Growth of the yeast Saccharomyces cerevisiae on a non-fermentable substrate: control of energetic yield by the amount of mitochondria

The purpose of this study was to investigate the long-term control of ATP synthesis during the course of Saccharomyces cerevisiae batch grown on lactate, a purely respiratory substrate. For this, we used a respirometric and on-line calorimetric approach to analyse the energetic balances and the control of energetic metabolism during growth. Enthalpic growth yields assessed by enthalpy balance (taking account of substrate consumption, by-product accumulation, biomass formation and heat dissipation) remained constant during the entire exponential growth. Moreover, at the same time, a parallel decrease in basal respiratory rate and enthalpy flux occurred. It is shown that the decrease in respiration corresponds to a decrease in the amount of mitochondria per cell but not to a change of steady state of oxidative phosphorylation. Taking into account the part of energy used for maintenance, it can be concluded that mitochondria by themselves are the major heat dissipative system in a fully aerobic metabolism, and that the decrease in the amount of mitochondria when growth rate decreases leads to an enthalpic growth yield constant.     

Heat flow measurements in aerobic microbial growth processes with a nonisothermal calorimeter operating directly in the fermenter

A nonisothermal flow calorimeter operating directly in the fermenter was used for heat flow measurements of aerobic microbial growth processes with high biomass productivities. The measuring arrangement makes it possible to describe transitional stages of aerobic yeast cell growth by the ratio of heat production to oxygen consumption (oxy-caloric coefficient). The oxy-caloric coefficient was not constant under the described conditions. The results refer to the existence of an additional energy-delivering mechanism in microbial systems with aerobic carbon source utilization. The mechanism can involve polyphosphate bond division coupled to biomass synthesis.     

Application of macroscopic principles to microbial metabolism

General expressions for mass, elemental, energy, and entropy balances are derived and applied to microbial growth and product formation. The state of the art of the application of elemental balances to aerobic and heterotrophic growth is reviewed and extended somewhat to include the majority of the cases commonly encountered in biotechnology. The degree of reduction concept is extended to include nitrogen sources other than ammonia. The relationship between a number of accepted measures for the comparison of substrate yields is investigated. The theory is illustrated using a generalized correlation for oxygen yield data. The stoichiometry of anaerobic product formation is briefly treated, a limit to the maximum carbon conservation in product is derived, using the concept of elemental balance. In the treatment of growth energetics the correct statement of the second law of thermodynamics for growing organisms is emphasized. For aerobic heterotrophic growth the concept of thermodynamic efficiency is used to formulate a limit the substrate yield can never surpass. It is combined with a limit due to the fact that the maximum carbon conservation in biomass can obviously never surpass unity. It is shown that growth on substrates of a low degree of reduction is energy limited, for substrates of a high degree of reduction carbon limitation takes over. Based on a literature review concerning yield data some semiempirical notions useful for a preliminary evolution of aerobic heterotrophic growth are developed. The thermodynamic efficiency definition is completed by two other efficiency measures, which allow derivation of simple equations for oxygen consumption and heat production. The range of validity of the constancy of the rate of heat production to the rate of oxygen consumption is analyzed using these efficiency measures. The energetic of anaerobic growth are treated—it is shown that an approximate analysis in terms of an enthalpy balance is not valid for this case, the evaluation of the efficiency of growth has to be based on Gibbs free energy changes. A preliminary analysis shows the existence of regularities concerning the free energy conservation on anaerobic growth. The treatment is extended to include the effect of growth rate by the introduction of a linear relationship for substrate consumption. Aerobic and anaerobic growth are discussed using this relationship. A correlation useful in judging the potentialities for improvement in anaerobic product formation processes is derived. Finally the relevance of macroscopic principles to the modeling of bioengineering systems is discussed.     

