Effect Of Medium Composition Research Articles

Effect of medium composition on pentachlorophenol removal by Amylomyces rouxii in solid-state culture

Pentachlorophenol (PCP) degradation by fungi was studied in solid state cultures (SSC); these cultures can be used in different bioremediation strategies. We assessed the effect of medium composition and PCP initial concentration on growth of Amylomyces rouxii and PCP degradation. This fungus displayed a high degradation rate on PCP when tyrosine was added to the culture medium, 0.030 mg PCP mg −1 biomass h −1, as compared against a culture without tyrosine (0.017 mg PCP mg −1 biomass h −1). Besides, results showed that growth and specific degradation were higher at high initial PCP concentration; at 100 mg PCP l −1, 100% of initial PCP was degraded at 72 h, and 50% of PCP was degraded when initial PCP was 12.5 mg l −1. In these cultures, specific degradation rates were 0.081 and 0.026 mg PCP mg −1 biomass h −1, respectively.

Nutritional requirements of mycelial growth of Cordyceps sinensis in submerged culture

The nutritional requirements for mycelial growth of Cordyceps sinensis in semi-synthetic liquid media were investigated. The results provide a basis for further physiological study and industrial fermentation of the fungus. Nutritional requirements, including 17 carbohydrates, 16 nitrogen compounds, nine vitamins, four macro-elements, four trace-elements and eight ratios of carbon to nitrogen, were studied for their effects on the mycelial growth in submerged cultures of C. sinensis by using one-factor-at-a-time and orthogonal matrix methods. Among these variables, sucrose, peptone, folic acid, calcium, zinc and a carbon to nitrogen ratio 12 : 1 were identified as the requirements for the optimum mycelial growth. The concentrations of sucrose, peptone and yeast extract were optimized and the effects of medium composition on mycelial growth were found to be in the order sucrose > yeast extract > peptone. The optimal concentration for mycelial growth was determined as 50 g l(-1) sucrose, 10 g l(-1) peptone and 3 g l(-1) yeast extract. Under optimal culture conditions, over 22 g l(-1) of mycelial biomass could be obtained after 40 days in submerged cultures. Cordyceps sinensis, one of the most valued medicinal fungi, is shown to grow in axenic culture. This is the first report on nutritional requirements and design of a simplified semi-synthetic medium for mycelial growth of this psychrophilic species, which grows slowly below 20 degrees C. The results of this study will facilitate research on mass production of the fungus under defined culture conditions.

Quantifying the Effect of Medium Composition on the Diffusive Mass Transfer of Hydrophobic Organic Chemicals through Unstirred Boundary Layers

Unstirred boundary layers (UBLs) often act as a bottleneck for the diffusive transport of hydrophobic organic compounds (HOCs) in the environment. Therefore, a microscale technique was developed for quantifying mass transfer through a 100-microm thin UBL, with the medium composition of the UBL as the controllable factor. The model compound fluoranthene had to (1) partition from a contaminated silicone disk (source) into the medium, (2) then diffuse through 100 microm of medium (UBL), and finally (3) partition into a clean silicone layer (sink). The diffusive mass transfer from source to sink was monitored over time by measuring the fluoranthene content of the source and sink disks. The diffusive flux of fluoranthene was slightly higher for air than for water. Cyclodextrin, humic acids, and micelles of sodium dodecyl sulfate (SDS) enhanced the diffusive flux of fluoranthene in water by more than 1 order of magnitude. These results demonstrate that medium constituents, which normally are believed to bind hydrophobic organic chemicals, actually can enhance the diffusive mass transfer of HOCs in the vicinity of a diffusion source (e.g., contaminated soil particles). The technique can be used to evaluate the effect of natural fluids on diffusive mass transfer, as it integrates the different processes, partitioning and diffusion, in one laboratory model.

