Saccharomyces's blog en Yeast Cultures are Like Nuclear Weapons <div class="field field-name-body field-type-text-with-summary field-label-hidden view-mode-rss"><div class="field-items"><div class="field-item even" property="content:encoded"><p>Back in the bad old days, a home brewer was happy just to have a reliable yeast culture to pitch into his/her wort. The average home brewer today is no longer content with having access to yeast cultures that get the job done without leaving a trail of metabolic trash that is a mile wide. He/she wants to be able to compute and hit the exact number of cells needed to ferment a given batch of wort. The cold hard truth is that this level of precision is neither obtainable, nor is it necessary in a home brewery. A yeast culture is like a nuclear weapon in that a brewer only needs to be within a reasonable distance of his/her target in order to complete the task at hand. In this blog entry, we will cover how the yeast biomass in a starter or fermentation grows, and why home brewers are placing emphasis on precision where it is not needed.</p> <p>Brewing yeast is a mystery to many home brewers. It is easily the most technically complex part of the brewing process, after all, brewers make wort, yeast makes beer. Fermentation is little more than controlled spoilage. In the case of beer, the spoilage microorganism is the yeast culture that we pitch. We want the pitched yeast culture to “own” the wort, and we want to ensure that it owns the wort quickly enough to prevent any wild microflora (yeast, bacteria, mold) that may have hitched a ride on airborne particulate matter from gaining a foothold in our fermentation. We accomplish this task by pitching a large number of active yeast cells while practicing good brewery hygiene.</p> <p>Yeast cells go through three distinct phases during fermentation before entering a state known as quiescence. The phase that we will be discussing in this blog entry is known as the exponential phase (also known as the logarithmic phase). The exponential phase is where the yeast biomass grows. This phase is called the exponential phase because the cell count grows exponentially at a rate of 2<sup>n</sup>, where n is the number of minutes that have elapsed since the culture entered the exponential phase divided by the replication period in minutes (computer scientists who are reading this blog entry will recognize this growth pattern as O(2<sup>n</sup>), or binary exponential growth). The reason why the yeast biomass grows at a rate of 2<sup>n</sup> is because each mother cell buds a daughter cell during a replication period.</p> <p> With the above said, let’s examine the basic formula for approximating the cell count at a given number of minutes into the exponential phase. yeast_cell_count_at_time_t = initial_yeast_cell_count * 2<sup>n</sup>, where n equals the number of minutes that have elapsed since the beginning of the exponential phase (time_t) divided by the replication period (replication_period) Let’s apply the formula shown above with time_t equal to 90 minutes and replication_period equal to 90 minutes.</p> <p>time_t = 90 minutes into the exponential phase</p> <p>initial_cell_count = 200 billion</p> <p>replication_period = 90 minutes</p> <p>n = 90 / 90 = 1 replication period</p> <p>yeast_cell_count_at_time_t = 200 billion * 2^1 = 400 billion cells, where the symbol "^" denotes raised to the power of</p> <p>After ninety minutes of exponential growth, the culture has doubled in size.</p> <p>Let’s extend time_t to six hours, which equals three hundred and sixty minutes.</p> <p>n = 360 / 90 = 4</p> <p>yeast_cell_count_at_time_t = 200,000,000,000 (200 billion) * 2^4 = 3,200,000,000,000 (3.2 trillion) cells</p> <p>After four replication periods, the cell count is now sixteen times larger than it was when it was pitched. Herein, lies the explosive power of exponential growth.</p> <p>We can determine the number of replication periods necessary to reach a target cell count given an initial cell count by re-writing the equation to solve for n. We will refer to the variable n as the number_of_replication_periods and the variable cell_count_at_time_t as target_cell_count in our re-written equation.</p> <p>number_of_replication_periods = log (target_cell_count / initial_cell_count) / log(2)</p> <p>Let’s set target_cell_count to 3.2 trillion and initial_cell_count to 200 billion to verify that the formula produces the number 4 for the number of replication periods.</p> <p>number_of_replication_periods = log (3,200,000,000,000 / 200,000,000,000) / log(2) = 4</p> <p>With that said, the yeast calculator that we used for our latest recipe determined that we needed to pitch 200 billion cells. Our culture only contains 150 billion cells. How much impact will underpitching by 50 billion cells make in the amount of time necessary to reach maximum cell density for 5 gallons, which is approximately 3.8 trillion cells?</p> <p>number_of_replication_periods = log (3,800,000,000,000 / 200,000,000,000) / log(2) = ~4.25</p> <p>number_of_replication_periods = log (3,800,000,000,000 / 150,000,000,000) / log(2) = ~4.66</p> <p>As long as there is enough oxygen in solution to support cellular health, the difference in exponential growth time between pitching 150 billion cells and 200 billion cells is 4.66 – 4.25 = 0.41 * 90 = ~37 minutes.</p> <p>Okay, let’s pitch half of the number of cells that our yeast calculator computed.</p> <p>number_of_replication_periods = log (3,800,000,000,000 / 100,000,000,000) / log(2) = ~5.25</p> <p>Once again, as long as there is enough oxygen in solution to support cellular health, the difference in exponential growth time between pitching 100 billion cells and 200 billion cells is 5.25 – 4.25 = 1.0 * 90 = 90 minutes.</p> <p>As one can clearly see, underpitching by as much as 50% only lengthens the exponential growth phase by 90 minutes. The key is to ensure that there is adequate dissolved oxygen to support cellular health when underpitching, as there is almost always enough carbon (sugar is carbon bound to water; hence, the term carbohydrate). While brewing species within the Saccharomyces genus do not respire in brewer’s wort due to being Crabtree positive, they do use oxygen for ergosterol and unsaturated fatty acid (UFA) biosynthesis by shunting oxygen and a small amount of carbon to the respirative metabolic pathway during the lag phase. These compounds are used by cells to maintain their plasma membranes. Plasma membrane health determines how well a yeast cell can take in nutrients and expel waste through its cell wall.</p> <p>If yeast cultures are like nuclear weapons, why do we have pitching guidelines? Well, most pitching guidelines are for slurry, not laboratory grown yeast. Slurry is a mixture of various age cells that have been through one or more fermentations. These cells have been subjected to ethanol and brewery-related environmental stress. Most starters are grown from laboratory-cultured yeast. Laboratory-prepared growth media and environmental conditions are designed to maximize biomass growth while minimizing stress.