Alcohols produced by yeast during fermentation can also contribute to the aroma and flavor of beer, mainly by varying the alcoholic perception, which on the higher end can increase the warming of the mouthfeel. Generally, I tend to like beers with very little of this warming effect. The main higher alcohols called fusel alcohols in beer contributing to beer flavor are propanol, isobutanol, active amyl alcohol, and isoamyl alcohol. The thresholds for these fusel alcohols can be found in the chart above. It’s important to point out that different yeast strains across both ale and lager strains are an important factor in determining beer alcohol and ester ratios in fermented beer. It’s also important to keep in mind that in each paper detailed below different yeast strains were used and the results could vary, however, I think it’s fair to use the information to form a high-level understanding of esters and alcohols and use the information to increase the likelihood of brewing beer closer to the desired goal.

Although the biochemical background of ester and alcohol production is very interesting, I chose to leave this discussion out of this post mainly because after reading through the studies, I cannot even pretend I understand the process enough to write a comprehensible synopsis.

Temperature

It is already generally understood that higher fermentation temperatures lead to increases in ester production, however, amounts can vary greatly across different strains and the particular individual esters produced.

Enari showed that the amounts of fusel alcohols increased “markedly” with increasing fermentation temperature from 50°F to 59°F. Interestingly, the same study also found that the more flocculent yeast strain (PBL 20) had much less fusel alcohols created than a non-flocculent strain (PBL 19). Although a slightly dated study, I’m curious if this same concept applies to the smoothness of NEIPAs. Commonly fermented with advertised flocculent yeast strains (despite not dropping clear after dry hopping) if these are producing fewer fusel alcohols, this could help explain the smoothness and lack of heat on the palate.

Increased temperatures leading to increased esters was also found by Hiralal in 2013 where increasing the fermentation temperature from 64.4°F to 72.5°F resulted in increased acetate ester concentration by 14% and total ethyl ester concentration by 63% (with the Safale S-04 yeast strain).

Trub

A 1982 study looked specifically at the role trub (a mixture of cold and hot break) played during beer fermentations. Pilot scale fermentations were conducted in two separate tanks with trub ranging from 0.00 to 2.66% of volume/volume. The study found that esters and fusel alcohols formed during fermentation were influenced by trub levels. Specifically, the levels of esters in the beer fermented with no trub were higher than the beers fermented with the trub added. Further analysis showed that the wort lipids in the beers with trub were significantly higher than the clear no-trub worts (more on this below). Interestingly, the study also found that the beers with trub fermented substantially faster (1.75 days faster) and had a more vigorous fermentation with a higher suspended yeast count during fermentation. On the other hand, the formation of fusel alcohols slightly increased in beer fermented with high levels of trub. Also a little off topic but interesting, the study found that the no-trub wort with a high level of dissolved oxygen fermented at the same rate as a high-trub wort with low levels of dissolved oxygen. Maybe most important, however, is that panelist demonstrated a significant preference for the beer produced from clarified wort.

A follow-up study looking at the potential effects of trub during fermentation was conducted in Japan with Saccharomyces cerevisiae BH-84 (commercial lager yeast). Three separate tanks were filled with cooled unfermented wort with varying amounts of trub ranging from 0.13 mg/ml to 15.9 mg/ml. The beers were then fermented and tested for the esters ethyl acetate and isoamyl acetate. The authors found that when the total trub content in the wort increased, both ethyl acetate and isoamyl acetate were significantly decreased.



Yeast Nutrients

A relatively recent paper looked at the role of two yeast supplements (zinc sulphate (ZnS04) and the amino acid L-leucine) and tested their effects on ester production in beers fermented with the Safale S-04 yeast strain. For reference, Servomyces is a commonly available yeast nutrient that contains zinc and L-leucine is an α-amino acid (α-amino acids are commonly found in commercial yeast nutrients like WLN1000 from White Labs). I reached out to White Labs to find out the exact contents of their yeast nutrient but wasn’t able to get an answer. The paper found that the ester compounds producing during fermentation were greatly increased with the addition of ZnS04 and L-leucine. Specifically, 0.12 g/l of ZnS04 resulted in a 27% increase in acetate esters and 123% increase in total ethyl esters compared to unsupplemented sample. The addition of 0.750 g/l of L-leucine resulted in a 41% increase in total acetate ester concentration and 84% increase in total ethyl ester concentration compared to unsupplemented sample.

The addition of amino acids of valine, leucine, and isoleucine (total nitrogen added = 95 mg/L) was found to strongly increase the production of fusel alcohols (isobutanol, isoamyl alcohol, and amyl alcohol). Specifically, 60-70% of the added leucine and isoleucine were transformed into isoamyl alcohol and amyl alcohol, and the all the valine was transformed into isobutanol. A potential downside of no added amino acids (yeast nutrient) to the ferment was a slightly slower time frame to reach the end of fermentation, in this case, the difference was only about 10 hours.

