Yeast Cultivation

Course 27033 BioCentrum-DTU Technical University of Denmark January 2006 By, Marta Karolina Mustafa Shanshan Vijaya Krishna

Introduction
Types of cultivation

- Batch - Chemostat Pulse input Glucose repression Crabtree effect

Batch Cultivation
Cultivation conditions

- 4L working volume; pH around 5; 10g/L initial glucose conc.; 10g/L initial galactose conc.; 600rpm; air flow 1 v/v/min; temperature 30C - Standard CBS Medium

Yeast Strains
CEN.PK-113-7D (haploid lab strain) as wild

type organism
- OD600 of shake flask was 2,835 - Inoculum volume was 175 ml

snf1 (CEN.PK-506-1C) as mutant

organism
- OD600 of shake flask was 2,915 - Inoculum volume was 175 ml

Measurements
Cultivation time was 24h

Samples were taken every hour between t=

0-10 and after 24h Samples at t= 1-10 and 24 data were taken for OD, pH, CO2, O2, and at t= 4-10 and 24 data were taken for DW Samples at t= 0-10 and 24 were analyzed by HPLC

Glucose Repression

Crabtree Effect
C6H12O6 + O2 Cells + CO2 (Respiration)

C6H12O6 Cells + CO2 + C2H6O (Fermentation)

Batch Cultivation Results

Method
Two strains one wild-type and one of the

glucose repression mutant

Batch cultivation performed in a 5L reactor

Results from CEN-PK-113-7D (WT)

Dynamism of various components

Amount glucose; galactose; Ethanol
Glucose ; Galactose; Ethanol concentration(g/L)
12.0000 10.0000 8.0000 6.0000 4.0000 2.0000 0.0000 0.0 10.0 20.0 30.0 time(h) glucose galactose Ethanol acetate Glycerol

Linear relation - OD vs Dry weight

OD vs. Dry weight
0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 0 10 20 OD600 30 40
Dry weight (g)

Gas analysis
CO2 vs time
10 y = 0.008e 0.3662x
CO2%

1 0 0.1 y = 0.0341e 0.4265x 0.01 time(h) 5 10 15 20 25 30

m= 0.2172
specific growth rate from OD
0.6 0.4 y = 0.2172x - 1.0346 0.2 0 2.0 4.0 6.0 -0.2 0.0 -0.4 -0.6 -0.8 -1 Time(h)

log(OD)

8.0

m= 0.3751
specific growth rate from DW
0 -0.5 0.0 2.0 4.0 6.0 8.0 10.0

logDW

-1 -1.5 -2 -2.5 -3 Time(h) y = 0.3751x - 5.3132

Specific growth rate

method CO2 DW OD m 0.427 0.375 0.217

Ysx=0.3341; Yse=0.369
Ysx and Yse

[Biomass](g/l);[Ethanol ](g/l)

3 2. 5 2 1. 5 1 0. 5

y = 0. 369x - 0. 0546

y = 0. 3341x - 0. 6458

0 0. 0000

2. 0000

4. 0000

6. 0000

8. 0000

C gl ucose g/ L) s(

Result for CEN.PK-506-1C (mutant)

Biomass, glucose, galactose and ethanol concentration as a function of time

12
Glucose, galactose and ethanol concentration g/L

2.00 1.80

10

1.60 1.40 Biomass 1.20 concentration g DW/L 1.00 0.80

0.60 0.40 0.20

glucose

0 0 4 8 12 time [h] 16 20 24 28

0.00

galactose ethanol biomas biomass

Linear relation - OD vs Dry weight

Biomass concentartion as a function of optical density
2 1.8 1.6
Biomass concentration [g DW/L]

1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 6 7 8

OD

OD

Gas analysis
CO2 production as a function of time
1 0.9 0.8 0.7 0.6
%CO2

y = 0.0279e0.0063x

0.5 0.4 0.3 0.2 0.1 0

200

400

600

800 time [s]

1000

1200

1400

1600

Specific growth rate

Optical density and biomass concentration as a function of time
8 7 6 1.4 5 4 y = 0.0289e 0.4614x 3 2 1 0 0 5 10 15 time [h] 20 25 30 OD BIomass y = 0.182e 0.3423x 1.2
Biomass
OD

