Estimating cell and product yields

The balance equations for carbon, reduction potential, and energy during cell growth and product formation are rederived in a general form. Cells are treated simply as a very complex product, and the Y(ATP) concept is extended to products. Limitations on the theoretical yield are discussed for different product types. Simple aerobic products cannot be energy limited unless the maintenance requirement is large, while complex products cannot be reduction limited. A maximum yield is defined for products much more oxidized than their substrate (carbon limited) because the theoretical yield conditions may violate the energy balance. For reduced complex products the yield on available electrons is related to Y(ATP), the P/O ratio, and the product composition. Narrow bounds are established on the actual yields in simple anaerobic fermentations, and the significance of the yields in the linear growth equation is discussed.     

Modeling aerobic carbon oxidation and storage by integrating respirometric, titrimetric, and off-gas CO2 measurements

A method for detailed investigation of aerobic carbon degradation processes by microorganisms is presented. The method relies on an integrated use of the respirometric, titrimetric, and off-gas CO(2) measurements. The oxygen uptake rate (OUR), hydrogen ion production rate (HPR), and the carbon dioxide transfer rate (CTR) resulting from the biological as well as physicochemical processes, coupled with a metabolic model characterizing both the growth and carbon storage processes, enables the comprehensive study of the carbon degradation processes. The method allows the formation of carbon storage products and the biomass growth rates to be estimated without requiring any off-line biomass or liquid-phase measurements, although the practical identifiability of the system could be improved with additional measurements. Furthermore, the combined yield for biomass growth and carbon storage is identifiable, along with the affinity constant with respect to the carbon substrate. However, the individual yields for growth and carbon storage are not identifiable without further knowledge about the metabolic pathways employed by the microorganisms in the carbon conversion. This is true even when more process variables are measured. The method is applied to the aerobic carbon substrate degradation by a full-scale sludge using acetate as an example carbon source. The sludge was able to quickly take up the substrate and store it as poly-beta-hydroxybutyrate (PHB). The PHB formation rate was a few times faster than the biomass growth rate, which was confirmed by off-line liquid- and solid-phase analysis. The estimated combined yield for biomass growth and carbon storage compared closely to that determined from the theoretical yields reported in literature based on thermodynamics. This suggests that the theoretical yields may be used as default parameters for modeling purposes.     

Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Y(psm') g/mol) are 80 < Y(psm) < 95 when product is formed from glucose and 50 < Y(psm) < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO(2) production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Y(spm). Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.     

The auxiliary substrate concept — an approach for overcoming limits of microbial performances

In biotechnological processes, fundamental performances of microorganisms are used. The economy of these processes is essentially determined by the efficiency, velocity (productivity) and quality of the products. Therefore it is a permanent task and challenge for basic and biotechnological research to seek out measures for improving the actually attained parameters. The auxiliary substrate concept supplics an approach. It is based on the fact that chemo-organo-heterotrophic substrates differ in the carbon: energy ratio, thus, growth yield is limited in energy and/or reducing power. It says that, by simultaneous utilization of physiologically similar substrates (mixed substrates), the growth yield increases. The substrates are to combine in such a way that with their simultaneous utilization a minimum of carbon is dissimilated merely for the purpose of the generation of biologically useful energy and/or reducing power. Since all chemo-organo-heterotrophic substrates are more or less energy-deficient, an increase in growth efficiency can be expected if the individual substrates of the mixture are assimilated more efficiently than the respective substrates alone. This may result, for instance, from an immediate assimilation of a substrate (according to the “manner of finished part construction”). An increased growth rate is rather the rule than the exception in mixed substrate utilization. In product syntheses the substrates are, depending on the concrete product and metabolic pathway, either energy-excess or energy-excess or energy-deficient. or, in other words, the processes are energy-generating or energy-consuming, respectively. If this is responsible for discrepancies between the possible yields determined by the carbon metabolism and the experimentally obtained yields, the discrepancies should be able to be decreased and the yields increased by mixing substrates. The substrates are to choose and combine so that, due to simultaneous utilization, the product formation process becomes energy neutral. As a rule, the enhanced efficiency is accompanied by an increased velocity. This does not only apply to syntheses, but also to degradation (and detoxification) reactions. Even supposedly inert compounds or persistent substances can be activated by simultaneous (co-)metabolization of another (an auxiliary substrate, victim substrate or co-substrate) and converted at a considerable rate. It is of interest for syntheses of products but in particular for degradation and decontamination of harmful and waste products in the environment that the residual concentrations of the substrates are smaller than those achieved if the compounds of a mixture are metabolized separately. The auxiliary substrate concept has proven to be fruitful, both for theoretical and practical questions. It was practically already being used before it was formulated (mixed substrate utilization, cometabolism). However, an abundance of regulatory and energetic aspects are waiting to be investigated in more detail.     

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.     

