In ethanol production, yeast (Saccaromyces cerevisiae) is used to convert sugar into ethanol. Other microorganisms can compete with yeast for the sugars. These microorganisms include but are not limited to lactic acid and acetic acid bacteria. When acid bacteria grow, they compete with the supply of sugar resulting in less sugar for ethanol production. Also, acid-forming bacteria can create low pH conditions that tend to inhibit the growth of ethanol producing yeast. To control the growth of acid producing bacteria, many ethanol plants add antibiotics to the fermentation tanks. The antibiotics kill much of the acid bacteria, but the antibiotics apparently do not harm the yeast. The current method calls for 3 to 5 pounds of antibiotic, usually Virginiamycin, per 500,000 gallons of corn mash in the fermenter. Actual dose of antibiotics is determined by the level of lactic acid in the corn mash during the first 30 hours of fermentation. Antibiotics, though generally effective, have several major disadvantages. The main disadvantage of antibiotics is that they only work in the fermenter.  Biofilms that grow in the mash cooler and consume sugars are not arrested and, as a result, ethanol yield is compromised. A second disadvantage of antibiotics is that acid bacteria can become resistant over time rendering the use of antibiotics less effective, resulting in ethanol production losses.  In order to further improve ethanol yield, DeLasan CMT, a patented system comprised of 22% peracetic acid (PAA), distributed by DeLaval, and hydrogen peroxide (HP) were blended in line and added to the fermenter via the mash cooler. The primary objective of the study was to determine the most economical dose of DeLasan CMT and to determine the relationship between lactic and acetic bacteria reduction, ethanol yield improvement, and dose. Secondary objectives included measurement of residual levels of PAA, HP, and stabilizers in the distillers grains. Background The ethanol plant in the study is a 55 MMgy continuous plant located in Illinois. The facility’s management desired to improve ethanol yield, save money on their antimicrobial program and produce distillers grains  that are free of antibiotics. The plant process flow is shown in Figure 1. The corn mash enters the stage-one mash cooler at 180 degrees F and is cooled to 140 degrees F. It then enters the stage-two mash cooler and is cooled to 90 degrees F. Mash flow through the cooler is 650 gallons per minute (gpm). From the stage-two cooler the mash is sent to the fermenter. Once in the fermenter, fermentation begins to accelerate, releasing heat which is dissipated in the pre-fermenter cooler. The corn mash is then sent to the beer well at the end of fermentation. System parameters are shown in Table 1. During fermentation, carbohydrates, ethanol, and organic acids are routinely monitored to insure that the fermentation process is occurring normally and to insure that undesirable bacteria are kept under control. Also, the mash temperature is kept in a range of 90 F to 95 F. Typical end fermenter process parameters are shown in Table 2. Study Procedure DeLasan CMT and 34% HP were added at a rate of 2.2 and 3.0 gallons per hour, respectively, to a stainless steel pipe that carried the mixture to a header located between the first and second stage mash cooler. At the point of injection, the mash temperature was 140 F and the mash flow rate was 650 gpm. The pumps used to pump both the HP and DeLasan CMT were ProMinent diaphragm pumps fitted with Teflon liquid ends. The dose of the DeLasan CMT was 15 ppm (as PAA) and the dose of the HP was 30 ppm (as peroxide). However, the DeLasan CMT and HP were fed for only half of the fermenter fill time, making the overall dose 7.5 ppm of PAA and 15 ppm of HP. Samples were taken every hour at the mash cooler exit and fermentation tank. The samples were tested using a Hach DPD total chlorine test. Three milliliters of sample was added to a test tube, then one DPD powder pillow was added to the sample. The sample was then stirred gently for five seconds and a pink color was observed at the bottom of the test tube. PAA concentration was estimated based on the hue of pink color after 30 seconds. HP concentration was estimated after 6 minutes. Normal corn mash tests on carbohydrates, ethanol, and organic acids were taken by plant personnel every six hours. Results were compared with normal historical averages when antibiotics were used. Microtesting As part of the study, lactic acid bacteria (LAB) were plated in several locations including the mash cooler inlet, mash cooler outlet, and fermenter. The bacteria counts were analyzed as a function of program chemistry variations. All the LAB counts in the mash cooler inlet were zero and so those results are not shown. However, there were major differences in counts based on program chemical variations as shown in Table 3. The microtesting indicated that the only program that would reduce the LAB coming out of the mash cooler was the normal program of DeLasan CMT and peroxide.  Also the normal program (CMT + peroxide) reduced the LAB in the fermenter by two logs versus  the antibiotic program.   It is postulated that the peroxide increased the LAB in the mash exit by dislodging but not killing biofilm in the mash cooler (see Figure 2). Test Results Four years of operating results indicated the following. Organic acids: Lactic acid in the beer well was reduced from an average of 0.20% to 0.10%.  Backset was increased with no increase in lactic acid production (see Figure 3). Ethanol production: ethanol production was 1.5% higher with DeLasan CMT vs antibiotics. This result was due to the decrease in lactic acid production in the fermenter and decrease in biofilm activity in the mash cooler (see Figures 4 and 5). Conclusion Overall study results were extremely positive over a period of four years. The study indicated that the DeLasan CMT program is effective in preventing ethanol loss due to the formation of biofilm in the mash cooler and organic acids in the fermenter. The net ethanol yield increase was 1.5% over the study period. No negative effects of the program were noted based on the balance of carbohydrates, ethanol, and organic acids. In addition to helping the plant achieve ethanol yield goals, the DeLasan program met the following objectives: • Cost effective: Because the DeLasan CMT is effective at very low dose, the program cost was less than the cost of the antibiotics. • Increased ethanol production: The DeLasan CMT program resulted in a net increase in ethanol yield of 1.5%. In a 55 MMgy plant, that amounts to increased profits of $1.5 million per year. • Improved distillers grains: Elimination of antibiotics resulted in more marketable coproduct. • Increased backset: Because the thin stillage had less organic acid content, backset was increased by 5%, thus reducing water, energy and nutrient costs. Author: Reed Semenza Technical Manager, DeLaval Reed.Semenza@DeLaval.com