Showing papers on "Growth medium published in 2012"

A prototrophic deletion mutant collection for yeast metabolomics and systems biology

Abstract: Auxotrophic markers - mutations within genes encoding enzymes in pathways for the biosynthesis of metabolic building blocks, such as an amino acid or nucleotide, are used as selection markers in the vast majority of yeast genetics and genomics experiments 1-3. The nutritional deficiency caused by the mutation (auxotrophy) can be compensated by supplying the required nutrient in the growth medium. This compensation, however, is not necessarily quantitative because such mutations influence a number of physiological parameters and may act in combination 2,4,5 . The construction of a prototrophic derivative of the parent strain of the widely used genome-scale yeast deletion collection1 has confirmed the need to remove auxotrophic markers in order to reduce bias in physiological and metabolic studies 2 . Moreover, flux balance analyses using a genome-wide metabolic model (Yeast 5)6 indicate that the activity status of some 200-300 reactions changes between different auxotrophic strains and the wild type. To alleviate this bias we have constructed a version of the haploid deletant collection restoring prototrophy in the genetic background, such that influence of auxotrophy to the phenotype of a given gene deletion is prevented. This new deletant library facilitates the exploitation of yeast in both functional genomics and quantitative systems biology. The physiological impact of auxotrophy was assessed by monitoring the growth of 16 yeast strains carrying all possible combinations of the markers (histidine (his3Δ1), leucine (leu2Δ), methionine (met15Δ) and uracil (ura3Δ) used in the MATa version of the yeast deletion collection1. All markers and their combinations affected yeast growth, but without altering the adenylate (ATP, ADP, AMP) energy charge (Fig 1 a). As the most critical phenotypic quantity, the maximum specific growth rate (μmax) varied between 0.125 h−1 (leu2Δ) and 0.20 h−1 (his3Δmet15Δura3Δ), rendering quantitative comparisons between these strains impossible (Fig 1 a, Suppl. Table 1). These growth differences were not explained by the different media supplementations, as i) prototrophic yeast exhibited a different and substantially less diverse growth pattern in the 16 minimal media (Fig 1c, left panel; media recipes are given in the Supplementary material); and ii) growth differences where altered, but not abrogated, when other proteogenic amino acids were supplemented as well (synthetic complete (SC) medium; Fig 1b). Importantly, on both types of media, complex interactions between all auxotrophic mutations were observed. For instance, restoring MET15 in leu2Δura3Δhis3Δmet15Δ (0.185 h−1 → 0.164 h−1) or leu2Δura3Δmet15Δ (0.162 h−1 → 0.149 h−1) had a negative effect on μmax, but surprisingly promoted growth in leu2Δhis3Δmet15Δ (0.136 h−1 → 0.173 h−1) (Fig 1a); restoring LEU2 in leu2Δura3Δhis3Δ (0.164 h−1 → 0.185 h−1) or leu2Δura3Δmet15Δ (0.136 h−1 → 0.161 h−1) had a positive effect, but not in leu2Δura3Δhis3Δmet15Δ (0.185 h−1 → 0.186 h−1) (Fig 1a; Suppl. Table 1). Thus, although blocking different pathways, all markers influence each other, indicating that they have a wide-ranging and combinatorial influence on the metabolic network. Figure 1 The combinatorial impact of yeast auxotrophic markers In batch culture experiments, further problems arise from the unequal consumption of amino acid supplements resulting in cultivation phase-dependent starvation. Growth of BY4741 (the auxotrophic parent of the standard yeast gene-deletion collection 1) in SC media depleted nutrients in a way they became first limiting for met15Δ, then for leu2Δ, his3Δ1 and finally for ura3Δ auxotrophic yeast (Fig 1d). This effect could not be compensated by increasing amino acid supplementation(s), as this inhibited cell growth (Fig 1c, right panel). Chronological lifespan (CLS) is a phenotype that is profoundly influenced by both nutrient supplementation and growth rate. Indeed, we observed an increase in stationary phase survival in YPD media upon restoring prototrophy. In a competitive growth experiment, auxotrophic cells had lost their colony-forming capacities within 10 days, but their prototrophic counterparts were perfectly viable for more than 20 days (Fig 1e). Longer CLS of prototrophic versus auxotrophic yeast was also reported for other backgrounds, and in synthetic media nutrient starvation shortened the lifespan of auxotrophic cells 7,8. Restoring protrotrophy is thus, to our knowledge, one of the most powerful genetic modifications for extending CLS. Hence, as auxotrophic markers have substantial and combinatorial influences on fundamental biological parameters such as growth and ageing, auxotrophic genome resources introduce bias for analyzing physiological parameters and even more to quantitative studies addressing the metabolic network. We would thus encourage the yeast community to switch, where possible, to prototrophic yeast for experiments in transcriptomics, proteomics, and metabolomics. To create a prototrophic resource for genome-scale experiments, we re-introduced auxotrophic markers into the MATa versions of the S288c-based deletion collection (5185 strains)1 and the titratable promoter essential collection (839 strains)3. These strains were transformed with a centromere-containing single-copy vector (minichromosome), containing the chromosome VI centromere, the autonomous replication sequence of HHF1 (ARSH4)9, and the marker genes HIS3, URA3, LEU2, and MET15 under control of their endogenous promoter sequences (pHLUM; Suppl Fig 1, Addgene ID 40276). Under non-selective conditions, the vector was transmitted in 99.15% of cell divisions (0.85% segregation mean over 20 generations). After 20 days, all cells were found prototrophic due to their positive selection (Fig 1e), facilitating screens on both selective and non-selective media. Furthermore, pHLUM- transformed BY4741 derivates wild type for HIS3, LEU2, MET15 or URA3 grew similar as BY4741 pHLUM (Suppl. Fig 2), indicating that the minichromosome fully restored prototrophy. The titratable-promoter essential collection3 was exploited to demonstrate screening capacities. By replicating original and prototrophic strains onto doxycycline-containing media, we found that 13 of the 370 lethal phenotypes were compensated (Fig 1 f, Suppl. Table 2). Thus, auxotrophic markers do not only influence physiological parameters, they are also responsible for a number of essential phenotypes. Since all strains possess a native metabolic network, the new library reduces bias from the use of auxotrophic markers in functional genomics and metabolic systems biology. Based on the pHLUM minichromosome, which is counterselectable, the new resource retains full compatibility with the popular S288c knock-out and essential collections 1,3. However, the use of a plasmid will introduce confounding factors to those mutants which have deficits in plasmid stability and segregation. The library is distributed as arrayed on 96-well plates (Euroscarf, Frankfurt), and contains a deep-red coloured and counter-selectable mutant (ade12Δ) on both universal and plate-specific positions, which simplifies plate orientation and identification, and can serve as replicate-control in quantitative metabolomics experiments (Suppl. Fig 3).

