Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: I. EFFECT OF MAGNESIUM AND CALCIUM IONS

Sporulation in a strain of the wild yeast, Hansenula saturnus,was investigated. The yeast was found to form spores even indistilled water. The sporulation rate (percentage of ascus-bearingindividuals) in this case was found to be markedly affectedby the cell concentration adopted in the test. The addition of inorganic nutrients to the sporulation mediumstimulates sporulation. The yeast requires either magnesiumor calcium for growth and sporulation. Higher concentrationsof these ions are required for sporulation than for growth.In both cases magnesium is effective at more dilute concentrationsthan calcium. Under the conditions of the experiments, in which the yeastforms a pellicle, the sporulation rate in the pellicle far exceedsthat in the sediment. The effects of environmental factors on the sporulation wasconsidered in relation to growth. It was found that, under theconditions of poor growth in the sporulation culture, no exogenousmagnesium and calcium are required for sporulation. In suchcases, the yeast cells are inferred to have an endogenous stockof magnesium and calcium enough for the sporulation. ¹ Present address: Laboratory of Microbiology, Department ofAgriculture, Tôhoku University, Sendai. (Received May 4, 1961; )     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: II. INFLUENCE OF CARBON-NITROGEN RATIO OF CULTURE MEDIUM

1. Several factors affecting sporulation of a wild yeast, Hansenulasaturnus, especially carbon sources and the carbon-nitrogenratio of sporulation medium were studied.
2. The sporulationis stimulated at a certain definite C/N ratioof glucose medium.
3. Several carbon sources such as ethanol, acetate, lactate,glycerol,succinate, glucose, gluconate and citrate are utilizedby theorganism both for growth and sporulation.
4. The numberof spores in an ascus depends on the C/N ratio ofthe medium.An increase in the ratio stimulates the yield of2-and 3-sporedasci, especially of the former. One-spored ascibecome abundantas this ratio decreases.
5. Lysine promotes sporulation in anacetate medium, and its presencein a large amount in glucosemedium also stimulates sporulation,while a small amount isinhibitory. When lysine was employedas the sole nitrogen source,most of the asci were 1-spored.
6. It is discussed that sporulationof yeast is induced by a balanceof metabolism, rather thanby one definite "sporulation substrate".

¹ Present address: Laboratory of Microbiology, Department ofAgriculture, T{circumflex}hoku University, Sendai. (Received May 23, 1961; )     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

Effect of yeast extract and succinic acid on growth and sporulation of alternaria species

Summary Alternaria solani andA. nyctanthi, these pathogens causing leafspot disease were able to metabolize a variety of nitrogen compounds when grown on different culture media. The amount of growth varied with the nitrogen source. Peptone produced the best zonation when added in definite proportion to the yeast extract medium. Ammonium compounds were found to be moderately effective for growth but poor for sporulation. The effect of adding succinic acid in media containing ammonium sources and the role of pH in the utilization of nitrite nitrogen was investigated.The fungus gave more vegetative growth on a mixture of aminoacids than in culture media in which the same amino acids were supplied singly to study the effect produced on growth and sporulation.     

Production ofPaecilomyces Fumosoroseus conidia in submerged culture

Mexican isolates ofPaecilomyces fumosoroseus (Wize) Brown & Smith virulent to nymphs and adults ofBemisia tabaci Gennadius (Homoptera: Aleyrodidae) were screened in terms of spore production in submerged culture. Effects of light, temperature stress and yeast extract on sporulation were studied. Cycles of 12 hours light/12 hours dark increased spore production as well as an incubation for 24 hours at 37°C prior to incubation at 30°C. In absence of organic nitrogen both fungal growth and sporulation were very low. Spore production in fermentors with a culture media of a C:N ratio of 25 was doubled as compared to a media with a C:N ratio of 11. Both conidia and blastospores were produced. Production of conidia directly from blastospores through microcyclic sporulation was observed. The proportion of conidia obtained under optimal conditions was 88.8%. Submerged culture ofP. fumosoroseus seemed advantageous compared to ricefilled plastic bags production method because of shorter fermentation times, higher spore yields and substantially higher volumetric spore productivity. Results indicated that careful manipulation of nutritional and environmental conditions allowed for production of conidia during submerged growth ofP. fumosoroseus, microcyclic sporulation being induced under a set of environmental conditions including temperature stress and nutrients limitation.     

