The heat generated by yeast cultures with a mixed metabolism in the transition between respiration and fermentation

The heat generated by both batch and continuous cultures of the yeast K. fragilis was studied using a modified Bench Scale Calorimeter. Batch cultures were used to measure the heat dissipation rates and the heat yields during fully aerobic and completely anaerobic growth, whereas continuous cultures enabled, in addition, a quantitative study of heat dissipation rates during growth on mixed metabolism. In this case, the extent of fermentation versus respiration could be specified and controlled by varying the degree of oxygen limitation. The heat dissipated per unit biomass formed was highest for fully respirative catabolism and fell continuously to a much lower value typical of anaerobic cultures as the catabolism was shifted increasingly to the fermentative mode. The heat generated per mole of oxygen taken up stayed quite close to the fully aerobic value of 506 kJ mol(-1) even when a sizable fraction of the substrate available to catabolism was fermented. If the fraction of respiration in the metabolism is lowered beyond a certain threshold, the ratio of the heat generation to oxygen consumption starts to increase dramatically and finally tends to infinity for fully anaerobic growth. All experimental results were quantitatively analyzed and explained on the basis of a simple model which formally describes the cultures in terms of two parallel "chemical" reactions. In simple cases such as the one presented here, the model enables calculation of the whole stoichiometry of the culture from a single measured heat yield.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

Metabolic uncoupling of Shewanella oneidensis MR-1, under the influence of excess substrate and 3, 3', 4', 5-tetrachlorosalicylanilide (TCS)

The dissociation between catabolism and anabolism is generally termed as metabolic uncoupling. Experimentally, metabolic uncoupling is characterized by a reduction in the observed biomass yield. This condition can be brought about by: (a) excess-substrate (as measured by S(0)/X(0)), and (b) addition of chemical uncouplers such as 3, 3', 4', 5-Tetrachlorosalicylanilide (TCS). An empirical model is proposed to quantify the uncoupling effects of both excess-substrate and uncoupler addition on the microbial cultures. Metabolic uncoupling of Shewanella oneidensis MR-1, under the influence of excess pyruvate and TCS, has been modeled using the proposed expression. The degree of uncoupling was measured as a fractional reduction in theoretical maximum observed yield. Excess-substrate was observed to successively reduce biomass yield as substrate concentration was increased. In the presence of TCS, conflicting trends were obtained for number yield and protein yield. This could, in part, be attributed to the observed increase in cellular protein content upon addition of TCS. Excess-substrate conditions dominated uncoupling, as compared to uncoupler addition. However, these two approaches were found to have additive effects and could, in conjunction, be employed to control biomass growth during microbial processes such as subsurface bioremediation and activated sludge treatment.     

A macroscopic model describing yield and maintenance relationships in aerobic fermentation processes

The present paper presents a generalized treatment of the principles of elemental and enthalpy balances which are applied to aerobic fermentation processes. It is shown that strict relations do exist between the various yield factors of biomass or product on substrate, oxygen, carbon dioxide, and between the various maintenance coefficients. These relations are confirmed from the existing body of literature data on yield and maintenance coefficients. Another consequences of the application of elemental balances is the existence of limits for the maximum biomass yield on substrate and oxygen, which depend on the degree of reduction of the substrates with different degree of reduction. It appears from this model that substrates with a high degree of reduction are C limited and substrates with a low degree of reduction are energy limited. Finally the effects of temperature on yield and maintenance coefficients are analyzed from the existing body of literature data. It can be concluded that the maintenance coefficients follow an Arrhenius type of relationship and that yield is temperature independent. The literature data seem to indicate that a degree of reduction of about 4 is optimal for the carbon and energy needs for biomass formation.     