</p> <p>With that said, there is a significant challenge that places a lower bound on our pitched cell count; namely, sanitation. No real-world brewery is sterile. Airborne microflora has an opportunity to contaminate a culture or a medium every time it is exposed to air. Residual surface contamination from less than adequate cleaning and/or sanitation increases the chance of wild microflora gaining a foothold in a fermentation. Bacteria are the biggest threat because they too grow exponentially, but their replication period is one third that of yeast; hence, the bacteria cell count increases by a factor of eight every time the yeast cell count doubles. If we normalize the bacteria growth model to that of the yeast growth model, we end up with the equation shown below.</p> <p>bacteria_cell_count_at_time_t = initial_bacteria_cell_count * 8<sup>n</sup>, where n equals the number of minutes that have elapsed since the beginning of the exponential phase divided by the yeast replication period</p> <p>Since the bacteria cell count doubles in one third of the amount of time that it takes the yeast cell count to double, the bacteria cell count grows at a rate of 2^3 every time the yeast cell count grows at a rate of 2^1.</p> <p>To give readers an idea of how this difference allows a tiny number of bacteria cells to overtake a larger number of yeast cells, let’s calculate the 2n and 8n multipliers out to 16 yeast eplication periods (n = 1 to 16).</p> <p>Cell count multiplier for the 2n growth pattern = 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, 8192, 16384, 32768, 65536</p> <p>Cell count multiplier for the 8<sup>n</sup> growth pattern = 8, 64, 512, 4096, 32768, 262144, 2097152, 16777216, 134217728, <a href="tel:1073741824">1073741824</a>, <a href="tel:8589934592">8589934592</a>, <a href="tel:68719476736">68719476736</a>, <a href="tel:549755813888">549755813888</a>, <a href="tel:4398046511104">4398046511104</a>, <a href="tel:35184372088832">35184372088832</a>, <a href="tel:281474976710656">281474976710656</a></p> <p>After sixteen yeast replication periods, the yeast cell count can be as much as 65,536 times larger than it was when we started whereas the bacteria cell count can be as much as a whopping 281,474,976,710,656 times larger than when we started, that is, if there is sufficient carbon, oxygen, and space to support that much growth. Herein, lies the reason why we need to pitch a large number of cells. We want our yeast culture to rapidly shutdown the replication of competitors by dominating a batch of wort. In modern vernacular, we want the culture to “own” the wort to the extent that nothing else stands a chance of tainting our controlled spoilage process. However, that feat can be easily accomplished with modern commercial yeast cultures without having to worry about pitching a precise number of yeast cells.</p> <p>In closing, hopefully readers have gained an understanding of the explosive nature of exponential growth. Exponential growth can basically erase a difference in cell counts that is less then a factor of two, and make up to a factor of four difference in cell counts insignificant, which is why yeast cultures are like nuclear weapons. There are even times when we want to purposely underpitch, but that is a topic for a future blog entry.</p> <p> </p> </div></div></div> Fri, 29 Jan 2021 01:09:40 +0000 Saccharomyces 798 at Shaken, not Stirred: The Stir Plate Myth Buster <div class="field field-name-body field-type-text-with-summary field-label-hidden view-mode-rss"><div class="field-items"><div class="field-item even" property="content:encoded"><h2>Introduction</h2> <p>When I started to brew in early 1993, no one I knew used a stir plate. That count included all of the hardcore amateur brewers I knew at that point in time and throughout my first pass through the hobby.  I brewed all-grain beer and maintained a yeast bank on agar slants for over a decade before taking a hiatus to focus on my family.  When I came back to the hobby in 2013, everyone was using a stir plate and proclaiming that a stir plate was a “must have” if one was going to make starters.  My experience with yeast cultures did not align with this assertion.  However, being inquisitive, I played along and purchased a stir plate and bar.  The performance of my cultures did not match what was promised. The media was so foul smelling after turning off the stir plate that I ditched it and went back to my old way of making a starter. </p> <p>This blog entry covers yeast propagation in general, claims made by proponents of stir plates, and my method of making and handling a starter.  My method is not the do all, be all yeast starter method, but it provides a simpler, lower cost way of making a starter that performs just as well, if not better than one made using a stir plate.</p> <h2>Yeast Propagation</h2> <p>The goal of yeast propagation is to transform a small number of yeast cells into a larger number of cells.  There are two basic types of propagation; namely, batch and continuous.  Propagation that is performed in steps with each step increasing the number of cells is known as “batch propagation.”  Propagation where an initial seed culture starts replication with a continuous flow of new culturing medium and O<sub>2</sub> entering the process with new yeast cells continuously existing the process is known as, wait for it, “continuous propagation.”  Continuous propagation is significantly more complex than batch propagation.</p> <h3>Batch Propagation</h3> <p>Brewers and liquid culture propagators use what is known as batch propagation.  Batch propagation is used when the medium is above the Crabtree threshold, which is 0.2% w/v (a specific gravity of 1.0008).  The Crabtree effect states that whenever the medium is above the Crabtree threshold, Crabtree positive yeast cells will replicate using their anaerobic (fermentative) metabolic pathway even in the presence of O<sub>2</sub>.  All brewing yeast strains are Crabtree positive.  Brewing yeast cells never truly respire when grown in a medium that is above the Crabtree threshold.  Instead, they shunt O<sub>2 </sub> and a small amount of carbon (sugar is carbon bound to water) to the aerobic (respirative) metabolic pathway for the production of ergosterol and unsaturated fatty acids (UFA) during the lag phase.  Ergosterol (the plant equivalent of cholesterol) and UFAs keep cell plasma membranes pliable, which, in turn, allow sugar and waste products to pass through these membranes.</p> <h3>Continuous Propagation</h3> <p>Unlike batch yeast propagation, dry yeast propagators use continuous propagation.  They do so using something known as a bioreactor.  A bioreactor can keep the medium in a steady state below the Crabtree threshold while continuously supplying O<sub>2 . </sub> The medium is stirred to keep it homogenous.  The stirring action creates shear stress, which is detrimental to yeast cell wall health.  However, it is necessary to keep the medium in a steady state.  Keeping the medium oxygenated in a steady state below the Crabtree threshold results in yeast cells using their much more efficient aerobic (respirative) metabolic pathway for reproduction.  Ethanol, higher alcohols, and esters are the result of the inefficient conversion of carbon to energy in fermentation.  These compounds are all carbon based; therefore, we can think of them as the yeast cell metabolism equivalent of incomplete combustion.  