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Closed Book, Closed Notes, Closed Neighbors The questions here were from Exam II and I have provided solutions - as detailed as I could here, to each question. Along with the solution, I have added comments reﬂecting some of the answers I have seen during grading. In addition to the questions that appeared in the exam, I have added a few more solutions at the end to questions from the back of the book, again with solutions. 1. (20 points) The maximum speciﬁc oxygen uptake rate of a particular organism is given as 5 mmol O2 g−1 h−1 . The organism is grown in a stirred fermenter to a cell density of 40 g/liter. The kL a under these conditions is 0.15 sec−1 . At the fermenter operating temperature and pressure, the solubility of oxygen in the culture liquid is 8 x 10−3 kg m−3 . Is the rate of cell metabolism limited by mass transfer or solely dependent on metabolic kinetics? (i.e. are you supplying oxygen at a rate suﬃcient to maintain the cells at the density required?)

Solution: We need to calculate the Oxygen transport rate (OTR) and Oxygen uptake rate (OTR). qO2 is given as 5 mmol Oxygen/(gm.hr) - you calculate OUR as 5.00 mmol/(gm.hr) multiplied by 40.00 gm/liter (cell density) and get 200.00 mmol/(liter.hour). Now OT R = kL a C ∗ − CL ) you get maximum OTR when CL is zero. Setting CL to zero you can calculate OTR. Since the OUR was calculated as 200.00 mmol/(liter.hour) we have to convert the OTR into those units. We multiply 0.15 sec−1 with 3600.00 seconds/hr to get 540.00 hr−1 We then multiply this with 0.0080 kg/(m3 sec) to get 4.320000 kg/(m3 .hr). We have to convery the kg of Oxygen into millimoles and m3 into liters so we can make a direct comparison. We know that the molecular weight of oxygen is 32 grams/mole and we have 1000 grams/kg and 1000 liters in 1 m3 . So by multiplying 4.32 kg/(m3 .hr) with with 1000.00 grams/kg (4320.000000) and 0.0010 m3 /liter (4.320000) and divided by 32.00 grams/mole we get OTR to be 0.135000 moles or when multiplied by 1000.000 millimoles/mole we get OTR to be 135.000000 millimoles. We had earlier calculated OUR to be 200.00 So the growth is limited by Mass Transfer

0 0.06 5.26 3.06 4.07 6. If the OD at 420 nm is linear in cell density with and OD of 0. (I do want to see a plot of the data).5 0.0 0.06 5.11 1. At time t = 0.37 3. the time lag and the overall yield factors (grams of cell per grams of substrate) assuming substrate exhaustion in each case.15 % lactose and the subsequent OD vs time data is given in column 2.10 6. approximate values are OK in exams .70 OD 2 Time (hrs) 0.88 0.0 0.00 1.as long as you clearly show how you arrived at your answer. about 1 hour (may be?) in the simple medium).08 1.00 Solution: The ﬁrst thing to do is plot the data. Explain the shape of the growth curve in each case.18 7.5 0.1 mg dry weight of cells per milliliter. I am going to assume that the exponential growth phase starts at 1.04 0.5 0.175 equal to 0.5 0. The same species is also cultured in a complex medium containing 0.5 0.04.14 and at 5. µmax = X ln X o t .52 0.14 2.0 0. calculate the maximum speciﬁc growth rate. almost 2 hours.5 hours .5 0. You can see this in the log plot clearly.0 0. Let us take the growth curve in the simple medium ﬁrst.0 0.32 OD OD 1 2 0. this culture is innoculated into a larger sterile volume of the identical medium The optical density (OD) at 420 nm (which you can consider to be proportional to cell density) versus time following innoculation is given in column 1 of the table.13 7.3 % (weight/volume) glucose.49 4.50 0.5 0.84 1.48 1.26 8.43 1. As I have always said in class. There is a lag in the growth curves (very pronounced in the complex medium.0 0.04 0.2.0 hours the OD value is 1.OD value of 0.0 0. (20 points) A culture is grown in a simple medium including 0.60 0.15 % glucose and 0. OD Time (hrs) 1 0 0.20 2.06 0.5 0. µmax . You do not have to convert the OD to cell density right away (you can do all the calculations and apply the calibration factor supplied).

00000 = 0.50000 hours you get the growth rate to be µ=0.10.00 and like earlier.57295 in OD units The conversion is 0.17500 OD units for 0. Doing these calculations for the complex medium we get (in a similar manner) I am going to assume that the exponential growth phase in this case starts at 2 hours . we notice that at 6 hours.00533 when divided by 3.let’s calculate Xo where both of them.52287 in OD units The conversion is 0.65393 and the ln 1.327401 0.30259 and so -1.32740 for the growth in simple medium.24911 for the growth in complex medium for the second phase in lactose. Here is a summary Medium Type Lag Phase Simple 1.57143 mg/ml per OD unit and we can recalculate µ as equal to 0.where the X is the value of the cell density at time t and Xo the cell density at time t = 0 (or the beginning) (here. our t = 0 is 2 hours and hence the t in the denominator is 3.10000 mg cells/ml or 0.10000 = −2.65393 when divided by 1.10000 mg cells/ml or 0. At 5.65393 AND their diﬀerence is 0. the growth rate in the ﬁrst phase for the second curve.56862 AND their diﬀerence is 1. As we can see.32740 For the growth phase in lactose.48 and like earlier. µmax = t where the X is the value of the cell density at time t and Xo the cell density at time t = 0 (or the beginning) (here. the OD value is 0. our t = 0 is 2 hours and hence the t in the denominator is 3.29878 is smaller than the growth phase for the ﬁrst curve 0.57143 mg/ml per OD unit and we can recalculate µ as equal to 0.5 hours) The ln 1.48000 = −0.29878 for the growth in complex medium for the ﬁrst phase in glucose.5 hrs Complex 2.5 hours) ln X The ln 0. our t = 0 is 6 hours and hence the t in the denominator is 1.OD value of 0.14 = −1.0 hours) ln X The ln 0. µmax = t the X is the value of the cell density at time t and Xo the cell density at time t = 0 (or the beginning) (here.298783 0.03922 and the ln 0.50000 hours you get the growth rate to be µ=0.04 = 0.56862 when divided by 3.0 hours.52000 = −0. 0.57143 mg/ml per OD unit and we can recalculate µ as equal to 0.17500 OD units for 0.52 and at Xo 7.17500 OD units for 0.00533 AND their diﬀerence is 2.00000 hours you get the growth rate to be µ=0.249114 .0 hrs Exp Phase I (µ1 ) Exp Phase II (µ2 ) 0.5 hours it is 1.96611 and so -2. the OD is 0.00000 and so 0.10000 mg cells/ml or 0.43595 in OD units The conversion is 0. We see however that there are two exponential phases .73397 and the ln 0.

The ∆S for glucose for complex medium is 0.57 mg/ml per OD unit you get 0.30000 % weight/volume and so the Yield Coeﬃcient is YX/S = 1.828571 Medium Type Y (glucose) Y (lactose) Simple 1.14 = 0.828571 .27 mg/ml cells. As you can imagine.714285 For the complex medium.10 = 0.15000 % weight/volume and so the Yield Coeﬃcient is YX/S = 1.90 in OD units and when multiplied by the conversion factor of 0.48 − 0. The ∆S for glucose for simple medium is 0. the other for glucose in complex medium and one for lactose in the complex medium) Let us do each. we get ∆X as equal to 0.48 in OD units and when multiplied by the conversion factor of 0.15000 % weight/volume and so the Yield Coeﬃcient is YX/S = 1.00 − 0.5 hours.22 mg/ml cells. We now need to calculate the overall yield coeﬃcients.51 mg/ml cells.38 in OD units and when multiplied by the conversion factor of 0.714285 Complex 1. The ∆S for glucose for complex medium is 0.57 mg/ml per OD unit you get 0.447619 1. you will have three (One for the curve in simple medium for glucose.Notice that there is a lag between the two phases in the complex medium of 1. we get ∆X as equal to 1. Let us calculate the ∆X for the exponential growth phase for simple medium and it is 1.57 mg/ml per OD unit you get 0.52 = 0.447619 and for the complex medium in lactose.04 − 0. for the glucose phase.

