Aerobic and Anaerobic Starvation Metabolism in Methanotrophic Bacteria

The capacity for anaerobic metabolism of endogenous and selected exogenous substrates in carbon- and energy-starved methanotrophic bacteria was examined. The methanotrophic isolate strain WP 12 survived extended starvation under anoxic conditions while metabolizing 10-fold less endogenous substrate than did parallel cultures starved under oxic conditions. During aerobic starvation, the cell biomass decreased by 25% and protein and lipids were the preferred endogenous substrates. Aerobic protein degradation (24% of total protein) took place almost exclusively during the initial 24 h of starvation. Metabolized carbon was recovered mainly as CO(inf2) during aerobic starvation. In contrast, cell biomass decreased by only 2.4% during anaerobic starvation, and metabolized carbon was recovered mainly as organic solutes in the starvation medium. During anaerobic starvation, only the concentration of intracellular low-molecular-weight compounds decreased, whereas no significant changes were measured for cellular protein, lipids, polysaccharides, and nucleic acids. Strain WP 12 was also capable of a limited anaerobic glucose metabolism in the absence of added electron acceptors. Small amounts of CO(inf2) and organic acids, including acetate, were produced from exogenous glucose under anoxic conditions. Addition of potential anaerobic electron acceptors (fumarate, nitrate, nitrite, or sulfate) to starved cultures of the methanotrophs Methylobacter albus BG8, Methylosinus trichosporium OB3b, and strain WP 12 did not stimulate anaerobic survival. However, anaerobic starvation of these bacteria generally resulted in better survival than did aerobic starvation. The results suggest that methanotrophic bacteria can enter a state of anaerobic dormancy accompanied by a severe attenuation of endogenous metabolism. In this state, maintenance requirements are presumably provided for by fermentation of certain endogenous substrates. In addition, low-level catabolism of exogenous substrates may support long-term anaerobic survival of some methanotrophic bacteria.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

Monensin has no effect on growth and metabolism ofMegasphaera elsdenii

A rumen strain ofMegasphaera elsdenii was grown on glucose and lactate in monensin-free and monensin-supplemented medium (10 mg/L). Monensin had no effect on growth rate, growth yields, metabolic pattern and composition of cells. Growth yields of dry matter and protein were higher in cultures supplied with glucose than in cultures supplied with lactate. The bacterium compensated the lower gain of energy from fermentation of lactate by rapid utilization of this substrate. Cells grown on glucose contained more saccharide and less protein than lactate-grown cells.     

Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> Si3

During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 °C), the freeze-drying survival of Lb. coryniformis Si3 was 67% (±6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 °C) did not influence freeze-drying survival, whereas mild cold- (26 °C), base- (pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 °C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 °C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.     

Lactate and Acrylate Metabolism by Megasphaera elsdenii under Batch and Steady-State Conditions

The growth of Megasphaera elsdenii on lactate with acrylate and acrylate analogues was studied under batch and steady-state conditions. Under batch conditions, lactate was converted to acetate and propionate, and acrylate was converted into propionate. Acrylate analogues 2-methyl propenoate and 3-butenoate containing a terminal double bond were similarly converted into their respective saturated acids (isobutyrate and butyrate), while crotonate and lactate analogues 3-hydroxybutyrate and (R)-2-hydroxybutyrate were not metabolized. Under carbon-limited steady-state conditions, lactate was converted to acetate and butyrate with no propionate formed. As the acrylate concentration in the feed was increased, butyrate and hydrogen formation decreased and propionate was increasingly generated, while the calculated ATP yield was unchanged. M. elsdenii metabolism differs substantially under batch and steady-state conditions. The results support the conclusion that propionate is not formed during lactate-limited steady-state growth because of the absence of this substrate to drive the formation of lactyl coenzyme A (CoA) via propionyl-CoA transferase. Acrylate and acrylate analogues are reduced under both batch and steady-state growth conditions after first being converted to thioesters via propionyl-CoA transferase. Our findings demonstrate the central role that CoA transferase activity plays in the utilization of acids by M. elsdenii and allows us to propose a modified acrylate pathway for M. elsdenii.     

Long-term nutrient starvation of continuously cultured (glucose-limited) Selenomonas ruminantium.

Selenomonas ruminantium, a strictly anaerobic ruminal bacterium, was grown at various dilution rates (D = 0.05, 0.25, and 0.35 h-1) under glucose-limited continuous culture conditions. Suspensions of washed cells prepared anaerobically in mineral buffer were subjected to nutrient starvation (24 to 36 h; 39 degrees C; N2 atmosphere). Regardless of growth rate, viability declined logarithmically, and within about 2.5 h, about 50% of the populations were nonviable. After 24 h of starvation, the numbers of viable cells appeared to be inversely related to growth rate, the highest levels occurring with the slowest grown population. Cell dry weight, carbohydrate, protein, ribonucleic acid (RNA), and deoxyribonucleic acid declined logarithmically during starvation, and the decline rates of each were generally greater with cells grown at higher D values. Both cellular carbohydrate and RNA declined substantially during the first 12 h of starvation. Most of the cellular RNA that disappeared was found in the suspending buffer as low-molecular-weight, orcinol-positive materials. During growth, S. ruminantium made a variety of fermentation acids from glucose, but during starvation, acetate was the only acid made from catabolism of cellular material. Addition of glucose or vitamins to starving cell suspensions did not decrease loss of viability, whereas a starvation in the spent culture medium resulted in a slight decrease in the rate of viability loss. Overall, the data indicate that S. ruminantium strain D has very little survival capacity under the conditions tested compared with other bacterial species that have been studied.     

Amino Acid Deamination by Ruminal <Emphasis Type="Italic">Megasphaera elsdenii</Emphasis> Strains

When ruminal fluid from a cow fed timothy hay was serially diluted (10-fold increments into anaerobic broth containing 15 mg ml⁻¹ Trypticase), the low dilutions (≤10⁻⁶) had optical densities greater than 2.0 and ammonia concentrations greater than 100 mM. The optical densities and ammonia concentrations of the 10⁻⁸ and 10⁻⁹ dilutions were very low, but large cocci were observed in the 10⁻⁸ dilution. The large cocci were isolated and identified by 16S rDNA sequencing as Megasphaera elsdenii. The freshly isolated strain (JL1) grew well on Trypticase, but less than 4% of the amino acid nitrogen in Trypticase was converted to ammonia. Optical density and ammonia production were twice as great if Casamino acids were provided, and similar results were obtained with seven other strains (B159, AW106, YT91, LC1, T81, J1, and YZ70). Specific activities of deamination (based on Casamino acids) of the eight strains ranged from 100 (strain JL1) to 325 (strain B159) nmol mg protein⁻¹ min⁻¹. None of the strains could utilize branched-chain amino acids as an energy source for growth, but specific activities of branched-chain amino acid deamination ranged from 15 to 65 nmol mg protein⁻¹ min⁻¹. All eight of the M. elsdenii strains grew well in the presence of 5 μM monensin, and only two of the strains were strongly inhibited by 20 μM monensin. On the basis of these results, it appears that M. elsdenii is deficient in peptidase activity and can utilize only a few amino acids. Some M. elsdenii strains produced ammonia and branched-chain volatile fatty acids nearly as fast as obligate amino acid-fermenting ruminal bacteria, but the extent of this production was at least fourfold lower. Because all of the strains could tolerate 5 μM monensin, it is unlikely that this feed additive would significantly inhibit M. elsdenii in vivo. Received: 12 December 2001 / Accepted: 5 February 2002     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Responses of Ruminococcus flavefaciens, a Ruminal Cellulolytic Species, to Nutrient Starvation

Ruminococcus flavefaciens strain C94, a strictly anaerobic, cellulolytic ruminal bacterial species, was grown either in batch or continuous cultures (cellobiose limited or nitrogen limited) at various dilution rates. Washed cell suspensions were incubated anaerobically at 39°C without nutrients for various times up to 24 h. The effects of starvation on direct and viable cell counts, cell composition (DNA, RNA, protein, and carbohydrate), and endogenous production of volatile fatty acids by the cell suspensions were determined. In addition, the effect of the pH of the starvation buffer on direct and viable cell counts was determined. Survival of batch-grown cells during starvation was variable, with an average time for one-half the cells to lose viability (ST₅₀) of 10.9 h. We found with continuous cultures that viable cell counts declined faster when the initial cell suspensions had been grown at faster dilution rates; this effect was more pronounced for suspensions that had been limited by cellobiose (ST₅₀ = 6.6 h at a dilution rate of 0.33 h⁻¹) than for suspensions that had been limited by nitrogen (ST₅₀ = 9.5 h at a dilution rate of 0.33 h⁻¹). With continuous cultures, viable cell counts in all cases declined faster than direct cell counts did. The rates of disappearance of specific cell components during starvation varied with the initial growth conditions, but could not be correlated with the loss of viability. Volatile fatty acid production by starving cells was very low, and acetate was the main product. Starved cells survived longer at pH 7.0 than they did at pH 5.5, and this effect of pH was greater for cellobiose-limited cells (mean ST₅₀ = 7.1 h) than for nitrogen-limited cells (mean ST₅₀ = 12 h). Although it has relatively low ST₅₀ values, R. flavefaciens has sufficient survival abilities to maintain reasonable numbers in domestic animals having maintenance or greater feed intake.     