Perhaps it’s worth experimenting with only supplementing yeast starters with nutrients if your goal is to keep the ester and fusel alcohol production in check. This supplemented starter would ensure you are pitching a healthy and adequate amount of yeast needed for fermentation, but the absence of additional nutrient in the fermenting wort may lead to lower ester and alcohol profile beer (depending on the goal). This would be most useful to people who harvest their yeast from starters and not from large fermentations, as yeast health would likely go downhill after numerous generations of unsupplemented fermentations.

Oxygen

Anaerobic and semianerobic fermentation conditions were tested in high gravity worts to find out the effects of esters and fusel alcohol production. The semianerobic fermentations were done with the use of a sterile foam plug (I’m guessing similar to what many homebrewers use on their starters) which resulted in drastically reduced levels of esters compared to the anaerobic ferment. This was true regardless of the amount of nutritional supplementation given to the beers. The fusel alcohols didn’t appear to be significantly influenced by either anaerobic or semianerobic conditions.

Other studies have also pointed to decreased ester production under conditions of increased wort aeration, even across numerous yeast strains tested and even at different pitching rates. Of course, the goal isn’t always reduced esters, but if that is a goal, mimicking open fermentations could be a way to reduce the levels. This can easily be done simply by covering the opening of a carboy with sterilized tinfoil during the active phase of fermentation, likely the first five days or so.

Top Pressure (Dissolved C02)

Similar to what I found in a previous article, top pressure or C02 pressure, can also have an effect on ester production. In an experimental brew I did for the article, I was underwhelmed by the overall flavor and aroma of the beer fermented under pressure, suggesting to me that even in extremely hoppy beers, a complete clamp on esters may not be ideal. One study looking at top pressure applied by sustained carbon dioxide to the fermenter (capped) and found that both esters and fusel alcohol concentrations were decreased as a result. The suggested reasoning for the decline was linked to the overall reduction of yeast biomass growth when under pressure (less yeast repopulation). Nakatani also found that when dissolved C02 concentration in fermenting wort was increased both ethyl acetate and isoamyl acetate were reduced. This is interesting because you would just assume if more oxygen decreased esters that no oxygen would increase them, but that’s not the case.

Because dissolved carbon dioxide can influence ester production, big commercial breweries with extremely large tanks may experience a loss in their complex ester profile because of the design of their fermenters causing higher hydrostatic pressure on the wort, particularly with tall fermenters.

Yeast Pitching Rates

A study as recent as 2015 looked at fermenting beer (pitched with saccharomyces carlsbergenis yeast W34/70) in three separate tanks and then dosed each tank with either 5, 7, or 9 million yeast cells per milliliter of wort. They then tested samples of the beer for 18 days. The authors found that increasing the yeast dose from 5-7 million yeast cells per/mL did not alter the amount of ethyl acetate formed, but increasing the dose to from 7-9 cells per/mL significantly increased the concentration by about 10%.

There seemed to be a few older studies that would both agreed and disagree with the findings in the study mentioned above. Krzysztof makes note in the study to explain that when the yeast pitching rate increases, the biomass or multiplication of yeast decreases because of a reduced availability of nutrients and oxygen in the wort.

Off topic, but another finding in the Krzysztof study I found interesting was that in sensory evaluations, the beers produced with the greatest amount of yeast were evaluated more poorly. I too have been unhappy with beers that I purposely overpitched (with WLP002), particularly with hoppy beers.

Beer pH

The Hiralal study also looked at the role of beer pH and its effect on ester production, which is something I hadn’t seen studied before. Three different beers were fermented each with a different starting pH (3.0, 5.0, and 7.0). As the starting pH increased, so did the esters formed during fermentation, but a low starting pH actually reduced the esters. The beer fermented at a pH of 7.0 resulted in the highest ester concentration with a 13% increase in total acetate esters and 7% increase in total ethyl ester production compared to the control (5.0 pH beer). On the other hand, the beer fermented at a low 3.0 pH resulted in an 18% decrease in total ester concentration.

This last part is something to keep in mind when brewing kettle sour beers, which requires dropping the beer pH into the 3.0’s with lactobacillus prior to pitching a strain of saccharomyces. To still get good yeast character in these beers, it might be best to try adding additional yeast nutrient and restrict the amount of oxygen added to the ferment to encourage a more complex ester profile.

