35 Efficiency of growth and product formation 3.0 Intrduction 3.1 Growth stoichiometry 36 36 3.2 Relationships between product formation and growth 3.3 Determination of maintenance energy requirement and maximum 42 biomass yield 48 3.4 Determination of P/O quotients 49 3.5 Metabolite overproduction and growth efficiency Summary and objectives 50 58 36 Chapter 3 Efficiency of growth and product formation 3.0 Introduction In order to develop a rational approach to improving rates of metabolite production, it is necessary to consider the fate of the nutrients that are required for its synthesis. However, overcoming the major flux control points within a metabolic pathway may not lead to metabolite overproduction if the energetic consequences of the alteration are unfavourable to the organism. In this chapter, we will consider growth and product formation from an energetics perspective. In the first part of the chapter, the stoichiometry of growth is considered in some detail. The relationships between product formation and growth are then described, together with approaches to determining key parameters of growth efficiency. Finally, classes of metabolites are defined, according to the relationships between energy and metabolite synthesis. Examples of commercially significant products in each class are also discussed. Since process technologists rely on quantitative relationships, many seemingly complex equations describing growth and product formation stoichiometry are presented in this chapter. These are intended to illustrate a quantitative approach to the study of efficiency of growth and product formation. You are not expected to recall all details of the equations, rather the factors that need to be considered in such a quantitative analysis. If this approach is entirely new to you, we recommend Chapters 8-10 of the BIOTOL text ‘Bioprocess Technology: Modelling and Transport Phenomena’, which deals with the modelling of growth and product formation. energetics perspective 3.1 Growth stoichiometry 3.1.1 Yield coefficients In any quantitative assessment of growth and/or product formation, it is essential to link formation of microbial biomass and products with the utilisation of substrate and nutrients. In the case of microbial biomass production, the total amount of cell mass formed is often proportional to the mass of substrate utilised. Mathematically this is expressed as the corresponding ratio, or yield coefficient: yield coefficient where AX = amount of biomass produced AS = amount of substrate mnsumed Efficiency of growth and product formation 37 Yield coefficients may be defined for different substrates in the medium and are usually based upon substrate change in units of mass or mol of substrate eg, Ydo2 is used to relate the amount of biomass formed to the amount of oxygen consumed, so: Organism Pseudomonas fluorescens Substrate YXJ. 9 9-’ g mol-’ glucose 0.38 68.4 y*2 Where yield coefficients are constant for a particular cell cultivation system, knowledge of how one variable changes can be used to determine changes in the other. Such stoichiometric relationships can be useful in monitoring fermentations. For example, some product concentrations, such as COZ leaving an aerobic bioreactor, are often the most convenient to measure in practice and give information on substrate consumption rates, biomass formation rates and product formation rates. In practice, variations in yield factors are often observed for a given organism in a given medium. For example, yield Coefficients often vary with growth rate. An explanation for these variations comes from a consideration of the fate of substrate in the cell, which can be divided into three parts: assimilation into cell mass; energy for growth; energy for maintenance. Energy for maintenance is the energy required for survival, or non-growth related purposes- It includes activities such as active transport across membranes and turnover (replacement synthesis) of macromolecules. Where a single substrate serves both as carbon and energy source, which is the case for chemoheterotrophic organisms used for biomass production, we can write: AS = ASassimilation + &growth energy + &aintenance energy where AS = the total amount of substrate consumed Asassimilation = amount of substrate assimilated AS~OW~ energy = amount of substrate consumed to provide energy for grwoth &,&tenan- energy = amount of substrate consumed to provide energy for maintenance Or, expressed as yield coefficients: 38 Chapter 3 where AX is amount of biomass produced Yx/s is the yield coefficient Whereas the amount of substrate assimilated per unit of biomass formed (AX/GaSMrmkt,,,,,) is constant regardless of the growth rate, the overall yield coefficient (Y,,J is variable and dependent upon the environmental conditions within the culture. To illustrate this growth rate dependence, consider two extremes: a culture growing at its maximum specific growth rate will use most of its substrate for assimilation and growth energy, whereas a stationary phase culture (non growing) will consume substrate for maintenance without any growth. From the biotechnological process point of view, yield coefficient variability is extremely important and yield coefficients must, of course, be optimised. Later in this chapter (section 3.2.1) we shall consider yield coefficients with respect to product formation. 