Optimizing Yeast Starters and Large-Scale Yeast Production

I received a question on my youtube channel from a viewer, asking for methods to step-up a yeast stored on a slant to a culture large enough for a 1 BBL (~150 L) batch of beer. I have done this a few times myself. In fact, I’ve provided local breweries with some of the rarer yeasts in my yeast bank. This is also a capacity a commercial brewery could easily add with only a minimal investment in equipment. With the caveat that it takes a lot of time and effort to produce yeast in these quantities. Even if you don’t need industrial quantities of yeast, this information may still be of interest. Optimizing yeast starters can improve the yield and vitality of yeast produced in your starters. At home, a few simple tweaks can improve your yeast yields in homebrew-scale yeast starters by 20-30%.


What About Fed-Batch?

The first part of the viewers question was whether he could use continuous aeration to promote aerobic growth. Aerobic growth produces 16-18 times the energy of fermentation, and correspondingly, greatly increases yeast yields per gram of sugar consumed. Sounds like a winner – but it won’t work. Simply pumping in air won’t give you growth much above what you would see with a conventional starter due to the “Crabtree effect”.

When yeast are exposed to sugar concentrations above 150 mg/L yeast will ferment the sugars, even if oxygen is present in large amounts. That is equivalent to a specific gravity of only 1.0011! This is an evolutionary approach used by yeast to “steal” sugars and convert them into ethanol, when sugars are plentiful. Fermentation is much faster than aerobic metabolism, allowing the yeast to eat all the sugar and to make ethanol to kill off competing organisms. The yeast are able to later metabolize the ethanol for additional energy. So continual aeration of a normal starter will not have much of an additive effect compared to conventional stirring.

There is a way to force yeast to grow aerobically, called fed-batch culture. In fed-batch you slowly provide the sugar to the starter. You need to provide the sugar at the same rate it is consumed, to keep sugar levels low enough to avoid the crabtree effect. As you can imagine, such a system is not really possible outside of a lab or commercial production facility. Moreover, the crabtree effect is a “genetic program” in yeast that is distinct from the genetic profile of fermentation. Yeast made by batch-fed tend to have long lag phases when pitched, as they need to “rewire” their gene expression profile for fermentation. So the benefits of high growth are countered by the yeast not being in a fermentation-ready state.


Optimizing Yeast Starters

So what can we do to optimize yeast starters? The main thing is to manage nitrogen and temperature – two factors which greatly contribute to growth during fermentation.

Oxygen

First up though, this does not mean that oxygenation is not important. You still need oxygenation for production of sterols and unsaturated fatty acids. Continuous aeration or stirring is best.

Temperature

The single best thing you can do to maximize yields is to manage the temperature of your starters. Ale yeast grow best at temperatures of 30 to 32C, with temperatures above 36C beginning to kill the yeast. You can nearly double your yields by growing yeast at 30C, compared to growing them at room temperature (20C).

This temperature is not universal. Some lager strains will start to die at temperatures of just 28C, so optimum starter temperatures for these yeasts are closer to 24-25C. This is still far warmer than conventional lager temperatures (~10C), but is below the ale yeast optimum. Optimum temperatures are not as well established for non-conventional yeasts such as Brettanomyces. At least one study found 30C to be optimal for Brettanomyces bruxellensis. This suggests that Brettanomyces responds to temperatures similarly to ale yeast. It may be a good idea to be more conservative and to use a temperature of no more than 28C.

Nitrogen

Increasing nitrogen content has a dramatic impact on yeast growth, as nitrogen (which is used to make DNA and proteins) is often a limiting nutrient for yeast growth. A recent study found that using low-sugar (~1.008) wort with high nitrogen (~30g yeast extract [essentially Fermaid-O] per liter of wort) produced nearly 50% more yeast than the same gravity wort with minimal additional nitrogen added. For more details, see the episode 62 of the Bru-Lab Podcast.

At home (or in the brewery) this isn’t really a practical option, as yeast extract is not exactly cheap. But what this paper does show to us is that increasing nitrogen content will have a positive effect on yields. I add ~5 g/L yeast extract to my starters, at a gravity of 1.040, and see a yield increase of ~10%. Even just DAP, at 5X (or more) the manufacture’s recommended dose, can increase yields. If you do this in the final stage before pitching the yeast, be sure to remove as much of the wort as possible. Yeast can make some pretty unpleasant compounds under high nitrogen conditions.

