82 — 4 Kinotles — finding quantities for bloprocess reactions, 4.4 The yield coefficient — combining substrate turnover and growth ‘The question we are grappling with in this paragraph is how the cells ‘know’ how fast they can grow. Many environmental parameters like temperature, pH, or salinity of course play a role. Intracellular enzymatic steps also influence possible growth rates differently for different groups of organisms. Even in the case that all environmental parameters are at their respective optimum the cell is limited by intracellular steps e.g., ifan enzyme isat its maximum turnover rate. This gives reason to define a maxi- mum specific growth rate pmax [g/g/hl. Itis the task of metabolic modeling to find out the intracellular reasons for the value of imax for different classes of microorganisms. In the next paragraphs we focus on the role of substrate concentration. From previous experience it is known that substrate concentration is one ma- jor factor that can be influenced by process engineering means. In the discussion of medium design (Chapter 3) we already used the relationship between initial substrate and final biomass concentration. From the experiment (Figure 4.1) it becomes obvi- ouss that with progressively increasing biomass concentration substrate concentration also decreases faster, so we assume that growth and substrate uptake are coupled. In the time interval where cs approaches zero growth is no longer possible, implying that growth depends on the actual substrate concentration. The first attempt to find a more or less unspecific relation between growth and a limited environmental resource was formulated by Pierre-Francois Verhulst (1854). The background was the observation that growth, e.g., as predicted by Fibonacci, can- not go ad infinitum but has to stop somewhere. The imagination was a limited area offering enough space or food for a couple of animals but with a limited carrying ca pacity K of individuals. As this capacity becomes exhausted the animals have to mi- grate or do not get so many offspring due to starvation. Food like grass could further grow but is divided by more and more animals. Finally, each individual gets so little food that only survival but no propagation is possible. In addition to the linear growth model (4.29) Verhulst postulated that growth depends on the residual capacity (K-N)/K (normalized from 0 to 1) of the habitat: an “Gp = imax N (4.37) The solution of this differential equation with No being the initial population is: K-No-etnmt N= (4.38) K¥No- (et 1) This so called ‘logistic growth curve’ is a sigmoid function and a popular (as it has at least a closed solution) first trial to fit growth data. Nevertheless, the assumptions do not really represent the situation ina bioreactor. In our batch, for example, we see that growth ceases only very late, when glucose is nearly exhausted.4.4 The yleld coefficient — combining substrate turnover and growth —— 83 Fig, 4.23: The concept of substrate Umitation; an enzymatic transport step (symbolized by the turn: Ing enzyme) carries substrate Into the cell. This substrate flux determines the possible growth, The first one to describe growth as a function of substrate concentration was Jacques Monod in 1942 when he gave the famous Monod equation: és Wp GE «ss Monod won the Nobel Prize (Physiology or Medicine) in 1965 for the operon theory, not for this equation. Since then the Monod equation has been employed to describe myr- iad bioprocesses. The equation shows structural similarity to enzyme kinetics, so the assumption has been formulated that enzyme kinetics is an underlying mechanism. Substrate uptake as an enzymatic step in metabolism is the first candidate to investi- gate as shown in Figure 4.13. Understanding that the more biomass is present the more substrate will be taken up, it is sensible to define the specific substrate uptake rate rs in analogy to the specific growth rate: dms, ~ dtmx Ts (4.40) as a measurable property of the cells. Following the attempt to understand the Monod equation and in analogy to enzyme kinetics rs can be given asa function of the sub- strate concentration cs: « s Kg ves This is in accordance with the experiment in Figure 4.1 that at lower substrate concen- trations the substrate uptake rate is reduced. Substrate availability inside the cell then determines the possible growth rate. This concept is called ‘substrate limited growth’, The substrate is then converted by hundreds of enzymes inside the cell. However, growth is regarded as a thermodynamic process where the cell could make ‘the best of it’ under the restrictions of energy and material balances, stoichiometric relations, and other constraints. We have already discussed the elemental balance as such acon- straint. Biology has