SCIENCE BEHIND WINE FERMENTATION Sara Belchik 28 June 2014

Wine fermentation

Overview History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast

Wine predates written records

But we did not understand it 6000 BCE Earliest evidence of wine production 1680 AD Description of small spheres during fermentation

But we did not understand it 6000 BCE Earliest evidence of wine production 1680 AD Description of small spheres during fermentation 1830 AD Small spheres identified as living organisms

Pasteur experiment SterilizedNot sterilized

Pasteur experiment SterilizedNot sterilized

Yeast are responsible agents 6000 BCE Earliest evidence of wine production 1680 AD Description of small spheres during fermentation 1830 AD Small spheres identified as living organisms 1863 AD Yeast identified as microbe responsible for fermentation of grapes into wine 1890 AD Addition of pure yeast culture to help with fermentation 1965 AD Commercially available dehydrated yeast for sale

Overview History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast

What are yeast?

Yeast are eukaryotic cells Unicellular organisms Kingdom Fungi Complex interior allows for complex reactions to occur Yeast is umbrella term Almost 2000 species Earliest domesticated organism

Semi-permeable membrane Water-loving parts on exterior Water-hating portions on interior Difficult for many molecules to get through Require transport proteins to help get across membrane

Many yeast species

Wine yeast genera Predominant species on surface of grape berries Kloeckera Also found on grapes but to lesser extent Brettanomyces Candida Crytococcus Kluyveromyces Metschnikowia Pichia Rhodotorula Found in vineyards or on winemaking equipment Debaryomyces Saccharomyces Saccharomycodes Schizosaccharomyces Zygosaccharomyces

Overview History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast

Early earth had no oxygen

How did early microbes generate energy? Need to generate energy in absence of oxygen Life evolved to utilize glucose Glucose molecule has energy within its bonds Multiple enzymes adapted to produce maximum energy out of these bonds First part of glucose metabolism is glycolysis All life on earth uses glycolysis ENERGY

Glycolysis generates pyruvate and NADH Glucose  2 pyruvate 2 ATP generated Energy currency of the cell Needed for most cellular processes 2 NADH generated Electron carrier of the cell Needs to be oxidized to NAD + for glycolysis to continue Fate of NADH determined by environment

Glycolysis generates pyruvate and NADH Glucose  2 pyruvate 2 ATP generated Energy currency of the cell Needed for most cellular processes 2 NADH generated Electron carrier of the cell Needs to be oxidized to NAD + for glycolysis to continue Fate of NADH determined by environment

NADH and NAD + Need to regenerate this to continue using glucose

Regenerate NAD + to keep using glucose This pathway uses oxygen and generates lots of ATP Our muscles sometimes do this

Overview History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast

Yeast use ethanol fermentation

From glucose to ethanol

Glucose comes from the grape

Overview History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast

But…wine isn’t just ethanol

Glycerol in wine

Glycerol production by yeast

Organic acids Tartaric, malic, and citric acid present in grapes Tartaric and citric acid mostly stable throughout fermentation Malic acid is tart and considered undesirable in a red wine Luckily, bacteria (not yeast) can convert malic acid to more palatable lactic acid

Oenococcus oeni Certain bacteria are preferred over others based on byproducts of fermentation Products of malolactic acid fermentation Lactic acid Acetoin and diacetyl Acetic acid Various esters Higher alcohols

Overview History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast

What about before 1863? Microorganisms are ubiquitous and live in various environments Grapes are just one example of a habitat Yeast and certain bacteria live on grapes When grapes are crushed, the natural microbes mix with grape juice Called spontaneous fermentation

Spontaneous fermentation Low concentrations of microbes on grapes Fermentation takes longer to start Entire process requires more time Wild yeast may not have favorable traits Low alcohol tolerance Results in high sugar content Other microbes present Uncertainty of final product Off-aromas and weird esters

Overview History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast

Yeast added to control end product

Inoculated fermentation Added at high concentrations to grape must Inoculum outcompetes other microorganisms present Preferred yeast is Saccharomyces cerevisiae Multiple strains exist Different strains for different wines Other yeast can also be used Kloeckera Zygosaccharomyces

Overview History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast

Breeding to generate perfect yeast Yeast can be haploid (one copy of chromosomes) or diploid (two copies) Take two yeast with desirable traits and breed them Result is diploid cell containing genetic information from both parents

Not a perfect process

Target specific genes in yeast

Let’s target glycolysis Process involves a series of enzymes We can increase number of enzymes in the process Increased flux through glycolysis results in more pyruvate More pyruvate results in more ethanol Not much success Rate limiting step is getting glucose into the cell

Let’s target sugar transport Increase amount of glucose getting into cell Increased glucose in cell ultimately results in more ethanol Target glucose transporters Success!

Let’s target sugar transport Increase amount of glucose getting into cell Increased glucose in cell ultimately results in more ethanol Target glucose transporters Success!

Let’s improve ethanol tolerance Different yeast strains have different ethanol tolerance limits Ethanol destabilizes the membrane Target membrane fluidity or detoxification enzymes Many GMOs with increased tolerance

Other modifications Sulfur dioxide resistance SO 2 used to decrease unwanted microbes Higher SO 2 resistance in yeast means we can increase concentration and kill off even more unwanted microbes Nitrogen assimilation Yeast require carbon and nitrogen Nitrogen not abundant in grapes Increase nitrogen uptake or utilization to increase robustness of yeast

Yeast GMOs result in more wine varieties

Wine fermentation

Questions?