Production of melatonin by Saccharomyces strains under growth and fermentation conditions.

Abstract: Melatonin is a bioactive compound that is present in wine because it is contained in vinification grapes and synthesized by yeast during alcoholic fermentation. The purpose of this study was to determine the capacity of various Saccharomyces strains to form melatonin during its growth and alcoholic fermentation. A selection of yeasts including six S. cerevisiae (Lalvin CLOS, Lalvin ICV-D254, Enoferm QA23 Viniferm ARM, Viniferm RVA, and Viniferm TTA), one S. uvarum (Lalvin S6U) and one S. cerevisiae var. bayanus (Uvaferm BC) were tested to determine whether they produce melatonin in yeast extract peptose dextrose and synthetic must media in a variety of conditions. Two S. cerevisiae strains (ARM, and QA23), the S. uvarum and the S. cerevisiae var. bayanus, synthesized melatonin. The conditions in which they did so, however, were different: the QA23 strain produced melatonin best in a medium with a low concentration of reducing sugars and Lalvin S6U and Uvaferm BC required a synthetic must under fermentation conditions. Melatonin synthesis largely depended on the growth phase of the yeasts and the concentration of tryptophan, reducing sugars and the growth medium. These results indicate that melatonin may have a role as a yeast growth signal molecule.

A defined growth medium with very low background carbon for culturing Clostridium thermocellum

Abstract: A growth medium was developed for cultivation of Clostridium thermocellum ATCC 27405 in which “background” carbon present in buffers, reducing agents, chelating agents, and growth factors was a small fraction of the carbon present in the primary growth substrate. Background carbon was 1.6% of primary substrate carbon in the low-carbon (LC) medium, whereas it accounts for at least 40% in previously reported media. Fermentation of cellulose in LC medium was quite similar to Medium for Thermophilic Clostridia (MTC), a commonly used growth medium that contains background carbon at 88% of primary substrate carbon. Of particular note, we found that the organism can readily be cultivated by eliminating some components, lowering the concentrations of others, and employing a tenfold lower concentration of reducing agent. As such, we were able to reduce the amount of background carbon 55-fold compared to MTC medium while reaching the same cell biomass concentration. The final mass ratios of the products acetate:ethanol:formate were 5:3.9:1 for MTC and 4.1:1.5:1 for LC medium. LC medium is expected to facilitate metabolic studies involving identification and quantification of extracellular metabolites. In addition, this medium is expected to be useful in studies of cellulose utilization by anaerobic enrichment cultures obtained from environmental inocula, and in particular to diminish complications arising from metabolism of carbon-containing compounds other than cellulose. Finally, LC medium provides a starting point for industrial growth media development.

