Fermentation

00_185556An important step in brewing is the fermentation process. It is here, alcohol is formed together with various flavor substances from sugars in the presence of yeast. The initial sugar content, temperature, and yeast type dictates how the fermentation proceeds.

Although all strains of Saccharomyces will produce ethanol as a fermentation end product, in practice the strains employed in the production of beers worldwide are classified into the categories of ale and lager yeasts.

20140511_204054Ale yeasts, which are Saccharomyces cerevisiae strains, are the more diverse yeasts and have been isolated in innumerable locations worldwide. Such yeasts are often referred to as “top-fermenting” yeasts, insofar as in traditional open fermenters they rise to the surface of the vessel, facilitating their collection by skimming, ready for repitching into the next fermentation. The hydrostatic pressure in modern cylindroconical fermenters, many of which may contain up to 10,000 hl of fermenting beer, tends to overcome this tendency of ale yeast, which accordingly collects in the cone of the tank.

The nomenclature of lager yeast (“bottom-fermenting” yeast, on account of its tendency not to rise to the surface under any set of fermentation conditions) has evolved, passing through iterations of S. carlsbergensis and S. cerevisiae lager type to the currently accepted name, S. pastorianus. Irrespective of its name, lager yeast is a more complex organism than ale yeast, and it has been proposed that it arose in perhaps two separate steps involving the hybridization of S. cerevisiae with S. bayanus.

It has generally come to be considered that lager yeast (unlike ale yeast) is not readily isolable from nature, though it was recently proposed that the cryotolerant strain of yeast that melded with S. cerevisiae in domestication circumstances to produce S. pastorianus originated in southern beech forests in Patagonia and represents Saccharomyces eubayanus sp. nov.

There is far more diversity among ale strains than among lager strains. The latter can be divided into the Carlsberg and Tuborg types, based on chromosomal fingerprints, and there are comparatively minor differences between them. Casey suggests that this far greater diversity of ale strains reflects their isolation in multiple locations, whereas the lager strains emerged from a very limited locality.

The genome of S. cerevisiae has been sequenced fully. Whereas the strains used for sequencing were haploid, brewing strains of yeast are polyploid or aneuploid, with 3 or 4 copies of each chromosome. There is only limited information on the significance of this for yeast behavior, with one of the few studies being that of Galitski et al, who found very few effects.

It is generally believed that the multiplicity of gene copies makes for a more stable yeast organism, and there may be a boost of enzyme production leading to more rapid metabolism of wort components, e.g., maltose. There appear to be some fundamental differences between the chromosomes in haploid and polyploid strains. Despite the polyploid nature of brewing strains, there is evidence that there is chromosomal instability. Repercussions include changes in flocculation and utilization of maltotriose. Yeast drift can also arise through the partial or complete loss of mitochondrial DNA, leading to the production of so-called “petites”. Although alcoholic fermentation is anaerobic, meaning there is no role for a respiratory function in mitochondria, the latter organelles do have other metabolic functions in brewery fermentations.

Typing of YeastThe differentiation of brewing strains has been reviewed by Quain and Casey et al. Traditional approaches include examining colony morphology on plates, the ability of yeasts to metabolize melibiose (lager strains can do so due to their elaboration of an α-galactosidase, whereas ale strains cannot), temperature tolerance, flocculation tests, behavior in small-scale fermenters, and oxygen requirements. Latterly, the emphasis has been on DNA-based techniques, including restriction fragment length polymorphism analysis, PCR, karyotyping, and amplified fragment length polymorphism analysis. Additionally, pyrolysis mass spectroscopy, Fourier transform infrared spectroscopy, fatty acid methyl ester profiling, and protein fingerprinting are other possibilities.

Yeast Resources and Handling

Several yeast culture collections and providers are available. Larger brewing companies, however, tend to manage their own in-house strains, including the storage of master cultures. Back-ups of these organisms are deposited with third parties. Storage of cultures in liquid nitrogen is deemed preferable in terms of survival, shelf life, and genetic stability compared to storage on agar, in broth, or by lyophilization.

While there are still brewers who simply repitch yeast from one fermentation to the next ad infinitum (“backslopping”), concerns about genetic drift and selection of variants mean that most brewers pitch with yeast newly propagated from the master cultures at intervals. 20151016_201423The frequency is typically 10 to 15 “generations” (this word in a brewing context refers to successive fermentation batches), though even this may be excessive in terms of yeast deterioration. The chronological events occurring in the life cycle of yeast in brewery fermentations and the consequences for population ageing have been addressed.

Yeast propagation, involving batches of successively increasing volumes, has been reviewed by Maule and Quain. Yields of biomass can be limited at the high sugar concentrations employed (Crabtree effect), and some have advocated fed-batch systems analogous to those used in the production of baker’s yeast. Gene transcription during propagation and fermentation has been investigated. Newly propagated yeast does not usually “perform” as expected in the initial commercial fermentation, in part due to a lack of synchronicity in the cell population.

An alternative approach to handling yeast that is attracting some attention in brewing but which is already applied widely in wineries is the use of dried yeast. Concerns include an impaired ability to handle vicinal diketones (VDKs see below), impaired flocculation of yeast, and deteriorating foam and clarity in the beer.

