Ethanol fermentation
The alcoholic fermentation (syn. ethanol fermentation, ethanol fermentation) is a biochemical process in which carbohydrates are broken down, mainly glucose, under anoxic conditions to ethanol ( " drinking alcohol " ) and carbon dioxide. Most microorganisms (microbes ) with the ability to use this alcoholic fermentation pathway only temporarily for energy when the oxygen required for normal cellular respiration is missing.
- 6.1 drinks
- 6.2 Other Foods
- 6.3 industry
History of discovery
The man took the alcoholic fermentation, for example in the production of beer or wine making for thousands of years, without knowing the exact biological processes. In 1815 the French chemist Joseph Louis Gay -Lussac presented for the first time, the gross chemical equation for the breakdown of glucose to ethanol. After that, different views regarding the expiry of fermentation developed. While attributing a catalysing effect in the 1830s Jöns Jakob Berzelius and Justus von Liebig with the " mechanistic theory of fermentation " certain substances, could Charles Cagniard -Latour, Theodor Schwann and Friedrich Traugott Kützing prove independently that living beings, namely yeasts are responsible for it. Louis Pasteur also postulated in 1857 the " vitalistic theory of fermentation ", according to which the alcoholic fermentation is only possible in conjunction with living cells. This controversy was decided on January 11, 1897 by Eduard Buchner, with a publication on the proof of alcoholic fermentation by cell-free yeast extract. He made the material zymase - according to current knowledge, a mixture of different enzymes - for the conversion of sugar to ethanol responsible and received the 1907 Nobel Prize in Chemistry "for his biochemical research and his discovery of cell -free fermentation ". Detailed studies by Arthur Harden and William John Young led to the discovery of a phosphorylated intermediate: the Harden -Young ester, now known as fructose-1 ,6 -bisphosphate. Together received Harden and Hans von Euler - Chelpin in 1929 for their "Research on the fermentation of sugar and their proportion of the enzymes in this process " also received the Nobel Prize for Chemistry. After piece by piece elucidated the partial reactions and schemes have been designed for the process of fermentation, Otto Warburg identified the cofactor nicotinamide adenine dinucleotide (NADH ) as an essential component of the fermentation process. As early as 1937 succeeded Erwin Negelein and Hans Joachim Wulff crystallization of fermentation enzyme alcohol dehydrogenase.
Today involved in the fermentation enzymes from different species have been isolated and characterized biochemically ( pH optimum, temperature optimum, reaction rate, conversion rate ). The crystal structure analysis gave a first insight into their molecular spatial structure. It has insights into the reaction mechanisms. All in all, one is thus able to draw comparisons between species. The decrypted genes contain the blueprints for these enzymes, shed light on the evolutionary origin and their possible original function.
The role in the metabolism
The alcoholic fermentation is mainly used by different yeast species for energy production. If oxygen is available, they build from sugar through cellular respiration and gain the energy needed for life. The sugars are doing by a long series of enzymatic reactions ( glycolysis - respiratory chain oxidative decarboxylation - - Citric Acid Cycle ) oxidized by oxygen consumption completely to carbon dioxide and water. If no oxygen is available, the yeasts in alcoholic fermentation have an alternative way to generate energy. But they may therefore - in comparison to the complete oxidation by cell breathing - gain significantly less energy in the form of adenosine triphosphate ( ATP) from glucose: For complete oxidation 38 molecules of ATP are produced from one molecule of glucose, wherein the alcoholic fermentation only 2 molecules of ATP. These 2 ATP are produced in glycolysis, the first step in the reaction sequence of both the respiration and fermentation. The two additional reaction steps to fermentation and thus ultimately the production of ethanol do not serve energy generation but the regeneration of the cofactor NAD is consumed by the enzymes of glycolysis. Since NAD is available only in limited quantities, it is by the fermentation enzymes in the reduced state (NADH) by oxidation with acetaldehyde again in the oxidized state (NAD ) are added, thereby reducing the acetaldehyde to ethanol.
Yeasts are facultative anaerobes. If oxygen is available, glucose is metabolized aerobically. The absence of air yeasts, however, must operate the alcoholic fermentation. Since this is far less energy is generated as a by aerobic respiration, the need of glucose increases significantly. This phenomenon is called Pasteur effect. Due to the limited energy yeasts to multiply the absence of air far less than those of air. In addition, the ethanol formed acts as a cytotoxin.
It has also been observed ethanol production in yeast, although sufficient oxygen was present. This happens when they grow in a sugary medium and the enzymes of cellular respiration are overloaded. The yeasts are constantly taking on the sugar and use it in addition in addition to the cellular respiration through fermentation. It is to the Crabtree effect.
