research on alcohol fermentation

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Alcohol Fermentation

Melissa martinez, introduction.

Alcoholic fermentation or ethanol fermentation is a biotechnological process in which sugars such as glucose, sucrose, and fructose are converted into ethyl alcohol and carbon dioxide in the presence of yeast ( S. cerevisiae) , some bacteria, or other microorganisms. 

Alcoholic fermentation is a type of fermentation process widely used in producing alcoholic beverages such as beer and wine. Fermentation is a metabolic process where enzymes or microorganisms induce the decomposition of organic substances. This metabolic process makes beneficial changes in the food and beverages that maintain a healthy gut and increase the shelf life of food. For instance, it makes the food more flavorful and nutritious and increases the shelf life. 

Ethanol fermentation is a complicated process that involves various chemical, biochemical, and physicochemical processes. First, the sugar breaks down to form pyruvic acid, which is then converted into ethanol and carbon dioxide. The regeneration of NAD+ provides the yeast with energy to convert pyruvate molecules into ethanol and CO2. Typically, yeast has the capability to function in the presence and absence of oxygen. However, alcoholic fermentation occurs in the absence of oxygen (anaerobic condition). Under anaerobic conditions, the fermentation takes place in the cytosol of yeast. (Lee, 1983)

The basic principle of alcohol fermentation is that it is carried out by living yeast cells under anaerobic conditions. These cells absorb sugar molecules and break them in the presence of oxidation and reduction enzymes; as a result, by-products such as ethanol, carbon dioxide, water, and heat are produced. 

Alcoholic fermentation takes place in two steps, i.e., glycolysis and fermentation. Glycolysis involves breaking down sugar to form pyruvate molecules in the presence of yeast. In this step, 2 pyruvic acid molecules are produced. It is followed by fermentation in which the 2 pyruvate molecules are converted into 2 ethanol molecules, 2CO 2 , and ATP. In the absence of oxygen, the pyruvate molecule is first transformed into acetaldehyde and CO2 in the presence of the pyruvate decarboxylase enzyme. At the same time, NADH regenerates NAD+ bypassing its electrons to acetaldehyde in the presence of alcohol dehydrogenase enzyme, and as a result, ethanol is formed. ( Walker & Walker, 2018 )

The overall chemical equation can be explained as follows:

C6H12O6 → 2 C2H5OH + 2 CO2

• Erlenmeyer flask 
• Delivery tube 
• Glass tube 
• Clamp stand
• Stirring rods
• Glucose powder
• Yeast powder
• Paraffin oil

– Prepare 5% glucose solutions by mixing 5 g of glucose powder in 10ml of lukewarm water in a flask. 

– Prepare that 10% yeast suspension in another flask by adding 5g of yeast in water. 

– Take the prepared glucose solution in a larger test tube and add the yeast suspension in a 5:1 ratio. 

– Now add liquid paraffin drops along with the inner side of the test tube so that it covers the surface of the glucose-yeast solution completely. 

– Cover the test tube with the cork containing the delivery tube. Ensure that the delivery tube’s end does not touch the soil surface. 

– Seal the cork with glycerol 

– Set the larger test tube properly on the clamp stand and set a smaller test tube containing lime water on the other end of the delivery tube. 

– Make sure that the tube is immersed well in the lime water.

– Allow this setup to stand for a few minutes until the air bubbles start to come out of the end of the delivery tube and the lime water turns milky. 

– At the same time, a frothy layer will form on top of the oil layer in the larger test tube. 

– Disassemble the apparatus as fermentation has occurred. 

• The contents in the larger test tube will give a strong ethanol smell, which suggests that alcohol has been produced due to fermentation. 
• The cloudiness of lime water indicates that the gas produced inside the test tube is carbon dioxide, a by-product of fermentation.

Applications

Alcoholic fermentation is used in industries to produce alcoholic beverages, bread, and vinegar. For instance, wine is synthesized by fermentation of natural sugars found in grapes. Similarly, rums are produced by fermenting sugar cane product molasses followed by distillation.

( Boeira et al., 2021 ) conducted a study to mitigate nivalenol using alcoholic fermentation and magnetic field application. The conditions set for this study were nivalenol (0.2 µg mL-1), magnetic field application (35 mT) along with simultaneous use of mycotoxin. The results showed that glutathione and enzyme peroxidase level was significantly increased during the experiment, and nivalenol was mitigated by 56.6%. 

Soursop fruit is highly nutritious yet perishable, so ( Ho et al., 2019 ) conducted a study to produce soursop wine through alcoholic fermentation. Two cultures, i.e., mushroom ( Pleurotus pulmonarius ) and yeast ( Saccharomyces cerevisiae ), were used together to determine fermentation effects on physiochemical and antioxidant activities of soursop wine. Temperature, pH, time, and culture ratio were optimized to maximize ethanol production. This alternative fermentation technique showed increased ethanol production with higher antioxidant activities. 

Strengths and limitations

Alcoholic fermentation is a simpler process that is carried out from renewable resources. It does not require high amounts of energy for fermentation, due to which this is a low-cost process. The average temperature required for fermentation is between 35 to 40°C. 

Limitations 

The major drawback of this alcoholic fermentation is that it slows down towards the end because of the increased concentration of alcohol in the medium, which is toxic to yeast. So, fermentation ceases even before the sugar is metabolized completely. This incomplete process has a high risk of bacterial spoilage. 

The final product produced is impure, and it requires more steps to obtain the purified alcohol. Moreover, it is a batch process that requires a large amount of time to produce sufficient alcohol. 

Precautions

• Ensure to clean all the apparatus properly to avoid any bacterial contamination. 
• Wear insulated gloves while handling hot materials
• Use latex gloves while handling chemicals and samples
• Wear proper clothes (long pants and closed-toe shoes), a lab coat, and safety glasses

– Alcoholic fermentation is a process in which glucose is converted into alcohol and carbon dioxide in the presence of yeast or other microorganisms. 

– It is a batch process that involves two steps. 

– In the first step, the glucose breakdown to form pyruvate molecules by glycolysis. In the next step, the pyruvate molecule gets converted into ethanol and carbon dioxide in the presence of NADH. 

– Fermented foods contain beneficial microorganisms and probiotics that maintain a healthy gut and increase the shelf life of food.

– Alcoholic fermentation is widely used in industries for making bread, alcohol, and other products.

• Lee, F. A. (1983). Alcoholic Fermentation.  Basic Food Chemistry , 323–341. https://doi.org/10.1007/978-94-011-7376-6_14
• Walker, G. M., & Walker, R. S. K. (2018). Enhancing Yeast Alcoholic Fermentations.  Advances in Applied Microbiology , 87–129. https://doi.org/10.1016/bs.aambs.2018.05.003
• ‌Boeira, C. Z., Silvello, M. A. de C., Remedi, R. D., Feltrin, A. C. P., Santos, L. O., & Garda Buffon, J. (2021). Mitigation of nivalenol using alcoholic fermentation and magnetic field application .  Food Chemistry ,  340 , 127935. https://doi.org/10.1016/j.foodchem.2020.127935
• Ho, C. W., Lazim, A., Fazry, S., Hussain Zaki, U. K. H., Massa, S., & Lim, S. J. (2019). Alcoholic fermentation of soursop (  Annona muricata  ) juice via an alternative fermentation technique .  Journal of the Science of Food and Agriculture ,  100 (3), 1012–1021. https://doi.org/10.1002/jsfa.10103

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Yeast Fermentation and the Making of Beer and Wine

Once upon a time, many, many years ago, a man found a closed fruit jar containing a honeybee. When he drank the contents, he tasted a new, strange flavor. Suddenly his head was spinning, he laughed for no reason, and he felt powerful. He drank all the liquid in the jar. The next day he experienced an awful feeling. He had a headache, pain , an unpleasant taste in his mouth, and dizziness — he had just discovered the hangover. You might think this is just a tale, but is it? Several archaeological excavations have discovered jars containing the remains of wine that are 7,000 years old (McGovern, 2009), and it is very likely that humankind's first encounter with alcoholic beverages was by chance. How did this chance discovery lead to the development of the beer and wine industry (Figure 1), and how did scientists eventually learn about the biological mechanisms of alcohol production?

The History of Beer and Wine Production

Over the course of human history, and using a system of trial, error, and careful observation, different cultures began producing fermented beverages. Mead, or honey wine, was produced in Asia during the Vedic period (around 1700–1100 BC), and the Greeks, Celts, Saxons, and Vikings also produced this beverage. In Egypt, Babylon, Rome, and China, people produced wine from grapes and beer from malted barley. In South America, people produced chicha from grains or fruits, mainly maize; while in North America, people made octli (now known as "pulque") from agave, a type of cactus (Godoy et al. 2003).

At the time, people knew that leaving fruits and grains in covered containers for a long time produced wine and beer, but no one fully understood why the recipe worked. The process was named fermentation, from the Latin word fervere , which means "to boil." The name came from the observation that mixtures of crushed grapes kept in large vessels produced bubbles, as though they were boiling. Producing fermented beverages was tricky. If the mixture did not stand long enough, the product contained no alcohol; but if left for too long, the mixture rotted and was undrinkable. Through empirical observation, people learned that temperature and air exposure are key to the fermentation process.

Wine producers traditionally used their feet to soften and grind the grapes before leaving the mixture to stand in buckets. In so doing, they transferred microorganisms from their feet into the mixture. At the time, no one knew that the alcohol produced during fermentation was produced because of one of these microorganisms — a tiny, one-celled eukaryotic fungus that is invisible to the naked eye: yeast . It took several hundred years before quality lenses and microscopes revolutionized science and allowed researchers to observe these microorganisms.

Yeast and Fermentation

Figure 1: Fermented beverages such as wine have been produced by different human cultures for centuries. Christian Draghici/Shutterstock. All rights reserved. In the seventeenth century, a Dutch tradesman named Antoni van Leeuwenhoek developed high-quality lenses and was able to observe yeast for the first time. In his spare time Leeuwenhoek used his lenses to observe and record detailed drawings of everything he could, including very tiny objects, like protozoa, bacteria , and yeast. Leeuwenhoek discovered that yeast consist of globules floating in a fluid, but he thought they were merely the starchy particles of the grain from which the wort (liquid obtained from the brewing of whiskey and beer) was made (Huxley 1894). Later, in 1755, yeast were defined in the Dictionary of the English Language by Samuel Johnson as "the ferment put into drink to make it work; and into bread to lighten and swell it." At the time, nobody believed that yeast were alive; they were seen as just organic chemical agents required for fermentation.