The byproducts of respiration are CO<sub>2 </sub>gas and water.  Yeast cells derive two ATP (adenosine triphosphate) per glucose via fermentation and eighteen ATP per glucose via respiration (ATP is what powers yeast cells).  In effect, the respirative metabolic pathway is nine times more efficient than the fermentative metabolic pathway.  What this increase in carbon utilization means to dry yeast producers is that they can use less carbon than liquid yeast propagators for any given cell count.  Not only that, yeast cells propagated via the respirative metabolic pathway have fully charged ergosterol and UFA reserves when they are pitched, which significantly reduces initial dissolved O<sub>2</sub> requirements, usually completely alleviating the need to oxygenate wort.</p> <h2>Claims Made by Proponents of Stir Plates</h2> <p>If I had a dollar for every time a new brewer was advised to purchase a stir plate for making yeast starters, I would be able to build a very nice custom brew house.  As mentioned above, a stir plate has become a “must have” piece of equipment after a new brewer advances beyond the kit beer stage.  In my humble opinion, a stir plate is a “not needed” piece of brewing equipment.   What bothers me are the claims made when discussing stir plates, none of which appear to be grounded in science.  That being said, let’s examine claims made by proponents of stir plates.  </p> <h3>Oxygenation</h3> <p>It is claimed that stir plates oxygenate yeast cultures.  However, anyone who has studied physics and chemistry knows that the shape of an Erlenmeyer flask does not lend itself to O<sub>2 </sub>absorption.  The conical shape of an Erlenmeyer flask combined with its rapidly narrowing cone leads to a small specific surface area in which O<sub>2 </sub>can be absorbed.  People claim that spinning the bar fast enough to create a vortex improves oxygenation.  To a point, that claim is true because it results in an increase in specific surface area.  However, doing so comes at a cost to yeast cell wall health due to shear stress caused by the spinning bar (i.e., the spinning bar is a source of friction for the yeast cells in a starter).  Shear stress is something that has been well studied when it comes to the production of dry yeast.</p> <h3>Cell Suspension</h3> <p>Many amateur brewers use the argument that stir plates keep yeast cells in suspension.  This claim is true, but the cells that a stir plate keeps in suspension are non-viable and cells prone to early flocculation, neither of which are desirable. The counter to this argument is that brewing yeast cells do not need to be stirred to remain in suspension because they express the NewFlo phenotype.  NewFlo yeast strains do not flocculate until they have consumed all of the mannose, maltose, sucrose, glucose and the sugars that they can reduce to one of these four sugars in the wort, including maltotriose, which is composed of three glucose molecules bound by two glycosidic bonds (a molecule of maltotriose is first reduced to a molecule of glucose and a molecule of maltose, which is then reduced to two molecules of glucose).</p> <h3>Higher Cell Count</h3> <p>The reality is that all propagation mediums have a maximum cell density that places an upper bound on the number of viable cells that can be produced given a specific volume.  The generally accepted rule of thumb in brewing is two hundred billion cells per liter. The amount of yeast sediment available after a yeast culture has entered quiescence and sedimented does not matter.  What matters is the number of viable cells in the yeast sediment.</p> <h3>Healthier Yeast</h3> <p>There is not a more troubling claim in amateur brewing than stir plates produce healthier cultures. Not only is this claim wrong, the practice of allowing a starter to enter quiescence and settle out, so foul smelling medium that is the result of stressed yeast cells can be decanted misses the point.  What we are doing when creating a starter and then pitching it into a larger batch of wort is known as step propagation.  Allowing a starter to ferment out places the cells in a quiescent state where they have undergone morphological changes to guard against starvation.  An important morphological change that occurs is thickening of the cell wall.  That morphological change occurs in order to extend the time between sedimentation and autolysis, which occurs when a cell wall breaks down and releases the contents of a cell.  Allowing a culture to enter quiescence extends lag time because the cells have to reverse the morphological changes they underwent before they can go about replenishing their ergosterol and UFA reserves. </p> <h2>Shaken, not Stirred</h2> <p>I remember the pushback I received when I introduced the Shaken, not Stirred method for making and pitching a starter on the American Homebrewers Association forum.  The response I received made me feel like I had slapped someone’s puppy.  People could not believe that something so simple could perform as well, if not better than a stirred culture.  However, one by one, forum members started to give my starter method a shot.  It was like the floodgates of acceptance opened after Denny Conn gave it a shot and wrote about his experience.  Let’s discuss how SNS came about and why it works so well while being simple and low cost.</p> <h3>Background</h3> <p>The Shaken, not Stirred (SNS) method of making a starter is something that I stumbled upon in the early nineties.  It was the result of being much stronger than I am today due to being a gym rat in my twenties and early thirties.  SNS started out as just a way to disperse yeast cells after inoculating a starter.  I was using a repurposed forty-eight fluid ounce Ocean Spray glass container to make quart-size starters. That container broke one day, so I used the only other glass container I had on hand with a screw on cap, which was a one-gallon glass jug.  </p> <h3>Specific Surface Area</h3> <p>After I switched to using a one-gallon jug for making quart-size starters, I noticed that the increased volume of the container combined with intense shaking resulted in a large portion of the medium being transformed into foam.  The difference in lag time and fermentation strength appeared immediately.  However, it took a couple of months before the engineering side of my brain recognized why the SNS method produced a higher quality starter. The secret sauce is the large amount specific surface area provided by foam, which allows for significantly more O<sub>2</sub> absorption than the standard air to liquid interface.  In essence, SNS is a poor man’s O<sub>2 </sub>bottle.  One can achieve the same result with an  O<sub>2  </sub>bottle and a diffusion stone.  It is just that SNS achieves this feat in a much simpler, lower cost way.</p> <h3>Pitch Timing</h3> <p>With that said, it is not just the shaking in the SNS method that leads to a higher quality starter.  It is the fact that the method involves pitching the entire starter at high krausen.  High krausen is the point where the culture switches over from exponential growth to the stationary phase where replication is for replacement only.  