Dynamics of growth in batch and continuous cultures of Saccharomyces cerevisiae during shifts from aerobiosis to anaerobiosis and reverse

Summary Saccharomyces cerevisiae H 1022 was cultivated in batch and continuous culture on a glucose substrate. The yeast was subjected to a sudden change from aerobic to anaerobic growth conditions by switching the inlet-gas stream from air to dinitrogen. The dynamics of growth and product formation during the periods of adaptation were studied. A structured growth model based on Monod-Blackman-kinetics was applied to simulate these shift-experiments. The immediate switch of the yeast to maximum anaerobic growth and ethanol production predicted by this model was confirmed very well in the chemostat-experiments. However, a slow adaptation was evident for the switch from anaerobiosis back to aerobiosis.Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig-Stöckheim, Federal Republic of Germany     

Involvement of Endogenous Abscisic Acid in Onset and Release of Helianthus annuus Embryo Dormancy

Mature seeds of Helianthus annuus L. exhibit dormancy that is eliminated during storage in dry conditions. In vitro culture of immature embryos isolated at different times after anthesis showed that the youngest embryos are able to germinate, but within the third week after pollination, dormancy progressively affected most of the embryos. A radioimmunoassay showed that the endogenous abscisic acid (ABA) level, which increased sharply in the first half of the development period, fell at precisely the moment when embryo dormancy became established. An application of fluridone, before the increase of ABA level, prevented both ABA synthesis and development of embryo dormancy. Applied later, after the rise of the ABA level, fluridone could not prevent embryo dormancy development. Dormancy thus appears to be dependent on ABA synthesis but not concomitant with its accumulation; it must therefore be induced by ABA during maturation. Furthermore, a preincubation in water allowed dormant embryos to germinate. This acquisition of germinability could not be directly related to a leaching of free ABA. Possible effects of this treatment are discussed.     

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.     

Conditions of induction of secondary dormancy in apple after breaking of primary dormancy by anaerobiosis and low temperature

Dormant embryos of Pyrus malus L. cv. Golden delicious, isolated from the fruits at harvest time or after a few months storage at 10 to 15°C, were kept under anaerobic conditions in order to eliminate primary dormancy. Germination tests were then carried out at different temperatures, using three modes of culture depending on the nature of the contact between the embryo and the medium. In CM the distal part of the two cotyledons was immersed in the medium. In RM only the embryonic axis was immersed. In C/2M the embryo was placed flat on the medium, the radicle and the external surface of one cotyledon being in contact with it.
Results showed that primary dormancy was released progressively depending on the duration of the anaerobic treatment. After a treatment of 11 or 13 days the last symptoms of primary dormancy were only apparent when germination tests were carried out at high temperatures (26–30°C) or in CM mode of culture.
When the embryos were kept at 4°C during 3 months inside the fruits, subsequent germination was inhibited at high temperature and in CM mode of culture. When the embryos were kept under anaerobic conditions (7 days) after the chilling treatmem inside the fruits, germination was no longer inhibited. It is concluded that the inhibition of germination at high temperature and in CM mode of culture is due to the persistence of traces of primary dormancy. Therefore, these conditions do not seem to induce secondary dormancy in apple embryos.
After elimination of primary dormancy by anaerobiosis. only application of (±) abscisic acid (3.8 and 19 μM) inhibited germination. These results support the idea that ABA is an important factor in the induction of dormancy. However, the question remains whether this secondary embryo dormancy has the same characteristics as the original primary dormancy.     

Effects of Chilling and ABA on [H]Gibberellin A(4) Metabolism in Somatic Embryos of Grape (Vitis vinifera L. x V. rupestris Scheele)

Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. × V. rupestris Scheele). We have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with [³H]GA₄ (of high specific activity, 4.81 × 10¹⁰ becquerel per millimole) for 48 hours at 26°C. Chilling had little effect on the total amount of free [³H]GA-like metabolites formed during incubation at 26°C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble [³H] metabolites formed at 26°C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogenous GA precursors (e.g., GA₁₂ aldehyde-, kaurene-, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with [³H]GA₄ treatment at 26°C, reduced the uptake of [³H] GA₄ but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26°C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water-soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs).     

Changes in Abscisic Acid Biosynthesis and Catabolism during Dormancy Breaking in Fagus sylvatica Embryo

At harvest, embryos of Fagus sylvatica are dormant. A cold pretreatment without medium at 30% moisture content allowed them to germinate. A comparison of the abscisic acid (ABA) content before and after the pretreatment has no significant relevance since dormancy is expressed during the culture at 23°C. During this culture, both de novo biosynthesis and conjugate hydrolysis contributed to maintain a high level of ABA in the dormant axis. The level of conjugates and the rate of hydrolysis were not modified substantially by the cold pretreatment. In contrast, the dormancy release was associated with a strong decrease in the capacity for ABA synthesis. Moreover, feeding (+)-[³H]ABA to untreated and pretreated embryos proved that the cold treatment also induced a hastening of ABA catabolism. Received August 15, 1996; accepted December 6, 1996     

Absorption, Distribution, Metabolism and Leaching of [14C] ABA during Culture of Apple Embryos

Barthe, Ph. and Bulard, C. 1987. Absorption, distribution, metabolismand leaching of [¹⁴C] ABA during culture of apple embryos.—J.exp. Bot. 38: 1002–1011. It has been known for some time that dormant embryos, laid flaton damp filter-paper one cotyledon only being in direct contactwith it (C/2M mode of culture), exhibit unequal growth and greeningof their cotyledons. The aim of this work was to investigatewhether this particular mode of culture led to detectable differencesbetween the two cotyledons in the distribution and metabolismof [¹⁴C] abscisic acid (ABA). Two different approaches wereused, the material in both cases being dormant embryos of Pyrusmalus L. cv. Golden Delicious cultured at 23°C in darkness.In a first experiment, the embryos were cultured directly inthe C/2M mode and in the presence of 10^–2 mol m^–3[¹⁴C] ABA. In these conditions marked differences in the distributionof radioactivity between the lower (LC) and upper (UC) cotyledonappear after 24 h of culture. After 5 d, amounts of total radioactivitywere four times higher in LC than in UC, and the level of ABAwas three times higher. Metabolism was extremely active in LC,since certain metabolites were found in relative percentages(percentages with respect to the total radioactivity of thecotyledon) equivalent to (esters, glucosides), or even higher(dihydrophaseic acid, DPA) than those found in UC. It is suggestedthat this high level of metabolism in LC limits the availabilityof the transportable molecule, that is to say ABA. In a secondexperiment double culturing was carried out. The embryos werefirst cultured in the presence of 10^–2 mol m^–3 [¹⁴C]ABA in conditions which ensured equal distribution of ABA andits metabolites between the two cotyledons. After 5 d they weretransferred to an ABA-free medium and cultured in the C/2M mode.After 2 d and 5 d of culture, dissymmetry between the two cotyledonswas again noted; UC this time containing greater amounts ofradioactivity than LC. The differences, however, were less markedthan in the first experiment. This dissymmetry is due to leachingof a certain amount of radioactivity from LC, which is onlypartially compensated for by migration from UC. Key words: ¹⁴C-ABA distribution, leaching and metabolism, embryo culture, embryo dormancy     

Oxygen taxis and proton motive force in Salmonella typhimurium

The aerotactic response of Salmonella typhimurium SL3730 has been quantitatively correlated with a change in the proton motive force (delta p) as measured by a flow-dialysis technique. At pH 7.5, the membrane potential (delta psi) in S. typhimurium changed from -162 +/- 13 to -111 +/- 15 mV when cells grown aerobically were made anaerobic, and it returned to the original value when the cells were returned to aerobiosis. The delta pH across the membrane was zero. At pH 5.5, delta psi was -70 mV in aerobiosis and -20 mV in anaerobiosis, and delta pH was -118 and -56 mV for aerobic and anaerobic cells, respectively. A decrease in delta p resulted in increased tumbling, and an increase in delta p resulted in a smooth swimming response at either pH. Inhibition of aerotaxis at pH 7.5 by various concentrations of KCN correlated with a decreased delta p, due to a decreased delta psi in aerobiosis and little change in delta psi in anaerobiosis. At concentrations up to 100 mM, 2,4-dinitrophenol decreased delta psi, but did not inhibit aerotaxis because the difference between delta psi in aerobic and anaerobic cells remained constant. Considered as a whole, the results indicate that aerotaxis in S. typhimurium is mediated by delta p.     

