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 carbon catabolism on the auxiliary substrate effect,Paper given at the Reinhardsbrunn Symposium “Phyusiology of Microbial Growth and Differentiation”, May 20–26, 1984

Although the carbon/energy ratios of heterotrophic substrates for microbial growth are different this is not reflected in biomass. Nevertheless the macromolecular composition of cells may vary in dependence on growth conditions this does hardly influence the elementary composition and the growth yield. The energy requirement for synthesis of biomass starting from a central precursor, e.g. phosphoglycerate, can be assumed to be constant, hence any differences in carbon conversion efficiency must be attributed to carbon catabolism up to this precursor. This sequence determines if and to what extent an auxiliary substrate effect is possible. However, one has also to consider changes of the P/O ratio due to simultaneous utilization of substratesd which may account for the increase in growth yield with Hansenula polymorpha growing on a methanol/glucose mixture.     

The Auxiliary Substrate Concept: From simple considerations to heuristically valuable knowledge

Microorganisms are used in biotechnology. They are either (i) aim and purpose of a process, e.g. with the production of single cell proteins, or (ii) mean to an end insofar as they serve as a catalyst or “factory” for syntheses (e.g. of products of primary and secondary metabolism, of enzymes and antibiotics) or for the degradation and detoxification of harmful organics and inorganics. In all cases, the efficiency and velocity, finally the productivity, are parameters which essentially determine the economy of the processes. Therefore, search for approaches to optimize these processes is a permanent task and challenge for scientists and engineers. It is shown that the auxiliary substrate concept is suitable to increase the yield coefficients. It is based on the energetic evaluation of organics, on the knowledge that organics as sources of carbon and energy for growth are deficient in ATP and/or reducing equivalents, and says that it is possible to improve the carbon conversion efficiency up to the carbon metabolism determined upper limit. The latter is determined by inevitable losses of carbon along the way of assimilation and anabolism and amounts to about 85% for so‐called glycolytic substrates, e.g. glucose, methanol, and to about 75% for gluconeogenetic substrates, e.g. C₂‐substrates (acetic acid, hexadecane). The approach is explained and some experimental examples are presented. By simultaneous utilization of an extra energy source (auxiliary substrate) the yield coefficient can be increased (i) in glucose from about 0.5 to 0.7 g/g (by means of formate), (ii) in acetate from 0.34–0.4 to 0.5–0.65 g/g (by means of formate and thiosulfate, respectively), and (iii) in hexadecane from about 0.94 to 1.26 g/g (by means of formate). The precalculated yield coefficients and mixing ratios agree well with the experimentally attained ones. The approach is easily feasible and economically valuable.     

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.     

Intensification of 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.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas I. Growth Yields and Patterns Under Dual Substrate Conditions

Auxotrophic mutants of Hydrogenomonas eutropha and H. facilis requiring utilizable amino acids were employed to demonstrate the simultaneous utilization of H(2) and an organic substrate for growth. The ratio of the cell yields under dual substrate conditions compared to heterotrophic conditions indicated the relative contributions of the autotrophic and heterotrophic systems to the growth of the organism. Wildtype H. eutropha grown under simultaneous conditions exhibited a dicyclic growth pattern, the first cycle representing either heterotrophic or simultaneous growth and the second cycle representing autotrophic growth. The duration of the changeover period was either very short with no plateau or long with a plateau up to 8 hr, depending upon the organic substrate. The growth rate under simultaneous conditions with some organic substrates was faster than either the autotrophic or heterotrophic rate, but was not the sum of the two rates. The data suggest that, in the presence of both organic and inorganic substrates, heterotrophic metabolism functions normally but autotrophic metabolism is partially repressed.     

Global physiological analysis of carbon- and energy-limited growing Escherichia coli confirms a high degree of catabolic flexibility and preparedness for mixed substrate utilization

