M.sc. Dissertation

Carbohydrate utilization in mixed culture fermentation
Amit Rajan Mishra H00023055 am720@hw.ac.uk
A dissertation submitted in partial fulfilment of the requirements for the degree of M.Sc. in Brewing and Distilling.
Dissertation supervisor: Dr. Annie Hill
The International Centre for Brewing and Distilling School of Life Sciences, John Muir Building Heriot-Watt University Edinburgh EH14 4AS 1
Declaration
I, Amit Rajan Mishra, confirm that I have read and understood what is meant by plagiarism and this M.Sc. thesis is my own and is expressed in my own words. Any uses made within it of the works of other authors in any form (e.g. ideas, equations, figures, text, tables, programs) are properly acknowledged at the point of their use and a full list of references cited is included.
Signature:
Dated: 30.12.2011
Acknowledgement
It is a pleasure to thank all who have helped me in making this thesis possible. Foremost, I would like to express my deepest gratitude to Dr. Anne Hill, my supervisor for her invaluable guidance, assistance and support in achieving the aims of this study. I would like to express my deepest gratitude to Mrs. Gina Shepherd, Mr. James Mackinlay and Ms. Vicky Goodfellow for their invaluable assistance and patience, without which all the laboratory project work would not have been possible. Also would like to thank Prof. David Hopkins who has given me this chance for which I am indebted. I would like to express my sincere gratitude to my family, and my friends for their unconditional love, care and support. Last, but not the least, a very special vote of thanks to my girlfriend Ipshita Chowdhury, Ph.D. student at School of Built Environment, who has guided and motivated me throughout this dissertation. Her confidence in my capabilities, enthusiasm and encouragement has been truly inspirational.
Contents
Declaration Acknowledgement 1. Abstract...9 2. Introduction11 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 Fermentation..... 15 Raw materials in potable alcohol production.17 Beer Production.18 Wine production19 Sugar uptake by yeast in pure cultures......21 Crab tree effect......21 Carbon catabolite repression..22 Genetic correlation of sugar transport and utilization in yeasts.....23 Monosaccharide transportation..17 Disaccharide transportation...26 Sugar uptake sequence in yeast..27 Mixed culture fermentations..28 Sugar Utilization in mixed culture fermentation...29 Scope of this study.31
3. Materials....32 4. Methodology..33 4.1 4.2 4.3 4.4 4.5 Yeast culture propagation..33 Yeast nitrogen base media preparation..34 Preparation of inoculums...36 Sampling and analysis38 Analytical techniques.40 4
5. Results....42 5.1 5.2 Tall Tube results 1-8..43 Observation of results....51 5.2.1 5.2.2 5.3 5.4 5.5 5.6 Pure culture analysis....51 Mixed culture analysis.52
Results of Small scale conical flask fermentations....53 Small scale conical flask fermentations result analysis.57 Head space Analysis..58 Evaluation of Head Space analysis results60
6. Discussion..61 7. References.64
List of Figures Figure 1: Factors influenced during brewing fermentation. Source: Munroe James H., Handbook of Brewing, Chapter 12, pg.49916 Figure 2: Schematic representation of red and white winemaking process. Source: Wine fermentation, Mills. et al., (2008), pp 164.. 20 Figure 3: A) Passive diffusion B. Proton Symport System. Source: Chemostat Cultivation as a Tool for Studies on Sugar Transport in Yeasts (Weusthuis et al., 1994) pp. 617... 25
Figure 4: The graph depicts the sugar utilization during a brewing fermentation, Source: Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation (Gibson et al., 2008).. 27 Figure 5: Graph depicting the sugar utilization in wines with different mixed cultures. Source: Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation (Howell et al., 2006) pp. 95..... 30 Figure 6: Aseptically inoculating strains for propagation in broth media.....33 Figure 7: Filter Sterilization of YNB media. 35 Figure 8: Tall Tube inoculated with different combination of yeast strains.37 Figure 9: Setup to withdraw samples from Tall tubes. 39 Figure 10: Graphical representation of the depleting sugars as determined by HPAE analysis in Pure Ale yeast S. cerevisiae culture. 43 Figure 11: Graphical representation of the depleting sugars as determined by HPAE analysis in Pure lager yeast S. cerevisiae culture fermentation......44 Figure 12: Graphical representation of the depleting sugars as determined by HPAE analysis in Pure Champagne yeast S. bayanus....45
Figure 13: Graphical representation of the depleting sugars as determined by HPAE analysis in Pure Wine yeast S. fructum culture.. 46 Figure 14: Graphical representation of the depleting sugars as determined by HPAE analysis in Wine yeast S. fructum and Ale yeast S. cerevisiae mixed culture....47 Figure 15: Graphical representation of the depleting sugars as determined by HPAE analysis in Champagne yeast S. bayanus and Ale yeast S. cerevisiae mixed culture48 Figure 16: Graphical representation of the depleting sugars as determined by HPAE analysis in Wine yeast S. fructum and Lager yeast S. cerevisiae mixed culture....49 Figure 17: Graphical representation of the depleting sugars as determined by HPAE analysis in Champagne yeast S. bayanus and Lager yeast S. cerevisiae mixed culture........ 50 Figure 18: Graphical representation of the depleting sugars as determined by HPAE analysis.. 53 Figure 19: Graphical representation of the depleting sugars as determined by HPAE analysis.. 54 Figure 20: Graphical representation of the depleting sugars as determined by HPAE analysis.. 55 Figure 21: Graphical representation of growth curves of pure and mixed cultures..56
List of Tables Table 1: Strain composition of yeast cultures used in mixed culture wine fermentation. Source: Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation (Howell et al., 2006). ..30 Table 2: Fermentation profile of ale yeast S. cerevisiae ...43 Table 3: Fermentation profile of lager yeast S. cerevisiae...44 Table 4: Fermentation profile of champagne yeast S. bayanus..45 Table 5: Fermentation profile of Wine yeast S. fructum...46 Table 6: Fermentation profile of mixed culture of wine yeast S. fructum ...47 Table 7: Fermentation profile of mixed culture containing Champagne yeast S. bayanus and Ale yeast S. cerevisiae .48 Table 8: Fermentation profile of mixed culture containing wine yeast S. fructum and lager yeast S. cerevisiae......................................... 49 Table 9: Fermentation profile of mixed culture containing Lager yeast S. cerevisiae and Champagne yeast S. bayanus. 50 Table 10: Sugar uptake data of S. cerevisiae lager strain mono culture as analysed via HPAE. .53 Table 11: Sugar uptake data of S. bayanus champagne strain mono culture as analysed via HPAE.. 54 Table 12: Sugar uptake data of S. cerevisiae lager strain and S. bayanus champagne strain mixed culture as analysed via HPAE....... 55
Table 13: Optical density measurements of Lager (pure), Champagne (pure) and Lager and Champagne (mixed) cultures at 660 nm 56 Table 14: Head space analysis reveal the concentration of some of the volatile compounds present in samples 59 8
Abstract: Wort is a complex blend of fermentable carbohydrates which when utilized by yeast under anaerobic conditions produces ethanol. Yeast strains differ in the type and rate at which they utilize the sugar, a direct influence of their genotype. As per the specification of the product that is to be created, we use a particular type of yeast to carry out fermentation of the raw material. Yeast plays an important role in imparting flavour to the product and the grand variety of yeast that is available in the market allows brewers and wine makers to experiment and create a range of alcoholic products. Craft brewing industries and wineries use multiple strains to enhance flavour and aroma profile, but apart from its influence on sensory notes; the impact it has on fermentable carbohydrate depletion rate and growth of strain is examined within this study. Objective: The purpose of this investigation is to understand the difference and compare single strain fermentation against that of multiple strains with respect to sugar intake and growth patterns. Background: Brewing and wine making industries rely on a single strain of yeast to brew their beers and wines in order to have a consistent characteristic flavour profile of the product. To make this flavour more complex, some of the craft brewing industries and wineries use multiple strains of yeast in a single batch or during secondary fermentation. Yeast strains are constantly being developed in order to perform according to brewer and winemakers specifications. Growing demands of production and limited opportunities to expand plant size are major reasons why higher performance is expected from the yeast strain. For this, yeast strains are being improved genetically in order to metabolize faster and more types of saccharides, limit production of unwanted by-products, increased ethanol tolerance etc. and so hybrid strain development is being achieved either through mating or mutating the genomic sequence of strains with the desired phenotype. This type of development however poses the risk of releasing unknown strains into the environment and as a consequence can lead to contamination of the gene pool.
To avoid this risk, one can look towards brewing a single batch with multiple types of yeast strains. Using multiple strains can be beneficial especially in terms of flavour, faster and more complete sugar utilization. Although the cons of this can be: Competition among the strains leading to stuck fermentations. Complex nutrient requirement. Effect on volatile components.
Methods: Fermentation with single strains and multiple strains were carried out in 8 tall tubes containing Yeast Nitrogen Base media along with five types of sugars namely Glucose, Sucrose, Fructose, Maltose and Galactose. Samples were collected and tested for sugar concentration and cell concentration via HPAE analysis and spectrophotometer respectively. Four varieties of yeasts a wine (Saccharomyces fructum), an ale (Saccharomyces cerevisiae), lager (Saccharomyces cerevisiae) and champagne strain (Saccharomyces bayanus) were used in this study. Results: Results showed considerable performance from mixed cultures of Saccharomyces cerevisiae (Lager strain), Saccharomyces bayanus and Saccharomyces fructum. Conclusion: It can be concluded that the results of a mixed culture does not give the desired results in terms of sugar utilization. Faster fermentation rates could not be achieved by coinoculating these strains, but however they do have quite an advantage over single strain fermentation such as greater substrate utilization and less production of volatiles.
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Introduction: Yeast, an essential organism considering our food and alcohol industries and the dependency of these on this organism has been the key in driving the research for obtaining better performance and fermented product quality. The important role of yeast in brewing and wine making industry is well established as the production of ethanol under anaerobic conditions by the utilization of carbohydrates present in the wort and musts. We must also note the essential fact that yeast is an important organism in maintaining the bodys internal flora; this makes it an ideal choice for potable fermentation products. Yeast is the machinery behind the fermentation process; a vessel containing enzymes which guides and catalyses biochemical reactions within so as to convert fermentable sugars into ethanol. Ethanol production is thus directly related to the concentration of the fermentable sugars present. Yeast and a number of bacteria e.g. Enterobacteriaceas, Spirochaeta, Bacteroides metabolize glucose by the Embden-Meyerhof pathway. However, not all varieties of yeast can be used for producing alcohol as only certain strains produce the right balance between flavours and ethanol. The selection of yeasts for alcohol production is done on the basis of genotype and the phenotypic expression of their genotype (Boulton, 2001). The genotype influences the fermentation activity in a major way as it is responsible for physiological and biochemical behaviour of the yeast. The rate of fermentation, types and quantities of alcohol produced, sugars that can be metabolized, aromas and flavours produced are all decided by genotype composition of the strain. However, fermentation conditions also play an important role in regulating the above factors and needs to be managed effectively so as to get the desired product. Most of the strains used in the potable alcohol production belong to the Saccharomyces genus. Other non-Saccharomyces strains are also used these days in wine making and in brewing special beers such as lambics. In wine making, Saccharomyces cerevisiae dominate the fermentation and is majorly responsible for converting sugars to ethanol. Juices and wines which are naturally inoculated contain a variety of other yeast strains such as Zygosaccharomyces,
Schizosaccharomyces, Debaromyces, Kluyveromyces, Torulaspora, Brettanomyces, Pichia and Candida (Fleet 2003; Loureiro and Maltfieto-Ferrira 2003 as cited by Mills et al., 2008). According to a study conducted on Bordeaux wines, yeast of the genera Rhodotorula, Pichia, Candida and Metschaikowia occurred at low levels in the grape extract but died off soon as 11
fermentation commenced (Fleet et al., 1984). Fleet (1984) later noticed that Kloeckera apiculata, Torulopsis stellata and S. cerevisiae were the dominant yeast which proliferated to conduct the initial fermentation but eventually even K. apiculata and T.stellata died off, leaving only S. cerevisiae as the dominant yeast. The major reason for this domination is that S. cerevisiae strains have a relatively versatile metabolism enabling them to adapt to a variety of condition as they can modulate their physiology according to the changes in the environment (Briggs et al., 2004). Answers to their abilities to switch between oxidative or fermentative mode and to oxidize a variety of organic molecules can be found in their genome. The genus Saccharomyces has been most studied due to its application in brewing and wine making. This genus is S. kudriavzevii, S. made up of six species S. cerevisiae, S. bayanus, S. cariocanus, mikatae, S. paradoxus and the hybrid taxon
S. pastorianus (syn. S. carlsbergensis) (Naumov et al., (2000); Kurtzman, (2003). as cited in Naumova et al., (2005)). Of which S. cerevisiae, S. bayanus and S. carlsbergnesis have been widely used for producing potable alcohol. Four types of yeasts species used in this study are as follows: 1. Saccharomyces cerevisiae Lager yeast Y 401 2. Saccharomyces cerevisiae Ale yeast. NCYC 1040 3. Saccharomyces fructum Wine yeast and 4. Saccharomyces bayanus Champagne yeast - Lalvin EC-1118 aka AWRI (Australia wine research institute)- 838 The above strains all belong to Saccharomyces genus and can be differentiated based on their application. Classifying strains on the basis of application makes it easier for brewer or winemaker to relate to the characteristics of the strain, making it easier to choose from. Under the classification of S. cerevisiae, there exists a plethora of strains that although closely related taxonomically, have constantly evolved through selection and have become adapted to mutually exclusive application (Boulton, 2001). S. cerevisiae contains strains that are used in baking (Bakers yeast), wine making, and those that are used specifically for brewing lagers and ales. This diversity makes it essential for strains to be recognised according to their application. Bakers yeast if used in brewing produces off flavours resulting in an unpleasant beer. Lager 12
strains have been isolated from breweries around the world because they are better suited for making crisp, clean and angular beers. Similarly strains used in brewing ales are better at producing fruity, complex and rounded beers. Wine and champagne yeast produce varying quantities of acetic, n-butyric, n-caprioc, n- caprylic, succinic, formic, butyric, isobutyric, 2methyl butyric, isovaleric, lactic and malic acids as well as higher alcohols (fusel oil) and esters (Berry and Watson (1987) as cited in Jacob (1993)). These compounds contribute to the distinctive flavour and aroma of wines and are thus more suitable for fermenting musts. Lager yeast (S. cerevisiae) is bottom fermenting yeast i.e. they settle down as deposits as fermentation progresses and operate best at low temperature of 8-150C. DNA similarity suggests that lager strains closely resemble Saccharomyces pastorianus however it has been incorrectly named as S. carlsbergnesis (Rodrigues de Sousa et al., 1995) but is being retained due to its industrial application. S. carlsbergnesis is a naturally occurring hybrid strain whos DNA when compared with that of S. cerevisiae and S. bayanus shows a lot of commonality (Vaughan-Martini and Kurtz-man; 1985).
Ale yeast (S. cerevisiae) is top fermenting yeast i.e. the yeast rises up as the fermentation proceeds to form a rich creamy layer on top and operates at higher temperature range than lager strains typically around 200C. Operating temperatures are mostly calculated according to the product requirements. Ales have fruity notes which are mostly associated with ester production. At higher temperatures yeast tends to release more esters as by-product. Similarly in lagers since these notes of flavour are less preferred, the fermentation takes place at lower temperatures in order to guide the production of byproducts. Research shows that lager strain (S. pastorianus) ferment faster at low temperatures than ale strains. Two ale and lager strains with similar maltose transport activities at 200C, when subjected to 00C showed five times greater fermentation activity in lager strains as compared to ale strains. (Vidgren et al., 2010)
A major difference between ale and lager strain apart from fermenting temperature is the ability to metabolize the disaccharide melibiose (Boulton, 2001). Lager strains can ferment melibiose whereas ale strains do not; an ability which is directly correlated to the
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genomic
differences
of
the
two
strains.
Wine yeast S. fructum strain ferments at very low temperatures and is used in the production of red wines.
Saccharomyces bayanus is vigorously fermenting type yeast. This strain is employed for rescuing stuck fermentations and for producing cider and dry wines. Advantageously, this yeast can ferment over a wide range of temperature 100C 300C. S. bayanus also has a good tolerance to high levels of alcohol, thus making it suitable for wines containing 1218% ABV.
A wide variety of Saccharomyces strains have been discovered and isolated for the purpose of brewing lagers and ale, vinification, baking and distilling whiskeys and hard liquor. Separate strains are employed for specific processes based on their performance under a given set of conditions and the type of the product to be fermented. For example wine yeasts such as Saccharomyces fructum ferment at very low temperatures and are specific to wine making as they have good tolerance to higher levels of alcohol than lager yeasts. Thus the choice of yeast from a brewers or winemakers point of view depends on the following factors: Aroma and Flavour: Each yeast strain is unique in terms of flavour and aroma it imparts to the brew. This can be due to the ester profile which differs from strain to strain. The secondary metabolites formed as a by-product of fermentation plays a major role in developing distinctive flavour and aroma. Fusel alcohols and diacetyl levels greatly enhance certain characteristics on the palette. Degree of Attenuation: Attenuation is the final specific gravity reached at the end of the fermentation. Some yeast strains have higher whereas some have lower attenuation levels. The lower the level of attenuation the lesser the extent of sugar utilization. Rate of Fermentation: The fermentation rates are different for different strains. Faster they ferment higher the attenuation they reach. Flocculation: Some yeast strains tend to flocculate more than others. Higher the flocculation, easier it is to process post-fermentation.
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Fermentation: Fermentation is known to mankind since the birth of civilizations. Today this process finds application in lots of areas most including products of pharmaceuticals, beverages, foods etc. Fermentation is a biochemical process during which oxidation of organic compounds takes place in order to generate Adenosine Tri Phosphate (ATP) which is a source of stored energy in yeasts cells, as a by-product of this pathway ethanol and carbon dioxide are produced. This energy is utilized in maintaining inner osmotic pressure, active transportation of compounds through the membrane such as endocytosis and exocytosis, signal transduction, enzymatic reactions, intracellular repair and maintenance, and for synthesis of DNA and RNA which play a vital role in growth, development and in reproduction of yeast cells. Fermentation usually occurs in the absence of oxygen i.e. it is an anaerobic process. However yeast cells have shown preference to fermentation in the presence of sugars under aerobic conditions (Arthur and Dickinson, 2004). The citric acid cycle is shut down in favour of fermentation to produce ethanol with the evolution of CO2. (Ndip et al., 2001) Below is the chemical reaction involved in the production of ATP, the following chemical pathway
C6H12O6 (Glucose) + 2 ADP + 2 Pi + 2 NAD+ 2 CH3COCOO (Pyruvate) + 2 ATP + 2 NADH + 2 H2O + 2H+
Following the production of 2 molecules of pyruvate,

