Structured Models of Metabolism and Growth 225 SPECIFIC OXYGEN CONSUMPTION Ima/9-08"1 SPECIFIC GROWTH RATE (ORV) Figure 3.21. Relationship between specific oxygen uptake rate relative to fresh weight and the specific growth rate of the fresh weight [from D. Drapeau, H. Blanch and C. Wilke, Biotech. Bioeng., 23, 1555 (1986)]. on SPECIFIC OIOSGENIN RATE (mg/o-08M1 ° da spectre ono mare far“ Figure 3.22. The specific diosgenin biosynthesis rate relative to fresh weight as a function of the specific growth rate of fresh weight (from D. Drapeau, H. Blanch and C, Wilke, Biotech. Bioeng., 23, 1555 (1986)). 3.5 Structured Models of Metabolism and Growth Unstructured models do not recognize the complex set of metabolic reactions occurring within the cell. Therefore we should not expect these simple models to be able to predict the dynamic behavior of cells subject to changing external conditions. In addition, unstructured models can predict intracellular concentrations only if we assume there is a constant fraction of the particular metabolite in the cell; for example, that the fraction of226 Structured Models of Metabolism and Growth RNA or DNA within a cell is constant. They thus have limited utility in guiding research aimed at understanding cellular regulation and dynamics. Such studies and the models derived from them are also required for implementation of sophisticated control of biological reactors or biological processes. A number of approaches have been developed to tackle the problem of developing more sophisticated models of cell metabolism and growth. We shall examine some simple approaches in the following sections. It is clear that attempting to model alll the reactions within a cell would be an extremely complex undertaking, particularly since we know rel- atively little about the kinetics and regulation of many of the enzyme-catalyzed steps involved. We can, however, group many of these reactions based on their characteristic time constants or relaxation times. The temporal organization within the cell can be described in terms of the following systems: Genetic system: the longest timescale, associated with changes in DNA composition due to random mutagenesis, mutagenesis arising in response to changing environmental parameters, or natural genetic transfer of new characteristics via a vector such as a virus. This timescale is in multiples of the organism's generation time. Epigenetic system: the intermediate timescale and is related to DNA transcription and translation. Enzyme induction (synthesis) and repression occur over this timescale. Metabolic system: the short timescale of individual enzyme reactions. Usually much shorter than the epigenetic system. The behavior of the genetic and epigenetic systems may be considered constant over the period of observation of metabolic events. In most situations, there is sufficient difference between these timescales that the behavior of other systems can be considered constant. For example, if we are examining the substrate and product concentration changes of a particular enzyme-catalyzed reaction within the cell, the enzyme concentration can be considered constant over the time period of our observations. Changing enzyme concentrations involves transcription and translation of DNA onatimescale of order tens of minutes or hours, while the enzyme-catalyzed reaction may have a relaxation time of order seconds or minutes. On the other hand, if changes occurring in the external environment are on the timescale of the epigenetic system, the behavior of the metabolic system will be very fast and we can consider it to be in a quasi- steady state. This approach greatly simplifies model development. Models which incorporate the details of intracellular metabolism are referred to as structured models. Such models attempt to account for unbalanced growth of microor- ganisms, i.e., when the composition of the major cellular constituents, such as RNA, enzyme concentrations etc. vary as a result of changing external conditions. Such conditions apply in batch growth, in fed-batch growth, and in transient situations in well-stirred tank reactors. The transient responses of cells to these changing external conditions can be modelledStructured Models of Metabolism and Growth 227 by analogy with classical reactor modelling using the transfer function approach. By using an appropriate forcing function and determining the transient response of the cells, the behavior of various cellular constituents can be modelled as first or higher order’. This approach has some advantages in developing and analyzing strategies for process control, but does not provide much insight into the factors that regulate metabolism. We shall now turn our attention to models which incorporate more of the features of microbial metabolism. 3.5.1 Compartmental Models The earliest attempts to include structure in models of cell growth and metabolism generally subdivided the cell mass into various components on the basis of the function of parts of the cell's internal machinery, We shall examine a simple two-compartment model to illustrate the features of this approach. The Model of Williams” The model of Williams divides the cell into two compartments, a synthetic one (k- compartment) that we can consider as consisting of RNA and pools of small metabolites, and a genetic one (g-compartment) consisting of DNA and protein. The third component is the external substrate concentration. A simple model based on these compartments can be developed as follows. If K and G are the concentrations of the components in the k- and g-compartments, written as mass per unit cell volume (V,), we can write mass balances for aconstant reactor volume (V,) as follows. A HT hSKVe ie. ap Thx (3.78a) dSV,_ 1 ds i £ 7 hx¥e ie. a (3.786) The rate of substrate uptake is assumed to be first order in substrate concentration S and in total cell concentration X (both S and X being expressed as mass/reactor volume). We assume that the structural-genetic compartment material is produced from the synthetic compartment at a rate that depends directly on the concentration of species in each com- partment. The mass balances are based on the reactor volume, thus the concentration per cell must be multiplied by the total cell volume per unit reactor volume, X/p,, where p, is the cell density (cell mass per unit cell volume). (31) See for example Young, T.B., Bruley, D.F. and H.R, Bungay, Biotech. Bioeng. 