provided different approaches to understand how much biomass (441) Ts = 1,max84 — 4 Kinetics — finding quantities for bloprocess reactions, can be built up by a given amount of substrate taken up. Of specific interest is the partitioning into anabolic pathways lumped as growth on the one hand and energy producing metabolic pathways on the other. A fundamental relationship determining the relation of growth and substrate uptake was found by S. John Pirt in 1965: ty= ore ttm (4.42) YES To get a consistent system of terms the historic symbol y/ is replaced by rx, where r is rate and X is biomass. To maintain a given specific growth rate the cell has to take upa proportional rate of substrate. An important point is that the proportional factor, interpreted as yield coefficient, is constant for many growth conditions. This has to be understood as an optimized carbon allotment. Metabolic energy is provided by a part of the substrate while the other part is used for growth under energy utilization. This balance is kept stable by the cells in order to avoid waste of energy and carbon. As Jong as the cell does not change this metabolic pattern this results in a constant yield. In addition, energy is partly used for nongrowth-associated purposes like movement, keeping the water balance, or maintaining membrane potential. This part of energy utilization is lumped into the ‘maintenance* term ry, (m in the original reference). In- deed, very often measurement data fit quite well to the linear relation between growth and substrate uptake. For clarity it is noted that yy.s = dry/drs isa kinetic parameter, while ¥x,s = Amx/Ams is a measured process characteristic over a longer cultivation period. About the inner connection between substrate uptake rate and growth rate more background is given in the next chapter. A first idea to describe biomass formation and substrate uptake simultaneously is simply combining the Monod and the Pirt equation ((4.33) and (4.37)). A short inspec- tion of this system of equations suggests that it is not a good idea. For cs = 0a positive substrate uptake rate is predicted, whichis physically not possible. Turning the cause- effect chain we set the substrate uptake asa primary limiting step and reformulate the resulting growth rate following Pirt’s linear relationship: Fonw es a re = Hames (oa te = Yas ts—tm 5) This is a first simple physiological model of microbial growth and will be used as an example throughout this book. According to the essence of kinetics these equations hold irrespective of the way cs changes during the process. Besides biomass formation and substrate consumption, product formation is of interest. As was done before we define a specific product formation rate: dmp tp (4.45) dt-mx4.4 The yleld coefficient — combining substrate turnover and growth —— 85 In addition, a formal relation with growth and/or substrate turnover is required. As the cell is highly stoichiometrically determined again a linear relation in analogy to Pirt’s equation can be a good start: rp =yox-re + kp (4.46) This equation was introduced to the field by Luedeking and Piret and bears their names. Itis a flexible tool to fit different kinds of product formation patterns. The yield coefficient yp. = drp/dry describes the ratio of product formed per biomass growth. Often product formation is coupled with catabolism, meaning substrate turnover. At least the carbon comes from there. For technical applications the ratio of product produced to substrate consumed yp,s = Amp/(ms is of interest. This is considered in the reformulation: rp = Yp.s°TS—7P,m (447) Here product formation is coupled to substrate uptake where a small constant part of the substrate is reserved for other purposes. This holds only for rs and rx over a given threshold, which is also the case for the Luedeking-Pirt equation. In the next chapter and some case studies the coupling of product formation to catabolism and anabolism (rs, rx) and the complexity of control patterns of the cells is analyzed in more detail. Before trying out the kinetic equations for our standard batch process good guesses for reasonable values of the kinetic parameters are required. Table 4.4 gives some typical values. Some trends can be observed from Table 4.4. Aerobic microorganisms can reach a yield of 0.5 g/g growing on glucose. Sometimes it is a bit lower, but never higher. The yield also depends on the substrate. Witha higher degree of reduction (ethanol) higher yields are obtained. Anaerobic growth on glucose results in a low yield of about 0.03- 0.05 g/g. Here, the product is always formed in a high yield product per substrate of e.g., 0.5g/g. As substrate uptake is often higher for anaerobic growth, similar growth rates as for aerobic growth are obtained. ‘Tab. 4.4: Typical