1. Microbial Synthesis of Succinic
Acid from Typha Grass Hydrolysate
and Its Application in Biopolymer
Synthesis and as Co-plasticizer
By
Yakindra P Timilsena (111332)
Examination Committee
Prof. Athapol Noomhorm
Prof. Sudip K Rakshit
Dr. Anil Kumar Anal

2. Main Idea
 Renewable/Green Chemicals
 Cheap and Under-utilized resources
 Biodegradable polymer with better properties

3. Introduction
 Succinic Acid is a C4 dicarboxylic acid with
molecular formula C4H6O4, molecular weight 118.09
and melting point 185 1900C.
 It is predicted to be one of the most widely used
platform chemicals which can be produced from
renewable feedstocks (Bechthold et al. 2008).
 Broad range of industrial applications -as a source
of food, pharmaceuticals, surfactants, detergents,
antifoam agents, in the production of resins,
polymers, paints, cosmetics and inks (Isar et al.
2006).
 Biobased succinic acid can be a suitable alternative to its
petrochemical equivalent.

4. Introduction
 Glycerol is a by-product of biodiesel
industries and can be used as a good
plasticizer
 Succinic acid can be used as an efficient co-
plasticizer in starch based polymer synthesis
 Blending process is one of the important
methods of modifying the polymer
characteristics
 PBS is a biodegradable polyester- synthesized
from SA platform

5. Problem statements
 Most of the research work on Green Technology
are patented and details of the invention is not
disclosed
 Currently succinic acid and synthetic polymers
in food packaging - produced from petroleum
based chemicals - two limitations: non
renewability and non biodegradability
 Typha grass-abundantly available all over the
world- not investigated as renewable raw material
for high value platform chemical

6. Problem statements
 PBS synthesized from SA- lacks flexibility.
Blending with starch help improve the
characteristics of polymer.
 Glycerol plasticized starch polymer (GTPS)
recrystallize on storage- requires a co-
plasticizer.
 Amine co-plasticizer-toxic. Biosuccinic acid -
food grade- can serve as an alternative co-
plasticizer.

7. Main objective
• To synthesize bio-succinic acid from
Typha grass hydrolysate using
microbial fermentation and its
application for the synthesis of PBS-
starch and SGTPS copolymers

8. Specific objectives
1. To determine the yield of bio- succinic acid
produced from Typha grass hydrolysate
using A. succinogenes
2. To optimize the ratio succinic acid as co-
plasticizer with glycerol to synthesize
SGTPS
3. To optimize the ratio of cassava starch and
PBS to synthesize copolymerized PBS.

9. Literature review
Production of Succinic Acid by Bacterial fermentation
Raw Materials Micro-organisms Result/Findings References
wood hydrolysate M. yield of 56% Kim et al. 2004
succiniciproducens Batch Fermentation
straw hydrolysate A. Succinogenes 80.7% Zheng et al. 2009
yield after 48 hrs of
fermentation
cane molasses A. succinogenes yield of 79.5% after Liu et al. 2008
48 hrs of batch
fermentation

10. Literature review
Polymer and copolymer synthesis and characterization
Raw Materials Polymerization Result/Findings References
method/
polymer
CA, glycerol, CGTPS by melt • Esterification and better cross- Shi et al. 2007
starch blending linking
• Decrease in MW/light
• Decreased Tg
• Change in crystal structure
(reduced retrogradation)
Starch, glycerol, TPS/PCL Blends • Decreased Tensile Strength Averous et al. 2000
PCL • Decreased Elongation at Break
Starch, glycerol, TPS/PCL Blends • Decreased Tc Huang et al. 1993
PCL • Decreased Mp
• increased % crystallinity
• Increased Relative crystallinity

11. Materials and Methods
Materials and chemicals
Typha grass hydrolysate, Microbial strain pure
culture (A. succinogenes), sodium/calcium
hydroxide, biobased SA, PBS, Cassava starch,
Glycerol
Micro-organisms
• Actinobacillus succinogenes
• Anaerobiospirillum succiniciproducens or
• Mannheimia succiniciproducens