De novo ABA synthesis and expression of seed dormancy in a gymnosperm: Pseudotsuga menziesii

Seeds of dormant Douglas-fir seeds germinated poorly when they were cultivated at 20–23 °C while isolated embryos germinated fully within two weeks. Seed dormancy was therefore imposed on the embryo by its surrounding structures. This physiological behaviour was well correlated with changes in ABA level during culture. Indeed, the ABA level decreased in isolated embryos while it increased in both embryo and megagametophyte during culture of whole seeds. The origin of this increase was analysed and the different ways by which seed coats could interfere with ABA accumulation are discussed.     

Importance of anaerobic superoxide dismutase synthesis in facilitating outgrowth of Escherichia coli upon entry into an aerobic habitat.

The manganese-containing isozyme of superoxide dismutase (MnSOD) is synthesized by Escherichia coli only during aerobiosis, in accordance with the fact that superoxide can be formed only in aerobic environments. In contrast, E. coli continues to synthesize the iron-containing isozyme (FeSOD) even in the absence of oxygen. A strain devoid of FeSOD exhibited no deficits during either anaerobic or continuously aerobic growth, but its growth lagged for 2 h during the transition from anaerobiosis to aerobiosis. Complementation of this defect with heterologous SODs established that anaerobic SOD synthesis per se is necessary to permit a smooth transition to aerobiosis. The growth deficit was eliminated by supplementation of the medium with branched-chain amino acids, indicating that the growth interruption was due to the established sensitivity of dihydroxyacid dehydratase to endogenous superoxide. Components of the anaerobic respiratory chain rapidly generated superoxide when exposed to oxygen in vitro, suggesting that this transition may be a period of acute oxidative stress. These results show that facultative bacteria must preemptively synthesize SOD during anaerobiosis in preparation for reaeration. The data suggest that evolution has chosen FeSOD for this function because of the relative availability of iron, in comparison to manganese, during anaerobiosis.     

Effect of Oxygen on Several Enzymes Involved in the Aerobic and Anaerobic Utilization of Glucose in Escherichia coli

By using the continuous culture technique, the transition from aerobiosis to anaerobiosis and its effect on a number of enzymes has been investigated in Escherichia coli K-12. A decrease in the oxygen partial pressure below 28.0 mm of Hg resulted firstly in an increase of the respiratory enzymes (reduced nicotinamide adenine dinucleotide [NADH] oxidase, 2.53-fold; succinic dehydrogenase, 1.4-fold; cytochrome b(1), 3.91-fold; and cytochrome a(2), 2.45-fold) before the electron transport system gradually collapsed as cytochrome a(2), followed by cytochrome b(1), succinic dehydrogenase, and finally NADH oxidase decreased in activity. The change from respiration to fermentation was initiated well before the oxygen tension reached zero by the increase in levels of fructose diphosphate-aldolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases and a decrease in 2-oxoglutarate dehydrogenase. Whem the dissolved oxygen tension reached zero, dry weight and CO(2) formation together with isocitrate dehydrogenase decreased, whereas acid production and phosphofructokinase synthesis started to increase. Enzymatic investigations revealed that the kinetics of the enzyme phosphofructokinase from strict aerobic cultures (6.9 ppm oxygen in solution) was adenosine triphosphate (ATP)-insensitive, whereas the same enzyme from anaerobic cultures was ATP-sensitive. A mechanism is proposed for the change from aerobiosis to anaerobiosis together with the occurring change in glucose regulation.     

Aerobic metabolism and oxidative stress tolerance in the Lactobacillus plantarum group

Aerobic metabolism and response to oxidative stress and starvation were studied in 11 Lactobacillus plantarum, L. paraplantarum and L. pentosus strains in order to assess the impact of aerobic metabolism on the growth and on the stress response. The strains were grown in aerobiosis without supplementation (AE), with hemin (AEH) or with hemin and menaquinone (AEHM) supplementation and in anaerobiosis (AN) in a complex buffered substrate. Growth rate, biomass yield, glucose and O₂ consumption, production of lactic acid and H₂O₂, catalase activity, oxidative and starvation stress tolerance were evaluated. Aerobic growth increased biomass yield in late stationary phase. Further increase in yield was obtained with both hemin (H) and menaquinone (M) addition. With few exceptions, the increase in biomass correlated with the decrease of lactic acid which, however, decreased in anaerobic conditions as well in some strains. Addition of H or H + M increased growth rate for some strains but reduced the duration of the lag phase. H₂O₂ production was found only for aerobic growth with no supplementation due to catalase production when hemin was supplemented. To our knowledge this is the first study in which the advantages of aerobic growth with H or H + M in improving tolerance of oxidative stress and long-term survival is demonstrated on several strains of the L. plantarum group. The results may have significant technological consequences for both starter and probiotic production.     