In the eighteenth and nineteenth centuries, chemists worked hard to decipher the nature of alcoholic fermentation through analytical chemistry and chemical nomenclature. In 1789, the French chemist Antoine Lavoisier was working on basic theoretical questions about the transformations of substances. In his quest, he decided to use sugars for his experiments, and he gained new knowledge about their structures and chemical reactions. Using quantitative studies, he learned that sugars are composed of a mixture of hydrogen, charcoal (carbon), and oxygen.

Lavoisier was also interested in analyzing the mechanism by which sugarcane is transformed into alcohol and carbon dioxide during fermentation. He estimated the proportions of sugars and water at the beginning of the chemical reaction and compared them with the alcohol and carbon dioxide proportions obtained at the end. For the alcoholic reaction to proceed, he also added yeast paste (or "ferment," as it was called). He concluded that sugars were broken down through two chemical pathways: Two-thirds of the sugars were reduced to form alcohol, and the other third were oxidized to form carbon dioxide (the source of the bubbles observed during fermentation). Lavoisier predicted (according to his famous conservation-of-mass principle) that if it was possible to combine alcohol and carbon dioxide in the right proportions, the resulting product would be sugar. The experiment provided a clear insight into the basic chemical reactions needed to produce alcohol. However, there was one problem: Where did the yeast fit into the reaction? The chemists hypothesized that the yeast initiated alcoholic fermentation but did not take part in the reaction. They assumed that the yeast remained unchanged throughout the chemical reactions.

Yeast Are Microorganisms

In 1815 the French chemist Joseph-Louis Gay-Lussac made some interesting observations about yeast. Gay-Lussac was experimenting with a method developed by Nicolas Appert, a confectioner and cooker, for preventing perishable food from rotting. Gay-Lussac was interested in using the method to maintain grape juice wort in an unfermented state for an indefinite time. The method consisted of boiling the wort in a vessel, and then tightly closing the vessel containing the boiling fluid to avoid exposure to air. With this method, the grape juice remained unfermented for long periods as long as the vessel was kept closed. However, if yeast (ferment) was introduced into the wort after the liquid cooled, the wort would begin to ferment. There was now no doubt that yeast were indispensable for alcoholic fermentation. But what role did they play in the process?

When more powerful microscopes were developed, the nature of yeast came to be better understood. In 1835, Charles Cagniard de la Tour, a French inventor, observed that during alcoholic fermentation yeast multiply by gemmation (budding). His observation confirmed that yeast are one-celled organisms and suggested that they were closely related to the fermentation process. Around the same time, Theodor Schwann, Friedrich Kützing, and Christian Erxleben independently concluded that "the globular, or oval, corpuscles which float so thickly in the yeast [ferment] as to make it muddy" were living organisms (Barnett 1998). The recognition that yeast are living entities and not merely organic residues changed the prevailing idea that fermentation was only a chemical process. This discovery paved the way to understand the role of yeast in fermentation.

Pasteur Demonstrates the Role of Yeast in Fermentation

Pasteur performed careful experiments and demonstrated that the end products of alcoholic fermentation are more numerous and complex than those initially reported by Lavoisier. Along with alcohol and carbon dioxide, there were also significant amounts of glycerin, succinic acid, and amylic alcohol (some of these molecules were optical isomers — a characteristic of many important molecules required for life). These observations suggested that fermentation was an organic process. To confirm his hypothesis, Pasteur reproduced fermentation under experimental conditions, and his results showed that fermentation and yeast multiplication occur in parallel. He realized that fermentation is a consequence of the yeast multiplication, and the yeast have to be alive for alcohol to be produced. Pasteur published his seminal results in a preliminary paper in 1857 and in a final version in 1860, which was titled "Mémoire sur la fermentation alcoolique" (Pasteur 1857).

In 1856, a man named Bigo sought Pasteur's help because he was having problems at his distillery, which produced alcohol from sugar beetroot fermentation. The contents of his fermentation containers were embittered, and instead of alcohol he was obtaining a substance similar to sour milk. Pasteur analyzed the chemical contents of the sour substance and found that it contained a substantial amount of lactic acid instead of alcohol. When he compared the sediments from different containers under the microscope, he noticed that large amounts of yeast were visible in samples from the containers in which alcoholic fermentation had occurred. In contrast, in the polluted containers, the ones containing lactic acid, he observed "much smaller cells than the yeast." Pasteur's finding showed that there are two types of fermentation: alcoholic and lactic acid. Alcoholic fermentation occurs by the action of yeast; lactic acid fermentation, by the action of bacteria.

Isolating the Cell's Chemical Machinery

By the end of the nineteenth century, Eduard Buchner had shown that fermentation could occur in yeast extracts free of cells, making it possible to study fermentation biochemistry in vitro . He prepared cell-free extracts by carefully grinding yeast cells with a pestle and mortar. The resulting moist mixture was put through a press to obtain a "juice" to which sugar was added. Using a microscope, Buchner confirmed that there were no living yeast cells in the extract.

Upon studying the cell-free extracts, Buchner detected zymase, the active constituent of the extracts that carries out fermentation. He realized that the chemical reactions responsible for fermentation were occurring inside the yeast. Today researchers know that zymase is a collection of enzymes (proteins that promote chemical reactions). Enzymes are part of the cellular machinery, and all of the chemical reactions that occur inside cells are catalyzed and modulated by enzymes. For his discoveries, Buchner was awarded the Nobel Prize in Chemistry in 1907 (Barnett 2000; Barnett & Lichtenthaler 2001; Encyclopaedia Britannica 2010).

Around 1929, Karl Lohmann, Yellapragada Subbarao, and Cirus Friske independently discovered an essential molecule called adenosine triphosphate ( ATP ) in animal tissues. ATP is a versatile molecule used by enzymes and other proteins in many cellular processes. It is required for many chemical reactions, such as sugar degradation and fermentation (Voet & Voet 2004). In 1941, Fritz Albert Lipmann proposed that ATP was the main energy transfer molecule in the cell.

Sugar Decomposition

Glycolysis — the metabolic pathway that converts glucose (a type of sugar) into pyruvate — is the first major step of fermentation or respiration in cells. It is an ancient metabolic pathway that probably developed about 3.5 billion years ago, when no oxygen was available in the environment . Glycolysis occurs not only in microorganisms, but in every living cell (Nelson & Cox 2008).

Because of its importance, glycolysis was the first metabolic pathway resolved by biochemists. The scientists studying glycolysis faced an enormous challenge as they figured out how many chemical reactions were involved, and the order in which these reactions took place. In glycolysis, a single molecule of glucose (with six carbon atoms) is transformed into two molecules of pyruvic acid (each with three carbon atoms).

In order to understand glycolysis, scientists began by analyzing and purifying the labile component of cell-free extracts, which Buchner called zymase. They also detected a low-molecular-weight, heat-stable molecule, later called cozymase. Using chemical analyses, they learned that zymase is a complex of several enzymes; and cozymase is a mixture of ATP, ADP (adenosine diphosphate, a hydrolyzed form of ATP), metals, and coenzymes (substances that combine with proteins to make them functional), such as NAD + (nicotinamide adenine dinucleotide). Both components were required for fermentation to occur.

The complete glycolytic pathway, which involves a sequence of ten chemical reactions, was elucidated around 1940. In glycolysis, two molecules of ATP are produced for each broken molecule of glucose. During glycolysis, two reduction-oxidation (redox) reactions occur. In a redox reaction, one molecule is oxidized by losing electrons, while the other molecule is reduced by gaining those electrons. A molecule called NADH acts as the electron carrier in glycolysis, and this molecule must be reconstituted to ensure continuity of the glycolysis pathway.

The Chemical Process of Fermentation

In the absence of oxygen (anoxygenic conditions), pyruvic acid can follow two different routes, depending on the type of cell . It can be converted into ethanol (alcohol) and carbon dioxide through the alcoholic fermentation pathway, or it can be converted into lactate through the lactic acid fermentation pathway (Figure 3).

Since Pasteur's work, several types of microorganisms (including yeast and some bacteria) have been used to break down pyruvic acid to produce ethanol in beer brewing and wine making. The other by-product of fermentation, carbon dioxide, is used in bread making and the production of carbonated beverages. Other living organisms (such as humans) metabolize pyruvic acid into lactate because they lack the enzymes needed for alcohol production, and in mammals lactate is recycled into glucose by the liver (Voet & Voet 2004).

Selecting Yeast in Beer Brewing and Wine Making

Humankind has benefited from fermentation products, but from the yeast's point of view, alcohol and carbon dioxide are just waste products. As yeast continues to grow and metabolize sugar, the accumulation of alcohol becomes toxic and eventually kills the cells (Gray 1941). Most yeast strains can tolerate an alcohol concentration of 10–15% before being killed. This is why the percentage of alcohol in wines and beers is typically in this concentration range. However, like humans, different strains of yeast can tolerate different amounts of alcohol. Therefore, brewers and wine makers can select different strains of yeast to produce different alcohol contents in their fermented beverages, which range from 5 percent to 21 percent of alcohol by volume. For beverages with higher concentrations of alcohol (like liquors), the fermented products must be distilled.

Today, beer brewing and wine making are huge, enormously profitable agricultural industries. These industries developed from ancient and empirical knowledge from many different cultures around the world. Today this ancient knowledge has been combined with basic scientific knowledge and applied toward modern production processes. These industries are the result of the laborious work of hundreds of scientists who were curious about how things work.

References and Recommended Reading

Barnett, J. A. A history of research on yeast 1: Work by chemists and biologists, 1789–1850. Yeast 14 , 1439–1451 (1998)

Barnett, J. A. A history of research on yeast 2: Louis Pasteur and his contemporaries, 1850–1880. Yeast 16 , 755–771 (2000)

Barnett, J. A. & Lichtenthaler, F. W. A history of research on yeast 3: Emil Fischer, Eduard Buchner and their contemporaries, 1880–1900. Yeast 18 , 363–388 (2001)

Encyclopaedia Britannica's Guide to the Nobel Prizes (2010)

Godoy, A., Herrera, T. & Ulloa, M. Más allá del pulque y el tepache: Las bebidas alcohólicas no destiladas indígenas de México. Mexico: UNAM, Instituto de Investigaciones Antropológicas, 2003

Gray, W. D. Studies on the alcohol tolerance of yeasts . Journal of Bacteriology 42 , 561–574 (1941)

Huxley, T. H. Popular Lectures and Addresses II . Chapter IV, Yeast (1871). Macmillan, 1894

Jacobs, J. Ethanol from sugar: What are the prospects for US sugar crops? Rural Cooperatives 73 (5) (2006)

McGovern, P. E. Uncorking the Past: The Quest for Wine, Beer, and Other Alcoholic Beverages. Berkeley: University of California Press, 2009

Nelson, D. L. & Cox, M. M. Lehninger Principles of Biochemistry , 5th ed. New York: Freeman, 2008

Pasteur, L. Mémoire sur la fermentation alcoolique .Comptes Rendus Séances de l'Academie des Sciences 45 , 913–916, 1032–1036 (1857)

Pasteur, L. Studies on Fermentation . London: Macmillan, 1876

Voet, D. & Voet, J. Biochemistry. Vol. 1, Biomolecules, Mechanisms of Enzyme Action, and Metabolism , 3rd ed. New York: Wiley, 2004

Classic papers:

Meyerhof, O. & Junowicz-Kocholaty, R. The equilibria of isomerase and aldolase, and the problem of the phosphorylation of glyceraldehyde phosphate . Journal of Biological Chemistry 149 , 71–92 (1943)

Meyerhof, O. The origin of the reaction of harden and young in cell-free alcoholic fermentation . Journal of Biological Chemistry 157 , 105–120 (1945)

Meyerhof, O. & Oesper, P. The mechanism of the oxidative reaction in fermentation . Journal of Biological Chemistry 170 , 1–22 (1947)

Pasteur, L. Mèmoire sur la fermentation appeleé lactique . Annales de Chimie et de Physique 3e. sér. 52 , 404–418 (1858)

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Why, when, and how did yeast evolve alcoholic fermentation?

