Brettanomyces spp. are known for their important role in the production of Lambic and specialty sour ales, along with the secondary conditioning of a particular Belgian Trappist beer (Van Oevelen et al., 1976; Marten 1996; Vanderhaegen et al., 2003). Over the past decade, Brettanomyces spp. have seen an increasing use in the craft-brewing sector of the industry with a handful of breweries having produced beers that were primary fermented with pure cultures of Brettanomyces spp. This has occurred out of experimentation as very little information exists regarding pure culture fermentative capabilities and the aromatic compounds produced by various strains. Dekkera/Brettanomyces spp. have been the subjects of numerous studies conducted over the past century although a majority of the recent research has focused on enhancing the knowledge of the wine industry. This thesis is intended to provide a greater knowledge of the Brettanomyces strains available in the brewing industry through focusing on strain specific fermentations and identifying the major compounds produced during pure culture anaerobic fermentation in wort.
Taxonomical Classification
The taxonomy of the genus Brettanomyces has been debated since its early discovery and has seen many re-classifications over the years. Early classification was based on a few species that reproduced asexually (anamorph form) through multipolar budding (Custers, 1940). Shortly after, the formation of ascospores was observed and the genus Dekkera, which reproduces sexually (teleomorph form), was introduced as part of the taxonomy (Van der Walt, 1984). The current taxonomy includes five species within the genera of Dekkera/Brettanomyces. Those are the anamorphs Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, and Brettanomyces nanus, with teleomorphs existing for the first two species, Dekkera bruxellensis and Dekkera anomala (Kurtzman and Fell, 2000; Cocolin et al. 2004; Oelofse et al., 2008). The distinction between Dekkera and Brettanomyces is arguable with Oelofse et al. (2008) citing Loureiro and Malfeito-Ferreira from 2006 when they affirmed that current molecular DNA detection techniques have uncovered no variance between the anamorph and teleomorph states. Modern use throughout the industry includes two species of Brettanomyces available from yeast companies. The two species are B. bruxellensis and B. anomalus with the majority of the strains being B. bruxellensis. Any other names used are synonyms derived from old nomenclature no longer recognized. Throughout this text, species are referred to as the original authors used in their publications, with all cultures used in this study referred to by the strain name designated by the yeast company they were sourced from. As this study pertains to the use of these yeasts in the brewing industry and the accumulation of biomass is through the asexual budding of yeast cells, it seems appropriate to refer to the genus as Brettanomyces, while technically incorrect.
Initial Discovery and Characterization
The earliest published account came from a paper presented to the Institute of Brewing in which Claussen (1904) described the discovery of a newly isolated yeast that he proposed be called Brettanomyces, and was responsible for the secondary fermentation and development of characteristic flavors and aromas of the finest English stock ales. The first systematic investigation was conducted by Custers and in 1940 he presented his findings on 17 strains of Brettanomyces spp., which he believed to only be found in English and Belgian beers (Henrici, 1941). Custers extensive studies revealed the fermentation of glucose to ethanol occurred more rapidly under aerobic conditions then anaerobic conditions, a phenomenon which he termed “negative Pasteur effect” (Henrici, 1941; Skinner, 1947; Barnet et al., 2005). Custers findings included observing considerable amounts of acetic acid, produced during aerobic fermentation, while no appreciable amounts formed during anaerobic fermentation. Further observations under anaerobic conditions led Custers to believe cells slowly became adapted to anaerobic conditions with a normal anaerobic fermentation ensuing (Skinner, 1947).
