Research in Bangkok

1. Cultural
Thai Culture is lively and vibrant
and varies based on the region.
Within the first week, our group
visited the Erawan National
Forest and trekked up a seven
layer waterfall. Not far from the
park was an animal sanctuary
where we fed tiger cubs and
giraffes. The biodiversity in the
north is dense yet broad. In
Bangkok, there is an effort to teach
the history of the Thai culture both to visitors and locals alike. At Sampran
Riverside we learned dance, craft and history alike. We volunteered at Friends
for All Children, an orphanage in Bangkok, where we played with the kids and
helped feed them. With a group from Chulalongkorn, we explored the old
capital city Ayutthaya and aided in cleaning an old lab that was located in the
region. We made our own food, Thai cookout style and played games with
new friends.
Hypothesis
The higher concentrations of medicines in the fermentation batch reactions will
increase the sucrose consumption due to their antibiotic properties reducing
competing bacterial contaminants and allowing the yeast to grow and perform at its
most efficient rate.
Conclusions
There are additional tests that must be performed in order to
differentiate the sucrose consumed by the yeast and by infectious
bacterial contaminants. This data would serve to further analyze
the effectiveness of the medicines used in the experiment and
could then pose as recommendations to fuel ethanol industries
looking to enhance their efficiency.
Discussion
• The increased consumption of sucrose as evident in the graph
of controls with the positive control suggests that the less
bacterial contaminants, the more the yeast cells are able to
consume the sucrose energy source and the more ethanol
would be produced. This is also supported by the trend that
the higher concentrations of Robenz and Avatec show a
greater decrease in sucrose concentration over time.
• Bacterial contaminants do use the sucrose energy source so it
is important to run further tests on ethanol concentrations
would give direct evidence as to the productivity of the yeast
cultures.
• The higher concentrations
of treatment correlated
with lass bacterial growth,
but also reduced the
growth of yeast making it
important to increase
sample size.
Results
Objectives and Methods
• To improve production efficiency through application
of antibiotics and allow ethanol to better compete in
the energy economy.
1. Agar slant Preparation
2. Stock culture preparation
3. Medium preparation
4. Cell cultivation and harvesting
5. Batch fermentation
6. Sugar analysis preparation
7. Sample treatment I & II (DNS analysis)
Materials
Potato Dextrose Agar (PDA)
70% isopropyl alcohol
Sugar cane molasses
Ammonium Sulfate
NaOH
HCl
DNS reagent
Avatec® - Lasalocid Sodium 15 %
Cygro® - Maduramicin Ammonium 1%
Robenz® - Robenidine Hydrochloride 6.6 %
Elancoban 200® - Monesine Sodium 20 %
S. cerevisiae M30
TOMY SS-325 autoclave
ISSCO VS-124 laminar flow hood
Innova 4330 refrigerated incubation shaker
Vortex mixer
Centrifuge
Spectrometer
Abstract
This study analyzes the effect of medicines commonly used in poultry farming on
ethanol fermentation of sugar cane molasses in batch reactions to improve
production efficiency and allow ethanol to better compete in the energy economy
as an alternative to fossil fuels. Bacterial infection in the industrial production
facilities causes yeast to compete for energy and produces degrading bi-products.
Avatec (lasalocid sodium), Cygro (maduramicin ammonium), Robenz (robenidine
hydrochloride), and Elancoban (monesine sodium) that serve as anticoccidials or
coccidiostats were tested in 250 ml batches for fermentation of S. cervisiae using
sugar cane molasses as the food stock for 72 hours at 33°C and 36°C. Of Avatec and
Robenz, the sucrose concentration decrease over time and little discernable
differences occurred due to treatment concentration or temperature. The bacterial
growth was minimized with samples Robenz 100 ppm/L at both 33°C and 36°C,
Avatec 1000 ppm/L at 33°C and Avatec 100 ppm/L at 36°C. However, the higer
concentrations of medication resulted in fewer yeast cultures.
Acknowledgements
The Chulalongkorn University Faculty and Frank Alexis are gratefully
acknowledged for the organization of this research experience and the
Clemson University Calhoun Honors College is gratefully acknowledged for
the encouragement to go abroad through the distribution of a travel grant.
MEDICINE
TYPE
CONCENTRATION
PPM/L
MASS
[G]
21% W/V
MOLASSE
[ML]
%5 CELL
[ML]
AVATEC 1000 0.2375 237.5 12.5
500 0.1188 237.5 12.5
200 0.0475 237.5 12.5
100 0.0238 237.5 12.5
CYGRO 50 0.0119 237.5 12.5
20 0.0048 237.5 12.5
10 0.0024 237.5 12.5
5 0.0012 237.5 12.5
ROBENZ 300 0.0713 237.5 12.5
100 0.0238 237.5 12.5
40 0.0095 237.5 12.5
20 0.0048 237.5 12.5
MONESINE 100 0.0238 237.5 12.5
20 0.0048 237.5 12.5
10 0.0024 237.5 12.5
5 0.0012 237.5 12.5
CONTROL - - 237.5 12.5
POS.
CONTROL
- - 237.5 12.5
0
5
10
15
20
25
0 20 40 60 80
SucroseConcentrationw/v[%]
Fermentation Time [hours]
Controls 33°C
Control
0
10
20
30
0 20 40 60 80
SucroseConcentrationw/v[%]
Fermentation Time [hours]
Avatec 33°C
A 100
A 200
A 500
Bacterial Control in Ethanol Fermentation Batch Reactions
Timothy Karmilovich, Elena Miyasato, Pornpicha Palm, Dr. Muenduen Phisalaphong, Jaygita Wikranvanich
Clemson University Department of Bioengineering, Chulalongkorn University Faculty of Chemical Engineering
ChemicalMicrobialIndustrial
0
10
20
30
40
50
Bacterialcount[cfu/ml·102]
Treatment
Bacterial Growth in Batch Reations
TVC 33°
TVC 36°

