Epilogue

Epilogue

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Many people avoid WINE CHEMISTRY, because it is considered too difficult and not necessary. Indeed, it needs some efforts, but it is absolutely doable. The French say it beautifully, << Qui veut peut >>. It couldn't be simpler and clearer. When I started my own research on WINE CHEMISTRY, my chemistry knowledge was limited to O₂, CO₂ and H₂O. My interest on WINE CHEMISTRY started when I learned that  6CO₂ + 6 H₂O  à C₆H₁₂O₆ + 6O₂  and  C₆H₁₂O₆ à

2 C₂H₅OH + 2CO₂.

There are many ‘questions’ and ‘mysteries’ in the study of wine. Why do yeast convert sugar into alcohol and bacteria turn malic acid into lactic acid ?  What are glycerol, fatty acids, high alcohols, esters,  diacetyl, and wine diamonds?  And where do they come from ?  Why is oxidation sometimes good, and sometimes bad in wine?

It is not easy to get answers for those questions. Wine is a bio-chemical product, so I think the answers must be found in Wine Chemistry. There are some WINE CHEMISTRY books, but they are more for professional wine makers. For a chemistry layman, like me,  they are difficult to follow. So I decided to ‘google’ for it. Step by step, piece by piece, learning by doing, I put the puzzles together. I cannot assure you that my findings are the accurate. It is my attempt to get an answer to those questions I had in the study of wine.

The beauty of WINE CHEMISTRY is that it, with its metabolic pathways, provides a clarifying explanation to the phenomena in wine. It also, with its molecular structures, gives a 'face' to the wine components so that we can see  their differences. WINE CHEMISTRY is  desirable if you want to have a good picture of the wine components. WINE CHEMISTRY is certainly needed if you want to give a judgement on what you are told. I see WINE CHEMISTRY as grammar. You can learn to speak a language fluently wthout learning its grammar, but it is unlikely that you can truly understand what is right or wrong. You can study wine without WINE CHEMISTRY, but then you have to take everything you are told for granted.

Here, I would like to express my gratitude to Google. All my findings are 'dug' out from Google. Without Google, there is no way I could have gotten access to all this information and knowledge. Without Google, it would have been impossible for me to share my findings with you. THANK YOU VERY MUCH GOOGLE! YOU ARE GREAT!

Finally, I would like to thank you for reading. If you find my blog informative, please share it with your wine friends. Share and discuss knowledge result to increase and improve knowledge. A joy shared is a joy doubled! 

                                                    

Part 13 - Sulfur dioxide & Sulfite

Part 13 – Sulfur dioxide & Sulfite

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v  Sulfur (or Sulphur) - S

Sulfur is commonly known as the substance used in matches and gunpowder. Over 2000 years ago the Romans discovered by accident that wines stored in vessels treated with burning sulfur candles did not develop a vinegar smell. Due to combustion, sulfur combines with oxygen and becomes sulfur dioxide (S + O₂ à SO₂). SO₂ is a colorless gas with a sharp characteristic smell. It is toxic. It kills the micro-organisms and thus disinfects the barrels. Today, most wine makers still use SO₂ or sulfite to stop the growth of harmful micro-organisms.

v Atom, molecule and ion

Sulfur (S) is a non-metallic atom with 16 protons and 16 electrons. Protons are positively charged and electrons are negatively charged. An atom with an equal number of protons and electrons is electrically neutral.

Sulfur dioxide (SO₂) is a molecule. A group of two or more atoms held together by chemical bonds is called a molecule.  A molecule is electrically neutral.

Ion is an atom or molecule which is not electrically neutral. In other words, when an atom or molecule has become electrically charged, it is called an ion. A positively charged atom or molecule is called a cation, a negatively charged atom or molecule is called an anion. Sulfite (SO₃^-2)  e.g.  is an anion.

v Sulfur dioxide (SO₂)  in cylinders

SO₂ is supplied in cylinders. The gas is colorless with a pungent odor and toxic. It works strongly irritating to mucous membranes and eyes. Major winemakers use this to determine the precise dosage and only pure SO₂ is added. However, SO₂ (gas) dissolves in water and becomes sulphurous acid (H₂SO₃).  Acid gives off H-protons  and release bisulfite anion (HSO₃^-) and sulfite anion (SO₃^-2). These reactions are reversible, they go right and left, back-and-forth.

