E25 Spring 2011 Homework 1 Due: Thursday, April 7, 2011, by 12:30 PM in Braun Auditorium Consulting solutions to homework sets

s or exams from previous years is considered an honor code violation. In order to earn full credit for each problem, you must show all of your work. Please post any questions you may have on the Forum for HW 1 on Coursework. Problem 1 (1 point) A) Ethanol production from glucose by Saccharomyces cerevisiae can be inhibited in the presence of what gas found in the atmosphere. This gas is not toxic to the cells. (You may have to do some independent research)? (0.25pt) Oxygen B) The Gibbs free energy is defined as G = H TS. For an enzyme catalyzed reaction that takes place in the human body, if H = 0, then S < 0. True or False? (0.25pt) False C) Find the G of the overall reaction: (0.5pt) ATP + H2OADP+Pi G = -7.3 kcal/mole C6H12O6 2C2H5OH+2CO2 G = -56.6 kcal/mole Xymomonas mobilis produces ethanol like this: C6H12O6 +ADP+ Pi 2C2H5OH+2CO2+ H2O + ATP G = ? C6H12O6 2C2H5OH+2CO2 G = -56.6 kcal/mole ADP+Pi ATP + H2O G = 7.3 kcal/mole Add up the two equations: -49.3 kcal/mole

Problem 2 (2 points) As we have discussed in class, wine is made when yeast undergoes fermentation to convert the glucose in grape juice to ethanol. The two top fuel ethanol producing countries are the United States and Brazil. In Brazil, most of their ethanol is produced by fermenting sucrose found in molasses or sugarcane. For this problem, lets evaluate a hypothetical strain of yeast that achieves the following net conversion: C12H22O11 + H2O C2H6O + CO2 The enthalpy change, H, for a reaction can be calculated in the same way as the Gibbs free energy change, G, using the enthalpies of formation, Hf, for each species. The superscript circle on the enthalpy and Gibbs free energy here denotes that these properties are measured at a standard, or reference, state. This standard state is at a temperature of 298 K (25C and a pressure of 1 bar (100 kilopascals)). Show work for answer involving calculations.

A) Balance the equation (solve for , ). (0.25 pts) C12H22O11 + H2O 4C2H6O + 4CO2 ==4 + 0.15 pts for work + 0.10 pts for correct answer B) Using the table given below, calculate H and G for the equation. (0.25 pts) H = Hf (products) Hf (reactants) = [4*(-277.7) + 4*(-393.5)] [(-2225.5) + (285.8)] = -173.5 kJ/mol G = Gf (products) Gf (reactants) = [4*(-174.8) + 4*(-394.4)] [(-1544.6)+( 237.1)] = -495.1 kJ/mol + 0.15 pts for work + 0.10 pts for correct answer C) Do you anticipate the entropy change, S, for the reaction to be positive or negative? Explain qualitatively in terms of increase or decreasing total number of molecules. (0.25 pts) Positive, because the net number of atoms is increasing, meaning they can occupy more positions in space and hence have more disorder/entropy. (CO2 will escape as a gas, giving it even more entropy, but we didnt cover that in class) + 0.25 pts for correct answer D) Calculate the entropy change, S, (in units of kJ/mol*K) for the equation using your answers from above, assuming a temperature of 298K. (0.25 pts) G = H TS S = (H G)/T = ((-173.5 kJ/mol) (-495.1 kJ/mol))/298K = 1.08 kJ/mol*K + 0.15 pts for work + 0.10 pts for correct answer Several bacteria of the genus Acetobacter convert the ethanol in wine into acetic acid in the presence of oxygen. This is one way in which vinegar is made, and it is also the reason why a bottle of wine can go bad. Winemakers often add sulfur dioxide to the wine as an antioxidant and antimicrobial agent to circumvent this secondary reaction. These Acetobacter bacteria perform this reaction via an acetaldehyde intermediate by the following general pathway: (1) C2H6O + O2 C2H4O + H2O (2) C2H4O + O2 C2H4O2 E) What is the balanced, net reaction for these two reactions? (0.5 pts) C2H6O + (1/2)O2 C2H4O + H2O C2H4O +(1/2)O2 C2H4O2 Then overall: C2H6O + O2 C2H4O2 + H2O + 0.25 pts for work + 0.25 pts for correct answer

