Comments on "In search of a thermodynamic description of biomass yields for the chemotrophic growth of microorganisms".

In a recent publication, Heijnen and van Dijken (Biotechnol. Bioeng. 39: 833-858, 1992) reviewed the state of the art regarding the use of macroscopic methods in the correlation of biomass yields in growing microorganisms. In their article, reference is made to this author's work of some 10 years ago.Heijnen and van Dijken introduce the Gibbs' energy dissipation as a concept with favorable characteristics compared with various other approaches, including thermodynamic efficiency, as introduced by this author.In this communication, it will be shown that the "dissipation" and the "thermodynamic efficiency" description are completely equivalent and that there can be no preference for one of these in terms of rigor or characteristics.

Fermentation kinetics of Zymomonas mobilis near zero growth rate.

The fermentation kinetics Zymomonas mobilis were studied near zero growth rate in fed-batch cultures and continuous cultures with complete cell recycle. The results show the ethanol enhances that specific substrate conversion rate under these conditions. The maximum achievable ethanol concentration in continuous cultures with cell recycle (66 g/L) was significantly lower than in fed-batch cultures (100 g/L). The results indicate that growth-rate-independent metabolism is not instantaneous and can lag behind steadily increasing ethanol concentrations in fed-batch fermentations. A model is proposed to account for this slow adaptation.

Fermentation kinetics of Zymomonas mobilis at high ethanol concentrations: Oscillations in continuous cultures.

Substrate-limited continuous culture results at 47 g/L ethanol show that the maintenance factor and the yield factor of an unstructured maintenance model are lower compared to the values at 23 g/L ethanol. Computing the results according to a structured two-compartment model predicts an enhanced turnover rate of the K-compartment (RNA fraction) by ethanol, resulting in a lower steady-state amount of K-compartment. This is in agreement with experimentally determined RNA fractions. The parameters of both models respond qualitatively in the same way to elevation of the ethanol concentration as to elevation of the temperature. In product-inhibited continuous cultures, at ethanol concentrations above 55 g/L, nearly sustained oscillations in biomass, substrate, and products were observed. The maximum ethanol concentration achieved in these continuous cultures was 70 g/L. The oscillations could be described by a structured mathematical model, in which ethanol inhibits the maximum specific growth rate indirectly by inhibiting the synthesis of an internal growth-rate-determining compound.

The inhibition of the maximum specific growth and fermentation rate of Zymomonas mobilis by ethanol.

The inhibition of the maximum specific growth and fermentation rate of Zymomonas mobilis by ethanol was studied in turbidostat cultures at constant and stepwise changed ethanol concentrations. Up to 50 g/L ethanol, the inhibition kinetics can be approximated by a linear relationship between the specific growth rate and the ethanol concentration. Above this level, deviations from this linearity are observed. The specific fermentation rates were less inhibited by ethanol than was the specific growth rate. The maximum ethanol concentration achieved was 72 g/L.The response time for the adaptation of a turbidostat culture to step changes in the ethanol concentration was markedly dependent on the concentration level, the response time being large at high ethanol concentrations.

Mathematical modelling of growth and substrate conversion of Zymomonas mobilis at 30 and 35 degrees C.

Zymomonas mobilis was grown in continuous cultures at 30 and 35 degrees C. The specific substrate consumption rates at 35 degrees C were higher than those at 30 degrees C. An unstructured mathematical model based on the linear equation for substrate consumption provided a statistically adequate description for cultures grown at 35 degrees C but not for cultures grown at 30 degrees C. A structured two-compartment model described growth and substrate consumption well at both temperatures. Some theoretical and practical aspects of the two-compartment model are discussed.

The growth of Saccharomyces cerevisiae CBS 426 on mixtures of glucose and succinic acid: A model.

Saccharomyces cerevisiae CBS 426 was grown in continuous culture in a defined medium with a mixture of glucose and succinic acid as the carbon source. Growth on succinic acid was possible after long adaptation periods. The flows of glucose, succinic acid, oxygen, carbon dioxide, and biomass to and from the system were measured. It proved necessary to expand our previous model to accommodate the active transport of succinic acid by the cell. The values found for the efficiency of the oxidative phosphorylation (P/O) and the amount of ATP needed for production of biomass from monomers gave the same values as found for substrate mixtures taken up passively.

Penicillin production: biotechnology at its best.

A brief description is given of the history of penicillin production in the Netherlands. The development of today's penicillin production technology is analysed in terms of changes in the quality and intensity of the production process. Technological as well as genetical developments are shown to be of influence on the quality and the intensity of the production process. The analysis is illustrated by a brief description of the productivity improvement of the penicillin fermentation as it occurred at Gist-brocades during the past 20 years.