Sofia dashko.

1 Wine Research Centre, University of Nova Gorica, Vipava, Slovenia

2 Department of Biology, Lund University, Lund, Sweden

Concetta Compagno

3 Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Milan, Italy

Jure Piškur

The origin of modern fruits brought to microbial communities an abundant source of rich food based on simple sugars. Yeasts, especially Saccharomyces cerevisiae , usually become the predominant group in these niches. One of the most prominent and unique features and likely a winning trait of these yeasts is their ability to rapidly convert sugars to ethanol at both anaerobic and aerobic conditions. Why, when, and how did yeasts remodel their carbon metabolism to be able to accumulate ethanol under aerobic conditions and at the expense of decreasing biomass production? We hereby review the recent data on the carbon metabolism in Saccharomycetaceae species and attempt to reconstruct the ancient environment, which could promote the evolution of alcoholic fermentation. We speculate that the first step toward the so-called fermentative lifestyle was the exploration of anaerobic niches resulting in an increased metabolic capacity to degrade sugar to ethanol. The strengthened glycolytic flow had in parallel a beneficial effect on the microbial competition outcome and later evolved as a “new” tool promoting the yeast competition ability under aerobic conditions. The basic aerobic alcoholic fermentation ability was subsequently “upgraded” in several lineages by evolving additional regulatory steps, such as glucose repression in the S. cerevisiae clade, to achieve a more precise metabolic control.

Introduction

Yeast fermentation of different plant carbohydrate sources is one of the oldest human technologies, and its origins date back to the Neolithic period. Even nowadays, yeasts are essential for many biotechnological processes, such as beer, wine, and biofuel fermentations. However, the complexity of gene expression regulatory networks behind the alcoholic fermentation is still far from being completely understood (reviewed in Compagno et al ., 2014 ). Similarly, the origin and the driving forces in nature determining the path and outcomes of the yeast evolutionary history, and the present day evolutionary trends, are still rather unclear. It is the main aim of this review to speculate on and propose evolutionary pathways, trends, and driving forces, which operated during yeast evolutionary history and resulted in the present aerobic fermentative capacity of Saccharomyces yeasts and a new lifestyle of these yeasts.

Crabtree effect

One of the most prominent features of the baker's yeast Saccharomyces cerevisiae is its ability to rapidly convert sugars to ethanol and carbon dioxide at both anaerobic and aerobic conditions. Under aerobic conditions, respiration is possible with oxygen as the final electron acceptor, but S. cerevisiae exhibits alcoholic fermentation until the sugar reaches a low level. This phenomenon is called the Crabtree effect ( De Deken, 1966 ), and the yeasts expressing this trait called Crabtree-positive yeasts. In contrast, “Crabtree-negative” yeasts lack fermentative products, and under aerobic conditions, biomass and carbon dioxide are the sole products. However, it is possible to obtain pure respiratory utilization of glucose by S. cerevisiae under aerobic conditions if the glucose concentration is kept very low, for example using a glucose-limited continuous culture operating below a certain strain-specific threshold value (called “critical” dilution rate) or using fed-batch cultivations ( Postma et al ., 1989 ). This glucose repression phenomenon in S. cerevisiae involves different signal transduction pathways activated by extracellular and intracellular levels of glucose and its related metabolites and/or their fluxes through the involved metabolic pathways (reviewed in Johnston, 1999 ; Westergaard et al ., 2007 ). However, the complexity of glucose repression regulatory networks is still far from being completely understood. Some of the regulatory activities operate at the transcriptional level, and some others operate directly on the involved enzymes. Important to note, so far it is not clear yet if the glucose repression mechanism was the original step to promote evolution of the Crabtree effect, or it has been “added” later during the evolution of some yeast lineages.

Different physiological and molecular approaches have been used as the background for the current definition of the Crabtree effect. The most accepted definition explains the long-term Crabtree effect as aerobic alcoholic fermentation under steady-state conditions at high growth rates. When S. cerevisiae is cultivated under glucose-limited conditions, the long-term effect appears when the dilution rate (or in other words: the glucose uptake rate) exceeds the strain-specific threshold value. The same effect is observed also when yeast cells are cultivated in batch cultivations. The molecular background for the long-term Crabtree effect has been explained as a limited respiratory capacity due to the repression of the corresponding respiration-associated genes ( Postma et al ., 1989 ; Alexander & Jeffries, 1990 ). On the other hand, the short-term Crabtree effect is the immediate appearance of aerobic alcoholic fermentation upon addition of excess sugar to sugar-limited and respiratory cultures. This effect has been explained as an overflow in the sugar metabolism and could be associated directly with the biochemical properties of some of the respiration-associated enzymes and their regulators ( Pronk et al ., 1996 ; Vemuri et al ., 2007 ). However, it is still unclear if the regulatory molecular mechanisms operating during the long-term and short-term Crabtree effect are indeed different from each other. A very interesting aspect is also the evolutionary and ecological background for the development of these regulatory mechanisms ( Piskur et al ., 2006 ; Rozpędowska et al ., 2011 ).

Ecology perspective and yeast lifestyle

Every autumn, when fruits ripen, a fierce competition for the fruit sugars starts within microbial communities. Yeasts, especially S. cerevisiae and its close relatives, usually become the predominant group in niches with freely available mono- and oligosaccharides. The fast sugar consumption, ethanol production, and tolerance, and the ability to propagate without oxygen, are likely some of the “winning” traits responsible for the competition outcome ( Piskur et al ., 2006 ). However, a great majority of yeasts, which we find in nature, has been only poorly studied in laboratory so far or even in their environmental context.

At least three lineages (Fig. ​ (Fig.1), 1 ), including budding and fission ( Schizosaccharoymces pombe ) yeasts, have apparently independently evolved the metabolic ability to produce ethanol in the presence of oxygen and excess of glucose (reviewed in Piskur et al ., 2006 ; Rozpędowska et al ., 2011 ; Rhind et al ., 2011 ). This metabolic »invention« (Crabtree effect) represents in nature a strong tool to outcompete other microorganisms. Both groups of ethanol-producing budding yeast, including S. cerevisiae and Dekkera bruxellensis , can also efficiently catabolize ethanol, and therefore, their corresponding lifestyle has been named as the “make-accumulate-consume (ethanol)” strategy ( Thomson et al ., 2005 ; Piskur et al ., 2006 ; Rozpędowska et al ., 2011 ). On the other hand, S. pombe can grow only poorly on ethanol as sole carbon source. In short, this life strategy is based on that yeasts can consume very fast more sugar than other species, convert it to ethanol to inhibit the growth of other species, especially bacteria, and then consume the remaining carbon once they have established competitive dominance in the niche.

Phylogenetic relationship among some yeasts. Note that some of the shown yeast lineages separated from each other many million years ago and have therefore accumulated several molecular and physiological changes regarding their carbon metabolism. However, during the evolutionary history, there have also been parallel events. Apparently, at least three lineages, Saccharomyces , Dekkera , and Schizosaccharomyces , have evolved (1) the ability to ferment in the presence of oxygen and (2) to proliferate under anaerobic conditions. This figure was adopted from Compagno et al . (2014) .

Ability to grow anaerobically

The availability of oxygen varies among different niches. One of the main problems an organism faces under anaerobic conditions is the lack of the final electron acceptor in the respiratory chain. This reduces or completely eliminates the activity of Krebs cycle, respiratory chain, and mitochondrial ATP generation. As a response to hypoxic and anaerobic conditions, organisms have developed several processes to optimize the utilization of oxygen and even reduce the dependence on the presence of oxygen. According to their dependence on oxygen during the life cycle, yeasts are classified as: (1) obligate aerobes displaying exclusively respiratory metabolism, (2) facultative fermentatives (or facultative anaerobes), displaying both respiratory and fermentative metabolism, and (3) obligate fermentatives (or obligate anaerobes) ( Merico et al ., 2007 ).

The ability of yeasts to grow under oxygen-limited conditions seems to be strictly dependent on the ability to perform alcoholic fermentation. In other words, enough ATP should be generated during glycolysis to support the yeast growth, and NADH generated during glycolysis gets re-oxidized. Apart from the energy and NADH/NAD redox problems, under anaerobic conditions, yeasts must also find a way to run various reactions independent of the respiratory chain and a normal Krebs cycle. In other words, substrates (intermediates) for de novo reactions, for example for the amino acid synthetic pathways, need to originate from a modified metabolic network. On the other hand, in yeast some compounds, such as unsaturated fatty acids and sterols, cannot be synthesized in the cell under anaerobiosis and must originate from the medium or from previous aerobic growth.

Apparently, the progenitor of Saccharomycetaceae was an aerobic organism, strictly dependent on oxygen. It seems that later several yeast lineages (Fig. ​ (Fig.1) 1 ) have evolved the ability to grow anaerobically, or at least can grow partially independently of oxygen. S. cerevisae and a majority of post-WGD yeasts, as well as some lower Saccharomycetaceae branches, such as the Lachancea yeasts, show a clear ability to proliferate without oxygen. Interestingly, two other lineages, D. bruxellensis ( Rozpędowska et al ., 2011 ) and S. pombe ( Visser et al ., 1990 ) have apparently also evolved the ability to propagate under anaerobic conditions. However, they need some extra supplements in the medium to be able to propagate without oxygen. It is interesting to point out, that the same three lineages, which can perform alcoholic fermentation under aerobic conditions can also proliferate in the absence of oxygen.