2. Bacterial Control in Ethanol
Fermentation:
Using chemical medicines to improve process efficiency
Elena Miyasato
Advised by Professor Muenduen Phisalaphong

3. Introduction
 Ethanol as an alternative energy source
 Utility and application
 Competition in modern economy
 Bacterial contaminants decrease efficiency
 Potential for application of antibiotics
http://www.cappersfarmer.com/-/media/Images/CFR/Editorial/Articles/Online-Articles/1991/08-01/Making-
Molasses/Sugar-Cane-jpg.jpg?h=589&la=en&w=400&hash=DFAC5D5D05506E568C0A0794B4B365C3F20DC0BD

4. Overview
http://nptel.ac.in/courses/103103029/module4/lec29/images/3.png

5. Objectives
 Effect of antibiotics on ethanol production
 Type, Temperature, Concentration
 Use the data to support company advertisement
 Effect of environment on sucrose concentration and cell growth
 Time, Type, Temperature, Concentration
 Use the data to compare with further testing

6. Background & Hypothesis
 S. cerevisiae
 Hypothesis:
 The higher concentrations of medicines in the fermentation batch reactions will increase
the sucrose consumption due to their antibiotic properties reducing competing bacterial
contaminants and allowing the yeast to grow and perform at its most efficient rate.
http://sciencegeist.net/wp-content/uploads/2011/02/fermentation.png

7. Methods: Overview
 Scope of Experiment:
 Agar slant Preparation
 Cell cultivation and harvesting
 Stock culture preparation
 Medium preparation
 Batch fermentation
 Sugar analysis preparation
 Sample treatment I & II

8. Methods: Overview (cont.)
TYPE CONCENTRATION
PPM/L
MASS [G] 21% W/V
MOLASSES
[ML]
%5 CELL
[ML]
AVATEC 1000 0.2375 237.5 12.5
500 0.1188 237.5 12.5
200 0.0475 237.5 12.5
100 0.0238 237.5 12.5
CYGRO 50 0.0119 237.5 12.5
20 0.0048 237.5 12.5
10 0.0024 237.5 12.5
5 0.0012 237.5 12.5
ROBENZ 300 0.0713 237.5 12.5
100 0.0238 237.5 12.5
40 0.0095 237.5 12.5
20 0.0048 237.5 12.5
MONESINE 100 0.0238 237.5 12.5
20 0.0048 237.5 12.5
10 0.0024 237.5 12.5
5 0.0012 237.5 12.5
CONTROL - - 237.5 12.5
POS. CONTROL - - 237.5 12.5
Avatec® - Lasalocid Sodium 15 %
Cygro® - Maduramicin Ammonium 1%
Robenz® - Robenidine Hydrochloride 6.6 %
Elancoban 200® - Monesine Sodium 20 %

9. Methods: Organism Specification
 Saccharomyces cerevisiae
 High fermentation rate
 Biproducts:
 CO2
 Ethanol
 Glycerol
 Cell Biomass
 Lactic Acid bacteria
 Lactobacillaceae
 Gram positive
 Batch fermentation 72 hours
 33°C and 36°C
https://upload.wikimedia.org/wikipedia/commons/thumb/9/95/Saccharomyces_cer
evisiae_SEM.jpg/240px-Saccharomyces_cerevisiae_SEM.jpg
https://drarossana.files.wordpress.com/2014/06/repor_1.jpg

10. Methods: Sample Treatment
 Hydrolyzed in acid solution using HCl
 Separate glucose and fructose
 3,5 Dinitrosalicylic acid (DNS) reagent gets reduced
 3-amino-5-nitro-salicylic acid (ANS)
 Absorption spectrum 500-540nm

11. Results: Standards
y = 32.091x
R² = 0.9958
0
5
10
15
20
25
30
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
ConcentrationofSucrose(%w/v)
Absorbance of 520 nm Light (%)
Standard Curve of Surcrose

