Introduction
Microbiology have played a major role in beer production ever since the old Egyptians came up with the first beer-like recipe. Throughout the years understanding and discovery of the microbiological processes in beer production been key to where we are today. In this report, I will study the major discoveries in microbiological processes since old age and how they have differentiated through time until today. I have selected this project because of my interest in microbiology but also learning how scientists through time have developed new techniques and if we are using some of the same techniques as we did in old age. When was yeast discovered as a major role in beer production and what types of microbes have and is still being used? Microbiology is used in every step of the initial beer production and it is essential that we treat every step with great care or the risk of beer spoilage will get bigger and bigger. When and how did we domesticate yeast and what challenges lies ahead?
The goal is to study different microbes, their domestication and the addition of hops used in beer production today.
The discovery of ATP
Around 1929, Karl Lohmann, Yellapragada Subbarao, and Cirus Friske discovered an essential molecule for life called adenosine triphosphate in animal tissues. ATP is a molecule used by almost all cells in living organisms. It is chemical energy and we use it for all physical movement. It is also required for many chemical reactions, such as sugar degradation and fermentation.
Glycolysis is the metabolic pathway that converts glucose into pyruvate. It is the first major step of fermentation or respiration in cells. The pathway was developed about 3.5 billion years ago when no oxygen was available from in the environment. The process glycolysis does not only occur in microorganisms but in every living cell. It’s an essential pathway for every cell and makes it possible for microorganisms to thrive in harsh environments.
Because the metabolic pathway is so important not only for microorganisms but for humans as well, glycolysis had very high priority to be understood by biochemists. Scientists faced a major challenge as they began analyzing the many chemical reactions that took place and in what order in the cell. The scientists found out that in glycolysis, a single molecule of glucose which has 6 carbon atoms is within the pathway transformed into 2 molecules of pyruvic acid, each with 3 carbon atoms. This means that there is no loss of atoms and therefor very efficient.2
To understand glycolysis, scientists began analyzing and purifying zymase by breaking down component of cell-free extracts. This was where they first detected cozymase which has a low molecular weight and is heat stable. Cozymase has later been referred to as Nicotinamide-adenine dinucleotide or NAD. Using chemical experiments, they concluded that zymase is a complex of different enzymes and cozymase is a mix of ATP and ADP together with NADH and NAD+ which are called coenzymes. ADP is a hydrolyzed form of ATP. They also found other components in cozymase such as metals and ions. All these components were required for fermentation to take place.
The full understanding of the glycolytic pathway was first fully understood and accepted in 1940. The pathway consists of ten chemical reactions where the result is 2 molecules of ATP for each molecule of glucose. Glycolysis consists of 2 redox-reactions where one molecule is oxidized by losing and electron and one molecule is reduced by obtaining the lost electron. NADH acts as an electron carrier and must be recomposed to contain the flow of the pathway.3
As mentioned above 1 glucose molecule is converted into 2 pyruvic acid during glycolysis. The cell can’t do much with pyruvic acid on its standard form but when oxygen is introduced pyruvic acid enters a cycle called the citric acid cycle also called Krebs cycle. It’s a series of chemical reactions used by all aerobic organisms to use stored energy via oxidation of acetyl-CoA which is derived from fats, proteins and carbohydrates into chemical energy and carbon dioxide. The chemical energy is in the form of ATP which is used for almost everything.4
In absence of oxygen or in anaerobic conditions there are 2 other pathways available for pyruvic acid. Depending on what type of cell it can be converted into alcohol through alcoholic fermentation or pyruvic acid can be converted into lactate through lactic acid fermentation. This was the problem when Buchner was helping Bigo with his fermentation problems.
Numerous microorganisms have been discovered and used to break down pyruvic acid to ethanol in brewing or wine making ever since Pasteur made his research public. Ethanol is not the only product that is used in the fermentation process. Also, the biproduct CO2 is used in many professions such as breadmaking and to carbonate beverages.
