1. WINTER Thesis Proposal TemplateExtractive fermentation of succinic acidusing an external precipitation technique Asst. Prof. Apichat BoontawanJiraphorn Lubsungnoen M5430116

2. Content01WINTERBackground TemplateResearch ObjectiveResearch HypothesisExperimental- Material & Method - Experimental setupExpected ResultResearch Plan 3. Background02 WINTER- Currently, the interest on development ofTemplatebiodegradable plastic since global warming problem has come.- Succinic acid (SA) is promising chemicalwhich has a wide range of applicationsin many industries. 4. Background 03WINTERTemplate 5. Background04WINTER- SA can be used as a precursor for synthesisTemplatebiodegradable plastic such as polybutyratesuccinate (PBS) and polyamide to produce bioplastics. 6. Background05 WINTER-Thailand is a large producer of raw materialTemplate to produce SA such as casava and sugarcane- Therefore, the country has high potential fordevelopment of SA production in order tosupport bioplastic industry. 7. Background06- Commercial scale production of SA ispetroleum-derived.-However, Fermentative production of SAfrom renewable resource has receivedincreasing interest .- SA can be produced by microbialfermentation using various microorganism. 8. Background 07-SA is a common intermediate in the metabolicTemplatepath way of several anaerobic and facultativemicroorganism .-Those organisms suitable for the efficientproduction of SA can be categorized intobacteria. 9. Background 08Template 10. Background09- Many different microorganism have beenTemplatescreened and studied for SA such as ;- Actinobacillus succinogenes,- Anaerobiospirillum succiniciproducens,- Mannheimia succiniciproducens and - Recombinant Escherichia coli. 11. Background10-Bio-based SA is potential substitute forTemplatethe current petroleum-based process.-However, compared with petrochemical-derived SA , biological SA production is still not economically competitive .-Because of its major draw backs:low product concentration in fermentationbroth and difficult product recovery. 12. Background11 WINTER-In addition, product inhibition is important Templatefactor for bioprocess development .-The product inhibition effect, which can bedescribed by specific growth rate equationai S P v = v max 1 S ( K + S + S2 /K i ) P m 13. Background 12WINTER- The cost of downstream processing canmake a very high Template total production portion in thecost, more than 50% .- It is necessary to reduce the production costof fermentation process in order to increaseits competitiveness with that of petrochemicalprocess. 14. Background13WINTER-The separation methods studied for SA Templaterecovery mainly include ;-Direct crystallization,-Precipitation,-Membrane separation,-Extraction,-Chromatography,-In situ separation.. 15. Background 14WINTERTemplate 16. Background 15WINTER-Formation of byproduct is a major problemTemplatethat has to be solved because it reducesSA yield and productivity, while increasesthe complexity and cost of SA recovery .-In situ product removal or extractivefermentation can be solved theaforementioned problem.. 17. Background 16WINTER- The advantage of in situ processTemplateseparation that can be improved the finalSA. concentration and reduces end-productinhibition.-Precipitation process is a very interestingtechnique due to its simplicity, and no doesnot require additional chemical or unitoperation. 18. Background 17 -This work, fermentation of SA will usedcommercial strain of SA producing bacteriaand using precipitation technique couple withfermentation SA .-Calcium carbonate will be used to control thepH of the broth. 19. Background 18- Submerge ceramic microfiltration membranewill be employed for take out the clarified brothprior to precipitation. -As a result, Calcium succinate will beprecipitate out , the remaining will then bere-circulated back into the fermenter.-The precipitate will be harvested ,washedand dried to produce a high purity calciumsuccinate 20. Research Objectives 19-To study the precipitation process ofcalcium succinate by using syntheticsolution.-To compare SA production performance interm of yield and volumetric productivitybetween this work and conventional batchfermentation. 21. Research Objectives20-To obtain a high purity of calciumsuccinate powder which facilitatessubsequent downstream processing. 22. Research Hypothesis 21- SA can be produced by usingActinobacillus succinogenes ATCC55618.- Precipitation of calcium succinate canbe achieved in the precipitation tank.- Product inhibition can be minimized andSA production is significantly increased. 23. Materials22-Actinobacillus succinogenesATCC 55618- Glucose- Calcium carbonate- Autoclave - 5 L Bioreactor- HPLC - Glucose analyzer 24. Methods23- Fermentation will be carried out in the absence of oxygen byflushing CO2 gas at the beginning of fermentation 25. Methods 24 26. Methods/ Batch fermentation 25 27. Methods/ Fed batch fermentation 26 - Fed batch fermentation will be started with1.2 L of culture medium and the concentratedculture medium will be gradually added. - pH of broth will be controlled at 6.8- Batch fermentation will be carried out first,feeding of the concentrated culture medium will bestarted when amount of glucose is nearly depletedor lower than 20 g.L-1. 28. Experimental setup 27Experimental setup for extractive fermentationof SA using external precipitation method.Calcium succinate 29. Analytical procedure 28WINTERTemplate 30. Analytical procedure29 WINTER-Reducing sugar will be measured byTemplateDinitrosalicylic (DNS) method accordingto Miller (1995)- Cell concentration will be determined byusing UVVIS Spectrometer. Cell viability will be accomplished using fluorescencemicroscopy.- Quantitative analysis of organic acids willbe analyzed by HPLC . 31. Expected results30 WINTER Template1.High yield and volumetric productivityof succinic acid will be achieved usingthe external precipitation method.2.This simple high efficiency methodcan be applied to pilot scale productionof succinic acid. 32. Research plan31 WINTERTemplate 33. WINTERTemplateTHANK YOU VERY MUCHJiraphorn Lubsungnoen M5430116