3.1 -2 Elemental material balances for growth The stoichiometry of growth and metabolism can also be described by elemental material balances. This approach can provide an insight into the potential of the organism for biomass or product production, and thus the scope for process improvement. An elemental material balance approach to growth stoichiometry requires an empirical formula for dry weight material: Ce Ha Op Ns growth rate dependence empirid fOm~Ia for dV biomass The ratios of subscripts in the formula can be determined if the elemental composition of an organism growing under particular conditions is known. A unique cell formula can then be established by relating elemental composition to one gram-atom of carbon, ie 8 = 1, then a, p, and 6 are set so that the formula is consistent with known relative elemental weight content of the cells. The formula can be extended to include other macro-elements, such as phosphate and sulphur, if elemental analysis shows these elements to be a significant proportion of cell material. n Complete the following statements: 1) One C-mol of cells is the quantity of containing one gram-atom of 2) One C-mol of cells corresponds to the cell weight with the carbon subscript (e) taken as The missing words are: 1) 'cells' and 'carbon'; 2) 'dry' and 'unity'. Use the data on elemental composition shown below to determine the empirical n chemical formula and the formula weight for the yeast. Efficiency of growth and product formation 39 composition (“A by weight) Empirical Fmuh CHNOPS chemical weight formula (C-mole of cells) Bacterium 47.1 7.8 13.7 31.3 666N0.2000.27 20‘8 Yeast 44.7 6.2 8.5 31.2 1.08 0.6 ? ? Atomic weights: C, 12.011; H, 1.00s; N, 14.008; 0,16.000; P, 30.98; S, 32.06. The empirical chemical formula is CH1.s N0.1600.52 Po.01 s0.00~ and the formula weight is 24.6. For example, the subscript for 0 in the chemical formula is determined as follows: (31.2/16)/(44.7/12.011). The formula weight is then calculated by multiplying the coefficients by the atomic weights and summing them. Thus (1 x 12.011) + (1.008 x 1.65) + (14.008 x 0.16) + (16 x 0.52) + (30.98 x 0.01) + (32.M x 0.005) = 24.6. Now lets consider the elemental approach to stoichiometry for a relatively simple situation: aerobic growth where the only products formed are cells, carbon dioxide and water. The following formulas can be used if we consider the four main elements: Cell material CHaOpNs Carbon source CH*o, Nitrogen source mmNn We can now write the reaction equation by introducing stoichiometric coefficients for all elements of the equation. stoichiometric coefficients a’ CH, 0, + b’ 02 + c’ I-h 0, N,, ---> CH, 0, Nc, + d‘ H20 + e’C02 E - 3.1 You should note that there are only five unkown stoichiometric coefficients (a’, b’, c’, d‘, e‘) since the coefficient of cells is taken as unity. The reaction equation can be used to establish relationships between the unknown coefficients by considering balances on the four elements, as follows: Ca’= 1 +e’ H. ak + c’I = a + 2d‘ Write material balances for the remaining two elements in the reaction equation n (E - 3.1). The material balances are: 0 a‘y + 2b’ + c’m = + d’+ 2e’ N: c’n = 6 40 Chapter 3 A bacterium is grown aerobically with glucose as sole source of carbon and armnoNum ions as nitrogen source. Experimental analysis shows that six moles of glucose are utilised for each mole of biomass produced. Write the reaction equation for growth if the elemental composition of the cells is CHI.& QZ NO^. (Hint: commence with the 'abstract' equation E - 3.1). ~~ For a more detailed stoichiometric representation for aerobic growth of a chemoheterotrophic organism we must consider: 0 the generation and utilisation of ATP; the oxidation-reduction balance of substrates and products. This allows an assessment of the relative efficiencies of the biochemical pathways involved in microbial growth and metabolism. It may be assumed that neither ATP nor NADH accumulates, ie formation must be balanced by utilisation. Let us first consider the formation and utilisation of ATP; we may write: energy ATP formation = ATPutilisation balance (substrate level phosphorylation) (biosynthesis) (oxidative phosphorylatioQ (maintenance and dissipation) For substrate level phosphorylation we can write: ES a (ADP + PI) + G a (ATP + Ha) E - 3.2 Where: cs = number of substrate-level phosphorylations per mole of carbon utilised. For oxidative phosphorylation we can write: 2b (P/O) (ADP + PI) + 2b V/O) (ATP + H20) E - 3.3 Where: P/O = is the number of ADP phosphorylations per atom of oxygen consumed. For biosynthesis (ATP utilisation) we can write: MWB (ADP+PJ MWB y ,,(ATP + HD) + - Y Inax ATP ATP Where: MWB = molecular weight of biomass; Y ATP = mass of cells formed per mol of ATP utilised in biosynthesis. E - 3.4 max Efficiency of growth and product formation 41 The Y Fg in the equation can be determined from growth yields and known mutes of max ATP synthesis. For growth of Exherich coli on glucose and mineral salts the Y value, estimated from known cell composition and known biosynthetic pathways, is 28.8 g dry weight mol-’ ATP. However, the Y determined experimentally from yield measurements is often around 50% of the theoretical (12 to 14 g dry weight mol-’ ATP). UlaX Explain the discrepancy between theoretical and experimentally derived values II max for Y ATP’ The discrepancy arises because ATP is used to drive processes which are not directly related to growth, eg membrane transport processes, protein turnover- These are dd the ’maintenance and dissipation’ demands for ATP. For maintenance and dissipation we can write, simply: c(ATP+HQ) -+ dADP+Pi) Now lets consider the balance for NADH, ie: bahnca for NADH E - 3.5 NADH formation = NADH utilisation (energy source dissimilation) (biosynthesis) (oxidative phosphorylation) To represent the balance for NADH using quantitative relationships, we must consider the degrees of reductance of substrate and products. The degree of reductance of material is the number of available electrons per atom of carbon and is determined using C(+4), H(+1), O(-2) and N(-3). So, for biomass with an empirical formula of CH1d02000.27, the degree of reductance ( $ is: degree of redmce (4) + (1 x 1.666) - (3 x 0.2) - (2 x 027) = 4526 n What are the degrees of reductance of (1) CG, (2) NH3 and (3) csS~Hsazols.~? The degrees of reductance are (1) 0, (2) 0 and (3) 3.93. The answer for (3) was determined as follows: 4 + (86.2/552 x 1) - (45.1/55.2 x 2) redudon balance For energy source dissimilation we can then write: CH, 0, + HQ + f NAD+ --> CQ + (NADH + H+) E - 3.6 Where: = the degree of ductance of carbon substrate. 42 Chapter 3 For biosynthesis we can write: (1 +dCH_I:Oy + -mOmNn n +? Cy,- *15(i+d---yN)(NADH+H+) 6 1 6 -> CH, 08 Na + a COZ + HzO E - 3.7 Where: y, = the degree of reductance of biomass; y, = the degree of reductance of nitrogen source; y = the degree of reductance of compound. For oxidative phosphorylation we can write: 2b (NADH + IT) + bQ -> 2b NAD' + 2bH20 E - 3.8 The coeffiaents u, b and c, which appear throughout these balance equations describe the extent to which these reactions occur relative to the growth reaction (ie 1 + a) and are written taken into account elemental balances for each reaction. n adapted for anaerobic fermentations? 1) By deleting OZ everywhere. 2) By dropping the oxidative phosphorylation reaction. How should the balance reactions already desaibed for aerobic metabolism be max Use the equations already given to predict how you might exped Y 'ys to be influenced by: 1) a decrease in the degree of reductance of substrate; 2) an increase in the efficiency of oxidative phosphorylation; 3) a decrease in energy demand for biomass synthesis? Give reasons for your responses. 3.2 Relationships between product formation and growth process khks When considering product fonnation stoichiometries it is essential to define the relationship between product formation and growth. Essentially, this classificatiun divides microbial product production processes into four types: Efficiency of growth and product formation 43 1) The main product appears as a result of primary energy metabolism. Examples: production of biomass, ethanol and gluconic acid. 2) The main product arises indirectly from energy metabolism. Examples: citric acid and some amino acids. 3) The main product is independently elaborated by the organism and does not arise directly from energy metabolism (the product is a secondary metabolite). Example: antibiotics such as penicillin and streptomycin. 4) Biotransformation, in which the main product is formed from substrate through one or more reactions catalysed by enzymes in the cells. Examples: steroid hydroxylation. Figure 3.1 illustrates the main patterns for batch fermentation process kinetics for type 1,2 and 3 processes. Figure 3.1 General patterns for batch fermentation process kinetics. In type 1 processes, substrate utilisation, biomass formation and product formation am linked in a simple chemical reaction. For example, if the product contains C, H, 0 only, we simply extend the biomass material balance equation used earlier: type 1 d CHI 0, + b’a + C% 0, N, - > U-LOpNa + dHzO+e’COr + j’CH,a E - 3.9 biomass product 44 Chapter 3 The product yield coefficient can then be calculated, taking into account the relative numbers of carbons in the substrate and product. The molar yield coefficient is then written as roductfod - f'n, - substrate used a'n, Yp/s = p (mols product formed/mols substrate consumed). Where: E - 3.10 ns = number of carbon atoms in the substrate molecule; np = number of carbon atoms in the product molecule. Determine the molar yield coefficient for exopolysaccharide production if 955 n mols of glucose (6H1206) are required to produce one mol of c65dk.Dfi.1. YP/. = (1 x 6) / (955 x 55.2) = 0.011 type 2 For type 2 processes, the simple stoichiometry (E - 3.9) does not apply. Here, product formation is not necessarily proportional to substrate utilisation or biomass formation. In these cases, we need to consider a product formation step in addition to the growth reactions considered earlier, ie the formation (or utilistion) of both NADH and ATP relative to product formation. d CHZoy + H20 + 2 (p - Z%)NAD+ > 1 E~~(ADP + Pi) > d (ATP + NO) Where: E - 3.11 E - 3.12 Z = the fraction of carbon substrate used for convertion to product; = the number of ATPs generated by product formation. If the coefficient ep in the product formation equation (E - 3.12) was negative, what n would this indicate? The ATP is utilised rather than