Isolated Yeast (Tree House): How to Identify and Characterize

isomerization

Long time lurker, finally decided to be productive and join the community.
So I am a biochemist/structural biologist (bacterial pathogenesis) and I love making/drinking IPAs (homebrewing for 2+ years) of all kinds.
I was drinking a Tree House Julius with a buddy of mine and we decided that we'd figure out what kind of yeast they use. So I harvested the dregs, got them growing and then plated out serial dilutions for isolation (single plate shown):

wqrfo2ecybj.jpg

Using a PCR method (https://suigenerisbrewing.blogspot....howComment=1490284392639#c8540474511060838192), I was able to amplify the ITS region (http://sites.biology.duke.edu/fungi/mycolab/primers.htm) of this yeast (my band is ~800 bp):

0se4xx2kyav.jpg

I then sequenced the amplicon, and used BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search for similar deposited sequences. I think this is where I've run into a wall, as the best hits I get back are only ~89% identical (they are S. cerevisiae). Last year White Labs (and others) published a really cool paper (http://www.cell.com/action/showMethods?pii=S0092-8674(16)31071-6) where they whole genome sequenced 157 S. cerevisiae yeasts. Those sequences are deposited, but I think my BLAST search is missing them for some reason.
Anyways, I am moving on the phenotype side of characterizing this yeast. Can anyone suggest experiments (lab side or fermentation) to figure more out about this isolate? I have a session NE IPA planned in the next couple weeks and can update more once that's done.

xerotime

I've been considered that the Treehouse yeast may be a blend of 2 different strains. Could that be why you are running into trouble identifying? It seems odd with all of these newer small yeast labs (Giga, Yeast Bay, etc.) that no one has grown it up for sale like what has been done with Conan.

isomerization

My only hold up with considering that they use a blend of yeast for fermentation is that would (seemingly) be really difficult to maintain over time. I'd expect growth rates to throw off the ratios over time and result in different fermentation/flavor/etc profiles. Something that is more likely (in my mind) is that they are using a genetic cross of two strains, something like the Conan x WLP644 strains (discussed here https://www.homebrewtalk.com/showthread.php?t=577066).
That doesn't explain why, as you mentioned, there haven't been any attempts to sell this strain by a commercial yeast lab. I've seen several mentions on HBT and other blog sites of people cultivating yeast from TH cans. I didn't test individual yeast colonies for genetic diversity though.
My issue with identification is that I don't have access to the right database (I think). I've contacted some companies that do contract work for professional brewers, but the fees aren't worth the information ($200). While I hate waiting for information, the best course in this case is to do some split batches and compare the yeast I harvested against WY1318, Conan and the like.

TheHairyHop

I don't think it's a hybrid. Treehouse started from really humble beginnings and they've been turning out the same beer, really. I think, if it would be a guarantee, you could find enough people to chip in for that $200. I would certainly be one of them

suregork

Unfortunately you won't be able to do any strain-level identification with the ITS sequence; it is only good for species-level identification. If you are willing to share the sequence, I have a local BLAST database with the 157 whole genome sequences from the Cell paper you linked to, and could search it with your sequence. But as I mentioned, I would not trust strain-level identification based on the ITS sequence (little variation between strains and more importantly it is only a small single region in the genome. E.g. of the strains in the Cell paper, many have identical ITS sequences, but belong to different clades).
You could instead try interdelta primers or RAPD primers:
http://onlinelibrary.wiley.com/doi/10.1016/S0378-1097(03)00205-2/full
You could then compare the electrophoresis band profile of your isolate with those you obtain from some different control strains (e.g. WLP001, WY1318, Conan, etc.). That should give you better idea of what its closest relatives are. If possible, include as many control strains as you can.

Sbe2

This is why I love HBT, structural biologist using their knowledge and sharing it with the rest of us minions.

isomerization

I was actually just going to email/PM you about this after reading through your blog post linked to the WLP644 x Conan thread.
I have the delta12 and delta 21 primers ordered, should arrive tomorrow. I have the following strains to test using this PCR method:
WY1056
WY1272
WY1318
WY1332
WY1968
WY3944
WLP802
Yeast Bay Vermont Ale
WLP670 (isolated each strain, one Saison and one Brett)
Julius isolate
If I remember correctly from the paper you cited, nothing should be amplified with the Brett strain, correct?
Once I have the gel image, would you be interested in helping interpret the results Suregork?

suregork

I'd be happy to help! I recommend using a higher agarose concentration (e.g. 2.5%) to help separate the smaller bands.

TheHairyHop

I will be watching this thread in anticipation! Be sure to let us know the results

glugglug

I feel like I'm at the opera. I don't understand any of this, but I love it!!!

Cainmos

As an avid fan of Tree House and IPAs in general I approve of this work! Interested to see the results.

5mooth0perator

I can't help with the BLAST results without the sequence or parameters, but I would recommend trying BLAT, by UCSC. I think they have a microbial version.
There is also
http://www.yeastgenome.org/blast-sgd
Looks like they use wublast.

isomerization

As I am now aware of, the stretch of DNA I originally amplified and sequenced is only reliable for identification across, and not within, species of Sacc. I am working toward a new technique (for me) of DNA fingerprinting (see posts by suregork). Hopefully should have something in the next week or so to analyze.
With that said, I went back and looked at the DNA chromatogram a bit closer, and there is a suspicious amount of overlapping peaks. Normally this would imply heterogeneity in the sample that was sequenced, but I'm honestly not sure at this point. I had a clear single band by PCR (image in first post). I'm a bacteria guy, not a yeast biologist, so again not sure why that happened.
I will post the DNA sequence when I'm back at work tomorrow, and can send the DNA peak file to anyone that wants to look at it.

isomerization

UPDATE:
10 strains of Sacc have been struck out:

xb14ebmh0u4.jpg

I then selected a single colony from each plate, grew the yeast in YPD for 24 hr at 30C and harvested the genomic DNA. Spectroscopic analysis indicated it was relatively pure and free from protein contamination:

ubczbgeicj0.jpg

Next week I will start on the DNA fingerprinting method and report back.

isomerization

I must admit, I almost gave up getting the PCR to work, but today was a good day (it is Friday after all)!
I stumbled across this technique in a paper (https://link.springer.com/article/10.1007/s10295-010-0837-z) as I was troubleshooting, instead of trying to isolate the DNA from a yeast culture, all you need to do is:
1.) pick a single colony (I suspect you could get away with a tiny amount of yeast from a liquid culture as well)
2.) drop it into sterile water in an eppendorf tube, close cap
3.) microwave on high for 5 min
4.) use 1 ul of this mixture in a PCR

o1s0khxndm3.jpg

Strain key is as follows:
1 - WY1056
2 - WY1272
3 - WY1332
4 - WY1318 (this culture did not like YPD media very well for some reason)
5 - WY1968
6 - WLP670 (saison isolate)
7 - WLP802
8 - Julius isolate
9 - The YeastBay Vermont Ale
10 - WY3944
Suregork, I am working on improving the quality of this image. Using less DNA dye (white bands across top and bottom of gel), more sample (15 ul here, but can load up to 50 ul on a larger gel), getting better bands for lanes 2 and 3, etc.
The only thing that really jumped out at me is WY1968 and Vermont Ale (aka Conan) have almost identical banding patterns! Any thoughts with the current image?

isomerization

I forgot to add that the Julius isolate is certainly unique via this DNA fingerprinting method!
So, what this really means is that I need more yeast samples to analyze, I'm looking into figuring out an easy way to mail samples through the postal service (only costs a stamp!), if anyone has suggestions and is interested in sending yeast that is not on my current image, please let me know!!

gromitdj

Not that I have tried it, but I've read the following on the Sui Generis Blog about mailing yeast samples.
Mailing Yeast
New Mailer System

Redtab78

wow, the amount of science in this amazes me!

TheHairyHop

Subbed! Always loved the biological aspect of brewing moreso than the chemistry or engineering that seems to encompass almost all homebrewing. Can't wait to see what you find out

isomerization

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suregork

I should have thought of that since I enjoy his blog so much!
If anyone is interested (and capable), I am more than willing to mail you autoclaved blotting paper for you to spot down yeast and mail back to me. Send me a PM if you have multiple strains not on the list above and are interested in swapping. I'm happy to send what I have access to as well.

isomerization

Will have a closer look at this tomorrow, but based on a quick glance it seems like your isolate is distinct from the other strains you've included and WY1056 is the closest relative. Interesting yes that the Vermont Ale and WY1968 seem closely related  

isomerization

First test using the isolated Julius yeast is done. Same recipes were used (albeit one week apart, so caveat there for sensory analysis below) and one batch was fermented with WY1318 while the other with the Julius isolate described here.