In addition to yeast species also some bacteria operate alcoholic fermentation. So Sarcina ulcer uses the same enzymatic pathway as yeast, Zymomonas mobilis during treading an alternative way. Likewise, low ethanol formation could be detected in a lack of oxygen in different plant.
Biochemical bases
Enzymatic reactions
The first step of fermentation are the glycolysis. In baker's yeast (S. cerevisiae) is the Embden - Meyerhof - Parnas pathway, while Zymomonas mobilis, a bacterium, the Entner- Doudoroff pathway used. In this case, one molecule of D-glucose is converted to two molecules of pyruvate. S. cerevisiae This produces two molecules of adenosine triphosphate ( ATP) from two molecules of adenosine diphosphate (ADP ) and two phosphate groups (P ) through substrate chain. In Z. mobilis only one molecule of ATP is formed. In addition, two molecules of NAD ( nicotinamide adenine dinucleotide ) are reduced to two molecules of NADH in two ways.
Thus, glycolysis can run again, NAD must be regenerated. This is done under anaerobic conditions now in the following fermentation reaction. From each molecule of pyruvate, a molecule of carbon dioxide by the enzyme pyruvate decarboxylase ( EC 4.1.1.1 ) is cleaved. Cofactors used in this reaction, thiamine pyrophosphate, a relative of vitamin B1, and two magnesium ions. The pyruvate decarboxylase should not be confused with the pyruvate dehydrogenase E1 ( EC 1.2.4.1 ) of the pyruvate dehydrogenase complex, which plays a central role in the aerobic degradation of pyruvate.
The heat generated in this step, acetaldehyde is highly toxic to the organism and is further reacted immediately in the following step. The catalyzing enzyme alcohol dehydrogenase ( EC 1.1.1.1 ) contains a zinc ion ( Zn2 ), which polarizes the carbonyl group on acetaldehyde. Thereby can be transmitted to the acetaldehyde two electrons and a proton from the NADH, thus reduced to ethanol and NAD is regenerated. Both glycolysis and the two subsequent reactions take place in the cell cytoplasm.
The net reaction equation is for baker's yeast as follows:
Glucose, 2 adenosine diphosphate and 2 phosphate react to form 2 ethanol, carbon dioxide 2, 2 adenosine triphosphate and 2 water
The enzyme alcohol is ethanol by reduction of acetaldehyde ago, but also catalyzes the reverse reaction. During the alcoholic fermentation runs for the most part from the reduction of acetaldehyde to ethanol. The ethanol formed is then discharged from the cells to the environment.
The oxidation of ethanol to acetaldehyde, for example, on the other hand takes place in the detoxification of ethanol in the liver. Acetaldehyde is toxic and fusel oils in addition to the main cause of headache and nausea after heavy alcohol consumption (the famous "hangover" ). Acetaldehyde is oxidized to acetic acid by the enzyme acetaldehyde dehydrogenase.
When the alcoholic fermentation by yeasts formed as unwanted by-products of methanol and accompanying alcohols such as butanol, amyl alcohol and hexanol. But your education does not proceed on the pathway described here, for instance through the breakdown of amino acids. In the body, methanol is oxidized to the highly toxic formaldehyde by the enzyme alcohol dehydrogenase. If you drink a lot of alcohol inferior (with high methanol content ), there arises in the body corresponding amount of formaldehyde, which then proteins, such as the highly sensitive sensors in the eye, damaging and at worst, muscle cramps, blindness and can eventually lead to death.
Regulation
The regulation, so switching between aerobic cellular respiration and anaerobic fermentation, is a current research topic. One can ' tilt switch when there is a lack of X' is no general regulation scheme according to the system set up. There is even variation between individual yeast strains, let alone of plants and bacteria. However, researchers are beginning to unravel the mystery. A major role of the oxygen content and the glucose levels.
There are also, for example, in S. cerevisiae, two genes for cytosolic enzyme alcohol dehydrogenase and characterized two slightly different enzymes, ADH1 and ADH2. Both enzymes can convert acetaldehyde into ethanol and vice versa. By small differences in its molecular structure, this is done at a different speed. ADH1 can quickly build ethanol, while ADH2 quickly degrades ethanol. The presence of the enzyme is regulated by transcription factors that control the reading of the respective gene. ADH1 for ethanol structure is always present. If the glucose levels decrease drastically, so the enzyme ADH2 is produced, can break down the ethanol for energy (if oxygen is present ) and thus the yeast alive. Yeast can thus build ethanol if enough sugar is present and even reduce them, ethanol later when urgently needed energy. Evolutionary seen it thus has an advantage: they poisoned all competitors for food with ethanol and then processed these self again. The emergence of the two genes for ADH1 and ADH2 is probably due to gene duplication of a common origin gene. In other species, there are, more than two alcohol dehydrogenases.
