Showing papers on "Growth medium published in 2014"

A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

Abstract: Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly,” that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27T and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.

Homo- and heterofermentative lactobacilli differently affect sugarcane-based fuel ethanol fermentation

Abstract: Bacterial contamination during industrial yeast fermentation has serious economic consequences for fuel ethanol producers. In addition to deviating carbon away from ethanol formation, bacterial cells and their metabolites often have a detrimental effect on yeast fermentative performance. The bacterial contaminants are commonly lactic acid bacteria (LAB), comprising both homo- and heterofermentative strains. We have studied the effects of these two different types of bacteria upon yeast fermentative performance, particularly in connection with sugarcane-based fuel ethanol fermentation process. Homofermentative Lactobacillus plantarum was found to be more detrimental to an industrial yeast strain (Saccharomyces cerevisiae CAT-1), when compared with heterofermentative Lactobacillus fermentum, in terms of reduced yeast viability and ethanol formation, presumably due to the higher titres of lactic acid in the growth medium. These effects were only noticed when bacteria and yeast were inoculated in equal cell numbers. However, when simulating industrial fuel ethanol conditions, as conducted in Brazil where high yeast cell densities and short fermentation time prevail, the heterofermentative strain was more deleterious than the homofermentative type, causing lower ethanol yield and out competing yeast cells during cell recycle. Yeast overproduction of glycerol was noticed only in the presence of the heterofermentative bacterium. Since the heterofermentative bacterium was shown to be more deleterious to yeast cells than the homofermentative strain, we believe our findings could stimulate the search for more strain-specific antimicrobial agents to treat bacterial contaminations during industrial ethanol fermentation.

Effects of growth medium on matrix-assisted laser desorption-ionization time of flight mass spectra: a case study of acetic acid bacteria.

Abstract: The effect of the growth medium used on the matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectra generated and its consequences for species and strain level differentiation of acetic acid bacteria (AAB) were determined by using a set of 25 strains. The strains were grown on five different culture media that yielded a total of more than 600 mass spectra, including technical and biological replicates. The results demonstrate that the culture medium can have a profound effect on the mass spectra of AAB as observed in the presence and varying signal intensities of peak classes, in particular when culture media do not sustain optimal growth. The observed growth medium effects do not disturb species level differentiation but strongly affect the potential for strain level differentiation. The data prove that a well-constructed and robust MALDI-TOF mass spectrometry identification database should comprise mass spectra of multiple reference strains per species grown on different culture media to facilitate species and strain level differentiation.

Recombinant protein expression in Lactococcus lactis using the P170 expression system

Abstract: The use of the Gram-positive bacterium Lactococcus lactis in recombinant protein production has several advantages, including the organism's long history of safe use in food production and the fact that it does not produce endotoxins. Furthermore the current non-dairy L. lactis production strains contain few proteases and can secrete stable recombinant protein to the growth medium. The P170 expression system used for recombinant protein production in L. lactis utilizes an inducible promoter, P170, which is up-regulated as lactate accumulates in the growth medium. We have optimised the components of the expression system, including improved promoter strength, signal peptides and isolation of production strains with increased productivity. Recombinant proteins are produced in a growth medium with no animal-derived components as a simple batch fermentation requiring minimal process control. The accumulation of lactate in the growth medium does, however, inhibit growth and limits the yield from batch and fed-batch processes. We therefore combined the P170 expression system with the REED™ technology, which allows control of lactate concentration by electro-dialysis during fermentation. Using this combination, production of the Staphylococcus aureus nuclease reached 2.5 g L(-1).

Utilization of Amino Acids and Other Nitrogen-Containing Compounds

Abstract: This chapter discusses the catabolism of amino acids and other nitrogen-containing compounds. Aspartate is transported into B. subtilis by two systems, a high-affinity system energized by the proton motive force and a low-affinity system. The enzymes of the arginase degradative pathway are found in B. subtilis and B. licheniformis. In B. subtilis and B. licheniformis, the proline-degradative enzymes are induced by proline. In B. subtilis, this induction is inhibited if the growth medium also contains glucose and amino acids. Hut expression in B. subtilis is induced by histidine and repressed by rapidly metabolized carbon sources such as glucose. Growth in the presence of amino acids severely inhibits synthesis of the Hut enzymes. Dehydrogenase enzymes may play a role in the degradation of phenylalanine in Bacillus badius, of valine in Streptomyces spp, and of leucine in B. cereus. Nitrate reductase activity is found in B. subtilis cells growing in the presence of nitrate under semianaerobic conditions. Amino acids and small peptides produced by the degradation of extracellular polypeptides can also supply B. subtilis with nutrients during growth and sporulation.