The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture

Abstract: Two recombinant strains of Saccharomyces cer- evisiae Y294 producing cellulase using different expression strategies were compared to a reference strain in aerobic culture to evaluate the potential metabolic burden that cel- lulase expression imposed on the yeast metabolism. In a chemically defined mineral medium with glucose as carbon source, S. cerevisiae strain Y294(CEL5) with plasmid-borne cellulase genes produced endoglucanase and β-glucosidase activities of 0.038 and 0.30 U mg dry cell weight −1 , respec- tively. Chromosomal expression of these two cellulases in strain Y294(Y118p) resulted in no detectable activity, al- though low levels of episomally co-expressed cellobiohy- drolase (CBH) activity were detected. Whereas the biomass concentration of strain Y294(CEL5) was slightly greater than that of a reference strain, CBH expression by Y294 (Y118p) resulted in a 1.4-fold lower maximum specific growth rate than that of the reference. Supplementation of the growth medium with amino acids significantly improved culture growth and enzyme production, but only partially mitigated the physiological effects and metabolic burden of cellulase expression. Glycerol production was decreased significantly, up to threefold, in amino acid-supplemented cultures, apparently due to redox balancing. Disproportion- ately higher levels of glycerol production by Y294(CEL5) indicated a potential correlation between the redox balance of anabolism and the physiological stress of cellulase pro- duction. With the reliance on cellulase expression in yeast for the development of consolidated bioprocesses for bio- ethanol production, this work demonstrates the need for development of yeasts that are physiologically robust in response to burdens imposed by heterologous enzyme production.

Influence of growth media and temperature on bacterial adhesion to polystyrene surfaces

Abstract: Bacterial adhesion to inert surfaces is a complex process influenced by environmental conditions. In this work, the influence of growth medium and temperature on the adhesion of Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Micrococcus luteus and Listeria monocytogenes to polystyrene surfaces was studied. Most bacteria demonstrated the highest adhesion when cultured in TSYEA, except S. marcescens, which showed to be positively influenced by the pigment production, favored in poor nutrient media (lactose and peptone agar). P. aeruginosa adhesion to polystyrene increased at low temperatures whatever the medium used. The culture medium influenced the surface properties of the bacteria as assessed by the MATS test.

Oleaginous Yeasts: Biochemical Events Related with Lipid Synthesis and Potential Biotechnological Applications

Abstract: The last years there has been a significant rise in the number of publications in the international literature that deal with the production of oils and fats deriving from microbial sources (the so called “single cell oils – SCOs”) that could be used as precursors for the synthesis of bio-diesel or as “tailor-made” lipids amenable for the replacement of expensive fatty materials found in the plant or animal kingdom [1,2]. These lipids are produced by the so-called “oleaginous” microorganisms (microorganisms principally belonging to yeasts, fungi and algae and to lesser extent bacteria, capable of storing quantities of lipids higher than 20%, wt/wt, in their dry weight) [1,3-5]. Remarkable differences in biochemical and kinetic level exist between the process of lipid accumulation when glucose or similarly metabolized compounds are used as substrates (“de novo” lipid synthesis) compared with that performed when hydrophobic materials are used as substrates (“ex novo” lipid synthesis). De novo lipid biosynthesis in the oleaginous microorganisms is non-growth associated process, conducted due to change of intra-cellular concentration of various metabolites after nitrogen depletion into the culture medium. Nitrogen exhaustion leads to a rapid decrease of the concentration of cellular AMP, which is further cleaved in order for nitrogen to be offered to the microorganism. Cellular AMP concentration decrease alters the Krebs cycle function; NAD + - (and in various cases NADP + isocitrate) dehydrogenase, allosterically activated by intracellular AMP, loses its activity and the carbon flow, hence, is directed towards the accumulation of intra-mitochondrial citric acid. When the concentration of citric acid inside the mitochondria becomes higher than a critical value, it is secreted inside the cytoplasm. Then, citric acid is cleaved by ATP-citrate lyase, enzyme-key showing the oleaginous character of the microorganisms, into acetyl-CoA and oxaloacetate and acetyl-CoA, by virtue of the action of fatty acid synthetase generates cellular fatty acids and subsequently triacylglycerols (TAGs), that are the most common form of lipophilic compounds found in the oleaginous microorganisms [1,3-5]. In the non-lipid producing microorganisms, nitrogen exhaustions provokes secretion of the previously hyper-synthesized citric acid into the growth medium (case of the fungus Aspergillus niger and many of the strains of the yeast Yarrowia lipolytica) or results in a block in the level of 6-phosphofructokinase (with mechanisms similar to the ones related with the decrease of activity of NAD

Synergistic action of both Aspergillus niger and Burkholderia cepacea in co-culture increases phosphate solubilization in growth medium.

Abstract: Co-inoculation of the fungus Aspergillus niger and the bacterium Burkholderia cepacia was undertaken to understand the interaction between different species of phosphate-solubilizing microorganisms (PSM). PSM were inoculated in a single or mixed (A. niger-B. cepacia) culture. During 9 days of incubation, microbial biomass was enhanced, accompanied with increases in the levels of soluble phosphate and titratable acidity, as well as increased acid phosphatase activity. Production of acids and levels of phosphate solubilization were greater in the co-culture of A. niger-B. cepacia than in the single culture. The quantity of phosphate solubilized by the co-culture ranged from 40.51 ± 0.60 to 1103.64 ± 1.21 μg PO(4) 3- mL(-1) and was 9-22% higher than single cultures. pH of the medium dropped from 7.0 to 3.0 in the A. niger culture, 3.1 in the co-culture, and 4.2 in the B. cepacia culture. On the third day of postinoculation, acid production by the co-culture (mean 5.40 ± 0.31 mg NaOH mL(-1)) was 19-90% greater than single cultures. Glucose concentration decreased almost completely (97-99% of the starting concentration) by the ninth day of the incubation. These results show remarkable synergism by the co-culture in comparison with single cultures in the solubility of CaHPO(4) under in vitro conditions. This synergy between microorganisms can be used in poor available phosphate soils to enhance phosphate solubilization.

