Yeast growth experiments – some early results

For a while now, actually ever since I got the microscope, I have been keeping track of yeast growth in starters, fast ferment tests and some beer fermentations. In some cases I was too lazy and did not record any data. But what I found is that the new growth per gram of extract (stuff dissolved in wort), measured in Billion per gram, was all over the place. Initially I thought I could come up with a simple formula to estimate cell growth based on wort volume and extract content. But that doesn’t seem to be the case.

So I started examining the issue in a more controlled environment. For my first set of experiments I prepared 6 l of ~10 Plato wort from old malt extract and some left over wort I had in the freezer. This was frozen in three 2 l soda bottles. I then grew some WY2042 from a slant to use in these experiments. WY2042, Danish Lager, works well since it doesn’t flocculate when its done fermenting. To conduct the experiment I would thaw one of the bottles with wort and keep it in the fridge. I then take ~230 ml of that wort innoculate it with yeast and place it on a stir plate covered with foil. The initial cell count was determined with a hemocytometer and the yeast was allowed to complete fermentation for 1 to two days. Then the final cell count is taken and the sample is allowed to settle in the fridge. For the next experiment some of the yeast sediment is kept in the flask and fresh wort is added. The amount of wort added is recorded as well as the initial cell count. Using this approach allows me to run a series of experiments with yeast of consistent freshness and only one stir plate. To eliminate age effects I went back and forth over the starting cell density range of 1 through 270 Million cells per ml. In the chart below you see Billion cells per liter, which is the same.

Here are the results:For reference I also added numbers I got from Jamil’s and the Wyeast pitching rate calculator.  The dashed lines show which experiment provided yeast for the next experiment.

There are notable differences at low and high starting cell densities.  In my experiments the specific growth I got at high starting cells counts (>150 B/l) was much less than the two pitching rate calculators predicted. The low growth makes sense since more sugar will be needed to feed the larger population during its lag phase. I think that this might be more pronounced with older yeast cultures where glycol reserved are lower (these yeast cultures sat only 1-2 days between propagation).

I also saw larger than expected growth at low starting densities.

Around initial densities of 100 B/l, the experimental results agree with the other two calculators which is good and means that there is some predictability.

You also see one data point for a 2nd series of the experiment. In that series I started a new culture from a slant and want to see if it behaves similarly.

The error bars get lager for higher starting densities since the error for counting the initial population plays a larger role when that population is large. E.g if the starting population is 1 Billion and has an error of 10% (+/- 0.1 Billion) and the final population is 100 Billion with a 1o% error (+/- 10 Billion) the error for the new growth of 99 Billion is ~ 10% or +/- 10 Billion. But if the starting population is 101 +/- 10 Billion and the final population is 200 +/- 20 Billion the difference is still 99 but with an error of +/- 22.

Stay tuned for more data as I have it ready for publishing.

Yeast un-flocculation for cell counting

One problem in cell counting is that the cell culture needs to be evenly suspended in its volume and any cell clumps need to be broken up. That is not an issue with poorly flocculating yeasts like German ale yeast. But heavy flocculators like English Ale yeast (WLP 002)  provide a challenge. I knew that any yeast can be un-flocculated in the presence of maltose since maltose inhibits flocculation. This makes sense since it it more advantageous for the yeast cells to float freely when food is available. However, adding fresh wort to a yeast slurry simply for counting its cells seems a bit wasteful.

So I asked White Labs about this and their response was to use sulfuric acid or EDTA (Ethylenediaminetetraacetic acid). A search on EDTA revealed that it is a chelating agent. This makes sense in the context of preventing yeast flocculation since it is able to chelate the calcium necessary for flocculation.  This gave me ideas for other chemicals that might work.

Tonight I spent some time in my lab/fermentation room to test a variety of chemicals for their ability to un-flocculate WLP002, the heaviest flocculator I have encountered so far. I had some WLP 002 sediment from the fast ferment test for a pale ale I brewed last weekend.