Metabolic capacity of Bacillus subtilis for the production of purine nucleosides, riboflavin, and folic acid

We developed a stoichiometric model of Bacillus subtilis metabolism for quantitative analysis of theoretical growth and biochemicals production capacity. This work concentrated on biochemicals that are derived from the purine biosynthesis pathway; inosine, guanosine, riboflavin, and folic acid. These are examples of commercially relevant biochemicals for which Bacillus species are commonly used production hosts. Two previously unrecognized, but highly desirable properties of good producers of purine pathway-related biochemicals have been identified for optimally engineered product biosynthesis; high capacity for reoxidation of NADPH and high bioenergetic efficiency. Reoxidation of NADPH, through the transhydrogenase or otherwise, appears to be particularly important for growth on glucose, as deduced from the corresponding optimal carbon flux distribution. The importance of cellular energetics on optimal performance was quantitatively assessed by including a bioenergetic efficiency parameter as an unrestricted, ATP dissipating flux in the simulations. An estimate for the bioenergetic efficiency was generated by fitting the model to experimentally determined growth yields. The results show that the maximum theoretical yields of all products studied are limited by pathway stoichiometry at high bioenergetic efficiencies. Simulations with the estimated bioenergetic efficiency of B. subtilis, growing under glucose-limiting conditions, indicate that the yield of these biochemicals is primarily limited by energy and thus is very sensitive to the process conditions. The maximum yields that can reasonably be expected with B. subtilis on glucose were estimated to be 0.343, 0.160, and 0.161 (mol product/mol glucose) for purine nucleosides, riboflavin, and folic acid, respectively. Potential strategies for improving these maximum yields are discussed.     

A metabolic network stoichiometry analysis of microbial growth and product formation

Using available biochemical information, metabolic networks have been constructed to describe the biochemistry of growth of Saccharomyces cerevisiae and Candida utilis on a wide variety of carbon substrates. All networks contained only two fitted parameters, the P/O ratio and a maintenance coefficient. It is shown that with a growth-associated maintenance coefficient, K, of 1.37 mol ATP/ C-mol protein for both yeasts and P/O ratios of 1.20 and 1.53 for S. cerevisiae and C. utilis, respectively, measured biomass yields could be described accurately. A metabolic flux analysis of aerobic growth of S. cerevisiae on glucose/ethanol mixtures predicted five different metabolic flux regimes upon transition from 100% glucose to 100% ethanol. The metabolic network constructed for growth of S. cerevisiae on glucose was applied to perform a theoretical exercise on the overproduction of amino acids. It is shown that theoretical operational product yield values can be substantially lower than calculated maximum product yields. A practical case of lysine production was analyzed with respect to theoretical bottlenecks limiting product formation. Predictions of network-derived irreversibility limits for Y(sp) (mu) functions were compared with literature data. The comparisons show that in real systems such irreversibility constraints may be of relevance. It is concluded that analysis of metabolic network stoichiometry is a useful tool to detect metabolic limits and to guide process intensification studies. (c) 1995 John Wiley & Sons, Inc.     

Production of crystalline surface-active glycolipids by a strain of torulopsis apicola

The conditions for the production of extracellular glycolipid with Torulopsis apicola IMET 43747 have been investigated. Different culture conditions resulted in the production of either water-soluble or crude crystalline glycolipids. Growth was always accompanied by a strong decrease of the pH-value in the reaction medium. Cultivation at pH-value below 2 yielded the water-soluble product. The addition of either sodium citrate or sodium hydroxide to correct the pH-value to 3, resulted in the formation of large amounts of crystalline glycolipids. Depending on the kind of carbon source and its relative concentration, the product concentration was 5–90 g 1⁻¹ with maximal yields of 0.46 g g⁻¹ (product per substrate) after growth on a mixture of plant oils and glucose. The crude crystalline glycolipid mixture can be separated from the culture medium by filtration. It is composed of 80% of one major crystalline glycolipid and different minor compounds. Purification by liquid chromatography on silica gel yields the pure compound. This main product is a nonionic glycolipid with remarkable interfacial activities.     

Proteomic and physiological experiments to test Thermotoga neapolitana constraint-based model hypotheses of carbon source utilization

Constraint-based models of biochemical reaction networks require experimental validation to test model-derived hypotheses and iteratively improve the model. Physiological and proteomic analysis of Thermotoga neapolitana growth on cellotetraose was conducted to identify gene products related to growth on cellotetraose to improve a constraint-based model of T. neapolitana central carbon metabolism with incomplete cellotetraose pathways. In physiological experiments comparing cellotetraose to cellobiose and glucose as growth substrates, product formation yields on cellotetraose, cellobiose, and glucose were similar; however cell yields per mol carbon consumed were higher on cellotetraose than on cellobiose or glucose. Proteomic analysis showed increased expression of several proteins from cells grown on cellotetraose compared with glucose cell cultures, including cellobiose phosphorylase (CTN_0783), endo-1,4-β-glucosidase (CTN_1106), and an ATP-binding protein (CTN_1296). The CTN_1296 gene product should be evaluated further for participation in cellotetraose metabolism and is included as one of two hypothetical gene-protein-reaction associations in the T. neapolitana constraint-based model to reinstate cellotetraose metabolism in model simulations.     