generated in connection with product formation. What does the coefficient d and the parameter yp denote in the product formation equation (E - 3.12)? The coefficient d denotes the extent to which the reaction occurs relative to the growth reaction. The parameter rp denotes the degree of reductance of product. Efficiency of growth and product formation 45 type 3 and 4 For type 3 processes, growth and metabolic activity reach a maximum early in the batch process cycle (Figure 3.1) and it is not until a later stage, when oxidative activity is low, that maximum desired product formation occurs. The stoichiometric descriptions for both type 3 and 4 processes depend upon the particular substrates and products involved. In the main, product formation in these processes is completely uncoupled from cell growth and dictated by kinetic regulation and activity of cells. Product formation stoichiometry can be used to estimate the upper bounds for product yields in processes. A relatively simple example is the anaerobic fermentation of glucose by yeast. Here, carbon dioxide and ethanol are the only products. Modification of (E - 3.9) then becomes: 3 3 E - 3.13 n For the ethanolic fermentation described by (E - 3.13), determine the upper bound max on the molar yield factor, ie Y . - 2x6 =2 3 1x2 From (E - 3.101, Y Ern = Yields much lower than the upper bound value indicate that there is significant substrate utilisation to support growth, maintenance or synthesis of other products. 3.2.1 Product yield considerations We can see that for type 1 processes, high growth rate is obligately linked to a high rate of product formation. Indeed, this is the case for all products produced by a fermentative mode of metabolism, eg ethanol, lactic acid, acetone. Chemostat studies have shown that for most aerobic processes when growth is limited by some nutrient other than the carbon source, the yield of product decreases with increase in specific growth rate (p or D; p = dilution rate (D) in chemostat culture). Conversely, both thq biomass yield and the specific rate of substrate utilisation (qs; g substrate g biomass- h-’) increase with specific growth rate. The relationships between specific rate of substrate consumption and dilution rate and between yield coefficients and dilution rates are shown in Figure 3.2. growth rates ad Product yields 46 Chapter 3 Figure 3.2 Theoretical relationships for (a) qs against dilution rate and for (b) Yps and YvS against dilution rate. The micro-organism is grown aerobically in a nitrogen limited chemostat culture. These observations can be explained by considering the substrate uptake rates. At high growth rates there is relatively little difference between the organisms potential substrate uptake rate and the substrate requirement for growth. However, at low growth rates relatively little substrate is required for growth but the potential substrate uptake rate is unaltered - this means that the organism can channel more of its substrate carbon into product. n Can you think of a disadvantage of operating a process at low dilution rate? The main disadvantage is that, although high yields of product might be achieved, it is difficult to achieve high productivities (kg product m-3 h-'1. High productivities at low dilution rate would require: expression of substrate uptake rates near their potential maximum; tight control of respiratory activity to ensure that substrate is converted to product and not to COZ. Ideally, we would wish for high substrate uptake in the absence of growth and in the absence of maintenance energy requirements. Since aerobic micro-organisms control their rates of substrate uptake when growth is slow or absent, manipulation of substrate uptake may be necessary. Efficiency of growth and produd formation 47 These comments also apply to batch cultures where product formation occurs after growth has ceased. The relationships between energy metabolism and product formation in an organism can be used to predict the influence of maintenance requirements on product yields. In aerobic fermentation, where type 3 and 4 product formation is often the goal, increased maintenance requirements decrease product yields by inaeasing substrate utilisation for energy production. However, the converse is true for some type 1 processes, for example the production of ethanol by anaerobic fermentation in a medium containing glucose and ammonia (as a nitrogen source). Here, substrate-level phosphorylation is the only sowce of ATP and a relatively high maintenance requirement means a relatively large amount of glucose will be metabolised. This in turn generates relatively more NADH, which is in excess to that used in biosynthesis. The NADH has to be oxidised to NAD' to ensure continued catabolism of glucose. This is achieved by increases in product formation (ethanol), ie the stored electrons in NADH are transferred to a product which is more reduced than the substrate and so the oxidation-reductance balance is maintained. Where biosynthesis of a product requires the net input of energy, the theoretical yield will be influenced by the P/O quotient of the process organism. Furthermore, where the formation of a product is linked to the net production of ATP and/or NADH, the P/O quotient will influence the rate of product formation. It follows that to estimate the potential for yield improvement for a given primary or secondary metabolite, it is necessary to determine the P/O quotient of the producing organism. We have seen that both the maintenance energy requirement and the P/O quotient of the process micmrganism influences the rate of product formation. In the following sections we will consider how these two factors can be determined, together with the maximum biomass yield. mantenance requimment PI0 quobent energy dissipation To which of the four types of processes do each of the following statements 1) High growth rate is obligately linked to a high rate of product formation. 