Culture of microalgae Chlorella minutissima for biodiesel feedstock production

Microalgae are among the most promising of non‐food based biomass fuel feedstock alternatives. Algal biofuels production is challenged by limited oil content, growth rate, and economical cultivation. To develop the optimum cultivation conditions for increasing biofuels feedstock production, the effect of light source, light intensity, photoperiod, and nitrogen starvation on the growth rate, cell density, and lipid content of Chlorella minutissima were studied. The fatty acid content and composition of Chlorella minutissima were also investigated under the above conditions. Fluorescent lights were more effective than red or white light‐emitting diodes for algal growth. Increasing light intensity resulted in more rapid algal growth, while increasing the period of light also significantly increased biomass productivity. Our results showed that the lipid and triacylglycerol content were increased under N starvation conditions. Thus, a two‐phase strategy with an initial nutrient‐sufficient reactor followed by a nutrient deprivation strategy could likely balance the desire for rapid and high biomass generation (124 mg/L) with a high oil content (50%) of Chlorella minutissima to maximize the total amount of oil produced for biodiesel production. Moreover, methyl palmitate (C16:0), methyl oleate (C18:1), methyl linoleate (C18:2), and methyl linolenate (C18:3) are the major components of Chlorella minutissima derived FAME, and choice of light source, intensity, and N starvation impacted the FAME composition of Chlorella minutissima. The optimized cultivation conditions resulted in higher growth rate, cell density, and oil content, making Chlorella minutissima a potentially suitable organism for biodiesel feedstock production. Biotechnol. Bioeng. 2011;108: 2280–2287. © 2011 Wiley Periodicals, Inc.     

The role of oxygen in yeast metabolism during high cell density brewery fermentations

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.     

Cultivation ofCandida lipolytica 4-1 on hydrocarbons

The length of the carbon chain of the hydrocarbon substrate affects markedly the fatty acid composition in the cell lipids of the yeastCandida lipolytica 4-1. During cell growth onn-hexadecane dissolved in deparaffinated gas oil, direct incorporation of palmitic acid into lipids takes place. During growth onn-dodecane, on the other hand, an immediate synthesis and incorporation into oleic acid is observed. The cells contain only little lauric acid (maximum 11%). During fermentation of then-alkanes dissolved in deparaffinated gas oil which contains a mixture of isoalkanes, alkylated aromatic and cyclic hydrocarbons, free fatty acids accumulate in the oil phase. The fatty acid composition in the oil differs markedly according to the growth stage of cells. At the beginning of the logarithmic phase of growth, the fatty acid composition in the oil phase reflects the acid composition in the cell lipids, toward the end of cell growth, the cooxidation products of the isoalkanes accumulate. The aqueous phase contains lower fatty acids and cooxidation products of isoalkanes and of alkylated aromatic and alicyclic hydrocarbons. Part III. Oxidation and Utilization of Individual Pure Hydrocarbons—seeFolia Microbiol. 14,334 (1969).     