Gravity

More food equals more growth, right? It works for us humans (as my waistline will attest), but does it work with yeast? At lower gravity things work as you’d expect – doubling gravity from 1.010 to 1.020 will roughly double yeast production. This improvement in yields continues up to gravities of 1.036 to 1.040. But above this we start to see diminishing returns. You will get more yeast at 1.050 than 1.040. But you’d get more yeast if you diluted that 1.050 wort to 1.040, and grew yeast in the larger volume.


Large-Scale Production at Home

So how can the homebrewer (or small commercial operation) produce enough yeast to pitch into 1 BBL or more of a moderate-gravity ale without needing a dedicated yeast-production plant? I’ve done this myself a few times for local breweries, and it isn’t too hard. As always, sterility and proper aseptic techniques are critical for limiting contamination.

Optimizing Yeast Starters will Maximize Yeast Yield (diagram of yeast cell division)
Optimizing Yeast Starters will Maximize Yeast Yield

I generally make these larger amounts of yeast, starting with a frozen culture. For all steps, other than the last, I typically use wort made from DME, 1.040 gravity, plus 5 g/L yeast extract or DAP. I pressure-cook the smaller starters to ensure sterility. The larger ones I boil for 12 minutes, as my 3L flask doesn’t fit into my pressure cooker.

Step 1

I start a small (5 to 10 mL) culture from my frozen stock. I add single microbiological loops worth of frozen yeast to start the culture. More than this can inhibit growth due to excess glycerol. I first incubate 25C (for ales) or 20C (for lagers) to allow the yeast to recover from being frozen. After 21 hours this I increase the temperature to 30C (for ales) or 25C (for lagers) to maximize growth. For oxygenation, I cap and shake the tube several times a day. Ideally, some sort of orbital shaker or rotary culture mixer would be used to provide continual aeration.

Step 2

Once the 5-10 mL culture has finished growing, I pitch the entire volume into a 200 to 250 mL (~1 cup) starter. I prepare this in a 0.5 L flask, as it fits in an instapot for sterilization. This starter is stir continually with a stir bar, on a stir plate. To maintain temperatures I place my stirplate and starter into a cardboard box equipped with a USB-powered “personal heater” controlled by an Inkbird temperature controller. I do not start at a lower fermentation temperature, and instead, the starter is maintained at its ideal temperature throughout. Again, that is 30C for ales and 25C for lagers. If you use a similar heating setup, tape the temperature probe to the flask. The starter can get warmer than the air due to heat from fermentation and the stirplate.

Step 3

I next pour the culture from step 2 into a 2L starter, again stirring the starter for aeration. As above, I use a temperature controller and heater in a cardboard box to maintain temperature. This produces enough yeast for a 20L/5 gallon batch of modest-gravity (upto 1.060) beer.

Step 4

This is where you have options. When I’ve produced yeast for a brewery, I simply brewed a 20L batch of ~1.040 gravity ale that I oxygenated well and fermented with the yeast. This will produce enough yeast for upto ~200 L (~1.25 BBL) of 1.060 gravity beer. It will be in a healthy state, and can be repitched multiple times by the recipient brewery.

To maximize yield and health at this stage you will not want to make beer, and instead you will want to make a 20L starter. Same starting gravity and nitrogen dosing as above, and same optimal temperature. The harder issue will be keeping yeast in suspension and well oxygenated. It is unlikely you’ll be able to do use with a stir plate. Instead, you will need some sort of a continuous aeration system. I’ve never done this, so I can only offer vague suggestions.

Off the top of my head, you’ll need a stainless steel or ceramic airstone and silicone tubing – this way you can sterilize the oxygenation rig in a pressure cooker. You’ll also need a bung or manifold to attach the airstone/tubing to your fermenter and a source of sterile-filtered air. I suspect that if you have the airstone at the bottom of the fermenter, and a high enough flow rate, the air will keep the yeast suspended – but some sort of additional stirring may be required. I imagine that some sort of foam control (e.g. Fermcap-S) will also be required.