Theory and applications of unstructured growth models: Kinetic and energetic aspects.

Microbial kinetics and energetics are discussed in connection with the formulation of unstructured growth models. The development of microbial energetics and the use of macroscopic methods in the study of microbial growth are briefly evaluated. The general approach to the modelling of microbial growth has been critically discussed and a strategy for the formulation of unstructured models is presented. A simple unstructured model based on Monod kinetics and the linear relation for substrate consumption is evaluated with reference to extensive experimental and simulation data obtained in batch, fed-batch, and continuous cultivation modes. Choice for a kinetic expression is discussed and has been shown not to be critical in most situations. It is shown that during growth in batch mode, the behavior of the system is rigidly fixed by the kinetic parameter: the maximum specific growth rate. The energetic parameters have minimal influence. In continuous cultivation the behavior is fixed by the energetic parameters: the maximum yield and the coefficient of maintenance. Implications of these observations have also been discussed. The linear relation for substrate consumption is tested with continuous culture data. It is shown that significant deviations at low growth rates cannot be fully accounted by the loss of viability. The situations where unstructured models will be adequate or not for system description, are evaluated and checked experimentally. Influence of an environmental factor, the temperature, on the unstructured model parameters is also quantitatively described. It is concluded that the art of unstructured model building has already reached its maturity and that now much effort should be channelled into the development and verification of structured models.

Modeling of bacterial growth; formulation and evaluation of a structured model.

Models which consider changes in the composition of biomass in response to environmental changes are called Structured models. They provide a more comprehensive description of microbial behavior than unstructured models. Compared with the unstructured modeling efforts, very little has so far been done on the theory and practice of structured model building. In most of the works reported so far, no experimental data were provided, and hence no means of testing the proposed models were offered. Others only reported macroscopic response data and not the cellular composition. In an attempt to fill some of the gaps in this field, in this work, first the general formal approach to structured modeling is developed in matrix notation. Then, a simple two-compartmental model, i.e., a structured model describing the activity of the biomass with two variables, is described. The cell is divided into two fractions, one of which relates to the RNA fraction. The proposed model was then critically evaluated with experimental data, including the RNA data, obtained from fed-batch and continuous-culture experiments. The importance of using cellular structure data for model verification, i.e., RNA data in this case, is shown. Shortcomings and capabilities of the developed model are discussed.

The growth of Saccharomyces cerevisiae CBS 426 on mixtures of glucose and ethanol: a model.

Saccharomyces cerevisiae CBS 426 was grown in continuous culture in a defined medium with a mixture of glucose and ethanol as carbon source. Growth on ethanol as the sole carbon source was only possible after the addition of a small amount of glutamic acid. The flows of glucose, ethanol, oxygen, carbon dioxide and biomass to and from the system were measured and a model for the growth of the yeast on the carbon sources constructed. The model is shown to allow independent estimation of YATP and P/O. YATP is not independent of the substrate used, but the amount of ATP used in the production of biomass from the monomers is approximately the same for growth on ethanol and on glucose.

A quantitative description of the growth of Saccharomyces cerevisiae CBS 426 on a mixed substrate of glucose and ethanol.

Saccharomyces cerevisiae CBS 426 was grown aerobically in continuous culture with a mixture of glucose and ethanol as the carbon source. The flows of biomass, glucose, ethanol, oxygen, and carbon dioxide were measured. A model for growth with two substrates was derived. Application of this model to the above-mentioned system yielded values for YATP and P/O. The joint confidence regions for these parameters were calculated. The relevance to industrial production of bakers' yeast is discussed.

Application of balancing methods in modeling the penicillin fermentation.

This paper shows the application of elementary balancing methods in combination with simple kinetic equations in the formulation of an unstructured model for the fed-batch process for the production of penicillin. The rate of substrate uptake is modeled with a Monod-type relationship. The specific penicillin production rate is assumed to be a function of growth rate. Hydrolysis of penicillin to penicilloic acid is assumed to be first order in penicillin. In simulations with the present model it is shown that the model, although assuming a strict relationship between specific growth rate and penicillin productivity, allows for the commonly observed lag phase in the penicillin concentration curve and the apparent separation between growth and production phase (idiophase-trophophase concept). Furthermore it is shown that the feed rate profile during fermentation is of vital importance in the realization of a high production rate throughout the duration of the fermentation. It is emphasized that the method of modeling presented may also prove rewarding for an analysis of fermentation processes other than the penicillin fermentation.