Time course gene expression profiling of yeast spore germination reveals a network of transcription factors orchestrating the global response.

Abstract: Spore germination of the yeast Saccharomyces cerevisiae is a multi-step developmental path on which dormant spores re-enter the mitotic cell cycle and resume vegetative growth. Upon addition of a fermentable carbon source and nutrients, the outer layers of the protective spore wall are locally degraded, the tightly packed spore gains volume and an elongated shape, and eventually the germinating spore re-enters the cell cycle. The regulatory pathways driving this process are still largely unknown. Here we characterize the global gene expression profiles of germinating spores and identify potential transcriptional regulators of this process with the aim to increase our understanding of the mechanisms that control the transition from cellular dormancy to proliferation. Employing detailed gene expression time course data we have analysed the reprogramming of dormant spores during the transition to proliferation stimulated by a rich growth medium or pure glucose. Exit from dormancy results in rapid and global changes consisting of different sequential gene expression subprograms. The regulated genes reflect the transition towards glucose metabolism, the resumption of growth and the release of stress, similar to cells exiting a stationary growth phase. High resolution time course analysis during the onset of germination allowed us to identify a transient up-regulation of genes involved in protein folding and transport. We also identified a network of transcription factors that may be regulating the global response. While the expression outputs following stimulation by rich glucose medium or by glucose alone are qualitatively similar, the response to rich medium is stronger. Moreover, spores sense and react to amino acid starvation within the first 30 min after germination initiation, and this response can be linked to specific transcription factors. Resumption of growth in germinating spores is characterized by a highly synchronized temporal organisation of up- and down-regulated genes which reflects the metabolic reshaping of the quickening spores.

L-carnosine affects the growth of Saccharomyces cerevisiae in a metabolism-dependent manner

Abstract: The dipeptide L-carnosine (β-alanyl-L-histidine) has been described as enigmatic: it inhibits growth of cancer cells but delays senescence in cultured human fibroblasts and extends the lifespan of male fruit flies. In an attempt to understand these observations, the effects of L-carnosine on the model eukaryote, Saccharomyces cerevisiae, were examined on account of its unique metabolic properties; S. cerevisiae can respire aerobically, but like some tumor cells, it can also exhibit a metabolism in which aerobic respiration is down regulated. L-Carnosine exhibited both inhibitory and stimulatory effects on yeast cells, dependent upon the carbon source in the growth medium. When yeast cells were not reliant on oxidative phosphorylation for energy generation (e.g. when grown on a fermentable carbon source such as 2% glucose), 10-30 mM L-carnosine slowed growth rates in a dose-dependent manner and increased cell death by up to 17%. In contrast, in media containing a non-fermentable carbon source in which yeast are dependent on aerobic respiration (e.g. 2% glycerol), L-carnosine did not provoke cell death. This latter observation was confirmed in the respiratory yeast, Pichia pastoris. Moreover, when deletion strains in the yeast nutrient-sensing pathway were treated with L-carnosine, the cells showed resistance to its inhibitory effects. These findings suggest that L-carnosine affects cells in a metabolism-dependent manner and provide a rationale for its effects on different cell types. © 2012 Cartwright et al.

A shift to 50°C provokes death in distinct ways for glucose- and oleate-grown cells of Yarrowia lipolytica

Abstract: Based on the observation that shocks provoked by heat or amphiphilic compounds present some similarities, this work aims at studying whether cells grown on oleate (amphiphilic pre-stress) acquire a tolerance to heat shock. In rich media, changing glucose for oleate significantly enhanced the cell resistance to the shock, however, cells grown on a minimal oleate medium lost their ability to grow on agar with the same kinetic than glucose-grown cells (more than 7-log decrease in 18 min compared with 3-log for oleate-grown cells). Despite this difference in kinetics, the sequence of events was similar for oleate-grown cells maintained at 50°C with a (1) loss of ability to form colonies at 27°C, (2) loss of membrane integrity and (3) lysis (observed only for some minimal-oleate-grown cells). Glucose-grown cells underwent different changes. Their membranes, which were less fluid, lost their integrity as well and cells were rapidly inactivated. But, surprisingly, their nuclear DNA was not stained by propidium iodide and other cationic fluorescent DNA-specific probes but became stainable by hydrophobic ones. Moreover, they underwent a dramatic increase in membrane viscosity. The evolution of lipid bodies during the heat shock depended also on the growth medium. In glucose-grown cells, they seemed to coalesce with the nuclear membrane whereas for oleate-grown cells, they coalesced together forming big droplets which could be released in the medium. In some rare cases of oleate-grown cells, lipid bodies were fragmented and occupied all the cell volume. These results show that heat triggers programmed cell death with uncommon hallmarks for glucose-grown cells and necrosis for methyl-oleate-grown cells.