Growth conditions for heterotrophic bacteria in the environment are characterized by low concentrations of carbon and energy sources and complex substrate mixtures. While mechanisms of starvation-survival in the absence of carbon substrates have been studied in considerable detail, information on the physiology of slow growth under oligotrophic conditions is limited. We intended to elucidate general strategies by which Escherichia coli adapts to low concentrations of a mixed carbon and energy source pool. A new screening method based on BIOLOG AN MicroPlates, which allowed us to distinguish repressed and induced catabolic functions in E. coli, was combined with the analysis of periplasmic high-affinity binding proteins. Extending previous findings for E. coli and other microbial species, we found that numerous alternative catabolic functions and high-affinity binding proteins are derepressed under either glucose- or arabinose-limited growth conditions, in spite of the absence of the respective inducers. Escherichia coli cells growing in carbon-limited complex medium chemostat cultures exhibited an even higher degree of catabolic flexibility and were able to oxidize 43 substrates. The BIOLOG respiration pattern indicated simultaneous dissimilation of diverse sugars, amino acids and dipeptides (mixed substrate growth). The observed physiological adaptations of E. coli to low concentrations of carbon and energy substrates presumably are advantageous in many natural growth situations and also offer an explanation why many heterotrophic bacteria have and maintain such a broad carbon substrate range.     

Improvement of Y-values of Hansenula polymorpha growth on methanol by simultaneous utilization of glucose

The simultaneous utilization of methanol and glucose by Hansenula polymorpha MH20 was investigated in chemostat (C-limited) cultivation. The mixed-substrate utilization results in biomass yields which are greater up to 20 to 25% as expected assuming an additive growth on both substrates. This is referred to as an auxiliary-substrate effect. Additionally, methanol can be utilized at higher growth rates in the presence of glucose compared to those obtained on this substrate alone. The extend of the auxiliary-substrate effect and the optimum ratio of substrates to reach this effect depend on dilution rate. The greatest stimulation in yield is obtained at D approximately 0.1 h-1, after raising the dilution rate this effect diminishes. At a rate of 0.1 h-1 the optimum mixed-substrate ratio of methanol: glucose is 7:1 (g). By increasing the growth rate the ratio changes toward glucose and reached a value of 1:1 (g) at D = 0.3 h-1. This change in the optimum ratio correlates with diminution in yield coefficient of methanol accompanying an increase in growth rate greater than 0.15 h-1. Energy balances of the utilization of the single substrates are used for interpretation of these results. From this it is evident that methanol does not play the role of an energy-rich substrate in the metabolism of yeast. Rather glucose is the energy-providing substrate in this combination.     

Thermodynamic evaluation of energy metabolism in mixed substrate catabolism: modeling studies of stationary and oscillatory states

Thermodynamic and kinetic calculations were performed in a model of mixed substrate metabolism. The model simulates the catabolic breakdown of a first substrate, glucose (S(1)), in the presence of a second substrate, formate (S(2)), which acts as an additional source of free energy. The principal results obtained with different relative rates of uptake of S(2) allow to predict and interpret the following experimental observations: (1) the existence of increased ATP yields by mixed substrate utilization with a maximum ATP yield and optimum input (or molar) ratio for both substrates; (2) a greater assimilation of S(1) which may be interpreted as a decreasing fraction of energy required for assimilation; (3) a decrease in ATP yields due to increasing energy demand for transport; (4) an increased assimilation of the carbon source (S(1)) as a function of increasing inputs of the additional energy source; (5) thermodynamic efficiency (eta) defined as the ratio between the output power of ATP synthesis and the input catabolic power, increases for S(2)/S(1) ratios ranging between 0.08 and 2 while for ratios higher than two a slight decrease of eta was noticed; and (6) the observed maximum in ATP yield for optimum molar ratio of the two substrates corresponds to high eta predicting that higher biomass yields may be obtained through a variable, high, eta by chanelling fluxes through catabolic pathways with different ATP yields. During oscillatory behavior, maxima in fluxes were almost coincident with maxima in forces. Thus, the pattern of dissipation was not so advantageous as in the single substrate model under starvation conditions.     

Kinetics and Yields of Pesticide Biodegradation at Low Substrate Concentrations and under Conditions Restricting Assimilable Organic Carbon

The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter⁻¹. We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.     

Metabolic regulation in bacterial continuous cultures: II

The transient behavior of a continuous culture of Klebsiella pneumoniae with mixed feed of glucose and xylose arising from step-up and step-down in dilution rates and from a feed-switching experiment is presented. he organism gradually switches from simultaneous utilization of the substrates at low growth rates to preferred utilization of the faster substrate (i.e, supporting a higher growth rate) at high dilution rates. The metabolic lags following a step increase in dilution rate and a significant accumulation of the slower substrate during the transient period result from the effects of metabolic regulation. The cybernetic modeling approach that successfully described the foregoing situations with single-substrate feeds is employed to describe mixed substrate behavior. The parameters in the mixed-substrate (glucose and xylose) model are the same as those in the single-substrate models with the singular exception of the rate constant for the xylose growth enzyme synthesis. The reason for this discrepancy is discussed in detail. It appears that the constitutive rate of enzyme synthesis for growth on a given substrate may be related to the past history of the organism in regard to whether or not the organism has been exposed to the particular substrate. Thus, the results further demonstrate the ability of the framework to effectively describe metabolic regulation in batch, fedbatch, and continuous microbial cultures.     