How to deduce the yeast evolutionary history

The onset of yeast genomics ( Goffeau et al ., 1996 ) has provided a tool to reconstruct several molecular events, which have reshaped the budding yeasts during their evolutionary history (reviewed in Dujon, 2010 ). Several molecular events have left a clear fingerprint in the modern genomes (Fig. ​ (Fig.2), 2 ), while the origin of more complex traits, such as the Crabtree effect, is often not easy to determine using only a genome analysis approach.

The Saccharomycetaceae family covers over 200 million years of the yeast evolutionary history and includes six post-whole-genome duplication (post-WGD) genera, Saccharomyces , Kazachstania , Naumovia , Nakaseomyces , Tetrapisispora , and Vanderwaltozyma ; and six non-WGD genera, Zygosaccharomyces , Zygotorulaspora , Torulaspora , Lachancea , Kluyveromyces , and Eremothecium . Hereby, we show a rough phylogenetic relationship among these genera. Two evolutionary events are shown, WGD, which took place app. 100 million years ago and the loss of Respiratory Complex I (which took place app. 150 million years ago). This figure was adopted from Hagman et al . (2013) .

The Saccharomycetaceae family covers over 200 million years of the yeast evolutionary history and includes six post-whole-genome duplication (post-WGD) genera, Saccharomyces , Kazachstania , Naumovia , Nakaseomyces , Tetrapisispora , and Vanderwaltozyma ; and six non-WGD genera, Zygosaccharomyces , Zygotorulaspora , Torulaspora , Lachancea , Kluyveromyces , and Eremothecium ( Kurtzman & Robnett, 2003 ; Casaregola et al ., 2011 ) (Fig. ​ (Fig.2). 2 ). The phylogenetic relationship among these genera is now relatively well understood. However, only a very few species are reported in literature for their carbon metabolism ( Merico et al ., 2007 ).

We have recently studied over forty yeast species, which in nature occupy similar niches and rely on glucose as the »preferred« substrate ( Kurtzman et al ., 2011 ) and analyzed their carbon metabolism using uniform experimental conditions all along the fully controlled growth in fermentors ( Hagman et al ., 2013 ).

The origin of the long-term Crabtree effect

The studied yeasts belonged to the Saccharomycotina family, including six WGD genera and six non-WGD genera, thus covering 200 million years of evolution (Fig. ​ (Fig.2). 2 ). The observed extent of the Crabtree effect in each species corresponds to its position on the yeast phylogenetic tree. In addition, the observed Crabtree effect is much more pronounced in a majority of WGD yeasts than in the ethanol-producing non-WGD species, suggesting at least a two-step »invention«. On the other hand, carbon metabolism in the »lower« branches of Saccharomycetaceae yeasts, belonging to modern Kluyveromyces and Eremothecium , is similar to other Saccharomycotina yeasts, such as Candida albicans , Yarrowia lipolytica , and Pichia pastoris , which are Crabtree-negative yeasts. Therefore, the origin of the “make-accumulate-consume” strategy/Crabtree effect could take place within the time interval spanning the origin of the ability to grow under anaerobic conditions, and the loss of respiratory chain Complex I, after the split of the Saccharomyces - Lachancea and Kluyveromyces - Eremothecium lineages, approximately 125 million years ago. On the other hand, the second step, leading toward even a more pronounced Crabtree effect, occurred relatively close to the WGD event (Wolfe and Shields, 1996), the settlement of rewiring of the promoters involved in the respiratory part of the carbon metabolism ( Ihmels et al ., 2005 ), and the settlement of the petite-positive character ( Merico et al ., 2007 ). There are also some other possible scenarios, which can be “deduced” from the Hagman et al . (2013 ) results. The origin of the long-term Crabtree effect could took place much before, coinciding with the loss of respiratory chain Complex I, but this trait was later lost in some lineages, such as Kluyveromyces - Eremothecium . The long-term effect may have even originated independently in several Saccharomycetaceae lineages. To clarify this point, in the near future, one would need to focus on some of the “early Crabtree positive” branches, such as Lachancea , and perform detailed carbon metabolism studies on these yeasts, including gene expression profiling, to deduce which regulatory circuits are already present in Lachancea , and which ones only in Saccharomyces .

The origin of modern plants with fruits, at the end of the Cretaceous age, more than 125 mya ( Sun et al ., 2011 ), brought to microbial communities a new larger and increasingly abundant source of food based on simple sugars. On the other hand, ancient yeasts could hardly produce the same amount of new biomass as bacteria during the same time interval and could therefore be out-competed. We speculate that slower growth rate could in principle be counter-acted by production of compounds that could inhibit the growth rate of bacteria, such as ethanol and acetate. However, what were the initial molecular mechanisms that promoted the evolution of the new “lifestyle” and rewiring of the carbon metabolism? Was competition between yeast and bacteria indeed the original driving force to promote evolution of the aerobic alcoholic fermentation?

The Crabtree effect, which is the background for the yeast »make-accumulate-consume« strategy, results in a lower biomass production because a fraction of sugar is converted into ethanol. This means that more glucose has to be consumed to achieve the same yield of cells (Fig. ​ (Fig.3). 3 ). Because only a fraction of sugar is used for the biomass and energy production this could theoretically result in a lower growth rate in Crabtree-positive yeasts. In nature, a lower growth rate would have a negative effect for the yeast during the competition with different yeasts species and between yeasts and bacteria. However, an increased glycolytic flow (achieved by increased uptake of glucose and its faster conversion to pyruvate and final fermentation products) could in principle compensate for the Crabtree effect and balance the growth rate providing the same number of cells during the same time interval. Just much more glucose would be consumed in this case (Fig. ​ (Fig.3). 3 ). What could be the original driving force that increased the flow through the glycolytic pathway?

Crabtree effect results in lower biomass production because a fraction of sugar is converted into ethanol. This means that more glucose has to be consumed to achieve the same yield of cells if comparing with Crabtree-negative yeasts. Because only a fraction of sugar is used for the biomass and energy production, this could theoretically result in lower growth rate in Crabtree-positive yeasts and these could then simply be out-competed by Crabtree-negative yeasts and other microorganisms. However, ethanol could be used as a tool to slow down and control the proliferation of other competitive microorganisms.

Short-term Crabtree effect: a strengthened glycolytic flow

The short-term Crabtree effect is defined as the immediate appearance of aerobic alcoholic fermentation upon a pulse of excess sugar to sugar-limited yeast cultures. In a recent follow-up ( Hagman, 2013 ; Hagman et al ., 2014 ) of the above study, ten different yeast species, having a clearly defined phylogenetic relationship, have been characterized for short-term Crabtree effect. These species very roughly cover the phylogenetic span of yeasts, which have been studied in the long-term experiments. Yeasts have been cultivated as continuous cultures under glucose-limited conditions, and upon a glucose pulse, their general carbon metabolism analyzed ( Hagman et al ., 2013 , Hagman, 2013 ). In pulse experiments, yeasts belonging to Pichia , Debaryomyces , Eremothecium , and Kluyveromyces marxianus have not exhibited any significant ethanol formation (just like they also do not show long-term Crabtree effect), while Kluyveromyces lactis behaved, surprisingly, as intermediate yeast. The Lachancea , Torulaspora , Vandervaltozyma , and Saccharomyces yeasts have, upon a glucose pulse, exhibited rapid ethanol accumulation. These yeasts are also long-term positive species ( Hagman et al ., 2013 ).

Roughly, long-term positive species in glucose pulse experiments behaved also as short-term positive. However, the results suggest that Kluyveromyces yeasts can be considered as intermediate in both phylogenetic position and their carbon metabolism. K. lactis is in fact on one hand a short-term Crabtree positive, but on the other hand a long-term Crabtree negative. Even if the number of studied yeasts is limited (only ten), one could still speculate that the time of origin of the short-term Crabtree effect and the time of origin of the long-term Crabtree effect seem to be very close to each other and may even overlap, coinciding with the horizontal transfer of URA1 and the ability to proliferate anaerobically ( Merico et al ., 2007 ).

However, one of the most surprising observations has been that when S. cerevisiae and its Crabtree-positive relatives grow in continuous culture below a sugar threshold with a respiratory metabolism, their fermentative pathways are fully expressed, whereas the respiration-associated parts are repressed when the sugar level overcomes a certain level. In other words, it seems that these yeast cells have all the time the capacity to ferment “switched-on”, while the respiration ability is strictly related to the amount of sugar availability. On the other hand, in several Crabtree-negative yeasts, it has been demonstrated that the fermentative pathway is “switched-on” only when oxygen becomes limiting ( Kiers et al ., 1998 ; Jeffries, 2006 ; Baumann et al ., 2010 ). Interestingly, in Crabtree-negative yeasts, the flow through glycolysis matches the respiration associated one, whereas in Crabtree-positive yeasts, the glycolytic flow is apparently “over-dimensioned”.

Hypothesis on the original driving forces

Why would the progenitor yeast initially benefit from the strengthened glycolytic and fermentative flow? It is apparent from previous studies that the ability to proliferate under anaerobic conditions originated at approximately the same time as the origin of the first modern fruits and aerobic alcoholic fermentation, upon the split of the Kluyveromyces-Eremothecium and Lachancea-Saccharomyces lineages ( Hagman et al ., 2013 ). It could be that regular exposure to poorly aerobic niches represented a selection pressure which promoted yeast “mutant” lineages with strengthened glycolytic and fermentation pathways, as well as having improved resistance toward ethanol. In these cells, up-regulation of the glycolytic-fermentative capacity should have provided sufficient carbon flow and energy yield even in the absence of oxygen. In other words, exploration of anaerobic niches could be a driving force to build up a carbon metabolism network, which is better adapted to ferment. However, in principle these yeasts could still alternate between aerobic and anaerobic niches.

At the same time, the yeast progenitor could also get remodeled several general metabolic pathways, not only the energy yielding ones, to be able to proliferate also under anaerobic conditions. For example, the fourth step of the de novo pyrimidine synthesis became, upon horizontal transfer of the URA1 gene, less dependent on the functional respiratory chain ( Gojković et al ., 2004 ). Note, that this step toward independence of oxygen occurred before the separation of the Kluyveromyces and Lachancea-Saccharomyces lineages and therefore likely before the origin of the aerobic fermentative-respiratory lifestyle.

Fast consumption of glucose through an increased uptake would simply “starve-out” other microbial competitors. The duplication of glucose transporter genes in the progenitor of the Lachancea-Saccharomyces lineages could represent one of the molecular backgrounds for the initial increased ability to consume glucose. However, the increased carbon flow through glycolysis generated an overflow and resulted in synthesis of fermentation products. When these metabolites, especially ethanol, were accumulated at high concentrations, they could impair the growth of other competing microorganisms. The fermentation products could thus become a new weapon to out-compete other microorganisms. At this point, the driving biological force for optimizing the ethanol fermentation pathway, even in the presence of oxygen, could be “to kill” other competitors. In parallel, yeasts had also to evolve the ability to better tolerate ethanol. Further on, the origin of glucose repression of the respiratory pathways under fully aerobic conditions, in the S. cerevisiae lineage, could represent a fine tuning mechanism, which increased the efficiency of ethanol production.