Allowing a culture to ferment beyond this point results in wasted ergosterol and UFA reserves.  These compounds are produced during the lag phase and are shared by mother cells with all of their daughter cells.  We do not want to allow a starter tor ferment past high krausen because doing so will result in extended lag times and higher initial O<sub>2 </sub>requirements in order to allow mother cells to rebuild the ergosterol and UFA reserves that were wasted on replacement replication during the stationary phase.  Remember, yeast cells are in a battle with bacteria cells for ownership of the wort and bacteria cells replicate three times in the period of time that is required for the yeast cells to replicate; therefore, anything we can do to shorten the lag phase is beneficial to fermentation.</p> <h3>Closing Remarks</h3> <p>The “stir plates produce higher viable cell counts and healthier yeast cultures than other methods” myth is just that, a myth.  Every starter volume has a maximum cell density, which if met before available sources of carbon have been depleted cannot be exceeded.  My suggestion is to give the SNS starter method a try.  All one needs is a sanitizable container that is at least four times the volume of the starter wort (I use a five liter media bottle for one liter starters).  The starter medium can be shaken before or after pitching.  It depends on one’s threshold for yeast cell stress becase the initial shake needs to be performed like the starter wort owes one money.  For people new to this technique, I recommend pitching after shaking.  If one pitches after shaking, the starter will need to be shaken a second time to disperse the cells, albeit much more gently.   The goal is to attempt to transform as much of the starter wort into foam as is humanly possible. The photo shown below is my old five-liter media bottle (there was a liter of liquid before the shake).</p> <p><img alt="" src="" /></p> <p> </p> <p> </p> </div></div></div> Thu, 17 Sep 2020 22:55:02 +0000 Saccharomyces 2038 at Have You Seen Ester? <div class="field field-name-body field-type-text-with-summary field-label-hidden view-mode-rss"><div class="field-items"><div class="field-item even" property="content:encoded"><h2>Introduction</h2> <p>I remember back to when I brewed my first batch of beer. It seems like yesterday; however, over two decades have elapsed since that faithful day. Much in the world of home brewing has improved dramatically during the last twenty years. An improvement that comes readily to mind is ingredient quality. Those of us who were participating in the hobby during the first home brewing boom can attest to having to work with hops that were often brown and malt that was past its prime. Small-scale brewers used to receive macro brewer cast-offs, and home brewers received the macro cast-offs that small-scale brewers rejected.</p> <p>While poor ingredient quality and selection are a thing of the past, there are areas of home brewing that have changed very little in the last twenty years. One such area is an understanding of fermentation byproducts. We have transitioned from a hobby with an incomplete understanding of fermentation byproducts that fermented at room temperature to a hobby that uses temperature-controlled fermentation chambers to mask our incomplete understanding of fermentation byproducts. The topics covered in this blog entry are fermentation byproducts and the role that they play in beer flavor.</p> <h2>Fermentation Metabolites</h2> <p>Brewers who are relatively new to brewing often treat esters and fusel alcohols (a.k.a. fusel oils) like they are the spawn of Satan. However, beer would not taste like beer without these compounds. In fact, suppressing the production of fusel alcohols and esters can often remove much of an individual yeast strain’s character, resulting in little to no change in flavor when changing yeast strains.</p> <h3>Fusel Alcohols</h3> <p>With that said, what are fusel alcohols? Fusel is a German word that translates to “bad liquor.” Another term for fusel alcohol is “higher alcohol.” Why are fusel alcohols referred to as higher alcohols? Well, the word “higher” refers to the fact that fusel alcohols contain more than two carbon atoms. If we examine the chemical formula for ethanol, we discover that it is most commonly written as CH<sub>2</sub>CH<sub>3</sub>OH. Another formula for this chemical compound is C<sub>2</sub>H<sub>6</sub>O, which makes it clear that ethanol contains two carbon atoms. Alcohols with more than two carbon atoms have higher molecular weights and boiling points than ethanol; hence, they are higher alcohols.</p> <p>One of the most commonly encountered higher alcohols in brewing is isoamyl alcohol. The chemical formula for isoamyl alcohol is (CH<sub>3</sub>)<sub>2</sub>CHCH<sub>2</sub>CH<sub>2</sub>OH. The formula for isoamyl alcohol is often written as C<sub>5</sub>H<sub>12</sub>O. As one can clearly see, isoamyl alcohol contains more than two carbon atoms. In fact, another name for isoamyl alcohol is isopentyl alcohol due to the fact that the compound contains five carbon atoms. Another frequently encountered higher alcohol is isobutyl alcohol. The chemical formula for isobutyl alcohol is (CH<sub>3</sub>)<sub>2</sub>CHCH<sub>2</sub>OH. The formula for isobutyl alcohol is often written as C<sub>4</sub>H<sub>10</sub>O. Once again, one can clearly see that this alcohol contains more than two carbon atoms.</p> <p>An alcohol that is often grouped in with fusel alcohols that is not a higher alcohol is methanol. The chemical formula for methanol is CH<sub>3</sub>OH, which is also written as CH<sub>4</sub>O. Methanol has a lower molecular weight and boiling point than ethanol. One will often hear the term “heads” used to describe the first condensate that is produced during alcohol distillation. This portion of the condensate is discarded. The reason being is that the heads are mostly methanol due to the fact that methanol makes the phase change from liquid to vapor before ethanol. Methanol is generally not a problem in beer because it exists at low levels. Methanol becomes a problem when we distill beer into whiskey, which is why one should stick with beer. Due to their higher molecular weights and boiling points, the true higher alcohols appear in the condensate known as the “tails.” Higher alcohols have an oily consistency, which is why they are referred to as fusel oils.</p> <h3>Esters</h3> <p>Okay, now that we now know that higher alcohols are alcohols that contain more carbon atoms than ethanol, what is an ester? An ester is the result of a condensation reaction between an alcohol and a carboxylic acid. A condensation reaction is a reaction where two compounds combine resulting in a new compound and a water molecule.</p> <p>A carboxylic acid is an acid whose formula ends in COOH. Esters are responsible for a large part of what we describe as beer flavor, especially ale flavor. A carboxylic acid that is commonly found in beer is acetic acid. Acetic acid production is integral to the yeast metabolic cycle. Every beer drinker who has tasted German-style hefeweizen has encountered an acetic acid-based ester that is available at above perception threshold levels. That ester is isoamyl acetate. Isoamyl acetate is the condensation reaction between isoamyl alcohol and acetic acid.