This is the 2nd series of experiments regarding the effects of various additives and fermentation conditions on fermentation performance and beer quality. This time I wanted to take a closer look at the impact of various levels of zinc additions on fermentation performance.

Zinc is an important co-factor in many enzymes and thus a requirement for yeast growth. Most yeast strains require 0.1 – 0.2 mg/l of zinc. Zinc levels greater than 0.6 mg/l can inhibit yeast growth (Priest, Handbook of Brewing).

Zinc may be the only yeast nutrient that even barley malt is deficient of. As a result fermentation performance may benefit from the addition of zinc. This is why I conducted this experiment.

In the end I was not able to find any significant difference between the control fermentation and the 4 different levels of zinc additions with the exception of yeast growth. The most yeast growth was observed for the fermentation that added 0.2 mg/l zinc. This would agree with the literature which mentions possible yeast growth inhibition at total zinc levels above 0.6 mg/l.

The absence of other differences may be because the wort (98% Pilsner malt wort) contained sufficient zinc for the yeast that was used (WLP 830) or that the yeast was not able to utilize the zinc that was added (in this case the yeast growth differences would have to be attributed to a random error). The zinc came from simple 50 mg zinc tablets that are available as dietary supplements in a grocery store.

Details of the experiment

The wort that was used was left over from a Pilsner brewed with

• 96% Best Pilsner Malt
• 2% Weyermann Sauermalz
• 1% Weyermann CaraMunich I
• 1% Weyermann CaraPils

Mashed with a Hochkurz mash

• 30 min at 65C
• 45 min at 72 C
• 15 min mash-out

moderately hopped with FWH Saaz and 60 min boil addition of hop extract

It was boiled and cooled before a stir plate and O2 were used to achieve a wort oxygen content of ~12 ppm. WLP 830 yeast was then added and allowed to un-flocculate completely before the pitching rate was determined with a hemocytometer. The pitching rate was about 9 Millon cells per ml.

About 413-125g of this pitched wort were poured into 5 500 ml Erlenmeyer flasks. In order to eliminate the effects of settling yeast during this process flasks were filled in random order and the cell density in the remaining wort was also determined. That cell density was around 10 M/ml. As a result there was no significant yeast settling that may have lead to different pitching rates.

Wort oxygenation while the oxygen content is monitored with a dissolved oxygen meter

To provide varying levels of zinc additions a 50 mg zinc tablet was dissolved in 185 ml water resulting in a 0.27 mg/ml solution. 0, 0.3, 1, 4 and 8 ml of this solution were added to the 5 different fermentation resulting in 0.0, 0.2, 0.7, 2.6 and 5.1 mg/l of zinc addition, respectively. To keep the addition of water the same DI water was added such that the water addition was 8 ml for each fermentation.

Zinc solution and Zinc tablets

The flasks were sealed with an airlock and their initial weight was determined before they were placed into a temperature controlled freezer chest. The initial ambient temperature was 7.5 C and was later raised to 11.8 C.

All five flasks in the freezer chest with the temperature probe in their middle

The flasks were weighed about twice per day for 15 days until the weight loss stabilized. At that point the extract content of the beer was determined with a precision 0.990 – 1.020 sg hydrometer before the beer was filled into a bottle along with 1.9 g of sugar and some of the yeast sediment.

The yeast sediment remaining in each flask after all beer was decanted was determined by weighing the flask and subtracting the empty weight of the flask.

About 1 oz of beer was left over which used to measure pH and these samples were also tasted.

These are the various metrics that were collected for all 5 fermentations:

1) refers to the extract loss per day during the most intense part of the fermentation and was determined from the slope in the fermentation profile

And the fermentation profile:

fermentation profile as percent weight loss over time

For some reason the “+8 mg/l Zn” fermentation showed a larger weight loss (measure of lost CO2) then the other fermentations, but that did not show up as higher attenuation or lower final extract. It is not known what could have caused this increased weight loss.

The fermentation metrics for all 4 beers are very much the same. The most yeast growth was achieved for the “+0.3 mg/l Zn” fermentation, while higher additions led to less yeast growth. This might be an result of total wort zinc levels in excess of 0.6 mg/l which is known to inhibit yeast growth. This yeast growth inhibition did not affect other fermentation parameters like extract drop per day during the most intense part of fermentation.

There were no taste differences that I detected between the beers. Tasting of samples was done after the measurements were taken and my taste perception could have been influenced by my expectation that there would be no difference.

It is possible that the zinc had no effect and that the yeast sediment weight differences are the result of a random error. On the left is a picture of the ingredients label. Zinc is present in these caplets as Zinc Gluconate which may not readily release Zn2+ into the wort. The use of zinc chloride would have been more reliable, but I didn’t have that at hand. Maybe for a later experiment I get ZnCl2.

I finally got around to conduct a new set of experiments. These experiments focus on fermentation. Rather than brewing full batches of beer and change parameters I’m using wort left over from full size batches to run a few small scale fermentation experiments.

The first set of experiments focused on pitching rate. Since pitching rate is a parameter that has been evaluated by many brewers I didn’t expect too many surprises. I was mostly interested in testing my approach, in particular using the weight change of the fermenting beer as an indicator of the progression of fermentation. This weight drop is the result of escaping CO2.