The experimentation set-up was simple. I added a little bit of WLP002 sediment, water and the de-flocculation agent I wanted to test to test tubes. Then I closed the test tubes and sloshed the contents around to see if the clumps are breaking up. When the sample was un-flocculated I inspected a sample under the microsope to check if there are truly no clumps of cells left. Here is what I found:

  • water: this was the control. While the yeast clump did break up it only broke up into small floccs of yeast. This was not good enough for counting.
  • fresh wort: it didn’t take long for the yeast clumps to break up into individual cells after brewing wort was added. This is the best option if the yeast sediment needs to be suspended anyway for pitching or as another stage of yeast propagation. This method does not kill the yeast and the viability can be assessed with methylene blue.
  • glucose: clumps broke up slowly. This method does not kill the yeast either, but it is not very practical since there are better options.
  • sulfuric acid: works very well. Clumps broke up very quickly and it doesn’t kill the yeast which allows for methylene blue staining. But sulfuric acid is a hazardous chemical and needs to be handled with care
  • PBW: Yes, Five Star’s Powdered Brewery Wash. I got the idea to use this since it also contains chelating agents. It works very well and the cells were quickly suspended as individual cells. It does not dissolve the yeast immediately but kills them. When stained with methylene blue a significant number of cells stained blue while the culture had a known viability of 95+%. PBW is much safer to handle than sulfuric acid and many brewers have it at hand.
  • GH test solution: since calcium chelation is the key for many of these agents and general hardness (GH) tests do just that I also gave this a try. It worked very well but is not practical due to its cost. The amount that can be found in a simple GH/KH test kit for $6 would only be enough to un-flocculate a few yeast sediment samples.
  • disodium EDTA: I haven’t tried this yet but plan to test it when I order from a place that sells it. I expect that it works and that it does not kill the yeast either.
  • Phosphoric acid: (mentioned by Northwestbeer in the comments for Yeast pitching by weight) Works very well, is generally available in a home brewer’s lab and does not kill the yeast. When I tried it I used about 2% phosporic acid (1 ml 10% phosphoric acid, 3 ml water and 1 ml yeast sample)

For now I’ll use fresh wort when I need to re-suspend the yeast in wort anyway and I’ll use PBW when I only need to count the cells in a given slurry of yeast without plans to use the yeast later.

Enzymes in the fermenter

You may remember the recent post about the Infinium’s patent pending brewing process and its mention of enzyme extracts in the fermenter. I recently brewed a Hopfen Weisse inspired Weissbier IPA where the wort fermentability was unexpectedly high. I decided that this was an opportunity to test the use of enzymes in the fermenter. So I took about 1800 ml of the young beer after primary fermentation and split it into three 600 ml batches. One became the control, one received an enzymatic malt extract and the last one got some Beano. I heard that Beano has the tendency to create “rocket-fuel” and I wanted to test this.

mashing the enzymatic malt extract

The instructions for the enzymatic malt extract came from this patent for low calorie beer where an enzymatic malt extract is prepared using a 30 min mash at 60 C. This kills most of the bacteria but keeps enough b-amylase active to be useful in the fermenter. Killing bacteria is important since the extract will be added directly to the fermenter without any boiling. I was curious if this really works since spent grain, which is raised to even higher temperatures for longer times during mashing, seems to be loaded with microbes. A fact for which I get a pungent reminder when I open a mash tun I forgot to clean on brew day.

The mash consisted of 70 g milled Pilsner malt and 360 ml reverse osmosis water. I was not too concerned about getting the correct mash pH. After all, I only needed enzyme extraction  and  some level of pasteurization. Set in a water bath I was able to hold a steady temp of ~60 C for 30 min. Temperature control of small volumes is challenging. A well insulated and pre-heated thermos may work too.

Filtering the enzymatic malt extract

The malt extract was filtered through a paper towel set into a funnel. Its extract content ended up being 10 Plato. This amounts to 75 % conversion efficiency. Not that this efficiency matters much, but I took the extract content of the malt extract into account when calculating the corrected starting extract for the beer. 53 ml of this malt extract was added to the 600 ml beer. About 8% of its volume. There was no science behind this ratio. Too much and I would dilute the beer too much and too little I would not add enough enzymes.

