Efficiency of growth and product formation 3. Introduction 3.2 Relationships between product formation and growth 3.3 Determination of maintenance energy requirement and maximum 3.4 Determination of P/o quotients 3.5 Metabolite overproduction and growth efficiency Summary and objectives
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
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. nergetics In this chapter we will consider growth and product formation from an energetics perspective 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 petween energy and metabolite synthesis. Examples of commercially significant products in each class are also discussed cquations describing growth and product formation stoichiometry are presented in this pter. 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 3.1 Growth stoichiometry 3.1.1 Yield coefficients In any quantitative ass of growth and /or pro on, 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 comew formed is often proportional to the mass of substrate utilised. Mathematically this is as the com responding ratio, or yield coefficient: △X x/s △s AX= amount of biomass produced As=amount of substrate consumed
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 yo2 is used to relate the amount of biomass formed to the amount of oxygen consumed, so 9 g glucose 684 oxygen 27.2 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 stbichiom tre stoichiometric relationships can be useful in monitoring fermentations. For example, some product concentrations, such as Co 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 Energy for maintenance is the energy required for survival, or non-growth related mantuan 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: ASassimilation+ ASgrowth energy ASmaintenance energy As= the total amount of substrate consumed ASassimilation amount of substrate assimilated Sgrowth energy=amount of substrate consumed to provide energy for grwoth Asmaintenance energy=amount of substrate consumed to provide energy for maintenance Or, expressed as yield coefficients Yx/s=△X/△ Assimilation+△X/△ Sgrowth energy+△X/△ maintenance energy
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:
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/ASassimilation)is constant regardless of the growth rate, the overall yield coefficient pendence (Yx/d is variable and dependent upon the environmental conditions within the culture To illustrate this growth rate dependence, consider two extremes: a culture growing at ts maximum specific growth rate will use most of its substrate for assimilation and rowth 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 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 An elemental material balance approach to growth stoichiometry requires an empirical formula for dry weight material 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 ieθ=1,thenα,阝,andδ 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 elements to be a significant proportion of cell matel lemental analysis shows these macro-elements, such as phosphate and sulphur, if 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' 'unity Use the data on elemental composition shown below to determine the empirical chemical form nd the formula weight for the yeast
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 mposition(% by weight) Formula C HN O Bacterum4717813.7313 208 44.7628.531.21080.6 ? ? Atomic weights:C12011;H1.008;N,14.008;O,16.000P30.98;532.06 The empirical chemical formula is CH1.65 No16 O0.52 Po.01 So.005 and the formula weight is 24.6. For example, the subscript for O in the chemical formula is determined as allows:(312/16)/(447/12011) The formula weight is then calculated by multiplying the coefficients by the atomic weights and summing them. Thus(1x12011)+(1.008x1.65)+(14008×016)+(16x0.52)+(30.9x001) +(3206x0.05)=246 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 CHoOB Ns Carbon source CHro Nitrogen source HomNn stoichiometric We can now write the reaction equation by introducing stoichiometric coefficients for dCH,O,+bO2+cH,.N->CH,O, Ns+d'HO+eCOL E-3.1 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 he four elements as follows: H:ax+cl=a+2d ∏I Write material balances for the remaining two elements in the reaction equation CE-31) The material balances are O:ay+2b+cm=阝+d+2e
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‘ n Write material balances for the remaining two elements in the reaction equation (E - 3.1). The material balances are: 0 a‘y + 2b’ + c’m = + d’+ 2e’ N: c’n = 6
Chapter 3 SAQ 3.1 a bacterium is grown aerobically with glucose as sole source of carbon and ammonium 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 CH16 Oz Nozo. (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 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 metabol 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 ergy ATP formation AtP utilisation (substrate level phosphorylation (oxidative phosphorylation (maintenance and dissipate For substrate level phosphorylation we can writ e。a(ADP+P)→Ea(ATP+H2O) E-3.2 Where: Es=number of substrate-level phosphorylations per mole of carbon utilised For oxidative phosphorylation we can write: 2b(P/O)(ADP +Pi)-2b(P/O)(ATP H2O) E-33 Where: P/O=is the number of ADP phosphorylations per atom of oxygen consumed For biosynthesis(ATP utilisation)we can write MW MW 又(ATP+H2O) (ADP+ Pi ATP ATP E-34 Where y me omass mass of cells formed per mol of atp utilised in biosynthesis