300 ml of 12 Plato beer contain about 36g of extract. With an apparent attenuation limit of 80% about 66% or 23.8g of this extract is fermentable. About 1/2 of its weight is converted to CO2 of which the majority escapes. This means during fermentation the beer looses about 11g or 4% of its weight. This weight loss can easily be followed using a scale with an accuracy of 0.1 g. The scale I use is a cheap kitchen type scale with a capacity of 2 kg and a precision of 0.1g. It has shown excellent repeatability of weight measurements.

Setup

For this experiment WLP 830 yeast sediment from a fast ferment test was added in different amounts to about 355g of 13.1 Plato Pilsner wort. The wort was aerated to about 7.2 ppm O2 before it was divided into the individual 500 ml flasks for the experiment. The yeast was brought into suspension and pitching rates were determined though cell counts.

The beer was not agitated during fermentation and the flasks were closed with an airlock. The mean ambient fermentation temperature was 18 C. Weight measurements were taken twice daily. After 7 1/2 days, once the weight stabilized and approached the expected weight loss, the extract was measured using a hydrometer and the beer was filled into individual 12 oz bottles. Sugar syrup and some yeast was added to carbonate the beers.

To asses the level of yeast growth that happened during fermentation all beer was decanted from the flask and the flask with sediment was weighed. The empty weight of all flasks used was known and hence the weight of sediment could be determined. To estimate cell count a sediment density of 4 Billion cells per gram was assumed. This is a number that I found true for yeast sediments from fast ferment tests or yeast propagation.

Results

The following chart shows the beer’s weight drop (in %) during fermentation for all 5 pitching rates:

The following table shows metrics that were measured

1) the extract drop per day was for the most active part of the fermentation

2) difference between limit of attenuation and actual attenuation

Discussion

The progression of the extract or weight drop during fermentation was as expected: The higher the pitching rate the faster the fermentation start and the greater the extract drop during the most active part of fermentation. This has been observed before and is related to the greater initial yeast population.

It is also well known that beers with lower pitching rates have a more difficult time or take longer to attenuate fully. This was observed as a higher final extract for the beers with the lowest pitching rates compared to the beers with higher initial pitching rates. The differences were not very dramatic, though.

The amount of yeast growth showed a correlation to pitching rate. Higher pitching rates lead to more yeast growth (the initial cell population was considered in this analysis) but the differences in yeast growth were not large. The highest pitching rate yielded about 25% more yeast growth. This might be due to the fact that yeast growth was limited by sterol reserves. A large initial population has more combined sterol reserves than a small initial population which means that it can sustain more cell divisions.

I have not yet tasted the finished beer and also plan to measure the pH of each of these beers at that point.

I finally ran a brewing experiment again and am dusting off this blog to post the results.

The experiment was a repeat of a standard experiment that evaluates yeast growth in still, intermittently shaken and stirred starters. I like how easy it is to set up and plan to repeat it again.

Setup

919g of 12 Plato wort were aerated to 100% air saturation on a stir plate. Then 7.8 g WLP 830 yeast sediment from a primary fermentation was added and allowed to un-flocculate completely. The cell density was determined as 22.3 Million/ml with a hemacytometer. If you do the math you find that the slurry contained a total of about 20 Billion cells which means its density was 2.63 Billion/g. Previous slurry density assessments for slurries from yeast propagation ranged from 4-5 Billion/g for this yeast. I assume that trub played a big role in the reduced cell count in this slurry.

Once the yeast was evenly distributed in the wort, the wort was evenly divided into 3 500 ml Erlenmeyer flasks. The flasks were labeled with their empty weight and covered tightly with aluminum foil. I didn’t tighten the cover intentionally. I noticed that this happens when I grab the flasks at their neck to shake them. I made sure that all of them had tightened aluminum foil caps, even though only one would be shaken intermittently.

All starters were allowed to complete fermentation. One sat still, one was shaken intermittently (1-2 times in the morning and 3-5 times in the evening) the last one was placed on a stir plate and stirred continuously. A vortex formed on the stir plate, but was eventually covered with foam when CO2 started escaping. The ambient temperature was around 18 C.

Data:

Once fermentation was complete the yeast was allowed to settle into a dense cake until the beer on top cleared. The beer was poured off and the flask’s weight with yeast and stir bar (in one of the flasks) was determined. From that, the empty weight and the stir bar weight the yeast weight was determined:

I only recorded the yeast weight and did not count the yeast with the microscope. Simply, because I did not want to spend too much time on this. To estimate the number of cells grown I assumed that the sediment had a density of 4.7 Billion/g, an average of the numbers I had assessed in earlier experiments.

Discussion and Conclusion:

The wort volumes for the three starters were not exactly the same, hence the difference in the extract weight.

I was surprised to see that the sill and shaken starters showed no difference in the amount of yeast that was gown from the available extract. This is different from the data reported by others (e.g. the great Maltose Falcon article on yeast propagation: Yeast Propagation and Maintenance: Principles and Practices) where a shaken starter grew significantly more than a still starter and a stirred starter outperformed a shaken starter by about 4x. The caption to the chart mentions that the data point for the still starter was taken from a different experiment, though.

Data I recorded on past yeast propagation steps (usually stirred)  and fast ferment tests (usually shaken) show more difference in extract utilization for yeast growth, but there are also a number of other parameters that have been different for those experiments. Most notably the starting gravity and the initial cell density.

I’ll have to repeat this experiment in the future to see what covering loosely with aluminum foil does and what an airlock would do to the extract utilization that can be achieved. And maybe I also find the time to count the final number of cells.