The Beano I used came in pill form and I dissolved one pill in 100 ml water. Beano’s strength is measured in GAU (GlucoAmylase Units). One Glucoamylase Unit (GAU) is the amount of enzyme activity that will liberate on (1) gram of reducing sugar as D-glucose per hour under the conditions of the GA Assay (Vri 511.002). (source http://vitallifeproducts.com). 22 ml of this solution went into one of the beer samples which added about 110 GAU per liter beer.

20 ml water were added to the control to lower its original extract since the other 2 experiments also lowered the starting extract of the beer.

The 3 fermentations immediately after being prepared

The following morning (about 10 hour later) I noticed strong fermentation activity in the fermentation with malt extract. The Beano fermentation showed some low activity. But since I did not control the actual amount of enzymes that went into each, it is perfectly reasonable that the Beano fermentation ended up will less enzymes than the malt extract fermentation.

10 hours later, the malt extract fermentation shows the most activity

After 9 days I concluded that all fermentations were done and I bottled the beers.

After 9 days

To carbonate the beer I added 4.3 g dissolved table sugar to each 500 ml bottle and some yeast.

I also got an opportunity to measure the current extract and pH. Most importantly I was able to taste uncarbonated samples. Here are the stats:


control malt extract Beano
corrected starting extract 17.8 17.7 17.7 Plato
days fermented 9 9 9 days
Final extract 4.3 2.5 2.2 Plato
ADF 75.9% 85.9% 87.6%
pH 4.48 4.4 4.35

As expected the addition of enzymes boosted attenuation. Beano seems to digest more dextins than a malt extract. I wonder to what extent the difference is caused by limit dextrins (1-6 glucose links), which can only be broken by limit dextrinase, an enzyme which likely did not survive the 30 min mash at 60 C. Beano’s glucoamylase is likely able to cleave both the 1-4 and the 1-6 links found in starch leaving only glucose and no limit dextrins.

It was surprising, however, that even with enzyme use during fermentation the apparent attenuation stayed in the mid 80’s. This suggests that the low wort fermentability may not have been the result of incomplete starch and dextrin break-down during the mash. After all, I was going for very fermentable wort and held 30 min rests at 55 C, 63 C and 65 C followed by a 45 min rest at 72 C. I was expecting to get an attenuation limit in the high 80’s with this mash schedule.

There was also a drop in pH. I contribute this to the extended fermentation activity, which was longer for Beano compared to the malt extract.

So much for the metrics, here are some brief tasting notes:

control: No noticeable off flavors. While this was a Weissbier yeast, it is a yeast that produces only limited amounts of phenolics. The mouthfeel is rather thick and the beer does not have the refreshing IPA character that I was going for

enzymatic malt extract: No off flavors. Whatever surviving microbes came in from the malt did not take hold and spoil the beer. I’ll have to see how this develops as the beer sits in the bottle for a month. The mouthfeel was much lighter and more  easy drinking than the control. This was the taste I was shooting for.

Beano: No off flavors either. The mouthfeel was even lighter than the malt extract version. But it was not the dreaded “rocket fuel” that others have gotten from Beano before. I think this version felt a bit too thin in its taste, though. I’ll have to defer to tasting a carbonated sample.

Conclusion

I think enzymatic malt extract in the fermenter works. It appears to be a viable option for creating highly attenuated high alcohol beers and I plan on using it on a full size batch in the future. I don’t know if it is a practical option for fixing low wort fermebtability in lower gravity beers since they may become too thin. After all, the enzymatic reactions will continue until all substrate, i.e. dextrines,  is gone.

Hops From A Can

Many brewers scoff at the idea of using hop extract. After all, it represents what’s wrong with industrial brewing these days: finding ways to cut corners and save money. For these purists even pellet hops are too much processing and only whole flower hops should be used. In a hypocritical move, the same brewer may also be adding anti-foam or mash pH stabilizer during the brewing process.

Hop extract is a processed form of hops. The two types that are allowed in German brewing are CO2 and Ethanol extracts. Because they use fermentation CO2 or ethanol, respectively, they comply with the Reinheitsgebot (purity law). Due to the extraction of chlorophyll, Ethanol hop extracts have a green color. CO2 extracts, on the other hand, appear yellow and seem to be more commonly in use. Both types of extracts contain the hop’s resins and aromatic oils.

Hop extract in a can

Hop extract in a can

One of the major benefit of hop extract to the home brewer (and commercial brewer) is the lack of vegetative matter. This means more alpha acids can be added to the boil without increasing hop trub. The other benefit of it is the reduction of polyphenols extracted from hop vegetative matter. According to Narziss/Back (German brewing authors) this leads to a cleaner bitterness. CO2 extracts don’t contain any hop polyhenols while Ethanol extracts contain some.

I got my hop extract as a 150 g alpha-acid can for $15 through a club bulk buy from North Country Malt.  I only knew the amount of alpha acid in the can and the alpha acid percentage of the extract had to be calculated based on the weight of the extract. In the end I determined that there are 54% alpha acid in the extract. Copying Northern Brewer’s Hop shot, I ordered 25 10 ml syringes to store the hop extract. The syringes were filled with hop extract heated in a water bath.

When using hop extract I squirt it onto a piece of aluminum foil while weighing it. The hop extract is then added to the wort at start of the boil. It slides right of the foil when held into hot wort. It has been my experience that the extract’s bittering “power” is not as much as pellet hops. That might be a result of the smoother bitterness or the fact that it doesn’t mix as well with the wort as pellet hops do.  After chilling I can see a lot of resin beads on the side of the kettle. Because of this I use bout 10-20% more alpha acid with hop extract than I would use with pellet hops. Your mileage may vary and you’ll have to experiment.

If you don’t want to buy a can of hop extract yourself, you can try Northern Brewer’s Hop Shot. They appear to be the only home brew store that sells hop extract.

CO2 hop extract

Facelift

I decided to give the blog a face lift and go with a simpler looking theme. I’ll have to spend some additional time to restore the links I list. Switching back to the old theme didn’t restore them either.

Yeast pitching by weight

For a while now I have been tracking yeast propagation data in a big spread sheet. But not every yeast propagation gets logged in there since I don’t always bother about taking cell counts.

However, I do have a few data points that are of interest when it comes to pitching yeast by weight and calculating starter volumes. The latter I’ll save for another day.

The idea of pitching by weight relies on having reasonably accurate idea of the cell count per gram of yeast slurry. With this the cell count can be estimated from the yeast slurry’s weight. Weighing of propagated yeast is simple if you know the weight of the propagation vessel (for most brewers that will be an Erlenmeyer flask). It’s a good idea to mark the empty weight on the flask. You should also note the weight of the stir bar if you are using one. You should stir your starters.
Simply decant the spent starter beer until left with yeast sediment and stir bar. This works best with strains that floculate well. Weissbier yeast, for example doesn’t settle into a dense sediment and you’ll loose some slurry during decanting or have to leave a fair amount of starter beer in the sediment.

Then weigh the flask, subtract flask and stir bar weight and you have the yeast sediment weight.

That also works for washed yeast. If you don’t wash yeast and want to use the yeast sediment from a primary, turn the carboy upside down and let the yeast drip into a sanitized cup or beaker.

But how much yeast is in that sediment?

Here is some data I have:

  • freshly propagated WLP 830 sediment contains about 4.5 Billion cells per gram. I have 4 data points ranging from 4.0 to 4.9 B/g. My starters have very little trub since I use leftover wort that has already been boiled for 60+ min
  • harvested WLP 830 contains about 2.5 Billion cells per gram. I don’t get much trub into the fermenter. Virtually no hot break and maybe 50% of the cold break.
  • I have only two data points for freshly propagated WLP 833. The average is 3.8 B/g. This can safely be rounded up to 4 B/g
  • WLP 001 and WY 1272, both American Ale type yeasts, showed about 2.5 B/g in freshly propagated yeast sediment. These yeasts flocculate well and the sediment was as dense as the sediment for WLP 830/833. The yeast cells might just be a bit bigger than lager yeasts. I haven’t compared them under the microscope.
  • The only top crop data I have is for WY1272 and that showed 2.7 B/g, which is close to the freshly propagated sediment. I would expect that washed yeast sediment has about the same density.