Effect of water activity and protective solutes on growth and subsequent survival to air-drying of Lactobacillus and Bifidobacterium cultures

Abstract: Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a w) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a w = 0.96 or 0.99 were then recovered by centrifugation, added to a sucrose–phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a w = 0.96. Growing the cells in complex media adjusted at a w = 0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose–phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.

Exogenously supplied osmoprotectants confer enhanced salinity tolerance in rhizobacteria

Abstract: The response of eight rhizobacterial isolates obtained from the rhizosphere of Salicornia brachiata to osmotic stress (salinity) in minimal medium M9 to evaluate their osmotolerance properties. These rhizobacteria could tolerate NaCl upto 0.714 M in M9 minimal medium. It was observed that all isolates demonstrated different response to salt stress in the presence of glycine, proline, betaine, glycerol and yeast extract in the growth medium. The maximum osmoprotective effect under salinity stress was registered by yeast extract followed by glycerol, proline, glycine and betaine. The present findings suggested that proline, glycine and betaine played a critical role in osmotic adaptation at high osmolarity. Among the rhizobacterial isolates, Zhihengliuella sp. and Brachybacterium sp. synthesized highest proline as osmoprotective substance under salinity stress.

Production of zinc-enriched biomass of Saccharomyces cerevisiae

Abstract: Zinc accumulation and the growth of Saccharomyces cerevisiae were investigated in a culture with zinc sulfate-supplemented medium. The cultivations were performed on Sabouraud dextrose broth medium in aerobic conditions, without the addition of zinc (control culture) and with the addition of zinc sulfate (5, 10, 15, 30 and 60 mg ZnSO4 l -1 medium) at 28°C for 72 hours. The results showed similar trends of yeast growth rates at 24, 48, and 72-hour interval, with concentrations above 10 mg l-1 ZnSO4 in the nutritional medium significantly decreasing the yeast growth rate and the biomass yield (P<0.05). Substantial differences between the initial ZnSO4 concentrations in the growth medium were demonstrated in the overall adsorption of Zn ions (Zn) in yeast cells by a colorimetric assay (P<0.05). Similarly, the content of total accumulated zinc, as well as the fractions of Zn present in cells depended mainly on the zinc concentration in the medium, as the total Zn accumulation and organically bound Zn fractions were increased by elevating the ZnSO4 supplementation in the culture medium up to 30 mg l -1, but gradually reduced by any further addition of ZnSO4 determined by an ICP-MASS assay (P<0.05). In the presence of 30 mg l-1 ZnSO4, the Zn content in the biomass increased by 24-fold, to 4132.34 mg g-1 in comparison to 171.9 mg g-1 achieved in the basal medium. Thus, the ability of S. cerevisiae to accumulate zinc can be used for production of a zinc-rich ingredient for functional food products.

Production of amylase from Aspergillus niger using a defined synthetic growth medium and also rice ( Oryza sativa ) as growth substrate

Abstract: Aspergillus niger grew in both a growth medium with rice as carbon and growth source and in a defined synthetic medium with varying carbon and nitrogen sources at 25 o C producing amylase. Optimum amylase activity in rice was expressed on the eighth day of incubation as 0.58 Units. In the synthetic growth medium with starch as carbon source and tryptone as nitrogen source, optimum amylase activity was expressed on the seventh day as 0.47 Units; with ammonium chloride as nitrogen source and maltose as carbon source of growth, optimum amylase activity was expressed on the ninth day as 3.525 Units. This investigation suggests a means of production of amylase for industrial purposes.

Media Compositions for Promoting Bacterial and Fungal Growth

Abstract: Methods and compositions for enhancing or promoting germination of bacterial spores, and yeasts are disclosed herein. The composition of the present invention comprises an extract obtained from banana or any member belonging to the genus Musa that may be used alone or in a growth medium to promote and enhance germination of bacterial spores, growth of bacterial, yeast, and fungal cell cultures.

Phosphate limitation induces sporulation in the chytridiomycete Blastocladiella emersonii

Abstract: The cell cycle is controlled by numerous mechanisms that ensure correct cell division. If growth is not possible, cells may eventually promote autophagy, differentiation, or apoptosis. Microorganisms interrupt their growth and differenti- ate under general nutrient limitation. We analyzed the effects of phosphate limitation on growth and sporulation in the chy- tridiomycete Blastocladiella emersonii using kinetic data, phase-contrast, and laser confocal microscopy. Under phosphate limitation, zoospores germinated and subsequently formed 2-4 spores, regardless of the nutritional content of the medium. The removal of phosphate at any time during growth induced sporulation of vegetative cells. If phosphate was later added to the same cultures, growth was restored if the cells were not yet committed to sporulation. The cycles of addition and withdrawal of phosphate from growth medium resulted in cycles of germination-growth, germination-sporulation, or germination-growth-sporulation. These results show that phosphate limitation is sufficient to interrupt cell growth and to in- duce complete sporulation in B. emersonii. We concluded that the determination of growth or sporulation in this microor- ganism is linked to phosphate availability when other nutrients are not limiting. This result provides a new tool for the dissection of nutrient-energy and signal pathways in cell growth and differentiation.