Enhanced production of napthoquinone metabolite (shikonin) from cell suspension culture of Arnebia sp. and its up-scaling through bioreactor

Abstract: Cell culture in shake flask and air-lift bioreactor was carried out to exploit the potential of Arnebia sp. for napthoquinone metabolite production. Cell suspension cultures of Arnebia were established from friable callus in liquid MS medium supplemented with 6-benzylaminopurine (BAP) (10 μM) and indole-3-butyric acid (IBA) (5 μM). Growth kinetic studies were done by using settled cell volume and fresh/dry cell weight method. Suspension cultures were maintained by sub-culturing at 10 days interval. A two-stage culture system is employed using growth medium (GM) and modified M9 medium (production medium) for cell biomass and naphthoquinone pigment production, respectively. Results showed that cultivation of cells under dark conditions at room temperature (25 ± 2 °C) enhanced the cell biomass from 100 to 625 g l−1. The pigment production was also found to be increased in dark conditions at room temperature. Alkaline pH found to have positive effect on pigment yield. In case of M9 medium constituents, absence of Na2SO4 does not affect the pigment yield. The current approaches have the cumulative effect to meet an increased level of (25.5 μg/ml) metabolite production in air-lift bioreactor.

An improved medium for the anaerobic growth of Paracoccus denitrificans Pd1222.

Abstract: Paracoccus denitrificans is a well studied model organism with respect to its aerobic and anaerobic respiratory enzymes. However, until now, the growth medium for this organism has not been optimized for anaerobic growth. In particular, the requirements of P. denitrificans for trace elements (TEs) are not well known. In the present study we aimed to improve growth rates of P. denitrificans Pd1222 on a defined medium under anoxic conditions. We designed media containing different combinations of TEs at various concentrations, and tested their performance against previously reported media. Our results suggest that growth rate and yield depend on the availability and concentration of TEs in the medium. A chelated TE solution was more suitable than an acidified TE solution. Highest growth rates were achieved with medium comprising the TEs iron, manganese, molybdenum, copper and zinc ranging from 0.1 to 9 μM. On this medium, P. denitrificans Pd1222 grew with a generation time of 4.4 h under anoxic conditions and 2.8 h under oxic conditions. Diauxic growth was clearly shown with respect to nitrate and nitrite reduction under anoxic conditions.

Nitrogen Utilization and Metabolism in Ruminococcus albus 8

Abstract: The model rumen Firmicutes organism Ruminococcus albus 8 was grown using ammonia, urea, or peptides as the sole nitrogen source; growth was not observed with amino acids as the sole nitrogen source. Growth of R. albus 8 on ammonia and urea showed the same growth rate (0.08 h(-1)) and similar maximum cell densities (for ammonia, the optical density at 600 nm [OD600] was 1.01; and for urea, the OD600 was 0.99); however, growth on peptides resulted in a nearly identical growth rate (0.09 h(-1)) and a lower maximum cell density (OD600 = 0.58). To identify differences in gene expression and enzyme activities, the transcript abundances of 10 different genes involved in nitrogen metabolism and specific enzyme activities were analyzed by harvesting mRNA and crude protein from cells at the mid- and late exponential phases of growth on the different N sources. Transcript abundances and enzyme activities varied according to nitrogen source, ammonia concentration, and growth phase. Growth of R. albus 8 on ammonia and urea was similar, with the only observed difference being an increase in urease transcript abundance and enzyme activity in urea-grown cultures. Growth of R. albus 8 on peptides showed a different nitrogen metabolism pattern, with higher gene transcript abundance levels of gdhA, glnA, gltB, amtB, glnK, and ureC, as well as higher activities of glutamate dehydrogenase and urease. These results demonstrate that ammonia, urea, and peptides can all serve as nitrogen sources for R. albus and that nitrogen metabolism genes and enzyme activities of R. albus 8 are regulated by nitrogen source and the level of ammonia in the growth medium.