That’s all the reliable data I have. I have used English ale yeast (WLP 002), but that one flocculates so strongly that I was not able to count yeast cells.

The little data I have suggests that clean lager yeast sediment contains almost twice the number of yeast cells than comparable ale yeast sediment and that harvested sediment has about 60% the cell count of clean sediment.

Feel free to use these numbers as guidelines and keep in mind that being off from the actual yeast count by +/- 25% should not be a big deal. Most pitching rate experiments where brewers evaluated under and over pitching where done with 0.5x, 1x and 2x the recommended pitching rate or an even greater spread.

If you have sediment density data of your own, that you are willing to share, feel free to do so.

Bottled this year’s Doppelbock tonight

Tonight I finally go around to bottling this year’s Doppelbock. I didn’t get to brew it until rather late. But what’s more important is that I started a repeat of the Doppelbock flavor and yeast experiment. Three 12 oz bottles were dosed with about 0.5 loose WLP 830 slurry. I estimate that this amounts to about 1-2 Billion cells. Three bottles are kept as control. In addition to that I purged the head space of 4 additional bottles with O2. Two of these O2 bottles also got 0.5 ml yeast sediment. The idea is that if the desired Doppelbock flavor is caused by oxidation, it should appear sooner in the bottled with O2 but no yeast.

Now I have to wait. I think I’ll crack the first bottles in about 3 months. The beer tastes good now. It’s clean and higher alcohols are low. But it is still missing this deep “dark malt” flavor.

I encurage others to try a similar experiment when bottling a beer, that’s to be aged, from a keg.

Five Things That Made Brewing Easier For Me

Most experienced brewers will have a list of things that made their brewing easier. And this list will not only change from brewer to brewer, brewers will also disagree on the value of certain techniques. Here is my list in no particular order. Feel free to disagree:

(1) Thinner Mashes:

Through German brewing texts I was introduced to thin mashes very early on. From a practical point they are easier to stir and do a better job at holding their temperature. From a beer quality point they force the brewer to less sparging and thus less extraction of unwanted husk compounds. And from an efficiency point of view they convert faster and more complete than thick mashes. I really miss the latter when I brew my Doppelbock, which needs a thicker mash due to the higher gravity and my limited mash-tun space

(2) settling trub before racking

The earliest problem I had in brewing was trub clogging the screen of the plastic funnel I was using to fill wort into the carboy.I was about to invest into a large conical screen from a restaurant supply house until I found the whirlpool technique and let the trub settle before racking. This was a big step towards more enjoyable brew days. Nowadays I don’t whirlpool anymore but let the trub settle while the kettle sits in an icebath in a large tub. The ice bath chills the wort to pitching temp after I brought it into the mid 20’s (Celsius) which is around 80 in Fahrenheit.  The tub and the kettle are elevated so that I can siphon the cooled wort directly into the carboy.

(3) Brew More Than Needed And Save The Rest For Starters

This goes along with letting the trub settle. In order to not pick up trub during racking about 2-4 qt of wort need to be left behind in the kettle. In my brewing this wort does not go to waste. I’ll filter this sludge overnight through a paper towel set on a screen in a large funnel. The next morning the clear wort is filled into 2 l soda bottles and stored in the freezer. When I need starter wort, and I always need starter wort, I thaw and boil to frozen wort. I haven’t bought DME in years. Another advantage of brewer’s wort for starters is that it has already been boiled and doesn’t boil over as easily or create a lot of hot break.

