Consequences of a Lower pH

At an ethanol plant experiencing bacterial contamination, lowering the pH in the propagator/fermenter is typically the first step used to control bacterial growth. Phibro’s Diagnostic Kit services show that over 80 percent of the bacterial contamination in ethanol plants is from the lactic acid bacteria (LAB) family. The pH for optimal growth of most LAB is between 5.5 and 5.8. In contrast, S. cerevisiae yeast can tolerate and multiply better than most bacteria in acidic environments—from pH 4.0 to 6.5.

Knowing this, it seems logical to lower the pH during contamination to inhibit the bacteria while allowing the yeast to grow. But should lowering the pH be the first option? Are there negative consequences to lowering pH for yeast and ethanol plants?

Following is a list of issues to consider, along with a somewhat surprising solution: Raise the pH.

Sulfuric Acid
Lowering the pH in a fermenter requires an increased amount of sulfuric acid. With a liquefaction pH of 5.7 and a fermenter volume of 750,000 gallons, the amount of sulfuric acid typically needed to lower the fermenter pH to 5.2 is 4,286 pounds. But, to adjust the same fermenter to a pH of 4.5 requires 8,573 pounds of sulfuric acid. Sulfur is already a problem for many fuel ethanol plants to meet specifications for distilled ethanol. Excess sulfur in dried distillers grains with solubles (DDGS) also increases the risk of the fatal disease polioencephalomalacia (PEM) when fed to animals.

Consequences for Yeast Growth
Yeast use pH as a signal for multiplication, fermentation and metabolism. In general, yeast multiply most efficiently at a pH higher than 5.0. In contrast, efficient fermentation is achieved at a pH lower than 5.0.  Yeast does not grow below a pH of 2.8, although its metabolic activity continues, albeit at a lower rate than normal. Yeast multiplication rate increases non-linearly as the pH increases, with optimal growth occurring at a pH of 5.5 to 6.0. During optimal propagation/fermentation, the pH is typically set to about 5.0. As the process continues, the pH naturally decreases, which also decreases the yeast growth rate.

Complicating the picture somewhat is the fact that yeast cells have been evolutionarily “tuned” to multiply not only when the pH is about 5.5, but also when there is a natural pH difference of 1 to 2 units from the start to end of propagation/fermentation. If this pH difference is cut short (either by starting at a lower absolute pH, or preventing the pH from reaching this difference), yeast multiplication is significantly curtailed. Nutrition is part of the explanation. Many of the yeast cell transporters that move nutrients such as carbohydrates, ions and amino acids from the outside environment into the yeast require that a pH gradient is maintained across the membrane with a lower pH outside than inside. In addition, each yeast transporter has a specific and optimal pH. Moving the pH away from these optimal transport values decreases nutrient uptake.

Organic Acid Inhibition
The primary function of the yeast cell membrane is to physically house all of the various cellular organelles, structures, enzymes and biochemical reactions inside the cell. Another function of the yeast cell membrane is to keep materials that inhibit multiplication and metabolism—lactic and acetic acids, fatty acids and fusels—out of the cell.

pKa and Inhibition
Lactic and acetic acids are two of the more common organic acids found within contaminated fermenters at fuel ethanol plants. They are classified as weak acids with specific pKa values (lactic acid pKa is 3.86, acetic acid pKa is 4.74). By definition, weak acids do not fully dissociate in solution like strong acids, such as hydrochloric acid, do. If any solution of lactic acid is adjusted to a pH of 3.86, exactly one-half of the total lactic acid will be undissociated, and one half will be dissociated. Only the undissociated form of weak acids can physically cross the yeast cell membrane to cause inhibition. If the pH of the same lactic acid solution is decreased below pH 3.86, a larger proportion of the total lactic acid will be undissociated, and if the pH is increased above 3.86, a larger proportion of the lactic acid will be dissociated.

In other words, when weak acids are present, decreasing the pH will increase the proportion of the undissociated form and thus increase the amount of yeast inhibition. So to fully understand how much yeast will be inhibited by weak acids, a plant must know both the pH and the amount of the acid using high performance liquid chromatography or gas chromatography.
Fatty acids (FA) can also accumulate at an ethanol plant from hydrolysis of corn oil, bacterial contamination, methanator process upset, or as a result of yeast stress. FAs also have pKa values and so behave similarly to weak organic acids. Lastly, some fusel compounds (produced by yeast and contaminating bacteria) also have specific pKa values.

The Solution: Raise the pH
Bacteria inhibit yeast in two ways. They compete with yeast for nutrients (especially micronutrients), limiting yeast’s growth by starving them of micronutrients. And they can also produce toxic organic acids that inhibit yeast.

The question becomes how to reduce one or both of these inhibition types on yeast. Antibiotics and antimicrobials can be added to yeast propagation and fermentation to limit the growth of bacteria. But what about the organic acids/FA already present even if 100 percent of the bacteria are theoretically dead? Raising pH can limit the organic acid/FA inhibition on yeast.

Phibro raised pH by 0.2 absolute at more than 43 fuel ethanol plants with contaminated fermenters that had been stalled for more than 32 hours. Yeast in these fermenters finished utilizing sugars and increased ethanol yield closer to levels of fermenters without bacterial contamination. This pH increase was achieved by adding into the fermenter either aqueous ammonia or caustic from clean-in-place (CIP) lines.

But, would caustic raise the sodium level in the fermenter and further inhibit the yeast? Technically, the sodium concentration will increase. But, at a lower pH in a contaminated fermenter, it is likely that the degree of inhibition on yeast from organic acids is much greater than inhibition by sodium. Also, to raise the pH by 0.2 in a 750,000 gallon fermenter requires approximately 1,500 gallons of 5 percent CIP, which increases the concentration of sodium only by 3 percent in the fermenter.

Would raising the pH allow contaminating bacteria to grow better and have a competitive advantage over the yeast? Technically, yes. But, in a contaminated, stalled fermenter, most of the inhibition on the yeast at that time is caused by organic acid/FA inhibition and not nutrient competition. Also, a pH increase of only 0.2 absolute was needed to decrease organic acid/FA inhibition on the yeast without increasing bacterial numbers. Larger pH increases will certainly provide an additional boost to bacterial growth, negating the benefits of reduced inhibition by organic acid/FA.

The routine practice of lowering pH to combat bacterial contamination does work. But it also increases the stress on the yeast by directly inhibiting the yeast, and it increases the chemical inhibition on the yeast from dissociated and undissociated forms of these acids. Raising the pH in more than 43 contaminated ethanol plants reduced sugars passed to distillation and partially recovered ethanol yield.


Author: Dennis Bayrock
Global Director Fermentation Research Lactrol
Phibro Ethanol Performance Group
651.788.0602
dennis.bayrock@pahc.com