Effect of anaerobiosis and anhydrobiosis on the extent of glycolytic enzyme binding in Artermia embryos

The effect of anaerobiosis and anhydrobiosis on the extent of binding of glycolytic enzymes to the particulate fraction of the cell was studied in Artemia salina embryos. During control aerobic development, trehalase, phosphofructokinase and pyruvate kinase showed an increase in the percentage associated with the particulate fraction which is consistent with the carbohydrate-based metabolism of Artemia embryos. However, anaerobiosis resulted in decreased enzyme binding for six glycolytic enzymes; hexokinase, aldolase, pyruvate kinase and lactate dehydrogenase were the exceptions. Decreased enzyme binding was also observed after exposure to dehydrating conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms in Artemia embryos. This reversible interaction of glycolytic enzymes with structural proteins may account for part of the metabolic arrest observed during anaerobic dormancy and anhydrobiosis.Abbreviation pH_i intracellular concentration of H⁺ ions     

Study on anaerobic and aerobic degradation of different non-ionic surfactants

Six alcohol ethoxylates (C5E2, C6E4, C7E4, C8E2, C8E4, C10E4) and two fatty acid esters were tested at lab-scale for degradation in anaerobic and aerobic conditions and oxygen uptake rate (OUR). Anaerobic removal of C5E2, C6E4 and C7E4 improved with increasing number of ethoxy groups (E) and decreasing length of the alkyl chain (C). Their aerobic removal was also great but lower than the anaerobic values. C8E2, C8E4 and C10E4 were adsorbed on sludge but not degraded in anaerobic conditions, while they were efficiently removed under aerobiosis. The fatty acid esters were removed to a level between the two alcohol ethoxylates groups in both anaerobiosis and aerobiosis. The measured OUR confirmed the different behaviours of the three groups of compounds.     

Effect of the activity of primary proton pumps on the growth of Escherichia coli in the presence of acetate

Escherichia coli batch cultures were grown under aerobic and anaerobic conditions on glucose with the substrate addition at pH 7.0. The cultures accumulated acetate in the medium at concentrations sufficient to inhibit the growth. This inhibitory effect of acetate was mediated apparently via its action on the intracellular pH. The inhibition of E. coli growth by acetate increased when the redox proton pump was switched off in the course of transition from aerobiosis to anaerobiosis and when the regulation of K+ fluxes was disordered in the presence of valinomycin. H+-ATPase was not essentially involved in maintaining the high rate of E. coli growth in the presence of acetate under aerobic conditions. If the activity of H+-ATPase was inhibited under anaerobic conditions at pH 7.0, the growth ceased after the dissipation of ionic gradients on the membrane. When CCCP was added under aerobic conditions, the growth did not stop at once if the medium had a pH of 7.6, but ceased immediately at pHout 7.0 in the glucose-salt medium.     

Abscisic Acid metabolism by source and sink tissues of sugar beet

The fate of exogenously applied, labeled abscisic acid (±)-(ABA) was followed in source leaves and taproot sink tissues of sugar beet (Beta vulgaris cv AH-11). The objective was to determine if differential pathways for ABA metabolism exist in source and sink tissues. Tissue discs were incubated for up to 13 hours in a medium containing 1 micromolar labeled ABA. At various time intervals, samples were taken for metabolite determination by reverse-phase high performance liquid chromatography. The labeled metabolites were identified by retention times using an online scintillation counter.

Dihydrophaseic acid (DPA) aldopyranoside, DPA, phaseic acid (PA), ABA glucose ester (ABA-GE), and two unidentified compounds were recovered from both tissues. An additional unidentified metabolite was also present in root tissue. Leaf tissue discs exhibited a higher capacity for ABA conjugation, and root discs showed a greater preference for ABA catabolism to PA and DPA. After 4 to 5 hours, ABA incorporation into the various metabolites was proportional to the external ABA concentration in both tissues. But the internal ABA pool size was independent of external concentrations below 10⁻⁶ molar. These results suggested that rates of ABA metabolism was proportional to the rates of uptake in both tissues.