Activation of Rhizobium tibeticum with flavonoids enhances nodulation, nitrogen fixation, and growth of fenugreek (Trigonella foenum-graecum L.) grown in cobalt-polluted soil

Abstract: The goal of this study was to investigate the response of activation of Rhizobium tibeticum with mixture of hesperetin and apigenin to improve growth, nodulation, and nitrogen fixation of fenugreek grown under cobalt (Co) stress. The current study showed that high concentrations of Co-induced noxious effects on rhizobial growth, nod gene expression, nodulation, phenylalanine ammonia-lyase (PAL) and glutamine synthetase (GS) activities, total flavonoid content, and nitrogen fixation. Addition of a mixture of hesperetin and apigenin to growth medium supplemented with different concentrations of Co significantly increased bacterial growth. PAL activity of roots grown hydroponically at 100 mg kg−1 Co and inoculated with induced R.tibeticum was significantly increased compared with plants receiving uninduced R.tibeticum. Total flavonoid content of root exudates of plants inoculated with activated R.tibeticum was significantly increased compared with inoculated plants with unactivated R.tibeticum or uninoculated plants at variant Co dosages. Application of 50 mg kg−1 Co significantly increased nodulation, GS, nitrogenase activity, and biomass of plants inoculated with either or uninduced R.tibeticum. The total number and fresh mass of nodules, nitrogenase activity, and biomass of plants inoculated with induced cells grown in soil treated with 100 and 200 mg kg−1 Co were significantly increased compared with plants inoculated with uninduced cells. Induced R. tibeticum with flavonoids significantly alleviates the adverse effect of Co on nod gene expression and therefore enhances nitrogen fixation. Induction of R. tibeticum with compatible flavonoids could be of practical importance in augmenting growth and nitrogen fixation of fenugreek grown in a Co-contaminated agroecosystem.

Production of recombinant protein by a novel oxygen-induced system in Escherichia coli.

Abstract: The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes. The soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence. By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW). Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery.

Effect of protein hydrolysates on growth kinetics and aminopeptidase activities of Lactobacillus.

Abstract: The goal of this study was to evaluate how two new hydrolysates from poultry by-products act on ten lactobacilli growth kinetics when supplemented to the growth medium. These effects were compared with ones induced by two most common commercial hydrolysates, i.e., tryptone and peptone. Growth medium, supplemented with one of new hydrolysates, 78T, as only nitrogen source, can sustain the maximum growth rate and the biomass yield in the same way of MRS, reach of different nitrogen sources. Moreover aminopeptidase activities (AA) of each strain were determined to investigate the effect of the growth condition on the modulation of aminopeptidase pattern. Five cell extracts of each ten strains, obtained from their cultivation in MRS and in the presence of the two common hydrolysates and the two new ones, were considered. AA was investigated against five different chromogenic substrates: β-naphthyl amide derivatives of L-anomers of leucine, lysine, proline, glycine-proline, and phenilalanine-proline. A great variability of AA was observed among the strains: also strains belonging to the same species showed peculiar AA profile.

Optimization of physical parameters of a-amylase producing brevibacillus borostelensis r1 in submerged fermentation

Abstract: Bacteria have been regarded as treasure of many use ful enzymes viz., amylases, proteases, lipases, hyd rolases and reductases. Among them amylolytic enzymes have great biotechnological applications and economic exploitations. The produ ction of α-amylases by fermentation had been thoroughly investigated and s hown to be affected by a variety of physicochemical factors, such as the composition of the growth medium, the type of strai n, cell growth, methods of cultivation, inoculum co ncentration, time of incubation, pH, temperature, salinity, carbon, nitrogen and min eral sources. The present study was carried out to optimize the α-amylase production of Brevibacillus borstelensis R1 using t en different media viz., Nutrient broth, Luria Bert ain broth, Clarks & Lub medium, Pikovskaya’s medium, Tendler's non-synthetic medium , Amylase production medium, Soluble starch beef ex tract medium, Soybean casein digest medium, Yeast extract peptone dextros e glucose medium and Tryptone glucose beef extract medium. Among these ten media, Pikovskaya’s (PK) medium proved to be optima l for α-amylase production (1861±17U/ml). The optimized α-amylase production in PK medium by submerged fermentation ( SmF) was subjected to varying physical parameters s uch as 24hrs incubation time, 2% inoculum size, 37 0 C, pH 7.0 and 1% NaCl. Alpha-amylase produced by B. borostelensis R1 have many applications in starch processing, desizing of textiles, paper sizing, detergent additive, bread improvement, ethanol product ion, sewage treatment, effluent treatment and other fermentation processes.