Influence of medium on growth and development of wild rose in vitro.

Abstract: This research was conducted during 2010 in controlled environmental conditions as two step production of wild rose rootstock. The aim of this study was to determine growth and development of wild rose at two different growth media, to preserve genotype characteristics and to shorten a multiplication time of virus-free plant material. Plants were cultivated on two different growth media. The first medium contained two hormones BAP (6-Benzylaminopurine) and IBA (Indole-3-butyric acid), and the second contained just hormone BAP. Plants that were grown on these growth media were transplanted in commercial substrate and treated with biostimulant Radifarm® by watering. Investigated parameters were shoot number, stem height, root length and stem and root mass. The results showed that addition of BAP hormone alone in the growth medium resulted in significantly longer stems, but it had no influence on shoot number. Application of the biostimulant had positive effect on shoot number and root weight. Investigation shows how the biostimulant application in R. canina transplants production improves growth and development of root and above-ground mass which is important for faster plant adaptation on stress during transplanting.

EPS Production, PHB Accumulation and Abiotic Stress Endurance of Plant Growth Promoting Methylobacterium Strains Grown in a High Carbon Concentration

Abstract: In this study, we compared growth pattern, floc yield, Exo-polysaccharides (EPS) production, Poly-β-hydroxybutyrate (PHB) accumulation, resistance to osmotic and acid stress in Methylobacterium strains CBMB20, CBMB27, CBMB35, and CBMB110. Modified high C:N ratio medium denoted as HCN-AMS medium was used with a C:N ratio of 30:1. The HCN-AMS medium favored increased growth in all the studied strains. All Methylobacterium strains tested positive for EPS production and showed positive fluorescence with calcoflour stain. Elevated levels of EPS production from 4.2 to 75.0% was observed in HCN-AMS medium. Accumulation of PHB in HCN-AMS medium increased by 3.8, 36.7, and 12.0% in strains CBMB27, CBMB35, and CBMB110 respectively. Among the abiotic stresses, osmotic stress-induced growth inhibition of Methylobacterium strains was found to be lowered when grown in HCN-AMS medium. Likewise, growth inhibition due to acid stress at pH 5.0 was lower for strains grown in HCN-AMS medium compared to growth in AMS medium. Enhanced survivability under stress conditions may be attributed to the high EPS and PHB production at increased carbon concentration in the growth medium.

The Effect of Growth Medium pH towards Trypsin-Like Activity Produced by Lactic Acid Bacteria

Abstract: In cases of pancreatic disease, trypsin deficiency often occurs due to reduced expression of trypsin in the pancreas. Patients with pancreatic problem can be treated with a supplement containing digestive enzymes, including trypsin. However, most of the enzymes currently used for the treatment are derived from porcine and bovine sources. On the other hand, lactic acid bacteria are also known to show trypsin-like activity. In the previous work, our group screened 11 lactic acid bacteria isolates, which had previously been proven to show serine protease activity, for trypsin-like activity. The strains were initially grown in MRS (de Mann, Rogosa and Sharpe) medium before being transferred directly to the production medium to produce trypsin. During the previous study, the initial pH of the production medium was set at 6 (the same as the MRS medium pH), which is the optimum pH for the cell growth of lactic acid bacteria. However, most trypsin has an optimum pH of around 8. In this study, we altered the production medium pH to 8 and we harvested the lactic acid bacteria from MRS medium by centrifugation prior to their inoculation to the production medium. Observation of the culture growth and enzyme activity indicated that the new strategy improved the enzyme activity expressed by some strains.

Comparison of two culture media for breaking seed dormancy and germination improvement in four species of Linum L.

Abstract: The aim of this study was to compare the effects of different treatments in breaking dormancy and to increase germination percentage and days to germination in two media of water with agar and Murashige and Skoog (MS) growth medium in four species of Linum L. namely, L. mucronatum, L. nervosum, L. album and L. austriacum. Stages of breaking dormancy were incorporated with the exogenous application of gibberellic acid (GA 3 ) (400, 800 and 1600 part per million (ppm) for periods of 72, 96, 144 and 168 h at 4°C; sulfuric acid (H 2 SO 4 ) (25, 40 and 50%) for periods of 10, 15, 20 and 25 min and washing in 70°C running water for 24, 48, 72 and 96 h and some combined treatments such as H 2 SO 4 with GA 3 and H 2 SO 4 with washing in 70°C running water treatments. Germination of four species of Linum significantly increased in higher concentrations of GA 3 (1600 ppm). ‘/ The best result in H 2 SO 4 treatments was recorded at 25% concentration for 25 min. Also, the percentage germination and days to germination in each of the four species were higher and faster in water with agar medium in comparison to MS growth medium. These results suggest that the Linum seeds have exogenous and endogenous dormancy. Key words : Dormancy, germination, MS media, Linum species, water + agar medium.