2 CH3COCOO(Pyruvate) 2 CH2OH (Ethanol) + 2 CO2.
The above fermentation reactions generate ATP molecules via electron transport chain in mitochondria and are stored for utilization during growth and maintenance of the cells. Fermentation is thus proportional to yeast growth and analysis reveal four stages of growth namely lag phase, logarithmic phase, and stationary phase. Lag phase is the duration the inoculated cells take to get adjusted to their environment, essentially sensing nutrients and activating enzyme synthesis for utilization of sugars and nutrients. The logarithmic phase is the rapid growth phase wherein cells multiply rapidly by consuming sugars and nutrients. This phase lasts for a short time and depends mostly on the sugar concentration which depletes as cell
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density increases. Rapid depletion of the most preferred sugars glucose, sucrose and fructose occurs during this log phase. After attaining a point of maximum growth rate, cells phase into stationary growth period and begin utilizing lesser preferred sugars such as maltose and galactose. Fermentation occurs at a fixed rate here after and is for a longer duration. Other factors influenced during fermentation are CO2 evolution, ethanol production, specific gravity and pH.
Figure 1: Factors influenced during brewing fermentation. Source: Munroe James H., Handbook of Brewing, Chapter 12, pg. 499.
The above graph shows us the direct correlation between fermentation, by products of fermentation i.e. ethanol and CO2 and cell growth. The increase in cell density is observed as yeast begins consuming sugars during log phase; an increase in the sugar consumption upsurges the release of the by-products of fermentation CO2 and ethanol. The consumption of sugars is translated into decreasing specific gravity and pH.
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Raw materials in Potable Alcohol Production:
A large production requirement of industrial ethanol has led to the use of chemical rather than biochemical method wherein petroleum feedstock is utilized for lower costs reasons and more effectiveness of the process. The sugars and nutrients utilized by yeast in brewing and winemaking however are provided through processing of agricultural products which act as the major raw material in potable alcohol production. These raw materials are rich in carbohydrates but also contain essential proteins, vitamins and minerals acting as a source of growth medium for the yeast. Agricultural products include a variety of cereal grains such as malted Barley (Hordeum vulgare) which is most widely used as a base raw material in brewing beers and in distilled alcohol production, inexpensive sources of raw material such as wheat (Triticum aestuvum), sorghum (Sorghum vulgare), rye (Secale cereal), oats (Avena sativum) and millet are also used in brewing. (Briggs, Boulton and Brooks, 2004). The latter are sometimes used as adjuncts to give desired characteristic to the product. Sugar in either crystallized or syrup form is also used as an adjunct to increase the alcohol content.
Fruits such as grapes, apples and pears are used as a source of fermentable sugars sometimes as a part of the product and sometimes wholly. Processing fruits for making wines and cider is easier as the hexose sugars are readily available unlike that in the grains where primarily sugars are stored as starch and other polysaccharide and have to be broken down by the action of ,amylase enzymes. Boulton et al., (1998) states that Wine industry of the world is built upon one species of grapes Vitis vinifera L.. Most of the famous wines are produced with this grape variety. Fruits are used in brewing, distilling and winemaking as they are a source of fresh juices easy to crush and extract and adds more complexity to the flavour profile. Fruits are rich in sugars, vitamins, minerals, anti-oxidants, and are low on protein and fat content however contain enough nutrient supply to support yeast growth.
Brewing and wine making is thus an art that requires processing of these raw materials with the purpose of extracting the maximum amount of fermentable sugar. This creates a blend of various sugars and nutrients which when supplied to yeast leads to propagation with alcohol production being a secondary response. An outline of brewing and winemaking process is mentioned next to understand the importance of each step required to produce potable alcohol.
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Beer production. In brewing, sugars are made available after processing the raw materials. The steps of which can be summarized as below. Preparation of European style beer productions involves (Eaton, 2006): The first and foremost step is malting of barley and other cereal grains. Malting is a process in which cereals are partially modified by controlled steeping, germinating and then kilning. This step increases the friability and enzyme levels of the grains. Steeping and germination are essential steps that help release gibberellin hormones that promote formation of enzymes and -amylases within the grain. These enzymes are responsible for breaking down the starchy endosperm into fermentable sugars during mashing. Kilning removes moisture, the lack of water inactivates enzymes and this prevents the malt from germinating further. The above process occurs during malting and such malt is then made available to breweries.
The enzymes released, partially degrade the cell walls of the grain and makes milling them easier. Milling converts the malted grains into flour (grist) which can be mixed with water.
During mashing the grist is incubated in warm water. Sometimes it is mixed with other starchy material (adjuncts) and enzymes. This step activates malt enzymes, essential for degrading starch and dextrins present in the malt into soluble fermentable sugars. Yeast cells can assimilate these free sugars during fermentation readily as compared to long chain polysaccharides such as starch. Study shows that standard laboratory strains of S. cerevisiae do not ferment starch. They differ from S. diastaticus as they lack structural genes STA 1, STA 2, STA 3 and STA 10 (Polaina and Wiggs, 1983).
Next step is wort clarification, here the wort is separated from the draff (undissolved solids) in lauter tuns or mash tuns or mash filters.
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The extracted wort is sweet in taste and can be now tested for the presence of starch by iodine test; no occurrence of indigo colour indicates all the starch has been completely broken down. The wort is then boiled to sterilize it completely along with hops, giving it bitterness, flavour and aroma.
After wort boiling the coagulated proteins and hop debris are removed in the whirlpool tank. This collected debris is called trub. This process further clarifies the wort.
Before the yeast is added to the wort it needs to be cooled to below 200C, since the yeast cells would perish at high temperatures and aerated with oxygen to ensure healthy yeast growth in the tanks. The cooling takes place in heat exchangers usually a plate heat exchanger is used in breweries. Chilled glycol and water is used to remove all the excess heat.
Once cooled and aerated the yeast is pitched accordingly and the wort is now allowed to ferment for 7-9 days. During this time, cells consume sugars and nutrients dissolved in the wort and produce ethanol and CO2. Yeast produces these compounds as by-products of the synthesis of compounds necessary for growth and metabolism
At the end of the fermentation, the yeast collected in the cones of the vessels is collected first and the immature beer is then filtered and further conditioned in Bright beer tanks. The conditioned beer is then finally packaged and distributed.
Wine Production: Wine and champagne production can be outlined according to the below mentioned flow chart. The main raw material for wine industry is grapes. Grapes are collected, destemed and crushed to produce grape extract. White wines are made from white grapes, whereas red wines from red grapes. This grape extract contains free fermentable sugars. The difference between the two processes can be seen in the chart. SO2 is added in both steps so as to sterilize the extract.
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Figure 2: Schematic representation of red and white winemaking process. Source: Wine fermentation, Mills. et al., (2008), pp 164. 20
Sugar uptake by yeast in pure cultures: Yeasts are chemoorganotropic unicellular fungi; this means that they obtain carbon and energy from compounds in fixed, organic linkages such as carbohydrates (Walker, 1998). ATP is provided as a result of oxidation of organic molecules which is then utilized for other functions. Yeast cells can obtain carbon via sources such as amino acids, alcohol and organic acids yet they prefer sugars over other sources. The fate of the sugar metabolized is decided by the conditions prevalent such as presence of oxygen and sugar concentration. 1. Under aerobic conditions and limited sugar concentration; sugars such as glucose are first converted to pyruvate via the Embden Myerhoff pathway (glycolysis) which is then converted to acetyl CoA. This is oxidized via the Tricarboxylic cycle (TCA) cycle and oxidative phosphorylation generating NADH, which when passed along an electron transport chain produces ATP. 2. Under anaerobic conditions yeast cells convert the pyruvate to acetaldehyde which is then converted into ethanol generating NAD+. However yeast strains such as S. cerevisiae show a strong tendency to undergo fermentative metabolism even in the presence of oxygen since respiration is also modulated by the concentration of sugar and nitrogen in the medium. Boulton (2001) quotes In brewery fermentation, respiration, in the true sense of complete oxidation of sugars to carbon dioxide and water, coupled to ATP generation via oxidative phosphorylation does not occur. Various studies have been conducted to study this unusual behaviour of yeasts. Below we elaborate on two theories that are directly related with yeast sugar metabolism and have been widely accepted.
Crab-Tree effect: One such theory called the Crab tree effect is currently being defined as the alcoholic fermentation that occurs under aerobic conditions (Pronk et. al 1996). S. cerevisiae catabolizes glucose mainly via fermentation process; this effect was termed as Crab tree effect (Swanson and Clifton 1948 as cited in Rodrigues et al., 2006). Yeast strains when subjected to aerobic conditions along with low concentration of sugars dissolved in the medium showed complete oxidation of the sugars via the TCA and oxidative phosphorylation. To further understand the 21
conditions under which fermentation occurs, the same medium when pulsed with high concentration of glucose resulted in yeasts immediately producing ethanol and CO2. Postma et al., (1989) suggested that at high glucose levels, above a certain critical concentration, Short term Crab tree effect occurs in yeast cells. Rieger et al., (1983) proposed that this short term crab tree behaviour could be due to the limited respiratory capacity of cells. An increase in the glucose flux results in the overflow of carbon due to the saturation of the respiratory pathway which is then channelled towards the fermentative pathway. Saturation can occur due to limited availability of respiratory enzymes, such as pyruvate dehydrogenase which catalyses the conversion of pyruvate to Acetyl CoA. However, Postma et al., (1989) concluded from the above conducted experiment that fermentation is not necessarily due to limited respiratory capacity of yeast cells, but also due to the uncoupling effect of certain organic acids produced as a result of respiration.
The crab tree effect has been divided into short term effect and long term effect. The short term effect occurs due to a sudden increase in glucose concentration which triggers alcoholic fermentation and the long term effect is observed as respiration-fermentative metabolism in batch fermentation conditions occurring at above critical concentration levels (Rodrigues et al., 2006).
Carbon catabolite repression:
The above mentioned crab tree effect is more concerned with the mechanisms occurring at enzymatic levels; however these enzymes are also modulated as per the environmental conditions present surrounding the yeast cells. Enzymes are proteins which are synthesized by a sequence of genes, each of which codes for a particular amino acid. The expression or repression of these genes modulates enzyme synthesis, this further decide the fate of metabolism of sugars. Enzymes not only help catalyse reactions but are also essential in transportation of nutrients. Sugars such as glucose have been noticed to have an influential role on yeast metabolism. S. cerevisiae cells can sense the nutrient environment around them and consequently activates or inactivates enzyme synthesis, so as to be able to assimilate those nutrients. Enzymatic activity is 22
thus modulated in yeast cells, either induction or repression of enzyme synthesis occurs due to the presence or absence of sugars.
S. cerevisiae prefers glucose and fructose over other sugars as carbon source. When these sugars are present, the enzymes for metabolism of other sugars are either synthesized at low levels or not at all. This phenomenon is termed Carbon Catabolite repression (Gancedo, 1998). Glucose when present in the medium acts as a metabolic signal and causes a decrease in the concentration of enzymatic mRNAs, their translation rate or triggering of inactivation and/or proteolysis of a number of proteins associated with metabolism of other sugars (Helmut, 1976). In a study, Saccharomyces cerevisiae cells growing on galactose were transferred onto a medium containing glucose and cycloheximide. Cycloheximide inhibits proteins synthesis. Galactose transporter in yeast cells is associated with GAL2 gene product; it was observed that glucose induced inactivation by degrading the GAL2p by ubiquitinating the molecule and thereby repressing galactose transport (Horak and Wolf, 1997). Glucose is the preferred substrate when present in the medium; it inactivates and represses other sugar carriers (Rosario, 1993). Thus repression due to the presence of glucose has been identified as the rate limiting step in yeast sugar uptake. Other sugars are metabolized only after considerable amount of glucose is depleted.
