Every ethanol producer understands that active and healthy yeast are an integral part of the fuel ethanol production process. Many of the duties of laboratory and production staff involve monitoring the activities of yeast in propagation and fermentation. One of the first steps to optimal fermentation involves proper yeast propagation, a process that differs in several ways from fermentation. Here we dissect the yeast propagation process, examine its purpose, common problems and how it can be optimized for best results.

Yeast propagation holds several purposes. It serves to rehydrate, condition and increase yeast populations using their natural reproduction capabilities as living organisms. Unlike the various chemicals that are used in the process of making fuel ethanol, it is important to appreciate yeast as living organisms and their requirement for an environment that supports their growth.

The first thing to consider before delivering or "pitching" yeast to the propagation tank is the status of the yeast itself. The yeast Saccharomyces cerevisiae is typically used in fuel ethanol production. Check the yeast's expiration date to make sure it isn't spoiled or otherwise expired. Make sure the yeast is from a reliable source and has been stored properly, avoiding high temperatures for example.

For active dried yeast this means checking that the yeast has remained vacuum-sealed to retain viability, and wet cake yeast has been properly refrigerated and does not emit any odors associated with spoilage or rot. Fresh yeast, whether active dried yeast or wet cake, has a characteristic smell that can be equated with bread making with a slight hint of molasses, the substrate most often used to produce yeast on an industrial scale. Once it has been determined that the yeast is of high quality and viability, it can be added to the propagation tank with confidence. Tank size is important for propagation and is normally between 2 percent and 5 percent of the fermentor size.

Optimal Conditions
The conditions of propagation are also critical for optimal yeast production and subsequent fermentation. They include adequate glucose, aeration, temperature and nutrient additions. First, the yeast needs a source of carbon, provided by adding liquefied mash, for cell wall biosynthesis and energy production. Without further enzymatic conversion, liquefied mash is low in glucose, typically below 0.5 percent. Glucose levels are elevated in propagation by adding the enzyme glucoamylase, similar to what occurs in fermentation.

Higher polymers of glucose (DP4+, DP3 (maltotriose), and DP2 (maltose)) are converted to individual glucose subunits (saccharification), which are subsequently utilized by the yeast. One major difference between propagation and fermentation is that a much lower level of glucose is preferred in propagation compared to fermentation. Glucose levels should be targeted at or just above 2 percent at the beginning of propagation because higher glucose concentrations can induce the yeast to produce ethanol, resulting in less energy production per molecule of glucose, rather than utilizing more of the available energy through aerobic respiration.

Saccharomyces yeast is good at producing ethanol, and higher glucose concentrations will cause a default metabolism leading to ethanol production, a process known as the Crabtree effect. The proper amount of glucoamylase to add to the propagation mix to achieve close to 2 percent glucose is dependent on several factors, such as choice of enzyme, solids concentration in the mash, mash-to-water ratio and temperature. For a typical corn-to-ethanol plant with slurry solids measured at 32 percent to 34 percent with a mash-to-water ratio of approximately 70:30, which is standard, the amount of glucoamlyase is typically 0.5 to 1 gallon.

Glucoamylase is typically added at the beginning of propagation at the same time as, or just prior to, yeast addition. Although quite easy to maintain at optimal levels, high glucose (5 percent and higher) in propagation is a chronic problem at fuel ethanol plants. One common misconception is that, compared to fermentation, glucose uptake is much slower in propagation. This is somewhat counterintuitive because of the increased energy production per molecule of glucose in propagation compared to fermentation. One must remember that close to 16 times more energy is generated during aerobic respiration per molecule of glucose when compared to fermentation. Another way to explain it is that during fermentation yeast uses only a portion of the energy contained in the glucose molecule, resulting in the high-energy byproduct ethanol, whereas yeast utilize much more of the energy when respiring aerobically during propagation. An analogy is taking only one bite out of a sandwich and leaving the majority behind (fermentation), compared to eating the whole thing (aerobic respiration), which takes more time and provides more energy.