2) Product formation is completely uncoupled from cell growth. 3) The main product is a secondary metabolite. 4) Growth, substrate utilisation and product formation time courses exhibit apply? coincident maxima. 5) The main product arises indirectly from energy metabolism. 48 Chapter 3 A 1 m3 aerobic bioprocess was operated in a continuous mode with nitr en as the growth limiting nutrient. The steady state biomass concentration 8 x , the biomass yield coefficient (YXl6) and the product yield coefficient (Yp/,) were determined at a low and at a high dilution rate (D). D = 0.1 h“ D = 0.6 h-’ - - x = 10 kg biomass ni3 YX/B = 0.25 kg biomass (kg substrate)-’ x = 8 kg biomass m-3 Yds = 0.5 kg biomass (kg substrate )-’ (kg substrate)- Y,, = 0.6 kg product (kg substrate)-’ Y@s = 0.2 kg yoduct Which of the two dilution rates should the process be operated at? (Hint: compare productivities for the product). Note that biomass productivity = D.2 1. 3.3 Determination of maintenance energy requirement and maximum biomass yield Cells need a certain amount of energy for maintenance. The maintenance energy is, for instance, needed for maintaining the proton motive force which is, among other purposes, used for maintaining the ion gradients across the cell membrane. Furthermore, energy is needed for the turnover of proteins and mRNA, for repair and for movement (if mobile). Maintenance energy requirements can be defined in terms of rate of substrate consumption per unit of biomass for maintenance: this is known as the maintenance coefficient (m). In the absence of product formation, the relation between the observed growth yield (Yrb) and the maximum growth yield (Y r) can be written as: 1 m - 1 --- Yr. YT + - CC E - 3.14 where: p = specific growth rate m = maintenance coefficient. This relationship is very useful experimentally because it can be used to determine both Y y and m. In practice, carbon limited chemostats are used and Yx/. is measured at different dilution rates (D). In a plot of - against 1/p, the intercept is - - and the yy. slope is m. (Note that at steady state p = D). intercept is 1 - YT 1 1 y Y Efficiency of growth and product formation 49 The observed values of YXl6 at the different dilution rates can also be used to determine Y do2 values. This is achieved by defining YxlS and Y in terms of their respective rates of production and taku\g into account the degrees of reductance. So. rate of biomass production "" = rate of substrateutilisation y do2 Combining these two rate equations and taking into account the degree of reductance, we have: dope is m do2 - rate of biomass production rate of oxygen consumption - x yY, - ys --*I. - *IX x 1% * y6 y do2 E - 3.15 (where y sigxufy the degree of reduction for 02, % for biomass, % for substrate) A bacterium was growth yield (YX16 r was measured at different dilution rates. wn as a glucose-limited chemostat culture and steady state D (h-') YdS (C-mol biomass/C-mol substrate) 0.9 0.475 0.4 0.470 0.2 0.485 0.1 0.455 0.05 0.426 Use the data to determine the maintenance coefficient (m) and the maximum growth yield Y r. Determine the Y cH1.d02~027). value for p = 0.9 h-' (the empirical formula for biomass is do2 3.4 Determination of P/O quotients As we noted earlier, P/O is the number of ADP phosphorylations per atom of oxygen consumed, ie #he amount of ATP produced per 05 moles 02. The quotient can be derived from the maximum yield for oxygen (Y equation: max 1 using the 02 50 Chapter 3 max Y,"" = YATP . P/O E - 3.16 carbon limited UJKIJ~~~ In practice, carbon limited chemostat cultures are used to estimate the P/O quotient. These conditions are used because they favour the most efficient conversion of the carbon substrate into cellular material, ie the highest efficiency of energy conservation. The steady state respiration rate (qs) is measured as a function of dilution rate (specrfic growth rate) and r can be obtained from the reciprocal of the slope of the plot. qs is also known as the metabolic quotient for oxygen or the specific rate of oxygen consumption. You will note that the equation used to determine P/O does not take into account ATP synthesis via substrate level phosphorylation, which is a limitation of the P/O estimation. n estimation? Substrate level phosphorylation leads to an overestimation of P/O by about 7% and 30% at P/O quotients of 3 and 1 respectively. The P/O quotient obtained in this way is therefore only an approximation. Nevertheless, the values can be used as a cmparitive measure of growth efficiency, provided organisms are grown under carbon limited conditions (difference in Y ATp is minimised). Such a comparison of the energetic efficiency would otherwise not be possible. A bacterium was grown as a glucose-limited chemostat culture and steady state respiration rate (qs) was measured at different dilution rates: substratehei phOSphO~latiOn How would you expect substrate level phosphorylation to affect the P/O max D (h-') qs (mmol g-' h-') 0.6 9.2 0.5 8.7 0.4 6.8 0.3 5.3 0.2 3.5 0.1 2.8 max Determine the P/O quotient for the bacterium if Y ATp during growth on glucose is 13.9 g dry wt mol-'. 