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Left is WY1318 and Right is Julius (poured from growlers that were filled earlier in the day, otherwise they have similar head retention). I stupidly didn't fill the glasses up evenly, but the color and haze appear pretty much identical to my eye. Both beers had the same OG (1.058) and almost the same FG (1.015 and 1.014 for WY1318 and Julius isolate, respectively. Beers were mashed at 156 F. If I mash lower (151 F) for WY1318, it usually drys out too much (1.010-1.012 range) for my tastes with this recipe (h/t Braufessor).
I'm terrible with flavor descriptors, but I'd say the biggest differences between the two beers were in the mouthfeel and hop burn, with the Julius isolate "beating" WY1318 in both categories. I could not detect any real flavor differences, but the typical stone fruit notes were there in both. I have tried this recipe with Conan (Yeast Bay) and preferred WY1318 in a head to head there, fyi.
Combine these observations with the fact that it doesn't gunk up my fermentor/hop bags like WY1318 (damn you crop topper) and I'm definitely going to use it again.

OdeCloner

Thanks! I appreciate your input greatly.
I have a new gel that I think provides (the potential for) more insight. I used two different primer pairs that have been reported in the literature, delta12-delta2 (left gel) and delta12-delta21 (right gel):

ixgiyjjmhmg.jpg

The strains are the same order as before with the addition of an 11th yeast (harvested from Tree House Double Shot).

Horseflesh

Subbed. Keep it up!

mikeroesoft

This is cool. I did biochem back in university, though I never worked in the field. Man, prepping an acrylamide gel was such a PITA back then. I hope it's easier these days.
Looking forward to more!

Biobrewer

I have to say this is one of the coolest threads I've seen on HBT. Awesome.

isomerization

Unsurprised that Vermont Ale resembles WY1968 on the level of DNA sequence as the source material we used to isolate Vermont Ale from uses a strain that was rumored to originally have British origins. I'd say that's where the similarities start and end! WY1968 is a lower attenuator, high flocculator, produces decent amounts of diacetyl and has a fruity but restrained ester profile. Vermont Ale is a higher attenuator (typically around 80%), low flocculator, does not produce diacetyl in any appreciable quantities and has a massive ester profile of peaches/apricots.

Biobrewer

Thanks for commenting! I do think it's a bit of an over generalization to just say both came from the U.K., so they're similar but the point is taken. I'd like to analyze other British strains if I can get my hands on some. It's still pretty interesting (to me at least) that they are so different phenotypically yet seem to share a lot of DNA similarity.
Since you'd be precisely to person to ask, why haven't we seen a commercially offered isolate from Tree House products? The yeast looks to be different than known NE IPA yeasts and performed favorably (n=1). I realize you may not want (or be allowed) to answer that, but I figured I'd ask anyways.

isomerization

My supposition was that they may have branched off from a common lineage, more recently than not. Can't really speak to the isolation efforts of others, and I'm not saying we're isolating this strain, but we are currently in the process of isolating a new strain that is similar. Just posted about it on The Yeast Bay Facebook page:
"Everything is back in stock on the online store! Also, Brettanomyces bruxellensis - Strain TYB261 will be available to homebrewers shortly, and we'll likely be posting stock next week.
We also have yet another beta in the works that hasn't even been assigned a TYB number yet. We just streaked out the source material and are excited to get into characterizing the many isolates we'll pick. This house yeast, isolated from another brewery in the Northeastern United States, is similar to the Vermont Ale strain though the ester profile is less peach/apricot driven and more closely resembles citrus/pineapple/mango/guava. Keep your eyes peeled for updates, we're hoping to get through the isolation and characterization of this strain and make it available to commercial brewers and homebrew folks by the middle-end of May.
Cheers!"

Warthaug

Very cool! I look forward to picking up a vial.

Biobrewer

As always, I'm late to the party. I'm Bryan of Sui Generis Brewing, which has been cited here a little. Anyways, some replies to some previous posts...

89% homology is awfully low for an ITS sequence; I typically see 99-100% homology for yeast. Did you trim your sequence to remove poorly resolved bases (usually ID'd as an 'N' in the chromatagram/sequence file)? The mixed peaks may be a sign of contamination, but assuming there are two strains and both strains are S. cerevesea, it shouldn't matter as ITS sequences are pretty well conserved between species. Unfortunatly ITS size isn't a useful measure either, as pretty much any Eukaryote (including yourself) will produce an ~800bp ITS band. As Surgork already mentioned, you cannot do strain ID with ITS and need to reply on other methods. Inter-delta is the norm, but as you've already experienced, it needs to be compared to a database of other fingerprints to ID the strain.

This is a good example of why even interDelta can be of only modest value. It doesn't take much to alter ester profiles or flocculation (and therefore attenuation) characteristics of yeast - there are many examples out there of single point mutations doing just that. interDelta can let you follow the parentage of yeasts, but some strain-specific changes can be "below detection" for even this method. Full genome sequencing is about the only way to get to that level of resolution, but the cost of that is still a bit beyond what is practical for the home brewer.
Just my $0.02
Bryan

isomerization

Well said Bryan, this is useful information that's worth far more than $0.02!

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xerotime

Regarding the ITS sequencing, I think it was a problem on the sequencing end, rather than a true heterogenous population. But, as many have pointed out on this thread now, ITS sequencing wasn't going to be helpful in this situation anyways. So I dropped it and switched to interdelta PCR, which I was not aware of until I started playing around with this strain.
Personally, I think interdelta PCR was very helpful here not because it revealed shared parental lineage, but because it didn't! It appears to suggest that the yeast isolated from a Tree House can is different than any of the other traditionally used (by homebrewers at least) NE IPA yeasts. I think that's pretty cool!
Phenotypic characterization is more important for us homebrewers anyways, but I think its still cool to ask and try to answer these types of questions.

isomerization

I'd really like to see how Fermentis S-04 and S-05 compare. If you want to PM me your address, I'll just order a pack of each and have them shipped to you.

5mooth0perator

PM sent. I also picked up T-58, S-23, W-34/70 and Danstar Munich dry yeasts to look at as well ($6 including shipping for all 4 since they're old).

Redlantern

Single point mutations can be huge, just look at teosinte vs corn, it only took like four mutations, but you wouldn't recognize thm as the same species. Same for grapes, people did some sequencing and it turns out many of the French grapes are literally siblings.
I think it helps though to know when you have essentially the same strain producing different flavors vs a strain that is close to a different species.

trav77

This has got to be the most interesting conversation despite 80% of it going straight over my head.
I am glad there are guys like you out there so I can brew.

trav77RevKev

It took only a small number of mutations to get rid of the rock-hard glume in teosinte and make it edible. After that there was an incredible amount of de novo variation and selection by humans to get to what is now corn.
But yes, a single point mutation can be huge in certain cases.

isomerization

Kind of late to the game here but have you looked into doing a genome wide SNP fingerprint? Pretty cheap these days. I haven't done much work with yeast but it's a model organism so no doubt there are vast resources out there to do this type of work. That would let you do a phylogenetic analysis and would give a better representation of relatedness than a handful of loci via PCR. Just my 2c. Cool project! Just went to a seminar about a month back in which the researcher at my university used qPCR to quantify bacterium and is working on viral applications. The work was in the field of environmental chemistry classifying different strains of bacteria that are markers for whether water pollution at beaches was at unsafe levels. Interesting stuff.
And props to you for using your knowledge base for brewing science, something that doesn't get enough exposure to the beer community. Me being an analytical chemist I grasp methods but I am likely unhelpful unless you need work done on mass spectrometers.. I have access to a wide variety (ICP, GC, LC, FTICR and Orbitrap).
I'll stay tuned as the bioanalytical work is a guilty pleasure of mine.

isomerization

The Siebel Institute offers these services (as I'm sure other companies do as well). I had emailed with them about a different (presumably less expensive) service and that was ~$200, so I don't think genome wide sequencing is quite at the level of homebrewer access yet!

isomerization

I would think that would be a very helpful experimental technique for homebrewers! I know it has been extensively applied to the study of hop oil compounds in beer and I'm sure there are other applications (yeast identification, other metabolites in finished beer, etc.) that it would be useful for as well.
Just wanted to add that Scott Janish's blogsite (http://scottjanish.com/increasing-bitterness-dry-hopping/) has some great reading if you're interested in the analytical side of homebrewing.

RevKev

***DNA fingerprinting update***

c2w0muxae4f.jpg

New strains analyzed in the right gel (old gel on the left for comparison purposes). Strain 1 (WY1056) was used as a control of sorts.
New strains (all are dry yeasts):
12: S-04
13: S-05
14: S-23
15: Danstar Munich
16: W-34/70
17: T-58
Nice to see S-05 and WY1056 have a very similar banding pattern, although this just further reinforces the previous commentary that similar strains at the genetic level can produce differences at the phenotypic level (e.g. S-05 krausen behavior v. WY1056).
S-23 and W-34/70 have very similar banding patterns as well. I haven't used either yeast, so can't say much there...
***REQUEST***
I am interested in testing more yeast strains, and would be willing to mail autoclaved blotting paper foil packets (see here: http://suigenerisbrewing.blogspot.com/2017/03/new-mailer-system.html) to those with yeast strains I have not tested yet. Shoot me a PM if interested.