Genetic correlation of sugar transport and utilization in yeasts: The uptake of sugars in yeasts has been found to be sequential, i.e. yeast cells tend to prefer certain sugars over others. In brewing worts and grape musts, various types of sugars at varying concentrations are present. As explained above, the carbon catabolite repression in yeasts creates a bottle neck effect due to the presence of glucose; other sugars such as maltose and galactose are taken up only after glucose is depleted. In general, the utilization of a particular sugar is controlled by glucose repression, which is a complex regulatory system that affects the expression of a multitude of genes including those involved in sugar uptake and fermentation (Ernandes, 1993). In Saccharomyces two genes have been found that are responsible for
repression, HXK2 and SNF1. HXK2 is a structural gene that encodes for PII (B) isozyme 23
(Carlson, 1987). Carlson and team also found a gene required to release glucose repression SNF1 which encodes for a protein kinase suggesting that protein phosphorylation plays a critical role in S.cerevisiae glucose repression (Carlson, 1987, Celenza and Carlson, 1986)
Sugars can be either monosaccharides such as glucose and fructose, disaccharides such as sucrose, maltose, lactose, trehalose and cellobiose or trisaccharides such as raffinose. Before these sugars can be utilized, they have to be transported across the membrane. The transportation and metabolism of these depends mostly on the genetic composition of the strain and this also decides whether or not a type of sugar can be utilized by a particular strain. Sugar utilization is governed by both genetic capability and regulatory mechanisms (Carlson, 1987). This is because certain sugars require specialized transporters and enzymes to break them down, the availability of which totally depends on the genotype of the strain. For example, S. diastaticus can metabolize polysaccharides such as wort dextrins due to the presence of the STA gene which is translated into glucoamylase whereas S. cerevisiae lacks this ability to utilize polysaccharides and dextrins due to the absence of this gene.
Transportation of sugar precedes metabolism in yeast cells. Due to the highly polar nature of sugar molecules, free diffusion across the membrane occurs at low rate when concentration is low (Lengeler, 1993 as cited in Weusthuis et al., 1994). However at high sugar concentration such as in worts and grape juices, facilitated diffusion allows passive transportation of sugars into the cell.
Below we discuss the main components of transportation associated with five types of sugars used in this experiment glucose, sucrose, fructose, maltose and galactose.
A point arrives when the concentration of sugars is equal on both sides. In such a case the active transportation occurs by coupling the intake of sugar molecule with uptake of one or more protons. This is known as proton symport system. Here the proton motive force acts as a driving force for the accumulation of sugars. This proton motive force requires the use of ATP hence the term active transportation (Weusthuis et al., 1994). and
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Figure 3: A) Passive diffusion B. Proton Symport System. Source: (Weusthuis et al., 1994)
Monosaccharide transportation: Two transporters for monosaccharides, the glucose and galactose transporters act by a facilitated diffusion mechanism. In the case of glucose transport, which also acts upon D-fructose and
D-mannose,
two components with high- and low-affinity constants have been identified
kinetically. Activity of the high-affinity component is dependent on the presence of active kinases whereas activity of the low-affinity component is independent of the presence of these enzymes (Rosario, 1993).
Monosaccharide transportation is governed by a set of genes, known as HXT genes. So far, 20 HXT genes have been identified of which HXT 1-7 are involved in transportation. HXT1 and HXT3 are low-affinity transporters and come into play when high concentration of glucose is present; HXT2, HXT6, HXT7 have been identified as high affinity transporters and are essential for hexose transport in low concentration conditions. A most common observation in wine fermentation has been rapid utilization of glucose over fructose. It was found that the enhanced fructose fermentation capacity of Fermichamp, wine yeast depended on the expression of a mutated HXT3 allele (Guillaume et al., 2007). However research carried out at the Institute of Wine Biotechnology, shows that the discrepancy between hexose sugar utilization; glucose and fructose depends not only on the genotype of the yeast strain but also on the external conditions such as assimilable nitrogen, the increase of which triggered rapid fructose intake (Berthels et al., 2004).
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Galactose utilization requires the Leloir pathway enzymes which are encoded by GAL1, GAL7 and GAL 10 genes and GAL2 which codes for galactose permease. We mentioned previously, how the presence of glucose degrades the GAL2 gene product and represses galactose intake. Expression of these genes is induced by the presence of Galactose and the gene responsible GAL4 encodes a protein which activates the enzyme synthesis essential for galactose catabolism.
Disaccharide transportation:
Disaccharide sugars are molecules which are made up of two monosaccharide units connected via a glycosidic bond. The utilization of such generally requires an enzyme that splits the molecule into respective monosaccharide units. Sugars such as sucrose which is the most preferred substrate for yeasts among all sugars is hydrolysed by extracellular periplasmic enzyme invertase which separates the 1,4 glycosidic bond separating D-glucose and D-fructose units and then transported further into the cell (D'Amore et al., 1989). The synthesis of enzyme invertase is regulated by a family of SUC genes (SUC 1, SUC 2, SUC 3, SUC4, SUC 5 and SUC 7), the regulation of which is solely governed by glucose repression (Carlson, 1987).
Disaccharide maltose transportation and metabolism has been widely studied as it happens to be a major brewing wort sugar. Three gene products, a specialized transporter, a maltose hydrolysing enzyme -glucosidase and an activator of transcription are involved in maltose uptake. Maltose permease is the transporter protein encoded by the MALT gene, the hydrolysing enzyme also known as maltase is encoded by MALS and MALR which encodes for an activator protein which catalyses maltose catabolism. (Needleman et al., (1984) as cited in Boulton, (2001)). Maltose uptake begins only after a considerable decline in glucose concentration is achieved.
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Sugar uptake sequence in yeast: When yeast is introduced in the wort or grape must, it is exposed to a vast array of nutrients available for its growth. Of these nutrients, 90% of the total wort concentration is carbohydrates (Gibson et al., 2008) and the other 10% consists of vitamins, proteins, dextrins, amino acids and nucleic acids. The carbohydrates present are majorly sucrose, glucose, fructose, maltose and maltotriose. Maltose and maltotriose being present at a greater fraction as compared to the rest in brewing wort, and in wine musts sugars such as glucose and fructose are present at a higher concentration. Grape musts contain equal amounts of glucose and fructose, with total hexose concentrations typically ranging from 160 to 300 g/L (Guillaume et al., 2007). When exposed to such a complex environment, the sugars present are utilized in a particular sequence with sucrose being given the highest preference. Glucose repression in yeasts causes sequential sugar uptake in yeasts. The sequence as sucrose being the most preferred substrate among all types, followed by glucose, fructose, maltose, maltotriose/galactose (Rosario, 1993).
Figure 4: The graph depicts the sugar utilization during a brewing fermentation, shows the concentrations of maltose ( ), maltotriose ( ), glucose ( ), fructose ( ) and sucrose (). Source: Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation (Gibson et al., 2008).
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It has been found that brewing yeasts utilizes several mechanisms for sensing the nutritional status of its environment and accordingly adapts its metabolism and uptake of nutrients. Galactose as a sugar is unusual in brewing worts or wine musts and is thus least preferred by the cells; however when other sugars are depleted, yeast cells activates GAL genes to metabolize the sugar.
Mixed culture fermentations: Mixed culture fermentations are fermentations carried out in batch or continuously by using multiple or more than one strain for inoculation. Such a culture would consist of bacteria, fungi of known and unknown strains and quantities. Mixed culture fermentation finds application in food and wine industry generally. Traditional foods such as kozi and miso are prepared by fermenting it with a fungi Aspergillus oryzae first which hydrolyses starch and protein and further fermentation is carried out by lactic acid and yeast (Hesseltine, 1992). Growing international competition and demands for better products in the wine industry are fuelling innovations for improved wine fermentation (Fleet, 2008). Winemakers are thus turning towards mixed culture fermentations as a source for better flavour profile and sugar utilization. Mixed cultures contain a combination of Saccharomyces and non-Saccharomyces species, some wines use pure isolated cultures whereas some use naturally occurring inoculations in wine. These combinations often result in unpredictable fermentation by-products which affects both chemical as well as aromatic composition in wines (Ciani et al., 2010). Fleet et al., (1984) observed that yeast species such as Pichia, Candida, Kluyveromyces and Metschnikowia existed in freshly extracted grape juices. These strains have been found to exist naturally as a part of the microbial habitat in wineries. Such species are mainly responsible for spontaneous fermentation in the initial stages but perish during mid-fermentation due to their sensitivity to rising alcohol levels and thus domination by S. cerevisiae occurs (Fleet, 2008). In brewing, most beers and ales are prepared by monocultures and some maybe even subjected to secondary conditioning by inoculating different strains from the one used for fermenting. Traditional beers and wines were all a product of mixed culture fermentation. Cantillon brewery makes some of the finest Belgium Lambic beers which are prepared by mixed culture 28
inoculation. These beers are prepared by spontaneous fermentation by diverse organisms that are naturally inoculated by exposing the wort in open shallow trays overnight (Oliver et al., 2011). The wort is fermented and aged for two years and this fermented product is called Lambic. Studies on lambic fermentation have found the following strains in the culture: Kloeckera apiculata, Saccharomyces spp, Brettanomyces spp, lactic and acetic acid bacteria. Microbial population of a lambic beer in the first month was found to consist of the following species Enterobacteriaceae Klebsiella aerogenes, Enterobacter cloacae, E. aerogenes, Escherichia coll, Citrobacter freundii, A-D group bacteria Hafnia alvei (Martens H. , 1991).
Sugar Utilization in Mixed Culture fermentations: Not many studies have been conducted that give us detailed information about sugar utilization. Reasons being different combinations of cultures behave differently when it comes to sugar uptake kinetics. Thus it can be hypothesized that sugar utilization kinetics completely depends on the types and number of species/ strains present in the culture. As mentioned earlier, uptake of sugar in yeast and other species is completely governed by the genotype which would greatly differ from strain to strain. Apart from individual genotypes, microbial interactions i.e. interaction between different strains also needs to be considered when referring to mixed cultures. There are six types of interactions such as commensalism, competition, predation, inhibition, parasitism and synergism which influence the microbial ecology (Gall, 1970). Thereby it can be said, in mixed cultures due to the unpredictability of microbial reactions and of cultures present it is difficult to have a consistent analysis. Dominance by one species is very common in mixed cultures. Studies report that dominance of a strain in wine making depends on the following: 1. Viability and the amount of inoculum added. 2. Metabolic and physiological characteristics of the selected yeast in the inoculum. 3. The technology used in the winemaking (e.g., temperature of fermentation clarification procedures and SO2 addition) (Amerine and Cruess, 1960; Benda, 1982; Reed and Nagodawithana, 1988; Ciani and Rosini, (1993) as cited in (Ciani et al., 2010).
29
However controlled mixed cultures have been created at laboratory scales so as to have desired output results in terms of flavour, aroma and substrate utilization. In a study, grape juices were fermented with four different groups of yeasts. Each group contained a different set of yeast strains. Below mentioned is the graph depicting sugar utilization in mixed culture fermentation (Howell et al., 2006) Fermentation Trail M1 M2 M3 M4 Combination of yeast strains AWRI 796, ICV D47, QA23 AWRI 796, ICV D47, AWRI 838 AWRI 796, ICV D47, AWRI 835 AWRI 1434, AWRI 1176, AWRI 838
Table 1: Strain composition of yeast cultures used in mixed culture wine fermentation. All strains used were of S. cerevisiae except for AWRI 1176 Saccharomyces bayanus. Source: Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation (Howell et al., 2006). Note: AWRI Australian wine research institute pp-93
Figure 5: Graph depicting the sugar utilization in wines with different mixed cultures. The sugars measured were Glucose and Fructose. The plots were made by taking an average of the HPAE values. Source: Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation (Howell et al., 2006) pp-95.
The above study results of M4 cultures closely resemble the results of our experiments below in a mixed culture of S. bayanus and S. cerevisiae. Mixed cultures are known to have better substrate utilization as compared to monoculture fermentation. 30
Mixed culture fermentations thus differ greatly in terms of microbial population, substrate utilization, product yield and by-products of metabolism from single strain fermentation. They have numerous advantages over single strain fermentation.
Scope of this study: Closely related Saccharomyces species differ greatly in their ability to utilize sugars- an effect that maybe due to the fact that strains of Saccharomyces carry different active members from a family of genes responsible for controlling sugar intake (Carlson, 1987). Based on the above theory we decided to test four different types of yeast by inoculating them in dual cultures i.e. containing two types of strain and comparing it with mono cultures in a media containing five different types of sugars. The yeast varieties chosen were on the basis of their different brewing and winemaking application. In brewing, lager and ale S. cerevisiae strains are used and in winemaking yeasts such as S. fructum and champagne yeast S. bayanus are employed. These strains were chosen as they differ greatly from each other in terms of sugar utilization, growth rate and ester production. Thus we carry out experiments by inoculating all the possible combinations in twos and carry out mono culture fermentation in tall tubes to compare our results. Combinations used were: Ale and Champagne yeast. Lager and Champagne yeast. Wine and Ale yeast Wine and lager yeast