Application of an enthalpy balance model of the relation between growth and respiration to temperature acclimation of Eucalyptus globulus seedlings

The enthalpy balance model of growth uses measurements of the rates of heat and CO(2) production to quantify rates of decarboxylation, oxidative phosphorylation and net anabolism. Enthalpy conversion efficiency (eta(H)) and the net rate of conservation of enthalpy in reduced biosynthetic products (R(SG)DeltaH(B)) can be calculated from metabolic heat rate (q) and CO(2) rate (R(CO2)). eta(H) is closely related to carbon conversion efficiency and the efficiency of conservation of available electrons in biosynthetic products. R(SG)DeltaH(B) and eta(H) can be used, together with biomass composition, to describe the rate and efficiency of growth of plant tissues. q is directly related to the rate of O(2) consumption and the ratio q:R(CO2) is inversely related to the respiratory quotient. We grew seedlings of Eucalyptus globulus at 16 and 28 degrees C for four to six weeks, then measured q and R(CO2) using isothermal calorimetry. Respiratory rate at a given temperature was increased by a lower growth temperature but eta(H) was unaffected. Enthalpy conversion efficiency - and, therefore, carbon conversion efficiency - decreased with increasing temperature from 15 to 35 degrees C. The ratio of oxidative phosphorylation to oxygen consumption (P/O ratio) was inferred in vivo from eta(H) and by assuming a constant ratio of growth to maintenance respiration with changing temperature. The P/O ratio decreased from 2.1 at 10-15 degrees C to less than 0.3 at 35 degrees C, suggesting that decreased efficiency was not only due to activity of the alternative oxidase pathway. In agreement with predictions from non-equilibrium thermodynamics, growth rate was maximal near 25 degrees C, where the calculated P/O ratio was about half maximum. We propose that less efficient pathways, such as the alternative oxidase pathway, are necessary to satisfy the condition of conductance matching whilst maintaining a near constant phosphorylation potential. These conditions minimize entropy production and maximize the efficiency of mitochondrial energy conversions as growing conditions change, while maintaining adequate finite rates of energy processing.     

Ethanol fermentation technologies from sugar and starch feedstocks

This article critically reviews some ethanol fermentation technologies from sugar and starch feedstocks, particularly those key aspects that have been neglected or misunderstood. Compared with Saccharomyces cerevisiae, the ethanol yield and productivity of Zymomonas mobilis are higher, because less biomass is produced and a higher metabolic rate of glucose is maintained through its special Entner-Doudoroff pathway. However, due to its specific substrate spectrum as well as the undesirability of its biomass to be used as animal feed, this species cannot readily replace S. cerevisiae in ethanol production. The steady state kinetic models developed for continuous ethanol fermentations show some discrepancies, making them unsuitable for predicting and optimizing the industrial processes. The dynamic behavior of the continuous ethanol fermentation under high gravity or very high gravity conditions has been neglected, which needs to be addressed in order to further increase the final ethanol concentration and save the energy consumption. Ethanol is a typical primary metabolite whose production is tightly coupled with the growth of yeast cells, indicating yeast must be produced as a co-product. Technically, the immobilization of yeast cells by supporting materials, particularly by gel entrapments, is not desirable for ethanol production, because not only is the growth of the yeast cells restrained, but also the slowly growing yeast cells are difficult to be removed from the systems. Moreover, the additional cost from the consumption of the supporting materials, the potential contamination of some supporting materials to the quality of the co-product animal feed, and the difficulty in the microbial contamination control all make the immobilized yeast cells economically unacceptable. In contrast, the self-immobilization of yeast cells through their flocculation can effectively overcome these drawbacks.     

Comparison of oxygen consumption rates in minimally transformed BALB/3T3 and virus-transformed 3T3B-SV40 cells

In the recent years, bioenergetics of tumor cells and particularly cell respiration have been attracting great attention because of the involvement of mitochondria in apoptosis and growing evidence of the possibility to diagnose and treat cancer by affecting the system of oxidative phosphorylation in mitochondria. In the present work, a comparative study of oxygen consumption in 3T3B-SV40 cells transformed with oncovirus SV40 and parental BALB/3T3 cells was conducted. Such fractions of oxygen consumption as “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration observed in the presence of protonophore were determined. Maximal respiration was shown to be only slightly decreased in 3T3B-SV40 cells as compared to BALB/3T3. However, in the case of certain fractions of cellular respiration, the changes were significant. “Phosphorylating” respiration was found to be reduced to 54% and “reserve” respiration, on the contrary, increased up to 160% in virus-transformed 3T3B-SV40 cells. The low rate of “phosphorylating” respiration and high “reserve” respiration indicate that under normal incubation conditions the larger part of mitochondrial respiratory chains of the virus-transformed cells is in the resting state (i.e. there is no electron transfer to oxygen). The high “reserve” respiration is suggested to play an important role in preventing apoptosis of 3T3B-SV40 cells.     