Conclusions

During the recent years, there has been more and more research focus on nonconventional yeasts, especially many of these yeasts got their genomes sequenced. These sequence data now help to deduce a reliable phylogenetic relationship among yeasts and provide us with a possibility to reveal evolution fingerprints, which have remained preserved in the genome. These data should now be complemented with physiology and molecular genetic studies on a variety of yeast species. This will open additional avenues in biotechnology and evolution research. In this review, we attempted to analyze the most recent results on yeast carbon metabolism and develop a hypothesis on the evolution of alcoholic fermentation. We speculate that the exploration of anaerobic niches and later on the competition with other microorganisms were the driving forces behind the remodeling of the yeast carbon metabolism.

Acknowledgments

The authors thank Swedish Research Council, EU ITN Cornucopia, ARRS (Slovenia) and Crafoord, Fysiografen, Lindström, and Soerensen Foundations for their financial support.

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Basic Food Chemistry pp 323–341 Cite as

• Alcoholic Fermentation
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Alcoholic beverages have one point in common. They all depend on the process of fermentation—the conversion of hexose sugars into alcohol and carbon dioxide. This is indeed a very important process and is basic to all of the industries involved. Alcoholic fermentation is an anaerobic process carried on by living yeast cells. The cells absorb the simple sugars, which in turn, are broken down in a series of successive changes in which action by oxidizing and reducing enzymes within the cell takes place. The final result is the formation of ethyl alcohol and carbon dioxide accompanied by the liberation of some energy in the form of heat. This process does not account for all of the original energy in the sugars, however, because a part of it is used for multiplication and metabolism of the yeast cells, and the process, therefore, changes compounds of higher energy to those of lower energy. Buchner (1897) showed that a cell-free extract from yeast cells induced fermentation, demonstrating that the enzymes and not the cells themselves produce the chemical action.

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Georgia Ch. Lainioti, George Karaiskakis, New Approaches to the Kinetic Study of Alcoholic Fermentation by Chromatographic Techniques, Journal of Chromatographic Science , Volume 51, Issue 8, September 2013, Pages 764–779, https://doi.org/10.1093/chromsci/bms257

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The kinetics of the fermentation process has gained increasing interest, not only in the scientific community, but in the industrial world as well. Information concerning the improvement of batch fermentation performance may potentially be valuable for the designing of scale-up processes. Intensive studies have been conducted with the use of various chromatographic techniques, such as conventional gas chromatography, reversed-flow gas chromatography (RFGC), high-performance liquid chromatography, field-flow fractionation and others. In the present study, specific focus is placed on the employment of RFGC, a method that can successfully be applied for the determination of physicochemical quantities, such as reaction rate constants and activation energies, at each phase of the alcoholic fermentation. In contrast to conventional chromatographic techniques, RFGC can lead to substantial information referring to the evaluation of fermentation kinetics at any time of the process. Moreover, gravitational field-flow fractionation, a sub-technique of field-flow fractionation, presents the ability to monitor the proliferation of Saccharomyces cerevisiae cells through their elution profiles that can be related to the different cell growth stages. The combination of the two techniques can provide important information for kinetic study and the distinction of the growth phases of yeast cell proliferation during alcoholic fermentations conducted under different environmental conditions.

Alcoholic fermentation

“Wine fermentation must be a decomposition that occurs when the sugar-fungus uses sugar and nitrogenous substances for growth, during which, those elements not so used are preferentially converted to alcohol,” explained Schwann in his paper entitled “A Preliminary Communication Concerning Experiments on Fermentation of Wine and Putrefaction” ( 1 ). Alcoholic fermentation is one of the oldest procedures in the history of humanity. Yeast converts grape sugar into roughly equal parts of ethanol and carbon dioxide, producing heat. Alcoholic fermentation is a combination of complex interactions involving the variety of must, microbiota and wine-making technology. The concept of inoculating grape juice with selected starter cultures of Saccharomyces cerevisiae to encourage rapid, consistent fermentation has become widely accepted within the wine industry ( 2 , 3 ). With the progress of biotechnology, many changes have been made to traditional wine-making procedures. Immobilized cells have been extensively used in food and fermentation industries due to their advanced properties toward free cell systems. The employment of biocatalysts has been important in many industries for centuries because they are attractive catalysts for a wide range of chemical transformations. This can be attributed to their advanced properties in comparison with free cell systems, such as improved productivity and products of higher quality. The support material on which the Saccharomyces cerevisiae strain will be immobilized is of utmost importance for an effective immobilization. Inorganic (γ-aluminum, kissiris or ceramic materials) ( 4–7 ) or organic (cellulosic materials, polyacrylamide, carrageenan or alginates) materials ( 3 , 8 , 9 ), in addition to fruit pieces (apple and quince) ( 10 , 11 ) have successfully been used as immobilization supports.

With respect to the properties of yeast, yeast cells typically operate under mild conditions of pH and temperature, leading to the formation of highly pure products. As discussed in the literature, the fermentation temperature can affect the microbial ecology of the grape must and the biochemical reactions of yeast cells ( 12 ). In addition to affecting growth and survival, temperature has many substantial effects on the metabolism of yeast. One of the most marked is its influence on the fermentation rate ( 13 ). Cool temperatures dramatically slow the fermentation rate and may cause its premature termination. Excessively high temperatures may also induce stuck fermentation by disrupting enzyme and membrane functions. Because yeast strains differ in this response, the optimum temperature for vinification can vary widely ( 13 ).

It is well known from the literature that the life cycle of yeast is activated from dormancy, when it is added to the fermentation liquid. Yeast growth follows four phases: the lag phase, which appears after the inoculation of yeast cells in the medium and represents the period of adjustment in their new environment; the growth phase, during which yeast cells multiply and the growth rate becomes maximum; the stationary phase, in which yeast cells develop their action as fermenting organisms; and finally, the decline or death phase, during which yeast cells die because of the high ethanol concentrations and the lack of nutrition substances in the fermentation liquid ( 14–16 ).

Alcoholic fermentation kinetics

With increasing interest in the industrial application of alcoholic fermentation, various kinetic models have been proposed in the literature for cells suspended in batches or continuous operations ( 17–21 ). Parameters such as maximum specific growth rate for growth phase have been measured, in addition to biomass concentration, fermentation and substrate uptake or dilution rate for steady-state fermentation. Kinetic models can provide valuable information when the cell composition is time-dependent. As stated before, the yeast growth cycle follows four phases, which are arbitrary because all of the phases may overlap in time. The development of appropriate mathematical models can be a good approximation for describing the cell growth cycle and the kinetics of fermentation.

In 1945, Monod reported an empirical equation to describe cell growth as a function of the limiting substrate concentration ( 22 ). Since then, various models have been reported in the literature describing the kinetics of alcoholic fermentation processes. A wide range of empirical models has been used to study alcoholic fermentation in terms of cell concentration, substrate utilization rate and ethanol production rate. The most widely used model ( 21 ) provides the coefficients for substrate uptake and product formation. The models of Monod ( 22 ) and Teissier ( 23 ) represent inhibition-free kinetics. Edwards ( 24 ) and Luong ( 25 ) provide equations for substrate inhibition effects, whereas the models of Hinselwood ( 26 ), Ghose and Tyagi ( 27 ) and Aiba et al. ( 28 ) include product inhibition kinetics.

Gulnur et al. ( 29 ) used the models proposed by Monod ( 22 ) and Hinselwood ( 26 ) for the description of immobilized Saccharomyces cerevisiae cells at low and high initial glucose concentrations. Blanco et al. ( 21 ) conducted a comparative study of alcoholic fermentation under similar pH and temperature conditions, but different initial glucose concentrations of culture medium. After testing various empirical models, the Hinselwood model ( 26 ) was found to be the best for this work.

Gulnur et al. ( 29 ) presented a mathematical description for the fermentation characteristics of Saccharomyces cerevisiae cells immobilized on Ca-alginate gel beads in a stirred batch system of different initial glucose concentrations. The experimental results were tested using 11 well-known mathematical models, taking into account the possibility of glucose or ethanol inhibition on both yeast cell growth and ethanol production.

Caro et al. ( 30 ) proposed a kinetic expression that accounts for the temperature dependence in batch alcoholic fermentations. According to that, the specific growth rates of Saccharomyces cerevisiae at 20 and 15°C were 0.090 and 0.024 h −1 , respectively. Moreover, Ozilgen et al. ( 31 ) proposed simple mathematical models for simulating microbial growth, reducing sugar utilization and increasing ethanol production and temperature in a spontaneous wine-making process. They calculated the rate constants for the third phase between 0.039 h −1 (fast fermentation) and 0.042 h −1 (slow fermentation).

Ciani et al. ( 32 ) determined the growth kinetics and fermentation behavior of five non- Saccharomyces yeast species associated with wine-making. The specific growth rates for Saccharomyces cerevisiae , calculated by cell number and dry weight, were 0.262 and 0.085 h −1 , respectively. Giovanelli et al. ( 33 ) reported specific growth rates of Saccharomyces cerevisiae equal to 0.13 h −1 under aerobic conditions and 0.07 h −1 under anaerobic conditions.

Furthermore, according to Birol et al. ( 34 ), the observed maximum specific growth rates were determined between 0.191 and 0.201 h −1 . Godia et al. ( 17 ) used a different technique, based on the integration of the different rate equations in the modeling of batch alcoholic fermentation using Saccharomyces cerevisiae at different temperature and initial sugar concentrations. The kinetic parameters for the maximum specific growth rate and the maximum specific fermentation rate were calculated using two mathematical models. Chen et al. ( 20 ) presented a modified kinetic model suitable for continuous cultures and ethanol fermentations under high gravity fermentation conditions, through which the specific growth rate of Saccharomyces cerevisiae and the ethanol production rate for different initial and residual glucose concentrations were determined. The maximum values of 0.146 and 1.172 h −1 , respectively, were illustrated at an initial glucose concentration of 200 g/L.

Differences observed in the proposed kinetic models confirm that the equation parameters depend not only on the species of the used microorganism, but also on other factors, such as fermentation temperature and glucose concentration.

Chromatographic techniques for the study of alcoholic fermentation

Chromatographic techniques are widely used for determining volatile compounds and ethanol content in wines and alcoholic beverages. Such compounds are important for the classification, quality control and sensory evaluation of wines ( 35 , 36 ).