A minimal growth medium for the basidiomycete Pleurotus sapidus for metabolic flux analysis.

Abstract: Pleurotus sapidus secretes a huge enzymatic repertoire including hydrolytic and oxidative enzymes and is an example for higher basidiomycetes being interesting for biotechnology. The complex growth media used for submerged cultivation limit basic physiological analyses of this group of organisms. Using undefined growth media, only little insights into the operation of central carbon metabolism and biomass formation, i.e., the interplay of catabolic and anabolic pathways, can be gained. The development of a chemically defined growth medium allowed rapid growth of P. sapidus in submerged cultures. As P. sapidus grew extremely slow in salt medium, the co-utilization of amino acids using 13C-labelled glucose was investigated by gas chromatography–mass spectrometry (GC-MS) analysis. While some amino acids were synthesized up to 90% in vivo from glucose (e.g., alanine), asparagine and/or aspartate were predominantly taken up from the medium. With this information in hand, a defined yeast free salt medium containing aspartate and ammonium nitrate as a nitrogen source was developed. The observed growth rates of P. sapidus were well comparable with those previously published for complex media. Importantly, fast growth could be observed for 4 days at least, up to cell wet weights (CWW) of 400 g L-1. The chemically defined medium was used to carry out a 13C-based metabolic flux analysis, and the in vivo reactions rates in the central carbon metabolism of P. sapidus were investigated. The results revealed a highly respiratory metabolism with high fluxes through the pentose phosphate pathway and TCA cycle. The presented chemically defined growth medium enables researchers to study the metabolism of P. sapidus, significantly enlarging the analytical capabilities. Detailed studies on the production of extracellular enzymes and of secondary metabolites of P. sapidus may be designed based on the reported data.

Use of stem cell conditioned medium to inhibit oxidation for anti-aging skin

Abstract: A use of a stem cell conditioned medium to inhibit oxidation for anti-aging skin. First, mesenchymal stem cells are cultured in a cell culture dish containing a complete growth medium. After mesenchymal stem cells are sub-cultured in the complete growth media for three times and transferred to a basal medium, a conditioned medium can be acquired from the basal medium.

Isolation and Characterization of Proteases Enzyme from Locally Isolated Bacillus sp.

Abstract: A bacterium was isolated from the natural source. Gram staining & spore staining showed that the organism is gram positive and forms spore during adverse condition in the growth medium. After various tests it was suggested and the features agreed with the description of Bacillus sp in Bergey’s Manual of Systematic Bacteriology [24]. It was also identified as Bacillus sp with 99.9% identity by API 50 CHB. In growth curve determination showed that the growth of the organism is increased with the increase of incubation period and the growth reached maximum at around 24 hours of incubation and the protease activity was the maximum of the 26 hours culture. This microbe has grown at high temperature and pressure. Its optimum pH and temperature were 8.5 and 60°C. It secretes an extracellular protease in the growth medium. The enzyme hydrolyses a number of proteins including azocasein which suggests that that it is an extracellular protease. The enzyme seems to be alkaline protease which is capable of De-Hairing from skin and hides. A number of companies such as NOVO chemicals started to produce NOVOzymes for tannery industries. The potential for use of microbial enzymes in leather processing lies mainly in areas in which pollution-causing chemicals are being used.