In addition to a carbon source, supplied by glucose, additional nutrients above what is naturally provided in the corn are added to optimize growth. Nitrogen in the form of urea is
most often used at a rate of between 300 parts per million to 500 parts per million or higher. Although ammonia is also a good nitrogen source for the yeast, it can be inhibitory to yeast during rehydration. Failure to add additional nitrogen can cause sluggish yeast growth, resulting in abnormally low yeast counts or slower metabolism. Additional ingredients like magnesium and zinc are sometimes added for additional benefit, but it is unclear whether this is necessary as the grain itself contains a certain amount of these elements.

Providing Proper Aeration
As mentioned earlier, propagation is an aerobic process, thus the propagation tank must be properly aerated to maintain a certain level of dissolved oxygen. Adequate aeration is commonly achieved by air inductors installed on the piping going into the propagation tank that pull air into the propagation mix as the tank fills and during recirculation. The capacity for the propagation mix to retain dissolved oxygen is a function of the amount of air added and the consistency of the mix, which is why water is often added at a ratio of between 50:50 to 90:10 mash to water. "Thick" propagation mixes (80:20 mash-to-water ratio and higher) often require the addition of compressed air to make up for the lowered capacity for retaining dissolved oxygen. The amount of dissolved oxygen in the propagation mix is also a function of bubble size, so some ethanol plants add air through spargers that produce smaller bubbles compared to air inductors. Along with lower glucose, adequate aeration is important to promote aerobic respiration, which differs from the comparably anaerobic environment of fermentation.

One sign of inadequate aeration or high glucose concentrations is increased ethanol production in the propagation tank. There will always be some ethanol produced during propagation, but limiting that is a good sign that the yeast is respiring aerobically as should be occurring during propagation.

Time and Temperature
Yeast requires a comfortable temperature for growth and metabolism. A good temperature for propagation is between 92 and 94 degrees Fahrenheit. Lower temperatures result in slower metabolism and reduced reproduction, while higher temperatures can cause production of stress compounds and reduced reproduction. Due to the small tank sizes relative to fermentation and the fact that most propagation tanks are indoors and protected from the insult of high summer or low winter temperatures, maintaining optimum temperatures of between 92 and 94 degrees is usually not a problem.

Another common question is how long to propagate yeast before adding it to the fermentor. Propagation times vary between ethanol plants, but most often range between six and 10 hours, which corresponds to the time it takes for the yeast to reach exponential growth phase. Longer propagation cycles can result in the yeast entering stationary phase or a stage of decline due to depletion of nutrients and accumulation of byproducts such as acetic acid, which can cause a subsequent lag in yeast performance once in the fermentor.

Shorter propagation cycles do not allow time for adequate doubling or reproduction of the yeast, one of the primary reasons for propagating in the first place. Determining optimal drop times for propagation may involve charting growth under the conditions described above and deciding when the yeast has reached exponential growth in relation to when it enters into the subsequent stationary or rapid decline phases.

Bacterial or wild yeast contamination is rarely a problem during propagation because yeast propagation tanks are smaller and can be more easily cleaned than fermentation tanks.

Propagation tanks are also indoors, allowing for better temperature control. Another factor contributing to lowered contamination is that propagation takes much less time than fermentation, thus contaminating microorganisms don't have adequate time to reach significant numbers. In addition, there is less glucose available for contaminating bacteria and wild yeast. If contamination does occur in the propagation tank it is almost always related to inadequate cleaning, malfunctioning or damaged equipment, or other error. Heat exchanger or piping issues are a leading cause of contamination in propagation tanks.

Apart from cleaning, antibacterial products are often added to prevent growth of unwanted microbes. Prefermentors at continuous plants are more likely to harbor contaminating microbes due to lengthy periods between cleanings, which can go on for more than a year in some cases.

In summary, yeast propagation is an integral part of the fuel ethanol production process. By following the aforementioned guidelines, propagation can be optimized and problems in fermentation minimized.EP

Patrick Heist is chief scientific officer for Ferm Solutions Inc. Reach him at eheist@ferm-solutions.com or (606) 218-5429.