Fernandes et al. ( 37 ) used different multidimensional chromatographic techniques to study wine aroma pattern changes during malolactic fermentation (MLF). Ethyl lactate enantiomeric ratios were determined using online multidimensional gas chromatography. The values corresponded with a spontaneous MLF. Offline multidimensional high-performance liquid chromatography/gas chromatography (HPLC/GC) was used to deconvolute and enrich the sample and to ease enantioselective chromatography. Aroma compounds were analyzed by HPLC.

Peinado et al ( 38 ) used an Agilent 6890 Series Plus gas chromatograph with electronic pressure control to develop and validate a simple method for quantifying many of the most important volatile compounds and polyols in wine in a single chromatographic run.

The composition and content of odor compounds determine the quality of alcoholic products. The smell of an alcoholic beverage is the effect of many chemical compounds with different properties (such as polarity or volatility) occurring at widely differing concentrations. The chemical composition of the odors depends on the quality and type of the raw materials and on the conditions of the fermentation process. Gas chromatography–olfactometry (GC–O) studies on the odor of alcoholic beverages usually have the goal of determining the relationship between the composition and the content of volatile compounds and the organoleptic properties of products such as beer ( 39 , 40 ), wines ( 41 , 42 ) and whiskeys ( 43 ), in addition to the identification and comparison of the compounds entering the aroma of different alcoholic beverages, such as wines ( 44–51 ) or cognacs ( 52 , 53 ).

Distilled alcoholic beverages, wines and beers, are often characterized by GC or gas chromatography–mass spectroscopy (GC–MS) analysis of fatty acids and their esters, which contribute to the taste and quality of these commodities. Fatty acids in wines are mostly straight-chain C6–C10 acids. They are derived from yeast during fermentation, and from the firm tissues of the grapes. As in distilled alcoholic beverages, fatty acids in wine occur as free acids and ethyl esters at low levels (mg/L). GC analysis of the major aroma components has been conducted by means of solvent extraction with 1,1,2-trichlorotrifluoroethane (Kaltron or Freon 113) using a large sample to solvent ratio (100:1) ( 54 ). The required sensitivity for GC–flame ionization detection (FID) analysis was achieved by large volume injection with solvent removal in a program temperature vaporizer (PTV) liner. Recently, the applications of solid-phase microextraction (SPME) to the GC analysis of wines have frequently been reported in literature. Vas et al. demonstrated ( 55 ) that SPME-GC–MS was a simple, quick and sensitive approach for characterization of wines. Regarding distilled alcoholic beverages, strong anion-exchange solid-phase extraction (SAX-SPE) has been used for the selective isolation of organic acids from wines. In the procedure reported by Deng ( 56 ), the ethyl ester derivatives of C4–C18 fatty acids and other organic acids were extracted by dichloromethane and reproducibly quantified by GC–FID using internal standards. The applicability of supercritical fluid extraction–gas chromatography (SFE–GC) for analyzing wine aroma has been evaluated by Blanch et al. , using PTV as an interface to trap the analytes ( 57 ).

The aim of this review, concerning the new approaches to the kinetic study of alcoholic fermentation, is to present the techniques of reversed-flow gas chromatography (RFGC) and field-flow fractionation (FFF) for the determination of physicochemical quantities related to the kinetics of the alcoholic fermentation and for the analysis of yeast cell proliferation during alcoholic fermentations conducted under different environmental conditions.

Reversed-flow gas chromatography

The principle on which the RFGC method is based is illustrated in Figure  1 A.

Schematic representation of the experimental arrangement of the RFGC system for the determination of ethanol concentration in the fermentation medium (A); sample peaks of ethanol at 30°C obtained by RFGC (B). From Ref.[ 78 ], with permission.

A long diffusion column, of length L 1 , containing the liquid at its bottom, is connected perpendicularly to the mid-point of a so-called sampling column, of length l ′ + l , and the whole cell is thermostated inside the oven of the chromatograph. The two ends of the sampling column are connected to the carrier gas inlet and the flame ionization detector through a four-port valve, as shown. By switching this valve from one position (solid lines) to the other (dashed lines), the direction of the carrier gas flow through the column of length l ′ + l is reversed, producing sample peaks in the recorder line.

From the slope − L 2 1 /4D of a plot of the left-hand side of Eq. 6 versus 1/ t 0 , the first experimental value is found for D . This can be used with the slope found from the plot of Eq. 5 to calculate a better approximation of the k c value, and this is used to replot Eq. 6 for a better approximation of the D value. These iterations can be continued until no significant changes result in the values of k c and D , but it is not usually necessary to go beyond the first iteration.

Experimental

As mentioned previously, the development of appropriate mathematical models can provide valuable information about the life cycles of yeast cells. The estimation of the duration of each of the previously discussed phases (lag, growth, stationary and decline) is of great importance because it allows the determination of the rate constants for each phase of the fermentation process. The aforementioned can be conducted by developing an appropriate mathematical model for the analysis of the experimental data obtained from a versatile gas chromatographic technique, RFGC. RFGC can be applied for the determination of ethanol production during alcoholic fermentation processes by measuring the ethanol produced at any time during the fermentation process. A gas chromatograph (Shimadzu-8A) equipped with an FID was used for the separation and quantitation of ethanol (Figure  1 A). The chromatograph was slightly modified to include a T-shaped cell constructed from a glass chromatographic tube inside the chromatographic oven and a four-port gas valve (Figure  1 A).

The oven of the system contained the sampling column, consisting of two sections of lengths l = l ′ = 100 cm and the diffusion column with length L 1 = 43 cm. At the lower end of diffusion column, a glass vessel ( L 2 = 5 cm) containing the fermentation mixture was connected. The two columns comprised the sampling cell, which was connected to the carrier gas inlet and the detector so that the carrier gas flow through the sampling column (no carrier gas flows through the diffusion column) could be reversed in direction at any time desired. This can be done by using a four-port valve to connect the ends of the sampling column to the carrier gas supply and the detector, as shown schematically in Figure  1 A. An analytical column (2 m × 1/4 in. × 2 mm glass) packed with 5% Carbowax-20 M and 80/120 mesh Carbopack BAW, which was kept at 115°C, was placed before the detector to separate ethanol from the by-products of alcoholic fermentation. The temperature of the detector was set at 150°C, whereas the temperature of the chromatographic cell was 75°C. The experiments were conducted under constant flow rate (20 mL/min) using helium as carrier gas.

An aliquot of 0.5 mL of the fermentation mixture was added to the glass vessel (Figure  1 A) at constant temperature and pressure. By the time a concentration-time curve appeared, the chromatographic procedure began by reversing the carrier gas flow direction through the Shimadzu valve for 6 s. This period of time was shorter than the gas hold-up time in both sections l and l ′. When the flow of the carrier gas was restored to its original direction, sampling peaks were recorded, like those shown in Figure  1 B.

Finally, solutions with different ethanol concentration (%, v/v) in triply distilled water were placed into the glass vessel at the end of the diffusion column and the values of H ∞ were measured. These values and their corresponding ethanol concentrations were plotted and a calibration curve (slope = 1.202 cm/M, intercept = 0.041 cm and R 2 = 0.999) was obtained. According to the calibration curve and by measuring the height of the sample peaks during the fermentation process, the unknown ethanol concentrations of each fermentation mixture can be found.

Applications on kinetic study of alcoholic fermentation

RFGC is a sub-technique of inverse gas chromatography, which was introduced in 1980 by Professor N. A. Katsanos ( 67 ) and his colleagues (at the University of Patras, Greece). RFGC involves the change of the carrier gas flow direction at various time intervals. It has a vast number of applications in a wide scientific field, such as the determination of diffusion coefficients ( 68–71 ), adsorption equilibrium constants ( 72–74 ), Lenard-Jones parameters ( 75 ), activity coefficients ( 76 ), rate constants for alcoholic fermentations, gaseous reactions ( 77–81 ) and heterogenous catalytic reaction ( 82–88 ), and molecular diameters and critical volumes ( 89 ).

The RFGC technique was first applied for the kinetic study of the alcoholic fermentation process on the industrial scale in conjunction with measurements of suspended particles in the fermenting medium by Economopoulos et al. ( 66 ). It was found that the overall process consists of four phases that have different first-order rate constants during ethanol formation. The RFGC technique provides an easy study of the chemical kinetics during each phase of the overall process.

As reported by Lainioti et al. ( 16 ), RFGC was one of the separation techniques that was used for the distinction of the growth phases of Saccharomyces cerevisiae cell cycles at different temperatures and pH values. As previously mentioned (in the “Introduction”), alcoholic fermentation is divided into four phases, which can be correlated to the growth cycle phases of AXAZ-1 cells ( 77 , 16 ). As a result, the four slopes in Figure  2 correspond to the lag, growth, stationary and decline phases of the alcoholic fermentation processes. The estimation of the duration of each of the fermentation phases is of great importance because it allows the determination of physicochemical parameters of crucial interest, such as rate constants and activation energies, for each phase of the fermentation process.

Variation of ln( H ∞ 0 – H ∞ ) with time for AXAZ-1 cells at pH 5 and T = 30°C for the first (diamonds), second (squares), third (triangles) and fourth (circles) alcoholic phases. From Ref.[ 16 ], with permission.

According to the slopes of Figure  2 , which result from Eq. 8, the values of reaction rate constants, k , for ethanol production can be calculated at the four phases of the alcoholic fermentations conducted with free and immobilized cells at various initial glucose concentrations.

The results presented in Table  I are mean values of the rate constants; their standard deviations are also given. The aforementioned slopes were confirmed from the R 2 values, which were within 0.960–0.999.

Rate Constants, k , with their Standard Deviations for the Fermentation Phases of AXAZ-1 Cells at pH 5.0 and at 30, 25, 20 and 15°C, Obtained by RFGC (From Ref.[ 16 ], with permission)

The greatest values of the reaction rate constants were observed at the second and fourth phases (growth and decline phase, respectively), whereas the lowest and almost stable values were observed at the first phase (lag phase), during which the total biomass concentration did not show any change.