(4) track pitched yeast amounts by weight

One part of consistent brewing is pitching consistent amounts of yeast. For a while I determined the yeast amount by settling yeast in a graduated cylinder. But that requires a number of yeast settling steps if the cylinder holds only 250 ml but the propagation volume was 2000 ml or even larger. In a forum discussion I was pointed to weighing the yeast sediment instead. So I noted the empty weight on all my flasks and after propagating the yeast I simply settle the yeast in the fridge and then decant the spent starter wort. The flask with yeast sediment it weighed and its empty weight plus stir bar weight is subtracted. From cell counts I know that there are about 4.5 Billion cells per gram of freshly propagated yeast sediment, at least for my most favorite lager yeast (WLP 830).

(5) siphon from keg to keg under pressure.

When I started out performing secondary fermentation and cold conditioning in kegs, transferring carbonated beer from one keg to another was a chore. I like to carbonate beer during secondary fermentation or cold conditioning b/c the beer is ready to drink once that process is done. The problem of transferring carbonated beer is that it has to happen under pressure. Initially I was doing this with a bleeder valve on the destination keg with which I would regulate the pressure in that keg while the beer is pushed in by pressure applied to the source keg. But with this set-up you cannot leave or must not forget amount it otherwise foam will come out of the bleeder valve when done. Then I found a post in a German home brewing forum that mentioned how to siphon beer under pressure: place the source keg higher than the destination keg. Connect both keg’s CO2 outlets with a CO2 line. Then connect the beer out lines with a beer line. Now vent the destination keg a little to get the siphon started (the CO2 line may have to be disconnected briefly). Since this is a simple siphon, that will stop when all beer has been transferred, you may leave and check back in 30 min. 30 min during which I can do something else and don’t have to worry about foam being spewed across the basement floor.

patent pending brewing process for Infinium

Infinium is collaboration between the Boston Beer Company and Weihenstephan brewery in Freising Germany. I had the beer once at a club meeting and didn’t think it was all that special. What interested me more, though, was the novel brewing process for which Boston Beer filed a patent application.

The problem in making a light high alcohol beer that complies with the German Purity Law (Reinheitsgebot) is that it has to be made from all malt and can’t use sugar to increase alcohol without adding body. Normal brewing procedures, even if aimed at high fermebtability, leave too much dextrins which would add too much body to the beer.

After not being able to find the patent or application on my own I asked around in my brewing club, with success:

United States Patent Application 20110274785 – METHOD AND SYSTEM FOR PRODUCING A MALT BEVERAGE HAVING A HIGH DEGREE OF FERMENTATION

The wort is brewed with a stepped mash that isn’t out of the ordinary. The patent application mentions long (150 min) rest at 63 C before a mash -out at 72 C is done. This will already produce a highly fermentable wort. Key to even higher fermentability, however, is the preparation of an enzyme rich solution from what they call “green” malt.  “green” malt, also known as air-malt is malt that has been germinated and dried, but not kilned. The absence of high temperatures ensures that it is extremely rich in enzymes.

The enzyme rich extract is prepared my mashing the green malt at 57 C to preserve limit detxtrinase and b-amylase. The temperature is high enough to kill most micro organisms. The resulting supernatant can be added to the 65 C first wort in the kettle where the enzymes are able to continue to break down dextrines that were not broken down in the mash.

This by itself does not yet lead to a satisfactory fermentability and 24 hrs after the addition of the yeast another green malt mash is prepared. This time the supernatant is added to the fermenting beer where it is able to break down more residual dextrines w/o the enzyme life being cut short by the head of a mash or a boil. The patent appilication claims that all these processes can lead to a beer with an attenuation of 96%.

While this process seems fairly ingenious, a number of similar patents have already been issued. A simple patent search for “low calorie beer”, the main application for enzyme additions in brewing, lead me to this old Anheuser Bush patent:

United States Patent 4,272,552 – Process for producing a low carbohydrate, low calorie beer

It also talks about adding the enzyme rich supernatant from a secondary mash to fermenting beer in oder to lower residual extract or boost fermentation. The key difference to Boston Beer’s patent application is the use of green malt by Boston Beer. But I don’t think that this is all that crucial and that an Infinium like beer can easily be brewed without green malt.

In addition to that at what drying temperature is malt no more “green” malt but kilned malt?