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A biotech view onGLYCEROLCorso di Biotecnologie Industriali- DCCI-Università di PisaDr.ssa. Federica Chiellini23th February 2012 - Greta Dalle Luche

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A biotech view onGLYCEROLApplications:-medical-chemical-food industry●Drugs●Cosmetic●Toothpaste●Paints●Cellophane●Dynamite

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A biotech view onGLYCEROLProduction:● Chemical●Biotech

DunaliellaGreen algae

Saccharomices cerevisiaeother yeasts

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A biotech view onGLYCEROLProduction:● Chemical●Biotech

Saccharomices cerevisiaeother yeasts

“Microbial production of glycerol has been known for 150 years, and glycerol was produced commercially during World War I. Glycerol production by microbial synthesis subsequently declined since it was unable to compete with chemical synthesis from petrochemical feedstocks due to the low glycerol yields and the difficulty with extraction and purification of glycerol from broth. As the cost of propylene has increased and its availability has decreased especially in developing countries and as glycerol has become an attractive feedstock for production of various chemicals, glycerol production by fermentation has become more attractive as an alternative route.”

Dunaliella Green algae

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A biotech view onGLYCEROLProduction by yeastNaHSO3pH=7osmotolerant yeastsSaccharomices cerevisiae

1ST

2ND

3RD

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Saccharomyces Cerevisiae(yeast)

●GRAS●Flexible metabolism●Easily genetically modifiable●Simple growth media●High ethanol content resistant

●Schizosacchromyces pombe●Kluyveromices lactis●Pichia-Hansenula●Yarrowia lipolytica

A biotech view onGLYCEROL

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Saccharomyces Cerevisiae(yeast)

●Budding●Flocculating

A biotech view onGLYCEROL

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A biotech view onGLYCEROLProtol® process● Chemical●Biotech Saccharomices

cerevisiaeADH

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Protol® process● Chemical●Biotech Saccharomices

cerevisiaeSaccharomices cerevisiaeSaccharomices cerevisiaeX

1ST) Trapping of the acetaldehydehexose + bisulfite -----> acetaldehydebisulfite + CO2+ H2O + glycerol

A biotech view onGLYCEROLADH

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A biotech view onGLYCEROLProtol® processConnsteinand Luidecke (Germany, 1919)Saccharomices cerevisiae

Anaerobic conditionsSubstrate=100g/L saccaroseInocule=1% v/vTime=2-3ggT=30-35 °CConversion efficiency= 25% (glycerol/amount of sugar consumed)

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A biotech view onGLYCEROLProtol® processNaHSO3pH=7osmotolerant yeastsSaccharomices cerevisiae

Anaerobic conditionsSubstrate=100g/LInocule=1% v/vTime=2-3ggT=30-35 °C

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A biotech view onGLYCEROLProtol® process● Chemical●Biotech Saccharomices

cerevisiae

2nd) Increasing actity of aldehyde dehydrogenase at pH of 72hexose -----> 2glycerol+ ethanol + acetic acid + 2CO2+ 2H2O

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● Candida glicerinogenes● Candida magnolia● Debaryomyces● Hansenula● Pichia● Saccharomyces● Schizosaccharomyces

3rd) Osmotolerant yeastsHexose + O2-----> + CO2+ H2O + glycerol+ polyols

A biotech view onGLYCEROLCandida magnoliae

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3rd) Osmotolerant yeastsHexose + O2-----> + CO2+ H2O + glycerol+ polyols

A biotech view onGLYCEROLAerobic conditionsSubstrate=200g/L glucose/sucroseTime=3-5ggConversion efficiency= 45% (glycerol/amount of sugar consumed)

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A biotech view onGLYCEROLSaccharomices cerevisiae

Anaerobic conditionsSubstrate=100g/LInocule=1% v/vTime=2-3ggT=30-35 °C

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A biotech view onGLYCEROLSaccharomices cerevisiae

Anaerobic conditionsSubstrate=100g/LInocule=1% v/vTime=2-3ggT=30-35 °C = μ f (s)

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A biotech view onGLYCEROLTechnical ProblemsLow glycerol yield as other by-products(ethanol, acetaldehyde)High amount of sulphiteExpensive recovery( vacuum distillation,exchange resins-ion and exclusion) Saccharomices cerevisiae

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A biotech view onGLYCEROLGenetic ImprovementOverespression of the genes associated with te glycerol producionChanneling the glycolitic flux towards glycerol formationDecreasing the activities of the pathways for dissimilation of glycerol Saccharomices cerevisiae

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A biotech view onGLYCEROL● massive accumulation of β-carotene● hypersaline environments

Dunaliella

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A biotech view onGLYCEROLDivision rate(as number of divisions per day) of different Dunaliella isolates belonging to several species, as a function of the NaCl concentration

“Dunaliella cells lack a rigid cell wall, and the cell is enclosed solely by a thin elastic plasma membrane. As a result, the cells' morphology is strongly influenced by osmotic changes.”

Dunaliella

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A biotech view onGLYCEROLDunaliellagrowing phaseNaCl 1,5Mstoring phase: NaCl 4 Mrecovery: osmotic shock Dunaniella salina

pH= 9-10Substrate=NaCl, CO2, NO2, PO3 T=10-40 °C

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A biotech view onGLYCEROLpH= 9-10Substrate=NaCl, CO2, NO2, PO3 T=10-40 °C

Dunaliella

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A biotech view onGLYCEROLProtol® processSaccharomices cerevisiae

References“Glycerol production by microbial fermentation: A review”, Zheng-Xiang Wan, Biotechnology Advances 19 (2001) 201–223“A hundred years of Dunaliella research: 1905–2005”, Aharon Oren, The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, IsraelImages from www.sciencephoto.com

Dunaliella salina

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