  

 The molecular sulfur dioxide (SO₂ ) works about 100x as strong as the sulfite anion (SO₃^-2) against micro-organisms. That’s because only the molecular SO₂ can pass through the cell wall, after which it destroys the enzymes and proteins in the cell. However, the availability of the molecular SO₂ is dependent on these anions. The sulfite anion (SO₃^-2)  love to bind with acetaldehyde, anthocyanins and sugar. The binding with acetaldehyde is permanent and so the sulfite is lost forever. The more acetaldehyde to be formed (e.g. by slow fermentation) the more sulfite anions are extracted from the wine. This will be increasingly SO₂  from the left "sucked" to the right, so to speak. In other words, the more acetaldehyde, the greater the need for sulfurizing. As acetaldehydes are binded by sulfites, yeasts will produce glycerol to compensate for the NAD deficit. That explain why addition of sulfite will lead to more glycerol formation (see Part 4).

The bindings with anthocyanins and sugar are temporary. From these bindings, sulfite anion, bisulfite anion  and eventually SO₂  can be free again. These loose bindings act as a buffer stock SO₂.

v  Sulfites ( or sulphites)

Smaller winemakers usually use sulfites : potassium bisulfite KHSO₃  or  potassium disulfite K₂S₂O₅ . Sulfites release sulfur dioxide SO₂ , which is the active component that helps preserve wine and food.

                          

One molecule potassium disulfite can release two molecules SO₂.  1 gram KDS releases roughly  0.57 grams of SO₂. This can be calculated as follows:   

v  pH influence

The SO₂ released from potassium disulfite ( like the SO₂ from cylinders) will dissolve in water and become HSO₃^-  and SO₃^-2 . The lower the pH in wine, the more SO₂ will be free again; the higher the pH in wine, the less.

It is generally accepted that for a good action against micro-organisms in wine, a minimum 0.8 mg/L molecular SO₂ is required. The table below shows that at pH-4, it needs 10x more sulfite in order to have the same amount of working molecular SO₂  as at  pH-3.

v  Wine diamonds

Sulfites release not only SO₂, but also potassium ions (K+), which react with tartaric acid. This causes the precipitation of potassium bitartrate, known as tartaric acid crystals (or wine diamonds), and some (minor) deacidification takes place (tartaric acid disappears from the wine). It crystallizes because it’s a salt.

                                                          

Some winemakers let their wines undergo cold stabilization, a process by which a wine is cooled down before it is bottled. The “crystallized tartaric acid” fall out and can be separated from the wine.

Some winemakers believe that cold stabilization influences the wine’s balance and taste. According to them, the wine is actually ripped apart; as the rapid cooling changes the wine’s colloidal structure. In these wines, wine diamonds may occur.

v  Sulfurization of grapes, must or wine aims to:

1. SO₂ stops growth of good yeast (Saccharomyces cerevisiae) and prevents spontaneous fermentation of grapes.

2. SO₂ kills the oxidation enzymes (laccase and tyrosinase) and prevents premature oxidation (no brown colors). 

3. SO₂ kills wild yeasts (Brettanomyces) and prevents bad aromas  'brett'  in wine.

4. SO₂ kills acetic acid bacteria and prevents excessive acetic acid and ethyl acetate (glue-like smell) in wine.

5. SO₂ binds to oxygen in the wine and thereby limits the oxidation of alcohol to acetaldehyde.

6. SO₂ binds to products such as aldehydes and diacetyl, which suppress the fruity aromas.

v Legal limit sulfite content

mg/l	red	white, rosé	bio-wine	sherry,port	mousser.	sweet		Eiswein
<5 g r/s	150	200	120	150	185(BOB/BGA)	Spätlese	Auslese	BA/TBA
>5 g r/s	200	250	170	200	235	300	350	400

l Total sulfite content = free  SO₂ + HSO₃^-  + SO₃^-2  + bound sulfite (see table above).

l Sugars bind sulfite. Wines with residue sugars have a higher content of bound sulfite. That's one of the reasons

     that the legal limits for total sulfite content for dessert wines is higher than for dry wines.

l Since 25-11-2005 wines containing more than 10 mg/l sulfite must state on the label "contains sulfites" or  

    "contains sulfur dioxide”. That's because sulfite is an allergen, which can be dangerous for asthmatics and people

    with allergies to aspirin.

v Wine completely without sulfite

No, that does not exist. In wine there is always 5-15 mg/L of sulfite produced by the yeast from the sulfur-containing amino acids: cysteine and methionine (see Part 6).  