</p> <p>As mentioned above, the chemical formula for isoamyl alcohol is C<sub>5</sub>H<sub>12</sub>O. The chemical formula for acetic acid is CH<sub>3</sub>COOH.</p> <p> </p> <p>Condensation reaction for isoamyl acetate C<sub>5</sub>H<sub>12</sub>O + CH<sub>3</sub>COOH → C<sub>7</sub>H<sub>14</sub>O<sub>2</sub> + H<sub>2</sub>O</p> <p>The reaction shown above reads one molecule of isoamyl alcohol plus one molecule of acetic acid yields one molecule of isoamyl acetate plus one molecule of water.</p> <p>Two other acetic acid-based esters that are commonly encountered in beer above perception threshold levels are ethyl acetate and isobutyl acetate. As one has more than likely assumed, ethyl acetate is the result of a condensation reaction between ethanol and acetic acid. It has the sweet smell of nail polish remover. Isobutyl acetate is the result of a condensation reaction between isobutyl alcohol and acetic acid. Isobutyl acetate smells like raspberries or pears.</p> <p>Other carboxylic acids that are frequently encountered in fermentation are hexanoic acid and heptanoic acid. The esters that are most commonly found in beer that are condensation reactions between these carboxylic acids and an alcohol are ethyl hexanoate and ethyl heptanoate, respectively. Ethyl hexanoate smells like red apple. Many brewers who are new to beer sensory evaluation mistake ethyl hexanoate for another yeast metabolic byproduct that smells like apple; namely, acetaldehyde. Acetaldehyde smells like tart green apple. The reason being that if we oxidize acetaldehyde, we obtain acetic acid. Ethyl heptanoate is my all-time favorite ale ester. It smells like one of those grape lollipops that were often given to children by bank tellers and medical office receptionists when I was young. Ales fermented with the Young’s Ram Brewery strain usually contain high levels of this ester when young, which is why I refer to ethyl heptanoate as the British lollipop ester.</p> <h2>Factors Affecting Metabolic Production</h2> <p>I mentioned in my introduction that we have transitioned from a hobby with an incomplete understanding of fermentation byproducts that fermented at room temperature to a hobby that uses temperature-controlled fermentation chambers to mask our incomplete understanding of fermentation byproducts. Quite frankly, fermenting ales at low internal temperatures in order to avoid unwanted fermentation byproducts is treating the symptoms instead of the problem. While fermentation temperature cannot be be ignored, the role of genetics and wort composition in the production of fusel alcohols and esters are equally important.</p> <h3>Temperature</h3> <p>Fermenting at low temperatures slows yeast metabolism, and anything that slows metabolism slows growth. Most of the esters and higher alcohols are produced during the growth phase. Slowing metabolism reduces metabolite production.</p> <h3>Genetics</h3> <p>An important thing to understand is that ester formation during fermentation occurs within the cell wall with the aid of enzymes. An important ester production-related enzyme is known as alcohol o-acetyltransferase (AATase). As I mentioned in the blog entry entitled “Carbon Credits,” an enzyme is a reaction catalyst. A reaction catalyst is a compound that increases the rate at which a reaction occurs. There are actually two AATase enzymes; namely, AATase 1 and AATase 2. Yeast cells contain two genes that are responsible for encoding these enzymes; namely, ATF1 and ATF2.</p> <h3>Wort Composition</h3> <p>Other than yeast genetics, the most important attribute in ester production is wort composition. The carbon-to-nitrogen (C:N) ratio plays a major role in ester production. As I mentioned in my blog entry entitled “Carbon Credits,” yeast cells do not consume sugar, they consume carbon, which they attempt to convert into energy. Sugar is carbon bound to water. All-malt wort has a lower C:N ratio than does wort that contains adjuncts. The amount of nitrogen that is available after dissolved oxygen is consumed determines the amount of acetyl CoA that is formed during the growth phase. Acetyl CoA is formed by combining acetic acid with coenzyme A; therefore, more acetyl CoA translates to higher acetic acid-based esters. The least desirable of is ethyl acetate.</p> <p>Surprisingly, the higher C:N ratio found in adjunct wort results in lower ester levels. Macro beer is maligned beyond belief within the home and craft brewing communities; however, the German brewmasters who were responsible for creating this style were nothing short of geniuses.</p> <p>American 6-row and 2-row barley have higher protein levels than continental and British barley. Higher protein levels translate to higher nitrogen levels. The addition of adjunct reduces the aggregate nitrogen level of the grist, resulting in lower nitrogen wort, which, in turn, results in lower ester levels. Protein levels also play a role in higher alcohol production. Higher alcohols are formed when amino acids are metabolized via the Ehrlich pathway.</p> <p>Finally, the type of sugar being metabolized plays an important role in the creation of higher alcohols, which, in turn, plays a role in ester production. Sucrose and fructose result in increased higher alcohol production, and so does glucose to an extent. Maltose metabolism results in considerably lower higher alcohol production than does glucose and fructose.</p> <h2>Applying Science to Beer Production</h2> <p>With this blog entry almost complete, how does one put this information to work in a home brewery? Well, as Denny Conn likes to say, “Wort wants to become beer.” This statement is absolutely true. What we are attempting to do by applying science to beer production is to gain a finer level of control over the finished product. There is no one size fits all approach to brewing. There are just too many variables involved in beer production to distill the process down into a repeatable cookbook process that works in all breweries with all styles and yeast strains.</p> <h3>Quality Ingredients and Proven Techniques</h3> <p>Due to lack of access to a fully-equipped quality control laboratory, home brewers work with an incomplete knowledge of their ingredients; therefore, one should start by selecting the highest quality ingredients available and applying brewing techniques that have stood the test of time. After the basics have been mastered and a considerable amount of data has been collected (i.e., a proper brewing log is a must), a brewer can start to alter the experiment one variable at a time while taking copious notes.</p> <h3>Adjusting Wort Composition</h3> <p>We know that the disaccharide sucrose and the monosaccharides fructose and glucose tend to translate to increased higher alcohol production; therefore, one strategy to reduce to higher alcohol production would be to avoid mash rest temperatures below 150F, especially when using high protein barley such as American 2-row. A second strategy would be to dilute the protein levels found in American 2-row with a low-protein adjunct such as flaked maize at the rate of 10% of the grist. I personally prefer to use low nitrogen continental and British barleys.