Energy balance
Since under anoxic conditions, the cellular respiration does not work with respiratory chain of ADP to ATP, the only energy source for yeast under these conditions, glycolysis with production of ATP by substrate phosphorylation. It provides per molecule of glucose 2 ATP molecules. In comparison, cellular respiration would produce 38 molecules of ATP. Would stop the degradation of glucose when pyruvate, the process would soon ground to a halt because a NAD deficiency would occur through the consumption of NAD in glycolysis. NAD is in the cell only in trace amounts and has to be regenerated continuously. For this is decarboxylated in the alcoholic fermentation, pyruvate and reduced period, the resulting acetaldehyde with NADH to ethanol to NADH is oxidized to NAD . Taking the entire reaction sequence from glucose to ethanol, so there is no high-energy NADH. Looking at the carbon, but then changed its oxidation number from 0 ( in glucose) once to 4 ( carbon dioxide ) and twice to -2 ( ethanol). So that the alcoholic fermentation is a disproportionation, a particular case of the redox reactions.
The change in free energy under standard conditions, but pH 7 instead of 0 for the alcoholic fermentation ΔG0 = ' - 218 kJ per mole of glucose in cell respiration - 2822 kJ per mole of glucose. As standard conditions were agreed upon: temperature 25 ° C, pressure 1.013 bar, concentration of the substances involved in the reaction ( reactants) 1 mol / L has been agreed with the exception of water, for the 55.6 mol / L (pure water), and which is agreed by gases, for a concentration in solution equilibrium with a partial pressure of 1 bar in the gas phase. In biological systems, however, not of living things not tolerated concentration of 1 mol / L corresponding to pH 0, but 10-7 mol / L is agreed according to pH 7 for the H ion concentration. If the actual conditions of these standard conditions, so too is the amount of free energy change someone else, he can deviate significantly from the standard value. In living systems, standard conditions are not usually given and often change during the material implementation. The amount of change in the free energy under standard conditions in animals thus provides only an indication of the energy released during a chemical substance implementation.
Other substrates
In addition to glucose and other simple sugars, may be processed by the glycolysis, and thus also through the alcoholic fermentation. However, most yeasts have a special affinity for glucose ( they are " glucophil " ), so that, for example, in the alcoholic fermentation of grape must which contains glucose and fructose in equal parts, preferably the glucose is broken down. Is the finished wine then restsüß, that is, has not been degraded to any sugar alcohol, is the majority of the residual sugar of fructose. This is especially important for diabetics is of interest.
D-fructose can be phosphorylated, such as glucose, and thus introduced into the glycolysis to one of a hexokinase, the first enzyme of the glycolysis, as well. An alternative route to the fructose is converted by the enzyme Fructosekinase to fructose -1-phosphate, which is further degraded by the fructose-1 - phosphate aldolase to dihydroxyacetone phosphate. This will in turn directly in glycolysis use.
D-galactose can be converted via the intermediates of galactose -1-phosphate and UDP -galactose to glucose, the flow as used in the glycolysis.
In addition to simple sugars and disaccharides can be processed, if enzymes are present, they split into its components. So sucrose is broken down by the invertase into its components glucose and fructose, which enter into the glycolysis as previously described. The same procedure is lactose, which is cleaved by the enzyme β -galactosidase into galactose and glucose. The same is true of polysaccharides. To use as starch from grains, the seeds were germinated. The plant's own enzyme amylase breaks down starch into maltose, which in turn can be processed by the yeast.
Alternative Way
The bacterium Zymomonas mobilis is also to be capable of producing, from glucose ethanol. However, it uses only a portion of the pathway described above. Instead of glycolysis, glucose is broken down here by the Entner- Doudoroff pathway to pyruvate and glyceraldehyde -3 -phosphate. Glyceraldehyde -3-phosphate can be introduced into glycolysis and also degraded to pyruvate. The last two steps are similar to those of alcoholic fermentation in yeast. Thus may be obtained from a molecule of glucose, only one molecule of ATP. The fermentation runs, but in this way more quickly than on the yeasts used and a higher yield. Z. mobilis is used for the production of pulque from agave juice.
Fermentation by-products
Fermentation by-products or alcoholic impurities arising addition to ethanol and carbon dioxide during alcoholic fermentation. Some of these by-products are referred to as fusel oils.
