Growth and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in an adenine-supplemented chemically defined medium

AIMS: To analyse the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in a chemically defined medium (CDM) and the effect of nutrients and stress culture conditions on cell growth and EPS formation. METHODS AND RESULTS: Cultures were conducted in CDM: (i) containing essential and nonessential bases and vitamins; (ii) without nonessential bases and vitamins [Simplified CDM (SCDM)]; (iii) SCDM supplemented individually with vitamins and bases. The influence of carbohydrates, pH and osmotic culture conditions on growth and polymer formation was analysed. Adenine and lactose stimulated both growth and EPS production. Constant pH fermentations (4.5 and 6.2) did not improve EPS synthesis while NaCl and glycerol were detrimental for growth and polymer formation. In all media the EPS monomer composition was glucose and galactose (2.5 : 1). CONCLUSIONS: A SCDM containing adenine and lactose was optimal for cell growth and EPS formation by Lact. helveticus ATCC 15807. Controlled pH (6.2 and 4.5) and osmotic stress culture conditions did not improve polymer production. The EPS characteristics were identical in all media. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a better knowledge on EPS synthesis by Lact. helveticus. A CDM to perform regulation studies on EPS production by Lact. helveticus species is now available.     

Factors affecting aflatoxin production by Aspergillus parasiticus in a chemically defined medium

The optimum levels of sucrose, (NH4)2SO4, MgSO4, KH2PO4 and ZnSO4 for aflatoxin production in a chemically defined medium have been established. The last two were found to be essential for fungal growth and aflatoxin production. The effect of various carbon sources on aflatoxin production was tested using the defined medium. Asparagine was found to be essential for aflatoxin production. Very little aflatoxin was produced in the absence of asparagine with any of the other inorganic nitrogen sources tested. Supplementation with yeast extract, Casamino acids, Casitone and peptone increased the aflatoxin yield, but omission of asparagine led to decreased aflatoxin yields even when complex nitrogen sources were present. Asparagine could be replaced by aspartic acid or alanine.     

Transferrin enhances lentoid differentiation in rat egg cylinders cultivated in a chemically defined medium

Rat egg cylinders at the primitive streak stage were grown in modified organ culture for 2 weeks using a chemically-defined medium. The purpose of the experiment was to determine whether the terminal tissue differentiation is modified by human transferrin. The control sets were grown in medium with or without rat serum. In explants treated with transferrin, groups of atypical cells of the ocular lens (lentoids) appeared more frequently than in both control sets; however neuroblasts were observed as often as in the serum-supplemented medium. Bovine serum albumin (BSA) stimulated the differentiation of neuroblasts but did not promote lentoid formation. We conclude that human transferrin does stimulate the differentiation of lentoids in rat embryonic explants, but the mechanism of its action remains unknown.     

A chemically defined medium for cephalosporin C production by Paecilomyces persicinus

A chemically defined medium was developed for the biosynthesis of cephalosporin C by Paecilomyces persicinus Nicot strain P-10. Glucose served as the major carbon source and nitrogen was supplied by five amino acids, l-arginine, l-aspartic acid, l-glutamic acid, glycine and dl-methionine. Omission of any of the first four diminished or prevented production of cephalosporin C; omission of methionine did not. Methionine is not critical for the production of cephalosporin C in this defined medium. Production of the antibiotic was affected by the concentrations of inorganic salts employed. Biotin was required for growth and cephalosporin C synthesis. The addition of l-lysine precursors to the medium did not influence cephalosporin C levels and l-lysine itself inhibited antibiotic production. Known precursors of -lactam antibiotics as well as oleic acid did not affect biosynthesis of cephalosporin C. Chemical changes occurring in the defined medium revealed that glucose was efficiently utilized after 96 hours incubation whereas total soluble nitrogen levels increased following an initial sharp decrease. Mycelial weight and cephalosporin C production were both maximal after 96 hours incubation. Mycelial nitrogen was highest after 48 hours incubation whereas mycelial lipid levels were greatest after 72 hours.     

Endocytosis in guinea pig seminal vesicle epithelial cells cultivated in chemically defined medium

We have developed a chemically defined monolayer culture system for guinea pig seminal vesicle epithelial cells (SVEP). The cells appeared as a polarized monolayer with apical microvilli, tight junctions and desmosome-like junctions, and often dilated intercellular spaces. SVEP expressed epithelial-specific cytokeratins as detected immunocytochemically. Growth was obtained during the first week of culture. In this period, the cells were exposed to unconjugated horseradish peroxidase (HRP), a ricin-peroxidase conjugate (Ri-HRP), or cationized ferritin (CF). HRP was endocytosed without binding to the SVEP surface (fluid-phase endocytosis) and was found mainly in multivesicular endosomes and lysosomes. Ri-HRP and CF, however, were endocytosed following binding to the cell surface. Initially these markers were present in multivesicular endosomes, but later also in smaller tubular and vesicular endosomes, some Golgi-associated elements (but not Golgi stacks), and lysosomes. We conclude that our SVEP culture system may be useful in further studies, on e.g. hormonal regulation of endocytosis and other processes of importance for SVEP maintenance and modulation of the seminal fluid in vivo.     