TheHairyHop

Yes I think I have stumbled upon that! I've been thinking about doing headspace injections with GC-MS to identify aromatic and volatile oils for SMaSH beers.. if I pay for the instrument time. Metabolomics is my field and I actually am working on 'leaf spray' ionization with HR-MS. I have some whole cone hops that could be cool to analyze and I bet that I could talk my PI into a little side study.. I mean for the love of beer!! (He's a big fan of craft also)
I'll have to post back when it comes summer time and I'll have some free time.. and thanks for the link.
I may have some Belle Saison and some wild fellers in the coming months

isomerization

It's hard to tell with those images, but is that T-58 close to the Tree House isolate? I've noticed that Tree House yeast starters smell very Belgian...

TheHairyHop

I wonder how WLP007 compares

phillip092

Wow, nice catch! I wasn't even thinking about that as a possibility, but I re-ran the samples side-by-side (Julius on the left, T-58 on the right):

alpequhodzn.jpg

There are a couple small differences, the main one just above the smallest ladder band (100 bp) in the T-58 sample, but wow, pretty darn close. It doesn't seem likely that they are adding yeast at canning right?

isomerization

I've suspected TH is using something (at least partially) Belgiany based on the esters I get in starters, and the well-noted "bubblegum" character of fresh TH. Perhaps a blend including T-58 fermented at lower temps? That said, I would not be at all shocked if they mix in other strains at canning to protect their IP--assuming it could be done in a way not to affect the quality of the beer inside. I've seen others claim an isoamyl-y banana flavor in their harvested yeasts.
Forgive my ignorance... Does the Julius sequence show any indication of being multiple strains, or is that not possible to tell with the PCR? What do you make of the differences between the Julius and Double Shot sequences? Thanks so much!

RevKev

T-58 is known for staying powdery in suspension, right? They could be using it to up the haze around canning time... maybe?

isomerization

This was my speculation as well, I cultured yeast from a can of haze, brewed two batches with it at the same time/temp, the only difference was I accidentally under pitched one of the two batches. The underpitched batch was supposed to be an IPA with a TON of hops, I couldn't taste anything but earthy/clove like belgian esters. The other one had hints of the same esters but they weren't nearly as prominent as the under pitched batch. I've since used it again, fermented on the cold side and tried to over pitch, still had hints of those same esters but turned out okay.
When I initially noticed the belgian-like esters, the first thing that came to mind was the possibility that they were adding yeast at canning, but I kinda dismissed that idea because I thought that you'd have to be really cynical to intentionally sabotage your beer in a way that could harm it, just to prevent home-brewers from culturing their yeast....especially since Nate used to be a homebrewer himself. It's definitely interesting to see other people propose that idea though.
On another note, I've recently cultured more of the yeast from a can of green, and I do not notice any of the belgian-like aroma or taste in the cultured starter. I have not used it to brew a batch yet though.

I am also curious to know this, is there any way to tell if there are two different strains of yeast? and if so, is there any way to tell them apart?

I have a few yeast that I would be willing to mail, would you be willing to test cultures from other breweries as well? Or just commercial yeast?

Apimyces

The short answer is, I was not expecting multiple strains in the can of Julius I harvested from, so I propagated in DME before streaking for isolation and genetic analyses. Next time (might be a bit since I am in Kansas!) I will streak out the dregs of a can and analyze 10-15 colonies from there. With that said, I dont think they are adding anything nefarious at canning. I am tasting a friends all-Citra APA made with this yeast tomorrow.
The DNA fingerprinting is super cheap and easy for me to do, so I am more sure than happy to test any samples sent my way!

isomerization

I've also seen that WLP 644 Sacch Trois may perform well in the New England style. Do you have access to test that strain in the next iteration?

Horseflesh

This guy has WLP644 that I will be putting in a German juice bomb of an IPA

stpug

I am hopefully acquiring 12 strains from Bryan/warthaug (http://suigenerisbrewing.blogspot.com/) through his yeast exchange program in the next month or so. WLP644 is on there!

TheHairyHop

Wait, are you saying the yeast companies rip off their customers and commercialize their private strains?

Sbe2

THE PLOT THICKENS

r0zbp4022yq.jpg

So, a buddy of mine brought a can of Doppelganger over last week (very pineapple forward), so we harvested the dregs and before propagating them, I struck out the yeast present (left image in pic above). I didn't notice this until after running the PCR, but you can clearly make out different colored yeast colonies, reflecting the diversity of strains present. I am streaking out my Julius isolate stock to see if something similar occurs.
Analyzing the DNA fingerprinting, there clearly appears to be 4 species present in the 10 colonies I picked! The majority of which contains a novel fingerprint signature (for my strains). The other 3 include the T58-like sample previously ascribed to the Julius strain, something very similar to S-04 and something somewhat similar to WY1272 (and WY1332 which looked the same) minus the high MW bands.
I will know more once I look at the streaked out Julius stock, but my friend used it to make an all Citra APA that turned out very nice. So whatever's in there makes beer!

isomerization

I continue to love this thread.

TheHairyHop

I've been quietly following along as well.

5mooth0perator

The plot has indeed become quite thick

isomerization

Fascinating, great job on your work.
I can't wait to see what becomes of this thread

xerotime

At this point I have more questions to answer than when I started, sounds like science! What are the chances that this was unintentional on the part of Tree House? Will need to analyze more/different cans to see if this is a trend or consistent practice. I also plan to make small starters with each of the different isolated colonies to see if any sensory clues jump out.

TheHairyHop

Nothing is unintentional with Tree House. The question is, are all strains pitched together, some later, and/or some at packaging? Incredibly fascinating. Thanks for taking us on this journey!

isomerization

yea, who knows. maybe some is pitched with dry hop additions? Is there a way to check the proportions?

couchsending

I wonder what happens if you isolate yeast from the outside of a hop. Come to think of it, hop with its strong abiotic properties would be a good vector for inoculating yeasts.
Wild Hopping, as it were.

isomerization

Okay, so was able to get my hands on another TH beer (Green). This one was a bit on the old side (by taste, the date was smudged) and didn't have nearly as much viable yeast as the other 2 cans I've streaked (only 3 colonies on first plate, normally have close to 100). I also went back and analyzed the Julius glycerol stock I have in the -80C freezer. Unfortunately, it looks like I took a single colony to make the stock, as all 5 colonies have the same fingerprint.

tvgkmi0wdau.jpg

First lane is a new yeast (Wyeast Forbidden Fruit), then next 5 lanes are different Julius colonies from main stock and then 8 colonies from Green colonies. As you can see all DNA fingerprints appear to be consistent with the 4 possible strains we saw in the Doppelganger can (gel on the right re-posted for convenience). This also means that the Julius yeast I (and my buddy) have used to make beer with is probably just S-04, haha!
My next plan is to streak out a new TH can (after centrifuging dregs to concentrate yeast) and analyze upwards of 50-100 colonies. This would enable a prediction as to what ratios are being used. Its possible TH uses different ratios for different beers. Also makes me want to re-check the Double Shot yeast stock (I saved a heterogeneous stock for everything after the Julius stock).
I'm also very interested to figure out what the red square strain is, as this consistently (n=2) has been the most abundant strain in the cans I've analyzed. I have saved this strain by itself, will need to brew a NE IPA with it soon. I have more yeast coming from 2 different people soon, so stay tuned!

skibb

That had been my suspicion for some time now. The stouts and Bright are probably S-05 since they were using that early on.

Carlscan26

I believe they meant that the colony that had been cultured was S-04. It seems there is more than one strain per can

isomerization

Re: Green, that beer has much less house yeast character than J, Haze, etc. It seems to be strong component of clean neutral yeast like Bright (probably S-05) with smaller portion of the unidentified "house" strain. Excited to see how the red square performs on its own.

isomerization

You are correct. Makes sense with the side by side we did (along with WY1318), as I thought they were pretty similar.
Also note that the previous Julius colony I analyzed was likely T-58 (or very close relative), so I think all 3 cans I've analyzed so far have been mixed populations.