We avoided using Lager and Ale strains together as we know in industrial application such fermentation is not possible as the two strains have a very different operating temperature. We hypothesized that since glucose repression causes preferential sugar uptake in single culture fermentation, carrying out fermentation with multiple yeast strains could make a difference in sugar utilization. When using single organisms for fermenting sugar mixtures, a major challenge could be that the organism might not be able to optimally adjust the sugar uptake rate to match the fluctuating sugar concentrations (Unrean and Srienc, 2010) and thus we used a co-culture of multiple strains and observe the difference in uptake rates. 31
Materials Yeast Nitrogen Base Media: Yeast Nitrogen base with amino acids (Difco) was used for classifying yeast based on carbon assimilation. Yeast Nitrogen Base is a preparation based on Wickerhams and Burkholders formula which is synthetic and a complete medium (Wickerham and Burton, 1948). This medium was developed as a result of Wickerhams research on classification of yeast. The only thing this medium requires is a carbon source i.e. Carbohydrates or sugars. YNB media was developed based on the nutritional requirement of yeast strains and contains all the essential salts, vitamins, amino acids such as histidine, methionine and tryptophan; a nitrogen source such as ammonium sulphate and asparagine to promote the growth of yeast strains (Walker, 1998). The carbon sources used in assimilation tests for yeast classification have been limited to glucose, fructose, mannose, galactose, maltose, sucrose, lactose, and ethyl alcohol and can be added to the base to a final concentration of 1%. Thus we use this media for determining the rate and sequence of sugar utilization of different yeasts. 1. Malt Extract Broth: Oxoid CM0057 Malt Extract Broth used for culturing yeast cells from petri plates into broth. This media was used to propagate strains from master cultures. 2. Sugars: Glucose (D-(+)-Glucose), Sucrose (- D- fructofuranosyl -D-glucopyranoside), Fructose (D-(-)-Fructose), Galactose (D-(-)Galactose) and Maltose (4-O--D-
Glucopyranosyl-D-glucose) 3. Yeast Strains: Saccharomyces cerevisiae Y 401 Lager yeast, Saccharomyces cerevisiae NCYC 1040 Ale yeast, Saccharomyces fructum Wine yeast, Saccharomyces bayanus EC 1118 Lalvin Champagne yeast.
4. Tall Tube fermenters: 8 Tall Tube fermenters of 2 L capacity each.
32
Methodology: I. Yeast Culture Propagation: 1. Pure cultures of the four yeast strains Saccharomyces cerevisiae Y 401 Lager yeast,
Saccharomyces cerevisiae NCYC 1040 Ale yeast, Saccharomyces fructum Wine yeast
and Saccharomyces bayanus EC 1118 Lalvin Champagne yeast were grown separately for 48 hours in petriplate to prepare master cultures. These master cultures were then propagated so as to achieve the cell count required for pitching in Tall Tube fermenters. 2. Propagation was achieved by inoculating the strains in a sterilized broth. The broth was formulated by dissolving 4 g of Malt Extract Broth in 200 ml of distilled water. 3. Since propagation of all the four strains of yeast had to be done separately, we distributed the broth into four separate conical flasks equally i.e. 50 ml in each. 4. The flasks were stoppered and autoclaved at 1150C for 10 minutes (as per the instructions) to completely sterilize the media. 5. The flasks were allowed to cool to 200C (room temperature) before aseptically inoculating the yeast strains Saccharomyces cerevisiae (Lager yeast), Saccharomyces cerevisiae (Ale yeast), Saccharomyces bayanus (Champagne yeast) and Saccharomyces fructum (Wine yeast) from master cultures in respective flasks. The broths were incubated at 200C in orbital shakers to achieve the desired cell count.
Conical flasks
Master cultures
Figure 6: Aseptically inoculating strains for propagation in broth media. 1. Saccharomyces cerevisiae (Lager yeast). 2. Saccharomyces cerevisiae (Ale yeast). 3. Saccharomyces bayanus (Champagne yeast). 4. Saccharomyces fructum (Wine yeast).
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II.
Yeast Nitrogen Base media preparation:
1. To prepare the medium for the tall tubes fermentation, we use Difco Yeast Nitrogen Base with amino acids; a media specially designed to measure the carbohydrate assimilation of the yeast strains. To prepare this culture we first calculate the required amount of media. In 8 tall tube fermenters of 2 liter capacity each, a liter of media was needed. To make 8 liters of media below are the calculations:
2. According to the media preparation guide: 6.7g of YNB media along with 5 g of sugar is to be dissolved in 100 ml of water. Of which 0.5 ml is dissolved in 4.5 ml of water to make up to 5ml of final medium.
3. Preparing the final media for tall tube:
As per the above equation to make 8 liters of media (1 L in each tube). 800ml of media in 7200ml of water will be required according to the above calculation. To make 8 liters of medium we have to dissolve (6.7g YNB + 5g of sugar in 100ml) x 8 = 53.6g of YNB + 40g of sugar in 800ml.
4. Sugar requirement. The sugars used were Glucose, Sucrose, Maltose, Fructose and Galactose. Total Sugar required / Types of sugars to be added = Amount of each type to be added 40g / 5= 8g of each type of sugar.
34
8gms of glucose, sucrose, maltose, fructose and galactose was measured and added in 800 ml of distilled water along with 53.6 g of YNB media. The complexity of the media is designed with carbohydrate concentration of wort in mind. Galactose is used instead of maltotriose as some stouts use milk as an adjunct which majorly contains lactose. Lactose is a disaccharide which is broken down into glucose and galactose. 5. This solution containing YNB and sugars was then filter sterilized since the media contains heat labile nutrients. For this we utilize vacuum (negative pressure) filtration
Media Feed Detachable hose creates negative pressure Flask containing filtered media Filter Housing (0.45m).
Figure 7: Filter Sterilization of YNB media. technique which is carried out in a filter flask. These
flasks have heavy walls so as to avoid breakage due to the vacuum created within. The flask comes with a detachable hose connection which can be connected to a running water tap which creates the vacuum required for filtration. The apparatus is fitted with a 0.45 m Whatman filter paper which acts a sieve for any form of microbial contaminants.
6. The filtered media was later added to 7.2 L of autoclaved water in a Pyrex 10L media bottle to prepare 8L of media. 1L of the media was measured and transferred in each of the 8 tall tube fermenters which were all pre autoclaved.
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III.
Preparation of Inoculums:
1. The 24 hour old conical flasks broths containing four types of yeast strains were transferred to four separate sterile centrifuge tubes; 50 ml in each and centrifuged at 30,000 rpm for 10 minutes. This allowed all the yeast cells to separate out of the broth and form a pellet at the base. The media was decanted slowly so as to not disturb the pellet. The pellets were weighed and tested for viability.
2. To test for viability, 0.1ml of broth was diluted with 9.9ml of distilled water in a test tube. 1ml of this dilution was then transferred to another test tube where 1ml of Methylene blue was added and held for 5 minutes. Cells showed 98% viability under the microscope and each tube contained 0.5 g of pure live yeast cells pellet. 3. 0.5g of each yeast pellet was dissolved in 5ml of autoclaved water hence the final concentration was 0.1g of yeast /ml of water. 1ml of this solution was added in pure culture tall tubes and 0.5ml+0.5ml for mixed culture tall tube was used to inoculate the tall tube fermenters i.e. a tall tube for lager and champagne mixed culture fermentation was inoculated with 0.5 ml of lager yeast solution and 0.5ml of champagne yeast solution so as to have 50% inoculation of each strain. A tall tube for pure culture fermentation with lager yeast was inoculated with only 1ml of lager yeast solution or 100% inoculation and so on, thus below mentioned are the tubes with inoculum
Tall Tube 1: Ale strain (pure). Tall Tube 2: Lager strain (pure). Tall Tube 3: Champagne strain (pure). Tall Tube 4: Wine strain (pure). Tall Tube 5: Wine strain + Ale strain (mixed). Tall Tube 6: Champagne strain + Ale strain (mixed). Tall Tube 7: Wine strain + Lager strain (mixed). Tall Tube 8: Champagne strain + Lager strain (mixed).
36
Figure 8: Tall Tube inoculated with different combination of strains Tall Tube 1: Ale strain (pure), Tall Tube 2: Lager strain (pure), Tall Tube 3: Champagne strain (pure), Tall Tube 4: Wine strain (pure), Tall Tube 5: Wine strain + Ale strain (mixed), Tall Tube 6: Champagne strain + Ale strain (mixed), Tall Tube 7: Wine strain + Lager strain (mixed), Tall Tube 8: Champagne strain + Lager strain (mixed).
37
IV.
Sampling and Analysis:
4. The tubes were inoculated and each day after that, samples were extracted at 24 hour intervals from the fermenters and subjected to analysis for cell density, specific gravity, pH and HPAE analysis for sugar concentration. 5. To draw out samples from tall tube fermenter is a difficult task especially when contamination has to be avoided at all costs and since the fermenters were being used at half capacity. 6. To resolve this we use the following setup: A tubing of 0.05mm diameter is fitted with a 1 ml glass pipette. The two are sealed in an autoclave bag and sterilized at 1210C for 15 min. The setup required using a yeast dosage pump through which the tubing is passed, the pipette end was immersed in the tall tube and the tube end was introduced in sterile centrifuged tubes where the samples were collected. Below is a picture of the setup, the motorized dosing pump system was set to deliver dosages accordingly. 50 ml sample was collected from each tall tube fermenter every day using the same setup. When switching from one to another care had to be taken so as to avoid cross contamination. To overcome this we use the glass pipettes which can be washed and rinsed with ethanol, autoclaved water and then incinerated in flame to remove any trace of living organisms
38
Rubber tubing Yeast dosage pump for delivering exact doses of sample
Glass pipette
Figure 9: Setup to withdraw samples from Tall tubes. 7. The samples collected were first analyzed for cell concentration and then centrifuged at 30,000 rpm for 10 minutes to separate out the yeast cells. The decanted solution was then tested for pH, specific gravity analyses. 1.5 ml of the decanted solution was stored in micro vials and used later to carry out HPAE analysis. 8. As per the results, we furthered this experiment by focusing only on the Lager and Champagne mixed culture fermentation. For this we carry out similar fermentation with the same media and sugar concentration but only on a much smaller scale i.e. 250ml conical flasks. These flasks were inoculated in the similar way and samples were collected for two days after every 2 hours. So that a more magnified picture in terms of sugar consumption and growth rate could be formed. 9. Four samples from conical flasks fermentation at 10th hour and 20th hour of pure S. bayanus culture and mixed S. bayanus and S. cerevisiae cultures were subjected to head space analysis via gas chromatography to analyze the volatile components of the media.
39
V.
Analytical Techniques:
Spectrophotometry: Using light absorbance measurement, we determine the cell density present in the samples. A Thermo Scientific GENESYS 20 visible spectrophotometer is used for indirect cell concentration measurements. The initial media prior to inoculation was used as a blank to set the spectrophotometer to 0. The principle of spectrophotometry is based on light absorbance or transmittance. As light passes through the samples, a scattering effect takes place which reduces the intensity of light reaching the detectors; the higher the cell count in the sample the more scattering of light that occur. Readings for all the 8 samples from each Tall tube was loaded in 8 cuvettes and measurements were taken at 660 nm at 24 hour interval.
HPAE analysis: To quantify the sugar uptake, we carried out High Performance Liquid Chromatography (HPAE) of the 8 samples collected every 24 hours. High Performance Liquid Chromatography (HPAE) was used to separate carbohydrates which when coupled with pulsed amperometric detection (PAD) permitted detection of carbohydrates with excellent signal to noise ratios down to approximately 10 picomoles (Dionex 20, 2011). Carbohydrates are weakly acidic and is selectively separated using eluents with high pH and a base stable polymer anion exchange, under such conditions carbohydrates disassociate and are separated as anions (Jahnel et
al., 1998). PAD detects carbohydrate by measuring the electrical current generated by their
oxidation
at
the
surface
of
gold
electrode
(Dionex
20,
2011).
pH measurements: A glass pH electrode with a temperature probe was used to measure pH. The pH of a solution indicates how acidic or basic (alkaline) it is. pH effects fermentation capability of yeast. Although ethanol is the principal, reduced fermentation product from the metabolism of glucose by S. cerevisiae, organic acids are also produced which lower the external pH of the fermentation broth (Dombek and Ingram, 1987). Thus pH measurements can give an insight into the fermentation rates and extent. Specific gravity measurements: A densitometer was used to measure the specific gravity of the samples collected. These densitometers are based on hydrostatic weighing, meters such as this determine the total weight of a fixed volume tube containing the liquid to be measured (Eren,
1999). Initially the specific gravity gives us the concentration of sugars present but as
40
fermentation progresses, ethanol and higher alcohols are produced as byproducts and this causes reduction in specific gravity as sugars get converted into alcohols. Since the ethanol produced has a much lower specific gravity than the sugars consumed by the yeast.
