Silage Presentation

1. Fermentation of Centipede
Grass to form Silage
Matthew Lawrence, Natalie Whitaker,
Sarah Langston

3. Introduction
• Silage: compressed and
fermented grass or green
fodder, stored
anaerobically, as in a silo.
• Preserves food for cattle
through winter as
pasturing availability
decreases
• Retains nutritional value
better than regular hay

4. Objectives and
Considerations

5. Objective
● Determine how much acid was produced by the bacteria in the silage
reactors along with how much hemicellulose (substrate) was consumed by
the bacteria under anaerobic conditions
○ How much of the grass would become fermented?
● One reactor had a sucrose solution and the other did not
○ Determine which reactor had the most acid production

6. Project Overview
Reactor Type: Batch
Reaction Environment: anaerobic
Culture type: Heterotrophic
Carbon source: Grass (cellulose)
Energy source: Chemotrophic
Electron donor: Grass
Reactor Type: Batch
Reaction Environment: anaerobic
Culture type: Heterotrophic
Carbon source: Grass (cellulose)+
added sugar
Energy source: Chemotrophic
Electron donor: Grass

7. Proposed Testing Techniques
● Technique used to measure carbon source/e- donor: COD
● Technique used to measure cell mass: Yield from measuring product form over time
● Technique used to measure products: Acidity (Acid titration)

8. Considerations
● How could the reactor
environment stay truly
anaerobic?
● Inoculation with bacteria
or use microbes already
on the grass?
● Use sucrose in one of the
reactors or an enzyme?

9. Materials and Methods

10. Materials
• Two paint cans
• Chopped grass
(centipede)
• Balance
• Titration equipment
• 0.02 N NaOH
• COD Vials
• Table sugar

11. Methods
• Started with finding the dry weight and
bulk density of the grass in the can
• Took measurements from Can 1 (no
sucrose added) and Can 2 (sucrose
added) every other day
• Total of 5 measurements
• COD and acidity titrations.

12. Experimental Setup

13. Results

14. pH and Acidity Titration Results
Table 1. Initial pH measurements of silos Table 2. Volume of 0.02 N NaOH added
in acid titration
Day Can 1 Can 2
0 7.67 7.47
1 7.60 7.97
2 8.30 8.44
3 8.01 8.77
4 8.74 8.79
5 8.89 8.93
Day Can 1, mL Can 2, mL
0 2.85 2.80
1 1.52 1.90
2 - -
3 0.39 -
4 - -
5 - -

15. COD Results
COD results from reactor 1
(no added sucrose)
COD results from reactor 2
(added sucrose)
COD Absorbance Values
Day Can 1 Can 2
0 0.422 0.478
1 0.548 0.470
2 0.330 0.284
3 0.466 0.400
4 0.396 0.500
5 0.288 0.458
Table 3: Absorbance values from COD tests in lab
Oxidation of organic compounds:
Colorimetric reaction (Reduction of Chromate):

16. COD Results
Figure 1. Standard Curve of COD versus Absorbance at 600 nm
Can 1 Can 2
Day Abs @
600nm
COD
conc.
Day Abs @
600nm
COD
conc.
0 0.422 7.604 0 0.478 8.613
1 0.548 9.874 1 0.47 8.468
2 0.33 5.946 2 0.284 5.117
3 0.466 8.396 3 0.4 7.207
4 0.396 7.135 4 0.5 9.009
5 0.288 5.189 5 0.458 8.252
Table 4. COD concentration of silos over time

17. No Added Sugar- Biomass formation
Figure 2: Stella model of substrate and
biomass concentration with time in reactor 1
(initial conditions shown far right)
Figure 3: Theoretical graph of
substrate and biomass formation in
reactor 1 based on Stella Model
Oxidation of glucose by heterotrophic bacteria under aerobic conditions:

18. Added Sugar- Biomass Formation
Figure 5: Theoretical Graph of substrate use and
biomass formation based on the Stella model
for reactor 2.
Figure 4: Stella model of substrate and
biomass concentration with time in reactor
2 (initial conditions shown far right).

