Internship Project

1. FERMENTATION ANALYSIS OF
CELLULOLYTIC MICROORGANISM
THERMOBIFIDA FUSCA
Engineering Researcher: Karan Sharma
Supervisor: Dr. Stephen S. Fong, PhD
Date: 09/26/2011
Graduation Date: May 2012

2. SYSTEMS BIOLOGICAL ENGINEERING LAB
 Number of PhD candidates: 3
 Number of Undergraduates: 5
 Research Topics:
 Whole Cell Modeling
 Genetic Programming
 Metabolic Pathway Engineering
 Laboratory Adaptive Evolution
 Organisms: Thermobifida fusca, E.Coli

3. LOCATION
 Fermentation Research Engineer
 Principle Investigator: Dr. Stephen S. Fong
 Systems Biological Lab
 Rm. 409 West Engineering Hall

4. BIOFUELS
 What is a Biofuel?
 A fuel that is derived from natural and biological raw
masses.
 Kind of biofuels:
 Ethanol
 Biodiesel
 Bio-butanol, propanol
 Other higher alcohols

5. AN ILLUSTRATION OF
HOW BIOFUELS ARE
PRODUCED TODAY!
Lawrence Berkeley National Lab.

6. WHAT IS MOST IMPORTANT IN THE PROCESS!
 Cellulose:
 is an organic compound. A polysaccharide consisting of
a linear chain of several hundred to over ten thousand
β(14) linked by D-glucose units.
 is the structural component of the primary cell
wall of green plants.

7. THERMOBIFIDA FUSCA
 An aerobic filamentous bacterium.
 Rod shaped.
 Major degrader of plant cell wall.
 Extremely high thermo-stability of Cellulases (up to
70°Celcius)
 Cellulases produced by T.fusca can degrade the
cellulose to sugars which are then used to make
biofuels.

8. THERMOBIFIDA FUSCA B6
 B6 is an engineered strain by T.fusca made Dr. Yu
Deng and
 Dr. S.S. Fong.
Systems
Biological
Lab

9. Systems Biological Lab

10. INTEREST – THE PROJECT
 Fermentation Engineering of T.fusca & B6
 To improve overall production and yield of Butyrate and
1-proponal.
 Parameters to improve
 Aeration Rate
 Agitation (Stir) Rate
 pH
 Different carbon sources (c.s.) used for growth
 (glucose, glycerol & cellbiose)
 Temperature
 Instruments used
 Bioreactor
 Flask Test

11. DATA ANALYSIS METHODS
 Data was analyzed by two different methods.
 HPLC – High Performance Liquid Chromatography
 is a chromatographic technique that can separate a mixture of
compounds to identify, quantify and purify the individual
components of the mixture.
Key Peaks in the HPLC
0.5% Butyrate 23.095
0.5% Glycerol 13.868
1% Propanol 25.328
Acetate 16.412 -- 16.147
Acetic Acid 15.927
Butyric Acid 18.227
Cellibiose 8.366
Propionic Acid 18.607 (Ri) -- 18.834
Sucrose 8.415

12. DATA ANALYSIS METHODS (CONTI.)
 Bradford Protein Assay
 is an analytical spectroscopic procedure used to measure the
concentration of a protein in a solution. It is dependent on the
amino acid composition of the measured protein.
4.000
3.500
3.000
2.500
2.000 Carbon Source
Glycerol
1.500 Butyrate (g/L)
O.D.
1.000
0.500
0.000
0 2 4 6 10 13 17 31.5 33 34 35 37.5
Time (hours)

13. DATA – EXPERIMENT 1-2 BIOREACTOR 4.000
3.000
 T.Fusca B6
2.000
 Stir Rate : 400rpms 1.000
 Temp: 46 ° Celsius 0.000
pH: 6.7
0
2
4
6
10
13
17
33
34
35
31.5
37.5

1.8
4.5
1.6 4
1.4 3.5
1.2 3
1 2.5
C.S: Glucose
0.8 (g/L) 2 Cellbiose (g/L)
Butyrate (g/L)
0.6 1.5
Butyrate (g/L)
0.4 1
0.2 0.5
O.D.
0 0
0 2 6 8 12 16 20 24 36 40
0
2
4
6
11
50
26.5
29.5
45.5
46.5
47.5
48.5
Time (hours)
Time (hours)

14. DATA – EXPERIMENT 3-4 FLASK 4.000
3.000
2.000
 T.Fusca YX 1.000
 Stir Rate : 200 rpm 0.000
0
2
4
6
10
13
17
33
34
35
31.5
37.5
 Temp: 55 ° Celsius
 C.S : Cellbiose
4.5
4 4.000
3.5 3.500
3 3.000
2.5 2.500
Cellbiose (g/L)
2
Glycerol (g/L) 2.000
1.5 Butyrate (g/L)
1.500
O.D.
1
1.000
0.5
0.500
0
0.000
0 2 4 6 10 13 17 31.5 33 34 35 37.5 42
Time (hours) Time (hours)

15. FUTURE WORK
 Explore future aspects of T.Fusca fermentation
engineering.
 More specific & different parameters
 Explore future aspects of metabolic engineering of
T.Fusca

16. ACKNOWLEDGMENTS!
 Dr. Stephen S. Fong PhD
 Systems Biological Engineering Lab
 Colleagues: Cindy Lovelace, Dr. Yu Deng, Dr. Chris
Gowen & Niti Vanee

17. QUESTIONS?