Aerobic Chemostat Cultures Research Articles

The Two Acetyl-coenzyme A Synthetases of Saccharomyces cerevisiae Differ with Respect to Kinetic Properties and Transcriptional Regulation

Saccharomyces cerevisiae contains two structural genes, ACS1 and ACS2, each encoding an active acetyl-coenzyme A synthetase. Characterization of enzyme activities in cell-free extracts from strains expressing either of the two genes revealed differences in the catalytic properties of the two enzymes. The Km for acetate of Acs1p was about 30-fold lower than that of Acs2p and Acs1p, but not Acs2p, could use propionate as a substrate. Enzyme activity measurements and mRNA analyses showed that ACS1 and ACS2 were both expressed during carbon-limited growth on glucose, ethanol, and acetate in aerobic chemostat cultures. In anaerobic glucose-limited cultures, only the ACS2 gene was expressed. Based on these facts, the products of the ACS1 and ACS2 genes were identified as the previously described "aerobic" and "non-aerobic" forms of acetyl-coenzyme A synthetase, respectively. Batch and glucose-pulse experiments revealed that transcription of ACS1 is subject to glucose repression. A mutant strain lacking Acs2p was unable to grow on glucose in batch cultures, but grew readily in aerobic glucose-limited chemostat cultures, in which the low residual glucose concentration alleviated glucose repression. Experiments in which ethanol was pulsed to aerobic ethanol-limited chemostat cultures indicated that, in addition to glucose, ethanol also repressed ACS1 transcription, although to a lesser extent. In contrast, transcription of ACS2 was slightly induced by ethanol and glucose. Absence of ACS2 prevented complete glucose repression of ACS1, indicating that ACS2 (in)directly is involved in the transcriptional regulation of ACS1.

Nitrification and denitrification by Thiosphaera pantotropha in aerobic chemostat cultures

Thiosphaera pantotropha has been reported to denitrify aerobically and nitrify heterotrophically. However, recent evidence has indicated that these properties (particularly aerobic denitrification) have been lost. The occurrence and levels of aerobic denitrification and heterotrophic nitrification by T. pantotropha in chemostat cultures have therefore been re-evaluated. Only low nitrate reduction rates were observed: the apparent nitrogen loss was of the same order of magnitude as the combined error in the calculated nitrogen consumption. However, 15N mass spectrometry revealed low aerobic denitrification rates (about 10% of the rates originally published by this group). Heterotrophic nitrification rates were about a third of previous observations. N2 and N2O were both produced from NH4+, NO3− and NO2−. Periplasmic nitrate reductase was present in aerobically grown cells.

Nitrification and denitrification by Thiosphaera pantotropha in aerobic chemostat cultures

Thiosphaera pantotropha has been reported to denitrify aerobically and nitrify heterotrophically. However, recent evidence has indicated that these properties (particularly aerobic denitrification) have been lost. The occurrence and levels of aerobic denitrification and heterotrophic nitrification by T. pantotropha in chemostat cultures have therefore been re-evaluated. Only low nitrate reduction rates were observed: the apparent nitrogen loss was of the same order of magnitude as the combined error in the calculated nitrogen consumption. However, 15N mass spectrometry revealed low aerobic denitrification rates (about 10% of the rates originally published by this group). Heterotrophic nitrification rates were about a third of previous observations. N 2 and N 2O were both produced from NH 4 +, NO 3 − and NO 2 −. Periplasmic nitrate reductase was present in aerobically grown cells.

Effects of growth conditions on mitochondrial morphology in Saccharomyces cerevisiae.

