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: