What do all these things have in common?
What do all these things have in common?
DETERMINING KINETIC PARAMETERS OF SACCHAROMYCES
CEREVISIAE GROWTH IN A
BATCH STIRRED-TANK REACTOR
Joyanne SchneiderCH EN 4903
November 28, 2006
OverviewOverview
•Problem Statement and SetupProblem Statement and Setup
•TheoryTheory
•Results and DiscussionResults and Discussion
•Conclusions and Conclusions and RecommendationsRecommendations
•Questions and AnswersQuestions and Answers
Problem Statement and SetupProblem Statement and Setup
Biochemical company wanted to obtain growth Biochemical company wanted to obtain growth kinetics of a genetically modified yeast strain kinetics of a genetically modified yeast strain analogous to analogous to S. cerevisae S. cerevisae for use in for use in recombinant technologyrecombinant technology::
volumetric mass transfer coefficient (kLa) volumetric mass transfer coefficient (kLa) specific respiration rate, OUR specific respiration rate, OUR yield coefficient Yyield coefficient YX/SX/S
maximum specific growth rate (μmaximum specific growth rate (μmaxmax) ) specific glucose uptake rate, Rspecific glucose uptake rate, Rvv
Problem Statement and Setup: Problem Statement and Setup: New Brunswick BSTR ApparatusNew Brunswick BSTR Apparatus
Problem Statement and Setup:Problem Statement and Setup: ConditionsConditions
Temperature: 37 degrees CelsiusTemperature: 37 degrees Celsius pH: 6.5pH: 6.5 Agitation Rate: 500 RMPAgitation Rate: 500 RMP Air Flow Rate: 800 cc/minAir Flow Rate: 800 cc/min Startup: 1.5 L Deionized water Startup: 1.5 L Deionized water 40 g/L glucose 40 g/L glucose 10 g/L of yeast extract10 g/L of yeast extract 20 g/L of Bacto Peptone 20 g/L of Bacto Peptone
Thoery: PhasesThoery: Phases
Lag Phase (minimize)Lag Phase (minimize)
Acceleration PhaseAcceleration Phase
Exponential Growth PhaseExponential Growth Phase
Deceleration PhaseDeceleration Phase
Stationary PhaseStationary Phase
Death PhaseDeath Phase
Theory: kTheory: kLLa without cellsa without cells
Using Henry’s Law: Using Henry’s Law:
After re-aeration begins, After re-aeration begins,
Oxygen Transfer Rate
0
20
40
60
80
100
120
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16Time, hr
% O
2 S
atu
rati
on
HxyP
)*( CCaLk
dtdC
Theory: kTheory: kLLa without cells, a without cells, cont.cont.
Dividing both sides by C*, separating Dividing both sides by C*, separating variables, and integrating:variables, and integrating:
Determining kLa
-7-6-5-4-3-2-10
-0.0018 -0.0016 -0.0014 -0.0012 -0.001 -0.0008 -0.0006 -0.0004 -0.0002 0
Time, hr
ln(1
-C/C
*)
)()*
1( taLk
CCLn
Theory: OUR and kTheory: OUR and kLLa with cellsa with cellsDuring de-aeration, During de-aeration,
During re-aeration, During re-aeration,
OUR and kLa Determination
0
10
20
30
40
50
60
0 0.05 0.1 0.15 0.2 0.25
Time, hr
% O
2 s
atu
rati
on
OURdtCCd *)/(
)*
1(*)/(
CCkLaOUR
dtCCd
Theory: OUR and kLa with cells Theory: OUR and kLa with cells cont.cont.
Once OUR is determined, kOnce OUR is determined, kLLa can be determined by plotting a can be determined by plotting change in percent saturation plus specific respiration rate change in percent saturation plus specific respiration rate versus one minus percent saturation, 1-C/C*.versus one minus percent saturation, 1-C/C*.
Determining kLa
0
500
1000
1500
2000
2500
0 10 20 30 40 50 60 70 801-C/C*
d(C
/C*)
/dt
+
OU
R
Theory: Yield CoefficientTheory: Yield Coefficient
Yield is given by,Yield is given by,
where where ΔΔX is the change in cell X is the change in cell
concentration and concentration and ΔΔ S is the change in substrate S is the change in substrate (glucose) concentration.(glucose) concentration.
Glucose and cell concentrations obtained every half-Glucose and cell concentrations obtained every half-hour using HPLC and spectrometry (absorbance), hour using HPLC and spectrometry (absorbance), respectively.respectively.
SX
SXY
/
Theory:Theory:μμmaxmax
Monod Equation:Monod Equation:
μμ is the specific growth rate is the specific growth rate μμmaxmax is the maximum specific growth rate is the maximum specific growth rate S is the substrate (glucose) concentrationS is the substrate (glucose) concentration KKss is the Monod constant is the Monod constant
μμmaxmax is the asymptote of is the asymptote of μμ plotted as a function plotted as a function
of Sof S
SsKS
max
Theory: Theory: μμmaxmax (cont.)(cont.)
