Production of Production of Ethanol and Ethanol and RR-phenylacetylcarbinol from-phenylacetylcarbinol from

Whole Cell Biocatalyst UtilizesWhole Cell Biocatalyst UtilizesCarbon Source from Dried LonganCarbon Source from Dried Longan

Amalia S. Agustina, Poonsiri Phrathong, Utoomporn Apiwongngam, Kitiya Laewongnin,

Peerawat Jaiwunglok, Krit Sittivangkul,Chartchai Khanongnuch, Noppol Leksawasdi

Brawijaya UniversityChiang Mai University

IntroductionIntroduction

LonganLongan

• 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

Alternative solutionAlternative 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

Biotransformation of PAC from Biotransformation of PAC from pyruvate and benzaldehyde pyruvate and benzaldehyde

ObjectivesObjectives

• 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

OverviewOverview

Screening of appropriatemicrobial strain & suitable dried longan

as cultivation medium

Perform biotransformation of PAC from selected microbial strain cultivated in

selected cultivation medium

MaterialsMaterialsandand

MethodsMethods

MicroorganismsMicroorganisms

• 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)

MediaMedia

• 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)

Cultivation of Microbial Culture in 100 ml scaleCultivation of Microbial Culture in 100 ml scale

Ethanol and Cell Biomass

24 h of cultivation in static condition(T = 25.6oC)

24 h of cultivation with aeration(T = 25.6oC)

100 ml of media 10 ml seedinoculum

Biotransformation of PACBiotransformation 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 MgSO4.7H2O

Benzaldehyde emulsion systemBenzaldehyde emulsion system

• 0, 300, 600, and 900 mM of phosphate buffer

• 150 mM of benzaldehyde

• 180 mM of pyruvate

• 8oC for 8 h

Biphasic/octanol phosphate buffer systemBiphasic/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

• 6oC for 24 h, 200 rpm

Analytical MethodsAnalytical Methods

• Dried biomass• OD600• pH• Total Soluble Solids• Sugars• Organic Acids• Ethanol• PAC

ResultsResultsandand

DiscussionsDiscussions

Screening of Carbon Source and Whole Cells Screening of Carbon Source and Whole Cells BiocatalystBiocatalyst

• Concentrated dried longanConcentrated dried longanaged 6 years old (100% (w/v))aged 6 years old (100% (w/v))

• Concentrated dried longanConcentrated dried longanaged 6 years old (100% (w/v))aged 6 years old (100% (w/v))

Concentrated dried longanConcentrated dried longanaged 6 years old (100% (w/v))aged 6 years old (100% (w/v))

Concentrated dried longanConcentrated dried longanaged 1 month old (30% (w/v))aged 1 month old (30% (w/v))

Selection of appropriate microbial strainsSelection of appropriate microbial strainsand cultivation mediaand cultivation media

Yeast Strain

(TISTR)

Extract of 1 Month Old Dried Longan

∆P (g/l) ∆X (g/l) ∆P*∆X

5020 45.0 ± 6.1 2.76 ± 1.10 124 ± 52

5606 37.8 ± 3.1 4.17 ± 2.46 158 ± 94

Dried longan aged 1 month old was considered as appropriate cultivation medium with S. cerevisiae TISTR 5606 as microbial strain

Biotransformation of PACBiotransformation 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

Benzaldehyde emulsion systemBenzaldehyde emulsion system

Phosphate Buffer (mM)

PAC(mM)

0 0.00 ± 0.00

300 0.00 ± 0.00

600 0.61 ± 0.61

900 1.19 ± 0.01

Biphasic/octanol phosphate buffer systemBiphasic/octanol phosphate buffer system

ConclusionConclusion

• 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

AcknowledgementAcknowledgement

• 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

Thank You Thank You For For

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