3.5 Metabolite overproduction and growth efficiency The theoretical yield for a given metabolite can be estimated provided the following are known: the biosynthetic pathway for synthesis of the metabolite; 0 the P/O quotient of the producing organism. Efficiency of growth and produd formation 51 The extent to which the yield of metabolite can be improved is indicated by the difference between the theoretical and observed yields. The latter must, of course, be corrected for substrates requirements of growth and maintenance. Clearly, the influence of the P/O quotient on the theoretical yield will depend on the relationship between energy and metabolite synthesis. Three classes of metabolite can be dassesof tmtaboh~ distinguished in this respect. 1) Metabolites whose biosynthesis is energy requiring, for example exopolysaccharides using certain substrates. Here, part of the substrate has to be oxidised to provide ATP for biosynthesis and thus the P/O quotient of the producing organism influences the theoretical yield. 2) Metabolites whose biosynthesis leads to the net production of ATP and/or reducing equivalents, for example organic acids and certain secondary metabolites. In these cases, the P/O quotient influences the extent to which energy can be dissipated. 3) Metabolites that are composed of structures of quite different oxidation states. Certain secondary metabolites and biosurfactants fall into this class since they have both carbohydrates and fatty acids in their structures. It should be noted that, for some metabolites, the class to which they belong depends on the fermentation conditions, for example the class to which certain antibiotics belong depends on the substrate(s) used for their production. It is obvious that rapid metabolite production requires high fluxes of carbon through the metabolic systems responsible for its synthesis. The rate of metabolite production, for a wide range of micm-organisms, has been shown to increase with decrease in growth efficiency (Yr ). In addition, micro-organisms with low growth efficiency have a far greater capacity to dissipate energy (turnover ATP) than those with high growth efficiencies. We will now consider the three classes of metabolites in relation to growth efficiency. In class 1 (ATP requiring), the rate of metabolite production is limited by the micro-organisms capacity to dissipate energy. In class 2 (ATP generating), the rate of metabolite production, and oxidation state, are inversely related to the growth efficiency. In class 3, the rate of metabolite production from a single substrate may be limited by the rate of ATP turnover. Provision of ready made precursors can inmase both the metabolite yield (final concentration) and rate of production by decreasing the requirement for ATP turnover during biosynthesis. In the following sections we will consider the production of each class of metabolite separately. dissipirationof energy growth effidmq ATP ~J~XH 52 chapters 1) Which of the following statements are applicable to class 2 metabolites? a) The P/O quotient influences the extent to which energy can be dissipated. b) The rate of metabolite production is inversely related to ys". c) Biosynthesis of the metabolite leads to the net production of energy. d) The rate of metabolite production increases with increase in growth efficiency. 3.5.1 Exopolysaccharide production In this section we will consider the energetics of exopolysaccharide production in some detail. We will see how chemostat (substrate limited) derived yield coefficients and elemental balances can be used to determm . e how the nature of the substrate influences rates of metabolite production, and to give an indication of the scope for improvement of the producing micro-organism. You should note that for most industrial bioprocesses, the unavailability of data in the primary literature would prevent such an analysis. Further aspects of exopolysaccharide production are covered in Chapter 7 of this text. The energetic requirements of exopolysaccharide production from variom carbon sources can be calculated if the P/O quotient during growth on the carbon substrate is known. Table 3.1 shows molar growth yields measured during carbon limitmi growth in chemostat culture. n limiting substrate. nahrre Of substrate Use the data given in Table 3.1 to calculate the P/O quotient with succinate as IILaX Yy=YATP . P/O P/O = (29/2)/10/1= 1.4 Now enter this value for P/O quotient into Table 3.1. We can see from Table 3.1 that the P/O quotient is virtually independent of the carbon source. We can therefore assume a constant P/O quotient when calculating the energetic consequences of exopolysaccharide production from different carbon sources. It is also reasonable to assume that the rate of ATP turnover is similar on different carbon sources. amtantp~ wolient Efficiency of growth and