Write down the mass balances for oxygen. (as we have done for oxygen) we know that the rate of transport is calculated by kL a C ∗ − C and the units are grams/(liter. write down the steady state balances for a sterile feed for cells and for substrate 1 and 2. VR gives you the rate of substrate being delivered in grams/hour. Assume that the oxygen and methane behave independently . deﬁne any variables you think you need to deﬁne. So . (Hint: Feed is sterile. You can assume that both the liquid and the gas phases are well-mixed.e.69 in your textbook in the context of a substrate that is in the gas phase. When we are bubbling a gas into the system. the relationship between the concentration of the component in the gas phase and the dissolved concentration (what you know as C ∗ ) is not aﬀected by the presence of the other component. Make any assumptions you may need to make. Assuming constant Yield Coeﬃcients for each substrate.hour) when multiplied by volume of the reactor.3. Remember we are writing in terms of amount per time for each substrate and biomass (In the usual notations). Let kL1 a be the overall mass transfer coeﬃcient for the transport of oxygen from the gas phase into the liquid phase (and kL2 a be that for the transport of methane from the gas phase into the liquid phase. sparged fermentor (sparging refers to the bubbling of oxygen (and methane in this case) into the reactor) is described by a double MichaelisMenten form of cell growth µ = µmax S2 S1 KS 1 + S 1 KS 2 + S 2 (1) where S1 is oxygen and S2 is methane.) Solution: The equation that you need in each case is what comes in must equal to what leaves and what is generated (or consumed) in the reactor. You can (if you want to) consider one or two input ports for each of oxygen and methane .i. (10 points) Consider the following case where the utilization of methane in the presence of oxygen in a continuous ﬂow. methane and biomass. Assume that you are bubbling in oxygen and methane using one port and taking the exit gases out through one port.just remember that both oxygen and methane are substrates for the organism and that you will have two substrate balances. Note the units of the terms in the equation 6. Henry’s Law is used to relate the concentration of the component in the gas phase to that in the liquid What we are doing is writing equation 6.69 which is written for a substrate in the solution. So is the concentration of substrate (grams/liter) coming in multiplied by ﬂow rate (liter/hour) gives you the rate of substrate being delivered (grams/hour).

At steady state. you can equate the transfer rate to the uptake rate due to growth .so one assumption you can make is that all of the methane (and oxygen) transported are consumed by the 1 where growing cells.So here are all the terms written for methane (similar for oxygen) (the S in the growth equation refer to the dissolved concentration of the methane (or oxygen) in the medium) Description What comes in What leaves What gets consumed Equation ∗ kL a CCH − CCH 4 VR 4 Nothing 1 VR µX Y . Rate of consumption of methane by the growing cells VR µX Y the µ is what is given in the problem statement .What about removal? Are these two substrates removed in the gas phase? Liquid phase? Not speciﬁed.∗ − CCH 4 VR The rate at which methane is being delivered to the reactor is kL a CCH 4 (and a similar term for oxygen) .

Maximum growth rate (µmax ) . In some cases however the growth may be inhibited at higher oxygen concentrations (substrate inhibition). batch fermentation of an aerobic bacterium growing on methanol gave the results shown in the table.1 from your text book that I have reproduced here and for which I have provided a solution A simple. (20 points) This is problem 6. One way to do this is by using pure oxygen instead of air for example. Calculate: 1. or what you know as C ∗ ).take a look at the equation 3. (10 points) Oxygen transport to growing cultures can be increased by augmenting the oxygen partial pressures in the bubbles (which will increase the dissolved oxygen concentration in the medium. At what value of γ will the growth rate be maximum? (Your answer should be in terms of S ) Solution: This is substrate inhibition .and calculate the substrate concentration that gives you the maximum reaction rate as Smax = Note that I substituted 1 for Km and 1 γ 1 1 γ (6) for KS1 5. Assume that the growth dependence on oxygen can be represented by µ= µmax S 1 + S + γS 2 (2) where γ is a constant and S is the concentration of oxygen in the medium.4.34 in page 72 of your book Vm S ν= (3) S2 Km + S + K S 1 The substrate concentration that gives you the maximum reaction rate is Smax = Rewrite the equation you have this way µ= µmax S 2 1 + S + S1 γ Km KS 1 (4) (5) Now you can do a direct comparison .