</p> <h3>Selecting for Character</h3> <p>While ester production is bounded by higher alcohol and carboxylic acid production, yeast genetics play a significant role because enzymes are proteins and proteins are encoded via a genetically controlled process known as transcription. We can adjust wort composition and fermentation temperature regulation to control higher alcohol and ester production, but yeast genetics play the ultimate role in the production of these compounds. I always say, “One should pick a yeast strain for the task at hand instead of attempting to trick a yeast strain into performing the task at hand.” If a yeast strain is not producing the sensory profile given by a yeast supplier when used within the given temperature range, then one needs to examine one’s wort composition and/or ensure that one’s thermometer is calibrated. Temperature measurements should be taken as close to the middle of the fermentation vessel as possible.</p> <h2>Closing Thoughts</h2> <p>In the end, brewing is a continuous learning experience. Home brewers have the luxury of being able to brew without having to maintain a profit margin; therefore, one should feel free to experiment with wort composition, temperature control, and different yeast strains while fine tuning one's brewery and brewing process.</p> </div></div></div> Sat, 12 Mar 2016 18:38:01 +0000 Saccharomyces 902 at Carbon Credits <div class="field field-name-body field-type-text-with-summary field-label-hidden view-mode-rss"><div class="field-items"><div class="field-item even" property="content:encoded"><p>Fermentation is an incredibly complex process that can be mind boggling at times. Brewers like to think of yeast as a microscopic lifeform that transforms the sugars found in wort into alcohol, carbon dioxide gas, and metabolic byproducts that add flavor to the final product. However, in reality, yeast cells do not consume sugar. Yeast cells consume carbon, which they attempt to transform into energy. Alcohol and metabolic byproducts are the results of an inefficient metabolic pathway. The topic of this blog entry is how yeast cells transform compounds collectively known as carbohydrates into energy.</p> <p>Brewers often hear the term “organic chemistry” used when describing fermentation. Organic chemistry is the study of carbon-based compounds. Sugar is carbon bound to water; hence, the term carbohydrate. All of the sugars found in wort are multiples of CH<sub>2</sub>O. The simplest sugars found in wort are known as monosaccharides. The monosaccharides commonly found in wort are glucose, fructose, mannose, and galactose. These sugars are also known as hexoses because they contain six carbon atoms. All of the hexoses share the shame chemical formula, which is C<sub>6</sub>H<sub>12</sub>O<sub>6</sub>. How the hexoses differ is in their linear form. </p> <p>The four hexoses commonly found in wort belong to two different types of simple sugar. Galactose, glucose, and mannose are aldoses. Fructose is a ketose. An aldose is a sugar that contains one aldehyde group per molecule. A ketose is a sugar that contains one ketone group per molecule. Aldoses differ from ketoses in the location of something known as a carbonyl group. A carbonyl group is a carbon atom that is double bound to an oxygen atom. The carbonyl group appears at the end of the carbon chain in aldoses whereas it appears in the middle of the carbon chain in ketoses. Ketoses where the carbonyl group appears at the end of the molecule can isomerize into aldoses. The carbonyl group in d-fructose appears at the end of the molecule; therefore, it can isomerize into an aldose.</p> <p>As we move up the scale in complexity from the monosaccharides, we discover a group of sugars known as disaccharides. A disaccharide consists of two monosaccharides bound by what is known as a glycosidic bond. The most abundant disaccharide found in wort is maltose. Maltose consists of two glucose molecules bound by a glycosidic bond. Sucrose is also a disaccharide found in wort, but to a lesser extent. Sucrose consists of a glucose molecule bound to a fructose molecule by a glycosidic bond. Another disaccharide that Saccharomyces pastorianus (S. pastorianus or simply lager yeast) can reduce to monosaccharides, but Saccharomyces cerevisiae (S. cerevisiae or simply ale yeast) usually cannot is melibiose. Melibiose consists of a glucose molecule bound to a galactose molecule by a glycosidic bond. </p> <p>What is a glycosidic bond? A glycosidic bound is a type of covalent bond. In the case of glycosidic bonds, the bond occurs when atoms in two different sugar molecules share what are known as valence electrons. A glycosidic bond is formed via what is known as a condensation reaction. The outcome of a condensation reaction is another compound and an H<sub>2</sub>O molecule. For example, as mentioned above, maltose is a disaccharide that contains two glucose molecules bound by a glycosidic bond. Maltose is formed via the following condensation reaction:</p> <p>C<sub>6</sub>H<sub>12</sub>O<sub>6</sub> + C<sub>6</sub>H<sub>12</sub>O<sub>6</sub> → C<sub>12</sub>H<sub>22</sub>O<sub>11</sub> + H<sub>2</sub>O</p> <p>By the way, like the monosaccharides, all disaccharides share the same chemical formula. The chemical formula for a disaccharide is C<sub>12</sub>H<sub>22</sub>O<sub>11</sub>.</p> <p>The most complex sugars found in wort that affect fermentation belong to a family of carbohydrates known as trisaccharides. A trisaccharide consists of three monosaccharides bound by two glycosidic bonds. All trisaccharides share the chemical formula C<sub>18</sub>H<sub>32</sub>O<sub>16</sub>. The ability to reduce trisaccharides to simpler sugars is one of the attributes that affects how well changes in saccharification rest temperature affect final gravity. For example, maltotriose is the most frequently occurring trisaccharide found in wort. Maltotriose consists of three glucose molecules bound by two glycosidic bonds. One of the reasons why Lallemand Windsor leaves a high terminal gravity is because the yeast strain is maltotriose challenged. One way to offset this weakness is to rest one’s mash at a temperature of 65°C/149° or lower to produce an extract that contains a lower percentage of trisaccharides and dextrins. Many brewers refer to this type of wort as a more fermentable wort. </p> <p>If yeast cells can only use monosaccharides directly, how do they reduce disaccharides and trisaccharides to monosaccharides? Yeast cells perform this feat via the inverse of a condensation reaction. The process is called hydrolysis. The roots of the word hydrolysis are from the Greek “hydros” for water and from the Latin “lysis” for break apart or deconstruct. Together, these words mean break apart via the insertion of water, and that is exactly what happens. </p> <p>While breaking the glycosidic bond in a disaccharide is a one-step process, breaking the glycosidic bonds in a trisaccharide requires two steps. In the case of maltotriose, the first step involves breaking a maltotriose molecule into one maltose molecule and one glucose molecule. </p> <p>C<sub>18</sub>H<sub>32</sub>O<sub>16</sub> + H<sub>2</sub>O → C<sub>12</sub>H<sub>22</sub>O<sub>11</sub> + C<sub>6</sub>H<sub>12</sub>O<sub>6</sub></p> <p>The maltose molecule is then split into two glucose molecules.