Gas Chromatography: To carry out head space analysis of selective samples we use gas chromatography. This is carried out in order to check for the concentration of volatile components in the sample. This data provides us with details regarding presence of ethanol and higher alcohols. Volatile compounds in samples are salted out in a sealed vial and the equilibrium headspace vapour is sampled and analysed by gas chromatography. The VDKs are measured by Electron Capture Detector (ECD), all other components are measured by Flame Ionisation Detector (FID). Samples are pre-heated at 60C for 90min in order to convert acetolactate and acetohydroxy-butyrate to diacetyl (2,3-butanedione) and 2,3 Pentanedione, respectively. (IBD, Recommended Method of Analysis).
41
RESULTS:
Samples collected every 24 hours for 3 days from Tall Tube fermenters 1-8 were monitored for essential factors that undergo changes during a typical fermentation such as pH, specific gravity, yeast cell biomass and sugar utilization. Results are presented in the following order:
Tall Tube 1: Ale
Tall Tube 2: Lager strain (pure) Tall Tube 3: Champagne strain (pure). Tall Tube 4: Wine strain (pure) Tall Tube 5: Wine strain + Ale strain (mixed). Tall Tube 6: Champagne strain + Ale strain (mixed). Tall Tube 7: Wine strain + Lager strain (mixed). Tall Tube 8: Champagne strain + Lager strain (mixed)
Note: Samples in HPAE and in Headspace analysis that were not detected are represented by ND in the tables.
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Tall Tube 1: Ale Pure Ale yeast S. cerevisiae culture fermentation profile OD at 660nm pH Specific Gravity 0.007 0.236 0.112 0.16 5.43 3.23 3.1 3.14 1.0055 1.0052 1.0045 1.0030
Days 0 24 48 72
Sugars in sample (mg/L) Galactose Glucose Fructose Sucrose Maltose
0 Hour 910 and 912.3 826.9 and 828.3 990.7 and 992.3 808.2 and 810.5 984 and 986
24 Hours 848.1 and 851.3 65.9 and 66.5 372.7 and 370.8 ND 136.7 and 138.6
48 Hours 739.6 and 741.2 ND ND ND ND
Table 2: Fermentation profile of ale yeast S. cerevisiae in terms of Specific Gravity, pH, Spectrophotometry and HPAE analysis of Sugar utilization at regular interval of 24 hours.
1200 1000 Concentration mg/L 800 600 400 200 0 0 24 Hours 48 72 galactose glucose fructose sucrose maltose
Figure 10: Graphical representation of the depleting sugars as determined by HPAE analysis in Pure Ale yeast S. cerevisiae culture
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Tall Tube 2: Lager strain (pure).
Hours 0 24 48 72
Pure lager yeast S. cerevisiae culture fermentation profile OD at 660nm pH Specific Gravity 0.027 0.014 0.01 0.427 5.42 3.73 3.29 3.25 1.0055 1.0055 1.0049 1.0035
24 Hours 806.2 and 808.4 816.9 and 818.3 984.7 and 982.1 63.3 and 65.2 874.6 and 873.5
48 Hours 528.9 and 530.2 354.6 and 351.8 611 and 614.3 ND 492.7 and 490.5
72 Hours 78 and 75.3 ND ND ND ND
Table 3: Fermentation profile of lager yeast S. cerevisiae in terms of Specific Gravity, pH, Spectrophotometry and HPAE analysis of Sugar utilization at regular interval of 24 hours.
Figure 11: Graphical representation of the depleting sugars as determined by HPAE analysis in pure lager yeast S. cerevisiae culture fermentation. 44
Tall Tube 3: Champagne strain (pure).
Pure Champagne yeast S. bayanus culture fermentation profile Hours OD @ 660nm pH Specific Gravity 0 0.015 5.39 1.0054 24 0.102 3.22 1.0051 48 0.44 3.16 1.0044 72 0.58 3.18 1.0032
24 Hours 906.3 and 904.6 19.3 and 20.56 143.4 and 145.6 ND 57.3 and 58.6
48 Hours 880.5 and 881.3 ND 12.8 and 11.7 ND ND
72 Hours 548 and 551.3 ND ND ND ND
Table 4: Fermentation profile of ale yeast S. bayanus in terms of Specific Gravity, pH, Spectrophotometry and HPAE analysis of Sugar utilization at regular interval of 24 hours.
Figure 12: Graphical representation of the depleting sugars as determined by HPAE analysis in Pure Champagne yeast S. bayanus. 45
Tall Tube 4: Wine strain (pure)
Hours 0 24 48 72
Pure Wine yeast S. fructum culture fermentation profile OD @ 660nm pH Specific Gravity 0.01 5.4 1.0054 0.011 3.3 1.0052 0.01 3.19 1.0048 0.473 3.11 1.0033
0 Hrs 910 and 912.3 826.9 and 828.3 990.7 and 992.3 808.2 and 810.5 984 and 986
24 Hrs 596.9 and 593.8 119.9 and 121.3 326.6 and 324.7 283 and 280 968.3 and 965.3
48 Hrs 338.1 and 335.9 58.1 and 58.8 250.3 and 252.1 48.2 and 47.3 953.2 and 951.4
72 Hrs ND ND ND ND 881.2 and 879.5
Table 5: Fermentation profile of Wine yeast S. fructum in terms of Specific Gravity, pH, Spectrophotometry and HPAE analysis of Sugar utilization at regular interval of 24 hours.
Figure 13: Graphical representation of the depleting sugars as determined by HPAE analysis in Pure Wine yeast S. fructum culture. 46
Tall Tube 5: Wine strain + Ale strain (mixed).
Wine yeast S. fructum and Ale yeast S. cerevisiae mixed culture fermentation profile Hours OD @ 660nm pH Specific Gravity 0.016 5.42 1.0053 0 0.12 3.2 1.0055 24 0.047 3.09 1.0047 48 0.274 3.09 1.0036 72 Sugars in sample (mg/L) Galactose Glucose Fructose Sucrose Maltose
24 Hours 831.2 and 833.6 138.7 and 135.9 534.3 and 536.1 11.9 and 13.2 406.6 and 407.9
48 Hours 423 and 421.8 ND 40.6 and 38.8 ND 73.3 and 75.6
Table 6: Fermentation profile of mixed culture of wine yeast S. fructum and Ale yeast S. cerevisiae in terms of Specific Gravity, pH, Spectrophotometry and HPAE analysis of Sugar utilization at regular interval of 24 hours.
1200 1000 800 600 400 200 0 0 24 48 72 maltose sucrose fructose glucose galactose
Figure 13: Graphical representation of the depleting sugars as determined by HPAE analysis in Wine yeast S. fructum and Ale yeast S. cerevisiae mixed culture. 47
Tall Tube 6: Champagne strain + Ale strain (mixed).
Champagne yeast S. bayanus and Ale yeast S. cerevisiae mixed culture fermentation profile Hours pH Specific Gravity OD @ 660nm 0.01 5.3 1.0056 0 0.178 3.25 1.0052 24 0.053 3.14 1.0040 48 0.515 3.13 1.0031 72 Sugars in sample (mg/L) Galactose Sample (mg/L) Glucose Fructose Sucrose Maltose 0 Hour 910 and 912.3 826.9 and 828.3 990.7 and 992.3 808.2 and 810.5 984 and 986 24 Hours 910 and 909.5 114.5 and 112.9 435.2 and 433.9 18.5 and 20.3 218.6 and 217.3 48 Hours 908.5 and 910.3 ND 18.6 and 17.8 ND ND 72 Hours 905 and 906.3 ND ND ND ND
Table 7: Fermentation profile of mixed culture containing Champagne yeast S. bayanus and Ale yeast S. cerevisiae in terms of Specific Gravity, pH, Spectrophotometry and HPAE analysis of Sugar utilization at regular interval of 24 hours.
1200 1000 Concentration in mg/L 800 600 400 200 0 0 24 Hours 48 72 galactose glucose fructose sucrose maltose
Figure 14: Graphical representation of the depleting sugars as determined by HPAE analysis in Champagne yeast S. bayanus and Ale yeast S. cerevisiae mixed culture.
48
Tall Tube 7: Wine strain + Lager strain (mixed).
Wine yeast S. fructum and Lager yeast S. cerevisiae mixed culture fermentation profile Hour pH Specific Gravity OD @ 660nm 0.009 5.4 1.0055 0 0.017 3.3 1.0053 24 0.005 3.21 1.0039 48 0.528 3.13 1.0033 72 Sugars in sample (mg/L) Galactose Glucose Fructose Sucrose
0 Hour 910 and 912.3 826.9 and 828.3 990.7 and 992.3 808.2 and 810.5
24 Hours 512.6 and 514.6 146.5 and 143.9 438.6 and 440.1 ND
48 Hours 241.8 and 242.2 21.2 and 20.3 168.8 and 166.9 ND
72 Hours ND ND ND ND
984 and 986 Maltose 686.5 and 683.6 491.6 and 489.6 310 and 312.1 SSample (mg/L) Table 8: Fermentation profile of mixed culture containing wine yeast S. fructum and lager yeast S. cerevisiae in terms of Specific Gravity, pH, Spectrophotometry and HPAE analysis of Sugar utilization at regular interval of 24 hours.
Figure 16: Graphical representation of the depleting sugars as determined by HPAE analysis in Wine yeast S. fructum and Lager yeast S. cerevisiae mixed culture 49
Tall Tube 8: Champagne strain + Lager strain (mixed)
Champagne yeast S. fructum Lager yeast S. cerevisiae mixed culture fermentation profile Hours pH Specific Gravity OD @ 660nm 0.01 5.4 1.0053 0 0.046 3.34 1.0050 24 0.034 3.25 1.0039 48 0.388 3.22 1.0030 72 Sugars in sample (mg/L) Galactose Glucose Fructose Sucrose
0 Hrs 910 and 912.3 826.9 and 828.3 990.7 and 992.3 808.2 and 810.5
24 Hrs 388.9 and 390.5 26.7 and 27.8 157.3 and 156.1 0
48 Hrs ND ND ND ND
72 Hrs ND ND ND ND
984 and 986 127.3 and 128.6 ND ND Maltose Table 9: Fermentation profile of mixed culture containing Lager yeast S. cerevisiae and Champagne yeast S. bayanus in terms of Specific Gravity, pH, Spectrophotometry and HPAE analysis of Sugar utilization at regular interval of 24 hours.
Figure 17: Graphical representation of the depleting sugars as determined by HPAE analysis in Champagne yeast S. fructum and Lager yeast S. cerevisiae mixed culture
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Observation of results: PURE CULTURE ANALYSIS Tall Tube - 1 S. cerevisiae Ale yeast. Ale fermentation progresses rapidly during the first 24 hours, during this time glucose and sucrose are utilized at a faster rate and near disappearance. The strain shows slight preference to maltose as it is depleted to a greater extent than fructose during the log phase. Major depletion of galactose is noticed from 48-72 hour as other sugars in the solution are completely exhausted.
Tall Tube - 2 S. cerevisiae Lager yeast. Lager strain fermentation shows a longer lag phase. During the first 24 hours, the strain prefers only sucrose and a very low overlap of other sugars is observed. Only after sucrose is utilized, are the other saccharides preferred. The cell concentrations were very low for the initial 48hours; Rapid growth begins thereafter. All the sugars are quickly depleted after the cells enter log phase. Among all the pure culture fermentation, Lager yeast S. cerevisiae showed a greater attenuation.
Tall Tube - 3 S. bayanus Champagne yeast. Faster growth rate is observed; cells grow rapidly and reach a higher cell concentration than other pure cultures. Strain shows major overlaps in sugar utilization, most sugars are utilized concomitantly except for galactose whose utilization only begins after all the other major sugars are depleted. A considerable amount of galactose remained at the end of the fermentation showing very low preference. The graph demonstrates the plummeting lines for all sugars except galactose, the slopes of which can be directly correlated to the rapid sugar consumption.
Tall Tube - 4 S. fructum Wine yeast. Growth in wine yeast is slow in the beginning due to the strain being adapted to low temperature fermentations. A very low pH is observed which shows the acidic character of the by-products. Sequence initially shows more preference to glucose than sucrose, followed by fructose and galactose. Graphically, galactose utilization can be seen to have been more consistent. Least preference is given to maltose according to our results and uptake only begins after all the other sugars are utilized. 51
MIXED CULTURE ANALYSIS Tall Tube 5 Wine yeast S. fructum and Ale yeast S. cerevisiae A better utilization of sugars is noticed in mixed cultures as compared to pure cultures of wine and ale yeast. In case of pure Ale S. cerevisiae and pure S. fructum yeast fermentation residual galactose and maltose was noticed respectively whereas the mixed culture had a lower. This has resulted in the pH to decline even further due to the greater extent of sugar utilization.
Tall Tube 6 Champagne yeast S. bayanus and Ale yeast S. cerevisiae A clear domination by S. bayanus is observed, the fermentation profile is a good indicator of this phenomenon. The pH, specific gravity and optical density measurements remain unchanged from that of the pure culture fermentation of S. bayanus. The sugar utilization data reveals similar results to that observed by pure S. bayanus strain. This shows domination because the vigorously growing S. bayanus yeast competes with the S. cerevisiae Ale yeast by utilizing sugar faster and thus dividing and dominating the population. Galactose assimilation is almost negligible in this case even though after 48 hours no other sugars had remained in the media.
Tall Tube 7 Wine yeast S. fructum and Lager yeast S. cerevisiae Comparing the pure and mixed culture fermentation, we can observe the two differences; reduction in the long lag phase from pure S. cerevisiae fermentation profile and better utilization of maltose from pure S. fructum fermentation. The low pH indicates sufficient wine growth and thus we can conclude that the culture has been symbiotic.
Tall Tube 8
Champagne yeast S. fructum Lager yeast S. cerevisiae
This mixed culture shows the most promising results in terms of sugar utilization. Sugars deplete rapidly and near disappearance at the end of 24th hour. No Sugars are detected at the end of 48th hour. Comparisons with pure culture fermentations of S. bayanus and S. cerevisiae shows incomplete fermentation in the former and longer lag phase which lasted for 24 hours in the latter. Also to be noted is the pH of 3.22, which seems to fall in between 3.18 (pure S. bayanus culture) and 3.25 (pure S. cerevisiae culture). Due to the faster utilization of sugars in this culture, we decided to conduct the same experiment but only with S. bayanus and S. cerevisiae strains on a smaller scale with sugar utilization measurements and optical density being recorded. 52
Results of Small scale conical flask fermentations of Lager (pure), Champagne (pure) and Lager + Champagne (mixed) yeast strains: Lager Yeast:
Hours after inoculation 0 2 4 6 8 10 20 22 24 26
Galactose
1192.9 & 1188.9 1058.2 & 1061.3 1040 & 1043.8 1039.6 & 1035.2 1027 & 1031 1024 & 1026.3 1016 & 1013 1012 & 1011 1004 & 1009 922.2 & 925.6
Glucose
1149.1 & 1147.6 1087 & 1089.6 1039.3 & 1036 1012 & 1015 970.1 & 975.6 911 & 906 898.4 & 895.1 868 & 873 819 & 814 768 & 774
Fructose
1254 & 1251.3 1191 & 1194 1170 & 1173 1166 & 1163 1158 & 1160 1143 & 1138 998.7 & 1003.2 921 & 925 892 & 889 814 & 817
Sucrose
991.8 & 993.6 858 & 863 693 & 696 442 & 446 319 & 323 245 & 241 ND ND ND ND
Maltose
1063 & 1060.8 976 & 985 957 & 962 443 & 446 937 & 942 933 & 930 927 & 924 910 & 913 898 & 892 849 & 852
Table 10: Sugar uptake data of S. cerevisiae lager strain mono culture as analysed via HPAE.
1400 1200 1000 galactose 800 600 400 200 0 0 2 4 6 8 10 Hours 20 22 24 26 glucose fructose sucrose maltose
Figure 18: Graphical representation of the depleting sugars as determined by HPAE analysis
Concentration mg/L
53
Champagne yeast:
Galactose
1192.9 & 1188.9 1051 & 1054 1033 & 1032 1019 & 1015 1011 & 1013 1003 & 1004 995 & 998 988 & 991 965 & 969 1051 & 1054
Glucose
1149.1 & 1147.6 985 & 988 886 & 888 847 & 839 627 & 633 327 & 324 ND ND ND ND
Fructose
1254 & 1251.3 1021 & 1027 1007 & 1012 975 & 970 937 & 943 721 & 720 110 & 114 ND ND ND
Sucrose
991.8 & 993.6 729 & 734 607 & 609 418 & 421 212 & 218 73 & 70 ND ND ND ND
Maltose
1063 & 1060.8 913 & 915 908 & 909 903 & 901 865 & 861 766 & 759 33 and 38 ND ND ND
Table 11: Sugar uptake data of S. bayanus champagne strain mono culture as analysed via HPAE.
1200 1000 Concentraiton mg/L 800 600 400 200 0 2 4 6 8 10 Hours 20 22 24 26
galactose glucose fructose sucrose maltose
Figure 19: Graphical representation of the depleting sugars as determined by HPAE analysis 54
Lager and Champagne yeast:

Galactose
1192.9 & 1188.9 1087 & 1093 1082 & 1078 1078 & 1076 1075 & 1072 1056 & 1063 1041 & 1045 1038 & 1033 987 & 984 790.8 & 793
Glucose
1149.1 & 1147.6 1125 & 1120 1082 & 1086 1068 & 1073 1059 & 1057 1037 & 1033 50 & 45.3 ND ND ND
Fructose
1254 & 1251.3 1204 & 1206 1114.9 & 1118 1081 & 1079 1039 & 1035 1018 & 1011 339 & 344 116 & 109 ND ND
Sucrose
991.8 & 993.6 960 & 966 934 & 937 788 & 783 622 & 619 508 & 502 ND ND ND ND
Maltose
1063 & 1060.8 989 & 984 980 & 978 974 & 970 965 & 963 929 & 924 105 & 111 ND ND ND
Table 12: Sugar uptake data of S. cerevisiae lager strain and S. bayanus champagne strain mixed culture as analysed via HPAE.
1400 1200 1000 800 600 400 200 0 0 2 4 6 8 10 Hours 20 22 24 26 Galactose Glucose Fructose Sucrose Maltose
Figure 20: Graphical representation of the depleting sugars as determined by HPAE analysis
Concentration mg/L
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Indirect cell density measurements at 660nm Hours after inoculation 2 4 6 8 10 20 22 24 26 S. cerevisiae 0.014 0.014 0.022 0.032 0.046 0.089 0.094 0.108 0.113 S. bayanus 0.069 0.069 0.076 0.152 0.273 0.574 0.58 0.612 0.598 S. cerevisiae + S. bayanus 0.006 0.009 0.013 0.028 0.05 0.575 0.607 0.624 0.621
Figure 13 Optical density measurements of Lager (pure), Champagne (pure) and Lager and Champagne (mixed) cultures at 660 nm.
0.7 0.6 0.5 Optical Density 0.4 L 0.3 0.2 0.1 0 2 4 6 8 10 Hours 20 22 24 26 C L+C
Figure 21 Graphical representation of the depleting sugars as determined by HPAE analysis 56
Small Scale fermentation results analysis:

S. cerevisiae (Lager yeast)
The fermentation study reveals the previously noted phenomenon in lager strain S. cerevisiae. The yeast shows one-sided preference to sucrose alone during the first 24 hours. Due to this other sugar concentrations have largely remained unchanged in the media. The growth curve shows a long lag phase extending throughout.
S. bayanus (Champagne yeast)
Sugars have been metabolized according to the sequence sucrose, glucose, fructose, maltose and Galactose. Sucrose depleted the fastest followed by glucose; Fructose and maltose are initially assimilated at low concentration. After the disappearance of the two preferred substrates; glucose and sucrose, uptake of maltose and fructose increase exponentially. The plunging sugar concentrations of fructose and maltose can be compared from the graph. The two sugars are assimilated in tandem as the two lines overlap. The cell density increases gradually and smoothly every hour as evident from the graph. No significant decrease in galactose is noticed even thou other sugars are completely utilized from the medium.
S. cerevisiae and S. bayanus (Lager and Champagne yeast)
The mixed culture on closer inspection reveals initial lag in sugar utilization and only sucrose has been utilized consistently as compared to other sugars. During the first 10 hours, cell proliferation rate is low and growth curve seems almost parallel to the x-axis. After the initial lag, proliferation of cells begin at a much higher rate than that observed in the above two pure cultures. The high proliferation of cells demands rapid sugar uptake, graphical lines of glucose and maltose decreasing concentration suggest the two sugars are simultaneously assimilated between the 10th and 20th hour. Reduction in galactose is noticed to begin only after all the other sugars are exhausted. However as compared to the pure cultures, assimilation of galactose begins much earlier after other sugars in the medium are utilized completely and a substantial reduction is observed. The cell growth density is observed to be higher than S. bayanus and S. cerevisiae pure culture.
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Head Space Analysis: Head space analysis was conducted via gas chromatography to determine the concentrations of various volatiles produced as by products during fermentation. The production of by-products is directly related to the sugar utilization and gives us an insight into the aromatic and flavor profile of the strain. Compounds such as esters, higher alcohol, sulphur compounds, and volatile fatty acids are derived from sugar and amino acid metabolism, and their production is dependent on species and strains (Swieger et al., 2005, Romano et. al., 2003 as cited in Corts and Blanco, 2011). Esters are formed intracellularly in an enzyme-catalyzed condensation reaction between two cosubstrates, a higher alcohol and an activated acyl-coenzyme A (acyl-CoA) molecule (Nordstrm, 1962, 1963 as cited in Verstrepen et al., 2003). These compounds are majorly responsible for flavor and aroma of fermented drinks. Lager strains fermentation samples were not subjected to analysis as we know that S. bayanus strain is largely responsible for high ester production. Also the growth of the lager strain was highly limited due to which production of such by products would have been present at a very low quantity. Samples from S. bayanus pure culture 10th (C-5), 20th hour (C-6) and S. bayanus and S. cerevisiae mixed cultures from 10th (LC 5), 20th hour (LC 6) were subjected to head space analysis to measure the concentration of certain volatiles.
58
Volatile measurements of samples from S. bayanus pure cultures and S. cerevisiae + S. bayanus mixed culture
Volatiles Measured Acetaldehyde acetone ethyl acetate iso butyl acetate ethyl butyrate propanol iso butanol iso amy acetate 2-methyl butanol 3-methyl butanol ethyl hexanoate ethyl octanoate butanedione
C5 10th H
4.59 & 4.68 ND ND ND ND 0.35 & 0.33 0.055 & 0.057 ND 0.066 & 0.061 0.46 & 0.48 ND ND 0.0567 & 0.0561 0.0464 & 0.0468
C6 20th H
5.93 & 5.99 ND 0.061 & 0.065 ND ND 1.11 & 1.18 0.270 & 0.276 ND 0.113 & 0.119 0.63 & 0.66 ND ND 0.0151 & 0.0156 0.0106 & 0.0111
LC5 10th H
2.48 & 2.41 ND ND ND ND ND ND ND ND 0.14 & 0.17 ND ND 0.0466 & 0.0469 0.0232 & 0.0238
LC6 20th H
5.50 & 5.58 ND 0.026 & 0.021 ND ND 0.80 & 0.82 0.185 & 0.194 ND 0.054 & 0.058 0.510 & 0.502 ND ND 0.0297 & 0.0294 0.0230 & 0.0224
pentanedione
Table 14: Head space analysis reveal the concentration of some of the volatile compounds present in samples
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Evaluation of Head Space analysis results: Samples only from 10th and 20th hour were analyzed because substantial changes occur during this period. Samples from pure champagne yeast S. bayanus from 10th and 20th hour samples show the presence of significant quantities of acetaldehyde. In mixed cultures, concentration of acetaldehyde is low initially as compared to that present in pure culture. However as the growth of cells surges and sugars are consumed rapidly, acetaldehyde production increases. A significant difference is measured in the production of ethyl acetate, the concentration of which is higher in pure culture fermentation of S. bayanus but much lower in mixed culture fermentation. Even though both cultures consumed almost equivalent amounts of sugar and the biomass concentration is noticed to be much higher in mixed cultures, the ethyl acetate concentration difference is significant. Quantitatively, the main ester is ethyl acetate derived from the ethanolysis of acetyl Co-A (de la Roza et al., 2003). The synthesis of ethyl acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to ethanol acetyltransferase (Lilly et al., 2000). This implies that higher levels of ethyl acetate are a result of low ethanolysis of acetyl Co-A. Ethyl acetate is also produced as a result of condensation reaction between ethanol and acetic acid, so this in turn implies that acetic acid production was much lower in S. cerevisiae and S. bayanus mixed culture. Concentration of higher alcohols such as propanol, iso butanol, 2-methyl butanol and 3-methyl butanol are produced in lower quantities in mixed cultures as compared to pure culture of S. bayanus. Butanedione and pentanedione quantities seem to decrease during this phase, except for pentanedione concentration in mixed cultures remained unaltered.
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Discussion: Our studies on mixed cultures show us a lot of variation in sugar utilization; indicating that genotype of the strain not only determines the sequence and preference for sugars but also its interaction with other related species. As per our hypothesis although we could not achieve faster rate of sugar utilization as we had predicted, but results show us improvements as compared to that of pure culture fermentation. Mixed cultures show promising results in terms of substrate utilization and have thus lower attenuation levels. Fermentation studies conducted on grape must which constitutes majorly of glucose and fructose showed 20 g L-1 of residual sugar in pure cultures whereas only 2 g L-1 was found in mixed culture at the end (Howell et al., 2006). Except for S. bayanus and S. cerevisiae (ale) mixed culture, attenuation has been higher in all mixed cultures as compared to pure cultures. Interestingly, the above exception although clearly was being dominated by S. bayanus an inhibition is noticed in its ability for galactose utilization which the strain possessed while in pure culture. We can conclude that byproducts secreted by S. cerevisiae ale yeasts could cause an inhibition in expression of GAL genes responsible for galactose assimilation in S. bayanus. In this case repression of galactose due to the presence of glucose can be ignored as the medium was found to be devoid of all the other remaining sugars, yet galactose assimilation did not occur after 48 hours. Mixed cultures containing wine yeast S. fructum was found to have comparatively faster and more complete fermentation in mixed culture with ale and lager yeast. Among the two mixed culture, fermentation carried out with ale yeast S. cerevisiae showed better results than culture studies carried out with S. cerevisiae lager yeast. Wine yeast when in pure culture fails to metabolize maltose; this can be due to the adaptive nature of yeast. Wine yeasts are seldom exposed to maltose sugar as grape juices contain majorly glucose and fructose. It is known that yeast cells can be trained to utilize sugars (Harden, 1910); the absence of exposure to maltose could have thus deactivated the synthesis of MAL genes responsible for its assimilation. In the case of S. bayanus and S. cerevisiae (lager yeast) mixed cultures, faster utilization does occur but only during the log phase. HPAE analysis reveal the initial period of strains adapting to each other during which sugar assimilation occurs at a very low rate. After overcoming this lag, 61
rapid growth of cells and assimilation of sugars occur. Our second stage of experiment with S. bayanus and S. cerevisiae reveal symbiotic interaction among the two strains. One fundamental reason could be due to the major genomic similarity possessed by the two strains. Introgression evidence between biological species of S. cerevisiae and S. bayanus for several strains of S. bayanus has been found containing subtelomeric SUC and Y sequence of S. cerevisiae ((Naumov et al., 1992 as cited in Naumova et al., 2005). The combination of S. bayanus and S. cerevisiae also works well as S. cerevisiae complements the short comings of S. bayanus in terms of fructose utilization. As discussed previously, sugar uptake is directly correlated to hexokinase encoded sequence. A study concerning fructose uptake capacity found that four sparkling wine strains of S. bayanus had remarkably lower fructose utilization capacity which was in contrast to that found in S. cerevisiae (Schtz and Gafner, 1995). Growth curves of pure S. bayanus and S. cerevisiae cultures when compared to that of mixed cultures shows us that initially only pure S. bayanus growth has been consistent. The enhanced growth of cell cultures in mixed environment could be associated with the production of essential secondary metabolites or growth factors by one strain that could be utilized by the other. Secondary metabolites would act as sources of carbon or nitrogen and produce the desired growth conditions, such as altering the pH and temperature (D.E.F, 1978).
Production of volatiles also appears to be affected in mixed culture of S. bayanus and S. cerevisiae, especially the production of ethyl acetate whose concentration levels were found to be less than half of that produced in pure S. bayanus culture. Chardonnay wines that were fermented with Saccharomyces bayanus, instead of Saccharomyces cerevisiae, produced higher levels of certain organic acids and new aroma descriptors. By this reaction we can conclude either the presence of S. cerevisiae affected the metabolism of sugars in S. bayanus at genetic or physiological levels or that metabolites produced by one were utilized by the other. It has been theorized in mixed cultures fermentation studies on volatile composition of wines that compounds produced by one strain could be taken up by another and in this way sharing of metabolites might have occurred (Howell et al., 2006).
In future work, using E.B.C tall tube fermenters instead of tall tube fermenters that have been designed to mimic the cylindro-conical fermenter vessels could provide us better insight into the 62
fermentation when it comes to industrial application. The tube is constructed out of glass which is surrounded with a cooling jacket through which the coolant can be continuously circulated. The tube ends in a cone with a tap attached at the bottom for cropping purposes and the sample port that is designed to collect samples through midway of the tube. This ensures samples are uniform and that temperature conditions can be controlled as growth of lager yeasts and production of esters have a direct influence of temperature. Reason for the lag times in lager yeast in pure and mixed culture could be due to the fact that temperature conditions were not regulated according to the requirements of the strain and actual fermentation occurred at 20 0 C which is quite high for industrial lager fermentation. Carrying out similar fermentation in E.B.C with wort as the medium rather than defined media would be interesting. Due to the complexity of this media, factors such as sugar utilization, yeast growth and volatile production would differ significantly. Our studies could also be applied in cider production as they are fermented by S. bayanus and controlling ethyl acetate production would greatly increase the quality of the product. Even though the large amount of ethyl acetate gives cider its characteristic flavor and aroma, higher quantities are considered to degrade the quality of product and thus their control during fermentation has become essential (de la Roza et al., 2003). In chardonnay wine fermentation study with S. bayanus authors established that S. bayanus could be a good candidate to replace, or compliment, S. cerevisiae inoculations (Eglinton et al., 2000).
With the advent of genetic engineering, strains are being developed with multiple or selective genomes which can imitate the production of flavors in mixed culture fermentation. However it seems quite a daunting task to gather genomic sequences for enzymes and using on/off codon to regulate their production so as to produce the desirable flavor profile.
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Carbohydrate utilization in mixed culture fermentation