F) Calculate G [in units of kJ/(mol C2H6O)] for the balanced net reaction you found in part (E). (0.5 pts) G = Gf (products) Gf (reactants) = (-389.9 +-237.1) (-174.8 + 0) = -452.2 kJ/mol C2H6O + 0.25 pts for work + 0.25 pts for correct answer Name Sucrose Ethanol Acetic acid Acetaldehyde Water Oxygen Carbon dioxide Problem 3 (0.5 points) In class, we have learned that entropy can be calculated using Gibbs Free Energy. We can also calculate the change in entropy of a chemical reaction by the combined entropies of the products minus the combined entropies of the reactants. This is shown in the equation below: Formula C12H22O11 C2H6O C2H4O2 C2H4O H2O O2 CO2 Hf (kJ/mol) -2225.5 -277.7 -484.5 -191.8 -285.8 0 -393.5 Gf (kJ/mol) -1544.6 -174.8 -389.9 -127.6 -237.1 0 -394.4

Determine the change in entropy for the following chemical reaction, using the equation above, occurring at standard pressure and temperature: 2H2 (g) +O2 (g) 2H2O (l) Does the entropy increase or decrease? Compound H2 (g) O2 (g) H2O (l) OH- (aq) Hf (kJ/mol) 0 0 -285.83 -229.99 Gf (kJ/mol) 0 0 -237.14 -157.28 S(J/molK) 130.68 205.14 69.91 -10.75

+0.25 points for work +0.25 points for correct answer Problem 4 (1.5 points)

Because it is so well studied and understood, Escherichia coli is commonly used in industry for pharmaceutical research and development. Preparing the liquid medium is thus a critical part of preparing E. coli for studies. Show work for each answer. A) Assuming carbon, hydrogen, oxygen, and nitrogen compose all of E. colis weight, what is E. colis (CH1.92O0.3N0.24) biomass formula weight (in units of grams/mol equivalent of C)? (0.5 pts) (12) + 1.92(1) + 0.3(16) + 0.24(14) = 22.08 g/mol +0.25 points for work +0.25 points for correct answer B) If we want achieve a cell density of 80 g cell/L using ammonium sulfate ((NH4)2SO4) and glucose (C6H12O6) for the growth medium, what are the ammonium sulfate and glucose requirements per liter of media (Give concentration in mol/L)? (0.5 pts) 12 g C / 22.08 g cells * (80 g cell/L) * (1 mol C/ 12 g C) = 3.623 mol C/L 3.36 g N / 22.08 g cells * (80 g cell/L) * (1 mol N / 14 g N) = 0.870 mol N/L MW (glucose) = 6(12) + 1(12) + 6(16) = 180 g/mol MW (ammonium sulfate) = 2(14 + 4(1)) + (32.1 + 4(16)) = 132.1 g/mol 3.623 mol C/L * (1 mol glucose/6 mol C) = 0.6038 mol glucose/L 0.870 mol N/L * (1 mol ammonium sulfate/2 mol N) = 0.435 mol ammonium sulfate/L +0.25 points for work +0.25 points for correct answer How many grams of each reagent should we add per liter of media (Give concentration in g/L)? (0.5 pts) 3.623 mol C/L * (1 mol glucose/6 mol C) * (180 g glucose/1 mol glucose) = 108.69 g glucose/L 0.870 mol N/L * (1 mol ammonium sulfate/2 mol N) * (132.1 g ammonium sulfate/1 mol ammonium sulfate) = 57.4635 g ammonium sulfate/L +0.25 points for correct glucose requirements per L media +0.25 points for correct ammonium sulfate requirements per L media Problem 5 (2.5 points) A pharmaceutical company has just hired you to oversee the production of their new antibiotic. The drug production uses an engineered E.coli that synthesizes your drug precursor. The cells grow on glucose (C6H12O6), ammonia (NH3) and oxygen and produce carbon dioxide and water. Assume that E.coli can be modeled by chemical formula CH1.92O0.3N0.24, with an overall equation of C6H12O6 + NH3 + O2 CH1.92O0.3N0.24 + CO2 + H2O You measure the respiratory quotient to be 1.5 A) Define the respiratory quotient. (0.25pt) RQ = / or number of moles of CO2 produced per mole of O2 consumed B) Write an equation that balances the degrees of reduction. (0.25pt) E coli: 4+1.92-0.3*2-0.24*3 = 4.6 glucose: 4*6+12-6*2 = 24 24 - 4 = 4.6