Effects of media composition on submerged culture spores of the entomopathogenic fungus, Metarhizium anisopliae var. acridum, Part 1: Comparison of cell wall characteristics and drying stability among three spore types

Metarhizium anisopliae var. acridum (IMI 330189) can produce at least three spore types in vitro; blastospores, submerged conidia, and aerial conidia, as defined by culturing conditions, sporogenesis, and spore morphology. This study compares morphological characteristics (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), and performance (germination rate and drying stability) among these three spore types. Submerged conidia and aerial conidia both possessed thick, double-layered cell walls, with hydrophobic regions on their surfaces. However, in contrast to aerial conidia, submerged conidia have: (1) a greater affinity for the lectin concanavalin-A; (2) more anionic net surface charge; and (3) a less distinct outer rodlet layer. Blastospores were longer and more variable in length than both submerged conidia and aerial conidia, and had thinner single-layered cell walls that lacked an outer rodlet layer. Also, blastospores had a greater affinity than either conidia type for the lectin, wheat germ agglutinin. Blastaspores lacked hydrophobic regions on their surface, and had a lower anionic net surface charge than submerged conidia. In culture, blastospores germinated the fastest followed by submerged conidia, and then aerial conidia. Survival of submerged conidia and aerial conidia were similar after drying on silica gel, and was greater than that for blastospores. We provide corroborating information for differentiating spore types previously based on method of production, sporogenesis, and appearance of spores. These physical characteristics may have practical application for predicting spore-performance characteristics relevant to production and efficacy of mycoinsecticides.

Effects of media composition on submerged culture spores of the entomopathogenic fungus, Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics, carbohydrate concentrations, drying stability, and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l−1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l−1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.

Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

Evaluation of the Saccharomyces cerevisiae ADH2 promoter for protein synthesis

The Saccharomyces cerevisiae ADH2 promoter (P(ADH2)) is repressed several hundred-fold in the presence of glucose; transcription is initiated once the glucose in the medium is exhausted. The promoter can thus be utilized for effective regulation of recombinant gene expression in S. cerevisiae without the addition of an inducer. To evaluate this promoter in the absence of plasmid copy number and stability variations, the P(ADH2)-lacZ cassette was integrated into the yeast chromosomes. The effects of medium composition, glucose concentration and cultivation time on promoter derepression and expression level were investigated. Maximum protein activity was obtained after 48 h of growth in complex YPD medium containing 1% glucose. The widely used S. cerevisiae GAL1 and CUP1 promoters both require the addition of an inducer [galactose and copper(II) ion, respectively] before regulated genes will be expressed. The strengths of these three different promoters were compared for cells containing one copy of an integrated lacZ gene under their control. The ADH2 promoter was superior for all induction strategies investigated.

Effect of media composition and in vitro activity of posaconazole, caspofungin and voriconazole against zygomycetes

The effect of different media and composition on the in vitro activity of posaconazole, caspofungin and voriconazole against 59 zygomycetes species was determined. The media tested were RPMI 1640 medium with and without 2% glucose, antibiotic medium 3 (AM3) with and without 2% glucose, and high resolution (HR) medium. Posaconazole was significantly more active than caspofungin and voriconazole, both in RPMI 1640 medium with 2% glucose and in HR medium. Adding glucose improved the determination of end points, but had only minor influence on the MICs. MICs evaluated in AM3 were lower than in RPMI 1640 medium or HR medium. The in vivo effect of posaconazole in zygomycosis needs further evaluation.

Effect of Media Composition and Growth Conditions on Production of β-Glucosidase by <I>Aspergillus niger</I> C-6

Effect of Media Composition and Growth Conditions on Production of β-Glucosidase by <I>Aspergillus niger</I> C-6

Optimizing medium composition for TaqI endonuclease production by recombinant Escherichia coli cells using response surface methodology

The effect of medium composition on the TaqI endonuclease production, by recombinant Escherichia coli cells carrying a plasmid encoding TaqI endonuclease, was investigated using response surface methodology. The concentration of glucose, di-ammonium hydrogen phosphate, potassium di-hydrogen and magnesium sulphate in media were changed according to a central composite rotatable design consisting of 29 experiments and enzyme yields were determined. The results were fitted to a second order polynomial with an R 2 of 0.828. The model equation was then optimized using the Nelder–Mead simplex method to maximize enzyme yield within the experimental range studied. The optimum medium composition was found to be 6 g L −1 glucose, 1.5 g L −1 (NH 4) 2HPO 4, 8 g L −1 KH 2PO 4, and 0.8 g L −1 MgSO 4·7H 2O. The model prediction of 179 × 10 6 U g DCW −1 enzyme yield at optimum conditions was experimentally verified. This value is higher than any value obtained in the initial experiments as well as in the previously reported studies. The response surface methodology was found to be useful in improving the production of recombinant TaqI endonuclease in E. coli.

Lipase-catalyzed degradation of poly(ε-caprolactone)

The lipase-catalyzed degradation of poly(ε-caprolactone) (PCL) is described in the present work. The enzymatic degradation of PCL, having number-averaged molecular weight ( M n) of about 87,000 g/mol and molecular weight dispersion ( M w/ M n) of 1.51, was carried out in organic solvent (toluene) and the effects of medium composition, temperature and source of lipase were evaluated and quantified. Three fungal lipases of Candida rugosa, Mucor miehei and Rhizopus delemar were tested to select the best biocatalyst for PCL degradation. The retention of catalytic activity of M. miehei lipase was determined in organic solvent at different temperatures by pH-stat titration, using olive oil as substrate. The experimental data of the activity coefficient versus time were then used to calculate, for each temperature, the corresponding value of the first-order denaturation constant ( k d) and to estimate the main thermodynamic parameters of the enzyme thermal denaturation. The results of the degradation reaction, followed by size exclusion chromatography (SEC), showed that M. miehei lipase was able to catalyze the degradation of PCL with a maximum conversion degree of about 70% only after 1 h, within the temperature range of 40–60 °C. The catalytic activity of this biocatalyst demonstrated to be nearly stable at 40 °C, whereas the enzyme half-life was less than 2 h at 60 °C.

Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells.

The pluripotency and high proliferative index of embryonic stem (ES) cells make them a good potential source of cells for tissue engineering purposes. We have shown that ES cells can be induced to differentiate in vitro into pulmonary epithelial cells (type II pneumocytes) using a serum-free medium designed for the maintenance of mature distal lung epithelial cells in culture (SAGM). However, the resulting cell cultures were heterogeneous. Our aim in this study was to attempt to increase pneumocyte yield and differentiation state by determining which medium components enhance the differentiation of pneumocytes and modifying the medium accordingly. Quantitative RT-PCR was used to measure changes in the expression of a type II pneumocyte-specific gene, surfactant protein C (SPC), in response to alterations in the cell culture medium. Results suggested that most individual SAGM growth factors were inhibitory for type II pneumocyte differentiation, with the largest increases in SPC expression (approximately threefold) being observed upon removal of retinoic acid and triiodothryonine. However, large standard deviations occurred between replicates, illustrating the highly variable nature of ES cell differentiation. Nevertheless, these observations represent an initial step towards achieving directed differentiation of pneumocytes from stem cells that could lead to their purification for tissue engineering purposes.

The effect of medium composition on interleukin-2 production by murine EL-4 thymoma cells

Due to the role of interleukin-2 (IL-2) in the mediation of immune response, this cytokine has been used in the treatment of some types of cancer and infectious diseases. However, relatively high levels of this cytokine are required to achieve significant activity. The aim of this work was to study a culture medium composition designed to increase the production of IL-2 by suspended murine EL-4 cells. The cultivations were carried out aiming at producing IL-2 in stirred bioreactors. The effects of concentration of glutamine, phorbol-12-myristate-13-acetate (PMA), concanavalin A (Con A), Pluronic F68, and fetal calf serum (FCS) on cell viability and IL-2 production were evaluated. PMA alone was more efficient in IL-2 production than it was in association with Con A. The maximum IL-2 production was around 162 ng/mL with 856 ng/mL PMA and 1.45% (v/v) FCS.

Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system.

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.

