Yeast growth, Fermentation and Carbonation

What is Fermentation in a brewery

1. respiration
2. fermentation
3. lagering
4. flocculation

• in Yeast and bacteria, it's fermentation producing acid, gases, and alcohol
• in our muscle cells, it calls Lactic acid fermentation
• the science of fermentation: Zymology

Respiration, lagering, flocculation

• cellular respiration: to convert biochemical energy from nutrients into Adenosine triphosphate(ATP), respiration+catabolic reactions 
• during lagering, yeast reabsorb by-products of fermentation and clean the beer of off-flavor chemical compounds
• flocculation: yeast aggregate and become separated from the liquid

What is yeast?

The cell cycle

• 2 successive cell cycles in the budding yeast

1. G0: resting phase
2. G1: increase in size
3. S: DNA replication
4. G2: cell continues to grow
5. M: mitosis. cell growth stops, divide into two daughter cells

Biochemistry 101

• metabolism: the conversion of food(fuel) to energy to run cellular processes/building blocks for proteins, lipids, nucleic acids, carbohydrates
• metabolism are organized into Metabolic pathways(a sequence of Enzymes)
• catabolism: breaking down large molecules, releasing energy
• anabolism: building up large molecules from small ones, consuming energy
• coenzyme
• ATP(unstable) is hydrolyzed to ADP(stable), free energy, inorganic phosphate(Pi)
• phosphoanhydride bonds: bonds between phosphate molecules, have big energy
• Nicotinamide adenine dinucleotide(NAD), this coenzyme has 2 forms: oxidized NAD+, reduced NADH

Biochemistry 101(Glycolysis)

• glycolysis: converts Glucose into 2 pyruvates(pyruvic acid), 2 ATP(invest -2, harvest +4), 2 NADH
• take place in the cytoplasm of cells
• this pathway can function with(out) oxygen
• in humans, aerobic conditions produce pyruvate/ anaerobic conditions produce lactate
• in yeasts, only pyruvate can be produced
• during glycolysis, pH decreases(5.3->4.3). 0.5pH down/12hrs

Biochemistry 101(Pyruvate oxidation)

• pyruvate translocase: transport protein that brings pyruvates into the mitochondria
• malate-aspartate shuttle
• malate
• oxaloacetate(the end of Krebs)+acetylCoA(from pyruvate oxidation)=>Citrate(the begin of Krebs)
• glycerol phosphate shuttle 
• pyruvate dehydrogenase complex
• {glycolysis}-{pyruvate->acetylCoA}-{Krebs cycle}-{Oxidative phosphorylation}

Biochemistry 101(The citric acid cycle aka Krebs cycle)

• 2 Krebs cycle per one glucose
• products are: 1*2 GTP(=ATP), (1+3)*2 NADH, 1*2 QH2(=FADH), 2*2 CO2
• 1NADH=3ATP, (1+3)*2 NADH=8*3ATP
• 2FADH=2ATP, 1*2 QH2(=FADH)=2*2ATP
• 30ATP+{2ATP+2*3ATP}*from glycolysis=TOTAL 38ATP

Biochemistry 101(Oxidative phosphorylation)

• Protons(H+) are passed from the membrane of mitochondria
• creating a difference of potential between in and out of the membrane
• ATP synthase uses that mechanical Energy, convert ADP into ATP(maximum 38ATP)

Wort composition and Carbohydrate composition

• Plato: 7.5~15
• pH: 5~5.3
• Dissolved solid: 7.5~15%(90~90% of solid are sugars, 4~5% of solid are nitrogenous components, rest is lipids, tannins...)
• nitrogenous compounds: 20% of protein, 22% of poly peptides, 58% of free Amino Acids and peptides(150~200mg/L of wort)
• free Amino Acids: lipids, tannins, minerals(calcium, manganese, copper...), sulphur compounds...

Why minerals? 

