Influence of cultivation conditions on lipid production by Cryptococcus albidus

Summary Cryptococcus albidus var. albidus CBS 4517 was able to accumulate lipid under nitrogen-limited as well as excess-nitrogen conditions. The highest lipid-producting capacity was, however, observed in nitrogen-limited cultivations. In nitrogen-limited batch cultures, a lipid content of 34% (w/w) in biomass and a maximum specific lipid productivity of 37 mg lipid/g lipid-free biomass·h, was determined. The yield of lipid from glucose was about 0.15 g/g in nitrogen-limited and 0.11 g/g in excess-nitrogen cultures.In a nitrogen-limited fed-batch culture, 12.4 g/l lipid was produced at 90 h of cultivation and the cells contained 46.3% (w/w) lipid.Higher lipid yield and cellular lipid content were observed when inorganic nitrogen sources were used compared with organic. The choice of carbon source was seen to influence growth as well as lipid production and the highest yields of lipid were obtained when glucose, maltose or mannitol was used.A cultivation temperature of 20°C provided the highest lipid productivity compared to 25°C and 30°C. Addition of citrate to the growth medium was seen to have a stimulating effect on the specific lipid productivity.     

Nature and timing of some sporulation-specific protein changes in Saccharomyces cerevisiae.

During meiosis and spore formulation in Saccharomyces cerevisiae, changes that occur in a/alpha diploids, but not in isogenic nonsporulating a/a diploids, have been detected in cellular polypeptides. These were found by the technique of prelabeling growing cells with 35SO4(2-) and suspending them in sulfur-free sporulation medium. Under the conditions used, about 400 polypeptides were detected by two-dimensional gel electrophoresis, and 45 were altered during sporulation; of these, 21 changes were specific to a/alpha strains. These alterations were mainly due to the appearance of new polypeptides or to marked increases in the concentrations of a few polypeptides produced during vegetative growth. They could have been due either to modifications of existing polypeptides present in growing cells or to de novo synthesis of new gene products. They occurred at characteristic times during sporulation; whereas the majority of changes took place early (within the first 6 h in sporulation conditions), there were several changes characterizing the later stages of sporulation. Ten of the 35SO4(2-)-labeled polypeptides were also labeled with 32P in the presence of [32P]orthophosphate; of these, three were previously found to be sporulation specific. One of these was phosphorylated at all stages of sporulation and was labeled when [32P]orthophosphate was added either during growth of the culture of 1 h after transfer to sporulation medium. Another was labeled in the same way by adding 32P at either time, so that by 7 h in sporulation medium it was phosphorylated, but was dephosphorylated by 24 h. The third sporulation-specific peptide was labeled in extracts prepared at 7 h in sporulation medium (but not at 24 h) when [32P]-orthophosphate was added during presporulation growth, but not when [32P]-orthophosphate was added 1 h after transfer of the culture to sporulation medium. This polypeptide appeared early during sporulation; it is probably phosphorylated as it appears and is dephosphorylated at some time between 7 h and 24 h of sporulation.     

Effect of actinomycin D on viability,sporulation and nucleotide pool ofBacillus megaterium

A transient 7-fold rise of ppGpp concentration, 2-3-fold increase of pppGpp concentration and 50 % drop of the concentration of GTP inBacillus megaterium cells immediately after their transfer to the sporulation medium were observed. Actinomycin D, in concentrations inhibiting RNA synthesis by 95%, blocked the rise of the (p)ppGpp pool and caused an instant several-fold increase of the GTP level. When the cells were exposed to actinomycin D in the sporulation medium for a 1-h period (time 0–1 h, 1–2 h or 2.20–3.20-h), they were able to form colonies on nutrient agar after being kept, in addition for 1–2 h in the sporulation medium free of the antibiotic. The ability of sporulation was, however, markedly limited. The share of cells that could sporulate increased when the irreversible sporulation phase was reached.     