Determining Max Growth Rate
0
0.2
0.4
0.6
0.8
0 10 20 30 40
Glucose Conc. (g/L)
Gro
wth
Ra
te
(hr-1
)
Theory: Theory: μμmaxmax (cont.)(cont.)
If few samples are taken, no asymptotic If few samples are taken, no asymptotic relationshiprelationshipBecause rate of cell growth isBecause rate of cell growth is
Separating variables and integrating gives:Separating variables and integrating gives:
Plotting gives a slope of uPlotting gives a slope of umax.max.
XdtdX
gr
tLnXmax
Theory: RTheory: Rvv
The volumetric glucose uptake rate (g/(L-hr) The volumetric glucose uptake rate (g/(L-hr) is given by:is given by:
Since it is just change in glucose Since it is just change in glucose concentration per time, can just be calculated concentration per time, can just be calculated from:from:
XSX
Y
u
vR
dtdS
/
max
0
0t
ft
SfS
dtdS
Results and Discussion: Results and Discussion: kkLLa without cellsa without cells
Agitator Agitator (RPM)(RPM)
Flow Flow (cc/min)(cc/min)
kLa value (hrkLa value (hr--
11))
(95%)(95%)
Variance Variance
500500 400400 36.3+/-0.80536.3+/-0.805 0.1690.169
500500 800800 66.3+/-1.2666.3+/-1.26 0.4150.415
500500 800800 60.8+/-0.77460.8+/-0.774 0.1560.156
500500 12001200 87.0+/-1.6987.0+/-1.69 0.7390.739
100100 800800 7.94+/-0.3217.94+/-0.321 0.02700.0270
300300 800800 19.9+/-0.22519.9+/-0.225 0.1320.132
700700 800800 111.7+/-1.71111.7+/-1.71 0.7640.764
850850 800800 158+/-2.63158+/-2.63 1.791.79
Results and Discussion: Results and Discussion: OUR and kOUR and kLLa with cellsa with cells
Run Run ##
OUROUR
(1/hr)(1/hr)OUROUR
VarianceVariancekkLLaa
(hr(hr-1-1))
kkLLaa
VariancVariancee
11 1.73 1.73
+/- 1.32+/- 1.320.4570.457 74.4 74.4
+/- 3.39+/- 3.393.013.01
22 12.412.4
+/- 5.17+/- 5.176.966.96 95.195.1
+/- 3.00+/- 3.002.352.35
Results and Discussion: Results and Discussion: μμmaxmax and Y and YX/SX/S
Run Run ##
μμmaxmax
(hr(hr-1-1))
μμmaxmax VariancVariancee
YYX/SX/S YYX/SX/S VariancVariancee
11 0.1700.170
+/- 0.221+/- 0.2210.01280.0128 0.2360.236 0.008710.00871
22 0.1030.103
+/- 0.163+/- 0.1630.00830.008344
0.1500.150 0.003650.00365
Results and Discussion: Results and Discussion: RRvv
Run #Run # RRv v (g/L-hr)(g/L-hr) VarianceVariance
11 0.240 +/- 0.240 +/- 0.4720.472
0.05800.0580
22 0.3190.319
+/-0.210+/-0.2100.04060.0406
Conclusions and Conclusions and RecommendationsRecommendations
Start with growth medium as close to growth Start with growth medium as close to growth conditions as possible.conditions as possible.
Using dissolved OUsing dissolved O22 probe and percent probe and percent saturation, can’t get accurate ksaturation, can’t get accurate kLLa with cells a with cells growing (should be lower with cells than without)growing (should be lower with cells than without)
To increase kTo increase kLLa, use higher air flow rate and a, use higher air flow rate and agitation speedagitation speed
To obtain maximum yield, don’t allow the oxygen To obtain maximum yield, don’t allow the oxygen to fall below the critical saturationto fall below the critical saturation
Use more trials to get more data pointsUse more trials to get more data points
ReferencesReferences
Atkinson, B., Mavituna, F. Atkinson, B., Mavituna, F. Biochemical Engineering and Biotechnology Biochemical Engineering and Biotechnology HandbookHandbook, 2, 2ndnd ed., 1991, Macmillion Publishers, Hampshire, England. ed., 1991, Macmillion Publishers, Hampshire, England.
Asenjo, J., Merchuk, J. Asenjo, J., Merchuk, J. Bioreactor System DesignBioreactor System Design, 1995, Marcel Dekker, New , 1995, Marcel Dekker, New York.York.
Bailey, J., Ollis, D. Bailey, J., Ollis, D. Biochemical Engineering FundamentalsBiochemical Engineering Fundamentals, International ed., , International ed., 1977, McCraw-Hill, Tokyo.1977, McCraw-Hill, Tokyo.
Shuler, M., Kargi, F. Shuler, M., Kargi, F. Bioprocess Engineering: Basic ConceptsBioprocess Engineering: Basic Concepts, 2, 2ndnd ed., 2002, ed., 2002, Prentice Hall, Upper Saddle River, New Jersey.Prentice Hall, Upper Saddle River, New Jersey.
Thank you for listening…Thank you for listening…
Any Questions?Any Questions?