Evaluation of growth yield of Spirulina (Arthrospira) sp. in photoautotrophic, heterotrophic and mixotrophic cultures

In microbial cultures, both cellular growth rate and yield (defined as the degree of substrate conversion into the biomass) are important. Although effect of culture conditions on growth kinetics has been well documented for various microbial strains, there is almost no literature concerning the effect of environmental conditions on growth equilibrium, expressed as biomass yield coefficients from substrate. The present paper discusses the effect of culture conditions: irradiance (physical substrate) and glucose concentration (chemical substrate) on biomass yield coefficients from two chemical substrates: glucose and nitrate-nitrogen in photoautotrophic, heterotrophic and mixotrophic culture of blue-green alga Spirulina (Arthrospira) sp. The efficiency of substrates incorporation into the biomass can be precisely determined only if the elemental composition of the biomass is known. The experimental results showed that culture conditions had a substantial influence on biomass yield coefficients (biomass yield from glucose and nitrate-nitrogen). It was found that, the increase of irradiance favoured increase of biomass yield coefficient from both, glucose and nitrate-nitrogen. However, in the case of yield from nitrogen in mixotrophic culture, the effect was opposite. The effect of glucose concentration was different: the higher the initial glucose concentration, the lower the biomass yield coefficients from chemical substrates.     

In search of a thermodynamic description of biomass yields for the chemotrophic growth of microorganisms

Correlations for the prediction of biomass yields are valuable, and many proposals based on a number of parameters (Y(ATP), Y(Ave), eta(o), Y(c), Gibbs energy efficiencies, and enthalpy efficiencies) have been published. This article critically examines the properties of the proposed parameters with respect to the general applicability to chemotrophic growth systems, a clear relation to the Second Law of Thermodynamics, the absence of intrinsic problems, and a requirement of only black box information. It appears that none of the proposed parameters satisfies all these requirements. Particularly, the various energetic efficiency parameters suffer from major intrinsic problems. However, this article will show that the Gibbs energy dissipation per amount of produced biomass (kJ/C-mod) is a parameter which satisfies the requirements without having intrinsic problems. A simple correlation is found which provides the Gibbs energy dissipation/C-mol biomass as a function of the nature of the C-source (expressed as the carbon chain length and the degree of reduction). This dissipation appears to be nearly independent of the nature of the electron acceptor (e.g., O(2), No(3) (-), fermentation). Hence, a single correlation can describe a very wide range of microbial growth systems. In this respect, Gibbs energy dissipation is much more useful than heat production/C-mol biomass, which is strongly dependent on the electron acceptor used. Evidence is presented that even a net heat-uptake can occur in certain growth systems.The correlation of Gibbs energy dissipation thus obtained shows that dissipation/C-mol biomass increases for C-sources with smaller chain length (C(6) --> C(1)), and increases for both higher and lower degrees of reduction than 4. It appears that the dissipation/C-mol biomass can be regarded as a simple thermodynamic measure of the amount of biochemical "work" required to convert the carbon source into biomass by the proper irreversible carbon-carbon coupling and oxidation/reduction reactions. This is supported by the good correlation between the theoretical ATP requirement for biomass formation on different C-sources and the dissipation values (kJ/C-mol biomass) found. The established correlation for the Gibbs energy dissipation allows the prediction of the chemotrophic biomass yield on substrate with an error of 13% in the yield range 0.01 to 0.80 C-mol biomass/(C)-mol substrate for aerobic/anaerobic/denitrifying growth systems.     

The Measurement of Electron Transport System Activity in Freshwater Benthic and Planktonic Samples

The measurement of electron transport system (ETS) activity in freshwater benthic and planktonic samples was examined. Significant improvements in sensitivity were obtained by increasing the concentration of substrates and of Triton X-100 in the extraction buffer and by changing the buffer system. The mechanical homogenization procedures which have been used previously to extract the enzymes were shown to be inadequate yielding, at best, 25% of the activity obtained by ultrasonication. The yield of enzymes from sediments was greatly affected by the degree of dilution used and previously published methods would appear to be inadequate. ETS activity correlated well with biological oxygen uptake and, in individual water and sediment profiles, with microbial numbers and biomass. At a more general level, however, the ETS: biomass ratio showed considerable variation.     

Does microbial life always feed on negative entropy? Thermodynamic analysis of microbial growth.