Timing of mitochondrial DNA synthesis during meiosis in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) synthesis was examined during meiosis in Saccharomyces cerevisiae using an aneuploid strain disomic (n + 1) for chromosome III. The aneuploid has the advantage over true diploid strains in that it completes early meiotic events, including premeiotic chromosome replication, but does not form mature ascospores. Thus, differential extraction problems, resulting from the simultaneous presence of both unsporulated cells and spores in the population, are eliminated. The kinetic of mtDNA synthesis was monitored by determining the actual mtDNA content of cells following analytical CsCl centrifugation of cell extracts. MtDNA synthesis started soon after the cells were placed in sporulation medium and continued at an approximately constant rate until 24 h, resulting in slightly more than a doubling of the mitochondrial DNA content per cell. [¹⁴C]uracil was incorporated into mtDNA during the entire developmental period. Extensive preferential labeling of mtDNA occurred between 24 and 50 h, when no net DNA synthesis was observed.     

Induction of sporulation in food-spoilage yeasts

Four yeasts, Hansenula anomala, Kluyveromyces fragilis, Lodderomyces elongisporus and Saccharomyces cerevisiae, were cultured in two presporulation media at 30 ° C. Media consisted of yeast extract — peptone — acetate and yeast extract — peptone — dextrose broths. Except for K. fragilis, the test yeasts reached a high degree of sporulation when transferred to acetate- and ethanol-supplemented sporulation media. The percentage of S. cerevisiae cells forming asci was as high as 79% after 24 h incubation. H. anomala and L. elongisporus sporulated more rapidly in ethanol- compared to acetate-containing medium. Within test parameters, the concentration of acetate or ethanol, _pH, and incubation temperature (25 ° C and 30 ° C) did not substantially influence the extent of sporulation.     

Alterations in L fibroblast lipid metabolism and morphology during choline deprivation.

The growth of L fibroblasts in suspension culture after transfer directly to choline-free medium without a rinse was reduced to zero after 72 h. Monolayer cultures similarly treated multiplied at control rates for 96 h; one rinse of the latter prior to incubation in choline-free medium reduced growth at 48 h to 75% of control cells. A decrease in the size of cells in these rinsed monolayer cultures was apparent within 12 h, and preceded any changes in cell lipid composition; further decreases in cell size were observed at 24 and 48 h. After 24 h in choline-free medium the percent phosphatidyl choline had decreased only slightly and even at 48 h remained at 72 % of the value of control cells. At this time, however, there was a 50% decrease in the total phospholipid (PL) content of the cells, and a coincident 5–10-fold increase in the amount of triglyceride. There was no adaptive increase in the other principal PL classes. Changes in the ultrastructure of mitochondria were observed as early as 24 h; after 48 h without choline there was a decrease in the relative mitochondrial volume to 50% of control values. Alterations in the adhesive properties of choline-deprived cells may be related to altered lipid content or composition of the cell surface.     

Effect of oxygen on growth,sporulation, and mosquito larval toxin formation byBacillus sphaericus 1593

The effect of oxygen on growth, sporulation, and mosquito larval toxin synthesis byBacillus sphaericus 1593 grown in a small fermentor was investigated. With air as the source of oxygen, about one-half of the cells sporulated and 1022 units of toxicity/mg of cell dry weight were formed. A shift to 100% oxygen in the gas stream maintained a higher level of dissolved oxygen in the medium, but this produced a late block in sporulation; however, toxin synthesis was normal. The mechanism of oxygen inhibition of sporulation byB. sphaericus is unknown, but the same effect was observed inB. subtilis 168. Stopping of the air flow at 8 h, after forespores were completed in about one-half the cells, inhibited the completion of sporulation, but did not decrease toxin production.     