9800 10. Speciﬁc growth rate (µnet at 10 hours Time (h) X (g/l) 0.300 hrs.0000 0.20998 .0770 0. Mass doubling time (td ) 4. Mass doubling time ln(2) is µ and is 3.8000 4.2000 2.one is in the actual data given and the other is a log transformed data versus time The log plot shows more clearly that the exponential growth starts at time 2.6000 16.0000 5. The 10 hour time point is midway in the fermentation run and so the growth rate is the same as before.2000 S (g/l) 9.3050 8.so you can see where the lag phase ends and where the growth phase begins .0700 8.65005.0000 Solution: It may be easier if you plot the data (even if not asked) .2000 14. Saturation constant (Ks ) 5.6000 0.9200 0.2.2300 9.0000 0.7700 12.0000 0.0000 0.0 hrs and ends at 14 hours .2100 9.0000 1.0000 3.1500 18.Take a look at these two plots for the growth curve . 0.0000 6. The calculation of the saturation constant is not easy max since you need value of the growth rate at diﬀerent substrate concentrations (so I will ignore that for now).2110 4.0000 6.so let’s calculate the µmax The value of the biomass concentration at the end of the exponential growth phase is 5.0300 6.60 and the natural log value is 1.72276 and the ∆t you need is 12 hours and so you can calculate µmax as equal to 0. Yield on substrate (YX/S ) 3.20998 hr−1 Yield on substrate can be calculated using the data at the extremes (values at time points of 18 hrs and that at 0 hrs) and you get 0.

they have started with the most general equation and then simpliﬁed to get the expressions for the substrate and the biomass concentration. (20 points) Write the steady state material balance on an ideal chemostat for both biomass and substrate. D = µ since X = 0.64 (I have used µ for the speciﬁc growth rate and dropped all the subscripts and such) The material balance on the cell concentration around the chemostat gives you −F X + VR µX = 0 and dividing by VR we get F F −V X + µX = 0 or X − V + µ = 0 which is written as X −D + µ = 0 where R R F D = VR and called the dilution rate. (This is section 6.4. You can also assume that the feed is sterile. In the book. Assume that the Monod equation can be used to describe the growth rate on the limiting substrate.we can Monod equation is written as µ = K s +S s +S Ks D solve for S from this equation to get S = µm −D .whatever you are comfortable with) Solution: We start with equation 6. Thus at steady state. yield coeﬃcients and so on. parameters from the Monod equation. The µm S µm S and so we can write D = K . Derive the expressions for the substrate and the biomass concentration in the reactor eﬄuent in terms of dilution rates. Now the yield coeﬃcient is deﬁned as YX/S = SoX (note: Xo = 0) and hence X = YX/S (So − S ) and substituting for S −S sD you get X = YX/S (So − µK ) m −D .3 in your text book. You can do that or write down the simpliﬁed equations directly . assuming that endogeneous metabolism or the death rate is negligible compared to the growth rate.6. incoming substrate concentration.