Microbial processes in beer production today
In 1950 two microbiologists called Green and Grey introduced the antibiotic cycloheximide that worked as a very powerful inhibitor of culture yeast. If cycloheximide was introduced to the fermenting beer wort the main organism which in this case was yeast could be suppressed. This allowed other contaminating microorganisms to grow and to be visualized in the culture media and act as an indicator for contamination. Cycloheximide was introduced into many selective media and did wonders for brewers who had problems with contamination from the 1950s through the 1980s. Many other types of growth media were developed during this period which was used to determine contamination of wild yeast and bacteria. Cycloheximide is now illegal to use in brewing. It is classified as an extremely hazardous substance due to its toxic side effects, including DNA damage and reproductive defects. Brewers will need to be aware of this and will now need to select the kinds of media that benefits the yeast culture best and reduces contamination. Today cycloheximide is used in biomedical research to inhibit protein synthesis and as a pesticide. Fortunately, the use of cycloheximide as a pesticide is decreasing as the health risks have become better known.5
Up until today we have developed new methods to optimize beer production and even industrialized all the processes. We have come a long way since Pasteur first discovered yeast cells and Emil Christian Hansen cultivated the first yeast cells. On a microscopic level, we now know the major parts of the fermentation processes and how each most yeast strains work. I will now go through most of the processes that the microorganisms accomplish during beer production.
While fermentation of cereal extracts by Saccharomyces is the most important part of beer brewing many other microbes affect the process. When you dispose of other microbes in every step of the barley to beer process it greatly influences the finished beer. Though all strains of Saccharomyces will produce ethanol during fermentation the different strains of Saccharomyces are classified into categories of lager and ale yeasts. Ale yeasts are Saccharomyces cerevisiae strains and are mostly referred to as top-fermenting yeasts. This strain has been isolated over the entire world and has pretty much capitalized the ale marked. Because the yeast rises to the top, its ready to be repitched for a new batch. The lager yeast strain is called S. pastorianus and is referred to as a bottom-fermenting yeast because it does not rise to the surface under any time during fermentation. 6
When it’s time for harvesting yeast cells a key mechanism is its flocculation behavior where yeast cells get attached to each other and form “balls” of yeast. This clumping of yeast cells involves binding of lectinlike proteins mannoprotein receptors which are promoted by calcium ions to overcome the negative potential. In certain malt types, there are factors present that may lead to premature flocculation of yeast while there may be antiyeast material in some malts as well. What modern techniques have also showed is the entire composition of not just CO2 and ethanol but also other side products that can affect flavor. It has been discussed that while there are many yeast strains, their gene complement does not vary widely from each other, and does not produce unique flavor components compared to each other. Variations are still present in levels of some products but there are very limited yeasts that produce flavoring compounds that other brewery strains do not. There is one exception though which is an ale strain used to produce hefeweizen products in Germany. This strain has a gene coding for ferulic acid decarboxylase, which converts ferulate from cereal cell walls to 4-vinylguaiacol which gives a spicy clove-like taste. All brewing strains produce glycerol, alcohols, esters, short-chain fatty acids, sulfur containing compounds and organic acids. The level of each category depends on the strain, but also the fermentation conditions. The fermentation itself is a monocultural microbial phenomenon that involves a succession of microbial steps that have big impact in the final product. Brewers now have the knowledge and can manage each step in the beer process which as a result have led to a big increase in beer quality. 6
Brewing microbiology starts in the barley field where microbial events both pre-harvest and post-harvest can have huge implications for brewing processes and beer quality. The microbes do not survive the malting and brewing process but the secretion from the organism can and may affect downstream quality. On barley fields, a vast range of fungi and microbes are present coexisting with one another. These organisms originate from the entire environment and are affected by weather and other conditions. If it is an unusually wet year this may result in a bigger microbial growth on the barley and lead to pathogenesis. After the harvest, the barley may be stored in antimicrobial conditions which prevents microbial growth since microbes persist after harvest. There are only a few plant-pathogenic fungi strains with relevance to beer quality. The fungus Fusarium and other fungal pathogens of barley can produce mycotoxins which are detectable in finished beer.