Schr?dinger stated in his landmark book, What is Life?, that life feeds on negative entropy. In this contribution, the validity of this statement is discussed through a careful thermodynamic analysis of microbial growth processes. In principle, both feeding on negative entropy, i.e. yielding products of higher entropy than the substrates, and generating heat can be used by microorganisms to rid themselves of internal entropy production resulting from maintenance and growth processes. Literature data are reviewed in order to compare these two mechanisms. It is shown that entropy-neutral, entropy-driven, and entropy-retarded growth exist. The analysis of some particularly interesting microorganisms shows that enthalpy-retarded microbial growth may also exist, which would signify a net uptake of heat during growth. However, the existence of endothermic life has never been demonstrated in a calorimeter. The internal entropy production in live cells also reflects itself in the Gibbs energy dissipation accompanying growth, which is related quantitatively to the biomass yield. An empirical correlation of the Gibbs energy dissipation in terms of the physico-chemical nature of the growth substrate has been proposed in the literature and can be used to predict the biomass yield approximately. The ratio of enthalpy change and Gibbs energy change can also be predicted since it is shown to be approximately equal to the same ratio of the relevant catabolic process alone.     

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.     

Data consistency,yield and maintenance coefficient for the growth of Candida lypoytica and Pseudomonas aeruginosa on n-hexadecane

Summary When more than the minimum number of variables are measured, and measurement error is taken into account, the results of parameter estimation depend on which of the measured variables are selected for this purpose. The reparameterization of Pirt's models for growth produces multiresponse models with common parameters. By using the covariate adjustment technique, a unit variate linear model with covariates is obtained. This allows a combined point and interval estimates of biomass energetic yield and maintenance coefficient to be obtained using standard multiple regression programmes. When this method was applied using form I and form II of the Pirt's models, good combined estimates were obtained and compared. Using data from the literature for Candida lipolytica produced reliable results. However, for Pseudomonas aeruginosa, which has been known to produce intermediate products, a modified Pirt's model is required for a good estimate of the biomass energetic yield.Nomenclature a Mole of ammonia per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - b Moles of oxygen per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - c Moles of water per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon; no of covariates included in model - d Moles of carbon dioxide per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - e _i Error terms in Eqs. (6–8) - l Atomic ratio of oxygen to carbon in organic substrate, dimensionless - m Atomic ratio of hydrogen to carbon in organic substrate, dimensionless - m _e Rate of organic substrate consumption for maintenance, g equiv. of available electrons in biomass (h) or kcal/Kcal of biomass(h) - n Atomic ratio of oxygen to carbon in biomass, dimensionless - p Atomic ratio of hydrogen to carbon in biomass, dimensionless - Q _{CO 2} Rate of evolution of carbon dioxide, g moles/g dry wt (h) - Q _{O 2} Rate of oxygen consumption, g moles/g dry wt (h) - Q _s Rate of organic substrate consumption g/g dry wt (h) - q Atomic ratio of nitrogen to carbon in biomass, dimensionless - r Atom ratio of hydrogen to carbon in products, dimensionless; the number of parameters of interest - s Atomic ratio of oxygen to carbon in products, dimensionless - t Atomic ratio of nitrogen to carbon in products, dimensionless - r Mean of k responses in Eq. (10) - x _ki Kth response in the ith observation - y _c Biomass carbon yield (fraction of organic substrate carbon in biomass), dimensionless - z _i Covariate matrix - z Fraction of organic substrate carbon in products, dimensionless - a _i Parameters associated with covariates - _s Reductance degree of biomass, equivalents of available electrons per gram atom carbon - Reductance degree of organic substrate, equivalents of available electrons per gram atom carbon - Fraction of energy in organic substrate which is evolved as heat, dimensionless - Fraction of available electrons transferred to biomass; biomass energetic yield - True growth yield - Specific growth rate, h^-1 - _p Fraction of available electrons incorporated into products; product energetic yield - Correlation coefficient - Mass fraction carbon - ² Mean square error of model (10)     

Quantum thermodynamics approach to phosphorylation and heterotrophic growth yields