12. Equipments
• Bioreactor, carbon dioxide cylinder,
• HPLC with sugar column
• High speed mixer,
• twin screw co-rotating extruder,
• Melt Blender,
• FTIR, NMR, GPC, DSC, SEM

13. Experiment for Objective 1
METHODOLOGY
Biomass (Typha
grass)
Drying, Powdering
Pre-treatment
(Alkali)
Preparation of Hydrolysate
Hydrolysis
(Enzyme/Acid)
The method developed by
Hydr Mr. Idi Audu Guga, an AIT
Fermentable Sugars olysa doctoral student, will be
(Glucose, Xylose) te followed till hydrolysis

14. METHODOLOGY contd……
Fermentation and Product Experiment for Objective 1
Recovery
Preparation for
Fermentation
370C, pH 6.5,
Fermentation Buffer MgCO3,
( by A. succinogenes) 12h, CO2
Product Recovery Precipitation
with NaOH
Succinic Acid/Sod.
Succinate

15. METHODOLOGY contd……
Co-polymerization and Characterization
Glycerol + Cassava
Starch + SA PBS
Water Starch
High speed blending
Experiment for Objective 3
Extrusion Co-
Experiment for Objective 2
polymerization
Melt Blending
SGTPS PBS-starch copolymer
Characterization
Physical Mechanical Biodegradability

16. COMPOSITION of SGTPS
Co-polymer Abbrevi Sample Weight Proportions
Name ation
Hydrous Glycerol SA
starch (20%
mc wb)
GTPS SA0 100 30 0
SGTPS1 SA1 100 30 1
SGTPS2 SA2 100 30 2
SGTPS5 SA5 100 30 5
SGTPS10 SA10 100 30 10

17. COMPOSITION of PBS-starch
Co-polymer Name Sample Weight Proportions
Hydrous starch (20% PBS
mc wb)
PBS 100 0
PBSS1 80 20
PBSS2 70 30
PBSS3 60 40
PBSS4 50 50

18. CHARACTERISTICS TO BE MEASURED
• Tensile Strength
• % Elongation at Break
• IR spectra by FTIR
• DSC Thermograms
• Thermogravimetric Analysis
• Inherent Viscosity (using available viscometer)
• Degree of Substitution and Esterification
(According to Santayanon and
Wootthirahokkam, 2003)
• Biodegradability (using lipase enzyme)

19. Work Plan
S. Activities Aug Sep Oct Nov Dec Jan Feb Mar Apr
N.
1 Literature review X
2 Procurements of X
pure culture,
chemicals and
equipments
3 Fermentation, X X X
Pdt Recovery
4 Blending, Co- X X X
polymerization,
characterization
5 Result X X
interpretation
and data analysis
6 Final reporting X X

20. Budget Estimation
S.N. Operational Activities Amount
(Baht)
1 Chemicals, Pure culture and Enzymes 5000
2 Equipments: 30000
(HPLC Column, Melt Blender)
3 Travel 2000
4 Miscellaneous 5000
Total 42000

21. References
 Bechthold I, Bretz K, Kabasci S, Kopitzky R, Springer A (2008).
Succinic acid: a new platform chemical for biobased polymers
from renewable resources. Chem Eng Technol 31:647-654.
 Takiyama, E.; Fujimaki, T. (1994). Bionolle biodegradable plastic
through chemical synthesis. In Biodegradable Plastics and
Polymers; Doi, Y., Fukuda, K., Eds.; Elsevier Science: Amsterdam,
The Netherlands, pp. 150-174.
 Mochizuki, M.; Mukai, K.; Yamada, K.; Ichise, N.; Murase, S.;
Iwaya, Y. (1997). Macromolecules, 30, 7403.
 Azim, H.; Dekhterman, A; Jiang, Z. and Gross, R.A. (2006).
Biomacromolecules, 7, 3093-3097
 Shi, R.; Zhang, Z.; Liu, Q.; Han, Y.; Zhang, L.; Chen, D.; Tian,
W. (2007). Characterization of citric acid/glycerol co-plasticized
thermoplastic starch prepared by melt blending. Carbohydrate
Polymers 69, 748–755

22. Thank you