Flocculation onset in Saccharomyces cerevisiae: the role of nutrients

AIMS: To examine the role of the nutrients on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]. For cells grown in chemically defined medium (yeast nitrogen base with glucose) or in rich medium (containing yeast extract, peptone and fermentable sugars: fructose or maltose), the onset of flocculation occurred after the end of exponential respiro-fermentative phase of growth being coincident with the attainment of the lower level of carbon source in the culture medium. Cells, in exponential respiro-fermentative phase of growth, transferred to a glucose-containing medium without nitrogen source, developed a flocculent phenotype, while these carbon source starved cells, in the presence of all other nutrients that support growth, did not flocculate. In addition, cells in exponential phase of growth, under catabolite repression, when transferred to a medium containing 0.2% (w/v) of fermentable sugar (fructose or maltose) or 2% (v/v) ethanol, showed a rapid triggering of flocculation, while when incubated in 2% (v/v) glycerol did not develop a flocculent phenotype. CONCLUSIONS: The onset of flocculation occurs when a low sugar and/or nitrogen concentration is reached in culture media. The triggering of flocculation is an energetic dependent process influenced by the carbon source metabolism. The presence of external nitrogen source is not necessary for developing a flocculent phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the role of nutrients on the onset of flocculation in NewFlo phenotype yeast strains. This information might be useful to the brewing industry, in the control of yeast flocculation, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality.     

Flower-Inducing Activity of Exogenous Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Rich Medium

Flower-inducing activity of lysine was examined in Lemna paucicostata151, a weakly responsive short-day plant, cultured on nitrogen-richmedium under long-day conditions (continuous light). Lemna paucicostata151 was homogenized in a solution of lysine and the homogenatewas centrifuged. The supernatant (lysine-containing extract)was added to nitrogen-rich medium after passage through a membranefilter to give various concentrations of lysine in the medium.Flowering was induced in plants grown for six days on mediumthat contained lysine at concentrations above 0.25 µM.In plants grown on medium that contained 1 µM lysine,a significant flowering response was observed on the fourthday of culture. However, the flower-inducing activity of lysinedisappeared when the lysine-containing extract was added tothe medium and the medium was then autoclaved, suggesting thatthe active principle is unstable to autoclaving. Among derivativesof lysine tested, lysine hydroxamate had the highest flower-inducingactivity and lysyl lysine had almost same activity as that oflysine. When added to the medium without homogenization withplant material, lysine and lysyl lysine had flower-inducingactivity but lysine hydroxamate did not induce flowering. (Received April 26, 1993; Accepted November 8, 1993)     

Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeasts.

To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30 degrees C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22 degrees C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sporulation of Saccharomyces cerevisiae in the Absence of a Functional Mitochondrial Genome

The role of the mitochondrial system during sporulation of Saccharomyces cerevisiae was studied. Addition of ethidium bromide (EthBr) to cells growing in acetate medium resulted in the quantitative (>98%) conversion of the culture to the petite genotype in one generation. The cells were respiratory active (derepressed) but contained no mitochondrial deoxyribonucleic acid (mtDNA) as demonstrated by analytical ultracentrifugation in CsCl. When transferred to acetate sporulation medium, the culture sporulated. Ascus production was only slightly below that of the control culture. Synthesis of mtDNA occurred during sporulation in the control but not in the EthBr-treated culture. Mitochondrial protein synthesis was virtually eliminated in the EthBr-treated culture. Therefore, completely derepressed cells can sporulate without a functional mitochondrial genetic system. When partially repressed cells were treated with EthBr, no ascus formation was observed after transfer to sporulation medium. Control cultures underwent respiratory adaptation in sporulation medium and then sporulated. Extensive derepression of the respiratory system is thus required for sporulation, and this adaptation is dependent on a functional mitochondrial system. Our results suggest that once the cells are fully derepressed no mitochondrial genetic information has to be expressed during meiosis and ascus formation.     