A model of cell growth is presented which is based on the double postulates of quantized loss of energy during phosphorylation and reversible biosynthesis of cell structure. An immediate consequence of the postulates is the identical value for the energy efficiency of the phosphorylation and for that of the whole growth process. Another consequence is the relationship between the energy level of the biomass and the phosphorylation potential as embodied in the equation: EO = gamma'M X EATP, where EO is the heat of transfer of a pair of electrons to oxygen, EATP, the molar heat of hydrolysis of ATP, and gamma'M, the degree of reduction of the biomass, gamma M being constant and equal to 5. The model predicts five levels of growth yields corresponding to five permissible values for the P/O ratio (r = 0, 1, 2, 3, and 4). Any growth process would be characterized by a set of two integers N and lambda; N is the maximal P/O ratio prescribed by the energy content of the substrate as compared with that of the biomass, and lambda the number of further downward quantum jumps of the P/O ratio resulting from the adversity of the growth condition (N - lambda = r). Under full aerobiosis, one has 0 less than or equal to lambda less than or equal to N less than or equal to 3. When growth is limited only by the energy content of the substrate (lambda = 0), the time-independent dispersion of N, owing to substrate-level phosphorylations and (or) dephosphorylations, leads to effective values which are higher than the nominal ones for the yield per mole of oxygen and the heat of transfer of a pair of electrons. Under adverse conditions (lambda greater than 0), the apparent variations of the yields and the P/O ratio in function of the growth rate are shown to be an effect of the random dispersion of lambda and of the existence of a maximal rate of substrate consumption. Statistical evidence for the macroscopic quantum effect in heterotrophic growth is presented.     

Physiology of yeasts in relation to biomass yields

The stoichiometric limit to the biomass yield (maximal assimilation of the carbon source) is determined by the amount of CO2 lost in anabolism and the amount of carbon source required for generation of NADPH. This stoichiometric limit may be reached when yeasts utilize formate as an additional energy source. Factors affecting the biomass yield on single substrates are discussed under the following headings: Energy requirement for biomass formation (YATP). YATP depends strongly on the nature of the carbon source. Cell composition. The macroscopic composition of the biomass, and in particular the protein content, has a considerable effect on the ATP requirement for biomass formation. Hence, determination of for instance the protein content of biomass is relevant in studies on bioenergetics. Transport of the carbon source. Active (i.e. energy-requiring) transport, which occurs for a number of sugars and polyols, may contribute significantly to the calculated theoretical ATP requirement for biomass formation. P/O-ratio. The efficiency of mitochondrial energy generation has a strong effect on the cell yield. The P/O-ratio is determined to a major extent by the number of proton-translocating sites in the mitochondrial respiratory chain. Maintenance and environmental factors. Factors such as osmotic stress, heavy metals, oxygen and carbon dioxide pressures, temperature and pH affect the yield of yeasts. Various mechanisms may be involved, often affecting the maintenance energy requirement. Metabolites such as ethanol and weak acids. Ethanol increases the permeability of the plasma membrane, whereas weak acids can act as proton conductors. Energy content of the growth substrate. It has often been attempted in the literature to predict the biomass yield by correlating the energy content of the carbon source (represented by the degree of reduction) to the biomass yield or the percentage assimilation of the carbon source. An analysis of biomass yields of Candida utilis on a large number of carbon sources indicates that the biomass yield is mainly determined by the biochemical pathways leading to biomass formation, rather than by the energy content of the substrate.     

Biochemical limits to microbial growth yields: An analysis of mixed substrate utilization

A theoretical analysis has been made of carbon conversion efficiency during heterotrophic microbial growth. The expectation was that the maximal growth yield occurs when all the substrate is assimilated and the net flow of carbon through dissimilation is zero. This, however, is not identical to a 100% carbon conversion, since assimilatory pathways lead to a net production of CO(2). It can be shown that the amount of CO(2) produced by way of assimilatory processes is dependent upon the nature of the carbon source, but independent of its degree of reduction and varies between 12 and 29% of the substrate carbon. An analysis of published yield data reveals that nearly complete assimilation can occur during growth on substrates with a high energy content. This holds for substrates with a heat of combustion of ca. 550 kJ/mol C, or a degree of reduction higher than 5 (e.g. ethane, ethanol, and methanol). Complete assimilation can also be achieved on substrates with a lower energy content, provided that an auxiliary energy source is present that cannot be used as a carbon source. This is evident from the cell yields reported for Candida utilis grown on glucose plus formate and for Thiobacillus versutus grown on acetate plus thiosulfate. This evaluation of the carbon conversion efficiency during assimilation also made it possible to compare the energy content of the auxiliary energy substrate added with the quantity of the carbon source it had replaced. It will be shown that utilization of the auxiliary energy source may lead to extreme changes in the efficiency of dissimilatory processes.     