Effect of Growth Rate and Starvation-Survival on the Viability and Stability of a Psychrophilic Marine Bacterium

Cell populations of the marine bacterium ANT-300, from either batch or continuous culture with dilution rates ranging from D = 0.015 h⁻¹ to D = 0.200 h⁻¹, were monitored for viability, direct counts, and optical density for 98 days under starvation conditions. Three stages of starvation survival were observed for each of the cell populations. Although direct counts remained at 2 × 10⁷ to 3 × 10⁷ cells ml⁻¹ throughout the starvation period, large fluctuations occurred in cell viability during stage 1 (0 to 14 days) of starvation survival. Stage 2 (14 to 70 days) involved an overall decrease in viability for each of the cell populations; the rate of viability loss was dependent upon the growth rate. Cell viability stabilized at approximately 0.3% of the direct count in stage 3 (70 to 98 days). Long-term starvation corresponded to the prolongation of stage 3 starvation survival. Cell volumes for each of the cell populations decreased with the length of the starvation period. However, the cell volume of starved cells was also dependent more on growth rate than on the length of the time starved. We hypothesize that the cell population with the slowest growth rate is most closely representative of cells found in the oligotrophic marine environment.     

Glucose deprivation induced upregulation of phosphoenolpyruvate carboxykinase modulates virulence in Leishmania donovani

Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP⁺ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.     

Cell wall and organelle modifications during nitrogen starvation in Nannochloropsis oceanica F&M-M24

Nannochloropsis oceanica F&M-M24 is able to increase its lipid content during nitrogen starvation to more than 50% of the total biomass. We investigated the ultrastructural changes and the variation in the content of main cell biomolecules that accompany the final phase of lipid accumulation. Nitrogen starvation induced a first phase of thylakoid disruption followed by chloroplast macroautophagy and formation of lipid droplets. During this phase, the total amount of proteins decreased by one-third, while carbohydrates decreased by 12–13%, suggesting that lipid droplets were formed by remodelling of chloroplast membranes and synthesis of fatty acids from carbohydrates and amino acids. The change in mitochondrial ultrastructure suggests also that these organelles were involved in the process. The cell wall increased its thickness and changed its structure during starvation, indicating that a disruption process could be partially affected by the increase in wall thickness for biomolecules recovery from starved cells. The wall thickness in strain F&M-M24 was much lower than that observed in other strains of N. oceanica, showing a possible advantage of this strain for the purpose of biomolecules extraction. The modifications following starvation were interpreted as a response to reduction of availability of a key nutrient (nitrogen). The result is a prolonged survival in quiescence until an improvement of the environmental conditions (nutrient availability) allows the rebuilding of the photosynthetic apparatus and the full recovery of cell functions.

     

Studies on the endogenous metabolism of Escherichia coli

1. The endogenous metabolism of Escherichia coli has been studied by examining changes in cellular composition and of the suspending fluid during starvation of washed suspensions of the organism, in water or in phosphate buffer, at 37° under aerobic and anaerobic conditions. 2. When E. coli is grown in glucose–ammonium salts media the cells contain glycogen, which is utilized rapidly during subsequent starvation of the cells. 3. Ammonia is released by starved cells only after a lag period, which corresponds to the time taken for the cellular glycogen to be almost completely utilized. 4. If cells are grown under conditions that permit incorporation of ¹⁴C into protein but not into glycogen and are then starved, release of ¹⁴CO₂ commences immediately and continues at a linear rate throughout the period of glycogen utilization; it is concluded that the presence of glycogen in the cell prevents the net degradation of nitrogenous materials but does not suppress protein turnover. 5. RNA is degraded by the cells immediately they are starved, ribose is oxidized and ultraviolet-absorbing materials are released to the suspending medium. 6. There is no significant utilization of lipid during the starvation of glucose-grown E. coli. 7. There is no loss of viability during the initial 12hr. period of starvation under either aerobic or anaerobic conditions, but thereafter the cells die more rapidly under conditions of anaerobiosis. 8. These results are discussed in relation to the known patterns of endogenous metabolism and survival of other bacteria.     

Nutritional Interdependence Among Rumen Bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus

Nutritional interdependence among three representatives of rumen bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus, was studied with a basal medium consisting of minerals, vitamins, cysteine hydrochloride, and NH₄⁺. B. amylophilus grew well in the basal medium supplemented with starch and produced branched-chain amino acids after growth ceased. When cocultured with B. amylophilus in the basal medium supplemented with starch and glucose, amino acid-dependent M. elsdenii produced an appreciable amount of branched-chain fatty acids, which are essential growth factors for cellulolytic R. albus. A small addition of starch (0.1 to 0.3%) to the basal medium containing glucose and cellobiose brought about successive growth of the three species in the order of B. amylophilus, M. elsdenii, and R. albus, and successive growth was substantiated by the formation of branched-chain amino acids and fatty acids in the culture. Supplementation with 0.5% starch, however, failed to support the growth of R. albus. On the basis of these results, the effects of supplementary starch or branched-chain fatty acids on cellulose digestion in the rumen was discussed.     