Malting is the process of 3 primary steps: steeping, germination and kilning. When the barley is steeped, microbial cells multiply fast in the water and on the grain, stimulated by moisture, dissolved nutrients, aeration and warmth. This step I often referred to as the most dangerous one for the grain since microbes on can compete with oxygen with the embryo in the grain and prohibit germination as well as decreasing α-amylase activity. Although kilning decreases the count of microbes, microbial activity during germination can influence the beer quality in a bad way. 6
Bacterial growth during mashing is decreasing by a large amount though thermotolerant microbes can stay active in the right environments. This may result in beneficial consequences but only to a certain extend. Acidification by lactic acid bacteria can improve the extraction, nitrogen yield of wort, fermentability, foam stability, color and flavor of the beer. Growth of bacteria may also cause serious problems during mashing. Bacillus spp. can cause excessive acidification and growth of Clostridium in the mash can produce high levels of butyric acid. The butyric acid can give the beer a cheesy aroma. Large amounts of bacterial growth on malt can terminate the mash filtration but suppression of bacterial growth has shown to improve the filtration, extraction efficiency, and nitrogen yield during mashing. To avoid bacterial growth the wort is boiled for an extended period which sterilize the wort. But once the wort leaves the kettle it will still be susceptible for contamination so it is important that the right percussions are taken. After the wort has cooled down strains of Saccharomyces are added to rapidly convert the wort to beer through fermentation of maltose and other sugar to ethanol and CO2. This results in a tough environment for other microorganisms which makes yeast the most dominating microorganism.
After fermentation, the environment consists of high ethanol concentration, CO2, low pH, oxygen, hop-derived antimicrobial compounds and residual nutrients though around 20% the sugars consist of oligosaccharides which is not utilized by Saccharomyces. This however, may contribute to the mouthfeel and flavor. The severe conditions of beer fermentation have selected for only a few groups of yeast and microbes that are able to grow in beer. Both gram-positive and negative bacteria are able to grow in beer fermentation. For gram-positive bacteria, they are unavoidable but thanks to the hop-derived compounds they are preventable to a certain degree. There are those who have adopted to the tough environment because they mostly have developed hop tolerance. The aerobic gram-negative acetic acid bacteria were once a major problem because oxygen exposure could not be avoided with the aging casks beer was stored in. Today we have the technology to avoid oxygen exposure and therefore avoid gram-negative bacteria.
During packaging and distribution beer faces the greatest challenges to keep the beer microbial stabilized. From all the previous processes, from wort boiling to cooling, the product has been contained in a very clean environment. During beer packaging the beer travels through a complex bottle filling system where the beer is exposed to the atmosphere and can become contaminated. Biofilm may also develop on the filler heads and spoil the beer. Kegs also pose a threat to spoil the beer since they circulate between breweries and are reused. The surface inside a keg is also hard to make it 100% clean because of the form of the keg. There has also been a higher demand of non-pasteurized kegs the last years and this further promotes microbial growth. 6
Domestication of yeast
Now, we know that many new microorganisms have been discovered and used in beer production through time. Since prehistoric times, humans have exploited the possibilities of the common baker’s yeast Saccharomyces cerevisiae to convert sugars into ethanol and desirable flavor compounds. We have also used yeast to obtain foods and beverages and prolong their shelf-life. The use of pure yeast cultures started after the pioneering work and research done by Pasteur and Hansen in the 19th century. Though early brewers, winemakers, and bakers had already learned that mixing unfermented foods with a small portion of already fermented food resulted faster and more predictable fermentations. The ‘‘backslopping’’ properly resulted in yeast lineages that kept growing in these man-made environments and after generations lost contact with their natural niches. This provided a perfect setting for domestication. However, it is still unclear since evidence for this hypothesis is still missing. Whether industrial yeast diversity was shaped by selection and domestication or neutral divergence caused by geographic isolation and limited biological dispersal is still a mystery.
Domestication is defined as breeding of wild species and human selection to obtain genetical variants of said species that thrive in man-made environments. If a domesticated species is reintroduced to nature it may behave sub optimally7. In some scenarios, the species will mutate and revert back to its wildtype. Typical signs of domestication are genome decay, polyploidy, gene duplications, chromosomal rearrangements, and phenotypes. These signs are resulting from human-driven selection which have been reported in livestock, crops and pets. After several studies which have investigated the S. cerevisiae population by sequencing the genomes of hundreds of different strains have providing a first look into the complex evolution of this species.
Most of these studies conducted have focused primarily on yeasts species from wild and clinical habitats and often only included a limited set of industrial strains originating from wine production. These studies mostly used haploid derivatives instead of natural strains and the result is lack of typical patterns of domestication like polyploidy, aneuploidy and heterozygosity. This have excluded a large fraction of industrial strains that have lost the ability to sporulate like most beer yeasts. Studies have also shown signs of domestication in wine strains where the strain have showed resistance to copper and sulfite. Both chemical element is used as a preservative.