Timing of Ester and Alcohol Production & Fluctuating Temperatures

A study with insight into when ester and fusel alcohol production occurs during fermentation was done in 2001 looking at ferments with top pressure (so keep that variable in mind). They found and described a three-phase timetable for production which is as follows: 1) alcohol accumulates alone; 2) both alcohol and ester accumulate; and 3) ester accumulates alone. Fusel alcohols were produced during sugar and FAN assimilations during their experiments, which is slightly earlier in the process than ester formation. Ester production rate reached a maximum and approximately 80% of the total fermentation time, depending on temperature. The initial time of ester production was generally around the 20-hour mark despite the amount of top pressure applied or temperature.

Examining the chart included in the study, it appears to me that fusel alcohols production begins in the first 10 hours of fermentation and really began to ramp up around the 24-hour mark, but at a much more drastic rate at warmer temperatures. Ugh oh, this could mean bad things for pitching hot! However, when the beers were pitched warmer in the study no yeast lagtime was experienced, which is likely why alcohols formed slightly faster (this case 60°F for a lager yeast strain).

The Enari study also looked at the timing of alcohol production in both aerated (stirred) and unaerated fermentations by hours into fermentation. Production started off fairly low and started to drastically increase alcohol production around the 50-hour mark. At 24 hours into the ferments, both already had around 20-25 mg/l fusel alcohols created, suggesting the early stage of the fermentation is important to time for creating fusel alcohols, especially when temperatures remain high.

It’s important to note that although the study found that the temperature had a significant effect on fusel alcohol production rate through increased sugar and FAN consumption rates, they fermented the beer at the constant hotter temperature. So the benefit of pitching hotter than the desired fermentation temperature could be no yeast lagtime, however at the expense of slightly more alcohol production, but likely less than the study found because the temperature would be lowering during this fusel production period, not remain at a constant warm temperature.

The time frame at which esters are created during fermentation seems to be less of an issue when pitching hot. In a review on yeast and esters and alcohols in the Applied Microbiology and Biotechnology Journal, it’s stated that esters are mainly formed during the vigorous phase of primary fermentation by “chemical condensation of organic acids and alcohols.” The vigorous stage of fermentation is rarely reached during the short time wort is still cooling in a fermentation chamber getting to the desired fermentation temperature.

Directly looking at this issue, a 1994 study by the American Society of Brewing Chemists analyzed ester formulation during the course of fermentation at different temperatures. Fermentation was carried out at three different temperatures (53°F, 59°F, 64°F) with a lager saccharomyces cerevisiae strain (NCYC 1324) and ester samples were recorded by headspace analysis. Greater ester content was found per degree fermented at a higher temperature, which is not surprising. However, as it relates to the time frame, esters (isoamyl acetate) really started to ramp up around hour 20 during fermentation, peaking around 2.7 days for the hottest ferment and 3.75 days in the coolest ferment. To understand just how drastic the temperature difference was, the coldest ferment finished around 3 mg liter, which was where the hottest ferment was already at around 15 hours in. So it does seem logical, at least for isoamyl acetate, that pitching hot might not have drastic effects, but it certainly seems possible you’re creating a pathway for slightly higher esters and with low thresholds, could have a taste impact, but remember this was for fermentation held at constant temperatures.

Adjusting the fermentation temperatures during the course of fermentation alters the ester and production, but it’s a complex function of the final temperature level, the direction of the change in temperature, and the status of the yeast at the time of the temperature adjustment a 1995 study concluded. A commercial lager yeast strain was tested in oxygenated wort in four separate ferments, one at a constant 52°F, one raising the temperature from 52°F-65°F around 60 hours into fermentation (after C02 evolution rate reaches maximum), one raising from 52°F-65 around 20 hours into fermentation (after stationary phase), and lastly one at a constant 65°F. In this test, the highest esters were for the beer fermented at the constant 65°F. The beer ramped to 65°F after stationary phase (20 hours or so) finished pretty close to the beer at the constant lower 52°F. And surprisingly, the beer ramped to 65°F around 60 hours or about 2.5 days into fermentation finished with the lowest ester concentration (approximately 8 mg/L less than the constant 52°F ferment). So it seems plausible now, that starting with a higher pitching temperature and gradually cooling it down during the first 10 hours or so, could be offset by ramping the temperatures after the first few days of fermentation.

I have always been a little leery of raising the fermentation temperature early on into the ferment because I had a basic understanding that esters were still being formed 2-4 days into fermentation, however, after learning that increasing the temperature during this vigorous phase actually can lead to less ester production, why not crank up the temperature and encourage a faster ferment?