A wine without  'added' SO₂, can be . The question is: how old can that wine be?

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P.S.

This is my last post. It is a very ‘limited’ blog. Only ‘fermentation’ and ‘wine components’ produced therefrom are discussed. ‘Wine components’ coming from grapes and from oaks, as well as ‘wine-aromas’ and ‘wine-faults’ were left out. Wine Knowledge is a very extensive study. Quite rightly, Clint Eastwood said in the movie ‘Magnum Force’: ‘A man’s got to know his limitations.’

Part 12 - Malolactic Fermentation

Part 12 - Malolactic Fermentation (MLF)

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v 2 significant differences between yeasts and  bacteria
Alcoholic fermentation is done by yeasts. They have mitochondria in cytoplasm. Yeasts can produce 38 ATP from one glucose (see Part 2 – TCA cycle).

Malolactic fermentation is done by bacteria. They have no mitochondria. The lack of mitochondria means that bacteria can only generate 2 ATP from one glucose in cytoplasm (see Part 1 – The alcoholic fermentation). Another difference is that bacteria can grow on glucose as well as on organic acids.

    

                        

v Bacterial growth during fermentation in 3 phases

Lactic acid bacteria (LAB) can grow on glucose and organic acids. The first bacterial growth takes place at the beginning of alcoholic fermentation. But soon, after a few hours, the bacterial growth is brought to a halt by the powerful yeast growth. In the presence of oxygen, yeasts break down glucose in cytoplasm and mitochondria to produce 38 ATP. Bacteria, by lack of mitochodria, can only break down glucose in cytoplasm to produce 2 ATP. The bacteria simply lost the battle to the yeasts.

After the alcoholic fermentation, the bacteria can grow again when conditions allow. In the absence of residual sugar bacteria now live on the malic acids in the wine. That is in fact the malolactic fermentation.

v Factors affecting  MLF

Temperature – The optimal temperature is between 20^o and 25^o C. Under 15^oC and above 30^oC, MLF is practically impossible. By regulating the temperature the winemaker can control if MLF will happen or not.

 Acidity (pH) – At pH below 3.3, MLB can live but do not grow. At pH above 3.6, they grow well and optimally at around 4.5. In general, the pH of red wines is rarely less than 3.4 and whites is rarely higher than 3.6. That partly explains why in red wines almost always MLF occurs while a Riesling, from a cool climate, almost never does. If anyone would apply MLF to a wine with a very low pH, he must first deacidify the wine (e.g. with calcium carbonate).

Sulfur dioxide (SO₂) – The presence of SO₂ can prevent MLF because SO₂ is a very effective inhibition agent against malolactic growth. The dosage is dependent on the pH. In principle: the higher the pH, the more SO₂ required.

v Malolactic fermentation (MLF)

MLF is the conversion of the tart ‘malic acid’ into the soft ‘lactic acid’ by the LAB : Oenococcus-oeni (former name Leuconostoc-oenos).  Oenococcus-oeni  take in malic acid and decarboxylate it with malolactic enzyme into lactic acid and carbon dioxide.

                                     

v Oenococcus-oeni convert malic acid into lactic acid to generate energy (ATP)
In chemistry, acid is defined as a compound which can release proton (H+ ion). When this happens, R-COOH will become R-COO^– (R = the rest of the compound). Malic acid outside malolactic bacteria, can release two H+. Malic acid inside malolactic bacteria, will be converted into lactic acid which can only release one H+. This way, malolactic bacteria create purposely a pH gradient. That means the H+ concentration in the wine is higher than inside the bacteria cell. With a pH gradient, H+ will diffuse from an area of high concentration to an area of lower concentration. It is  called chemiosmosis. This way, malolactic bacteria get the H+ in the ATP synthetase to generate ATP.