12. Results: Sucrose Concentration 33°C
0
5
10
15
20
25
0 10 20 30 40 50 60 70 80
SucroseConcentrationw/v[%]
Fermentation Time [hours]
Robenz 33°C
R 20 R 40 R 100 R 300
0
5
10
15
20
25
0 20 40 60 80
SucroseConcentrationw/v[%]
Fermentation Time [hours]
Avatec 33°C
A 100 A 200 A 500 A 1000
0
10
20
30
0 20 40 60 80
SucroseConcentrationw/v[%]
Fermentation Time [hours]
Controls 33°C
Control Positive Control

13. Results: Sucrose Concentration 36°C
0.000
5.000
10.000
15.000
20.000
25.000
0 20 40 60 80
SucroseConcentrationw/v[%]
Fermentation Time [hours]
Robenz 36°C
R 20 R 40 R 100 R 300
0.000
5.000
10.000
15.000
20.000
0 20 40 60 80
SucroseConcentrationw/v[%]
Fermentation Time [hours]
Avatec 36°C
A 100 A 200 A 500 A 1000
0.000
5.000
10.000
15.000
20.000
0 20 40 60 80
SucroseConcentrationw/w[%]
Fermentation Time [hours]
Controls 36°C
Control Positive Control

14. Results: Robenz vs Avatec 33°C & 36°C
0.000
5.000
10.000
15.000
20.000
25.000
0 10 20 30 40 50 60 70 80
SucroseConcentrationw/w[%]
Fermentation Time [hours]
36°C Sucrose Concentrations
R 20 R 40
R 100 R 300
A 100 A 200
A 500 A 1000
Control Positive Control
0.000
5.000
10.000
15.000
20.000
25.000
0 10 20 30 40 50 60 70 80
SucroseConcentrationw/w[%]
Fermentation Time [hours]
33°C Sucrose Concentrations
R 20 R 40
R 100 R 300
A 100 A 200
A 500 A 1000
Control Positive Control

15. Results: Temperature Effect
0.000
5.000
10.000
15.000
20.000
25.000
0 10 20 30 40 50 60 70 80
SucroseConcentrationw/v[%]
Fermentation Time [hours]
Temperature Effect on Sucrose Concentration
Control 33
Positive Control 33
Control 36
Positive Control 36

16. Results: Bacteria Count
0
5
10
15
20
25
30
35
40
45
50
R 20 R 40 R 100 R 300 A 100 A 200 A 500 A 1000 Control Positive
Control
Bacterialcount[cfu/ml·102]
Treatment
Bacterial Growth in Batch Reations
TVC 33°
TVC 36°

17. Results: Yeast growth
0
20
40
60
80
100
120
140
Yeastcount[cfu/ml·105]
Treatment
Yeast in Batch Reactions at 33°
0
100
200
300
400
500
600
700
800
900
R 20 R 40 R 100 R 300 A 100 A 200 A 500 A 1000 Control Positive
Control
Yeastcount[cfu/ml·107]
Treatment
Yeast in Batch Reaction at 36°

18. Discussion
 Less sucrose = more growth
 Limited to the cell counts after 72 hours
 Potential for acidity testing throughout
 Temperature
 No conclusive effect on bacteria count
 Yeast grew more at higher temperature
 Positive Control
 The ultimate goal.

19. Conclusions
 Conclusive
 Sucrose decreased over time
 The tested Avatec prevented more bacterial contamination than Robenz
 Yeast growth is augmented at 36°C compared to 33°C
 Require further testing
 Ethanol concentrations over time
 Bacterial and yeast growth over time
 Greater quantity of medicine concentrations
 Greater variance in temperature

20. References
 Kelly A. Skinner, Timothy D. Leathers. “Bacterial contaminants of fuel ethanol production.” Journal of
Industrial Microbiology & Biotechnology (2004): Volume 31, Number 9, Page 401
 Aquarone, Eugênio. “Penicillin and Tetracycline as Contamination Control Agents in Alcoholic Fermentation
of Sugar Cane Molasses.” Applied Microbiology 8.5 (1960): 263–268. Print.
 Vinay Kant et al. “Anticoccidial Drugs Used in the Poultry: An Overview.” Science International
(2013). Volume 1, Issue 7 “Anticoccidial Drugs.” KTT Projects.University of Guelph. 2012. Web. 30 July 2017.
 Y. Lin, W. Zhang, C. Li, et al. “Factors affecting ethanol fermentation using Saccharomyces
cerevisiae BY4742.” Biomass- Bioenergy, 47 (2012), pp. 395-401
 Wang, Nam Sun. “Sucrose Assay by the Dinitrosalicylic Colorimetric Method”. University of Maryland. Web.
23 July 2017. <http://eng.umd.edu/~nsw/ench485/lab9d.htm>

21. Cultural Experience

22. Thank you to Professor Muenduen, Palm, Jan, Timothy, Dr. Alexis for their guidance, teaching,
teamwork, collaboration and organization during this incredible experience.