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.& QZNO^. (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 Y ATP The Y arp in the equation can be determined from growth yields and known routes of max ATP synthesis. For growth of Escherichia coli on glucose and mineral salts the Y ATP value, estimated from known cell composition and known biosynthetic pathways, is 28.8 g dry weight" ATP. However, the YATP determined experimentally from yield measurements is often around 50% of the theoretical (12 to 14 g dry weight mol ATP Explain the discrepancy between theoretical and experimentally derived values for y The discrepancy arises because aTP is used to drive processes which are not direct related to growth, eg membrane transport processes, protein tumover. These are called the maintenance and dissipation demands for AtP. For maintenance and dissipation we can write, simply: c(ATP+HO)→c(ADP+P) E-3.5 balance for Now lets consider the balance for nadh, ie: NADH NADH formation (energy source dissimilation (biosynthesis) oxidative phosphoryiation) To represent the balance for NADH using quantitative relationships, we must consider e degrees of degree of The degree of reductance of material is the number of available electrons per atom of reductance carbon and is determined using C(+4),H(+1), 0(-2)and N(-3) So, for biomass with an empirical formula of CH1.666No2 Ooz, the degree of reductance()is: (4)+(1x1.666)-(3x02)-(2x027=4526 What are the degrees of reductance of (1)co, (2)NH, and (3) 2H862Oa5 1? The degrees of reductance are( 1)0,(2)0 and (3)3.93. The answer for(3)was determined 4+(86.2/552x1)-(45.1/552x2) reduction For energy source dissimilation we can then write: aCHO, +Ho+ aNAD*->aCO,+5 a(NADH+H) E-3.6 Where: s=the degree of reductance of carbon substrate
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 II Explain the discrepancy between theoretical and experimentally derived values 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) bahnca for Now lets consider the balance for NADH, ie: 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+o)CH Oy+=H O, 1 (Y-7(1+0)-)NADH+H) 23 →> CHa n+CO+H2O 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 phosphory lation we can write 2b(NADH +H)+bo -> 2b NAD+2bHO E-3.8 The coefficients a, b and c, which appear throughout these balance equations describe the extent to which these reactions occur relative to the growth reaction ie 1 +o)and re written taken into account elemental balances for each reaction How should the balance reactions already described for aerobic metabolism be adapted for anaerobic fermentations? 1)By deleting O everywhere 2) By dropping the oxidative phosphorylation reaction SAQ 3.2 Use the equations already given to predict how you might expect Yy to be 1)a decrease in the degree of reductance of substrate; 2)an increase in the efficiency of oxidative horvlation 3)a decrease in energy demand for biomass synthesis? Give reasons for your responses 3.2 Relationships between product formation and growth process When considering product formation stoichiometries it is essential to define the knetcs relationship between product formation and growth. Essentially, this classification divides microbial product production processes into four types
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 fomation 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 productis formed from substrate through or more reactions catalysed by enzymes in the cells. Examples: ste hoie hydroxylation Figure 3. 1 illustrates the main patterns for batch fermentation process kinetics for type AA Type 3 Time product formaton Figure 3. 1 General pattems for batch fermentation process kinetics type 1 In type 1 processes, substrate utilisation, biomass formation and product formation are linked in a simple chemical reaction. For example, if the product contains C, H,oonly, re simply extend the biomass material balance equation used earlier. d CH O +O+CHOm Nn-> CHa OB Na+d H2o +e CO2+f ow E-3.9 product
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
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 product formed f'n substrate used a′ (mols product formed / mols substrate consumed) E-3.10 Where 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 mols of glucose( C6H12 0 are required to produce one mol of Cs5 2 Ha.Os. Yp/s=(1x6)/(955x552)=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 CHoY+H2O+(·Z)NAD d CHoW+ d(1-Z)cO+da(y-ZY )(ADH +H) Ep d(ADP+ P->Ep d(ATP+ H2O) E-3.12 Z= the fraction of carbon substrate used for convertion to prod Ep= the number of ATPs generated by product formation If the coefficient E in the product formation equation(E-3.12)was negative, would this indicate? The ATP is utilised rather than generated in connection with product formation. at does the coefficient d and the parameter denote in the product formation onCE-312)? The coefficient d denotes the extent to which the reaction occurs relative to the growth reaction. The parameter Yp denotes the degree of reductance of 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
