1. Production of Ethanol and R-phenylacetylcarbinol from Whole Cell Biocatalyst Utilizes Carbon Source from Dried Longan Amalia S. Agustina, Poonsiri Phrathong, Utoomporn Apiwongngam, Kitiya Laewongnin, Peerawat Jaiwunglok, Krit Sittivangkul, Chartchai Khanongnuch, Noppol Leksawasdi Brawijaya University Chiang Mai University

2. Introduction

3. Longan Longan as important economic crops of Thailand – Pinapple (US$361 millions) – Durians (US$87 millions) – Young corns (US$58 millions) – Longan (US$57.8 millions) Fresh longan – 25.2% (w/v) of carbohydrate – 1.0% (w/v) of proteins – Minerals – Other vitamins Problem of longan production – Biennial bearing – Perishable properties

4. Alternative solution Bioconversion of leftover sugars to ethanol and pyruvate decarboxylase (PDC) from remnant microbial biomass PDC as biocatalyst for biotransformation of pyruvate and benzaldehyde to R-phenylacetylcarbinol (PAC) PAC: chiral precursor for the production of ephedrine and pseudoephedrine

5. Biotransformation of PAC from pyruvate and benzaldehyde

6. Objectives To screen an appropriate microbial strains for ethanol and biomass production To select a suitable dried longan for cultivation medium To perform biotransformation in the system which will be suitable to produce PAC with whole cell PDC

7. Overview Screening of appropriate microbial strain & suitable dried longan as cultivation medium Perform biotransformation of PAC from selected microbial strain cultivated in selected cultivation medium

8. Materials and Methods

9. Microorganisms Candida utilis (TISTR 5001, 5032, 5043, 5046, 5198, 5352) Escherichia coli (TISTR 361, 1261) Klebsiella sp. (TISTR 1383) Saccharomyces cerevisiae (TISTR 5020, 5339, 5606) Zymomonas mobilis (TISTR 405, 548, 550)

10. Media Inoculum media (10 ml) Concentrated dried longan extract aged 1 month old (30% (w/v)) (100 ml) Concentrated dried longan extract aged 6 years old (100% (w/v)) (100 ml)

11. Cultivation of Microbial Culture in 100 ml scale Ethanol and Cell Biomass 24 h of cultivation in static condition (T = 25.6 o C) 24 h of cultivation with aeration (T = 25.6 o C) 100 ml of media 10 ml seed inoculum

12. Biotransformation of PAC Benzaldehyde emulsion system (single phase) Biphasic/octanol phosphate buffer system (two phase) 3.06 g/l of whole cell PDC concentration 1 mM of TPP and 1 mM of MgSO 4.7H 2 O

13. Benzaldehyde emulsion system 0, 300, 600, and 900 mM of phosphate buffer 150 mM of benzaldehyde 180 mM of pyruvate 8 o C for 8 h

14. Biphasic/octanol phosphate buffer system Types of solvent in organic phase – C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, – DPG (dipropylene glycol), DPG+C1, DPG+C2, DPG+C3, DPG+C7, DPG+C8, DPG+C9 500 mM of benzaldehyde 600 mM of pyruvate 6 o C for 24 h, 200 rpm

15. Analytical Methods Dried biomass OD600 pH Total Soluble Solids Sugars Organic Acids Ethanol PAC

16. Results and Discussions

17. Screening of Carbon Source and Whole Cells Biocatalyst Concentrated dried longan aged 6 years old (100% (w/v)) Concentrated dried longan aged 6 years old (100% (w/v))

18. Concentrated dried longan aged 6 years old (100% (w/v))

19. Concentrated dried longan aged 1 month old (30% (w/v))

20. Selection of appropriate microbial strains and cultivation media Yeast Strain (TISTR) Extract of 1 Month Old Dried Longan ∆P (g/l)∆X (g/l)∆P*∆X 502045.0 ± 6.12.76 ± 1.10124 ± 52 560637.8 ± 3.14.17 ± 2.46158 ± 94 Dried longan aged 1 month old was considered as appropriate cultivation medium with S. cerevisiae TISTR 5606 as microbial strain

21. Biotransformation of PAC Conducted by using whole cell PDC from S. cerevisiae TISTR 5606 cultivated in 1 month old dried longan Performed in benzaldehyde emulsion system and biphasic/octanol phosphate buffer system

22. Benzaldehyde emulsion system Phosphate Buffer (mM) PAC (mM) 00.00 ± 0.00 3000.00 ± 0.00 6000.61 ± 0.61 9001.19 ± 0.01

23. Biphasic/octanol phosphate buffer system

24. Conclusion One month old dried longan had sufficient nutrition as cultivation media for microbial strain S. cerevisiae TISTR 5606 was able to produce ethanol as well as producing biomass Biphasic/octanol phosphate buffer system was acknowledged as the most appropriate system form PAC production with C8 as solvent of organic phase

25. Acknowledgement Department of Food Engineering and Department of Biotechnology Faculty of Agro-Industry, Chiang Mai University Graduate School, Chiang Mai University The Government of Republic Indonesia

26. Thank You For Your Kind Attention