2 1.8 1.6

1 comcentration
[gW/L]

0.8 0.6 0.4 0.2 0

Specific growth rate

method CO2 DW OD m 0.0063 0.461 0.342

Ysx= 0.0169; Yse= 0.3751

Cs Vs Biomass and ethanol
3 y = 0.3751x - 0.0212 2.5

Biomass; Ethanol

Ysx Yse Yse Linear (Yse) Linear (Ysx)

1.5 1

0.5 y = 0.0169x + 0.0102 0 0.0000

2.0000

4.0000

6.0000 Cs

8.0000

10.0000

12.0000

Comparision of Strains
glucose, galactose and ethanol concentration as a function of time for both the strains 12

Glucose, galactose and ethanol concentration g/L

10

2 glucose-506 galactose-506 0 0 4 8 12 16 20 24 28 ethanol-506 glucose-113 7D Galactose-113 7D time [h] Ethanol-113 7D

Yields for both the strains

Y_sx Y_se

CEN-PK-1137D (WT) CEN.PK-5061C (mutant)

0.3341

0.369

0.0169

0.3751

Specific growth rate

Strain
mutant wild

CO2
0.0063 0.427

OD
0.342 0.217

DW
0.461 0.375

Chemostat Pulse input

Strain used - Wildtype DGI 342 Pulse of glucose given to a chemostat running at

steady state
Conentration of pulse (2.5g/l glucose and 2.5 g/l

galactose)
Samples taken in defined time intervals for 180

minutes for analysis

Time Vs O.D
35 30 25
O.D

20 15 10 5 0 0 20 40 60 80 100 Time (min) 120 140 160 180 200

Time Vs substrates and metabolites

2.0000

[Glycerol];[Ethanol];[Glucose];[Galactose] (g/l)

1.8000 1.6000 1.4000 1.2000 1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 0 50 100 Time (min) 150 200 Glycerol Ethanol Ethanol Glucose Galactose Galactose

The filter has a holding volume. Glucose hydrolysed molecule (C6H12O6.H2O) Biomass yeild on ethanol - increase in biomass from 150 to 180 min to the decrease in ethanol in the same time interval

Previous Experimental Data

D VS S,X,P
3.5 3 2.5 2 dry weight 1.5 1 0.5 0 0 -0.5 D (h-1) 0.1 0.2 0.3 D=0.342 0.4 0.5 Ethanol Glucose
[dry weight](gDW);[Glucose](g/l));[Ethanol](g/l)

Yeild, rs VS Dilution
0.7 y = 1.8263x + 0.0164 0.6 2.5

Yeild (gDW/gGlucose), ,rs (gDW/gGlucose/hr)

2 0.5 0.4 0.3 0.2 0.5 0.1 0 0 0.1 0.2 0.3 0 1.5 Y_sx rs rs Linear (rs)

D=0.342

0.4

0.5

Dilution (h-1)

So, from the curve fitting, ms = 0.0164; Y_xs_true = 1.83263; So, Y_sx_true = 1/ Y_xs_true = 0.547555

Gas Analysis
Time Vs CO2
0.9 0.8 0.7 0.6 20.8 20.7 20.6 20.5 20.4 20.3 20.2 20.1 20 0 50 100 time 150 19.9 200 CO2 O2

CO2

0.5 0.4 0.3 0.2 0.1 0

Time Vs CO2
2 1.5 20.8 20.7 20.6 20.5 20.4 20.3 20.2 20.1 20 19.9 1200

CO2

1 0.5 0 0 200 400 600 time 800 1000

CO2 O2

Conclusions
No growth was seen on galactose for the mutant

strain (snf1) clear sign of the strain unable to use other carbon sources and glucose repression Glucose repression was obeserved for the wild type as the galactose was utilized only at low concentrations of glucose The yeild of biomass on glucose is observed to be less for the mutant strain From the chemostat, for the strain DGI 342(WT), more ethanol is produced because of the crabtree effect because of high dilution rate