Methanol bioconversion by Butyribacterium methylotrophicum--batch fermentation yield and kinetics

Butyribacterium methylotrophicum is an anaerobic bacterium that can convert methanol to butyrate. This ability to produce longer-chain carbon compounds from C(1) substrates could be of commercial significance. The fermentation rates and product formation depend on the methanol/bicarbonate ratios during fermentation. The kinetics of batch fermentation fit the Luedeking-Piret model with growth and maintenance associated product formation. Butyrate yield of 0.256 mol/mol methanol (ca. 85% of theoretical yield) has been obtained in batch fermentation.     

Metabolic study of the adaptation of the yeast Candida guilliermondii to sugarcane bagasse hydrolysate

Batch xylitol production from concentrated sugarcane bagasse hydrolysate by Candida guilliermondii was performed by progressively adapting the cells to the medium. Samples were analyzed to monitor sugar and acetic acid consumption, xylitol, arabitol, ethanol, and carbon dioxide production, as well as cell growth. Both xylitol yield and volumetric productivity remarkably increased with the number of adaptations, demonstrating that the more adapted the cells, the better the capacity of the yeast to reduce xylose to xylitol in hemicellulose hydrolysates. Substrate and product concentrations were used in carbon material balances to study in which way the different carbon sources were utilized by this yeast under microaerobic conditions, as well as to shed light on the effect of the progressive adaptation to the medium on its fermentative activity. Such a theoretical means allowed estimation for the first time of the relative contribution of each medium component to the formation of the main products of this fermentation system.     

Glucose and carbon dioxide metabolism by Succinivibrio dextrinosolvens.

Growth rates and culture conditions affect the molar yields of catabolic end products and cells of Succinivibrio dextrinosolvens growing on glucose. When growth in chemostats occurred, a trend toward decreased succinate and acetate formation, increased lactate formation, and a higher yield of cells correlated with an increase in the growth rate. End product and cellular yields on defined medium indicate a high maintenance requirement for S. dextrinosolvens and are consistent with energy conservation steps during the formation of acetate and succinate. Simultaneous carbon dioxide consumption and production were determined from batch studies with NaH14CO3, and the amounts were used to calculate a fermentation balance. These data also indicated that CO2 consumption lags behind CO2 production early in the growth phase, becoming equivalent to it toward stationary phase. Significantly more CO2 was fixed by S. dextrinosolvens when the organism was cultured in chemostats sparged with CO2. Formate is in part derived from free CO2 in the medium, as shown by 13C nuclear magnetic resonance studies, and may be sensitive to CO2 availability. Nuclear magnetic resonance data are consistent with the carboxylation of a C3 intermediate of the Embden-Meyerhof-Parnas pathway of glycolysis to a C4 compound to eventually form succinate.     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Analysis of exponential growth data for yoghurt cultures

Obtaining accurate estimates of maximum specific growth rate, growth yield, and product yield is important for many fermentation processes. A systematic procedure is presented to select the exponential growth region and estimate the maximum specific growth rate using the covariate adjustment method with all the available measured variables (i.e. biomass, substrate, and product). The procedure is applied to data collected during growth of pure and mixed cultures of Lactobacillus bulgaricus and Streptococcus thermophilus on 3% dry milk under anaerobic conditions. The estimation procedure gives good estimates with relatively narrow confidence intervals even though biomass concentration is measured by an indirect method. The estimated values of maximum specific growth rate range from 0.2805 h(-1) for S. thermophilus (ATCC-19258) to 0.4672 h(-1) for S. thermophilus (Microlife). Growth and product yields are estimated using regression analysis and the data for the exponential growth region. The growth yields are compared to their theoretical maximum values.     

Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.     

The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P(i) was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

Stoichiometric balance based macrokinetic model for Penicillium chrysogenum in fed-batch fermentation

A macrokinetic model describing mycelium growth and product formation in fed-batch fermentation of Penicillium chrysogenum is proposed on the basis of intracellular metabolic pathway networks found in the literature. The model is composed of the stoichiometric balance equations for carbon source, ATP, NADH and pyruvate. A regulator model is used as an auxiliary part to simulate the lag phase at the beginning of product synthesis. Combining the macrokinetic model with the bioreactor model, one yields the relationship between manipulating variables, i.e., the substrate feeding rate, and the other state variables such as biomass, substrate and product concentrations. The rolling parameter identification is then applied to online identify the most sensitive model parameters dealing with the model mismatch caused by time-variant kinetic changes. Model validation was performed with the data of 22 batches from industrial-scale penicillin cultivation. Finally, the predictions of the product concentration, up to 24 h ahead, are carried out with an average prediction error of less than 3.5%.     

CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli

Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.     

Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation.

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.