Novel Factors Affecting Shoot Culture of Chrysanthemum (Dendranthema × Grandiflora)

Abstract: Teixeira da Silva J.A., 2014: Novel factors affecting shoot culture of chrysanthemum (Dendranthema × grandiflora) [Alternatyvių standiklių, skystų terpės priedų, CO2 sodrinimo ir kitų faktorių įtaka chrizantemų (Dendranthema × grandiflora (Ramat.) Kitamura) ūglių kultūrų auginimui]. – Bot. Lith., 20(1): 27–40. Chrysanthemum (Dendranthema × grandiflora (Ramat.) Kitamura) continues to be one of the most important ornamental plants in the world. Although the tissue culture of chrysanthemum has been widely explored, several unexplored topics remain, and, in developing countries, there is always the constant search for reducing the cost of raising tissue cultured plants. In this study, by focusing on a leading market cultivar in Japan, ‘Shuhouno-chikara’, alternatives to agar (as the gelling agent) and sucrose (as the carbon source) for chrysanthemum tissue culture were sought. Both Gellan gum and agar resulted in greater shoot and root production than all other gelling agents tested, including Bacto agar, phytagel, oatmeal agar, potato dextrose agar, barley starch and corn starch. All of the alternative liquid-based medium additives tested (low and full fat milk, Coca-cola®, coffee, Japanese green, Oolong and Darjeeling teas) negatively impacted plant growth, stunted roots and decreased chlorophyll content (SPAD value) of leaves. There was no difference between plants grown on medium with refined sucrose or table sugar, although poor growth was observed when stevia (Stevia rebaudiana) extract was used. Photoautotrophic micropropagation increased significantly the shoot mass relative to control plants, even when the density of plants was doubled. Aeration improved plantlet growth. The tetrazolium test was a simple, but effective essay to see the intensity and strength of root growth in different basal media. MDH activity decreased in the root+shoot extract of plants grown on most alternative media, but remained high on TCSGM (Teixeira’s chrysanthemum shoot growth medium), Gellan gum, aerated and CO2-enriched cultures. A similar trend was observed for deaminating GDH, while an opposite trend was observed for aminating GDH activity. These experiments indicate that tissue culture research for chrysanthemum still provides a rich field for exploration with interesting and valuable results.

In Vitro Regeneration of Curcuma caesia Plantlets from Basal Part and via Somatic Embryogenesis

Abstract: Plantlets of Curcuma caesia were produced in vitro from newly sprouting vegetative buds of tubers. Segments of the plantlets from the junction between the root and the basal portion of the stem were subsequently used as explants to investigate factors affecting callus induction and plant regeneration via somatic embryogenesis. The explants were placed on Woody Plant Medium (WPM) together with different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D) and benzyl aminopurine (BAP) in the presence of light. The growth medium supplemented with 5 mg/L BAP and 2 mg/L 2,4-D promoted callus induction after 70 days of culture. Sub-culturing on the same medium enhanced the production of friable callus. Culture media containing higher concentrations of agar promoted the development of green somatic embryos from the callus. Respond of somatic embryogenesis was most successful with MS medium in 6.0 g/L agar supplemented with 5 mg/L BAP and 0.2 mg/L 2,4-D whereby the callus developed into green somatic embryos with an efficiency of 53%. This culture medium also produced the largest number plantlets.

Biocontrol and Environmental Studies on Paper Degrading Mycoflora Isolated from Sanganer Area, Jaipur, India

Abstract: Culture media and temperature significantly affect the growth, sporulation and conidial discharge of any mycoflora. The colony diameter, cultural characteristics (texture, surface and reverse pigmentation) and sporulation of fungi were greatly influenced by the type of growth medium used. The survival period of fungi in relation to different temperature and pH conditions, media and natural habitats differs from species to species.Variation in mycelial growth and fungal sporulation was observed with media tested. Colony radial growth and sporulation of soil fungi was found to be excellent on Potato Dextrose Agar followed by Malt Extract Agar at optimal environmental conditions. In the present study in vitro antagonistic activity using the dual culture technique was carried out and show ed that antagonist Trichoderma sp. grow faster than the isolated fungi and produced inhibition zones thereby limiting the growth of the fungi. Maximum growth inhibition by Trichoderma sp (biocontrol agent) were found in the following order i.e. Fusarium sp. (46.66% ), A. fumigatus (42.85%), A. flavus (40.00%) and A. niger (24.44%), were found with the use of Trichoderma sp in dual culture. The present investigation was conducted to examine the effect of different temperature, pH and nutritional media on the mycelial growth and fungal sporulation of three soil fungi i.e. Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, isolated from paper and pulp industry and biocontrol studies were also carried out to develop antagonist Trichoderma sp. so as to prevent paper degradation by fungi.

