Samples were taken at regular intervals from the erlenmeyer flask and the number of viable cells was measured by planting them in petri agar medium. In the study, the number of viable cells log10 (cfu/ml) was chosen as the response variable. As a result of the study, it was concluded that glucose and yeast extract are absolutely essential components in the medium. The optimum medium composition was found as 96.06 g/L glucose, 40.76 g L-1 yeast extract, 19.43 g L-1 inorganic salts, and 11.01 ml/L Tween 80. The production of the maximum L. fermentum strain was determined as 10.75 log10 (cfu ml-1). It is predicted that this study will make positive contributions to the fermentation conditions and medium optimization studies for production of lactic acid bacteria

Production and purification of extracellular fungal cellulases using agricultural waste

Media optimization studies and production of adenosylcobalamin (Vitamin B12) by environment friendly organism Rhizobium sppNeha Nohwar, Rahul V. Khandare, Neetin S. Desai

Media optimization studies and production of adenosylcobalamin (Vitamin B12) by environment friendly organism Rhizobium spp

Media optimization studies and production of adenosylcobalamin (Vitamin B12) by environment friendly organism Rhizobium spp

Statistical media optimization for the production of clinical uricase from Bacillus subtilis strain SP6

In this study, a potent uricase producing organism was isolated by a thorough screening and identified as Bacillus subtilis strain SP6 by using 16s rDNA sequencing. Response surface methodological optimization was employed for the enhanced production of uricase from newly isolated Bacillus subtilis strain SP6. In media optimization studies, Plackett Burman (PB) design was used for the selection of the critical media components; which were further optimized using central composite design (CCD). Lactose, soya peptone, uric acid and FeSO4.7H2O were found to be the critical factors influencing the enzyme production. Optimum uricase production with these factors was deduced using central composite design. Significant level of the factors were 12.2 g/L of lactose, 12.79 g/L of soya peptone, 2.55 g/L of uric acid and 0.00325 g/L FeSO4.7H2O. Use of statistical optimization upsurges uricase yield from 1.2 U/ml to 15.87 U/ml enhancing the overall production by 13.23 fold; which confirms that the model is effective for process optimization.

Inulinase production in shake-flask fermentations from Mucor circinelloides BGPUP-9

In the present study, an inulinase producing fungal isolate BGPUP-9 was isolated from inulin-rich root tubers of Asparagus racemosus. On the basis of morphological characterization and 18S rRNA gene sequencing, fungal isolate BGPUP-9 was identified as Mucor circinelloides. In shake-flask fermentations, agitation mode of cultivation showed a significant increase in inulinase production (7.10 IU/mL) from Mucor circinelloides BGPUP-9. Maximum inulinase production was observed after 4 days of cultivation at 30°C, under agitation (150 rpm). After medium optimization studies, medium containing: inulin, 2.5%, NH4H2PO4, 3.0%, KH2PO4, 0.2%, KCl, 0.1% and adjusted to pH 6.0, supported maximum (23.55 IU/mL) inulinase production. Thin layer chromatogram revealed exoinulolytic nature of the enzyme. This is the first report on inulinase production from Mucor circinelloides. The investigation depicts Mucor circinelloides BGPUP-9 as a potent candidate for inulinase production.

Screening and Selection of Medium Components for Cyclodextrin Glucanotransferase Production by New Alkaliphile Microbacterium terrae KNR 9 Using Plackett-Burman Design.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) production using new alkaliphile Microbacterium terrae KNR 9 was investigated by submerged fermentation. Statistical screening for components belonging to different categories, namely, soluble and raw starches as carbon sources, complex organic and inorganic nitrogen sources, minerals, a buffering agent, and a surfactant, has been carried out for CGTase production using Plackett-Burman factorial design. To screen out k (19), number of variables, k + 1 (20), number of experiments, were performed. Among the fourteen components screened, four components, namely, soluble starch, corn flour, yeast extract, and K2HPO4, were identified as significant with reference to their concentration effect and corresponding p value. Although soluble starch showed highest significance, comparable significance was also observed with corn flour and hence it was selected as a sole carbon source along with yeast extract and K2HPO4 for further media optimization studies. Using screened components, CGTase production was increased to 45% and 87% at shake flask level and laboratory scale fermenter, respectively, as compared to basal media.

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.