I think I agree with that statement, my palate is unfortunately not the best, but Green is definitely lower on my like list when compared to Julius, Haze and Alter Ego.
I'm planning a side by side with the red square (and hoping to possibly identify it with some new yeast strains I have coming) and WY1318 in an all Citra Oat SMaSH next month.

robopp

Is it possible that they are just naturally carbonating all of their beers with something similar to T-58? In the description of T-58 that is one of the recommended uses for it.
Propped up a starter from a 750 of SAP that I grabbed from the brewery. First few steps smelled totally normal but the last 2.5 step starter might have got a little warmer (had to move it to a different room) and it totally exploded everywhere (in a 5L flask) and smelled incredibly phenolic. Pitched it into a 1.068 wort (loose Swish clone). Kept
It at 65 for the first 4 days then raised it to 68 then 70. Only got to 1.022 after 8 days but it still fermenting and the sample was very peppery and phenolic (more pepper than anything).
For me one of the most distinct qualities of Treehouse is the mouthfeel. Although some people say Co2 is Co2 I sometimes wonder. As I've only ever force carbonated I haven't done a split batch side by side comparison but I might just have to. People always rave about the mouthfeel of HF and TH beers and I'm pretty sure that most if not all the HF beers are naturally carbonated....

isomerization

Anything is possible at this point! I completely agree that bottle condition-derived CO2 has a "creamier" texture than kegerator-derived CO2 (with the caveat that I haven't done a real side by side, so could be biased). With that said, I'd expect there to be a lot more sediment in the cans if they were really doing that. Maybe the extra yeast are added in a secondary or brite tank set up? I don't know enough about the logistics of that to know whether that's a silly idea or not.
If they are truly fermenting with multiple strains, then they almost have to be pitching known amounts/ratios and not propagating them together. I bet that is where a lot of people have strange cultures when trying to harvest from cans. Eventually one of the strains takes over (sounds like its often the Belgian one, based on a lot of the comments I've read RE phenols).

robopp

Any updates on this? I just read through the thread - fascinating stuff.
A couple things:
I know many people have commented that their Heady Topper strains have been quite Belgiany - even Jim has mentioned that you can get this character but HE knows how to ferment it so it doesn't happen with HT (I will try and search for the interview where he states this). Have you tested any strains since then that are purportedly from Alchemist or Conan?
I run a commercial brewery in KY and we have a microbiologist on site. I can definitely send you some strains to compare if you are interested. I can say from my experience with yeast providers, some *cough*wyeast*cough* are notorious for unclean pitches. Most breweries I know that have used them have since moved on. I would be curious if you have tested any recent cans of TH and have seen any change in yeasts collected.
Cheers!

skibb

Hi, so did you get this new strain out? What's it's number?

isomerization

I have recently analyzed two more TH cans (Julius and Alter Ego). I only looked at 10-12 colones each (realized it would take way too long to run gels if I analyzed 50+ colonies/can). All colonies were either the blue triangle (S-04), red square (mystery) or green circle (T-58). The ratios were 14 (S-04), 5 (mystery) and 3 (T-58). Propagating the blend over several generations and then reanalyzing might provide some insight into why some people's starters from can dregs "turn" Belgiany. It could be that that strain grows faster than the others? I haven't analyzed any other breweries dregs (I live in KS, so difficult to get the NE IPAs).

TheHairyHop

Any progress identifying the red square? Have you propagated and used just that yeast on a batch yet? Thanks again!

skibb

I'm getting ready to analyze some new strains, but no evidence yet what the red square strain is. I've been talking with someone about how it could conceivably be S-33, a dry Belgian yeast that might actually be an English strain. It would fit with the other 2 primary yeasts that are dry (S-04 and T-58), just need to get down to the LHBS do buy some.
It might be a month away, but I'm going to use the red square strain in a NE IPA oat SMaSH (prob with Citra). Will update here once I do.

Ruckusz28

I can send you some money to offset your costs? I love everything about this thread and would like to support your research.

TheHairyHop

I appreciate the offer, but time is the real limiting factor for me right now. I will say that if you have a strain you'd like analyzed, send me a PM to discuss shipping. Its also possible to order yeast and have it shipped to me if you aren't currently set up for "home wrangling".

isomerization

I just read in a post that TH growlers say they are naturally carbonated? I'm now wondering if the T-58 like yeast is added along with a dose of fermentable sugar under pressure. They could dry hop at this time and get bio transformation as well along with O2 scavenging. This would possibly suggest they use a less complicated yeast blend for primary.
Anyone thoughts or personal experience with something like this in a NE IPA?
I don't believe they are... I did purchase four last time I went. Unless they add the yeast and sugar mix into their keg.
All that is possible, but the beer would be very vegetal if heavy keg dry hop, I don't remember that. Maybe a hopback is used.

RevKev

Where did you read that? It's the first I've heard that they naturally carbonate their beers.

quijiba

I highly doubt they are naturally carbonating their IPAs - I'm not sure any brewery would want their fresh/hoppy beer 'aging' just to get carbonation into it. I've never heard or met any brewery who naturally carbonates their super hoppy IPAs.

So you have determined that these strains are S-04 and T-58? If so, maybe the third mystery strain is also a dry yeast from Fermentis too? Doing yeast blends would be substantially easier with dry yeast and not terribly expensive.
Since it's nearly impossible to maintain yeast blend proportions throughout subsequent generations, I don't think this is too much of a stab in the dark. I mean, practically speaking, this just makes sense to me that it would be another dry yeast strain, and more likely than not, a Fermentis one to boot.

stpug

On a post from Mike Strassers blog: https://strasserbrewing.wordpress.com/2017/06/01/6-1-2017-creamsicle-milkshake-ipa/

I can't say I'm an expert on brewery practices, but I'd think it would actually be an efficient process and help lower DO in the final product.
I found this comment in an old thread:
https://www.homebrewtalk.com/showpost.php?p=465869&postcount=9
RE dry yeasts, I agree with you and have had discussions with others in a similar vein. Keep in mind that the genetic fingerprint doesn't conclusively "say" the yeast I've isolated is S-04 or T-58, just that they're closely related. I am thinking S-33 could be the red square mystery strain. That is supposedly an old English strain. I wouldn't be surprised if they were pitching heavy ratios towards the English yeasts and trying to get elevated glycerol/bubblegum esters from the Belgian strain. Would be an easy test on the home brew scale...

TheHairyHop

I disagree. If you hop with some extract left, naturally carbing is the logical next step. The whole process of hopping during fermentation and naturally carbing takes about a week.

StinkyBeer

My comment was more-so on adding another strain of yeast to naturally carbonate. If they were adding a conditioning strain it would not be in a primary/uni-tank, and the beer would not be still fermenting.
It's not as logical as you would think. Nearly every FV jacket is rated to 14.9 PSI. You cannot get 2.5-2.8 volumes of CO2 (2.8 being the usual target for a beer going into a can) into a beer under 15 psi unless the beer is ~45 F (i.e. not still fermenting). That being said, they could have 30 PSI rated tanks, but it's unlikely.
Capping a fermenter after dry-hopping while fermentation finishes (something I personally do) does end up providing close to 1.5 volumes of CO2 in my beer. I then cold crash and force carbonate.

StinkyBeer

It's printed on their growlers.
I had a reader reach out a few months ago and was discussing the possibility for natural carbonation of the tree house beers. Going by their word, I would suspect that the beer would be transferred to a bright tank a few points north of terminal gravity where it is dry hopped and sealed to allow the final period of fermentation to carbonate the beer. Depending on what yeast is used for primary fermentation, a secondary yeast that is more attenuative could be used to finish the beer. Being under pressure would force the dissolved aromas from the yeast esters and the dry hops into the beer as opposed to allowing them to escape through the airlock.
My last batch demonstrated an improvement in this area, however it was not done to the best of my ability. I used a strange experimental yeast and my fermentation keg had a stuck poppit that was leaking some pressure.
This is my best guess to their process.
Thanks @isomerization for your work here. This is one of the best threads I've read.

isomerization

I believe that S-04 is 5% more attenuative than T-58. I have also thought of this terminal OG -> dry hop + more attenuative strain. It seems a bit of trouble to do as generations change in attenuation and you could just transfer with gravity left, or add sugar after terminal and before/during transfer

isomerization

Thanks, I'm glad you enjoyed reading it, I should say that I enjoy your blog posts as well!
What are your thoughts on glycerol production. This is a somewhat elusive trait to find information on with specific strains, but it seems that saison yeasts are known for making more glycerol than others (helpful when your FG is below 1.006!). I'm assuming that's why the T-58 like yeast is in the cultures (albeit at what looks like a low ratio). This raises a good question though, "Is T-58 a saison yeast?" From NB's website: "A specialty ale yeast selected for its estery, somewhat peppery and spicy flavor." That sounds like a Saison yeast description to me!
My plan for my next NE IPA is to ferment in my conical, dry hop on day 2 (high krausen) and then say 2-3 days later once fermentation is slowing down, rack to a CO2-purged corny with the second dry hop already in there, attach the spunding and (hopefully) fully carb. I think 30 psi is needed at RT.
Its possible that one could use their standard NE IPA yeast in primary, rack to the keg, add a more attenuative Saison strain (T-58?) along with the keg hop and a spunding valve. In theory the Saison yeast would get to FG, carbonate and scavenge any residual O2.