Glycerol from novel strains of halophytic microalgae

Abstract: Glycerol is a new biofuel which underpins a commercial CHP technology as a result of the novel McNeil combustion cycle patented by Aquafuel, UK. This research explored glycerol production in new strains of halotolerant microalgae Dunaliella (T35, T36 and T37) and Asteromonas (T33a, T33b and T33c) obtained from Namibia. The aim was to: (a) Determine the optimum conditions of pH value, temperature and salinity on the specific growth rate and doubling time and parameters of glycerol production, (b) Investigate metabolites associated with glycerol accumulation and dissimilation in Dunaliella cells exposed to salinity stress under continuous light or dark regimes. (c) Maximize glycerol recovery from microalgal cultures using novel supercritical carbon dioxide (SC-CO2) extraction technology. Dunaliella and Asteromonas strains were shown to withstand large variations in the salinity of the growth medium 0.5 - 4.0 M NaCl. The optimum conditions for their growth were 1.0 M NaCl, 30 oC, pH 7.5 to 9.0 and 45 μmol m-2 s-1. Cultivation at 1.0 M NaCl produced 46.1 - 65.2 pg/cell of glycerol in Dunaliella and 53.8 to 67.1 pg/cell of glycerol in Asteromonas, but transfer of cells from 1.0 to 4.0 M NaCl for 240 min maximized glycerol production to 179.5 - 237.6 pg/cell in Dunaliella and 128.7 - 184.2 pg/cell in Asteromonas. The amount of glycerol produced by Dunaliella isolated from Namibia was between 3.3 and 5.2 fold greater than other species investigated (D. salina, D. quartolecta, D. parva and D. polymorpha). Transfer of cells to high salinities did not disrupt cell integrity, although cells shrank by 35 to 62 % of their original volume due to water efflux from the cell. The starch and glycerol pools of Dunaliella T35 were investigated after 24 h exposure to 1.0 and 4.0 M NaCl in dark and light regimes in order to investigate how starch degradation contributed to glycerol production. In 1.0 M NaCl, the amount of starch was 45 and 222 μg/mg of dry biomass under dark and light regimes respectively, and glycerol, 163 and 174 μg/mg of dry biomass. Under parallel conditions at 4.0 M NaCl, the amount of starch decreased to 27 and 146 μg/mg of dry biomass and glycerol increased to 592 and 706 μg/mg of dry biomass in the dark and light, respectively. This suggested that the contribution of starch breakdown to glycerol synthesis increased with increasing salinity stress in the Namibian strain of Dunaliella. Hyperosmotic shock decreased the pool size of proline and increased the pools size of pyruvate and glycerol. The degree of cell rupture after SC-CO2 extraction of glycerol from Dunaliella was dependent on the applied pressure. Cells were ruptured and glycerol was released at 100 bar and the degree of cell rupture increased with pressure to 200 bar in the presence of chloroform. At 350 bar in the absence of chloroform SC-CO2 extraction of glycerol produced ~6 % higher glycerol levels than sonication. Namibian strains of Dunaliella and Asteromonas T33c investigated are best suited for glycerol production compared to Asteromonas T33a, T33b and other strains of Dunaliella (D. salina, D. quartolecta, D. parva and D. polymorpha). Cells cultivated in 1.0 M NaCl to generate biomass followed by transfer to hyperosmotic stress for glycerol accumulation was shown to be optimum for glycerol production. SC-CO2 is shown to be an effective, ‘green’ technology for glycerol extraction from Dunaliella.