C) Solve for , , , , and . (1pt) Equations C: 6=1+ H: 12+3=1.92+2 O: 6+2=0.3+2+ N: =0.24 RQ: 1.5=/ DR: 24 - 4 = 4.6 Solution: 1.5= (RQ equation) 6 = + 1.15 6 = + 1 = 1.5 + 1 (Carbon and RQ equation) 6 = + 1.15 = 1.5 + 1 = 0.3 = 0.45 = 0.2417 = 0.24 = 0.85 (from Hydrogen or Oxygen equation) Solution using linear algebra A= 6 0 0 -1 0 12 3 0 0 -2 6 0 2 -2 -1 0 1 0 0 0 0 0 1.5 -1 0 24 0 -4 0 0 = 1 1.92 0.3 0.24 0 4.6 X = A\B: 0.2417 0.2400 0.3000 0.4500 0.8500 D) What is the cell yield in grams of cells per gram of glucose? (0.5pt) MW of cell mass = 22.08g/mol YP = 1/a (22g / 1mol cell mass) (1 mol glucose / 180g) = 0.5057 E) Your goal is to maximize cell yield. Do you want the respiratory quotient to increase or decrease? (0.5pt)

Increasing the RQ will increase the cell yield -They must provide some justification Bonus question: Plot the change in cell yield vs. respiratory quotient for RQ = 1.1 to 3 (0.25pt)

0.664 0.662 0.66 0.658 0.656 0.654 0.652 0.65 0.648

1.2

1.4

1.6

1.8

2.2

2.4

2.6

2.8

clear all; A =[6,0,0,-1,0;12,3,0,0,-2;6,0,2,-2,-1;0,1,0,0,0;24,0,-4,0,0;0,0,1.50000000000000,-1,0;]; B= [1;1.92000000000000;0.300000000000000;0.240000000000000;4.60000000000000;0;]; RQ = 1.1:.01:3; for k=1:length(RQ) A(5,3)=RQ(k); X = A\B; Yp(k) = 1/X(1)*22.08/180; if min(X) < 0 Yp(k)=0; end end plot(RQ,Yp) Problem 6 (1 point) You are planning to grow E. coli, and we want to make as many cells as possible. You use a basic medium that provides for glucose-limited growth. 30 mM glucose is the only carbon source. 1/3 of the carbon in the medium ends up as carbon dioxide. As we discussed in class, the fermentation can be described as: Glucose + ammonia + oxygen new cells + carbon dioxide + water + heat C6H12O6+ NH3+ O2 CHaObNc+ CO2+ H2O

A) Given that the elemental composition of E. coli cells is CH1.77O0.49N0.24, what is the theoretical maximum yield (i.e. you know you definitely cannot get more than this amount) you could expect (in g cells/liter)? (0.5 pts) MW per C E. coli = 12(1) + 1(1.77) + 16(0.49) + 14(0.24) = 25 g/mol (6)(2/3)=1 =0.25 mol glucose / mol C cells (30 mM) = 0.03 mol/l (0.03 mol glucose/l)(mol C cells/0.25 mol glucose)(25 g cells/mol C cells) = 3.0 g/l cells B) For your medium to be glucose-limited and not ammonia limited, the ammonia must be greater than what concentration (in mM)? (0.5 pts) 3.0 g/l cells =0.24 (30 mM glucose)(0.24 mol NH3 /0.25 mol glucose) = 28.8 mM NH3 28.8 mM NH3 Problem 7 (1.5 point) Using the convention from class, the degree of reductance also approximately tells you the number of electrons that can be transferred to oxygen during the making and breaking of bonds involved in aerobic respiration (or combustion). The amount of electrons transferred is proportional to the energy generated. For the following substrates, calculate the degree of reductance and rank the substrates in order of decreasing energy density per carbon atom. Natural gas or methane, CH4 Fat, C55H98O6 Starch, C60H10O50 Protein, C5978H9285 O1941N1835S31 Solution (no units OK since we did not emphasize the units in class): (0.25 pts) DR CH4 = (1)(4)+(4)(1) = 8 e-/mol DR/C CH4 = 8/1 = 8 e-/mol C (0.25 pts) DR C55H98O6 = (55)(4)+(98)(1)+(6)(-2) = 306 e-/mol DR/C C55H98O6 = 306/55 = 5.6 e-/mol C (0.25 pts) DR C60H10O50 = (60)(4)+(10)(1)+(50)(-2) =150 e-/mol DR/C C60H10O50 = 150/60 = 2.5 e-/mol C (0.25 pts) DR C5978H9285 O1941N1835S31 = (5978)(4)+(9285)(1)+(1941)(-2)+(1835)(-3)+(31)(6) = 23996 e-/mol DR/C C5978H9285 O1941N1835S31 = 23996/5978 = 4.0 e-/mol C

(0.5 pts) Methane>Fat>Protein>Starch