Effects of growth conditions on mitochondrial morphology were studied in living Saccharomyces cerevisiae cells by vital staining with the fluorescent dye dimethyl-aminostyryl-methylpyridinium iodine (DASPMI), fluorescence microscopy, and confocal-scanning laser microscopy. Cells from respiratory, ethanol-grown batch cultures contained a large number of small mitochondria. Conversely, cells from glucose-grown batch cultures, in which metabolism was respiro-fermentative, contained small numbers of large, branched mitochondria. These changes did not significantly affect the fraction of the cellular volume occupied by the mitochondria. Similar differences in mitochondrial morphology were observed in glucose-limited chemostat cultures. In aerobic chemostat cultures, glucose metabolism was strictly respiratory and cells contained a large number of small mitochondria. Anaerobic, fermentative chemostat cultivation resulted in the large, branched mitochondrial structures also seen in glucose-grown batch cultures. Upon aeration of a previously anaerobic chemostat culture, the maximum respiratory capacity increased from 10 to 70 mumole.min-1.g dry weight-1 within 10 h. This transition resulted in drastic changes of mitochondrial number, morphology and, consequently, mitochondrial surface area. These changes continued for several hours after the respiratory capacity had reached its maximum. Cyanide-insensitive oxygen consumption contributed ca. 50% of the total respiratory capacity in anaerobic cultures, but was virtually absent in aerobic cultures. The response of aerobic cultures to oxygen deprivation was qualitatively the reverse of the response of anaerobic cultures to aeration. The results indicate that mitochondrial morphology in S. cerevisiae is closely linked to the metabolic activity of this yeast: conditions that result in repression of respiratory enzymes generally lead to the mitochondrial morphology observed in anaerobically grown, fermenting cells.

Metabolic uncoupling in Saccharomyces cerevisiae

Aerobic chemostat cultures of S. cerevisiae were performed at different diln. rates under energy (glucose) limitation or at various extents of energy excess imposed by a N limitation. Energy excess induced metabolic uncoupling under steady-state conditions. The sp. EtOH prodn. rate was always higher the lower the feed medium N concn. throughout the whole range of diln. rates tested. In addn., the respiratory rate also increased under N limitation, for at least as long as the sp. O2 consumption rate was below the max. capacity. These results imply that the ATP yield (YATP in g biomass per mol ATP) and/or the amt. of ATP produced, i.e. the P/O ratio, must be able to change. By assuming a const. YATP of 16 g/mol, a decrease in the P/O ratio from 1 to 1.5 at low diln. rates and glucose limitation down to only 0.1-0.2 at high diln. rates and N limitation could be calcd. If, instead, a fixed P/O ratio of 1.0 was assumed, the YATP decreased from .apprx.20 down to <10 g/mol during these different conditions. The heat yield values, i.e., the amt. of heat produced per amt. of biomass formed, increased dramatically when the cells were subjected to a N limitation, indicating less efficient growth in terms of conserving substrate energy as biomass under energy excess compared to energy-limiting conditions. [on SciFinder (R)]

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.

Energy metabolism of Kluyveromyces marxianus in deproteinated whey. Chemostat studies. Modelling

The behavior of Kluyveromyces marxianus in aerobic chemostat cultures of deproteinated whey was studied in order to determine the characteristics of energy metabolism. The combined results show that K. marxianus presents a different pattern of behavior compared to Saccharomyces cereuisiae. A mathematical model has been constructed where energy metabolism is separated into two subunits: (a) a glycolytic subunit and (b) a respiratory subunit, through which the substrate C-flux (gram-atom g −1 h −1 is calculated. The coupling of fluxes assumes the existence of a respiratory pattern. Oxidoreductive metabolism appears as a consequence of an overflow effect resulting from relative increases in the substrate flux through the glycolytic subunit with respect to the respiratory unit, independent of the maximum respiratory capacity of the yeast. The model is able to reproduce the behavior of K. marxianus in the experimental conditions of culture implemented.

The role of alcohol dehydrogenase in the fermentation of D-xylose byCandida shehatae ATCC 22984

When the oxygen supply to an aerobic chemostat culture ofCandida shehatae growing on D-xylose was reduced to oxygen-limited and anoxic conditions, accumulation of ethanol occurred, the specific activity of alcohol dehydrogenase (ADH) increased up to four-fold and the number of ADH isozymes detected increased from one to three. ADH in crude extracts prepared from anoxically-grown cells had a lower affinity for but was more tolerant to ethanol than in extracts prepared from aerobically grown cells. ADH activity appeared to be sufficient for ethanol production byC. shehatae under anoxic conditions.