The conversion will stop when the H+ concentration in the wine and in the bacteria is at equilibrium point (=in balance condition). That means the bacteria can never convert all the malic acid into lactic acid.

v Oenococcus-oeni also convert citric acid into acetic acid, acetoin and diacetyl
The citric acid is first converted to acetic acid and oxaloacetic acid. The oxaloacetic acid is decarboxylated to pyruvate. The pyruvate binds with ethanal to become acetolactic acid, which will then be decarboxylated and reduced/oxidized to acetoin, butanediol and diacetyl. Acetoin and especially diacetyl give off a buttery smell that may contribute to a wine’s aroma. 2.3-butanediol is virtually odorless (We have seen this already in Part 11).

v MLF : YES or NO

(1) MLF can lead to considerable deacidification. One gram of malic acid is converted roughly into 0.67 grams of  lactic acid and 0.33 grams of CO2. It increases the pH by 0.1 to 0.3 units. MLF is beneficial for high acid wines,  but undesirable for low acid wines which, after MLF, will be more flat and unbalanced. Deacidification is also beneficial

to tannic wines since acids aggravate the astringency of tannins. Reason why most red wines go through MLF.

(2) MLF can contribute “buttery” aroma to wine due to the extra diacetyl and acetoin. This buttery aroma was once the hallmark of the California Chardonnay.  However, for fruity, fresh style wines; like Riesling, Sancerre and most Chablis,  MLF is not desirable as MLF might change their fruity and fresh character.

(3) MLF can improve microbial stability because the lactic acid bacteria have consumed many of the leftover nutrients that other spoilage microbes could use to develop wine faults. However MLF increases also pH level and acetic acid, which might make the wine vulnerable. For unsulphured wines this might not be beneficial.

v Spontaneous MLF or  Use of Selected Starter Cultures

Lactic acid bacteria (LAB) found in wine belong to three genera: Lactobacillus, Pediococcus and Leuconostoc. MLF is mainly performed by Oenococcus oeni (former name Leuconostoc-oenos), a species that can withstand the low pH (<3.5), high ethanol (>10 vol.%) and high SO₂ levels (50 mg/L) in wine.

Induction of MLF can be spontaneous or by the use of selected starters. The latter gives a better control on the fermentation of: the start, its progress and the strain that completes this proces. In fact, the inoculum of selected bacteria prevents the development of bacteria belonging to the genera Lactobacillus and Pediococcus. These are regarded as “bad guys”. These contaminating species can produce high concentration of acetic acid that can impair the organoleptic quality of the wine and substances that may be hazardous to human health (such as ethyl carbamate and biogenic amines)

v Moment of MLF

Traditionally, the MLF occured after the alcoholic fermentation, usually in spring, when temperatures rose again. Problem was that in all that time between, the wine was very vulnerable to all kinds of bacteriological contaminants. Interim sulfite addition was not an option, because sulfite will inhibit the growth of LAB. With the progressive knowledge of winemaking, it is possible to have the MLF taken place right after, or even during the alcoholic fermentation by creating the right conditions or by using a starter culture. This is possible, because LAB can grow on malic acid while yeast grow on glucose. The purpose of this is primarily to neutralize the production of diacetyl. The yeasts will then reduce the buttery diacetyl to the less fragrant acetoin and the virtually odorless 2,3-butanediol.

               

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P.S.

In this post, we see two important USES of WINE CHEMISTRY:

1. WINE CHEMISTRY makes it easier to understand ‘why’ and ‘how’ malolactic fermentation occurs.

2. WINE CHEMISTRY makes it easier to see the differences between wine components: citric acid (with 3 acid groups –COOH) is tarter than malic acid (with 2 acid groups), with lactic acid (with 1 acid group) being the softest.

  

In next and last post, we’ll take a look at how sulfite (= sulphite) works.