In a later study carried out by Wiken et al. (1961), the existence of a negative Pasteur effect was observed in young cells of all Brettanomyces spp., as glucose metabolism occurred at a higher rate in the presence of oxygen while a decrease in glucose consumption was measured when switching the cultures to anaerobic conditions. In the same year, Scheffers (1961) found anaerobic fermentation could be stimulated by minute amounts of O2 or the addition of H+-acceptors such as acetaldehyde, acetone, pyruvic acid and other carbonyl compounds. The term “Custers effect” was later introduced for negative Pasteur effect in Brettanomyces spp. by Scheffers and Wiken (1966; 1969), and further expanded by Scheffers and Misset in 1974 who proposed Custers effect occurred as a result of the net reduction of NAD+ to NADH and was due to an oxidative side reaction causing the formation of acetic acid (Scheffers, 1979). The metabolism of acetaldehyde in Brettanomyces spp. under anaerobic conditions has not been thoroughly studied, while under aerobic conditions Carrascosa et al. (1981) found support for the mechanism of Custers effect being linked to the formation of acetic acid through the oxidative conversion of acetaldehyde. Custers effect can then best be interpreted in terms of glycolytic activity, as cells are switched from aerobiosis to anaerobiosis, glycolysis is temporarily static as is shown from the lack of glucose consumption and CO2 production (Wijsman et al., 1984). Building on Scheffers earlier studies, Wijsman et al. (1984) found when Brettanomyces cells were introduced to an anaerobic environment a transient lag phase of up to ten hours was observed before the slow dissimulation of glucose and subsequent production of CO2 resumed. The mechanism responsible for the slow adaptation of the metabolism is not understood, although these findings are in agreement with other studies that have suggested anaerobic fermentation is possible with sluggish or slow activity occurring (Custers, 1940; Scheffers, 1961; Martens, 1996; Ciani and Ferraro, 1997; Aguilar Uscanga, 2003; Passoth et al., 2007; Garcia Alvarado et al., 2007).
Glycerol Production
The lag phase and subsequent Custers effect is created through the continued drainage of NAD+ by way of the irreversible conversion of acetaldehyde to acetic acid bringing glycolysis to a stop due to the lack of NAD+ (Wijsman et al., 1984). Wijsman (1984) further concluded that Custers effect observed in Brettanomyces spp. was caused by the inability of cells to restore the redox balance via production of reduced metabolites, specifically glycerol, possibly attributed to the absence of glycerol 3-phosphate phosphatase activity. The production of glycerol has been shown to be important in metabolizing NADH and restoring the redox balance during anaerobic fermentation in other yeasts (Oura, 1977). Further it is known that glycerol produced by yeast has a secondary role in enhancing the perception of body and mouthfeel in beer when 1-2 g/l are present (Boulton and Quain, 2006, p.120). Anecdotal reports of beers lacking body and mouthfeel when fermented with pure culture strains of Brettanomyces have been an issue and is possibly caused by the lack of glycerol produced during fermentation. A recent study by Aguilar Uscanga et al. (2003) observed slight glycerol production occurring under anaerobic conditions within a single strain of Brettanomyces bruxellensis. Glycerol production might then be variable between strains indicating the ability of the redox to be restored more rapidly, in which case strains would likely be capable of full fermentation with a decrease in the time required.
Growth Related Factors
The role of oxygen on growth and acetic acid production by Dekkera/Brettanomyces spp. has been carefully studied in wine and industrial alcoholic fermentations (Ciani and Ferraro, 1997; Abbott et al., 2005a,b). Moderate to low amounts of oxygen has been shown to encourage cell biomass production, with semi-aerobic conditions yielding the greatest cell growth (Aguilar Uscanga et al., 2003). The levels of ethanol and acetic acid produced during aerobic batch culture are dependent on the levels of aeration with increased concentrations of oxygen lowering growth rates (Aguilar Uscanga et al., 2003). Brettanomyces spp. have been observed to utilize both glucose and ethanol in producing acetic acid under increased levels of oxygen, although Freer (2002) showed not all strains could use both as carbon sources, with high variability seen in the levels of acetic acid produced by different strains. Temperature has also been shown to have an impact on growth, with higher temperatures decreasing the time required to reach maximum cell concentration, with optimal growth rates observed between 25 and 32°C (Brendam et al., 2008). Further analysis by Brendam et al. (2008) showed that increasing the temperature changed the speed at which metabolic compounds were produced but did not change the quantity of metabolites produced.
Fermentation Capabilities and the Enzymes Involved
Brettanomyces spp. are associated with highly attenuated beers and are known as over attenuating yeasts (Shantha Kumara et al., 1993). Not all strains appear to be capable of over attenuation as Custers observed strains of Brettanomyces anomalus that were unable to ferment maltose (Gilliland, 1961). Gilliland (1961) further observed an assortment of Brettanomyces strains capable of fermenting simple monosaccharides and some disaccharides, but unable to ferment maltose, asserting these strains were most like Brettanomyces anomalus in behavior. In a table produced by Gilliland (1961), the closely related Brettanomyces claussenii strains were shown to be capable of fermenting maltose and shared a common component with the Brettanomyces anomalus strains in that both were capable of fermenting lactose and cellobiose. The current taxonomical system makes no distinction between these species, and it would appear current Brettanomyces anomalus strains differ in their ability to ferment maltose. Spindler et al. (1992) observed a Brettanomyces custersii strain capable of fermenting cellobiose to ethanol, exhibiting a higher utilization then other Brettanomyces claussenii strains used in the study. The presence of b-glucosidase, the enzyme responsible for the hydrolysis of cellobiose in Brettanomyces spp. has been shown by Daenen et al. (2007) to be present at varying levels in Brettanomyces spp. This same enzyme is also capable of hydrolyzing flavor active compounds from glycosides present in beer from hops or fruit and shows great potential for use in bioflavoring (Daenen, 2007). Brettanomyces spp. are most commonly cultured from beer conditioned in oak barrels and a symbiotic relationship has been assumed given the longevity of their existence in the barrels, yet most of the Brettanomyces spp. which dominate during Lambic brewing do not have the necessary b-glucosidase enzyme present to hydrolyse cellobiose (Vanderhaegen, 2003). A Brettanomyces lambicus strain isolated from a typical over attenuated Lambic beer was shown to possess both extracellular and intracellular a-glucosidase activity (Shantha Kumara et al., 1993). The a-glucosidase enzymes from B. lambicus were capable of hydrolyzing wort dextrins with up to 9 degrees of polymerization while both the extracellular and intracellular enzymes showed similar activity for a range of carbohydrates (Shantha Kumara et al., 1993). These enzymes appear to be responsible in part for the over attenuation observed by Brettanomyces yeast.
Production of Secondary Metabolites
The ability of Brettanomyces spp. to influence the flavor and aroma of beer is well known (Gilliland, 1961), though the characteristic compounds produced during pure culture fermentation have been relatively under studied. Many organoleptic descriptors are used to describe the often-pungent aromas of Brettanomyces spp. Those include clove, spicy, horsey, barnyard, smokey, medical, band-aide, metallic, cracker biscuit, goat-like, apple, floral, tropical fruit and citrus. While these descriptors are generally associated with Brettanomyces yeast, research from the wine industry has explained the origin of a few of these compounds. They have shown these compounds are formed from specific components present based on raw materials, and their production by Brettanomyces spp. can vary greatly.
Volatile phenolic compounds are responsible for some of the most recognized aromatic characteristics associated with Brettanomyces species. Heresztyn (1986b) found 4-ethylguaiacol and 4-ethylphenol formed during the fermentation of grape must with Brettanomyces spp. along with trace amounts of 4-vinylguaiacol. The wines contained strong spicy and clove aromas while GC effluent sniffing revealed spicy, clove-like aromas and spicy, smokey aromas which corresponded to the retention times of 4-ethylguaiacol and 4-ethylphenol respectively (Heresztyn, 1986b). It was noted during the study that in regions of the chromatogram where the spicy odor was fading, an apple cider-like character was often perceived. Further research by Heresztyn (1986b) showed the hydroxycinnamic acids, p-coumaric acid and ferulic acid, were metabolized to 4-vinylphenol, 4-vinylguaiacol and 4-ethylphenol, 4-ethylguaiacol respectively by Brettanomyces spp. Chatonnet et al. (1995) observed Brettanomyces spp. were capable of producing 4-ethyl phenolic compounds under conditions with little residual sugar during the later maturation process. Recently two enzymes responsible for the phenolic aromas associated with Brettanomyces spp. have been characterized. The first is a phenolic (cinnamic) acid decarboxylase, responsible for the decarboxilation of hydroxycinnamic acids into the respective 4-vinyl derivative and the second, a vinyl phenol reductase responsible for the reduction of the 4-vinyl derivative into its respective 4-ethyl derivate (Godoy et al., 2008).