I think the key here (to this hypothetical situation we have created) is that TH would be using dry yeasts, therefore, they wouldn't need to harvest yeast. Also on a brewery scale, imagine how much money would be wasted on priming sugar.

suregork

I feel dumb now. Lol it is right on there.
View attachment 1497558725789.jpg

StinkyBeer

Not gonna lie, that blows my mind. Does that mean all of their products are naturally carbonated? That seems mildly insane/impressive that they are naturally carbonating that much beer!

melville

Actually, natural carbing is both easier and faster than force carbing with canned co2 - plus you get the added benefit of no additional O2 introduction (given a solid process) as well as the natural oxygen scavenging behavior of live yeast. Spunding with residual extract is probably the most effective method, but keg/fermenter priming is acceptable as well. That natural benefit of near-zero packaged oxygen is stability of the beer.

isomerization

I've also wondered about glycerol. It would be cool to apply a similar technique towards finding strains that produce an abundance of it. I've read that some white wine yeasts are good producers.
T-58 isn't particularly attenuative (I think it's around 70%). I would imagine it's not the yeast that they finish with, if they're also using S-04. At least, they aren't finishing with only T-58.
As far as dropping the hops into a keg to be spunded, I've definitely thought of that! I was considering giving it a try, but was worried about keeping hops in a warmish tank for so many days. Since hop flavor extraction seems to occur more rapidly than previously thought, I was think of my last hopping being at like 4 or 5 points from terminal gravity, and then racking to the carbonation/serving keg at 3 points from terminal.
By adding sugar, you're also bumping ABV. I wouldn't call that a waste. By no means do I operate a brewery, but when considering the cost of sugar vs entirely new yeast, I'd imagine sugar is less expensive. Trillium has sugar listed as an ingredient in basically every beer that they make. My hypothesis was that they may be using a continuous drip of sugar to keep the the environment continuously low O2 during dry hopping.

Ruckusz28

Hello everyone! What an ace thread...so cool.
We are packaging 2800litres of a Citra IPA today. Combined 2 FV's in to 1 BBT. Exact same recipe except one was fermented with S-04 and one with T-58.
The signs are that this is an absolute banger of a beer. Opaque, juicy, fruit bomb. Sweetshop and sherbet on the nose and huge mango flavour with bread malts. Thick and creamy.
Next experiment is pitching S-04/S-33/T-58 at a ratio of 40/35/25 in another IPA.
Regarding natural carbonation...I seem to remember Hill Farmstead saying their beers were naturally carbed (which isn't the case)...but what I think is happening is that brewers are partially carbonating through spunding and then finishing off the process via a stone (or possibly head pressure) in the same tank. That way they can indeed say the beer is 'naturally carbonated'...and they get to emblazon the word 'naturally' on their growlers!
So cynical.
S-33 sounds interesting. Looking to add some apricot and a little banana bubblegum with this to the tart, juicy fruity T-58. S-04 is fairly restrained IMO...a little tangy fruit but mainly quite doughy.
T-58 absolutely is adding considerable mouthfeel.

marshallb

Is it cool to post links? Apologies if not...please delete.
http://trinitybrewers.com/brews/ipa/julius-clone-treehouse-brewing-ipa/

Ruckusz28

I don't think that's an apples-to-apples comparison. If they are using multiple yeasts, then that's already part of the equation. So, the sugar is indeed an extra cost.

Well this is an interesting post.
2800 Liters each!? That's a lot of beer on an experiment. Or have you done this before?
I have a lot more questions if so, but what temp did you ferment the T-58 at? The lower attentuation of T-58 and/or increased mouthfeel could easily balance another yeast that finished at a lower FG. I'm very intrigued to try this for sure, could easily do two 3 gallon ferments with different yeast and combine in the keg!

marshallb

***DNA Fingerprint Update***
I have some new yeast to analyze, courtesty of @suregork and @robopp, thank you both!
In the first gel, I am showing you the 4 strains that have been found so far in various TH cans (Julius, Green, Alter Ego and Doppleganger). Next to that gel (same DNA reference ladder) is a gel with WLP644, F1, F1/C4 and Conan (TYB), see this thread for more info: https://www.homebrewtalk.com/showthread.php?t=577066. You can clearly see common bands between F1 and Conan, and that F1/C4 is quite different than F1. WLP644 doesn't seem to match up too well with either hybrid though?
Third gel to the right has 5 new dry yeasts provided by @robopp. You can see that S-33, Windsor and London ESB are all pretty darn similar. I believe all 3 are British in origin? WB-06 (a Hefe yeast I believe) looks pretty similar to the gold star yeast from TH, could possibly be a match. Unfortunately, no match yet for the red square strain, the search continues!

2314pj4wvai.jpg

I've also included the previous DNA gel that has the other 17 yeast strains I've analyzed, for reference:



Strain key:
1 - WY1056
2 - WY1272
3 - WY1332
4 - WY1318
5 - WY1968
6 - WLP670 (saison isolate)
7 - WLP802
8 - TH Julius isolate (single colony)
9 - The YeastBay Vermont Ale
10 - WY3944
11 - TH Double Shot isolate (single colony)
12 - S-04 (Fermentis)
13 - S-05 (Fermentis)
14 - S-23 (Fermentis)
15 - Munich (Danstar)
16 - W-34/70 (Fermentis)
17 - T-58 (Fermentis)

isomerization

Cool, didn't realize you were in the latest send-out
What primer pair did you use for these? Here is my gel with delta12 and delta21 with the isolates of WLP644 and Conan that I used as the parent strains:

2cvy5ayapqb.jpg

Edit: Hybrid H1 = F1

TheHairyHop

One FV was fermented with S-04 pitched at 19 let rise to 21. The other FV was fermented with T-58 pitched and held at 25. Both had about 16HL of wort and we lose about 200 after dry hopping and transfer to bright. So combined they form 28HL ready to package. This is showing excellent promise thus far.
We regularly use: US-05, S-04, WY1318, Conan (BTW expect this in dry form very soon sshhhhhhhhhhhhh).
I remember reading somewhere that Treehouse use Fermentis yeasts and pitch fresh each brew. So I would say your red square strain will come from Fermentis. Strains from them you are yet to test:
F-2
BE-256
S-189
My hunch is possibly F-2. If however the red square doesn't show up from Fermentis...then I'm certain it will be from a dry yeast from another company. It makes no financial sense to be co-pitching wet and dry yeasts each brew.
Have you tested a can of Bright yet? To me that was very similar to Alchemist profile. Bready, peachy, bitter and clean. Don't think it is Conan but something slightly less estery. Wasn't so sure it was US-05 either.
Anyway...yeast is so much fun!

melville

Love to hear your thoughts on how this blend turned out? Was it fruit forward as far as esters go? Did you notice any spice? Was it soft like Tree House beers tend to be, and how'd it compare to 1318?
Oh and any pics!?

melville

To your observation that the gold star could be WB-06 hefeweizen (?!), others have reported excellent results using just hef yeast:
http://trinitybrewers.com/brews/ipa/julius-clone-treehouse-brewing-ipa/

melville

Yep, I am planning on using F1/C4 in a Pineapple Berliner soon, hope the descriptions from others are accurate!
I used delta2 and delta12 primers, so should be pretty similar to what you used. My gel isn't as nice, so could be some visual error there. Also, my strain of Conan might be from a different source (the Yeast Bay) than yours. WLP644 was obtained in a yeast share, so I can't 100% say it is WLP644, could have been a mistake on either end of shipping/receiving.
To clarify, the first hybrid in your gel is F1? What lane is F1/C4 then?

I will have to check out those dry yeasts then, their descriptions don't sound expected, but then again I would have never guessed T-58. Speaking of that yeast, I can't believe you are able to ferment that high and have the final product fit with the NE IPA flavor profile! It must not be a "traditional" Belgian yeast, maybe something closer to a Witbier strain? I seem to come across tart, fruity and bubblegum descriptors a lot when looking a yeasts to use for Witbiers (e.g. Wyeast Forbidden Fruit WY3463).
I haven't come across a can of Bright since I started harvesting yeast, but will keep that in mind.

That was my first thought after I had opened the link! This is weird, but my email notification of posting on this thread said @StinkyBeer had posted this link, did that get deleted? Or rather, is TrinityBrewers a user posting in this thread?

isomerization

Ohhhhhhhh, I like this  

isomerization

Hello guys. I've read through this thread twice now as I'm so geeked out on what you're doing here. Trinity Brewers is my website, I haven't posted in here.
I just went through a good amount of TH beers and left Bright out of the sampling because to me, it tasted very clean like an American Ale (thoughts were 001 or 05). I will say that Bright is where I finally was able to make the Biscuit and Victory combo of grains in my recipe.
What's up @Ruckusz28 haha, haven't talked to you in a while!