Age-specific decrease in aerobic efficiency associated with increase in oxygen free radical production in Drosophila melanogaster

This study was designed to test the free radical theory of aging by using Drosophila melanogaster as a model system. Oxygen free radicals are generated by mitochondria during the process of normal oxidative metabolism. Age-specific measurements of oxygen consumption, heat production and anti-oxidant enzyme activity were obtained from two inbred lines of male flies, one selected for longevity and one normal-lived. The findings of this study demonstrate that although oxygen consumption remains relatively constant over the majority of the life span of each line of flies, aerobic efficiency declines with advancing age. This loss of aerobic efficiency manifests itself as a decline in total body metabolism as measured by heat production, and appears to be associated with an age-specific increase in damage inflicted upon mitochondria by oxygen free radicals.     

Utilization of mass–energy balance regularities in the analysis of continuous-culture data

Material and energy balances for continuous-culture processes are described based on the facts that the heat of reaction per electron transferred to oxygen for a wide variety of organic molecules, the number of available electrons per carbon atom in biomass, and the weight fraction carbon in biomass are relatively constant. Energy requirements for growth and maintenance are investigated and related to the biomass energetic yield. The consistency of experimental data is examined using material and energy balances and the regularities identified above. When extracellular products are absent, the consistency of yield models containing separate terms for growth and maintenance may be investigated using organic substrate consumption, biomass production, oxygen consumption (or heat evolution), and carbon dioxide evolution rate data for a series of dilution rates. The consistency of continuous-culture data in the published literature is examined.     

Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux

Calorimetric and respirometric studies of cultured cells show that both neoplastic and non-neoplastic cell types maintain an anaerobic contribution to their total heat flux. In many mammalian cells this can be explained quantitatively by lactate production observed under fully aerobic conditions. Uncoupling and enhanced futile substrate cycling increase the ratio of heat flux to oxygen flux, the calorimetric-respirometric (CR) ratio. The interpretation of calorimetric and respirometric measurements requires an energy balance approach in which experimentally measured CR ratios are compared with thermochemically derived oxycaloric equivalents. The oxycaloric equivalent is the enthalpy change per mole of oxygen consumed, and equals -470 kJ/mol O2 in the aerobic catabolism of glucose, assuming that catabolism is 100% dissipative (the net efficiency of metabolic heat transformation is zero). CR ratios more negative than -470 kJ/mol O2 have been reported in well-oxygenated cell cultures and are discussed in terms of integrated aerobic and anaerobic metabolism.     

Generation of superoxide anion by mitochondria and impairment of their functions during anoxia and reoxygenation in vitro

A small portion of the oxygen consumed by aerobic cells is converted to superoxide anion at the level of the mitochondrial respiratory chain. If produced in excess, this harmful radical is considered to impair cellular structures and functions. Damage at the level of mitochondria have been reported after ischemia and reperfusion of organs. However, the complexity of the in vivo system prevents from understanding and describing precise mechanisms and locations of mitochondrial impairment. An in vitro model of isolated-mitochondria anoxia-reoxygenation is used to investigate superoxide anion generation together with specific damage at the level of mitochondrial oxidative phosphorylation. Superoxide anion is detected by electron paramagnetic resonance spin trapping with POBN-ethanol. Mitochondrial respiratory parameters are calculated from oxygen consumption traces recorded with a Clark electrode. Respiring mitochondria produce superoxide anion in unstressed conditions, however, the production is raised during postanoxic reoxygenation. Several respiratory parameters are impaired after reoxygenation, as shown by decreases of phosphorylating and uncoupled respiration rates and of ADP/O ratio and by increase of resting respiration. Partial protection of mitochondrial function by POBN suggests that functional damage is related and secondary to superoxide anion production by the mitochondria in vitro.     