</p> <p>C<sub>12</sub>H<sub>22</sub>O<sub>11</sub> + H<sub>2</sub>O → C<sub>6</sub>H<sub>12</sub>O<sub>6</sub> + C<sub>6</sub>H<sub>12</sub>O<sub>6</sub></p> <p>Brewers who have delved into this area of fermentation have heard that S. pastorianus can use raffinose as a carbon source while S. cerevisiae can only partially metabolize raffinose. This limitation is due to the same limitation that prevents most S. cerevisiae strains from using melibiose as a carbon source. Raffinose consists of two glucose molecules and one galactose molecule. What happens when S. cerevisiae attempts to break the glycosidic bonds that hold raffinose together is that the raffinose molecule is split into one melibiose molecule and one glucose molecule. Unable to break the bond that holds melibiose together, this disaccharide is left undigested. Raffinose is lost during the malting of barley; therefore, it is absent from wort.</p> <p>The rate at which hydrolytic reactions occur is shortened by the creation of enzymes. Enzymes are reaction catalysts. A reaction catalyst is a compound that increases the rate at which a reaction occurs. The enzyme responsible for catalyzing the hydrolysis of maltose into two glucose molecules is called maltase.</p> <p>Enzymes are proteins, and proteins are encoded by cells via a process known as transcription. A cell’s DNA provides the blueprints for transcribing proteins. If one has ever wondered why different yeast strains yield different levels of attenuation given everything else equal, herein lies the reason. A yeast cell’s DNA controls the enzymes that can be encoded as well as the level at which the enzymes can be encoded.</p> <p>What happens after the higher-level saccharides are broken down into monosaccharides? Well, the yeast cell goes about performing something known as catabolization. Catabolism is a metabolic process where the yeast cells attempt to turn carbon-based compounds into energy.</p> <p>The primary catabolic process that occurs in yeast cells is called glycolysis. Once again, we see a word that ends in “lysis;” therefore, we know that this process involves the breaking apart or deconstruction of a compound. In the case of glycolysis, the compound is glucose. Glucose is the primary monosaccharide found in wort. It is also a building block for the most common disaccharides and trisaccharides found in wort. The goal of glycolysis is to turn glucose into a compound known as adenosine triphosphate (ATP). ATP is the fuel source for a cell. The transformation of glucose into ATP in the less efficient anaerobic metabolic pathway results the production of ethanol, higher alcohols, diketones, and organic acids. We can look at these metabolic byproducts as the yeast equivalent of incomplete combustion, as all of these compounds contain carbon.</p> </div></div></div> Thu, 26 Nov 2015 00:06:02 +0000 Saccharomyces 807 at There’s Gold in Davis, California <div class="field field-name-body field-type-text-with-summary field-label-hidden view-mode-rss"><div class="field-items"><div class="field-item even" property="content:encoded"><p>On January 24, 1848, James W. Marshall found gold at Sutter’s Mill in Coloma, California. That discovery set into motion the 1849 California Gold Rush. Over 300,000 people migrated to California to seek their fortune, many traveling all of the way from the East Coast in covered wagons. Today, there is a different kind of gold in California. It is a type of gold that is precious to brewers, a microscopic gold. The topic of this entry is the wealth of Saccharomyces and non-Saccharomyces yeast species held by the University of California, Davis. The University of California, Davis (UC Davis) is home to two yeast collections. The larger of the two collections is the Phaff Yeast Culture Collection, which is named after Dr. Herman Jan Phaff. Dr. Phaff was born in the Netherlands in 1913. He migrated to California to attend graduate school at the University of California, Berkeley (UC Berkeley) when he was 26 years old. Dr. Phaff’s interest in enology and brewing was kindled at his family’s winery. After moving from UC Berkeley to UC Davis in 1954, Dr. Phaff went about building the existing UC Davis yeast culture collection into one of the largest in the world. Today, the Phaff collection holds 800 of the 1,600 known species, including those useful to brewers and vintners. The current curator of the Phaff Culture Collection is Dr. Kyria Boundy-Mills. Dr. Boundy-Mills was fortune enough to be able to work with Dr. Phaff before he passed away. A second yeast culture collection is held by the Department of Viticulture and Enology. While many of the strains held in this collection are also held in the Phaff Collection, it does contain a few brewing strains that were either culled from or apparently never held in the Phaff Yeast Culture Collection. The curator of this collection is C.M. Lucy Joseph, M.S. In addition to caring for the Enology Culture Collection, Ms. Joseph is a published expert on the Brettanomyces genus. With the above said, the cultures held at UC Davis are not for budget conscious brewers, nor are they for brewers who are not versed in aseptic transfer technique, which is a topic for a future blog entry. However, for amateur and professional brewers who are comfortable wielding an inoculation loop, the cultures held at UC Davis offer a unique opportunity to work with heirloom strains that have been forgotten by history. One of the first cultures in the UC Davis culture collections to capture my attention was UCDFST 40-219/UCDVEN 1219. UCDFST 40-219/UCDVEN 1219 is the production yeast culture that was used at the defunct Acme Brewing Company in San Francisco. The deposit was made in 1942, which makes the culture 73 years old. A little known fact is that the yeast currently used to ferment Anchor Steam has only been employed at Anchor since the mid-seventies. It is an old Wallerstein Laboratories strain. UCDFST 40-219/UCDVEN 1219 was used to produce lager beer in San Francisco at least 73 years ago. Since the Acme Brewing Company survived prohibition, it is not out of the realm of possibility that UCDFST 40-219/UCDVEN 1219’s use in San Francisco predates the Volstead Act. Another interesting fact is that Leopold Schmidt founded the Acme Brewing Company after founding the Olympia Brewing Company in Tumwater, Washington; therefore, it is also not out of the realm of possibility that UCDFST 40-219/UCDVEN 1219 is a descendant of the original Olympia production yeast strain. In use, I am not going to sugar coat things. UCDFST 40-219/UCDVEN 1219 is not much fun to grow on solid media. The colony-forming units on a plate are tiny enough to be mistaken for petite mutants. I had to contact Ms. Joseph when I went to subculture the slant on which the strain arrived from UC Davis because it appeared to be blank. According to Ms. Joseph, the culture is a diploid yeast strain. Most brewing strains are polyploids, which is yet another topic for a future blog entry. I wound up using the add a few milliliters of autoclaved 5% weight by volume (w/v) wort to the culture tube, suspend the cells that are available, and then pitch