Carbon concentration and oxygen availability affect lipid and carotenoid production by carob pulp syrup‐grown Rhodosporidium toruloides NCYC 921

The simultaneous effect of oxygen availability and carbon source concentration on yeast lipid and carotenoid production has never been studied before. In this work, a Doehlert distribution design was used to study the simultaneous effect of carbon concentration and oxygen availability on Rhodosporidium toruloides NCYC 921 carotenoid and lipid production. A cheap industrial byproduct was used as carbon source (carob pulp syrup). A total sugar concentration of 106.3 g/L and a medium volume of 0.120 L induced the highest total carotenoid and total fatty acid productivities (4.60 μg/Lh and 0.029 g/Lh, respectively). Flow cytometry was used to assess yeast stress response under different cultivation conditions. The highest proportion of cells with permeabilised membrane (>20%) was induced when the cultivations were carried out at the highest sugar concentration studied (130.0 g/L) or when the culture reached the minimum final medium pH (4.60). The results showed that the total sugar concentration had a positive influence on the yeast biomass and carotenoid content, while the oxygen availability had little influence on the biomass concentration, but had a slight positive influence on the carotenoid content. Regarding the fatty acids, the two factors had a negative impact on the synthesis of these compounds.     

Lipid Synthesis During Sporulation of Saccharomyces cerevisiae

Lipid synthesis was studied in both sporulating (diploid) and nonsporulating (haploid) cells of Saccharomyces cerevisiae. Two phases of lipid synthesis occur in diploid cells transferred to sporulation medium. Phase I, which occurs during the first 12 h of exposure to sporulation medium, was also observed in the haploid strains. Phase II, occurring from the 20th to the 25th h, coincided with the appearance of mature asci and was observed only in the diploid cells. The majority of phospholipid synthesis took place during period I, whereas neutral lipid synthesis occurred during both periods. Phospholipid synthesis was virtually identical in both type and quantity in the sporulating and nonsporulating strains.     

Degradation and replenishment of the labile protein fraction in non-growing cells of the asporogenic mutant ofBacillus megaterium

Kinetics of degradation of labelled proteins was followed in two asporogenic mutants ofBacillus megaterium during incubation in a sporulation medium. Both the mutant producing exocellular protease (KM 1prn ⁺) and the mutant not producing the enzyme (KM 12prn) were found to contain a labile protein fraction, whose proportion decreases with prolonged time of labelling and whose half-life is about 1 h. Most proteins were relatively stable and were degraded at a rate of 1 %/h and 2 %/h in strains KM 1 and KM 12, respectively (half life 70–80 h and 35–40 h in strains KM 1 and KM 12, respectively). The intracellular proteolytic activity of the KM 12 mutant remains practically the same during incubation in the sporulation medium or slowly increases. The labile protein fraction practically disappears from the cells after a 3.5-h incubation. When such a culture is then subjected to a shift-up and transferred again to the sporulation medium, the rate of protein turnover temporarily increases. The temporary increase of the turnover rate is caused by a partial replenishment of the labile protein fraction rather than by an accelerated degradation of the relatively stable fraction. The intracellular proteolytic activity does not increase under these conditions. The wild sporogenic strain ofB. megaterium also contains the labile protein fraction. Its half protein life is 1 h or less. However, the second protein fraction is degraded much more rapidly than in the asporogenic mutants and its half life is 6–7 h.     

Integration of biological waste conversion and wastewater treatment plants by microalgae cultivation

In this study, growth performance and lipid content of two microalgae species Neochloris oleoabundans and Chlorella vulgaris are monitored by using three different types of sludge waste feedstocks obtained from the water treatment plants located in Bedonia, Borgotaro and Fornovo (Montagna2000 Spa, Province of Parma, Italy). The sludge waste is optimized in order to achieve microalgal growth media and dispose of the sewage sludge produced at the wastewater treatment facilities. Both photoautotrophic and heterotrophic growth conditions are applied to the microalgal cultivations. The growth parameters of microalgae strains such as cell concentration, growth rate, optical density, cell biovolume, photosynthetic pigments and lipid contents are monitored. The amounts of total dried lipid biomass, obtained by the biological conversion of the wet sludge waste, are determined. Lipid production of microalgal cells grown in the medium optimized from sludge waste from the Fornovo site provides the highest amount of microalgal lipid content for N. oleoabundans and C. vulgaris photoautotrophic cultivations, while sludge waste from the Bedonia site provides for N. oleoabundans heterotrophic cultivation.     