STUDIES ON THE PRODUCTION OF α- AMYLASE BY Bacillus subtilis

Abstract: Cell growth of Bacillus subtilis and the production of α-amylase in the medium were examined. Based on the amylase productivity level in shake flask cultures after 72 hours of growth, the growth medium containing starch and peptone was selected as the best medium. Our results show that the amylase production is higher in the presence of optimum Carbon and Nitrogen ratio. The production of the enzyme was maximum (370U/mg) at 72 h after inoculation. The effect of incubation period, pH of the medium and incubation temperature was optimized. The maximum production of enzyme was obtained at 30°C and pH 7.

Modification of MCF-10A cells with pioglitazone and serum-rich growth medium increases soluble factors in the conditioned medium, likely reducing BT-474 cell growth.

Abstract: In the present study, we aimed to preincubate MCF-10A cells with pioglitazone and/or serum-rich growth media and to determine adhesive and non-adhesive interactions of the preincubated MCF-10A cells with BT-474 cells For this purpose, the MCF-10A cells were preincubated with pioglitazone and/or serum-rich growth media, at appropriate concentrations, for 1 week The MCF-10A cells preincubated with pioglitazone and/or serum-rich growth media were then co-cultured adhesively and non-adhesively with BT-474 cells for another week Co-culture of BT-474 cells with the preincubated MCF-10A cells, both adhesively and non-adhesively, reduced the growth of the cancer cells The inhibitory effect of the preincubated MCF-10A cells against the growth of BT-474 cells was likely produced by increasing levels of soluble factors secreted by the preincubated MCF-10A cells into the conditioned medium, as immunoassayed by ELISA However, only an elevated level of a soluble factor distinguished the conditioned medium collected from the MCF-10A cells preincubated with pioglitazone and serum-rich growth medium than that with pioglitazone alone This finding was further confirmed by the induction of the soluble factor transcript expression in the preincubated MCF-10A cells, as determined using real-time PCR, for the above phenomenon Furthermore, modification of the MCF-10A cells through preincubation did not change the morphology of the cells, indicating that the preincubated cells may potentially be injected into mammary fat pads to reduce cancer growth in patients or to be used for others cell-mediated therapy

A simple physiologically relevant double-agar-layer method for post-germination treatment of seedlings

Abstract: Arabidopsis thaliana is frequently grown on semisolid medium in Petri dishes, for various experiments that usually consist of two stages on two distinct growth media. Seedlings are germinated under favorable conditions followed by their transfer to another medium containing the given treatment(s). This often causes secondary effects on seedlings due to root shock, or direct and unavoidable contact of the shoot with the second medium. We have developed a simple and efficient method for the transfer of seedlings grown on semisolid medium with minimal damage. In this double-agar-layer method, seeds are germinated on a thin growth-medium-containing agar layer. Subsequently, medium blocks containing the embedded seedlings are excised and placed on the second semisolid medium supplemented with the treatment agent. Differential agar concentrations allow easy penetration of the roots into the second medium, but do not allow the shoots to come into contact with it. This unique method offers several advantages over others that are in common use, in which the seedlings are individually transferred to the second medium or alternatively grown on transfer-carrier matrices, such as filter paper, mesh and cellophane. In the presented method, the entire root system faces the growth medium, the shoots are surrounded by air at all growth stages and transfer of the seedlings is much easier. In addition, a large number of seedlings can be transferred in a single step, without stressing the plants or damaging the delicate root system. This method can also be applied to other plant species grown on semisolid media.

Carbon dioxide production in wild type and PDC1 mutant Saccharomyces cerevisiae undergoing the Crabtree effect

Abstract: The production of carbon dioxide (CO2) was measured under various glucose concentrations in mutant yeast, Saccharomyces cerevisiae (YLR044) and wild type yeast by the use of respirometers. We investigated the effect of high glucose concentrations on yeast cells— referred to as the Crabtree effect. Three different glucose treatments were tested; the concentration found in the growth medium (0.11 M), the optimal concentration for gas production (0.50 M) and the concentration at which the Crabtree effect takes place (1.00 M). We also determined how much CO2 was produced by the wild type yeast compared to the mutant under high glucose concentrations (≥ 0.50 M). The Crabtree effect was observed in the wild type yeast as 3.4 times less CO2 was made per cell at 1.00 M glucose than at 0.11 M. This effect may be due to an evolutionary adaptation in yeast cells to allow competition with other microorganisms for food. The Crabtree effect was not observed in the mutant yeast even though 1.4 times less CO2 was made per cell at 1.00 M glucose than at 0.11 M because this difference was not statistically significant. The final CO2 concentration for mutant and wild type yeast cells show that there was a greater production of CO2 in wild type as compared to mutant yeast, which is presumed to be the result of the low pyruvate decarboxylase activity in the PDC1 mutants.