19. No Added Sugar- Acid Production
Figure 6: Stella model showing
product formation and substrate
utilization with time in reactor 1
(initial conditions specified on
slide 18)
Figure 7: Graph of product formation and
substrate utilization based on the Stella model
for reactor 1

20. Added Sugar- Acid Production
Figure 8: Stella model showing
product formation and substrate
utilization with time in reactor 2
(initial conditions specified on
slide 19)
Figure 9: Graph of product formation and substrate
utilization based on the Stella model for reactor 2

21. Data Analysis
• pH increased with time
• Titrations showed organic acid in reactors for days 0-1, while increased pH
on days 2-5 indicated no acid production
• COD results showed organic compounds were oxidized, however, COD
concentrations differed day-to-day
• Assumptions were made to model the biomass formation of heterotrophic
bacteria and acetic acid product formation under the conditions of the
reactors
• 1000 C6H12O6+ 1740 O2 + 852 NH3 = 852 C5H7O2N + 1740 CO2 + 4296 H2O
• Used this formula to represent cell mass formed in order to get Yb, which would
be used in the Monod Model
• Adding sugar resulted in higher acid production and product formation

22. Final Thoughts

23. Conclusion
● Fermentation did not go as planned
○ pH increased with time and COD absorbance readings did not follow a trend
● Data used to model assumed aerobic growth of bacteria and anaerobic
production of acids

24. Conclusion
Max Biomass Yield-
No added sugar
Max Biomass Yield-
Added sugar
Max Product Yield-
No added sugar
Max Product Yield-
Added sugar
4500 mg/L 5000 mg/L 10,525 mg/L 11040 mg/L
Table 5: Approximate maximum biomass and product yields for theoretical simulations of
both silos

25. Possible errors
Possible errors occurred
due to:
• Paint can rusting
• Paint residue in can
• Opening can for testing

26. Appendix

27. Mass of grass data

28. Calculating substrate concentration and Yb

29. Stella Tables
Left table: No Added Sugar- Biomass
Production
Right table: Added Sugar- Biomass
Production

30. Stella Tables
Left table: No Added Sugar-
Product formation
Right table: Added Sugar-
Product formation

31. QUESTIONS?

32. References
Drapcho, C. 2020. Anabolic Reactions for Heterotrophic, Organotrophic Microbes, Unpublished Class Notes, BE 4100, Clemson
University, Clemson SC.
Drapcho, C. 2020. Stoichiometry of microbial reactions, Unpublished Class Notes, BE 4100, Clemson University, Clemson SC.
Drapcho, C. 2020. Modeling Microbial Growth with Monod Model, Unpublished Class Notes, BE 4101, Clemson University,
Clemson SC.
Drapcho, C. 2020. Lab 6: Methods of Organic Substrate Determination: COD, Unpublished Laboratory Notes, BE 4101, Clemson
University, Clemson SC.
https://extension.okstate.edu/fact-sheets/solid-state-anaerobic-digestion.html
https://www.researchgate.net/publication/310441768_Chapter_8_Grass_Silage_for_Biogas_Production
https://qualitysilage.com/interperting-silage-analysis/
https://www.megalac.com/about/news/146-super-greens-why-silage-is-great-for-dairy-cows-and-dairy-farmers

33. References (photos)
https://extension.okstate.edu/fact-sheets/solid-state-anaerobic-digestion.html
https://www.labdepotinc.com/p-15110-cellulase?utm_source=google&utm_medium=cpc&utm_campaign=smart-
shopping&gclid=CjwKCAiA8Jf-BRB-EiwAWDtEGq3IilHl6Fbyv1F79yTbyTjJU_edxx_tJ_Y66UND6wzxc0riviFh3RoCn4IQAvD_BwE -
cellulase picture
https://phys.org/news/2019-05-mbl-microscopy-reveal-bacteria-wide.html - bacteria
https://1.bp.blogspot.com/_W19ZkFqGT-M/SXq5pRLD4MI/AAAAAAAAAx8/IGXa25IZNpw/s400/Cows+eat+trough+SM.jpg
https://cdn.pixabay.com/photo/2014/05/26/10/43/cow-354428_640.jpg
https://wellentheorie.wordpress.com/2017/12/02/ufo-ufologi-e-extraterrestri/
https://www.therebels.com/resources/fescue-what-you-need-to-know - grass picture
https://lallemandanimalnutrition.com/en/indonesia/whats-new/preserve-your-grass-silage-protein-content/ -grass picture on intro
slide