Mitophagy plays an essential role in reducing mitochondrial production of reactive oxygen species and mutation of mitochondrial DNA by maintaining mitochondrial quantity and quality in yeast

In mammalian cells, the autophagy-dependent degradation of mitochondria (mitophagy) is thought to maintain mitochondrial quality by eliminating damaged mitochondria. However, the physiological importance of mitophagy has not been clarified in yeast. Here, we investigated the physiological role of mitophagy in yeast using mitophagy-deficient atg32- or atg11-knock-out cells. When wild-type yeast cells in respiratory growth encounter nitrogen starvation, mitophagy is initiated, excess mitochondria are degraded, and reactive oxygen species (ROS) production from mitochondria is suppressed; as a result, the mitochondria escape oxidative damage. On the other hand, in nitrogen-starved mitophagy-deficient yeast, excess mitochondria are not degraded and the undegraded mitochondria spontaneously age and produce surplus ROS. The surplus ROS damage the mitochondria themselves and the damaged mitochondria produce more ROS in a vicious circle, ultimately leading to mitochondrial DNA deletion and the so-called "petite-mutant" phenotype. Cells strictly regulate mitochondrial quantity and quality because mitochondria produce both necessary energy and harmful ROS. Mitophagy contributes to this process by eliminating the mitochondria to a basal level to fulfill cellular energy requirements and preventing excess ROS production.     

31P-NMR saturation transfer measurements of phosphate consumption in Saccharomyces cerevisiae

³¹P-NMR measurements of saturation transfer have been used to measure phosphate consumption in respiratory competent cells of the yeast Saccharomyces cerevisiae. Measurements of oxygen consumption and maintenance of the cells in a metabolic steady state during the NMR experiments were facilitated by immobilisation of the cells in an agarose gel matrix which could be perfused in the NMR spectrometer. The contribution of glycolysis to the observed rate of phosphate consumption was estimated by simultaneously measuring glucose consumption and ethanol production in the perfusion buffer. The remaining phosphate consumption, which was attributed to flux through the reaction catalysed by the mitochondrial ATP synthase, combined with measurements of oxygen consumption allowed estimation of a P:O ratio (mol ATP synthesised:atoms oxygen consumed) which was close to 3.     

P-NMR saturation transfer measurements of phosphate consumption in Saccharomyces cerevisiae

³¹P-NMR measurements of saturation transfer have been used to measure phosphate consumption in respiratory competent cells of the yeast Saccharomyces cerevisiae. Measurements of oxygen consumption and maintenance of the cells in a metabolic steady state during the NMR experiments were facilitated by immobilisation of the cells in an agarose gel matrix which could be perfused in the NMR spectrometer. The contribution of glycolysis to the observed rate of phosphate consumption was estimated by simultaneously measuring glucose consumption and ethanol production in the perfusion buffer. The remaining phosphate consumption, which was attributed to flux through the reaction catalysed by the mitochondrial ATP synthase, combined with measurements of oxygen consumption allowed estimation of a P:O ratio (mol ATP synthesised:atoms oxygen consumed) which was close to 3.     

Growth of a Catharanthus roseus cell suspension culture in a modified chemostat under glucose-limiting conditions

Summary A system for the continuous cultivation of plant cells has been developed, based on a commercially available 3–1 turbine-stirred fermentor. A special device was constructed to provide for homogeneous effluent from the culture at low dilution rates. Two steady states with Catharanthus roseus cells growing under glucose limitation are described with respect to biomass yield on the carbon and energy source glucose, specific oxygen consumption, specific carbon dioxide production and (by)product formation. From a carbon balance for each steady state it is shown that the flow of carbon to the culture (as glucose) practically equalled the flow of carbon from the culture (as biomass, carbon dioxide and (by)product). Biomass yields on glucose were 0.31 g/g and 0.35 g/g at dilution rates of 0.0060 l/h and 0.0081 l/h respectively. The striking difference between the obtained yield coefficients and biomass yield commonly found for batch-cultured plant cells is discussed.     

Calorimetric and stoichiometric analysis of growth of Kluyveromices fragilis in continuous culture: nitrogen limitation imposed upon carbon-limited growth