Medium Optimization, Purification, Characterization and Specificity Studies of Extracellular RNase from Streptomyces sp. (NCIM 2081)

Medium Optimization, Purification, Characterization and Specificity Studies of Extracellular RNase from Streptomyces sp. (NCIM 2081)

Enhancing exopolysaccharide antioxidant formation and yield from Phellinus species through medium optimization studies

The study was conducted to enhance exopolysaccharide (EPS) antioxidant formation (as measured by Trolox-Equivalent Antioxidant Capacity, TEAC) by Phellinus sp. P0988 through medium studies, while jointly increasing EPS yield. Out of seven medium components, four significant variables were identified based on linear models with statistical significance (p<0.05), namely rutin, FeSO4, and aspartate, which influenced TEAC, and malt extract, which influenced yield. Based on regression analysis of the experimental results from central composite design in response surface models, two second-order polynomial models adequately representing the relationship between these variables and EPS TEAC and yield were obtained, and the optimum concentration of these variables were determined and validated. Variable combinations for high EPS TEAC and yield were optimized through a desirability function test. The maximum EPS TEAC and yield reached 8.49±0.32mM and 10.92±0.68g/L, respectively, in a 7L bioreactor under the optimum variable combinations.

Optimization of culture media for L-asparaginase production by newly isolated bacteria, Bacillus sp. GH5

The enzyme L-asparaginase has been extensively studied by many researchers mainly because of its considerable therapeutic properties. Producing a convenient quantity of L-asparaginase can be conducted either by discovering new microbial sources with higher enzyme production or by manipulating the medium components for known microbial sources. The present paper discusses the studies carried out in order to enhance the production of L-asparaginase by newly isolated bacteria, Bacillus sp. GH5. Based on the results obtained from media optimization studies, a modified media was developed for optimal L-asparaginase production. Concisely, screening of the nutrients using a proper statistical design showed that tapioca starch, gelatin, ammonium oxalate, CaCO3, and L-asparagine were respectively the most important sources for carbon, organic nitrogen, inorganic nitrogen, mineral salt, and amino acids. The composition of the optimized medium was the following (per 1 L): 5.0 g L-asparagine; 0.5 g MgSO4 · 7H2O; 6.0 g NaHPO4 · 2H2O; 3.0 g (NH4)2C2O4; 0.5 g CaCO3; 0.014 g CaCl2 · 2H2O; 2.0% w/v tapioca starch; 5.0 g gelatin; and 15.0 g agar.

Medium optimization and proteome analysis of (R,R)-2,3-butanediol production by Paenibacillus polymyxa ATCC 12321

Paenibacillus polymyxa can produce the (R,R)-stereoisomer of 2,3-butanediol (2,3-BDL) which is industrially very useful. Two important factors affecting (R,R)-BDL production by P. polymyxa ATCC 12321, medium composition, and addition of acetic acid to the culture were investigated in this study with accompanying comparative proteomic analysis. For this purpose, a simple control strategy of O(2) supply was applied on the basis of an optimized basal medium: after a short period of batch cultivation with relatively high O(2) supply, the culture is switched into strong O(2) limitation, thereby promoting BDL formation. Three parallel fed-batch cultures starting from the same batch culture in an early stationary phase were then comparatively studied: the first one was running as control with the only change of O(2) supply; the second was, in addition, supplemented with 0.5 g/L yeast extract; and the third one was further added with 6 g/L acetate. Proteomic analyses of the three fed-batch cultures identified more than 86 proteins involved primarily in the central carbon metabolism, amino acid biosynthesis, energy metabolism, and stress responses. The examination of expression patterns of selected proteins, especially combined with fermentation data, gave valuable insights into the metabolic regulation of P. polymyxa under the different given conditions. Based on the proteomic analysis and further medium optimization studies, methionine was identified as one major growth-limiting factor in the basal medium and explains well the effect of yeast extract. Acetic acid was found to trigger the so far less studied acetone biosynthesis pathway in this organism. The latter is suggested in turn to enhance the switch from acidogenesis to solventogenesis. Thus, these findings extended our knowledge about BDL formation in P. polymyxa and provided useful information for further strain and process optimization.

Optimization of Human Granulocyte Macrophage-Colony Stimulating Factor (hGM-CSF) Expression Using Asparaginase and Xylanase Gene’s Signal Sequences in Escherichia coli

The toxicity of the recombinant protein towards the expression host remains a significant deterrent for bioprocess development. In this study, the expression of human granulocyte macrophage-colony stimulating factor (hGM-CSF), which is known to be toxic to its host, was enhanced many folds using a combination of genetic and bioprocess strategies in Escherichia coli. The N terminus attachment of endoxylanase and asparaginase signal sequences from Bacillus subtilis and E. coli, respectively, in combination with and without His-tag, considerably improved expression levels. Induction and media optimization studies in shake flask cultures resulted in a maximal hGM-CSF concentration of 365 mg/L in the form of inclusion bodies (IBs) with a specific product yield (Y (P/X)) of 120 mg/g dry cell weight in case of the asparaginase signal. Culturing the cells in nutrient rich Terrific broth maintained the specific product yields (Y (P/X)) while a 6.6-fold higher volumetric concentration of both product and biomass was obtained. The purification and refolding steps were optimized resulting in a 95% pure protein with a fairly high refolding yield of 45%. The biological activity of the refolded protein was confirmed by a cell proliferation assay on hGM-CSF dependent human erythroleukemia TF-1 cells. This study demonstrated that this indeed is a viable route for the efficient production of hGM-CSF.