TheHairyHop

Hi Marshall! @marshallb
I stopped up at adventures in homebrewing today and picked up every fermentis dry ale yeast I could find. Tomorrow I'll brew a mainstay pale ale of mine and substitute the yeast so I can reflect on the new vs. the standard California ale V. Hooray for birthday brewing.
I picked up us04 us05 w33 t58 wb06 k97 packets.
I will pitch ratios of 50%us04, 25%w33, 25%t58 to get this rolling.

StinkyBeer

Lots of awesome here! You guys have me wanting to try a variant of these yeasts next brew.

isomerization

I look forward to reading it! Just to clarify, the mystery red square isn't S-33, or any other Fermentis yeast that I've tested.
I am propagating my harvested blend from Doppleganger and will try to follow the yeast strains as they change in ratios. I'm also going to try and do some test ferments with the red square strain to try and narrow in on a yeast strain family.

RevKev

The growth rate of each strain will be different, right? It sounds like you may have to analyze your own batches once you've identified all the strains in order to see if you're hitting the "correct" final ratios

isomerization

Love this thread. Ordered some S04, T58, and WB-06 and gonna give them a whirl this weekend 60/20/20. Does T58 have a known liquid equivalent? Just saying it is possible that they are using WLP007 and other white labs equivalents and we/you might be going down a rabbit hole with the dry yeast.

dcbw

I do wish the other unconfirmed strain/s were ones that made more sense. I keep getting stuck on the T-58 — most accounts I read about it do seem to confirm some spice, and as a somewhat frequent Tree House drinker, I don't get any spice at all in there, nor is spice mentioned in any of their descriptions (well one does "spicy grapefruit"). Maybe it really is there just for the natural carbonation (some say T-58 is used to bottle condition Tank 7)

Ruckusz28

Just poking around the internet to see what Nate's influences were:
"Nate Lanier: My a-ha beer was Rochefort 8. It blew my mind at the time, and I still love it today. Several others followed… Rodenbach, Supplication, Old Rasputin… dozens of other gateway beers.
My first Heady. Coming in gassed off of the slopes at Stowe and having a rip of Heady fresh at the pub is something I wish everyone could experience.
My first Hill Farmstead. I had a Harlan at Three Penny Taproom, served by the one and only Scott Kerner. It was transcendent."
https://www.pastemagazine.com/artic...-brewing-talks-malt-the-black-market-and.html
If one were going to clone Rochefort 8, one might use T-58 (or BE-256). One of the other strains could be in Hill Farmstead (yes, I know we think it's 1318, but we also all assumed Tree House was 1318 or Conan, etc.)

TheHairyHop

That's a great point (RE pitching specific ratios and seeing what comes out in the end). I was hoping that I could get a feel for what the starting ratio was by seeing how the dregs propagated, but w/o knowing when the yeasts were introduced, that is probably not going to work. I wanted pure cultures of each isolate anyways, so not a waste of time hopefully!

StinkyBeer

Anything is really possible, but I do hang my hopes on the dry yeast angle for a variety of reasons, primarily its cheaper on a commercial scale (e.g. plausibility) and it creates less yeast strains to try and screen (genetically or in test batches) on our end.
RE: T-58, I'm not sure anyone can agree what type of Belgian yeast strain it is, could be witbier, saison or something else. I've never used it in primary, but I plan on taking the TH isolate and fermenting small starters at different temps, then taste testing. I don't really have the setup right now at home to bother with anything less than 5 gallons. So, hopefully some insight will come out of this approach.

isomerization

I know that I get large amounts of bubblegum from Julius if my brain is in the right place or it's less than fresh. It's probably a bit of mental bias, but since the first time I tasted it, I can pick it up each time now. I wouldn't be surprised if T-58 is used in such a way to minimize spice and accentuate some of the more fruity byproducts. Who knows, perhaps the yeast does odd things in the presence of super fruit hops like Citra and Moasaic?

TheHairyHop

T58 at 25 degs is fruity and tart. Bubblegum too.
WB-06 gets fruity and bubble gummy above 23.
S-04 I always find has a slight sherbet/bubblegum character plus bread
Don't be afraid to experiment!
I would highly recommend a 20 litre batch of:
50% S-04
25% T-58
25% WB-06
Pitch and ferment at 25 degs. I've pushed S-04 to 26 with no ill effects.
Don't oxygenate.
Another option is simultaneously ferment 3 identical brews bar the yeast then combine the beers at different ratios in to cornies and force carb/test and reassess.
The red square yeast puzzles... Does it seem to resemble a group of strains at all? Could you narrow down the style of yeast?
It could be that they are using a single wet strain for certain brews (red square) and cutting this strain in to some of their other brews i.e. Julius etc. They will be harvesting so much yeast that throwing some in with the dry blend and then down the drain is not a financial concern.
You need to test all the Lallemand strains too.
I can't find the thread but basically someone in 'the know' said he has it on good authority Treehouse use Safale/Fermentis yeast and pitch fresh each time.

isomerization

I'm certainly going to try that, as well as an equal blend (25% each) of the 4 strain isolates, separately propagated. I figure an even blend might enable an easier time figuring out which strain is over represented in flavor profile and then I can also monitor the dregs as well.
On the red strain, I have tested several Lallemand dry strains, but not BRY-97. I bring those two up because BRY-97 is mentioned in the fruity yeast section in this article: http://www.thebeerfiles.com/american-yeast-strains-part-2-ale-yeasts/?cb=04830155076515602
The WY1272 profile was clearly not a match, but it makes a great NE IPA on its own.
This is interesting, maybe WY3944 (https://www.homebrewtalk.com/showpost.php?p=8022874&postcount=97), which is strain 10 in the lower gels could be close? That's a witbier strain as well, which would be weird given the presence of T-58. I will look into how to assign phylogeny from the DNA patterns, but I'm sure Suregork knows more about this than me.
Just need to get some more dry yeast to test!!

Ruckusz28

BRY-97 in my opinion suppresses hop flavor, I used it in what I guess would classify as an india red ale and honestly would never use it again. Had nice esters but the character I thought I would get was completely different.

melville

Well that's disappointing!

melville

Here's another thought...
Tree House cans their stuff.
Cans by definition allow more dissolved oxygen during the canning process because the lid is larger in diameter than a bottle neck.
Tree Houses stuff has great aroma and flavor. Oxygen destroys those things.
So they decide to counteract that by adding a specific small amount of yeast at canning time. Not for carbonation, just for O2 scavenging due to the worse DO characteristics of cans.
At HomeBrewCon the oxygenation presentation on Friday described exactly that process at New Belgium.
Even if Tree House doesn't filter their beer like NB does, and thus if yeast is still present, maybe not enough of a dose to scavenge O2 quickly enough is present and thus they add more.
And if that is a different strain than primary fermentation, , you now have another strain in the isolation tests.

Ruckusz28

The T-58 is either for natural carbonation or has to be blended in after ferment. I have a hard time believing that they would be copitching yeasts that benefit from such wide range of ferm temps. T-58 fermented cold is definitely spicy and while I've never tried S-04 warm everything I've read says it gets really messy. Maybe start the ferment wit T-58 and when it stalls (which it notoriously will) pitch in some S-04 at a lower temp to finish it out. Not sure if droppin the temp would produced Diacetyl or not and if it did would the S-04 clean it up...
I'm convinced that both HF and Treehouse naturally carbonate their beers. I just don't think there is any way you can get that soft feel without it. Everyone thinks it's not good for hoppy beers but that usually refers to bottle conditioning. In a keg (or bright tank) it makes much more sense and if you think about it, it pulls any available oxygen out of the beer. I've got two beers on tap now. One fermented with 007 and then naturally carbonated with T-58 and dextrose. The other one is 75% S-04 and 25% T-58 then naturally carbonated with bagged hops in the keg. Just used Dextrose for carbonation and it didn't quite get there. Next round I'm going to try carbonating with highly hopped Gyle and T-58 to see affect that might have on the aroma...
I also built up some HF yeast harvested from a growler of S&S9. Willing to bet there are a few different yeasts in there too. It's doesn't smell as phenolic as the starter I built from the growler of Sap but it has a distinct Saison aroma to it. Pitched it in a 5g batch of fresh 2017 Galaxy hopped IPA this afternoon. Interested to see what happens.
One other suggestion I might have for the mystery yeast is 1450. Whatever was in that Sap harvest produced the thickest mouthfeel Of any beer I've ever made. Nothing I did was different than normal and I've never had that type of mouthfeel in any beer I've made even a 158 mashed 1.024 FG Porter. FG of that beer was 1.016. One of the biggest descriptors of 1450 is mouthfeel. T58 - Pitched and fermented at 25degs, heavy dry hop produces an intensely fruity and tangy beer. Minimal spice. It does throw some sulphur but by day 5-6 it has gone. I can't say it simpler than that really. I'm sure it's also a great yeast to can/keg/bright tank condition/carbonate too...but so are many many other strains. It also produces a beer with a lovely creamy thick mouthfeel.
I would suggest trying the above before making any conclusions regarding primary fermentation yeasts.
I still stand by my 'spunding' for 'natural carbonation' proclamations. It is common practice to dry hop and cap to achieve some CO2 dissolution and then to finish off through force carbonation.
The S-04/T58 50/50 beer I recently made is a mango and pineapple bomb with slight spice, tangy finish, huge murk, slight bubblegum.