A new study has sequenced 157 S. cerevisiae strains used for industrial production of beer, saké, spirits, bread, bioethanol and wine. The purpose of the study is to shed light on the origins, evolutionary history and phenotypic diversity of industrial yeasts and at the same time provide a resource for selection of more evolved and superior strains. The data revealed that industrial yeasts are genetically and phenotypically different from wild type strains and originate from a limited set of ancestral strains that have adapted to manmade environments. These strains were split into 5 different groups: one including Asian strains such as saké yeasts, one mostly containing wine yeasts, a mixed clade that contains bread and other yeasts, and two separate families of beer yeasts. Most groups did now show any geographical substructure but one beer strain was recognized to be used in England, central Europe and the United States dating back to the colonization. The strains from Europe and the United States, interestingly exhibit more clear hallmarks of domestication. The change from harsh and variable environment to more stable and nutrient-rich beer medium favored beer yeasts that was somehow specialized but this also led to genome decay, loss of a functional sexual cycle and aneuploidy where there are fewer or more chromosomes than usual. 7
Human selection
Evidence was also found for human selection, demonstrated by more efficient fermentation of beer-specific carbon sources. This is also known as convergent evolution. The more efficient way to utilize carbon sources results mainly through mutations and duplication of the maltose genes and nonsense mutations in PAD1 and FDC1 genes that produce and undesired flavor in beer. This also suggests that beer domestication was initiated hundreds of years ago, after the first production of beer but before discovering microbes. Today’s industrial yeasts are the product of many years of human domestication and without domestication we would still be struggling with beer stabilization, flavor and impurities. This experiment showed that today’s industrial S. cerevisiae yeasts are genetically and phenotypically separated from wild stocks because of trafficking and human selection. The thousands of industrial yeasts that are available today may stem from a few ancestral strains that somehow chosen or by coincident got into food fermentation and later evolved into new lineages. These lineages are now used in beer production for different purposes. 7
Lager strains and domestication
The main strain that is used when producing lager beers is called S. pastorianus. This strain is further divided in two main distinct lineages, which are referred to as “Saaz” and “Frohbjerg”. It could be suggested that the existence of these 2 lineages is the result of convergent domestication but the precise evolution and ancestry of the 2 lineages is still questionable.
S. pastorianus is the most used and dominating strain in the lager brewing industry and this suggests a strong selective advantage of the hybrid over the strains parental species. Studies have shown that S. pastorianus may be a hybrid between S. cerevisiae and S. eubayanus. It has also been argued that parts of the S. eubayanus genome promotes enhanced cold-tolerance and the S. cerevisiae subgenome have other advantages in brewing such as efficient fermentation and the exploitation of maltotriose. However, experiments show that maltotriose transporters from S. eubayanus and not S. cerevisiae enables some Saaz type lager yeasts to utilize maltotriose.10 Other studies have shown that certain S. cerevisiae strains also have enhanced adaptation to cold environments which must mean that the enhanced coldtolerance does not 100% originate from S. eubayanus, and that the origin of lager yeasts may have obtained different traits caused by a different ancestral foundation. Recent papers suggest that increased fitness of yeast hybrids can also be due to genetic disorders that confuses safeguard mechanisms that would normally limit growth in the parental strains. This could lead to hybrids that divide more and therefore have higher fitness in tough and stressful environments like beer wort at low temperatures. Specific roles of lager yeast subgenomes in beer production still require more research to untangle.7
The addition of hops
Hop is a plant that is thought to have originated in China and from there spread to the moderate climatic zones. Through the years hops have been used as a medicinal plant against constipation and blood purification.11 There have been found many old writing through the years explaining hops’ antimicrobial effect when added to food but also when used in brewing. Today hops is widely known for it’s role in beer. The main part that is used in beer are the cones containing α- bitter acid and β-bitter acid. Both compounds are hydrophobic and responsible for the bitterness and hoppy aroma in beer. The most important compound of the two is the α-bitter acid which is secreted from the lupulin glands inside the hop cones as a yellow powder. The reason why the α-bitter acid is so important is the isomerization of the compound, the iso-α-bitter acid. The isomerization of β-bitter acid is not nearly as important since it’s still insoluble in water. β-bitter acid still have biological activities that is used in medical applications.8 Though Iso-α-bitter acid is thought to be the most important compound in hops in beer production because of the iconic bitter flavor It adds to beer and it’s antimicrobial properties, the normal form of α-bitter-acid and β-bitter-acid help the spoilage of beer caused by unwanted microbes as well. The mechanism behind the inhibition of beer spoilage bacteria has been studied and is now known. It is affecting beer spoilage bacteria such as Lactobacillus, Streptococcus, Bacillus and staphylococcus which are known for causing unwanted aromas such as diacetyl that is the butter-milk-like flavor.9 The compounds are generally speaking very effective against gram-positive bacteria.11 The β-bitter acids and α-bitter acid is incorporated into the cell membrane of the bacteria where gates for the exchange of protons, catalyzing proteins and other important complexes are found. This may lead to a decrease in the proton motive force which affects uptake of nutrients and as a result the cell will experience starvation and in the end, death.