Fermentative production of self-toxic fungal secondary metabolites

Fungi are well known for their vast diversity of secondary metabolites that include many life-saving drugs and highly toxic mycotoxins. In general, fungal cultures producing such metabolites are immune to their toxic effects. However, some are known to produce self-toxic compounds that can pose production optimization challenges if the metabolites are needed in large amounts for chemical modification. One such culture, LV-2841, was identified as the lead for one of our exploratory projects. This culture was found to be a slow grower that produced trace amounts of a known metabolite, cercosporamide, under the standard flask fermentation conditions, and extensive medium optimization studies failed to yield higher titers. Poor growth of the culture in liquid media was attributed to the self-toxicity of cercosporamide to the producing organism, and the minimum inhibitory concentration (MIC) of cercosporamide was estimated to be in the range of 8-16 microg/ml. Fermentations carried out in media containing Diaion HP20 resin afforded significantly higher titers of the desired compound. While several examples of resin-based fermentations of soil streptomyces have been published, this approach has rarely been used for fungal fermentations. Over a 100-fold increase in the production titer of cercosporamide, a self-toxic secondary metabolite, was achieved by supplementing the production medium with a commercially available neutral adsorbent resin.

Screening and Selection of Marine Isolate for l-Glutaminase Production and Media Optimization Using Response Surface Methodology

The current work details the screening of about 400 marine isolates from various marine niches, from which one isolate was finally selected based on the productivity of glutaminase (71.23 U/l). Further, biochemical identification tests and 16S rRNA sequencing identified this isolate to be Providencia sp. This isolate was taken up for further media optimization studies by using one-factor-at-a-time approach and subsequently by response surface methodology. A face centered central composite design was employed to investigate the interactive effects of four variables, viz., concentrations of glucose, methionine, urea, and succinic acid on glutaminase production. A significant influence of urea on glutaminase production was noted. Response surface methodology showed that a medium containing (g/l) glucose 10.0, urea 5.15, methionine 3.5, succinic acid 6.0, ammonium sulfate 2.5, and yeast extract 6.0 to be optimum for the production of glutaminase. The applied methodology was validated using this optimized media and enzyme activity 119 +/- 0.12 U/l and specific activity of 0.63 U/mg protein after 28 h of incubation at 25 degrees C was obtained.

Tetramethylpyrazine production from glucose by a newly isolated Bacillus mutant

2,3,5,6-Tetramethylpyrazine (TTMP) was produced using a newly isolated Bacillus mutant. Culture medium optimization studies showed that soytone, an enzyme-hydrolysate of soybean meal, with the supplementation of vitamins, can fully replace yeast extract plus peptone in supporting TTMP production from glucose. In a 5-l fermenter, using the optimized medium which contained 20% glucose, 5% soytone, 3% (NH(4))(2)HPO(4), and vitamin supplements, fermentations were carried out with stirring at 700 rpm, air flow at 1.0 vvm, controlled pH at 7.0, and temperature at 37 degrees C. TTMP reached 4.33 g l(-1) after 64.6 h cultivation. A product recovery method was described, which involved evaporation, crystallization, and lyophilization. The product purity was 99.88%, determined by GC with the normalization method. The main impurities were 2,3,5-trimethylpyrazine (0.09%) and 2-ethyl-3,5,6-trimethylpyrazine (0.02%), which were identified by GC/MS. (13)C NMR determination also gave a consistent result. Natural and high purity of the product and the utilization of cheap green renewable materials make this process promising to compete with TTMP chemical synthetic methods.