product formation 53 Carbon Source Carbon Limitation Glucose Gluconate Sorbiiol Xylose Glycerol Succinate Ethanol C6H1206 CtiHiz07 C6Hi406 C5H1005 C3H& C4H604 ClFnaX 0-9 0.41 Biomass (g dry 3.2 YX/S (g dry wt mol-’) 80.0 Ya (g drywt mol-’) 31.0 max ATP [g dry wt 13.9 (mol ATP)-’] PI0 quotient (mol 1.1 ATP per 0.5 mol 02) Nitrogen Limitation Biomass (g dry 1.16 qs (m mol g-’ h-’) 2.72 qo2 (m mol g-’ h-’) 2.66 Exopolysaccharide 7.86 qP (9 9-1 h-7 0.24 wt 1-1) wt I-’) (9 1- ) 0.25 2.1 7 69.4 27.3 13.9 1 .o 1.66 2.1 0 2.40 5.2 0.15 0.18 2.88 73.3 23.6 13.9 0.9 2.09 1.62 2.86 6.38 0.12 0.21 2.46 52.0 22.0 12.7 0.9 1.53 2.37 2.27 5.86 0.15 0.1 6 2.69 41.8 29.2 12.7 1.1 1.59 2.12 3.37 2.07 0.06 0.32 2.46 41.9 29.0 10.1 1.49 4.03 4.40 6.57 0.18 ~ C2H6O 0.16 3.43 30.4 16.6 7.17 1.2 1.28 1.5 3.3 0.09 0.002 Table 3.1 Parameters of growth and exopolysaccharide production for Agrobacrenum radiobactergrown in chemostat culture on various carbon sources. Data obtained from Linton J. D. et al(1987) Journal of General Microbiology 133.2979-2987. How would you expect the growth efficiency to be influenced by the P/O n quotient? The growth efficiency <YF ) is proportional to the P/O quotient; this is expected from the equation used to calculate P/O (section 3.4, E - 3.16). We already know that product formation is enhanced when substrate other than the main carbon source limits growth. Further, during nitrogen limited growth in chemostat culture, exopolysaccharide has been shown to be the only product excreted. We can see from Table 3.1 that, under nitrogen limited growth, the speafic rate of exopolysaccharide production (q,,) is highest with glucose as substrate. During exopolysaccharide production on glucose, gluconate and xylose the respiratory rates (qs) are similar. However, respiratory activity increases during growth on carbon sources either more reduced or oxidised than glucose. An explanation for this can be obtained from a consideration of the energy requirements of exopolysaccharide production. respiratory 54 Chapter 3 1) Determine the degrees of reductance of the carbon substrates shown in Table 3.1. Which substrates are more reduced and which are more oxidised than glucose? 2) Examine Table 3.1. What is the relationship between specific rate of exopolysaccharide production and growth efficiency? 3) Use the data in Table 3.1 for glucose limited growth to calculate q, at a dilution rate of 0.2 h-', in units of g g" h-'. (Molecular weight of glucose = 180). The ATP requirement for exopolysaccharide production can be twice that required for cell biosynthesis. High rates of ATP synthesis are therefore required to support high rates of exopolysaccharide production. However, whereas synthesis of the sugar backbone of exopolysaccharide is energy requiring, the production of the oxidid parts of the molecule (eg acetate and uronic acids) is energy generating. Optimal yield of exopolysaccharide occur, therefore, when carbon and energy fluxes are integrated. The extent of such integration depends on: 0 the degree of reductance of the exopolysaccharide; 0 the degree of reductance of the carbon source; 0 the P/O quotient of the producing micro-organism. For substrates (glucose, gluconate and xylose) that are around the same degree of reductance as exopolysaccharide (ie around 3.91, virtually all of the energy generated above that required for growth goes into exopolysaccharide production. However, the rate of exopolysaccharide production decreases with more oxidised (succinate) or more reduced (sorbitol, glycerol, ethanol) substrates because carbon and energy fluxes are not favourable. For example, in the case of sucanoglycan biosynthesis form ethanol (Table 3.2), there is a massive overproduction of ATP and high rates of exopolysaccharide production would require some form of energy dissipation. *Peof reductance Carbo hydrate Net ATP produced/untt Rate of succino-glyyn glucose (C6H1206) -1 8.25 0.24 gluconate (c6H1207) -0.2 0.15 succinoglycan production (g g h ) SOrbfiOl (C6H1406) 4.91 0.1 2 ethanol (C2H6O) +91.3 0.002 glycerol (c3H603) +12.95 0.06 Table 3.2 Rates of succinoglycan synthesis from carbohydrates and net ATP production during synthesis. Data obtained from Linton J. D. (1 990) EMS Microbiu/~y Reviews 75, 1-1 8. In order to quanw the scope for improvement of exopolysaccharide production, it is first necessary to correct the observed yields of exopolysaccharide for the amount of carbon substrate and oxygen required for cell production. The corrected yields are then compared with the theoretical calculated from the P/O quotient for the producing micro-organism. Such a comparison is made in Table 3.3. scope br hprovemmt 56 Chapter 3 The requirement for oxygen and carbon source for cell biosynthesis are calculated using mass balance equations for growth during exopolysaccharide production (nitrogen-limited cultures). These balances are derived from experimentally determined values of substrate and oxygen consumption; exopolysaccharide production; intracellular polyglucan production; bacterial biomass production; CO2production; cellular composition of C, H, 0 and N. The mass balance equation and YXls and YQ values of corresponding carbon-limited cultures are then used to determine the carbon substrate and oxygen requirements for cell production. The yield of exopolysaccharide (corrected for cell production) is then compared to the theoretical yield. The latter being calculated from the mean observed P/O quotient of carbon-limited cultures. Experimentally determined yields of exopolysaccharide have been found to be 70% of the theoretical. This suggests that exopolysaccharide production is an efficient process with little scope for mapr yield improvements. niwo*n-ii~M cunures carbon-limited cu'tures Y"'d mmparmn To which class(es) of metabolite, based on the relationship between energy and metabolite synthesis, would you expect exopolysaccharides to belong? Explain your reasoning. 