As reported by Lainioti et al. ( 77 ), RFGC was applied for the kinetic study of the alcoholic fermentation process conducted with cells of the alcohol-resistant and psychrophilic Saccharomyces cerevisiae AXAZ-1 yeast strain, either free or immobilized on wheat, barley and corn grains or on potato pieces. Repeated alcoholic fermentations with must of varying initial glucose concentrations were performed to estimate the catalytic efficiency of the biocatalysts used in the present study. In this work, each fermentation process consisted of three phases, which corresponded to the growth, stationary and decline phases of the alcoholic fermentation. The literature shows that the duration of the lag phase is highly dependent on the fermentation efficiency of yeast cells, the conditions of the fermentation procedure, the immobilization carrier and other factors. Considering the great fermentation efficiency of AXAZ-1 cells immobilized on wheat, barley, corn grains and potato pieces, and their quick adaptation in the fermentation liquid, the lag phase is not easily observed. In the present work, the rate constants k 1 and k 2 , which correspond to the growth and stationary phases, were decreased when the glucose concentration increased. This can be attributed to the negative influence of the osmotic stress that high sugar contents of the fermentation liquid exert in the growth of yeast cells. The values of k 3 , which correspond to the decline phase for all systems, reached a maximum value at an initial glucose concentration of 205 g/L, which indicated that this glucose concentration comprised the best environment for them to ferment. Additionally, the values of k 3 rate constants for ethanol production in the decline phase were of the same order of magnitude as those observed at the growth phase. This behavior may be because once the decline phase begins, yeast cells have already reached their maximum values. This means that the production of ethanol occurs at a high rate because there are large numbers of cells in the fermentation liquid at this moment. Consequently, what is called the decline phase in the present work corresponds to an initial stage of the decline phase of the growth cycle of yeast cells. The decline phase was not completed due to the great fermentation efficiency of AXAZ-1 cells, which managed to rapidly terminate the fermentation processes.

In another work by Lainioti et al. ( 78 ), RFGC was employed for the determination of the alcoholic fermentation phases and kinetic parameters for free and immobilized cell systems at different initial glucose concentrations and temperature values. The immobilization of the wine yeast Saccharomyces cerevisiae AXAZ-1 was accomplished on wheat and corn starch gels to prepare new biocatalysts of great interest for the fermentation industry. With great accuracy, resulting from a literature review, RFGC led, to the determination of reaction rate constants and activation energies at each phase of the fermentation processes. With respect to many factors (immobilization carrier and fermentation conditions), yeast cells may have a quick and easy adaptation in the fermentation environment. In addition to this, the lag phase in many cases is too short to be detected. Thus, in this work, three phases appeared in the diagram showing the variation of ln( H ∞ 0 – H ∞ ) versus t x , corresponding to the growth, stationary and decline phases, respectively. Rate constants k 1 and k 3 , which correspond to growth and decline phases, reached a maximum value at initial glucose concentration of 205 g/L for all systems in which the higher number of yeast cells was observed, as shown from the results of Table  II .

Rate Constants, k , with their Standard Deviations, for Each Phase of Alcoholic Fermentation in the Presence and Absence of an Immobilization Carrier (Wheat and Corn Starch Gels) at Different Initial Glucose Concentrations (From Ref.[ 78 ], with permission)

This behavior indicates that this glucose concentration comprised the best environment for cells to ferment. After the concentration of 205 g/L, a decrease in the rate constants ( k 1 and k 3 ) was observed in free and immobilized systems with an increase in glucose concentration. This can be attributed to the negative influence of the osmotic stress that high sugar contents of the fermentation liquid exert in the growth of yeast cells. The rate constant k 2 , which corresponds to the stationary phase, was decreased with the increased glucose concentration in all systems. Comparing free and immobilized systems, the rate constants for immobilized cells were higher than those observed in free cells. By summarizing the previously mentioned results, the increase in glucose concentrations had a negative influence on the growth of AXAZ-1 cells and rate constant values. Moreover, the decrease of fermentation temperature caused substantial reductions in the viability of immobilized cells and in rate constant values.

Because the rate constants for ethanol production have been calculated by the RFGC technique, activation energies for each phase of the alcoholic fermentations can be determined from the Arrhenius equation (Eq. 2 ).

Through the slope of the graphical representation of ln k versus 1/ T (Figure  3 ) as reported by Lainioti et al. ( 78 ), corn starch gel presented lower values of activation energies than those of wheat starch gel, as shown in Table  III .

Arrhenius plots of ln k versus 1/ T × 10 3 (1/ K ) for the evaluation of the activation energy of each phase of the alcoholic fermentations conducted with cells immobilized on wheat (triangles), corn starch gels (squares) and free cells (circles). From Ref.[ 78 ], with permission.

Activation Energies, E a , with their Standard Deviations, for Each Phase of Alcoholic Fermentations in the Presence and Absence of the Immobilization Carrier (From Ref.[ 78 ], with permission)

However, the two supports presented higher catalytic efficiency than free cell systems, because they presented 32 and 39% reduced activation energies for the first (growth) phase of the alcoholic fermentations, 22 and 32% for the second (stationary) phase and 17 and 23% for the third (decline) phase, when wheat and corn starch gel were used, respectively.

Moreover, considering the results of a previously mentioned work ( 16 ), the activation energy for the first (lag) phase of the alcoholic fermentation process was 18.4 ± 0.4 kJ/mol, whereas for the remaining phases (second, growth; third, stationary; fourth, decline), higher values were observed: 90.5 ± 0.2, 94.5 ± 0.3 and 95.6 ± 0.5 kJ/mol, respectively.

The results of activation energies, given in Table  III , are in the same order of magnitude with previous studies published in literature ( 60 , 79 ), in which the activation energy of AXAZ-1 cells immobilized on corn starch gel and potato pieces was 62.2 and 61.1 kJ/mol, respectively, for the whole fermentation process. This agreement of bibliographic and experimental values confirms the reliability of the RFGC method for the kinetic study of the alcoholic fermentation process and the great catalytic activity of the immobilized AXAZ-1 cells. Moreover, it shows that the immobilization carrier may act as a catalyst or a promoter of the catalytic activity of the enzymes involved in the fermentation process.

Field-flow fractionation

FFF is classified as a one-phase chromatographic technique in which an externally adjusted force field is applied to the suspended particles under motion in a channel. The particles are pushed to one of the channel walls so that they occupy a thin layer close to the accumulation wall. The thickness of the layer is an explicit function of the applied field, particle size and density of both particles and carrier solution. It is an elution technique, and as in classical chromatography, sample components elute at retention volumes that are related, and often rigorously predictable, to various physicochemical properties of the retained species.

The FFF technique is ideally suited to analytical-scale separation and characterization of particles ( 80 , 81 ). Different kind of fields have been applied to FFF. They can be a sedimentation field or a thermal gradient, an electrical field or a secondary flow. The type of field defines the FFF sub-techniques, which, therefore, shows wide applicability. Gravitational field-flow fractionation (GrFFF) is the simplest and cheapest among FFF techniques. It is a subset of sedimentation field-flow fractionation (SdFFF) that is suitable for the separation and characterization of various micrometer-sized particles of different origin ( 90 , 91 ). It employs Earth's gravitational field applied perpendicularly to a very thin, empty channel. GrFFF is very appealing in the field of biological applications. It has been applied to living samples such as parasites ( 92 ) and blood cells ( 93 ) because of its simplicity, low cost and reduced risk of sample degradation. It has also been used in the characterization of albumin ( 94 ), liposomes ( 95 ) and colloidal materials ( 96–101 ).

Schematic representation of the experimental arrangement of the FFF technique.

The GrFFF system used for the kinetic study of the alcoholic fermentation, which has been described in detail elsewhere ( 103 ), has the following dimensions: length, l = 48.3 cm; breadth, a = 2.0 cm; thickness, w = 0.021 cm. The channel void volume, V 0 , measured by the elution of the non-retained sodium benzoate peak, was found to be 2.025 cm 3 . A Gilson Minipuls 2 peristaltic pump was used to pump the carrier solution and the sample to the channel, whereas a Gilson model 112 UV/VIS detector operated at 254 nm, and a Linseis model L6522 recorder were also used for sampling analysis. Polystyrene particles of different diameters (nominal 2.013 ± 0.025, 4.991 ± 0.035, 9.975 ± 0.061, 15.02 ± 0.08 and 20.00 ± 0.10 µm) from Duke Scientific Corporation, dispersed in triply distilled water, were used for the optimization of the elution conditions. The carrier solution was triply distilled water containing 0.5% v/v FL-70 (Fisher Scientific Co.), a low-foaming, low-alkalinity and phosphate-, chromate- and silicate-free mixture of anionic and cationic surfactant and 0.02% (w/v) sodium azide (Fluka AG) as bacteriocide. An aliquot of 25 µL was drawn from the fermentation mixture and injected into the channel by a microsyringe. Following injection, the longitudinal flow (50 mL/h) was stopped for 10 min to allow sample relaxation. The general form of fractograms from which the data were obtained is illustrated in Figure  5 , showing the detector signal.

Fractograms obtained by GrFFF, showing the peak profiles of: corn (A); wheat starch granules (B). From Ref.[ 110 ], with permission.

The employment of analytical techniques has rapidly increased in terms of the separation and characterization of macromolecules and micro-sized particles. One of the most widely used separation techniques is FFF, which has proved, over more than three decades, the ability to characterize supramolecular species in a size range spanning many orders of magnitude ( 104 ). In industrial fermentation processes, such as baking, wine making, brewing and potable and fuel grade alcohol production, yeasts are added as promoters. In the past few years, the FFF technique has been applied for the study of yeasts. More specifically, GrFFF has been applied for the characterization of yeast cells ( 105 , 106 ) and the study of the viability and activity of Saccharomyces cerevisiae strains during wine fermentation ( 107 , 108 ).

Sanz et al. ( 107 ) used GrFFF to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus . It was demonstrated that during the wine fermentation process, differences in the elution curves and peak profiles during fermentation can be related to the different cell growth stages. Moreover, the different fermentation kinetic profiles of yeast strains could be correlated with the corresponding fractograms monitored by GrFFF.

The effective capability of GrFFF to characterize wine-making yeast was further investigated by Sanz et al. ( 109 ) on four different types of yeast (Cryoaromae, Fermol Rouge, Awri 350 and Killer). Figure  6 A shows the fractogram of the yeast sample Fermol Bouquet, which shows a broad profile with two maxima that can be ascribed to a multimodal character of the distribution of the Fermol Bouquet yeast population. Figure  6 B shows a bimodal distribution of the size of the sample population.

Fractogram of the yeast sample Fermol Bouquet showing the three collected fractions: Fraction 1 from 4.5 to 10 min, Fraction 2 from 10 to 12 min, Fraction 3 from 12 to 17 min (A); size distribution and scanning electron micrograph of the Fermol Bouquet population, obtained under the conditions described in the “Experimental” section (B). Total cells counted from 2 to 7 mm: 11,618. The fractions in Figure 6A are tentatively indicated on the Coulter size distribution pattern. From Ref.[ 109 ], with permission.

Lainioti et al. ( 108 ) used GrFFF to study the effect of temperature (15, 20, 25 and 30°C) on the fermentation kinetics and the proliferation of the alcohol-resistant and psychrophilic Saccharomyces cerevisiae (AXAZ-1) yeast strains in the presence or absence of wheat starch granules as immobilization carrier. As shown (Figure  7 ), fermentations at 30°C for immobilized cells indicated a monomial yeast cell size distribution, for 0 h < t < 2 h, which was kept almost constant, according to the values of retention volume ( V r ) which correspond to the maximum points of the resolution peaks.