Part 11 - Diacetyl, Acetoin & Butanediol

Part 11 – Diacetyl, Acetoin & Butanediol

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Diacetyl or 2.3-butanedione, (butane = 4xC, di =2, one = ketone groups C= 0, 2.3 = position of ‘one’, see molecular structure in the image underneath)  gives off a buttery smell

Acetoin  also gives off a buttery smell, but to a lesser extent

2.3-Butanediol (diol = 2 alcohol groups –OH) is virtually odorless.

1.  Produced by yeast from pyruvate in alcoholic fermentation

These compounds are produced by yeasts during alcoholic fermentation from two pyruvates or one pyruvate and one ethanal. The acetolactic acid is turned into diacetyl in one or more steps (see image underneath). Normally, the amount produced here is not enough to have a real impact on wine aroma. 

2. Produced by LAB from citric acid in malolactic fermentation

Considerable more diacetyl can be produced by lactic acid bacteria during malolactic fermentation from citric acid. Citric acid is first divided into acetic acid and oxaloacetic acid. The oxaloacetic acid is decarboxylated to pyruvate. The pyruvate binds with ethanal to become acetolactic acid (see image underneath). The acetolactic acid is turned to diacetyl in one or more steps ( see image above)

The production of diacetyl, and hence the strength of the buttery flavor, has to do with, among other things, the pH of the wine. The higher the pH (= the less acid the wine), the greater the increase of lactic acid bacteria and the higher the potential of diacetyl forming. It's one reason why a mature Chardonnay from Napa usually has a much stronger buttery flavor than a Chalis.

v ‘Malo’ during or right after alcoholic fermentation

Diacetyl and acetoin can displace the fruity aroma. That buttery smell can be limited by the malo to take place during or immediately after the alcoholic fermentation and allow the wine to ripen ‘on lees’ (‘sur lie’). The lees will then reduce the buttery diacetyl to the less fragrant acetoin and the virtually odorless 2,3-butanediol.

               

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P.S.

Next post, the malolactic fermentation, with 2  interesting questions :

What is the most significant difference between yeasts and bacteria?

Why do malolactic bacteria convert malic acid into lactic acid ?

Part 10 - Aldehyde and Acetaldehyde

Part 10 – Aldehyde and Acetaldehyde

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v  Aldehyde

Aldehyde is an organic compound containing a  –CH=O  group. A few examples of aldehydes:

  

v  Acetaldehyde (or Ethanal)

The main aldehyde in wine is acetaldehyde, also called ethanal (CH₃-COH). Ethanal is an intermediary of alcholic fermentation obtained by decarboxylation of pyruvate (see Part 1, step 11 or image below). Ethanal is mainly reduced to ethanol, but small quantities of it may be released into the wine. A high concentration of ethanal gives off a characteristic aroma that contributes to the perception that the wine is oxidized. Normally alcoholic fermentation produces only small amounts of ethanal (average 70 mg/L) that remains below its detection threshold (125 mg/L).    

                          

  

v  Chemical oxidation

However, in the presence of oxygen, more ethanal can be formed by ethanol oxidation. For this reason, ethanal is also defined as ‘oxidized alcohol’. In the bottle, the limited oxygen oxidizes the ethanol in the wine into ethanal. This happens very slowly. It is a non-enzymatic oxidation because no enzyme is involved. It is also called a chemical oxidation. In the long run, if additional oxygen can get into the bottle (e.g. the cork dries out), ethanal will be oxidized into acetic acid, making the wine smell like vinegar and completely out of balance. This explains why an open wine can not be kept long.

                               

  

v  Biological oxidation

Some wines, such as Fino and Manzanilla from Jerez, or wine Jaune from Jura, give off a particular aroma which is characterized by a high concentration of ethanal. These wines are obtained by aging the wine under aerobic conditions and under a film of flor yeasts. The flor-film protects the wine from oxidation. Since the ‘fermentation yeasts’ have consumed all the sugars, the ‘flor yeasts’ have to use the ethanol, glycerol and acetic acid in the wine and the oxygen in the air to grow. Inside the flor yeasts, the ethanol is oxidized into ethanal, catalysed by enzyme. It is a “biological oxidation”. Some of the ethanal escapes from the flor yeasts into the wine. It gives a characteristic aroma to the wine (see below the general scheme of Peinado and Mauricio). That’s what makes biological oxidation beneficial to these wines. However, in bottle, without the protection of the flor, these wines are subject to chemical oxidation as well.