The influence of NaCl on the degradation rate of dichloromethane byHyphomicrobium sp.

The degradation of dichloromethane by the pure strainHyphomicrobium GJ21 and by an enrichment culture, isolated from a continuously operating biological trickling filter system, as well as the corresponding growth rates of these organisms were investigated in several batch experiments. By fitting the experimental data to generally accepted theoretical expressions for microbial growth, the maximum growth rates were determined. The effect of NaCl was investigated at salt concentrations varying from 0 to 1000 mM. Furthermore the dichloromethane degradation was investigated separately in experiments in which a high initial biomass concentration was applied. The results show that microbial growth is strongly inhibited by increased NaCl concentrations (50% reduction of _max at 200–250 mM NaCl), while a certain degree of adaptation has taken place within an operational system eliminating dichloromethane. A critical NaCl concentration for growth of 600 mM was found for the microbial culture isolated from an operational trickling filter, while a value of 375 mM was found for the pure cultureHyphomicrobium GJ21. The substrate degradation appears to be much less susceptible to inhibition by NaCl. Even at 800 mM NaCl relatively high substrate degradation rates are still observed, although this process is again dependent on the NaCl concentration. Here the substrate elimination is due to the maintenance requirements of the microorganisms. The inhibition of the dichloromethane elimination was also investigated in a laboratory scale trickling filter. The results of these experiments confirmed those obtained in the batch experiments. At NaCl concentrations exceeding 600 mM a considerable elimination of dichloromethane was still observed for during several months of operation. These observations indicate that the inhibition of microbial growth offers a significant control parameter against excessive biomass growth in biological trickling filters for waste gas treatment.     

The reduction of xylose to xylitol by Candida guilliermondii and Candida parapsilosis: Incidence of oxygen and pH

Summary The ability of C. guilliermondii and C. parapsilosis to ferment xylose to xylitol was evaluated under different oxygen transfer rates in order to enhance the xylitol yield. In C. guilliermondii, a maximal xylitol yield of 0.66 g/g was obtained when oxygen transfer rate was 2.2 mmol/l.h. Optimal conditions to produce xylitol by C. parapsilosis (0.75 g/g) arose from cultures at pH 4.75 with 0.4 mmoles of oxygen/l.h. The response of the yeasts to anaerobic conditions has shown that oxygen was required for xylose metabolism.Nomenclature max maximum specific growth rate (per hour) - qSmax maximum specific rate of xylose consumption (g xylose per g dry biomass per hour) - qpmax maximum specific productivity of xylitol (g xylitol per g dry biomass per hour) - Qp average volumetric productivity of xylitol (g xylitol per liter per hour) - Y_P/S xylitol yield (g xylitol per g substrate utilized) - Y_P'/S glycerol yield (g glycerol per g substrate utilized) - Y_X/S biomass yield (g dry biomass per g substrate utilized)     

Application of mass and energy balance regularities in fermentation. Reprinted from Biotechnology and Bioengineering, Vol. XX, No. 10, Pages 1595-1621 (1978)

Material and energy balances for fermentation processes are developed 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. Mass-energy balance equations are developed which relate the biomass energetic yield coefficient to sets of variables which may be determined experimentally. Organic substrate consumption, biomass production, oxygen consumption, carbon dioxide production, heat evolution, and nitrogen consumption are considered as measured variables. Application of the balances using direct and indirect methods of yield coefficient estimation is illustrated using experimental results from the literature. Product formation is included in the balance equations and the effect of product formation on biomass yield estimates is examined. Application of mass-energy balances in the optimal operation of continuous single-cell protein production facilities is examined, and the variation of optimal operating conditions with changes in yield are illustrated for methanol as organic substrate.     