Optimization of Process Parameters for the Production of Inulinase from a Newly Isolated Aspergillus niger AUP19

Fifty strains were isolated from different soil samples on synthetic medium containing inulin as a sole carbon source for the production of extracellular inulinase. Of them, five isolates showed high inulinase activity and one of them was selected for identification and medium optimization studies. The isolate was identified as Aspergillus niger. Various physical and chemical parameters were optimized for inulinase production. Maximum productivity of inulinase (176 U ml−1) was achieved by employing medium containing 5% (w/v) inulin, galactose as additional carbon source, corn steep liquor and (NH4)H2PO4 as nitrogen sources, incubation period of 72 h, incubation temperature of 28 °C, pH 6.5, inoculum load at 10% (v/v) level and medium volume to flask volume ratio of 1:20 (v/v) with indented flasks.

Effect of the availability of magnesium ions in κ-carrageenan gels on the formation of conidia by Coniothyrium minitans

An isolate of Coniothyrium minitans did not sporulate on media to which 0‐05 mm magnesium was added when j-carrageenan was used to solidify the medium. Normal conidiation was observed when a technical grade agar or agar-agar was used as the gelling agent. The j-carrageenan and agars used in this study contained significant amounts of Mg. In the agar media all the Mg present was available to the fungus. By contrast, in the j-carrageenan gel a large portion of the Mg was bound by the gel and not available. Sporulation of C. minitans was only observed on media containing & 0‐17 mm of unbound Mg. The possible role of Mg in the initiation of conidium formation by C. minitans is discussed. Most microbial cultivations in industry are done in submerged culture. For some processes, however, cultivation on solid substrates in the absence of free-flowing water (solid-substrate cultivation) is advantageous. The production of high quantities of Coniothyrium minitans W. A. Campb. conidia, for instance, in stirred-tank reactors has heretofore not been reported. Numerous conidia, however, can easily be obtained when C. minitans is grown on various solid substrates (McQuilken & Whipps, 1995). These conidia are a promising biocontrol agent to treat Sclerotinia sclerotiorum contaminated soils and plants (Whipps & Gerlagh, 1992). The prospects of a biocontrol agent based on C. minitans are promising, provided that an ecient production process is developed. Optimization of the solid substrate with respect to spore formation is essential for the development of a cost-eective biocontrol agent. Quantification of biomass dry weight is important in such optimization studies. Direct gravimetric quantification of biomass is usually not possible when natural solid substrates are used, due to the close association of the microorganism with the substrate. During the development of an alternative model system based on j-carrageenan which facilitates the quantitative recovery of biomass dry matter (Weber, Tramper, & Rinzema, 1999), we found that magnesium is very important for sporulation. The present study evaluates the eect of several minerals in combination with various gelling agents on sporulation of C. minitans. We demonstrate that the amount of freely diusible magnesium strongly aects the spore yield. Furthermore, we demonstrate that the gelling agent used to mimic solid substrate cultivation may produce artefacts in medium optimization studies.

Fermentation studies of rustmicin production by a Micromonospora sp.

The antifungal antibiotic rustmicin was detected in the fermentation broth of the actinomycete MA 7094 as a specific inhibitor of sphingolipid biosynthesis in Candida albicans and as a potent fungicidal agent against Cryptococcus neoformans. Taxonomic characterization by both classical means and PCR fingerprinting supported the assignment of the producing culture to the genus Micromonospora. Fermentation medium optimization studies showed that the concentration of tomato paste in the medium was critical to increased production of rustmicin by MA 7094. The stimulatory effect of tomato paste in the medium on rustmicin production appeared to be related to the maintenance of pH at or below a value of 6.0. Addition of the antifoam agent P-2000 to the fermentation was found to dramatically reduce the rustmicin titer, while substitution of another antifoam agent, UCON-LB625, resulted in a 100% increase in the amount of rustmicin detected. After fermentation optimization studies and the generation of a non-sporulating mutant of MA 7094, the rustmicin titer was increased from an initial titer of 10mg/liter to 145 mg/liter.

On the topological features of optimal metabolic pathway regimes.

In this work, the stoichiometric metabolic network of Escherichia coli has been formulated as a comprehensive mathematical programming model, with a view to identifying the optimal redirection of metabolic fluxes so that the yield of particular metabolites is maximized. Computation and analysis has shown that the over-production of a given metabolite at various cell growth rates is only possible for a finite ordered set of metabolic structures which, in addition, are metabolite-specific. Each regime has distinct topological features, although the actual flux values differ. Application of the model to the production of 20 amino acids on four carbon sources (glucose, glycerol, lactate, and citrate) has also indicated that, for fixed cell composition, the maximum amino acid yield decreases linearly with increasing cell growth rate. However, when the cell composition varies with cell growth rate, the amino-acid yield varies in a nonlinear manner. Medium optimization studies have also demonstrated that, of the above substrates, glucose and glycerol are the most efficient from the energetic viewpoint. Finally, model predictions are analyzed in the light of experimental data.