Cybernetic modeling of microbial growth on multiple substrates

The internal regulatory processes, which underlie a variety of behavior in microbial growth on multiple substrates, are viewed as a manifestation of an invariant strategy to optimize some goal of the cells. A goal-seeking or cybernetic model is proposed here, with the optimization obased on a short-term perspective of response to the environment. The model parameters are determined from the growth data on single substrates. The model predicts the entire range of microbial growth behavior on multiple substrates from simultaneous utilization of all sugars to sequential utilization with pronounced diauxic lags. It is shown to predict the many variations of the diauxic phenomenon in different growth conditions. The transients in continuous culture growth on mixed substrates caused by varying the feed strategies are easily simulated by this model. The framework of this model can be applied to batch or continuous culture growth of many bacteria on different combinations of substrates.     

Effect of fluid dispersion on cybernetic control of microbial growth on substitutable substrates

Many fermentation media contain two or more substrates, which a microorganism utilizes for similar purposes. Depending on the conditions prior to and during a fermentation, the substrates may be utilized in succession or simultaneously. Since it is difficult to portray this behavior through mechanistic models, a cybernetic method was proposed earlier. Here the microorganism chooses the mode of substrate utilization that maximizes its own survival, usually expressed by the growth rate. In a fully dispersed bioreactor, simultaneous utilization generates higher growth rates but leads to low biomass concentrations since this utilization pattern is preferred at low concentrations of the substrates. In this study it has been shown that by allowing less than complete dispersion in the broth it is possible to shift from sequential to simultaneous utilization at high concentrations, thereby enabling both high growth rates and large biomass concentrations. This strategy thus allows the natural incomplete dispersion in large bioreactors to be gainfully exploited.     

Modeling bisubstrate removal by biofilms

A bisubstrate secondary utilization model is based on the concept that an individual substrate can be utilized not only by the biomass by its utilization but also by the biomass made from the utilization of the other substrate. When substrate concentrations are low, a key factor is having sufficient substrate to initiate biofilm growth. Modeling results for three characteristic cases demonstrate that satisfying a total S(min) concentration for a bisubstrate system is the necessary condition for initiating biofilm growth and simultaneous utilization of both substrates. Because having more than one substrate supporting biofilm growth enhances the removal of each compound, the utilization rate of a specific compound can be increased by the concentration of other compounds, and the total S(min) concentration can be less than the weighted average of individual S(min) values.     

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems

Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.     

Some theoretical considerations on overflow production of metabolites

The overflow production of metabolites appears to be an energy spilling process in terms of life because part of the energy of the primary substrate remains in the metabolite produced. The other part of energy, which is liberated as reducing equivalents and/or ATP along the way to the product, must be wasted. This part is discussed to be responsible for the discrepancies between the theoretically possible and experimentally obtained product yields, because for the wasting process substrate or product are consumed. By reducing the amount of this superfluous energy the product yield should be increased. The auxiliary substrate concept occurs to be an appropriate method.     

Formate as an auxiliary substrate for glucose-limited cultivation of Penicillium chrysogenum: impact on penicillin G production and biomass yield

Production of beta-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol.mol(-1), an increasing rate of formate oxidation via a cytosolic NAD(+)-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol.mol(-1), the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as beta-lactams.     

Are microbes intelligent beings?: An assessment of cybernetic modeling

Microorganisms growing in a multi-substrate medium have different and varying preferences for the various components of the medium. The preferences depend on the operating conditions and the substrates may be utilized sequentially or simultaneously. Sometimes an organism may change its preferences among substrates and/or switch between sequential and simultaneous utilization. These aspects are difficult to describe through models based on chemical and physical laws alone. Cybernetic modeling ascribes to microorganisms the ability to perceive their environment (i.e. the growth medium) and make 'intelligent' choices regarding substrate utilization to maximize an objective, which is usually the growth rate. This article reviews the development of cybernetic modeling since it began in 1982. Different workers have suggested different perspectives of how microbes make optimal use of their resources. These are discussed and future directions for improvement are indicated.