                                              

v Hate and love relationship between oxygen and wine

Oxygen is not always the enemy of wine. Especially to red wine, oxygen is a friend. The polymerization (= clumping) between anthocyanin molecules and tannin molecules and the polymerization of the tannin molecules themselves needs oxygen.

The color of red wine is determined by the anthocyanin and tannin molecules.The action of oxygen during the ripening promotes this polymerization process, which takes place at tank and especially in the slightly air-permeable wooden barrels. Polymerization of tannin molecules softens the astringent taste. That explains why young tannic red wine needs aeration after opening.

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P.S.

Buttery smell was once the trademark of California Chardonnay. Responsible voor this is the 'diacetyl' . Next post we'll take a look at how it is formed.

Part 9 - Acetic acid

Part 9 – Acetic acid

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v Acetic acid (CH₃-COOH)  is the main volatile acid in wine. Its presence at high concentrations gives off a vinegar odor and a disagreeable sensation in the mouth. Acetic acid can be produced by <1> yeasts, <2> acetic acid bacteria and <3> lactic acid bacteria.

<1>  Yeasts produce acetic acid in 2 ways : by ethanol oxidation and by acetyl-coA hydrolysis. If there are no problems during alcoholic fermentation, yeasts produce only small quantities of acetic acid (200-300 mg/L), which is far below its detection threshold (800 mg/L).

            

<2>   Acetic acid bacteria (AAB) ,  genus Acetobacter aceti, are aerobic, which means that they need oxygen to grow. In the presence of oxygen, they convert ethanol into  acetic acid. In proper fermentation, the AA-bacterial growth is minimum because all the oxygen is consumed by yeasts. By stuck and sluggish fermentation, they get a chance to develop. They use ethanol for their energy source to grow. The ethanol is oxidized via alcohol dehygogenase (ADH) and aldehyde dehydrogenase (ALDH) into acetic acid which comes in the wine. The removed hydrogen protons (H⁺) are pumped into the periplasm and come back in the cytoplasm via ATPase to form ATP. The hydrogen electronen are transported by the ubiquinol oxidase (Ox)  and finally captured by oxygen (O) which combines with 2H⁺  and become H₂O  (see also Part 3 ETC).

<3>   Lactic acid bacteria (LAB)   found in wine belong to three genera: Leuconostoc, Lactobacillus and Pediococcus. They are anaerobic, which means they don’t need oxygen to grow. They grow on sugars and acids by converting them into lactic acid. That’s why they are called lactic acid bacteria. Like yeasts, LAB can also break down glucose in cytoplasm by glycolysis to produce 2 ATP. Yeasts reduce aldehyde to ethanol and LAB  reduce pyruvate to lactic acid. They both do that for the same purpose: to regenerate NAD+.

                              

 Like Acetic acid bacteria, LAB live in the must, but can not grow because of the powerful yeast growth. By stuck and sluggish fermentation, they get chance to develop, especially Lactobacillus and Pediococcus, which are regarded as “bad guys”. They change pyruvate into acetic acid.

          

v Acetic acid  & ethyl acetate

l Acetic acid (threshold 800 mg/L) smells like vinegar. A normal wine contains 200-300 mg/L acetic acid.

l Part of the acetic acid in wine can combine with ethanol to form esters, called ethyl acetate (see Part 7)

l  Ethyl acetate (threshold 150 mg/L) smells like glues and nail polish removers.

l  In wine ethyl acetate is always accompanied by acetic acid. At a value of 150-180 mg/L of ethyl acetate combined with 700-800 mg/L of acetic acid, there is a vinegar sting (piqûre acétique). This is a wine fault, and the cause is bacterial contamination.

l  The legally permitted maximum volatile acid content (e.g. acetic acid, lactic acid and ethyl acetate)  is 1080 mg/L in  

    white wines, 1200 mg/L in red wines and 1500 mg/L in botrytis wines.

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P.S.

In the next post, we'll take a look at the formation of acetaldehyde (or ethanal) and the difference between chemical and biological oxidation.