Amit Rajan Mishra H00023055 am720@hw.ac.uk

Dissertation supervisor: Dr. Annie Hill

Following the production of 2 molecules of pyruvate,

Raw materials in Potable Alcohol Production:

Carbon catabolite repression:

4. Tall Tube fermenters: 8 Tall Tube fermenters of 2 L capacity each.

Yeast Nitrogen Base media preparation:

3. Preparing the final media for tall tube:

Sampling and Analysis:

Tall Tube 1: Ale

Sugars in sample (mg/L) Galactose Glucose Fructose Sucrose Maltose

48 Hours 739.6 and 741.2 ND ND ND ND

72 Hours 388.7 and 389.6 ND ND ND ND

Tall Tube 2: Lager strain (pure).

Sugars in sample (mg/L) Galactose Glucose Fructose Sucrose Maltose

72 Hours 78 and 75.3 ND ND ND ND

Tall Tube 3: Champagne strain (pure).

Sugars in sample (mg/L) Galactose Glucose Fructose Sucrose Maltose

48 Hours 880.5 and 881.3 ND 12.8 and 11.7 ND ND

72 Hours 548 and 551.3 ND ND ND ND

Tall Tube 4: Wine strain (pure)

Sugars in sample (mg/L) Galactose Glucose Fructose Sucrose Maltose

72 Hrs ND ND ND ND 881.2 and 879.5

Tall Tube 5: Wine strain + Ale strain (mixed).

72 Hours 140.6 and 138.9 ND ND ND ND

Tall Tube 6: Champagne strain + Ale strain (mixed).

Tall Tube 7: Wine strain + Lager strain (mixed).

Tall Tube 8: Champagne strain + Lager strain (mixed)

Champagne yeast S. fructum Lager yeast S. cerevisiae

galactose glucose fructose sucrose maltose

Lager and Champagne yeast:

Figure 21 Graphical representation of the depleting sugars as determined by HPAE analysis 56

Small Scale fermentation results analysis:

S. bayanus (Champagne yeast)

S. cerevisiae and S. bayanus (Lager and Champagne yeast)

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