They can be found even in the fermentation of pure glucose solution. When brewing beer the taste difference between the wort and green beer or beer indicates that fermentation by-products are formed. For example, they include higher alcohols such as n-propanol, i- butanol, 2- methylbutanol - 1, 3 -methylbutanol -1, and aromatic alcohols such as 2 -phenylethanol, tyrosol or tryptophol. Besides occur esters such as ethyl acetate, phenyl acetate and i- amyl acetate. Also carbonyl compounds such as aldehydes, such as acetaldehyde, propanal, butanal or furfural as well as ketones and diketones.
Sulfur compounds such as H2S, SO2, ethyl mercaptan and methyl mercaptan, occur in small amounts.
Furthermore, organic acids such as acetic acid, lactic acid, pyruvic acid, 2 -acetolactate and fatty acids are found (C4 - C12)
Also polyhydric alcohols such as glycerol, 2,3- butanediol and 2,3- pentanediol occur as fermentation by-products.
The listed materials are the most important representatives of each group.
Natural occurrence
Everywhere in Nature finds microorganisms. So even fruit is coated with bacteria and yeast, which can not be completely removed by simple washing. If fruit after harvest longer in a warm environment, these microorganisms multiply. They break down cellular structures and also penetrate into the interior of the populated fruit. It is the example as a soft spot or brown spot right on an apple. During this decomposition process, it may at times, especially coming inside the fruit to oxygen deficiency. The working there yeast then make their metabolism to alcoholic fermentation. So it is possible that spoiling fruits contain alcohol.
Until the 20th century, no additional yeasts were added in the winemaking process. By the middle of the 19th century was not even known that there are yeasts that produce alcohol. You just used the natural event that sugary liquids started after some time, to form alcohol. In wine making, which is partly still left to nature. After the pressing of grapes to mash the originally seated on the skins yeasts distribute in the liquid and begin with the spontaneous fermentation. Without the addition of yeast that lasts a little longer, since the concentration of the natural yeast is initially very low, but the wine receives thereby a more personalized touch. The yeast strains differ depending on the growing region of the grapes, which means that you can assign to wine produced in this way taste their zone. Frequently occurring yeasts are Kloeckera apiculata and Saccharomyces exiguus. Who rely on natural yeasts winemakers, however, he runs the risk that other living on the grape skins yeasts and bacteria out of hand during the manufacturing process and the slurry spoil. For a long time because of different yeast strains are bred to produce its typical aroma of each wine. These yeasts only consist of one yeast strain and often specialize in a grape variety. Since you can use the freeze-drying for the preservation of yeasts, such yeasts many months are commercially readily in large quantities, durable and easy to handle. If they are right at the beginning of fermentation, so the alcohol level rises rapidly and the harmful microorganisms die off.
Use by humans
Drinks
There are a vast number of alcoholic beverages whose alcohol content always goes back to alcoholic fermentation. Requirement is a sugary feedstock.
Other foods
One of the most important applications is the bakery. Baker's yeast (S. cerevisiae) is almost all bread and rolls as well as used in the manufacture of traditional cake with yeast dough for Teigauflockerung. While the dough is "going", produced by alcoholic fermentation, the gas carbon dioxide, which is finely dispersed in the dough and can increase its volume considerably. The resulting ethanol is the subsequent baking process, the yeast dies off due to the high temperatures at its beginning, evaporated.
An alcohol- containing food is made from milk kefir. For its preparation are Kefirknollen, a mixture of different types of living symbiotic yeasts and bacteria. The lactose contained in milk is broken down by the bacteria to lactic acid fermentation to lactic acid and alcoholic fermentation of the yeasts over to ethanol. Kefir has an alcohol content of less than one percent. In Asian steppe peoples traditionally Kumys, fermented mare's milk, drinking.
Industry
In times of scarcity of raw materials and in the foreseeable exhaustion of oil reserves wins ethanol as a vehicle fuel increasingly important. In addition, it is used in many industrial processes, is the starting material for chemical syntheses or used for disinfection. This ethanol is also produced by alcoholic fermentation of yeast. Raw materials are here cheap grain or potatoes, whose strength is cleaved only by large- engineered enzymes to sugar. Again, by fermentation not exceed a maximum alcohol content of 23 %. Subsequent distillation column provides a content of 96 % ( azeotrope ). Since alcohol produced would be enjoyable in this way, he is also subject to the tax on spirits in Germany. An exception is the ethanol used for fuel production, subject to stringent regulatory controls. Also no liquor tax must be paid on denatured ethanol. It is a small extent of methyl ethyl ketone and other substances to which make it unfit for human consumption and eliminates the enjoyment.