Protease Use for FAN as Urea Substitute Poses Challenges

Understanding yeast physiology and preferences for utilizing free amino nitrogen is important to avoid yield-robbing problems. This contribution appears in the June print edition of Ethanol Producer Magazine.
By Dennis Bayrock | May 19, 2017

Proteases have been a valuable tool to the fuel ethanol industry in aiding increased corn-oil extraction, but when used to provide yeast nutrition, many nuances need to be considered. Amino acids from proteases have the same potential as urea/ammonia to provide yeast with the nutritional nitrogen they need, but with significant differences. A proper understanding of yeast nutrition, proteases and process operations is vital to efficient protease application.

Corn mashes lack sufficient free amino nitrogen (FAN), which is defined as the biological portion of nitrogen that can be utilized by yeast. This is not to be confused with measurements of total or percent nitrogen in mash or in various chemicals. The proper amount of FAN is critical to the yeast, so much so that FAN levels should be checked with each and every change in process volumes, flow rates, composition or dilution of mash, and yeast pitching rates at the plant. Yeasts require a minimum of 300 parts per million (ppm) FAN in a typical 750,000 gallon fermentor of corn mash at 32 percent solids.

FAN Sources
In addition to FAN dietary requirements, the type of FAN also impacts yeast nutrition. The yeast transports only certain nitrogen-containing compounds across its cell membrane. Amino acids and di-peptides cross the cell membrane by active transport and facilitated diffusion, mechanisms that require metabolic energy. Ammonium ions, in contrast, enter the cell by passive diffusion and do not incur a metabolic penalty. Individual amino acids present in many commercial yeast foods can be utilized, along with small amounts of N-containing nucleic acids in corn. The protein in corn mash, however, cannot be used directly by yeast.

When dissolved in water, ammonia and aqueous ammonia form ammonium ions. Ammonia salts, such as di-ammonium phosphate and ammonium sulfate, fully dissociate into the ammonium cation and the respective phosphate or sulfate anion. Urea must first be broken down to ammonia and carbon dioxide by the yeast via the enzyme urease before the ammonia in urea can be used.

If given equal concentrations, does the yeast have a preference for which order it utilizes these FAN sources? It turns out it does. Aqueous ammonia is the yeast’s first choice, as this source of FAN incurs no metabolic penalty, leaves behind no inhibiting anions, freely diffuses into the yeast and is a central part of yeast metabolism. Urea is the next choice, and it, too, leaves behind no inhibiting anions. However, the yeast must first produce the enzyme urease, spending energy and materials to do so.
Amino acids are the next choice for FAN. One would expect that since yeast is always undergoing protein synthesis, the required amino acids should be easily acquired by scavenging the amino acids provided in the mash. If the demand can be satisfied with an existing amino acid in the media, the yeast will incorporate it without any penalty and save itself the cost of producing it from scratch. If, however, the yeast is faced with a delay for an amino acid, the yeast will instead metabolically de-aminate any available amino acid to form ammonium and a carbon skeleton. These carbon skeletal remains are toxic, so the yeast converts them to less-toxic fusel compounds. A build-up of fusels can create havoc in the ethanol process.

Ammonium salts are last in preference for FAN. Residual anions pose a significant challenge for yeast health. Further complicating the story for amino acids is that not all 20 in a mixture are used equally by the yeast. Although most amino acids are nearly completely utilized, the length of time to complete fermentations can increase nearly four-fold, depending on the type of amino acid provided as FAN.

What does this mean at the fuel ethanol plant? If faced with stressed yeast or a stalled fermentation, ammonia/aqueous ammonia addition gives the greatest and fastest benefit to the yeast for FAN with practically no biological side-effects. An additional benefit to using ammonia/aqueous ammonia is the simultaneous rise in pH, which alleviates stresses on the yeast from lactic and acetic acids. If faced with out-of-control, hot fermentors, changing the FAN source and timing of addition are options to slow down the yeast.

In corn-based ethanol fermentation running at 33 percent solids for 55 to 75 hours, the yeast are most metabolically active between 6 and 24 hours—the time frame when the yeasts have the highest demand for nutrients.