Modelling fungal solid-state fermentation: the role of inactivation kinetics

The theoretical mathematical models described in this paper are used to evaluate the effects of fungal biomass inactivation kinetics on a non-isothermal tray solid-state fermentation (SSF). The inactivation kinetics, derived from previously reported experiments done under isothermal conditions and using glucosamine content to represent the amount of biomass, are described in different ways leading to four models. The model predictions show only significant effects of inactivation kinetics on temperature and biomass patterns in the tray SSF after long fermentation periods. The models in which inactivation is triggered by low specific growth rates can predict restricted biomass evolution in combination with a fast temperature increase followed by a slower temperature decrease. Such inactivation might occur when substrate is limiting or products are formed in toxic concentrations. Temperature is predicted to be the key parameter. Oxygen concentration is predicted to become limiting only at high heat conduction and low oxygen diffusion rates. Desiccation of the substrate is predicted not to occur.     

Intensification of the exopolysaccharide synthesis by Acinetobacter sp. on an ethanol-glucose mixture: aspects related to biochemistry and bioenergetics

The possibility of intensifying the synthesis of microbial exopolysaccharides (EPS) by a strain of Acinetobacter sp. grown on a mixture of two substrates nonequivalent in terms of bioenergetics (ethanol + glucose) was shown. Based on theoretical calculations of the energy requirements for biomass and EPS synthesis from the energy-deficient substrate (glucose), the supplementary concentration of the energy-excessive substrate (ethanol) was determined that prevents the loss of glucose carbon that occurs when glucose is oxidized to CO2 to obtain energy for the processes of constructive metabolism. This made it possible to increase the efficiency of conversion of the substrate carbon to EPS. The introduction of ethanol into glucose-containing medium at a molar ratio of 3.1:1 allowed the amount of the EPS synthesized to be increased 1.8- to 1.9-fold; their yield relative to biomass increased 1.4- to 1.7-fold, and the yield of EPS relative to the substrate consumed increased 1.5- to 2-fold as compared to growth of the producer on single substrates. These results form the basis for the development of new technologies for obtaining secondary metabolites of practical value with the use of mixed growth substrates.     

Application of mass and energy balance regularities in fermentation

Material and energy balances for fermentation processes are developed 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. Mass–energy balance equations are developed which relate the biomass energetic yield coefficient to sets of variables which may be determined experimentally. Organic substrate consumption, biomass production, oxygen consumption, carbon dioxide production, heat evolution, and nitrogen consumption are considered as measured variables. Application of the balances using direct and indirect methods of yield coefficient estimation is illustrated using experimental results from the literature. Product formation is included in the balance equations and the effect of product formation on biomass yield estimates is examined. Application of mass–energy balances in the optimal operation of continuous single-cell protein production facilities is examined, and the variation of optimal operating conditions with changes in yield are illustrated for methanol as organic substrate.     

The calorimetric-respirometric ratio is an on-line marker of enthalpy efficiency of yeast cells growing on a non-fermentable carbon source

Although on-line calorimetry has been widely used to detect transitions in global metabolic activity during the growth of microorganisms, the relationships between oxygen consumption flux and heat production are poorly documented. In this work, we developed a respirometric and calorimetric approach to determine the enthalpy efficiency of respiration-linked energy transformation of isolated yeast mitochondria and yeast cells under growing and resting conditions. On isolated mitochondria, the analysis of different phosphorylating and non-phosphorylating steady states clearly showed that the simultaneous measurements of heat production and oxygen consumption rates can lead to the determination of both the enthalpy efficiency and the ATP/O yield of oxidative phosphorylation. However, these determinations were made possible only when the net enthalpy change associated with the phosphorylating system was different from zero. On whole yeast cells, it is shown that the simultaneous steady state measurements of the heat production and oxygen consumption rates allow the enthalpy growth efficiency (i.e. the amount of energy conserved as biomass compared to the energy utilised for complete catabolism plus anabolism) to be assessed. This method is based on the comparison between the calorimetric-respirometric ratio (CR ratio) determined under growth versus resting conditions during a purely aerobic metabolism. Therefore, in contrast to the enthalpy balance approach, this method does not rely on the exhaustive and tedious determinations of the metabolites and elemental composition of biomass. Thus, experiments can be performed in the presence of non-limiting amounts of carbon substrate, an approach which has been successfully applied to slow growing cells such as yeast cells expressing wild-type or a mutant rat uncoupling protein-1.