Lovastatin biosynthesis by Aspergillus terreus in a chemically defined medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium

The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.     

Growth of neural cell cultures in a chemically defined,serum-free culture medium

The composition of a serum-free, completely chemically defined culture medium which supports active growth of dissociated neural-cells in culture is described. This serum-free medium can also be used to grow many types of human cell lines without modification. It is the first report which describes the development of a wholly chemically defined, synthetic culture medium for growth of neural cells.     

Growth requirements and the establishment of a chemically defined minimal medium inZymomonas mobilis

Summary A chemically defined minimal medium which fulfils the growth requirements of differentZymomonas mobilis strains has been established. The kinetics of ethanol production of the strains ATCC 10988, CU1, CP4 and 11163 grown on the minimal medium at different glucose concentrations were measured. All strains produced ethanol at rates similar to those on complete medium. The minimal medium described is suitable to study spontaneous metabolic deficiciencies and regulation of enzyme activities inZ.mobilis.     

Growth of NS0 cells in protein-free, chemically defined medium

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.     

Copper but not iron limitation increases astaxanthin production by Phaffia rhodozyma in a chemically defined medium

The influence of copper (0–32 M) and iron (0–108 M) on growth and astaxanthin production by Phaffia rhodozyma was studied. Copper below 3.2 M increased the astaxanthin content of the cells (from 220 to 287 g g^–1) but at the expense of a slightly decreased growth (from 11.3 to 10.2 mg ml^–1). In contrast, iron below 1 M decreased both the growth and astaxanthin content of the cells. Using copper limitation instead of toxic respiratory inhibitors to improve astaxanthin production has obvious advantages from the product quality, environmental and process operation points of view.     

Growth characteristics of the yeast phase of Paracoccidioides brasiliensis in a chemically defined medium

The growth of four clinical strains of Paracoccidioides brasiliensis was investigated using the chemically defined medium of McVeigh and Morton. Emphasis was placed upon controlling conditions of inoculum preparation, age of inoculum used, and the homogeneity of samples used for analysis. The medium was evaluated for its ability to support growth of the yeast phase of P. brasiliensis at 37 degrees C. Cultures were followed for 240 h and growth patterns were determined by measuring optical density, dry weight, nucleic acids and protein. The medium is excellent for growing the yeast phase of P. brasiliensis. The exponential phase lasted an average of 135 h and the stationary phase 72 h; a decline began after 207 h. This defined medium supports abundant growth of the yeast phase of P. brasiliensis and may thus prove useful in the preparation of yeast-phase antigens.     

Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.     

Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.     

Cultivation of Pasteurella haemolytica in a chemically defined medium

A chemically defined medium was developed for the aerobic cultivation of Pasteurella haemolytica. Studies on the growth of strain H44L were conducted in a medium consisting of 15 amino acids, inorganic salts, citrate, nicotinamide, pantothenate, thiamine or thiamine monophosphate, and carbon sources. The amino acids were provided as l isomers, because racemic mixtures of some amino acids inhibited growth. The carbon source consisted of a mixture of 1.0% d-galactose and 0.1% d-glucose. Culture populations of strain H44L reached 2 x 10(10) cells per milliliter after 16 hr of incubation at 37.5 C. Other strains of P. haemolytica, from a wide variety of sources, were tested for growth in the medium, and 23 of 24 strains grew well. Five strains of P. haemolytica var. ureae failed to grow in the medium.     

Development of a chemically defined medium for the production of the antibiotic platensimycin by Streptomyces platensis

The actinomycete Streptomyces platensis produces two compounds that display antibacterial activity: platensimycin and platencin. These compounds were discovered by the Merck Research Laboratories, and a complex insoluble production medium was reported. We have used this medium as our starting point in our studies. In a previous study, we developed a semi-defined production medium, i.e., PM5. In the present studies, by varying the concentration of the components of PM5, we were able to develop a superior semi-defined medium, i.e., PM6, which contains a higher concentration of lactose. Versions of PM6, containing lower concentrations of all components, were also found to be superior to PM5. The new semi-defined production media contain dextrin, lactose, MOPS buffer, and ammonium sulfate in different concentrations. We determined antibiotic production capabilities using agar diffusion assays and chemical assays via thin-layer silica chromatography and high-performance liquid chromatography. We reduced crude nutrient carryover from the seed medium by washing the cells with distilled water. Using these semi-defined media, we determined that addition of the semi-defined component soluble starch stimulated antibiotic production and that it and dextrin could both be replaced with glucose, resulting in the chemically defined medium, PM7.