The Sporulation of Penicillium notatum Westling in Submerged Liquid Culture: II. THE INITIAL SPORULATION PHASE

The initial sporulation time of Penicillium notatum Westlinggrown in shaken submerged culture inoculated with spores orvegetative mycelia is inversely proportional to the logarithmof the inoculum load, with a minimum time of 17.5 hours in theformer and 8 hours in the latter cultures. The development ofcultures is divisible into two stages. Firstly, there is a periodof vegetative growth, the duration of which depends on the inoculumload, after which the culture can be described as mature. Calciumis not required for the development of this maturity. Secondly,the cultures when mature develop phialides and spores if calciumis added to the medium. The development of phialides and thefirst spores takes 6 hours from the time of adding calcium andthis period is not influenced by the inoculum load of the culture.The medium from a mature culture promoted more rapid sporulationof vegetative mycelia placed in it than did a fresh medium,indicating the presence of a sporulation factor(s) in maturemedium. Similar activity was also demonstrated in media whichhad supported growth of any one of five other Penicillium speciesor Aspergillus niger. The nature of the sporulation factor isso far unknown.     

Optimization of growth medium for <Emphasis Type="Italic">Sporosarcina pasteurii</Emphasis> in bio-based cement pastes to mitigate delay in hydration kinetics

Microbial-induced calcium carbonate precipitation has been identified as a novel method to improve durability and remediate cracks in concrete. One way to introduce microorganisms to concrete is by replacing the mixing water with a bacterial culture in nutrient medium. In the literature, yeast extract often has been used as a carbon source for this application; however, severe retardation of hydration kinetics has been observed when yeast extract is added to cement. This study investigates the suitability of alternative carbon sources to replace yeast extract for microbial-induced calcium carbonate precipitation in cement-based materials. A combination of meat extract and sodium acetate was identified as a suitable replacement in growth medium for Sporosarcina pasteurii; this alternative growth medium reduced retardation by 75 % (as compared to yeast extract) without compromising bacterial growth, urea hydrolysis, cell zeta potential, and ability to promote calcium carbonate formation.     

Effect of yeast extract,peptone, and certain nitrogen compounds on sporulation ofSaccharomyces cerevisiae

Summary Aeration of cells for 24 hrs. previous to placing them in 0.1% sodium acetate solution diminished sporulation, but this decrease was overcome by the addition of 0.1% yeast extract to the acetate solution. Cells starved by growth on Czapek solution agar +0.03% peptone formed very few ascospores in acetate solution. The addition of yeast extract or peptone in low content to the acetate solution increased the yields. However, the cells did not form as many ascospores as well-nourished cells in acetate solution.A comparison was made of the sporulation of cells from basal presporulation medium containing, separately, 18 nitrogen sources. In general, nitrogen sources that supported growth gave cells that sporulated well. Tyrosine and tryptophan were exceptions.Cells multiplied in basal medium with the nitrogen source deleted formed no asci in 0.1% acetate solution. When nitrogen sources were added to the acetate solution, many stimulated sporulation. Yields of asci in these sporulation cultures were, however, lower than the yield obtained from well-nourished cells in 0.1% acetate solution.Based on a thesis submitted byJ. H. Tremaine in May, 1953, to McMaster University in partial fulfilment of the requirements for the degree of Master of Science.     