The calorimetric response of the yeast Kluyveromices fragilis was investigated for growth in continuous culture where nitrogen limitation was imposed on a carbon-limited culture. Calorimetric measurements were combined with off gas analysis, measurements of biomass, substrate and product concentrations, elemental biomass composition, and heat production to study the physiological response of K. fragilis. Regions where both carbon and nitrogen limited growth, were found over a broad range of dilution rates and feed carbon-to-nitrogen ratios. The principle mechanism by which K. fragilis accommodated regions of dual carbon and nitrogen limitation was by partial decoupling of the anabolic and catabolic pathways. When the culture was only nitrogen-limited, increased decoupling of the two pathways was observed. The principal effect of the decoupling was an increased catabolic consumption of glucose, generating an increased heat yield. The preferred way to process the excess glucose was through respiration but the cells were also capable of fermenting a small percentage of the excess glucose in specific cases where the dissolved oxygen partial pressure approached zero. In addition, these results were qualitatively compared to similar studies on Saccharomices cerevisiae. The two yeasts were similar in their ability to accommodate dual limitation by uncoupling anabolic biomass formation from substrate consumption. The two yeasts were dissimilar in how the catabolic substrate was processed. For S. cerevisiae the presence of a bottleneck in the respiration pathway dictated that the majority of the catabolic glucose consumption was by fermentation. For K. fragilis, the lack of a bottleneck in the respiration pathway dictated that the majority of catabolic glucose substrate consumption was by respiration.     

Thermogenesis of white adipocytes: a novel method allowing long-term microcalorimetric investigations

A microcalorimetric method was developed to facilitate long-term assessment of energy balance in isolated fat cells. White rat adipocytes were primary cultured in glass ampoules with a matrix of agar-gel. Heat production was measured continuously over 3 days with an LKB BioActivityMonitor. In order to assist interpretation of the microcalorimetric measurements, glucose consumption and lactate and pyruvate production were determined in parallel cultures. Heat production, glucose consumption and lactate production were in an apparent steady state throughout the study whether employing aerobic (94 pW, 0.50 and 0.44 pmol/cell. h, respectively) or partial anaerobic experimental conditions (39 pW, 0.41 and 0.57 pmol/cell.h, respectively). However, oxygen availability influenced the apparent heat production and glucose and lactate metabolism. With partial anaerobiosis a 59% lower heat production, an 18% lower glucose consumption and a 30% higher lactate production than by employing aerobic experimental conditions were observed.     

Estimation of heat production by cultured cells in suspension using semi-automated flow microcalorimetry

Modifications to a heat conduction flow microcalorimeter are described which allow registration of heat production by cells cultured in suspension. LS cells produced 34 +/- 3 pW per cell. Over an 8.5 h period, cell numbers increased by 9% and heat production per cell by 18%. Oxygen consumption per cell was 0.244 +/- 0.02 mumol min-1 per 10(8) cells and the enthalpy change was -836 kJ/mol O2. An automated pumping system allowed sequential registration of heat production by untreated cells and those exposed to a metabolic inhibitor. The results showed that 0.1 mM 2,4-dinitrophenol caused a greater increase in power (+65% at 1.5 h) than in oxygen consumption (+36%). The opposite occurred in the case of cells treated with 1 mM potassium cyanide, heat dissipation being depressed (-48%) slightly less than oxygen uptake (-52%). The results illustrate the potential of careful calorimetric determinations in studying metabolic events in the growth and division of cells in culture.     

Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of ≥ 1% propionate, ≥ 3% acetate and ≥ 5% lactate in broth during incubation at 35°C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.     

Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris

Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanol-induced AOX1 promoter. Hence, the accepted production procedure has two phases: In the first phase, the yeast utilizes glycerol and biomass is accumulated; in the second phase, the yeast utilizes methanol which is used both as an inducer for the expression of the recombinant protein and as a carbon source. Since the yeast is sensitive to methanol concentration, the methanol is supplied gradually to the growing culture. Three methanol addition strategies were evaluated for the purpose of optimizing recombinant endostatin production. Two strategies were based on the yeast metabolism; one responding to the methanol consumption using a methanol sensor, and the other responding to the oxygen consumption. In these two strategies, the methanol supply is unlimited. The third strategy was based on a predetermined exponential feeding rate, controling the growth rate at 0.02 h(-1), in this strategy the methanol supply is limited. Throughout the induction phase glycerol, in addition to methanol, was continuously added at a rate of 1 g L h(-1). Total endostatin production was similar in all three strategies, (400 mg was obtained from 3 L initial volume), but the amount of methanol added and the biomass produced were lower in the predetermined rate method. This caused the specific production of endostatin per biomass and per methanol to be 2 times higher in the predetermined rate than in the other two methods, making the growth control strategy not only more efficient but also more convenient for downstream processing.