Enzyme profiles of Thiobacillus versutus after aerobic and denitrifying growth: Regulation of isocitrate lyase

The specific activities of the tricarboxylic acid (TCA) cycle enzymes in Thiobacillus versutus were invariably lower after aerobic growth as compared to denitrifying growth in acetate- or succinate-limited chemostat cultures. Of the glyoxylate cycle enzymes, isocitrate lyase (ICL) activity was nil during aerobic and 76 nmol·min-1·mg-1 protein during denitrifying growth on acetate whereas malate synthase (MS) did not change. In succinate-grown cells ICL was always near nil. The change in ICL and MS was followed after pulse additions of acetate and nitrate to an aerobic acetate-limited chemostat culture made anaerobic prior to the first pulse. ICL remained nil during denitrifying growth after the first pulse but increased to 47 and 81 nmol ·min-1·mg-1 protein after the second and third pulse, respectively. MS remained unaltered. The appearance of ICL was dependent upon de novo protein synthesis. During transition in a steady state culture on acetate from oxygen to nitrate as terminal electron acceptor, denitrifying growth started after 0.6 volume replacements. The resumption of growth was concomitant with an increase in TCA cycle enzyme activities. ICL was observed only after two volume replacements. During the reverse transition, ICL disappeared at a rate twice the dilution rate. SDS polyacrylamide gelectrophoresis of cell-free extracts containing ICL showed a major protein band with a Rf value identical to purified ICL and a mol·wt. of 60,000. ICL from T. versutus was inhibited by 1.5 mM itaconate but not by 10 mM phosphoenolpyruvate. Its activity was dependent upon the presence of Mg2+ and cysteine.

Isocitrate Lyase Activity in Thiobacillus versutus Grown Anaerobically on Acetate and Nitrate

SUMMARY: In cell-free extracts of Thiobacillus versutus, an organism which has been reported to be isocitrate lyase negative, an isocitrate lyase activity of 52 ± 18 nmol min−1 (mg protein)−1 was observed after anaerobic growth in a chemostat on acetate plus nitrate, i.e. during denitrification. Following growth on succinate plus nitrate, isocitrate lyase activity was only 1 ± 2 nmol min−1 (mg protein)−1. In cell-free extracts derived from aerobic chemostat cultures isocitrate lyase activity was always nil. The identity of the enzyme was analysed using a number of different methods, namely (a) three different enzyme assays, (b) 13C-NMR spectroscopy of the reaction products, (c) HPLC analysis of the reaction products, (d) mass spectrometry of derivatized glyoxylate enzymically produced from isocitrate and (e) radiography of derivatized glyoxylate enzymically produced from [14C]citrate. All these methods gave results consistent with the enzyme-catalysed conversion of isocitrate to glyoxylate and succinate.

Mixotrophic Growth of Thiobacillus A2 in Chemostat Culture on Formate and Glucose

In aerobic chemostat culture Thiobacillus A2-GFI grew autotrophically on formate and heterotrophically on glucose with maximum specific growth rates (μmax) of 0·21 and 0·33 h−1, respectively. At dilution rates of 0·1 and 0·18 h−1, it grew mixotrophically on formate + glucose mixtures, completely consuming both substrates. Ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase were present at high specific activity in autotrophic and mixotrophic cultures, but were repressed in cultures on glucose alone. A greater proportion of added glucose was assimilated in mixotrophic culture than in heterotrophic culture. Raising the dilution rate of a mixotrophic culture from 0·1 or 0·18 to 0·3 h−1 resulted in washout (with an apparent μ max for mixotrophic growth of 0·25 h−1) and the establishment of a culture dependent on glucose for growth. Growth yields on formate and glucose were, respectively, 3·3 and 100 g dry wt (mol substrate consumed)−1. Steady state biomass production in mixotrophic culture indicated additive growth yields. The biomass produced in cultures on formate + glucose at a dilution rate of 0·3 h−1 suggested that growth only occurred on glucose, but organisms still contained high activities of ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase. At a formate: glucose ratio (mm) of 100:1, some formate was oxidized and CO2 was fixed, but formate was not used when this ratio was 50:5. Formate-glucose mixotrophy benefits Thiobacillus A2-GFI when substrates are limited at low growth rates (&lt;μ max for formate), but is characterized by a μ max below that possible on glucose. Physiological behaviour at high growth rates was influenced by the formate:glucose ratio, resulting under some conditions, at least, in loss of mixotrophy and the establishment of heterotrophic growth.