The Sablayrolles study also looked at the effect on ester and fusel production when cooling the fermentation temperature. When the fermentation temperature was decreased from 65°F to 52°F after about 2.5 days (again, after C02 evolution rate reached maximum) the resulting ester levels were higher than for any other recorded variable. So lowering the temperature during peak fermentation causes higher ester levels than fermenting at a consistent cooler 52°F and a constant 65°F. Again, the lowest ester level during this particular test was when the fermentation temperature was raised from 52°F-65°F. This could mean ambient conditions for ferments in the winter could be a problem if your home fluctuates greatly throughout the day and night.

As far as fusel alcohol production in the two test described above, they didn’t change as much with the temperature adjustments. In general, there was a slight increase with higher fermentation temperatures and when raising the temperature mid-ferment resulted in a lower increase in alcohols versus a consistent hotter ferment. When the temperature was raised later into fermentation rather than early (approximately 20 hours vs. 60 hours) the alcohols were increased to a lesser extent.

Wort Sugar Profile and Gravity

Fermentations carried out in high gravity worts are known to lead to beers with an overproduction of acetate esters (overly fruity and solvent). One study found a fourfold increase in ethyl acetate and isoamyl acetate production when the gravity was increased from 1.042 to 1.083.

The type of assimilable sugars in the wort can also play a role in determining ester levels. It’s common in high gravity brewing to supplement the wort with sugar via syrup of some kind to bump up the fermentables. In one study, the influence of maltose and glucose levels in high gravity worts were tested to see the effects on acetate esters. The beers brewed with elevated maltose concentrations produced lower levels of acetate esters than similar gravity worts with the higher glucose or fructose. So if your plan is to bump up the alcohol of a big beer, but your want to avoid the possibility of increased esters, try experimenting with maltose syrups.

Wort Lipids

Awhile back, I wrote an article making a case for brewing beers with oats, one of the reasonings (if this is your end goal), is because oats are high in lipids which may suppress ester formulation (more in-depth look at this topic can be found in the article linked above). One such suggestion to reducing the overproduction of esters in high gravity beers is to create cloudy worts using adjuncts like oats that are high in fatty acids and lipids tame down the overall ester production.

Beer Aging

The ester profile of beer can change significantly during beer storage by yeast in the bottle (bottle refermentation) or by spontaneous chemical condensation of organic acids with ethanol. Some esters as isoamyl acetate can be hydrolyzed during beer storage in unpasteurized or bottle conditioned beers. Specifically, it was found that after 6 months of storage with an ale yeast strain at 75°F (whether yeast was in the bottle or not), lost about 35% of the original isoamyl acetate (fruity, banana) concentration. Interestingly, a lager strain tested only lost 10%.

Because acetate esters help provide the “fresh” beer aroma, they can actually aid in masking the perception of other flavor staling compounds. Low initial concentrations of esters in a beer could then limit this masking effect, especially after acetate ester degradation during beer storage. It then might be worth experimenting with yeast strains known for producing higher acetate ester production if the beer is intended to be aged (or any of the other processes to encouge mroe esters).

A study looking directly at the aging of top-fermented ales and the effect on esters was done in 2003. Sensory analysis revealed that beers stored at 104°F for 6 months lost their initial fruity estery flavor and developed strong port-like flavors with a solvent and minor papery flavor. The acetate esters were also measured and showed that all acetate esters decreased during the aging process, likely do to ester hydrolysis. Ethyl esters were also shown to hydrolyze during aging. Also of interest was that vicinal diketones (off flavors) and diacetyl clearly increased during this extreme beer aging Vanderhaegen, B., Neven, H., Coghe, S., Verstrepen, K. J., Verachtert, H., & Derdelinckx, G. (2003). Evolution of Chemical and Sensory Properties during Aging of Top-Fermented Beer. J. Agric. Food Chem. Journal of Agricultural and Food Chemistry, 51(23), 6782-6790. doi:10.1021/jf034631z–keep your beer cold!

The Brulosophy team released a great experiment testing the ability of tasters to distinguish beers made from either decanted yeast starters or from full yeast starters pitched directly into wort. They found that the panelist were unable to distinguish a differences from the two beers in triangle tests. Learning that increased oxygen from having a starter on a stir plate can actually decrease ester production combined with the ability of esters to hydrolyze during fermentation, the Brulosophy sensory findings seem to align with the literature. Maybe decanting the yeast really isn’t a necessary step before pitching. If anything you avoid dumping any yeast down the drain that was still in suspension and the added volume of liquid may offset gravity readings taken throughout the process.

In regards to starters being heavily oxygenated thus having suppressed esters, this is likely why when I taste my starters, they are very bland and yeasty tasting. If you are using your starter to gauge whether or not to pitch a certain yeast strain, it may not be a fair way to evaluate the yeast capabilities. In this case, you might be better off treating the starter like a typical ferment, without a stir plate and give it more time to ferment out before tasting, which will hopefully give you a better representation of the yeast’s ester potential.

Summary of the Research