Effect of starvation length upon microbial activity in a biomass recycle reactor

The kinetics of substrate degradation and bacterial growth was determined in a microbial community from a biomass recycle reactor that had been deprived of substrate feed for 0–32 days. Starvation caused changes in bacterial numbers, community composition, and physiological state. Substrate starvation for less than 1 day resulted in modest (less than threefold) changes in endogenous respiration rate, ATP content, and biomass level. During a starvation period of 32 days, there were substantial changes in microbial community composition, as assessed by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR amplicons of a portion of the 16S rDNA or by phospholipid fatty acid (PLFA) analysis. When the starved communities were stimulated with organic nutrients, the growth kinetics was a function of the length of the starvation period. For starvation periods of 2–8 days prior to nutrient addition, there was a phase of suboptimal exponential growth (S-phase) in which the exponential growth rate was about 30% of the ultimate unrestricted growth rate. S-phase lasted for 2–8 h and then unrestricted growth occurred at rates of 0.3–0.4 h⁻¹. At starvation times of 12 and 20 days, a lag phase preceded S-phase and the unrestricted growth phase. Received 04 January 2002/ Accepted in revised form 08 August 2002     

Adenosine triphosphate pool levels and endogenous metabolism in Arthrobacter crystallopoietes during growth and starvation

The adenosine triphosphate (ATP) content of Arthrobactery crystallopoietes was measured during growth, starvation and recovery from starvation. During exponential growth of the cells as spheres in a glucose salts medium, the level of ATP per cell remained constant at 8.0×10^-10 g/cell. Morphogenesis to rodshaped cells and an increased growth rate following addition of casein hydrolysate was accompanied by an almost two-fold increase in the ATP level. As division of the rod-shaped cells proceeded, the level of ATP declined. After growing as rods for 12–14 h the cells underwent fragmentation to spheres during which time the ATP level again increased to the original value of 8.0×10^-10 g/cell. As the spherical cells resumed growth on the residual glucose, their ATP content declined for a short period and then remained relatively constant. During starvation of sphere or rod-shaped cells for one week, the ATP level declined by approximately 70% during the first 40–50 h and then remained constant. The endogenous metabolism rate of spherical cells declined during the first 10–20 h of starvation and then remained constant at approximately 0.02% of the cell carbon being utilized per h. Addition of glucose to spherical cells which had been starved for one week increased both the ATP content per cell and their rate of endogenous metabolism. The ATP content fluctuated and then remained at a level higher than maintained during starvation while endogenous metabolism quickly declined.Non-Standard Abbreviations ATP adenosine triphosphate - GS glucose mineral salts - HC casein hydrolysate - PVP polyvinylpyrrolidone - DMSO dimethylsulfoxide - MOPS morpholinopropane sulfonic acid - EDTA ethylene diaminetetraacetic acid     

Supercritical fluid extraction of lipids from the heterotrophic microalga Crypthecodinium cohnii

Microalgae biomass can be a feasible source of ω‐3 fatty acids due to its stable and reliable composition. In the present study, the Crypthecodinium cohnii growth and docosahexaenoic acid (DHA, 22:6ω3) production in a 100 L glucose‐fed batch fermentation was evaluated. The lipid compounds were extracted by supercritical carbon dioxide (SC‐CO₂) from C. cohnii CCMP 316 biomas, was and their fatty acid composition was analysed. Supercritical fluid extraction runs were performed at temperatures of 313 and 323 K and pressures of 20.0, 25.0 and 30.0 MPa. The optimum extraction conditions were found to be 30.0 MPa and 323 K. Under those conditions, almost 50% of the total oil contained in the raw material was extracted after 3 h and the DHA composition attained 72% w/w of total fatty acids. The high DHA percentage of total fatty acids obtained by SC‐CO₂ suggested that this extraction method may be suitable for the production of C. cohnii value added products directed towards pharmaceutical purposes. Furthermore, the fatty acid composition of the remaining lipid fraction from the residual biomass with lower content in polyunsaturated fatty acids could be adequate for further uses as feedstock for biodiesel, contributing to the economy of the overall process suggesting an integrated biorefinery approach.