Effect of medium composition on the production of anthocyanins by hairy root cultures of Ipomoea batatas

We obtained hairy roots of Ipomoea batatas cv. Ayamurasaki by infection of Agrobacterium rhizogenes A13. Accumulation of anthocyanins in hairy roots was observed when they were cultured under continuous light irradiation, whereas it was not observed when they were cultured in the dark. The amount of accumulated anthocyanins in hairy roots increased as a result of choosing the medium and increasing the sucrose concentration in the medium. Accumulation of anthocyanins was also observed in the hairy roots cultured in liquid medium in the dark by choosing medium.

The alcohol acetyl transferase gene is a target of the cAMP/PKA and FGM nutrient-signalling pathways

The ATF1-encoded Saccharomyces cerevisiae yeast alcohol acetyl transferase I is responsible for the formation of several different volatile acetate esters during fermentations. A number of these volatile esters, e.g. ethyl acetate and isoamyl acetate, are amongst the most important aroma compounds in fermented beverages such as beer and wine. Manipulation of the expression levels of ATF1 in brewing yeast strains has a significant effect on the ester profile of beer. Northern blot analysis of ATF1 and its closely related homologue, Lg-ATF1, showed that these genes were rapidly induced by the addition of glucose to anaerobically grown carbon-starved cells. This induction was abolished in a protein kinase A (PKA)-attenuated strain, while a PKA-overactive strain showed stronger ATF1 expression, indicating that the Ras/cAMP/PKA signalling pathway is involved in this glucose induction. Furthermore, nitrogen was needed in the growth medium in order to maintain ATF1 expression. Long-term activation of ATF1 could also be obtained by the addition of the non-metabolisable amino acid homologue beta-L-alanine, showing that the effect of the nitrogen source did not depend on its metabolism. In addition to nutrient regulation, ATF1 and Lg-ATF1 expression levels were also affected by heat and ethanol stress. These findings help in the understanding of the effect of medium composition on volatile ester synthesis in industrial fermentations. In addition, the complex regulation provides new insights into the physiological role of Atf1p in yeast.

The effect of medium composition on the structure and physical state of sophorolipids produced by Candida bombicola ATCC 22214

Candida bombicola was grown using a variety of lipophilic carbon substrates. Most of the hydrocarbon and carboxylic acid substrates resulted in a mixture of sophorolipids consisting of free acids and the more desirable lactones. The ratio of diacylated lactone to free acid in these mixtures was a maximum when produced using hexadecane and heptadecane. All of the other lipophilic substrates resulted in significant amounts of free acids being produced. These lactone products were unique in that they precipitated as crystals, which were easily separated from the culture medium. All of the other products were isolated as oils as is usually reported in the literature. Finally, the amounts of these crystals recovered were significantly higher than those observed for any of the oily products. It was possible to determine the degree of direct incorporation of the lipophilic substrates into the sophorolipids for a homologous series of alkanes. The amount of direct incorporation increased with increasing chain length to a maximum for pentadecane, hexadecane and heptadecane. As the length of the alkane substrate increased further, the amount of direct incorporation then decreased until there was no apparent incorporation for eicosane.

Effect of medium compositions on biosensing of benzene derivatives using recombinant Escherichia coli

TOL plasmid pWW0 of Pseudomonas putida mt-2 encodes a series of enzymes for the degradation of benzene derivatives. In the presence of benzene derivatives, the complex formed from the regulating protein XylR and Ps promoter controls the expression of these enzymes in P. putida. For detecting of benzene derivatives, the plasmid called as pTS301-GFP has been constructed by introducing a gene encoding green fluorescent protein (GFP) into the downstream of the Ps promoter gene. GFP functioned as a reporter protein is transcribed by activation of the XylR complex. In the present study, the effects of the composition of culture medium and ATP addition on fluorescence of transformant were investigated. The optimal culture medium was found to NZM, and presence of casamino acid decreases the sensitivity for detection of chemicals. Furthermore, it was observed that the addition of 200 μM ATP was effective for detecting of fluorescence of transformants exposed to the low concentration (0.05 mM) of benzene derivatives for 4 h. Applying these results for the detection of the benzene derivatives, it will be expected that transformant with pTS301-GFP can be useful for environmental water evaluation.