Discussion
It has been demonstrated that beer yeasts have been domesticated by continuing growth in man-made fermentation environments. There has been a strong selecting pressure on yeast over many generations and this has contributed to the selection of desirable phenotypes. It has also altered the genomic structure and genome stability of the domesticates which can be both bad and good for the yeast. Backslopping is not common to do anymore in most of today’s industrial breweries and instead, brewers get rid of their yeast culture after a few fermentations have been carried out and use a new stock culture for brewing afterwards. As brewers continue to reverse to the same yeast stock it ensures consistency of their product, but it also prevents further evolution of the beer yeast. That is also why evolution and domestication of beer yeasts within breweries almost have come to a stop. If brewers do not backslop, there is no way for a specific lineage to evolve and adapt to other nutrients or environments. The process has now moved to specialized labs, where new tools is invented and our knowledge of biochemistry and biology keep expanding. This opens new windows to develop new superior variants of Saccharomyces specifically tailored to different environments while also keeping it’s optimal production rate. Experimental evolution which is similar to the process in traditional brewing, can be used in together with techniques like crossing, marker-assisted breeding and mutagenesis. This can result in new phenotypic variants and combinations. These new phenotypes need to be tested to check if they have the desired traits for the brewers. Centuries after the first yeast domestication, industrial brewing companies have been introduced to biotechnology which have sparked a beer revolution which is now driving a new era of beer yeast domestication and evolution.7
Hops is effective against gram-positive beer spoilage bacteria but not against gram-negative and mutated bacteria. These microorganisms have developed mechanisms to increase their resistance against α-bitter acid and β-bitter acid. The gram-negativ bacteria have a much thicker cell membrane which results in reduced permeability of nonpolar compounds. This means that gram-negative bacteria are almost completely resistant to the hop compounds. Some bacteria have also mutated and have developed mechanics like a multidrug resistance pump, a proton motive force dependent transporter and with the help of ATPase to pump protons out of the cell. Also changes in the lipid complexes in the cell membrane of lactobacillus can cause resistance to hop compounds. Luckily Saccharomyces cerevisiae, the most popular yeast in brewing, is resistant to the antimicrobial effects of hops in moderate concentrations. This is an advantage when brewing beer because it keeps the beer relatively safe from beer spoilage caused by other microbes. 8
Conclusion
Beer production though the years have changes dramatically since, perhaps by incident, the first fermentation of fruits were discovered. Since then we have developed better equipment and discovered ways to improve the production. Ever since Pasteur discovered yeast cells other scientists were drawn to the subject to further investigate the processes behind fermentation. Today we know a whole lot more about what microbes have to do with fermentation but also how to prevent undesired microbes. New yeast strains have been discovered and developed to produce certain beer styles. All strains that are used today commercially have at some point in history been domesticated. If they were to be reintroduced to nature they would not survive since they have adapted to a very different environment. Specific strains have also been genetically modified to produce a certain type of beer though the most common strains may have been a subject to human selection to obtain the best traits for beer production. The addition of hops has not only contributed to the great taste of beer but also kept the beer “safe” from other beer spoilage microbes. We have come a long way since the discovery of fermentation and as we keep optimizing conditions, environments, production and strains, beer production will only become better in the future.