Effect of Growth Media and Incubation Time on the Culturability of Soil Bacteria

Abstract: The effect of growth medium and incubation time were investigated by determining the number of colonies that were visible on triplicate plates of three different media on daily intervals. The viable counts were significantly different between the three different media even after the first day and this continued for seven days. Colony counts on the three media reached their maximum on the third day of incubation. The use of Nutrient agar (NA) and Plate count agar (PCA) resulted in increasing counts with increasing incubation time respectively. Counts obtained from the Nutrient agar were higher than that of Plate count agar followed by the soil extract agar (SEA) which had the lowest count and a longer bacteria lag phase due to the low nutrient content in the soil extract agar. The Plate count agar formed more distinct colonies than the Nutrient agar and Soil extract agar. During the incubation period, colonies appeared on the Nutrient agar even on the seventh day. These characteristics of the Nutrient agar to support a higher number of colonies for a long period of time could be attributed to its nutrient composition. The colony counts upon incubation for seven days ranged from a mean of 3.0x10 5 cfu/g on Soil extract agar, 6.1x10 5 cfu/g on Plate count agar and 7.6x10 5 cfu/g on Nutrient agar for example A which was a cultivated loamy soil and 1.3x10 5 cfu/g also on Soil extract agar, 3.9x10 5 cfu/g on Plate agar and 5.6x10 5 cfu/g Nutrient agar for soil sample B which was a non-cultivated sandy soil. Longer incubation time increased colony count on the different growth media because it was so obvious on the Soil extract agar. No visible colonies were observed from the first to the third day on the Soil extract agar, visible colonies appeared on the fourth day of incubation. Also in the other growth media, there was an increase in colony count on the third day upon incubation.

Antibacterial Activities of Stationary Phase Culture Filtrates of Aspergillus fumigates

Abstract: Our previous studies performed for the development of polymicrobial biofilm showed that simultaneous coculturing of Aspergillus fumigatus conidia or sporelings with Pseudomonas aeruginosa resulted in the killing of A. fumigatus cells whereas hyphae older than 12h were recalcitrant to the bacterial fungicidal activity. Since A. fumigatus produces an array of antimicrobial molecules, we examined the antibacterial activity of A. fumigatus culture filtrates on bacterial growth to examine the possible role of such small molecules minimizing the fungicidal activity of P. aeruginosa by spectrophotometric and colony forming unit assays. Culture filtrates collected from 48h stationary phase cultures inhibited the growth of P. aeruginosa and S. aureus, but not that of Candida albicans. Colony forming unit assay of culture filtrate-treated P. aeruginosa cells showed that the effect was bacteriostatic. Culture filtrate incubated in a boiling water bath for 15min retained its antibacterial activity compared to that of the unboiled culture filtrate. These results show that stationary phase liquid cultures of A. fumigatus produce a heat stable bacteriostatic compound(s) in liquid culture that is released into the growth medium inhibits the growth of P. aeruginosa.

Glutathione Deficiency Leads to Riboflavin Oversynthesis in the Yeast Pichia guilliermondii

Abstract: The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3–4 and 5–6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism.

Optimization of culture condition of human bone marrow stromal cells in terms of purification, proliferation, and pluripotency.

Abstract: Human bone marrow stromal cells (hBMSCs) possess multilineage differentiation potential and play an important role in modern tissue engineering. However, the development of culture media to maintain hBMSCs in an undifferentiated, self-renewing state during their robust proliferation remains a challenge. We developed and tested modified growth medium [medium 1: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), low glucose, 2% fetal calf serum (FCS)] on hBMSCs by comparing primary cell isolation, multipassage expansion, culture morphology, proliferation, and cellular phenotype, and performing an expression analysis of intrinsic-regulated genes to other two media. Cell morphology, proliferation, and phenotype varied among the media, while cells cultured in medium 1 displayed small, spindle-shaped morphology with the highest rate of growth capacities and the expected phenotype. RT-PCR analysis showed that medium 1 displayed the lowest expression levels of osteogenic genes, chondrogenic genes (osteonectin, runt-related transcription factor 2, cartilage oligo matrix protein, and SOX9), and adipogenic genes (lipoprotein lipase). The expression of another adipogenic gene, peroxisome proliferator-activator receptor-γ2, was higher in medium 1 but did not reach significance. In addition, hBMSCs expanded in medium 1 showed the highest expression ratio of self-renewing-related genes Kruppel-like factor 2 (KLF2) and KLF5. In conclusion, medium 1 allows for better expansion and pluripotency maintenance of hBMSCs and serves as a preferred alternative to traditional serum-containing media for research applications and future clinical use.