Microcalorimetric investigations of the metabolism of yeasts. growth in batch and chemostat cultures on ethanol medium

The heat evolution of aerobic batch and chemostat cultures of growing yeast (Saccharomyces cerevisiae), in ethanol medium was investigated with a flowmicrocalorimeter. A balance for energy, carbon and oxygen was established in the chemostat cultures. Acetate which was presumably formed during the degradation of ethanol accounted for about 24% of the energy content of ethanol, but was not metabolized. Furthermore, only 45% the energy produced was stored in cell material, whereas 55% appeared as heat. From this and from the carbon balance it is concluded that the production of yeast cell material from ethanol is an exothermic process.

Kinetics of biphasic growth of yeast in continuous and fed‐batch cultures

AbstractThe influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose‐limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr−1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr−1. Some supplementary data were collected from an ethanol‐limited aerobic chemostat culture and a glucose‐limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed‐batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.

The effect of 2,4-dinitrophenol on the growth ofKlebsiella aerogenesNCTC 418 in aerobic chemostat cultures

The effect of 2,4-dinitrophenol on the growth of<i>Klebsiella aerogenes</i>NCTC 418 in aerobic chemostat cultures

The effect of 2,4-dinitrophenol on the growth of Klebsiella aerogenes NCTC 418 in aerobic chemostat cultures

The effect of 2,4-dinitrophenol on the growth of Klebsiella aerogenes NCTC 418 in aerobic chemostat cultures

The role of energy-spilling reactions in the growth of Klebsiella aerogenes NCTC 418 in aerobic chemostat culture.

When cell-saturating amounts of glucose and phosphate were added to steady state cultures of Klebsiella aerogenes that were, respectively, glucose- and phosphate-limited, the organisms responded immediately with an increased oxygen consumption rate. This suggested that in neither case was glucose transport the rate-limiting process, and also that organisms must possess effective mechanisms for spilling the excess energy initially generated when a growth-limitation is temporarily relieved. Steady state cultures of mannitol- or glucose-limited organisms also seemingly generated energy at a greater rate than was required for cell synthesis since gluconate-limited cultures consumed oxygen at a lower rate, at each corresponding growth rate, than did mannitol- or glucose-limited cultures, and therefore expressed a higher YO value. Thus, mannitol- and glucose-limitations must be essentially carbon (and not energy) limitations. The excess energy generated by glucose metabolism is one component of "maintenance" and could be used at lower growth rates to maintain an increased solute gradient across the cell membrane, imposed by the addition of 2%, w/v, NaCl to the growth environment. The maintenance rates of oxygen consumption of K. aerogenes also could be caused to increase by adding glucose discontinuously (drop-wise) to a glucose-limited chemostat culture, or by exchanging nitrate for ammonia as the sole utilizable nitrogen source. The significance of these findings to an assessment of the physiological factors circumscribing energy-spilling reactions in aerobic cultures of K. aerogenes is discussed.

Growth and physiology of Candida utilis NCYC 321 in potassium-limited chemostat culture.

When grown in a defined simple salts medium, plus vitamins, Candida utilis displayed an absolute requirement for potassium. But the potassium content of this yeast was exceedingly variable and, with aerobic chemostat cultures (grown at a dilution rate of 0.1 h-1; 30 degrees C; pH 5.5), was low (less than 0.2%, w/w) when they were potassium-limited and high (greater than 2%, w/w) when glucose-limited. With potassium-limited cultures, the cell-bound potassium content also varied markedly with growth rate, though hardly at all with glucose-limited cultures; magnesium- and phosphate-limited cultures gave intermediate responses. Changes in cell-bound potassium content correlated only weakly with changes in the cellular contents of magnesium, phosphate and RNA, but strongly with changes in both the Yglucose and Y0 values, indicating an involvement of potassium in the generation of energy by oxidative phosphorylation reactions and/or the utilization of this energy for growth processes. Studies with isolated mitochondria revealed that potassium-limited organisms had a changed content of cytochrome b relative to cytochrome a, and lacked coupling at either site 2 or site 3 of the respiratory chain. These results are discussed in relation to the reported functions of potassium in the growth of micro-organisms, and the organizational differences between prokaryotic and eukaryotic cells.