Nutritional requirements of Brettanomyces bruxellensis: growth and physiology in batch and chemostat cultures

The nutritional requirements of Brettanomyces bruxellensis have been investigated. Batch culture and chemostat pulse techniques were used to identify growth-limiting nutrients. The study included determination of the essential components of the culture medium and quantification of the effects of the components. Among the components tested, ammonium sulfate and yeast extract had a significant effect on glucose consumption, growth, and ethanol production. However, if the ammonium sulfate concentration is above 2 g/L, an inhibitory effect on B. bruxellensis growth is observed. The yeast extract appears to be the most important and significant component for growth. The maximum amount of synthesized biomass is proportional to the concentration of yeast extract added to the culture broth (in the tested range). Magnesium and phosphate ions are probably not essential for B. bruxellensis. These ions appear to be supplied in sufficient amounts by the yeast extract in the culture medium. Brettanomyces bruxellensis appears to have very low nutritional requirements for growth.     

球孢白僵菌继代培养中菌落局变现象及环境影响因素的研究

球孢白僵菌[Beauveria bassiana(Bals.)Vuill.]野生型多孢株以及从其上分离的单孢株在不同培养基、光照、温度和湿度条件下继代培养,结果显示:无沦是多孢株还是单孢株,在传代中均发生显著的菌落局变,并产生形态各异的分离子,多表现为菌苔瘠薄或气生菌丝徒长,肉眼可见产孢量减少;尤其是单孢株菌落局变产生的分离子往往伴随着棕黄色或红棕色色素的分泌。培养基、温度、湿度和光照等环境因素对菌落局变产生具明显诱导作用。以SDAY培养基、偏低的温湿度和全光照条件下产生局变频率较低。大批量生产中以麦麸作原料较适宜。提出了菌落局变发生的几种可能的遗传机制。     

Effect of cyclic AMP,theophylline and caffeine on the glucose repression of sporulation inSaccharomyces cerevisiae

Summary Repression of the sporulation ability ofSaccharomyces cerevisiae by glucose present in the presporulation medium was studied. Glucose lowered sporulation ability when added to the presporulation medium containing yeast extract but did not do so when added to the presporulation medium without glucose. The glucose-repressed sporulation ability was recovered by the addition of cyclic AMP, and theophylline or caffeine to the presporulation culture. Theophylline promoted the action of cyclic AMP, but caffeine did not. The effect of caffeine to reverse glucose repression was greater than that of cyclic AMP and theophylline.     

Production of type 5 capsular polysaccharide by Staphylococcus aureus grown in a semi-synthetic medium

The concentration of the type 5 capsular polysaccharide (CP) antigen of Staphylococcus aureus can be measured directly in cultures or cell suspensions by a two-step inhibition enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies. CP was synthesized during growth on a variety of carbon substrates and its production was not affected by the nature of the carbon source. High levels of yeast extract inhibited CP formation. CP was synthesized in batch culture at the same rate during exponential growth as in the post-exponential phase. Post-exponential CP production contributed at least half the final amount of CP measured. This phenomenon was observed in different culture media, although the specific yield of polysaccharide varied from one medium to another. Post-exponential CP production was observed in the pH range 6-7, but not at pH 8. Post-exponential production was strictly dependent on oxygen availability and did not occur under anaerobic conditions.     

Growth and sporulation of Entomophthora virulenta on semidefined media in liquid culture

Entomophthora virulenta was tested for growth and sporulation under various nutritional conditions in shake cultures containing combinations of dextrose and protein hydrolysates. Darkness, a temperature of 25°C, and a pH near 6.5 induced the formation of the greatest number of zygospores when the carbon and nitrogen sources were held constant. The influence of the total concentration of nutrients and of the ratio of the carbon and nitrogen sources depended on the nitrogen source used: The highest amount of spores under these conditions was produced with yeast extract as nitrogen source, a carbon/nitrogen source ratio of 2, and a 15% total nutrient concentration. The influence of the preinoculum and inoculum on sporulation is discussed.     

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.
Methods and Results:  A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased.
Conclusions:  A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process.
Significance and Impact of the Study:  The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.     

Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B.]

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.