Seminar IV: Fermentation

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Seminar IV Fermentation
FAS Center for Systems Biology Harvard University
Freshman seminar 220 Principles of Industrial
fermentation
Chris Brown, Marcelo Vinces Kevin Verstrepen
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Fermentation, lagering, filtration, bottling
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Brewers yeast- Saccharomyces cerevisiae
C6H12O6 to 2CO2 2 CH3CH2OH
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Saccharomyces cerevisiae
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Saccharomyces cerevisiae
Saccharomyces sensu stricto cerevisiae,
paradoxus, bayanus, pastorianus
(carlsbergensis), mikatae, cariocanus,
kurdiavzevii Ale versus lager yeast Saccharomyce
s cerevisiae versus Saccharomyces
pastorianus Lager larger genome, ferments
melibiose, grows OK at 6-12 Celcius, but not
above 35 Celcius GRAS status 5 micron Eukaryote
with some prokaryotic characteristics
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Saccharomyces cerevisiae life cycle
Homothallic
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Mating type switching
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Mashing making wort making yeast food

• Nutrition for cells
• C,H,O,N,S,P
• Na, Ca, Mg, K, Fe
• Zn, Cu, Co, Mn, Mo, Se, Ni
• Vitamins
• O2
• Carbon/organic molecules Prototroph (
  autotroph) Auxotroph
• Energy Phototroph, Chemotroph, Litotroph,
  Organotroph

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Fermentation vs. respiration
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Weird yeast effects
Pasteur effect Crabtree effect
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Yeast growth curve
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Variables during fermentation
Apparent vs real extract
Temperature lager vs. ale
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Yeast metabolism - glycolysis
aldolase
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Yeast metabolism - glycolysis
Glycerol
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Yeast metabolism - gluconeogenesis
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Yeast metabolism alcohol dehydrogenase
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Fermentation in humans
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TCA / Krebs cycle
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TCA / Krebs cycle
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Electron transport chain
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Cellular uptake mechanisms
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Catabolite repression
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Catabolite repression

• Glucose
• Sucrose
• Fructose, Galactose, mannose
• Maltose, maltotriose
• Glycerol, ethanol, lactate

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Maltose uptake
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Uptake of amino acids
Yeast can synthesize all 20 AAs no essential
AAs NH4 needed, represses GAP but NOT specific
active uptake GAP System 1 groups A and C, but
A first System 2 Slow uptake of group B Group
A Glu, Asp, Asn, Gln, Thr, Lys, Arg Group B
Val, His, Met, Leu, Ile Group C Gly, Phe, Tyr,
Trp, Ala Group D Pro Valine uptake blocked by
Thr --- diacetyl
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Diacetyl
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Diacetyl
No Thr, no Val Val synthesis
Val uptake No synthesis
Thr present Val synthesis

• Low FAN level in wort long second synthesis
  phase
• Too much yeast growth (O2, glucose, mineral rich
  wort) -2nd peak occurs
• Flocculent yeast less reduction
• Certain yeast strains more production, less
  reduction
• Lack of warm rest before lagering less
  reduction
• Addition of alpha acetolactate decarboxylase
• Genetic engineering

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Flavor-active fusel alcohols in beer
Example isoamylalcohol (alcohol,
solvent) Leucine transamination Ox.
Decarboxylation reduction Mostly linked to
yeast growth increasing alcohols with
increasing oxygen, trub/lipids, CCTs,
Drauflassen, temperature and lower hydrostatic
pressure Yeast strain
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Flavor-active fusel alcohols in beer
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Flavor-active esters in beer
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Flavor-active esters in beer
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Cylindroconical tanks (CCTs)
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Cylindroconical tanks (CCTs)
25 m 80 ft
1.76 m 5.80 ft
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Cylindroconical tanks (CCTs)
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Yeast cell wall, old style
Beta glucans
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Yeast flocculation
cell wall mannan
lectin (e.g. Flo1p)
Beta glucans
Ca2
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Yeast flocculation
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temperature
ethanol
factors affecting FLO gene activity
?
nutrition
sensory mechanisms
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genetic background of specific strain
FLO genes
transcription translation
Flo proteins
incorporation into cell wall
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flocculin
factors affecting cell-cell interactions and
flocculin activation
Ca2 conc.
???
agitation
ionic strenght
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Controlling flocculation
Experimental observations

• Glucose inhibits flocculation
• Maltose, maltotriose ???
• Nitrogen ambiguous
• Nitrogen addition SOMETIMES delays flocculation
• Growth factors (Zn, vitamins) ambiguous
  addition SOMETIMES delays flocculation
• Better aeration sometimes improves flocculation
• pH, temperature, ethanol -mixed effects

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Selection during yeast cropping
Yeast cropping discrete layers of yeast
population in the cone of CCTs
Selection !
Younger, less flocculent cells Older, more
flocculent cells
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Genetic modification
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Lagering
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Beer centrifugation / filtration
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Bottling
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Pasteurizing
1 Pasteurization unit 1 minute at 60 Celsius
(140 F) 10 x faster at 70 Celsius Kegs vs.
bottles Flash vs. Tunnel (batch) UHT for milk
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Secondary fermentation / refermentation
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Keg tap
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Acknowledgements

• Verstrepen Lab
• - Matthieu Legendre
• Marcelo Vinces
• Nathalie Pochet
• Marina Caldara
• Chris Brown
• Theodore Pak
• Bianca Calderon
• Scott Smukalla
• Bena Chan
• Chen Yan
• Courtney Fiske

• K.U.Leuven
• - Johan Thevelein
• Jos Vanderleyden

• M.I.T.
• - Narendra Maheshri
• Gerald Fink
• An Jansen

Weizmann Institute - Naama Barkai
Tel Aviv University - Nir Osherov

• Sanger Institute
• - Anton Enright
• Stijn van Dongen

Harvard University - Andrew Murray - Erin O
Shea - Rich Losick - Alex Schier

• Institut Pasteur
• - Anne Beauvais
• Jean-Paul Latgé

University of Washington - Christine Queitsch
Amsterdam University - Frans Klis