melville

I brewed a session ipa batch yesterday featuring centennial/cascade/simcoe. OG 1.044. I did not oxygenate the wort. I did not use any fining agents. I treated the mash with chloride only. Mash of 150 for 60 minutes at a ph of 5.2. Grist of pilsner, carafoam, and 2% honey malt.
At the time of pitching I added half of my dry hops.
I direct pitched the equivalent of 1 sachet by weight consisting of 50% US04, 25% T58, 25% S33. Fermenting at ambient temperatures in my home with the AC on. After about 5 hours, the entire house smells like a tropical oasis. Peach, banana, gooey fruit, insert ambiguous fruit descriptor.
Updates to follow.

isomerization

Was S-33 pegged as being in a Tree house culture? I may have missed that bit

melville

S - 33 was a red square 'hunch'. So far it is not a Treehouse yeast. It does appear in the right hands to throw some pleasant fruity esters though.
Ruckusz28 - Can't wait to see how that one finishes up! Any idea what temp it's at?

isomerization

As StinkyBeer mentioned, it does not appear to be one of the 4 yeasts. My current hypothesis is that the red square could be a strain added after the initial pitch (something like F-2), which could account for the higher ratio we see with this strain. The primary dry yeasts would then be S-04, T-58 and WB-06. Needs to be tested though.

Ruckusz28

Word. Thanks. This thread is especially hard to follow on mobile haha. I have a NEIPA that I fermented with 1318 as a test of my new system. A 50/50 of T-58 and S-04 is my next move. Unfortunately, I no longer have temp control for fermentation. I'll be sure to add to the reports

isomerization

Well the advice with that yeast combo is to ferment at 25 C, which is around 77 F, so I think you're ok w/o temp control!

Ruckusz28

70f ambient so likely north of 74f inside the vessel.

melville

Mind sharing your carbonation technique? How much t-58 do you add? How much sugar, can you do it without a spunding valve?
Also – Did you note any differences between 007 and S-04?

isomerization

Did you rehydrate or sprinkle in?

cegan09

@Melville
007 is quite different than us04. I recently did a side-by-side comparison on a Old Ale period one half of the batch using 007 and the other half using us 04. The batch using 007 is much thinner dryer and allows more of the roasty flavors to come through. The batch using Uso for is a little sweeter has a better mouth feel and has all kinds of vanilla toffee caramel notes. I'm not saying that one is better than the other, however I am saying that they are very different and I do prefer us 04.

skibb

Went with a 50:30:20 blend of S-04:WB-06:T:58 in IPA with Citra, Galaxy, and Motueka last night. Woke up and bubbling quite nicely.

cegan09

Sweet, good to see we have multiple experimental batches going!

TheFlyingSparge

Several breweries within the NE-style dry hop under pressure, so my guess would be that they carb it at the same time as they do that.

melville

Took a gravity reading this a.m. (still under 48 hours of fermentation) and the 50:30:20 combo has brought things down from 1.068 to 1.022. Smells nice, I do get a bit of something saison-y in there when tasting it. First round of dry hops going in.

isomerization

Is anyone interested in asking TreeHouseNate on Twitter whether they blend their yeast pre-pitch or post fermentation? He seems to respond to a lot of questions, so his answer might be helpful.

mosaicbrewer

I blended pre pitch, but multi step yeast additions during fermentation do make more sense IMO. Timing and temperature for each variety and addition become individually manipulatable. Temperature being most important in coaxing out the desired esters and suppressing the undesirable.

TheHairyHop

They also become more difficult to reproduce! Or do we think that on a commercial scale, fermentation will proceed in equivalent fashion, say down to the hour, if all variables are kept the same from batch to batch?

Ruckusz28

I suppose that all depends. For me, I would measure the amounts of yeast and contribute them to the batch when the beer hits landmarks such as gravity or time. Gravity would be the best indicator IMO. As long as its the same recipe and equipment, I'd imagine things to be fairly direct to replicate as long as you have consistent measurement and monitoring practices in place.

melville

It seems... counterintuitive to give successive yeast strains a crack at wort/beer with less and less to sugar to work with. In my mind I would think esters and phenolics would be a byproduct of conversion, the more there is to convert the more esters etc. (I have no idea if this is actually how it works.)
This is sort of what makes me so suspicious of T-58 as anything other than a carbonation strain.

cegan09

The way I see it, is there are 3 (plausible) options here to end up with 4 different yeast strains in the bottom of TH cans.
1.) Yeasts are pitched into the same wort at controlled ratios
2.) Individual yeast strains are used to ferment various volumes of (the same?) wort and blended in a bright tank
3.) Yeasts are added at various times in the process, could be as either Strasser or Melville describe (e.g. staggered during primary or in the bright tank to help carbonate)
RE ester generation, its always been my understanding that esters/phenols are most abundantly generated during yeast propagation. This is why temp control within the first few days is so important, and also why you can use WY3711 to finish off difficult fermentations and not end up with a saison.

whovous

This is a crazy interesting thread. I look forward to seeing it progress.
I have heard that Nate From TH is crazy detail oriented, and meticulous about making sure everything is done the same every time. (so the opposite of me). That may or may not lend some credit to the idea of yeast being added at different times if he's gotten to the point of controlling what goes into fermenters and knows where in the fermentation process they are.
but at the same time, it's easier to control things with less moving parts. So trying to add different yeast mid fermentation just seems to be asking for variation.

melville

As stated in my follow up post I was referring to adding a bottling/conditioning yeast strain and carbonating that way. They may carbonate completely with spundigs and high-pressure rated tanks - might have to take a tour to find that out.
I do the same kind of half-way carbonation at my brewery: "Capping a fermenter after dry-hopping while fermentation finishes (something I personally do) does end up providing close to 1.5 volumes of CO2 in my beer. I then cold crash and force carbonate."

couchsending

No tours at tree house. It's show up, get in line, buy your beer, and get out. However the brewery is within full view while inside. You'd have to know what you're looking for. View is generally this. Far right are the kettles cropped out, and far left is generally obstructed by the canning line.

azrmnzqly5n.jpg

New brewery opening soon too.

prosserbrewer

subscribed. This is by far one of the most interesting threads I've read on this forum. Lovinit.

bierhaus15

Another gravity reading (I swear I'm not normally like this, just curious how things are going). Now at 1.015 at just under 3 days so easily on target for 1.013 I think. One unusual aspect is that the trub is well over the spigot on the Speidel, that hasn't happened before. Smells fruity and tastes fruity (its galaxy, citra, and motueka, so yeah...), spicy but hop spicy, not yeast spice. Don't get that Saison-y thing now. Does seem a bit hot ABV-wise, but again that could be the hops.

couchsending

Did you say what fermentation temp? Are you going to spund? And any banana notes from the WB-06?

isomerization

I have read most of this thread at this point. Has anyone considered that the T-58 may be added to the beer in small quantities to discourage people from propping up the yeast at home? I propped a bottle of everett a few years ago and it was awful. I could see using a blend of 05 and 04 or something to produce specific results, but this ratio conspiracy seems a little insane. I know people think there is some magic going on down there, but blending multiple dry yeast strains seems like a stretch.

melville

it's far less of a stretch than throwing in T-58 to throw us off from propagating S-04 and one other strain. T-58 also doesn't have the best attenuation.

cegan09

Today after work I'll be taking a gravity reading and a sample. It's been 4 days. With an OG of 1.062 it's probably finished and should be cleaned up. If all things are go, I'll rack to the dry hop keg, pressurize, and chill. I estimate transferring to the serving keg Friday and hitting my belly on Saturday.
For the record, here's my recipe. Yes - I know there are no flaked anythings.