6 thoughts on “Optimizing Yeast Starters and Large-Scale Yeast Production

  • September 27, 2022 at 2:37 am
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    Hi Bryan,
    In this article you mention: ” I add single microbiological loops worth of frozen yeast to start the culture. More than this can inhibit growth due to excess glycerol.”
    I use to freeze yeast in half filled 15ml test tubes with glycerol concentrations you suggest in your yeast freezing article (20%).
    I put the entire tube in a 500ml starter, then decant and add the slurry to a 1.5L starter.
    I always thought that in this process the glycerol could aid in yeast membrane formation. Was I wrong? Can I be putting stress on yeast this way?
    Thanks, Luca

    Reply
    • September 27, 2022 at 7:26 am
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      The answer is “its complicated”. Some non-saccharomyces yeasts can metabolize glycerol very efficiently, and use it both as an energy source and as a basis for lipid synthesis. While S. cerevisiae has the genes for this, its ability to utilize glycerol is limited. The main reason for this is where and why brewers yeasts use glycerol. Under oxidative conditions glycerol is used by brewers yeast to maintain redox balance between mitochondria and the cytosol, and it is under these conditions that the glycerol can be metabolized to make lipids or to produce energy. But, because brewers yeasts are crabtree-positive, this pathway is unused as the yeast suppress oxidative metabolism (e.g. mitochondrial function) and engage in fermentation when sugars are present – even if lots of oxygen is around. Under fermentative conditions there is no need to balance redox potentials between mitochondrial and the cytosol, and as such, the lipid synthesis and energy generation pathways are largely shut down.

      Glycerol does have a role during fermentation, and this is where the issue with excess glycerol in media can become a problem. Glycerol is the only osmotically active molecule used by yeasts to control their osmolarity (water balance). Glycerol is typically synthesized and secreted during fermentation to help the cell resist the osmolarity changes which occur as sugars are fermented to alcohol. High levels of glycerol in the medium can impair this process, making yeast more susceptible to osmotic stress – especially in the early stages of fermentation.

      Your process is likely okay as you are decanting, so while there would be some inhibition of growth during the early stages, this would be eliminated once you decant and transfer to a fresh starter. In the lab we will typically use either a loopful of yeast to initiate a small starter, or we will centrifuge the yeast and remove the medium before suspending the yeast in fresh growth medium.

      Reply
  • September 20, 2022 at 11:35 pm
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    Hi Bryan,

    Thanks for this post. I have just embarked on a yeast isolation/propagation journey, so this comes very timely. What I’m curious about is in your given growth medium at the given temperatures and aeration regimes, how long does it take on average for your 10ml inoculum to ‘finish growing’, and then when you pitch that into 250ml of your propagation medium, how long does this take to finish. I generally brew in 4L experimental batches, but for the benefit of others who brew in larger volumes and also for future me: How long are the times for larger sized starter batches?

    I’m sure this will be individual for each strain, but given my currently available equipment, it is much easier for me to measure time and temperature than it is to measure cell count. Do you have a ballpark number for Ale vs Lager yeasts, and/or is there any particular yeast in your collection that stands out as it is much faster/slower?

    Thank you!

    Reply
    • September 21, 2022 at 7:07 am
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      The first 10mL tube often takes 5 or so days to grow completely – the cells are shocked from being frozen, and can be quite slow to start growing. Once growing, they will typically finish in 24 hours. After that first “step”, the rest usually complete in 24 hours – e.g. when you add the tube of yeast to the 200mL starter, that 200mL starter is done a day later. You add that to a 2L starter, and that 2L starter will be done the next day. Lager strains may be closer to 36 hours/step.

      Reply
  • September 20, 2022 at 11:07 pm
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    Thanks for the post, Sui! I’m the viewer haha. I think I’ll end up doing just what you suggest.

    What I plan to do for the last step is use a corny keg with an additional ball lock post on the lid. Use its gas out as a blow off/airlock, liquid out to take samples, and the additional port to a silicone tube with an aereation stone that goes to the bottom of the keg. Aereate through a sterile filter with an aquarium pump connected to a smart plug. The smart plug will be programmed to turn on for a minute every 15 minutes during the first 24 hours, and for one minute every five minutes after the first 24 hours (per Kunze 4.2.3). I might also give it a nice shake every time i pass by. Probably going to be less than 5 gals to leave some headspace in the corny, I’ll count my cells at least the first time to verify if it’s enough to pitch my bbl. My only worry is that I can’t sterilize the corny. What do you think about this plan?

    Reply
    • September 21, 2022 at 7:05 am
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      Seems reasonable to me, although an anti-foam will be critical or all your starter will come out of the gas-out!

      Reply

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