Most yeast FAN calculators in the field (including Phibro’s) calculate required ppm of FAN based on a full fermentor, with the assumption that any chemical added for FAN is instantly available to the yeast. In most situations, these assumptions and calculations are valid. However, with the use of proteases to provide FAN, this may not be the case.

Protease Use
On the surface, it makes sense to convert protein to amino acids for use by the yeast as FAN. Corn typically contains 11 percent dry-weight (DW) protein and wheat 13 percent. In a 750,000 gallon fermentor operating at 33 percent solids, there is approximately 220,000 pounds DW protein that can potentially be used as a source of FAN for the yeasts.

Initially used to aide corn-oil extraction, more recently ethanol producers are utilizing proteases to offset or replace urea. Offsetting urea is reasonable, but totally displacing it poses some risks.
First there is potential for stuck and sluggish fermentations. Chemical FAN additions are nearly instantaneously available to the yeast. Proteases however, ideally, should complete all of the required amino acid FAN production prior to peak FAN demand, which is not always the case. As noted, yeast require most of their FAN between 6 and 24 hours into fermentation. Unfortunately, information on protease catalytic reaction rates at various mash temperatures, pH, etc., is difficult to obtain, so full understanding of FAN availability over time is unknown.

A second risk is the production of toxic fusels. Regardless the source, fusel production by yeast is maximized if all FAN is depleted when the yeast is still fermenting, often at about 32 hours. Starved of FAN, the yeast cannibalizes itself and hydrolyzes internal proteins to amino acids, converting the remaining toxic carbon skeletons into fusels.

In mash fermentations where FAN is mostly provided by amino acids, either directly or indirectly through proteases, fusel production increases. Numerous ethanol plants have documented large increases in fusel production and large increases in yeast inhibition to the point of complete metabolic stalling when FAN was provided by amino acids. At plants where proteases don’t replace, but only offset about 30 percent of the FAN provided from urea/ammonia, fusel increases were marginal.

A third issue surrounds potential impacts on distillers grains. In a 750,000 gallon fermentor, the total protein content is approximately 220,316 pounds. To provide sufficient FAN, the protease would need to hydrolyze 8,786 pound of protein. This corresponds to a bulk loss of 4 percent protein in the fermentor, which would arguably correspond to a similar, or possibly larger, reduction in the DDGS.

One cannot assume all amino acids generated by protease become yeast protein, as some will be shunted towards fusel production. In addition, when fermentable carbohydrates are depleted in late fermentation, amino acids can be used for energy production, which takes precedent over protein production. Furthermore, digestibility issues are known for yeast whole-cell protein additions to feed and it cannot be assumed that the total protein content within the yeast cell is available for the animal.

The fourth potential impact may be a loss of ethanol yield and increased Mailliard reactions. The Mailliard reaction occurs between amino nitrogen-containing chemicals, primarily amino acids, and reducing sugars, such as carbohydrates, glucose and maltose, to form a family of amino-sugar chemicals. The reaction rate and product formation rapidly accelerates at temperatures above 160 Fahrenheit.

Mailliard reaction products influence yeast in three ways. As little as 1 percent, visible as increased darkening in solution, can inhibit yeast metabolism by up to 10 percent. In addition, the FAN and sugar components within Mailliard reaction products cannot be metabolized by the yeast, and thus represent a loss of FAN, sugar and, subsequently, ethanol. While yeast cannot metabolize Mailliard reaction products, many bacteria can.

In mash, proteins have only one amino nitrogen unit that can participate in Mailliard reactions, although reducing sugars as carbohydrates are plentiful. However, once the 4,000-plus amino acids within each protein are liberated by a protease, each amino acid contains at least one amino nitrogen unit. Together with the 180F or better liquefaction temperatures and 3 to 4 hours of holding time, Mailliard reaction products rapidly accumulate and cause a noticeable darkening of the mash.
Normally, Mailliard reaction products are associated with distillation. With the large amount of amino acids in the mash when using proteases and elevated temperatures combined with the large difference in processing time in liquefaction, serious attention should be paid to the loss of fermentable sugars, FAN, ethanol yield, and potential increase in bacterial contamination. 

Author: Dennis Bayrock
Global Director Fermentation Research
Phibro Ethanol Performance Group