Ball & Chain Pale Ale
Author: Mike Strasser
Brew Method: All Grain
Style Name: American Pale Ale
Boil Time: 60 min
Batch Size: 3.5 gallons (fermentor volume)
Boil Size: 5 gallons
STATS:
Original Gravity: 1.062
Final Gravity: 1.014
ABV (standard): 6.28%
IBU (tinseth): 73.31
SRM (morey): 5.76
FERMENTABLES:
7 lb - German - Pilsner (84.8%)
0.75 lb - American - Carapils (Dextrine Malt) (9.1%)
0.5 lb - Canadian - Honey Malt (6.1%)
HOPS:
0.5 oz - Centennial, Type: Pellet, AA: 10, Use: Boil for 60 min, IBU: 29.87
0.5 oz - Cascade, Type: Pellet, AA: 7, Use: Aroma for 5 min, IBU: 4.17
2 oz - Cascade, Type: Pellet, AA: 7, Use: Whirlpool for 20 min at 170 °F, IBU: 14.98
1 oz - Centennial, Type: Pellet, AA: 10, Use: Whirlpool for 20 min at 170 °F, IBU: 10.7
1 oz - Simcoe, Type: Pellet, AA: 12.7, Use: Whirlpool for 20 min at 170 °F, IBU: 13.59
1 oz - Simcoe , Type: Pellet, AA: 11, Use: Dry Hop for 7 days
1 oz - Cascade, Type: Pellet, AA: 7, Use: Dry Hop for 7 days
1 oz - Centennial, Type: Pellet, AA: 10, Use: Dry Hop for 7 days
2 oz - Simcoe , Type: Pellet, AA: 11, Use: Dry Hop for 3 days
1 oz - Centennial, Type: Pellet, AA: 10, Use: Dry Hop for 3 days
MASH GUIDELINES:
1) Infusion, Temp: 150 F, Time: 60 min
FULL VOLUME MASH - NO SPARGE
OTHER INGREDIENTS:
1 tsp - Calcium chloride , Time: 1 min, Type: Water Agt, Use: Mash
1 tsp - yeast nutrient, Time: 15 min, Type: Other, Use: Boil
YEAST:
Fermentis / Safale - English Ale Yeast S-04 (50% composition)
Starter: No
Form: Dry
Attenuation (avg): 75%
Flocculation: High
Optimum Temp: 54 - 77 F
Fermentation Temp: 70 F (ambient first floor - carboy heater not working)
Additional Yeast: T56 (25% composition) w33 (25% composition)

isomerization

Temp range is 68-72. Not going to spund, no banana notes and even had a Weihenstephaner at the same time to reference banana.

skibb

At the rate they're pumping out beer, I kind of doubt they're doing anything to sabotage. They'll literally be selling beer as it's coming off the canning line.
What's the simpler explanation? That they're purposely sabotaging their beer at the risk of changing it so that a few home brewers won't build up their yeast? Or that they are using a yeast blend, which is something that a lot of breweries do?

melville

Subbed.
Thoroughly confused, but subbed. Osmosis, do your stuff!

isomerization

First I would say nothing about this thread makes any sense, that's what makes it so fun. Still, your suggestion seems the least plausible, though it is still possible. We know they naturally carbonate, some brewers use T-58 to condition — for me it makes the most sense that T-58 is used in this regard. The only other definite match is S-04. That of course makes a bunch of sense as their primary yeast strain. Then we have two mysteries (one sort of resembles wb-06) — if we could nail these down maybe we'd have something that made more sense.
After reading a bit more on T-58, I've noticed many describe or complain about fusels/hotness/alchohol, and I feel like I picked up on that tasting the hydrometer sample, which only heightens my suspicion that it's more for carbing the beer. Of course I could go the other way — why add a yeast/priming sugar when it'd be easier to spund a fermenter.
Also given everything we've seen on Brulosophy — does any of this stuff make a difference? Would tasters notice a difference between S-04 and a S-04 mix? Some didn't even notice a difference between Conan and S-05.

isomerization

Been doing a lot more scouring of the internets and a few more test batches with a lot more to come...
So the one yeast "looks" like S-04 however S-04 is supposedly the dry strain of whitbread 1098? People say it's the version of 1099 which is also 007 but it doesn't seem to get quite the same attenuation as 007 does it? Also one thing about S-04 is that it produces lactic acid and you get that slight twang from it, which I would assume is due to the lower PH? Got a beer in fermentor now with S-04 that is down to 4.19 PH. We know their beers generally have PH in the range of 4.5 (Haze sample I sent to Ward was 4.6). I've yet to measure a final beer after dry hopping. Will dry hopping up it that much? .3?
Also the other yeast "looks" like T-58 which is suppose to be dry DuPont? WLP-565. However could WLP-566 "look" similar? 566 is supposed to be more fruity than spicy/peppery. Also been reading a lot about 3711 and it's citrus/fruity characteristics at high temps (75-79). 3711 also happens to be the one of the highest (if not the highest) glycerol producing yeast strains available. To me one of the biggest areas where Treehouse separates itself is the mouthfeel of their beers. Everyone's knee jerk reaction to more mouthfeel is flaked adjuncts, Oats in particular but Nate has stated they aren't using any in any of their core beers. If you think about it blending in beer higher in glycerol and with some potential citrus/fruity esters is just going to potentially magnify the highly hopped base beer.
in the photos of the brewery those are small fermenters at the front closest to the area you purchase cans correct? Could those be used for fermenting smaller portions of the same wort that goes into the larger tanks with a different yeast?
You don't get as many highly ranked beers as they do by doing things the standard way. Sure a little hype helps but from my personal experience I don't think anyone comes close to matching the complete balance of their hoppy beers from aroma, flavor, mouthfeel, bitterness, etc. They only sell direct to consumer which gives them the ability to do different things than your average Brewery. They can take their time, only produce certain beers when it's optimal, etc. that's the beauty of their business model.

TheHairyHop

I'm WAY late to this thread party (and apologize in advance if I missed mention of this already as I admit skipping a couple pages). At any rate, I came across an interesting beer yeast identification project today and the associated results, protocols, etc. that they used - looks like it was just posted 8 or 9 days ago. These folks out of Switzerland sequenced the various yeasts and fungi found in 39 beers (Orval and 2 Chimay beers among them) and identified the species, if not strain, of what they found. The project is called BeerDeCoded and website is www.genome.beer
From Protocol document:
"ITS mapping analysis
We downloaded the curated set of ITS sequences from the Refseq database (https://www.ncbi.nlm.nih.gov/refseq/targetedloci/). We used these sequences to build an ITS index for the Burrows-Wheeler Aligner. We used BWA to map the reads of each beers from the fastq files to our ITS index. Subsequently, we counted the number of ITS per beer and per species and we kept only the species where we found more than 10 reads. We used R statistical software in order to plot the results and to perform the hierarchical clustering analysis with the ape library. Finally, we compared our results with the user friendly pipeline of One Codex using their targeted loci analysis. "

At any rate, I found the project interesting and hope they continue on with what was originally a kickstarter funded project. I'll attach the results they posted here so folks can take a look. As an aside, I found all this while looking up various protocols and methods for identifying yeast and bacteria as it's been a long time since I played around with DNA identification in college. I just enlisted some PhD's that work mostly with wine to help me investigate the pros and cons of wild sour beer fermentation locations. Figured I should get more up to speed but I'm very glad I don't have to be the final lab expert in these areas! Hopefully this source helps or at least you folks find it mildly interesting.
Cheers!
Peter
View attachment beer_analysis of yeast varieties in 39 Belgian Beers.pdf

isomerization

Just to clarify, S-04 is the Whitbread B strain (1098/007) and while it does produce more lactic acid than many yeasts, the amount is not enough to significantly change final beer pH. Moreover, T58 is very close to the Ardennes strain and is often recommended as a substitute for probrewers.
Per glycerol production, some yeasts do produce more, although a bigger impact on mouthfeel is residual sugar. TH beers often finish north of 1.012, sometimes around 1.015. That high FG would seem cloying, if not for a relatively high bittering charge.
Moreover, if TH is using blends of dried yeast, they are certainly not storing or reusing the yeast. The whole point of dry yeast in a production setting is that you don't need yeast storage or management equipment. And considering dry yeast mutates readily after one generation, it doesn't make sense to re use it.

xerotime

I've made plenty of hoppy beers that finishes between 1.014 and 1.017 with over 100 theoretical IBUs and tons of dry hopping that don't have the mouthfeel I get from Treehouse beers.

phillip092

***Red Square Update***
BOOM! I feel pretty confident that we have identified the 4th yeast that I've pulled out of TH dregs. I ran out of the previous DNA ladder I had been using, so the first two lanes are two different ladders, just FYI.

m4uzomtdxh4.jpg

My current hypothesis is that they are using CBC-1 (or possibly Fermentis F-2, I couldn't get my hands on that yeast w/o paying through the nose for shipping from the UK) during the dry hop/spunding portion of fermentation. This would then imply that the other 3 yeasts (S-04, T-58 and WB-06 like strains) go into primary (separately or not is still a question).
Shout out to @StinkyBeer for the suggestion of trying the CBC-1/F-2 type strains, I don't think I would have gone down that route ($5 a piece!) w/o the suggestion.

melville

Nice!!!