specific growth rates of different microorganisms and substrates. Organisms ‘Substrate r5.max Pama ks Yes Remarks fe/(g- te/e-hy)—tg/__ta/et Sacch. cerev. Glucose 2.52 60 0.180 0.485 Aerobie Ethanol 14 0.753 Anaerobic Glucose 0.53-0.63, E.coli Glucose 05-10 0.004 0.5 — Aerobic Aspergillus oryzae Glucose 0.005 Acroble Klebsiella aerogenes Glucose 0.009 Aoroble Glycerol 0.009 Virt. generic Glucose 1.0 10 0.5 Aerobic Virt. generic Glucose 1.0 1.0 0.05 — Anaerobic86 — 4 Kinoties — finding quantities for bloprocess reactions, 4.5 The batch process — the simplest form of a bioprocess Now alll the information can be assembled to get a simple model to describe a batch process. Remembering the physiological model (4.48) (a9) (4.50) the concentrations cx and cg have to be calculated to havea complete set of equations. This has to be done by setting up the material balances for biomass and substrate. For the batch process the balance equations are in the form of simple differential equa- tions of accumulation and reaction terms: ax ex (451) aes = is cy (4.52) ft =p ex (4.53) These reactor equations together with the physiological equations form a complete model to represent a batch cultivation. Note the minus sign for substrate to get 2 decreasing substrate concentration for a positive turnover rate. Even for more com- plicated cases the biological submodel and the reactor submodel should always be strictly separated during building up and for writing them down. Unlike in setting formal kinetics, e.g., only for biomass, these models cannot be solved explicitly but must be numerically integrated. A simulation is shown in Figure 4.14. Batch processes are usually divided into several phases. During the lag phase the cells have to adapt to the environmental conditions inside the reactor, which may be different from the parameters in the preculture. The specific growth rate increases only after some time, e.g., one hour, to the value predicted by kinetics. This can happene.g., in cases where the medium in the preculture contains supplementary compounds that are not present in the technical medium or vice versa. Adaptation is not represented in the model equations. As the lag phase is unproductive it should be kept as short as possible e.g,, by adjusting the conditions in the shaking flasks or the cultivation in the stage before. During the ‘exponential phase’, p is close to but not equal to pfmax for several hours as substrate concentration is far in the saturation range of Michaelis-Menten kinetics. Do not use the outdated term “logarithmic phase’. This is actually the state of the reac- tor we want to achieve as long as possible. This is no longer the case in the ‘stationary phase’, where substrate concentration falls to the order of magnitude of the limitation constant and becomes therefore the decisive value for growth. In many cultivationsac- cumulation of an (possibly unknown) inhibiting product could also contribute to the4,5 The batch process ~ the simplest form of a bloprocess —— 87 120 g 04 we Z| & fos : = 3 3 a o . “ 02 6s 8 e S a = . o1 2 2 = ce pee & 2 5 . 00 B ee . ee? “04 0 $3 8s * * at 04234567 8 8 01 1213 14 15 Cultivation time [h] Fig, 4.24: Simulation of a batch cultivation with the parameter set for the model organism Virtuella generica (Table 4.4) showing the course of the concentrations and the specific turnover rates. stationary phase. After the substrate is completely exhausted a decrease of cell dry mass is observed, which is also visible by the negative specific growth rate. That does not mean that the cells really perish in the ‘clecay phase’. In fact, they fuel their main- tenance demand by respiration on intracellular materials like storage compounds, a process called ‘endogenous respiration’. The optimum harvesting point is shortly be- fore entering the decay phase. We started this chapter by evaluating a batch process in terms of macroscopically observable volumetric rates R and yields Y (capital letter). These characteristic values have to be understood as integral values over a longer time interval. Enzyme kinetics could be deduced from the mass action law being a basic principle in chemical re- actions. Starting from the biological observation of propagation by cell division and doubling, the specific growth rate ry was introduced. Like the other specific rates r this is understood as a differential value being valid at all time instances. The same holds for the model parameters k and y (lowercase letter). Kinetics in combination with ma- terial balances are the key to formulate descriptions of a growth process with at least some mechanistic justification. The basic assumption was the translation of enzyme kinetics to growth kinetics. In the next chapters we will see how far we can go with this daring idea.