Case Study of Acetic Acid Bacteria Ionization Time of Flight Mass Spectra: a - Matrix-Assisted Laser Desorption Effects of Growth Medium on

Abstract: The effect of the growth medium used on the matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) massspectra generated and its consequences for species and strain level differentiation of acetic acid bacteria (AAB) were determinedby using a set of 25 strains. The strains were grown on five different culture media that yielded a total of more than 600 massspectra, including technical and biological replicates. The results demonstrate that the culture medium can have a profound ef-fect on the mass spectra of AAB as observed in the presence and varying signal intensities of peak classes, in particular when cul-ture media do not sustain optimal growth. The observed growth medium effects do not disturb species level differentiation butstrongly affect the potential for strain level differentiation. The data prove that a well-constructed and robust MALDI-TOF massspectrometry identification database should comprise mass spectra of multiple reference strains per species grown on differentculture media to facilitate species and strain level differentiation.

Isolation and partial purification of toxin from colletotrichum falcatum: the causal agent of red rot in sugarcane

Abstract: The red rot fungus (Colletotrichum falcatum) spores were isolated from infected stalk pieces of sugarcane and grown on Czapek Dox agar medium to obtain pure culture. Spores were obtained from the pure culture and grown in Czapec Dox liquid medium for further studies. It was observed that the fresh and dry weights of fungal mycellial mat significantly increased with time. In addition, fungal growth resulted in a significant change in the pH of the Czapec Dox growth medium over a period of 35 days and gradually increased from 6.5 in the control medium to 8.4. The fungal toxin was extracted from the growth medium and used to treat cell suspension of a red rot resistant sugarcane cultivar HSF-240. The maximum toxin was produced after 28 days of fungal growth in the growth medium which was ascertained by the dry weight of residue obtained from the ethyl actate fractions, as well as percent mortality of sugarcane cells in suspension. The LD50 value (50% cell mortality) of toxin in ethyl acetate (ETA) fraction was 22.03 days while in water it was 30 days. The fungal toxin extracted in ETA fractions was more effective than the one extracted in water. It was mainly due to high solubility and potency of the toxin in ETA than in water. In general, the maximum toxicity was noted at the 4 th week (28 days) which decreased at 5 th and 6 th week of mycelial growth.

Enhancement of Antioxidant Glutathione Production by Saccharomyces cerevisiaescc Growing under Stressful Condition

Abstract: 2 Abstract: The cultivation of Saccharomyces cerevisiae(SCC) in a medium containing molasses as carbon source gave low content of glutathione (GSH) (antioxidant compound). The highest antioxidant level was observed on basal medium containing ethanol as a sole carbon source followed by medium containing glycerol being 423 and 335µ mol/g dry cell, resulting to 17.62and 13.95 µ mol/g/h for antioxidant productivity. The sequence of total antioxidant capacity values were: ethanol > glycerol > glucose > sucrose > molasses. The cultivation of S. cerevisiae SCC in a medium containing 0.1 or 0.5 mg/ml furfural gave the highest values of antioxidant capacity being 345 and 335 µ mole/g, respectively. Examined yeast isolate was grown on media containing different concentrations of sodium chloride. At 1 and 2 % NaCl, cell dry weight of SCC was slightly affected when compared with control. The maximum content of glutathione (18 mg/g dry cells) was recorded at 1% NaCl. On the other hand, glycerol and gassing power were increased by increasing NaCl concentration in a growth medium and reached the maximum of 220 mg/g dry cell and 44 cm at 2 % NaCl, 3 respectively. In the same trend, the increasing of osmotic stress in the medium increased glutathione content, glycerol and the gassing power. The highest values of GSH content was recorded after 120 min in a medium containing 10 % glucose followed by 20 % being 35.32 and 26.0 mg/g dry cell, respectively. The maximum glycerol content was recorded at 90min in a medium containing 30 % glucose being 230 mg/g dry cell. The high values of gassing power were recorded to be 66 cm after 30 min and 71 cm after 60 min incubation. 33 This condition improved the GSH antioxidant, glycerol content and gassing power of the studied yeast isolate.