3.5.2 Citric acid production (class 2 metabolite) The production of organic acids by micro-organisms, and especially citric acid, is considered in detail in Chapter 4. In this section therefore we will only briefly consider citric acid production, from an energetics perspectwe. During citric acid production, ATP and NADH are generated via glycolysis and the tricarboxylic acid cycle. Reoxidation of NADH occurs via the respiratory chain, which can lead to the synthesis of ATP by oxidative phosphorylation. The major allosteric control enzyme in glycolysis is phosphofructokinase, which is inhibited by both ATP and NADH. Clearly, for metabolite production to proceed rapidly there must be a means of dissipating energy (ATP) and NADH. It has been found that organisms with low P/O quotients have a greater capacity to dissipate energy than those with high P/O quotients. In the case of industrial citric acid production, the process micmrganism (Aspghs niger) has a relatively high P/O quotient when not in citric acid production mode. However, during citric acid production the organism is able to reoxidise NADH via an additional electron transport chain, which is not coupled to oxidative phosphorylation. In essence this allows dissipation of energy and ensures that high carbon fluxes through glycolysis can be maintained leading to citric acid production. energy dissipahn addtional &dm "z: high carbon ""xes Efficiency of growth and product formation 57 Which one of the following organisms is likely to produce gluconic acid at the n fastest rate? Organism w A 2.7 B 2.1 C 2.8 D 2.3 Organism B because it has the lowest Yr, ie lowest growth efficiency or highest capacity to dissipate energy. 3.5.3 Sophorolipid production (class 3 metabolite) Sophorolipid is a glycolipid, ie it is composed of carbohydrate and lipid. It therefore contains moieties of widely different oxidation levels and its synthesis from single carbon sources has a high ATP demand. However, the demand for ATP is reduced if a mixture of glucose and C-18 alkane is used. If glucose and fatty acid is used the ATP demand is reduced further and relatively high specific production rates can be achieved. hgh ATP demand substrate Glucose 25.5 C-18 alkane 19.1 1 Glucose + 6 C-18 fatty acid ATP demand per molecule of sophorolipM Why is there a high ATP demand for sophornlipid synthesis from 1) alkane n substrate and from 2) glucose substrate? 1) The need to produce the carbohydrate moiety from alkane. 2) The need to produce the alkane moiety from glucose. 1) Explain why the use of A. nip to produce citric acid might be regarded to 2) Is a low or a high growth efficiency for the producing organism desirable for be far from ideal. sophornlipid production? Explain your reasoning. 58 Chapter 3 Summary and objectives Overcoming the major flux control points within a given metabolic pathway may not, by itself, lead to rapid metabolite overproduction if the energetic coIlsequences of the alteration are unfavourable. We have seen that in order to improve the yield of a given metabolite in a rational way, it is necessary to describe growth and metabolism by elemental material balances. Further, when considering product formation stoichiometries it is essential to define the relationship (four types) between product formation and growth. The theoretical yield of a given metabolite can be estimated provided 1) the biosynthetic pathway for its synthesis and 2) the P/O quotient of the producing organism is known. The extent to which the yield of metabolite can be improved is then indicated by the difference between the theoretical and observed yields. Three main classes of metabolite can be distinguished according to the relationship between energy and metabolite synthesis. We have also seen that the capacity to sustain large fluxes of carbon and energy required for rapid metabolite production is inversely related to the growth efficiency of Now that you have completed this chapter you should be able to: micro-organiSmS. use stoichiometric data and knowledge of the elemental composition of the cell to write reaction equations for growth; interpret stoichiometric representations of aerobic growth; classify bioprocesses according to process kinetics; use yield coefficients and productivities to determine the best operating conditions for bioprocesses; determine P/O quotients, maintenance coefficients and maximum biomass yield coefficients from continuous culture data; determine the degrees of reductance of carbon substrates; classify metabolites according to the relationships between energy and metabolite synthesis; appreciate how growth efficiency of organisms influence product formation rates.