Metabolic rates during sporulation of Saccharomyces cerevisiae on acetate

We have quantified yeast carbon and oxygen consumption fluxes and estimated anabolic fluxes through glyoxylate and gluconeogenic pathways under various conditions of sporulation on acetate. The percentage of sporulation reached a maximum of 55% to 60% after 48 h in sporulation medium, for cells harvested from logarithmic growth in acetate minimal medium. When cells were harvested in the stationary phase of growth before transfer to sporulation medium, the maximum percentage of sporulation decreased to 40% along with the occurrence of meiosis as could be judged by counting of bi- and tetra-nucleated cells. In both experiments, the rates of acetate and oxygen consumption decreased as a function of time when exposed to sporulation medium. Apparently, the decrease of metabolic rates was not due to alkalinization. By systematically varying the cell concentration in sporulation medium from 1.4×10⁷ to 20×10⁷ cell ml^-1, the percentage of sporulating cells was found to decrease in parallel with the rate of acetate consumption. When the sporulation efficiency attained under the different experimental conditions was plotted as a function of the rate of acetate consumption, a linear correlation was found. Anabolic fluxes estimation revealed a decrease of the rate through gluconeogenic and glyoxylate pathways occurring during sporulation progression. The pattern of metabolic fluxes progressively evolved toward a predominance of more oxidative catabolic fluxes than those exhibited under growth conditions. The results obtained are discussed in terms of a characteristic pattern of metabolic fluxes and energetics, associated to the development of yeast sporulation.Abbreviations DAPI 4,6-diamidino-2-phenylindole - dw dry weight - OD₅₄₀ optical density at 540 nm - SEM standard error of the mean - RQ respiratory quotient     

Characteristics of plasmid stability in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer

The stability of plasmids pC194 and pUB110 was investigated in an antifungal antibiotic iturin producer, Bacillus subtilis NB22. Although plasmid pC194 was maintained stably over a hundred generations in five successive cultivations in iturin-production no. 3 medium, significant curing was observed when the cultivation was prolonged for 10 d in the same medium. When the transformant of pC194 was cultivated in Schaeffer's sporulation medium, drastic curing took place in accordance with the occurrence of sporulation, even in the presence of an antibiotic for selective pressure. In the case of pUB110, in sharp contrast to the result with pC194, high stability was observed in the sporulation medium, but significant curing was observed during prolonged incubation in no. 3 medium.     

Effect of culture conditions on growth and sporulation ofBacillus thuringiensis subsp.israelensis and development of media for production of the protein crystal endotoxin

Summary The influence of medium composition on the inoculum and production stages of theBacillus thuringiensis subsp.israelensis bioinsecticide fermentation was investigated. Media which inhibited sporulation were selected for inoculum development stages. Bioinsecticide production media were designed to produce high cell counts and >90% sporulation in a 48h fermentation. Maximum insecticidal activity occurred at the point of maximum bacterial cell lysis/spore release. A process involving two inoculum stages and a 48h production stage in a 40 l fermenter yielded a viable cell count of 6.5 x 10⁹/ml with greater than 95% sporulation. Good correlation existed between spore counts and bioinsecticide activity.     

Turnover kinetics of proteins labeled in different sporulation phases ofBacillus megaterium

Bacillus megaterium was labeled by 10-min pulses of¹⁴C-leucine at the end of the growth phase or at 1, 3.5 and 5 h after transfer to a sporulation medium. Proteins labeled during growth or reversible sporulation phase were degraded in two-phase kinetics,i.e. a decreasing degradation rate was followed by its substantial increase. Proteins labeled during the irreversible sporulation phase were degraded at a continuously decreasing degradation rate only. However, when the amount of degraded proteins was expressed as a portion of proteins degradable during the whole sporulation cycle, the degradation was rapid and followed similar kinetics irrespective of the time of labeling. The degradation constants fluctuated in this case between 0.207/h and 0.275/h. The protein fraction insensitive to turnover increased with the time of incubation in the sporulation medium in parallel to the amount of proteins appearing in spores.     