The saturation constant using this substrate is 1.hr) You need to carefully keep track of variables and what is required of you in the problem.88200 g cell/liter.46 g cell/g acetate.8Dmax 3.6 from your text book that I have reproduced here and for which I have provided a solution Pseudomonas sp has a mass doubling time of 2. max you can calculate µmax (You may ﬁnd the text confusing at times .00000 minus 5.20000 grams/l Cell productivity is DX which is equal KS D to DYX/S So − µmax and we can calculate that.look at the equation for calculating cell concentration X = KS D YX/S So − S which can be written (using the expression for S = µmax as follows −D KS D X = YX/S So − µmax and since D = µmax you can write X = YX/S So − KS −D 2 and calculate X to be equal to 16.14440. (20 points) This is problem 6. The dilution rate for calculating cell concentration is half of the maximum growth rate 0.what we need is the maximum speciﬁc growth rate for these calculations) and so we calculate µ as 0.10628 and hence we get DX to be equal to 3.79999 multiplied by DYX/S which is 0. Substrate concentration when the dilution rate is 00.20000 which is 32.8µmax 0.8Dmax ln(2) Solution: We know that doubling time t = µ and so knowing doubling time.8µmax (which is 0.3 g/l (which is unusually high) and cell yield on acetate is 0. Let us ﬁrst calculate So − S −D as 38.4 hrs when grown on acetate.23104) you get S = µmax or simply S = 4KS −0.6931 divided by 2. Cell productivity at 0.8µmax and you get S as equal to 5.23104 multiplied by 0.28881 which is 0.46000 which is 0.4000 hrs and we get 0. . Cell concentration when the dilution rate is one half of the maximum 2.2888 hr−1 . But notice something .48606 gcell/(l. If we operate a chemostate on a feed stream containing 38 g/l acetate ﬁnd the following 1. Maximum dilution rate 4. When the dilution rate is KS 0.8Dmax or 0.7.

001 and get 0.5 hr−1 KS 30 mg/l. (20 points) This is problem 6.hr 2 which is greater than 10 and hence suﬃcient oxygen cannot be provided Y .7829596 g/l Now we need to calculate the X KS D from X = YX/S So − µmax 10608. We wish to operate the chemostat with an ethanol concentration in the feed of 22 grams/liter. YX/S 0. Determine the required dilution rate and whether suﬃcient oxygen can be provided. Let us calculate KS Remember to convert 22 grams/liter Dopt using the formula Dopt = µm 1 − KS + So to 22000 mg/liter (units of KS ) and you will get 0.9596609 in mg/liter and convert that to grams/liter by multiplying by 0. X/S Solution: Notice that you are not given YX/O2 which can be calculated as YO (as 2 /S the ratio) and we can calculate YX/O2 equal to 0.4815489 Now calculate KS D S = µmax −D as equal to 782.10 from your text book that I have reproduced here and for which I have provided a solution Ethanol is to be used as a substrate for single cell protein production in a chemostat.5201695 in mg/liter and you get 10.6085201 in −D µX g/liter Calculate OUR as OU R = YX/O So OUR equals 20.5 cells/g ethanol and YO2 /S 2 g O2 /g ethanol. We also wish to maximize the biomass productivity and minimize the loss of ethanol in the eﬄuent.4340848 gmO2 /liter.25000 gcells/gO2 . The available equipment can achieve an oxygen transfer rate of 10 g O2 /liter of liquid per hour.8. Assume the kinetics of cell growth on ethanol is of the Monod type with µm 0.

OD versus time for growth in Simple and Complex Medium 1.1 0 0 2 4 Time (Hours) 6 8 10 'simple.7 0.dat' using 1:2 'complex.3 0.1 1 0.9 0.dat' using 1:2 .8 0.6 OD 0.4 0.5 0.2 0.

5 -1 log_e(OD) -1.dat' using 1:(log($2)) 'complex.5 'simple.dat' using 1:(log($2)) -3 0 1 2 3 4 Time (hours) 5 6 7 8 .5 -2 -2.log_e(OD) versus time for growth in simple and complex medium 0 -0.

1 Growth Curve 7 6 5 4 X (g/l) 3 2 1 'exam2r.Problem 6.dat' using 1:2 0 0 2 4 6 8 10 12 14 16 18 Time (hrs) .

1 Growth Curve (in log) 2 1.Problem 6.5 1 natural log of X (g/l) 0.5 'exam2r.5 0 -0.dat' using 1:(log($2)) -2 0 2 4 6 8 10 12 14 16 18 Time (hrs) .5 -1 -1.

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