Effect of Medium Composition and Explant Source on the Distribution Profiles Selected Micronutrients in Mistletoe Tissue Cultures

In order to study the pattern of in vitro accumulation of selected micronutrients in mistletoe (Viscum album L.) callus cultures, we inoculated leaf and stem explants on a Murashige and Skoog (MS) medium supplemented with different combinations of plant growth regulators at various concentrations. The accumulation of manganese (Mn), zinc (Zn), copper (Cu), and boron (B) in mistletoe callus cultures was significantly affected by both plant growth regulator (PGR) treatment and explant source, as well as their interaction. However, neither of these factors affected Fe accumulation in a statistically significant sense. The average concentration of iron (Fe), Mn, Zn, and Cu in vitro was higher in stem‐derived callus cultures than in leaf‐derived calli, and much higher than in mistletoe plants and in mistletoe hosts, such as fir, oak and chestnut. For stem‐derived calli and with the exception of B, application of 4.65 µM potassium (K) was superior to other PGR treatments leading to the maximum in vitro accumulation of the selected micronutrients. Except for B, the accumulation of micronutrients in stem‐derived callus cultures was highly correlated with dry weight concentration; a high positive correlation was also observed among Fe, Mn, Zn, and Cu concentration, while B accumulation was slightly negatively correlated with the other micronutrients. On the contrary, in leaf‐derived cultures, a high positive correlation was observed only between Mn and Cu concentration. The concentration of the selected micronutrients fluctuated during a total incubation period of 10 weeks on MS + 4.95 µM α‐naphthylacetic acid (NAA), with an increasing trend after the eighth week (B concentration being again an exception). Finally, the addition of ascorbic acid to the culture medium generally promoted micronutrient accumulation, in particular Mn and Cu in stem‐derived cultures and Fe, Zn, and B in leaf‐derived calli. This effect was correlated with an increase in total free phenolics substances, and further discussed in view of various plant antioxidant systems in vitro.

Optimization of submerged culture conditions for mycelial growth and exo-biopolymer production by Paecilomyces tenuipes C240

This paper is concerned with optimization of submerged culture conditions for mycelial growth and exo-biopolymer production by Paecilomyces tenuipes C240 by one-factor-at-a-time and orthogonal matrix methods. The one-factor-at-a-time method was adopted to investigate the effects of medium components (i.e. carbon, nitrogen, and mineral sources) and environmental factors (i.e. initial pH and temperature) on mycelial growth and exo-biopolymer production. Among these variables, glucose, KNO 3, K 2HPO 4, and MgSO 4 were identified to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature and initial pH for mycelial growth and exo-biopolymer production were 28 °C and 6.0, respectively. Subsequently, the concentration of glucose, KNO 3, K 2HPO 4, and MgSO 4 were optimized using the orthogonal matrix method. The effects of media composition on the mycelial growth of P. tenuipes C240 were in the order of glucose>K 2HPO 4>KNO 3>MgSO 4, and those on exo-biopolymer production were in the order of glucose>K 2HPO 4>MgSO 4>KNO 3. The optimal concentration for enhanced production were determined as 4 g/l glucose, 0.6 g/l KNO 3, 0.1 g/l K 2HPO 4, and 0.1 g/l MgSO 4 · 5H 2O for mycelial yield, and 3 g/l glucose, 0.4 g/l KNO 3, 0.1 g/l K 2HPO 4, and 0.1 g/l MgSO 4 · 5H 2O for exo-biopolymer production, respectively. The subsequent verification experiments confirmed the validity of the models. This optimization strategy in shake flask culture lead to a mycelial yield of 10.18 g/l, and exo-biopolymer production of 1.89 g/l, respectively, which were considerably higher than those obtained in preliminary studies. Under optimal culture conditions, the maximum exo-biopolymer concentration in a 5 l stirred-tank bioreactor was 2.36 g/l.