Key to successful storage and handling of brewing yeast, irrespective of whether it is handled as a slurry or as a dried product, are the storage carbohydrates that it elaborates. Glycogen has attracted much study as an important carbon and energy reserve in brewing yeast, while the importance of trehalose as a stress protectant is well studied.

Fermentation control in pursuit of a constant fermentation performance, brewers seek to achieve consistent fermentations, which demands control of the key variables of yeast quantity and health, oxygen input, wort nutritional status, temperature, and yeast-wort contact (mixing).

While traditional techniques for counting yeast, such as counts with a hemocytometer, are still widely applied, there is increasing use of instrumental approaches, often inserted in-line to achieve automated pitching control. Devices include those operating on the basis of assessing capacitance/permittivity and according to principles of light scatter.

The viability of yeast has long been assessed by staining of cells with methylene blue; however, other staining approaches have been proposed. While these techniques inform about whether cells are alive or dead, they do not gauge the healthfulness (vitality) of the cells. Diverse procedures have been nominated for assessing this parameter, but none has been adopted universally. Techniques include assessments of glycogen, sterols, ATP , oxygen uptake rate, and acidification power, as well as modifications of the methylene blue viability test.

While it has long been recognized that a proportion of oxygen is needed by all yeast cells to support the production of the sterols and unsaturated fatty acid components of the cell membranes, there is a less-than-clear appreciation of why different yeast strains vary considerably in the amount that they demand. Traditionally, the oxygen is introduced to the wort, although there have been proposals to pitch unaerated wort with yeast that has been supplied directly with oxygen. Ensuring contact of all yeast cells with oxygen when yeast is present at a high density is important. On the other hand, oxygen represents one of the stress factors encountered by yeast, while others include ethanol, which limits the practical alcohol concentrations that can be achieved in brewery fermentations. Accordingly, there is interest in the development of yeast strains with greater tolerance of high-gravity conditions. A review of all the stresses likely to be encountered by brewing yeast has been provided by Gibson et al. There is extensive use of high-gravity brewing in commercial brewing, with the attendant osmotic and alcohol stresses.

20151021_123515One major variable that perhaps receives less detailed analysis and control than others in fermenter control is actually the wort composition. Most brewers simply regulate the strength of the wort (degrees Plato) and pitch on that basis, assuming that the relative balance of the diverse nutrients within the feedstock is consistent and modulated by the malt selection and how that malt is processed in the brewhouse. To a first approximation, this seems to be a reasonable situation on an experiential basis, although there are two variables that many brewers do seek to regulate more closely, i.e., the clarity of the wort and the concentration of zinc ions, although other additions to promote fermentations, particularly those with higher-strength wort, may be employed. The presence of insoluble particles in wort (which are derived in the brewhouse and are present at a level in inverse proportion to the extent that they are removed in clarification stages prior to fermentation) promotes yeast action by their ability to nucleate carbon dioxide, thereby releasing bubbles. Two effects may be at play, namely, the increased resulting tendency of yeast to be moved through the fermenter and the impact that this has on lowering dissolved CO2 levels in the wort from inhibitory concentrations.

The contact of yeast and wort in fermentation is not inconsequential. Often, huge fermenters are filled with several batches of wort, leading to quandaries over precisely when the yeast should be added to the fermenter and how to ensure homogeneity of yeast-wort contact throughout the vessel. Mechanical mixing is uncommon but advocated.

20151021_210737Fermentations may be monitored in various ways, including measuring the decrease in specific gravity of the wort (including in-process measurements), CO2 evolution, the pH decrease, and ethanol formation, as well as camera-based observation of events in the fermenter.

At the completion of fermentation, yeast is recovered either for disposal (commonly to animal feed or production of yeast extracts) or for repitching. For open fermenters, ale yeast is skimmed from the surface of the vessel, but for closed cylindroconical vessels the yeast is harvested from the cone. The population of yeast cells differs in the cone, with stratification such that older cells are located beneath the younger, more vital ones.

Harvested yeast may either be pumped to the next fermenter filling with fresh wort (cone-to-cone pitching) or stored in either a pressed or slurry form. It may receive acid washing to kill any bacteria that may have developed in the slurry. Its collection from fermenters is often through the use of centrifuges, creating damage that has implications for subsequent performance. The impact of serial repitching was addressed by Jenkins et al., who showed that extents of deterioration vary between yeast cells.

Flocculation

A key influence on harvesting of yeast is its flocculation behavior. The flocculation of brewer’s yeast was recently reviewed by Soares , Vidgren and Londesborough, and Verstrepen et al. The clumping of yeast cells involves the binding of lectin-like proteins to mannoprotein receptors, promoted by calcium ions to overcome the negative zeta potential. The surface hydrophobicity of the cell is also important, and this may relate to the tendency of cell aggregates to migrate to the surface of a fermenter (top-fermenting yeast). There are factors present in certain malts that lead to the premature flocculation of yeast (see below), and meanwhile, there may be additional antiyeast materials in malt.

Products of Yeast Metabolism in Brewery Fermentations

During fermentation, yeast excretes a range of molecules, in addition to ethanol and CO2, that can affect flavor. While there are diverse brewing yeast strains, it has been argued that the vast majority do not differ very widely in their gene complement such that they produce unique flavor components. Strain-to-strain variation exists in the levels of some products, but there are extremely limited instances of brewing yeasts procuring flavor-active species that are not produced to at least some extent by other brewery strains.

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