the liquid in the culture tube into a slightly larger amount of autoclaved 5% w/v wort technique because I was unable to harvest a significant yeast scrap from the slant. The truly strange thing about UCDFST 40-219/UCDVEN 1219 is that it does not behave like a petite isolate when pitched into wort. Attenuation proceeds at a pace that one would expect from any other production strain. The strain is very flocculent. The yeast aggregates into pea-sized flocs, resulting in rapid sedimentation at the end of fermentation. After struggling to get this strain to grow on slant, I was truly astonished to see how well it behaved after being grown into a culture large enough to pitch into a batch of wort. I have never experienced this kind of behavior with a yeast strain since plating my first brewing strain almost twenty-three years ago. The initial batch of wort used for experimentation with UCDFST 40-219/UCDVEN 1219 was a Pre-Prohibition-style Pilsner with an original gravity of 1.062 and a grist composed of 85% domestic 2-row and 15% flaked maize. The beer was hopped twice with Liberty. The boil length was 90 minutes with a hop addition at 60 minutes before the end of the boil and another hop addition at knockout. Primary fermentation was conducted at 15°C/59°F. The resulting flavor was very rich for such a well-attenuated beer. This strain quickly became a “keeper” in my bank. Another interesting culture that I obtained from UC Davis is UCDFST 40-420/UCDVEN 1420. UCDFST 40-420/UCDVEN 1420 was deposited by Dr. Catherine Roberts in 1947. This strain is from Kongen’s Bryghus (King Christian IV’s brewhouse) in Denmark. Dr. Roberts was quite a remarkable woman who was way ahead of her time. At a time when job opportunities for women were very limited, she was pioneering the field of yeast genetics with Dr. Øjvind Winge at Carlsberg Laboratory in Copenhagen, Denmark. Dr. Roberts earned her Ph.D. at UC Berkeley, which I assume is how the culture eventually wound up at UC Davis. If I had to describe UCDFST 40-420/UCDVEN 1420, I would say that it is like Wyeast 1007 with better flavor. Like Wyeast 1007, UCDFST 40-420/UCDVEN 1420 produces a huge head during fermentation. Apparent attenuation is very high (&gt;80%), but flocculation is low, resulting in slow sedimentation. The strain is suitable for top cropping. UCDFST 40-420 is fantastic strain for those looking to produce Northern European ale styles. It is crisp and clean with a nice subtle candy-like ester profile that works very well with Pilsner malt and continental and British hop varieties. In closing, the cultures held at UC Davis offer advanced amateur and professional brewers an opportunity to work with heirloom strains that have been forgotten by time. There are many other brewing yeast strains that are not as old with respect to deposit date, but are equally intriguing, including Wallerstein strain #36C4 (better known as the yeast strain that was selected for use at New Albion) and old lager cultures from the Lucky Lager Brewery in Azusa, California and Liebmann Breweries in Brooklyn, New York. Brewers should not expect to receive much in the way of brewing data when digging deeply into the UC Davis collection. Opening up the vault is experimental brewing in the truest form because one never knows what one is going to get with culture collection strains. If any commercial yeast propagators are reading this blog, I have attempted to convince Chris White to license UCDFST 40-219/UCDVEN 1219 from UC Davis. He does not appear to be interested in licensing the culture. Here is an opportunity to propagate a true turn-of-the-century San Francisco yeast strain. I know that Dr. Boundy-Mills has shown interest in working with yeast propagators.</p> </div></div></div> Tue, 03 Nov 2015 04:15:37 +0000 Saccharomyces 799 at The Great Yeast Culture Adventure <div class="field field-name-body field-type-text-with-summary field-label-hidden view-mode-rss"><div class="field-items"><div class="field-item even" property="content:encoded"><p>First off, I would like to take a minute to thank the Experimental Homebrewing team for extending the opportunity to blog on their site. I have considered creating my own blog since re-entering the hobby a few years ago. However, seeing that my hiatus was due to severe burnout, I wanted to avoid having home brewing become the obsession that it became during my first pass through the hobby. Blogging here will allow me to share what I know with others in one convenient place without having to maintain my own site.</p> <p>With the above said, what brought me back to home brewing after a hiatus that was long enough to place home brewing in my rear view mirror was my love for all things Saccharomyces. I have brewed almost exclusively with home cultured yeast since the beginning. When I first started to brew in February of 1993, being able to plate and slant yeast was much more of a survival skill than it is today. The dry yeast cultures available at the time were unreliable and liquid yeast was difficult to obtain due to the relative immaturity of the market. White Labs did not exist, and the Wyeast catalog could be easily memorized due to the small number of available cultures. As the hobby matured and high-quality yeast became more readily available, my desire to have regular access to high-quality yeast morphed into the desire to have greater control over the final product. The difference between an okay beer and a very good to great beer often lies in biological quality control because brewers make wort, yeast makes beer.</p> <p>My blog will deal almost exclusively with the care and feeding of the Saccharomyces genus. While I am not into wild yeast and bacteria, much of what will appear on my blog will apply to wild cultures as well. I do not work with wild yeast and bacteria because I maintain a yeast bank, and these microbes are known as beer spoilage microflora for a reason. White Labs maintains two separate propagation facilities. The Saccharomyces facility is held under positive pressure to keep things out whereas the Brettanomyces and bacteria facility is held under negative pressure to keep things in. </p> <p>In closing, I would like to give credit to three people who appear to be no longer active in the hobby, but whose contributions should not go unrecognized. Dr. Maribeth Raines' pioneering work in collecting and isolating yeast cultures and Jeff Mellem's entrepreneurial skills brought us BrewTek. There are several cultures available today because of the work performed by these two pioneers. One culture that readily comes to mind is Wyeast 1450 Denny's Favorite 50. This culture was first introduced to the home brewing community on mini-slant as BrewTek CL-50 California Pub Brewery Ale. It is still with us due to Denny Conn's effort to keep CL-50 alive after BrewTek went out of business. Another pioneer in this area of home brewing was Dr. Daniel McConnell. Dr. McConnell transferred several hundred brewing cultures that he had collected over the years to White Labs when he shuttered the Yeast Culture Kit Company. The hobby is much richer because of the contributions made by these three individuals.</p> </div></div></div> Thu, 29 Oct 2015 17:00:56 +0000 Saccharomyces 797 at