Two forms of substituted tetrahydropyridines, 2-ethyltetrahydropyridine (ETHP) and 2-acetyl-3,4,5,6-tetrahydropyridine (ATHP), can be produced by Brettanomyces spp. Their metabolism involves the amino acid L-Lysine and ethanol with oxygen having a stimulatory effect in their production (Heresztyn, 1986a; Snowdon et al., 2006; Oelofse et al., 2008). The aromas associated with ATHP are similar to cracker biscuit, although under low pH conditions can be described as metallic or bitter (Oelofse et al., 2008). Little is known about these compounds and their production by Brettanomyces spp.
Esterases present in Brettanomyces spp. have been shown to have ester synthesizing activity along with the observed ability of these yeasts to accumulate high concentrations of the C8 to C12 fatty acids (Spaepen et al., 1978; Spaepen and Verachtert, 1982). In Lambic beer the higher fatty acids become esterified to ethyl esters during the same period Brettanomyces spp. dominate as the most prominent active yeast (Spaepen et al., 1978). Further analysis confirmed that the esterases present in Brettanomyces spp. are responsible for the formation of ethyl acetate, ethyl lactate and phenethyl acetate, along with the hydrolysis of isoamyl acetate (Spaepen and Verachtert, 1982). These esters represent the typical aromas and flavors of Lambic and are influenced by Brettanomyces yeast during the mixed yeast and bacterial fermentation. During pure culture fermentation less acetic acid and lactic acid are present therefore the ester fraction could be less typical then described during mixed microbial fermentations (Van Oevelen et al., 1976; Martens, 1996).
Project Scope
Most recently, delicate fruit descriptors have become sought after aromas having anecdotally been reported from wort fermentations using pure cultures of Brettanomyces spp. It is not known which strains create the pineapple-like ester characteristics, although it has become the goal of a handful of brewers to recreate techniques that have resulted in these light fruit attributes. A handful of theories exist for the possible source of the unique aromas with no study previously concentrating on a wide variety of secondary metabolites produced by Brettanomyces yeasts. It is well understood that raw materials exhibit a great influence over the behavior of yeasts and for that reason this study concentrates on the overall performance of these yeasts in association with malt based wort used in brewing. The aim of this study was to increase the knowledge of the Brettanomyces genus through providing information regarding fermentation performance and metabolite production during anaerobic fermentation. It was first necessary to study various agar medias that could be used for culture detection and enumeration of strains throughout the study. Next step was to observe growth during batch culture to predict the correct propagation methods used to maximize cell production. Two variables were observed during pure culture anaerobic fermentation, pitching rate and initial lactic acid concentration. Three different pitching rates were studied: 6×106, 12×106, and 18×106 cells/ml and five initial lactic acid concentrations: 0, 100, 500, 1,000, and 3,000 mg/l in order to evaluate their influence on the over all fermentation performance. The information provided during this study regarding strains available from commercial yeast companies allows conclusions to be made about the best use of each strain and methods which might maximize their ability to produce various aromatic compounds including the occasionally described pineapple and tropical fruit characteristics. Increased knowledge of the Brettanomyces genus will improve the utilization of this organism, whether it is for pure culture use, mixed culture use, or as a secondary conditioning yeast.