• calcium, copper, zinc, etc... 
• calcium is important in flocculation
• manganese can stimulate yeast growth
• magnesium is important in ATP synthesis
• zinc is the most likely to be deficient and can be added during the boil(zinc salt)

Observing a typical fermentation in a lab

• respiration, synthesize ATP: small drop in gravity, sharp plunge in DOT
• prep for copying: increase in biomass(yeast) up to 40hrs  
• temperature: sudden drop at 160hrs, dead yeast come out(bad flavor, need to clean out)
• flocculation: begin at 100hrs, end at 168hrs(1 week)
• the alcohol production starts when the quantity of O2 is near zero

• during fermentation, daily check list is: 

1. temperature: determine when the fermentation is complete
2. pressure: no explosion!
3. pH: whether to be infected
4. gravity
5. volume of yeast

The 4 phases of wort fermentation

1. Lag phase: the yeast is waking up and prepares for growth(respiration)
2. Exponential growth: create Ethanol and CO2
3. Stationary phase: absorb unwanted chemicals
4. Decline phase: goes back to sleep and flocculate

Sucrose, maltose, maltotriose conversion to glucose

• increase of fructose: glucose converted into fructose during the glycolysis 
• end up using glucose, fructose, sucrose(take 20hrs)->begin to use maltose->begin to use maltotriose(plus 3 days after arrival to the terminal gravity)
• it's important for multi-batch: multi-batch must complete within 20hrs, oxygen is provided at the first batch only
• should not add more glucose(more wort) in your fermentors once all glucose has been metabolised
• if you do more than one batch of beer, you have a 16hrs window to add your worts to the fermentors, otherwise incomplete fermentation
• once the respiration in complete, fermentation starts. add your yeast and oxygen during the first batch.

a FAN of beer

• free Amino Acids are consumed mostly during the respiration, beginning at the fermentation
• during the respiration, FAN used for the production of Sterols and Unsaturated fatty acids
• sterols help yeast budding
• unsaturated fatty acid are produced until no more oxygen 

Creating energy reserves

• yeast need reserves for the lean time: Glycogen and trehalose
• depletion of nutrients and excess sugar trigger glycogen intake by yeast

Ethanol and yeast

• ethanol=toxic to yeast
• higher gravity beers needs more yeast
• higher magnesium is good for high gravity beers
• too fast fermentation, warmer temperature to be bad for ethanol production
• acetic acid>acetyldehide>ethanol(reduced)
• acetic acid<acetyldehide<ethanol(oxidize)
• higher-order alcohols=fusel alcohols
• by-products of amino acid metabolism or via pyruvate
• linearly related to yeast growth 
• fusel alcohols occur: at higher temp/ at lower pH/ yeast acitivity is limited by low nitrogen content

Esters in beer

• important esters include: 

1. ethyl acetate(chemical solvent)
2. isoamyl acetate(banana)
3. isobutyl acetate(floral/fruity)
4. ethyl caproate(apple)

• yeast growth<->ester formation
• factors: wort gravity, oxygen availability, temperature
• activated acid(from pyruvate)+alcohol=esters
• when there is oxygen, activated acid are used in yeast growth

Carbonyl in beers

• influence on the flavor stability of beer
• consist of various aldehydes and diketones(notably diacetyl)
• excessive concentration of carbonyl cause stale flavor
• vicenal diketones(VDKs): butter-like flavor, diacetyl is one of them
• diacetyl levels is the factor which determines when the beer may be moved to the conditioning phase
• reduction of diacetyl requires the presence of adequate yeast

Acethyldehide 

• grassy, green apple flavor
• accumulate during the period of active growth
• decline in the stationary phase
• elevated wort oxygen, pitching rate(too many yeast) and temperature promote accumulation
• premature separation of yeast from wort prevent reutilisation of acethyldehide

Sulphur by-products

• hydrogen sulphide(rotten egg), sulphur dioxide(burnt match)
• peak at the second of third day of fermentation
• adequate(not excess) amount of oxygen at the time of pitching is key factor
• remove via vigorous fermentation(carbon dioxide stripping)

aging

• the second fermentation: lagering for ale yeasts. for ale yeast, impossible to ferment a large size unfermentable sugar so adding extra sugar or sweet wort is necessary.
• lagering: lager yeasts can ferment larger size sugar left unfermented(and other by-products) in the first fermentation. 
• conditioning: let yeasts absorb oxygen dissolved when bottling(or kegging) process.