Fractograms obtained by GrFFF at T = 30°C and pH5.0 for: free cells at t = 0, 6.5 and 18 h (A); cells immobilized on wheat starch granules at t = 0 h, 6.5 and 12 h (B). From Ref.[ 108 ], with permission.

This fact reveals an initial lag phase during which yeast is adjusting to its new environment and beginning to grow in size. Fermentations at 15, 20 and 25°C began more slowly, as observed by their longer lag phase (monomial distribution until 4.5 h) and the slower fermentation rate. As the fermentation proceeded at 30°C, the yeast cells tended to cluster together (flocculate) and a bimodal size distribution was observed. At this point, the yeast began to reproduce rapidly and the number of yeast cells increased (thus known as the growth phase). However, the growth phase was retarded at 15, 20 and 25°C. At 30°C, and for t > 8.5 h, a monomial distribution appeared again, showing a steady-state period in which the size of the strains remained almost constant. According to the values of V r for free cells, the way of cell proliferation proved to be almost similar to that of immobilized cell systems. The differences in the form of the fractograms are primarily due to the absence of the wheat starch granules in the culture medium. The results, in general, indicated that both systems (free and immobilized cells) performed better at 30°C, whereas at lower temperatures, decreases were observed in the fermentation rate and in the number of viable cells. The activation energy for the fermentation process was reduced in the case of immobilized cells compared with free cells.

GrFFF was also applied to study the influence of pH and initial glucose concentration on the growth of the Saccharomyces cerevisiae yeast strain in the presence or the absence of corn and wheat starch granules as immobilization carriers ( 110 ). Fermentations were conducted at different values of pH (3.0, 4.0, 5.0 and 6.0) and initial glucose concentrations (177, 205, 247 and 300 g/L) to find the most favorable situation for the growth and proliferation of Saccharomyces cerevisiae cells. The results indicate that the growth of yeast cells was enhanced at pH 5.0 and glucose concentrations of 177 and 205 g/L. Higher glucose concentrations (247 and 300 g/L) acted as inhibitors to cell proliferation. Immobilization on wheat starch provided wider peak profiles, suggesting a broad size of cells and lower concentrations of haploid cells than cells immobilized on corn starch granules. The distinction of the phases of the yeast cell cycle was also accomplished through the GrFFF technique. For further confirmation of the results, the Michaelis-Menten model was used to calculate the Michaelis-Menten constant, K m , and the maximal velocity, V max , which are indicative factors for the affinity of the enzymes or cells to the substrate and the number of enzymatic or cellular molecules. The change of yeast populations was studied through the variation of the peak profiles in the fractograms, because the GrFFF technique is able to detect changes during the cell cycle. Differences observed in the elution curves can be related to the different cell size distribution, the unlike cell density and the morphology and/or shape ( 93 ). Figure  8 shows some selected fractograms for free and immobilized cells at different fermentation time periods during the alcoholic fermentations at pH 5.0 and glucose concentration of 205 g/L.

Fractograms obtained by GrFFF at pH 5.0 and glucose concentrations of 205 g/L at t = 2h, 6.5 and 8.5 h for free (A) and immobilized cells: on corn (B); wheat starch granules (C). From Ref.[ 110 ], with permission.

The fractograms of Figure  8 show that during the alcoholic fermentations of free cells at glucose concentrations of 177 and 205 g/L (Figure  8 A), the growth phase appeared at 4.5 h, whereas the stationary and decline phases appeared at 8.5 and 12.0 h, respectively. However, with the increase of glucose concentrations (247 and 300 g/L), the phases were slowed due to the growth-inhibitory effect. During the fermentations of cells immobilized on corn starch (Figure  8 B), a bimodal yeast cell size distribution (lag phase) was observed at 0 h < t < 2.0 h for a glucose concentration of 205 g/L, which was kept almost constant. As the fermentation proceeded, the growth phase (trinomial distribution) appeared at 2.5 h < t < 7.5 h, and it was followed by a stable distribution, which represented the stationary phase (8.0 h < t < 10 h). The decline phase followed from 10.5 h until the end of the reaction, with a bimodal distribution taking place again. Fermentations at 177 g/L proceeded almost the same as that at 205 g/L, whereas fermentations at 247 and 300 g/L began more slowly, shown by their longer lag phases (lag phase until 6.5 h for 247 g/L and 10 h for 300 g/L). The growth phase was slowed (7.0 h < t < 12 h for 247 g/L and 10.5 h < t < 20 h for 300 g/L), and it was followed by a stable trinomial distribution, revealing the stationary phase at 12.5 h for 247 g/L and at 20.5 h for 300 g/L. The decline phase appeared at 19 and 29 h for concentrations of 247 and 300 g/L, respectively. For cells immobilized on wheat starch at glucose concentration of 205 g/L (Fig.  8 C), the growth phase appeared at 2.5 h < t < 6.5 h, stationary phase at 7.0 h < t < 9.0 h and decline phase at 9.5 h, which lasted until the end of the reaction.

In the fractograms of free cells, the second elution peak corresponds to the daughter cells, which have been separated from mother cells (first elution peak) during the cell reproduction. To relate the different GrFFF profiles with the kinetics of the fermentation processes, the latter were monitored by the determination of fermentation time and reducing sugar contents (determination of Michaelis-Menten constants) at different time periods. The lowest value of Km for free and immobilized cells was found at pH 5.0 showing the high affinity of cells to the substrate at this pH value.

Combination of RFGC and FFF with other chromatographic techniques

The combination of RFGC with GrFFF can enrich the information concerning the kinetics of the fermentation process. In a study conducted by Lainioti et al. ( 16 ), the GrFFF and the RFGC techniques were used for the kinetic study and distinction of the growth phases of AXAZ-1 cell proliferation during alcoholic fermentations conducted under different environmental conditions. The use of GrFFF for the determination of elution curves and retention volumes, in combination with RFGC, through which reaction rate constants were calculated, showed that the distinction of the phases of the AXAZ-1 cell cycle with the GrFFF presented similar times to the distinction of alcoholic fermentation phases conducted with the RFGC technique.

Moreover, by the combination of the previously mentioned techniques (GrFFF and RFGC) with HPLC ( 16 , 110 ), through which sugar consumption was determined, the maximal fermentation rates and the time of maximal populations were determined, which appeared in the growth phase. Finally, the application of both GrFFF and RFGC techniques in combination with HPLC led to the determination of the ideal experimental conditions (temperature and pH) for alcoholic fermentation with AXAZ-1.

Advantages of RFGC

A detailed kinetic study of alcoholic fermentation in a factory, following the various steps that lead to the final product, may provide valuable information about the mechanism of the whole process. This could help to reduce the total time of the process, thus lowering the cost of the ethanol produced. A mechanistic study of this kind cannot be based on conventional methods of chemical analysis, like distillation or traditional GC, because these methods may alter the composition of samples taken at intermediate times, which will lead to erroneous conclusions regarding the mechanism. Two possible sources of error are the relatively high temperature used in the analysis and the effects of the filling materials used to pack the chromatographic columns on the analysis mixture. The RFGC technique does not suffer from these two sources of error and can be used for the present purposes. It is a flow perturbation method that has been reviewed ( 61 , 111 ) and described in more detail in two books ( 63 , 112 ).

Moreover, although conventional GC with FID or MS detection has repeatedly been used for measuring both ethanol concentration and by-products during the fermentation process, RFGC can successfully be applied for the determination of not only the ethanol concentration, but also of physicochemical quantities such as reaction rate constants and activation energies related to the kinetics of each phase of the alcoholic fermentation. This will lead to substantial information referring to the evaluation of the catalytic activity of biocatalysts.

Advantages of GrFFF

GrFFF is a simple, accurate and low-cost technique, with the ability to monitor the proliferation of Saccharomyces cerevisiae cells through their elution profiles. As it has been demonstrated ( 107 ), differences in the elution curves and peak profiles during fermentation can be related to the different cell growth stages.

Because the GrFFF technique has a significant number of applications on yeast cells, it has also proved to be a useful tool for the study of the effects of experimental conditions (pH and temperature) on AXAZ-1 growth kinetics.

GrFFF is suitable to determine the variation of the size distribution of a cell. In combination with other analytical techniques, such as RFGC and HPLC, useful information can be drawn for the monitoring of Saccharomyces cerevisiae (AXAZ-1) cell proliferation and the kinetic parameters during alcoholic fermentation.

The present work gives a broad overview of the methodologies used for the kinetic study of alcoholic fermentation. The increasing interest in the industrial applications of alcoholic fermentation has led to the employment of various kinetic models for cells suspended in batch or continuous operations. Parameters such as maximum specific growth rate, biomass concentration, fermentation and substrate uptake and dilution rate, have been measured for steady-state fermentation. For many batch processes, it is very important to define the optimum conditions to achieve sufficient profitability. For this reason, experiments have been conducted with specific kinetic models testing pH and temperature conditions, initial glucose concentrations of culture medium, the growth of yeast cells, ethanol production and many other parameters. The development of appropriate mathematical models can be considered a good approximation for describing the cell growth cycle and the kinetics of fermentation.

In recent years, intensive studies have been conducted regarding the kinetic study of alcoholic fermentation by chromatographic techniques. Conventional GC and HPLC have been extensively used for ethanol concentration and sugar uptake. The composition and content of odor compounds that determine the quality of alcoholic products have been characterized by GC–MS–O. Distilled alcoholic beverages, wines and beers, are often characterized by GC or GC–MS analysis of fatty acids and their esters, which contribute to the taste and quality of these commodities.

Specific attention is also placed on some new approaches for the kinetic study of alcoholic fermentation. The review presents RFGC, a simple and precise technique, which leads with great accuracy to the determination of reaction rate constants and activation energies at each phase of the alcoholic fermentation process, characterizing the catalytic activity of yeast cells. Moreover, GrFFF has been applied for the characterization of yeast cells and the study of the viability and activity of Saccharomyces cerevisiae strains during wine fermentation. Differences in the elution curves and peak profiles during fermentation can be related to the different cell growth stages. The combination of RFGC and GrFFF with other chromatographic techniques can provide valuable information about ethanol fermentation, growth cycles of yeast cells, sugar consumption and other parameters of vital important for the continuous fermentation process.

Although many chromatographic techniques and appropriate mathematical models have been used for the kinetic study of alcoholic fermentation, there is still need for improvement in recognizable techniques. Further research is still required for the optimization of functional parameters that are very important, considering the possibility of implementing new approaches for the kinetic study of the fermentation processes into industrial practice.

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