Turnover of proteins in asporogenicBacillus megaterium. Evidence for a gradual decrease of the turnover rate

The rate of protein turnover in asporogenicBacillus megaterium decreases continuously during incubation in a sporulation medium. The capability of equilibration of external amino acids with amino acids in the metabolic pool of non-growing cells was retained for at least 5 h. Leucine, while repressing the synthesis of the exocellular protease, does not significantly influence the course of protein degradationin vivo. Transfer of non-growing cells after 4 h to a fresh sporulation medium does not influence the rate of protein degradation. The gradual decrease of the rate of protein turnover in non-growing cells of the asporogenic variant is thus not an artifact caused by a decreased uptake of amino acids by cells or by conditions under which the protein turnover is determined.     

Combined effect of growth medium,age of cells and phase of sporulation on heat resistance and recovery of Hansenula anomala

Effects of two growth media, age of cells and phase of sporulation on heat resistance of Hansenula anomala were determined. Cells were grown on two solid media, McClary's acetate and V8 juice agars, at 21 ° C for 16 days. Heat resistance of cells was determined in 0.06 M potassium phosphate buffer at 48 ° C. Heat-stressed cells were plated on four recovery media: yeast extract-malt extract-peptone-glucose (YMPG), pH 7.0; YMPG, pH 3.5; YMPG containing 6% NaCl, pH 7.0; and YMPG containing 20% sucrose, pH 7.0. The composition of sporulation medium influenced the extent of sporulation and the relative heat resistance of sporulating cells. One-day-old cells were the most sensitive to heat. The heat resistance of cells was generally increased as the incubation time was extended to 16 days. Heat treatment caused a greater increase in sensitivity to NaCl than to sucrose or acid pH in recovery media. Young cells were more sensitive to NaCl than were older cells.     

Microbial interactions: Population and sporulation studies ofBacillus sphaericus grown in association withErwinia atroseptica

A study was made of a phenomenon, previously reported, in whichBacillus sphaericus failed to sporulate in the usual peptone media, but would sporulate in these media when grown in association withErwinia atroseptica.Pure culture studies withB. sphaericus indicated that the stimulus driving the cells toward further vegetative growth and the resulting failure of the cells to sporulate was associated with the peptide fraction of peptone media; inhibition of sporulation could be reversed by reduction of the peptone level of the medium or by replacement of the peptone with known amino acids, with known amino acids and short-chain peptides, or with complete hydrolysates of casein.Population and sporulation studies were performed onB. sphaericus cultured inE. atroseptica spent medium and on mixed cultures of the two organisms. A variety of population and sporulation responses were obtained through alteration of the chemical and physical nature of the media byE. atroseptica, cultured alone or in mixed culture withB. sphaericus.It is suggested that removal of pro-vegetative peptides byE. atroseptica is responsible for the enhancement of sporulation observed inB. sphaericus in peptone media.From a thesis submitted to the Graduate School of the University of Maryland, by the senior author, in partial fulfillment of the requirements for the Ph. D. degree.     

Correlation among turnover of nucleic acids,ribonuclease activity and sporulation ability of Saccharomyces cerevisiae

The turnover of nucleic acids and changes in ribonuclease activity during sporulation of Saccharomyces cerevisiae were studied. In the sporulating strains, 37–58% of vegetatively synthesized RNA were degraded during the sporulation process. The degree of degradation of vegetative RNA was proportional to the sporulation ability. In the non-sporulating strains, the degradation of vegetative RNA was less than 28% in the sporulation medium. Accompanied by the degradation of vegetative RNA, a ribonuclease activity increased several times during sporulation. We have found a close relation among the sporulation rate, the degree of the degradation of vegetative RNA and the increase in ribonuclease activity in the sporulation medium, using cells of which sporulation ability was repressed by changing the age or carbon source in various degrees.