12, 747 (1970). (32) A review of some of the many compartment models is provided in Harder, A. and J.A. Roels, Adv. Bio- chem, Engng., 21, 55 (1982). (33) Williams, F.M., J. Theoret. Biol. 15, 190 (1967)228 Structured Models of Metabolism and Growth dG(X/p.) dt Expanding the left hand side yields =kGK(X/p,) a(Xip.) dt (Xip.) So +G =k,GK(X/p,) dG 1dX ie k,GK-G Xa and combining this result with Eq. (3.78a) dG pT hGK ~ (kG (3.79) We have assumed that the density of the cell, p, , is constant. The synthetic portion of the biomass is produced at a rate that is first order in substrate concentration and depends on the cell density p, (equal to the sum of K and G): dKX/p, Go k,S(G +K) (Xip.) —kGK(X/p,) dK Lax . Lax | GF TRSG+K)-KGK-KYF and noting that ¢ 7 =kS IK wk $0,-hGK-kKS (3.80) We note that equations (3.79) and (3.80) can be added to show that the relationship below holds: dK 4G i 5p.-kS{K+G)=0 (as K+G=p,) 81) dt de BSP e . Equations (3.78) and (3.79) can be added to eliminate X d(X +¥,8) ease a0) thus X4Y,5 =Xq4 ¥5p (3.82) dt Equation (3.78) can now be solved for $ +38) ma Lara (3.83) 1 +7 exp{k (Xo VsSuV Ys) If we assume that the cell number depends only on the amount of genetic component present (ie., G), then the cell number will be proportional to GX/p, cells/reactor volume. The cell volume will change as a reflection of the changing amounts in the genetic and synthetic compartments, hence the cell sizé will be proportional to (K+G)/G, ie. p,/G. The behavior of the model is shown in Figure 3.23.Structured Models of Metabolism and Growth 29 batch growth 2.000 1.500! coll size 5 g 2 : 1.000 5 € 3 cell mass 0.500 substrate 0.000 + 0 20 40 60 80 100 Time Figure 3.23. Simulation of the two-compartment model of Williams for batch growth with the following initial conditions: X,= 0.05 mg/ml, S>= 1.0 mg/ml, Ky= 0.6 mg/ml, Gy= 0.4 mg/ml. Time is expressed in hours, concentrations in mg/ml of cell (for g-mass) or reactor volume (for cell mass and substrate). Values of the constants are k,= 0.0125 (mg/ml)'.hr', k,= 0.025 (mg/ml)''.hr', ¥=0.5 mg/mg, p, = 1.0 gm/ml. Cell number is proportional to the g-mass. The compartment model of Williams illustrates some important properties of cell growth, It predicts the existence of a initial lag phase (see the cell mass curve in Figure (3.23) and, if the inoculum is not fully adapted (ie. Ky and Gy do not correspond to values found in exponential growth), cell mass will increase (due to an increase in the K com- partment), while the cell number will not change initially (the cell density p, remains constant). During the growth phase, both cell mass and cell numbers increase exponentially The stationary phase is attained with respect to cell mass prior to cell number (as illustrated in Figure (3.23) by the curves of g-mass and cell mass). These phenomena are typically observed in batch culture experiments with bacteria. The model can be refined by changing the linear dependence of the rate of substrate uptake and growth from first order to a Monod type in equation (3.78). As can be seen from the batch simulations above, the concentration of the g-compartment approaches the cell density pat long times, while that-of the k-compartment approaches zero. Neither of these predictions is consistent with experimental observation. The inclusion of maintenance in the formulation of the model would change this inconsistent result and improve the model.230 Structured Models of Metabolism and Growth The Model of Ramkrishna et al. An analogous compartment model to that of Williams has been developed by Ram- krishna et al. The cell is divided into two compartments, G-mass, comprising RNA and DNA, and D-mass, which mostly consists of proteins. An inhibitor (T) is produced during growth which converts both G- and D-mass to inactive forms of biomass. The reactions assumed are the following: G+a,S+D32G+D+a,T G+a,/5+D 52D+G+a,'T GtTNgt(It+a,)T D+T Ny +(1+ay/)T In the first reaction, D-mass catalyzes the formation of G-mass, consuming a, units of substrate and producing a units of an unidentified inhibitor T. In the second reaction G-mass catalyzes the formation of D-mass. The last two equations show that both G- and D-mass are deactivated by inhibitor (producing inactive forms Ng and Np). The rate expressions assumed in the model for production of G- and D-mass are of the double substrate form of equation (3.44), while those for the deactivation reactions are assumed to be first order in each reactant. Mass balance equations for batch and continuous stirred tank reactors can be written and the model predictions can be compared with experimental data. This model can predict oscillatory behavior about a steady state. 3.5.2 Models of Cellular Energetics and Metabolism As we move beyond the compartmental models of the type above, the level of metabolic detail required increases. At this point, it will be useful to consider the types of reactions that occur within the cell. Metabolic pathways can be distinguished as catabolic and ana- bolic. In catabolism, energy-containing molecules, such as carbohydrates, hydrocarbons and other reduced carbon-containing compounds are degraded to CO, or other oxidized end-products and the energy is stored in ATP, GTP, and other energy-rich compounds. In anabolism, intermediates and end-products formed from catabolism are incorporated into cell constituents (such as DNA, RNA, lipids, carbohydrates, etc.) and their intermediate precursors (amino acids, purines and pyrimidines, simple sugars etc.). Anabolic reactions generally require energy, which is supplied via ATP and other high-energy phosphates generated during catabolism. As the concentration of these high-energy intermediates within the cell is rather small, anabolism is linked to catabolism and ATP is rapidly turned over. This implies that energy producing and energy consuming processes must be tightly regu- (34) Ramkrishna, D., Fredrickson, A.G. and H. Tsuchiya, Biotech, Bioeng., 9, 129 (1967),