Production of salmosin, a snake venom-derived disintegrin, in recombinant Pichia pastoris using high cell density fed-batch fermentation

Abstract: Salmosin, a snake venom-derived disintegrin, was successfully expressed in the methylotrophic yeast Pichia pastoris and secreted into the culture supernatant, as a 6 kDa protein. High-cell density fermentation of recombinant P. pastoris was optimized for the mass production of salmosin. In a 5 L jar fermentor, recombinant P. pastoris was fermented in growth medium containing 5% (w/v) glycerol at the controlled pH of 5.0. After culturing for 21 h, glycerol feeding medium was fed at one time into the culture broth. After 7 h (a total of 28 h), induction medium that contained methanol was increasingly added until the culture time totaled 75 h. Finally, these optimized culture conditions produced a high cell density of recombinant P. pastoris (dry cell weight of 113.38 g/L) and led to the mass production of salmosin (a total protein concentration of 369.2 mg/L). The culture supernatant containing salmosin inhibited platelet aggregation, resulting in a platelet aggregation of 9% compared to that of 94% in the control experiment, without culture supernatant. These results demonstrate that recombinant salmosin in culture supernatant from high cell density fed-batch fermentation can serve as a platelet aggregation inhibitor.

Prokaryotic lipidomics: development of a method to study thephospholipids in E. coli and B. subtilis

Abstract: The way in which prokaryotes, particularly Gram-negative bacteria, regulate their lipid compositions in response to changes in their growth media can provide insights into how bacteria adapt to life in oligotrophic environments. In this thesis, it is reported how electrospray ionisation mass spectrometry (ESI-MS) techniques can be adapted to map the impact of growth conditions on the regulation of the phospholipid biosynthetic pathways in E. coli and B. subtilis. Reliable counting of bacterial number densities is one of the main barriers to achieving reproducible quantitative lipid composition profiles. Consequently an improved method has been developed, based on an automated cell counter, to characterize the bacterial number densities and cell size for each of the samples used in the ESI-MS experiments. This approach has enabled the observation of unexpected changes in both the relative and absolute amounts of the phospholipid species isolated from bacteria grown under different conditions. These studies have investigated the adaptation of E. coli and B. subtilis to a glucose deplete growth medium. In E. coli the effect of growth in a nutrient rich growth medium, phosphate depletion and CTAB addition was investigated. In B. subtilis initial studies were performed into nitrogen depletion, myristic acid addition and DOPC addition. The changes in phospholipid classes, as well as in the homologues within each class, that we have observed may be related to changes in the fluxes within the lipid biosynthetic pathway of the organism. The expectation is that studying the changes in growth conditions using E. coli and B. subtilis as a model organism could help with the understanding of biofilm formation and help towards a greater understanding of bacterial adaptation to stressful environments.

Enhancement of glutamine synthetase activity in Paenibacillus polymyxa by optimization of different growth conditions

Abstract: Biological nitrogen fixation is highly regulated at the transcriptional level by sophisticated regulatory networks that respond to multiple environmental signals. Glutamine synthetase (GS) occupies a central position in cellular metabolism and offer to the cell, a potential point for regulation of biosynthetic function. The aim of this work is to enhance the activity of GS in Paenibacillus polymyxa through altering the constituents of the growth medium, thereby increasing the nitrogen fixation capability. Two bacterial strains were identified as P. polymyxa by 16S rRNA gene (Accession No AB727983). High GS activity was recorded in the two strains, in presence of the divalent cations Mg +2 and Mn +2 . Western blot analysis confirmed the presence of the GS at approximately ~60 kDa. GS activity was found to be affected by growth medium, carbon source, nitrogen source and sodium chloride. LB supplemented with 7% glycerol, 0.4% asparagine and 0.15% sodium chloride gave the highest GS activity. Key words : Glutamine, Paenipacillus polymyxa , 16S rRNA, nitrogen fixation, western blot, Mg +2 and Mn +2 .

Studies on Colletotrichum capsici - a Plant Pathogenic Fungus: Selection of Culture Medium for Growth And Determination of Minimum Inhibitory Concentration of Fungicide

Abstract: The present study investigated the growth of Colletotrichum capsici in four different growth medium Potato Dextrose Agar (PDA), Czapek Dox Agar (CDA), Sabouraud Dextrose Agar (SDA) and Peptone Yeast Extract Dex- trose Agar (PYDA). Among the different media tested, PDA supported the maximum growth significantly compared to all other media. The two fungicides Indofil M-45 and Carbendazim were evaluated for MIC (minimum inhibitory concentration) activity against the fungi. The effect of Indofil M-45 and Carbendazim on the fungi was evaluated by the disc diffusion method and the inhibition zones showed that the diameter of the colony of the Colletotrichum capsici was reduced. The higher concentration gave the maximum effect which decreased with dilutions. Carbendazim showed maximum efficacy even at a very low concentration of 10 -9 mg/ml as compared to that of Indofil at 10 -3 mg/ml. The two fungicides Indofil M-45 and Carbendazim were found to inhibit the growth of the fungi. How-