yeast – The Big Room (and the little things in it)

Sophic Suds

(Image: “When Beer Ruled the Earth” – you should really click through and read the caption that goes with it…)

A fundamental aspect of my personal philosophy is this: If you cannot play with something, you have not mastered it, and if you do not play with it, you will not master it.

I can sit here and read for hours, but it’s time I actually put my hands on some brewing again. I have a pound each of wheat and amber dried malt extract, an ounce of a low-bitterness (~2.9% alpha-acids) pellet hops, a packet of medium-attenuation dried beer yeast, two 39 millihogshead plastic containers that I can use as fermenters, an early-20th-century hand-cranked blower/bellows, a hot glue gun, a gallon of pineapple juice, some air-line tubing, a cabinet full of spices (including, of course, ginger), at least one small room air-filter, several pounds of honey, perhaps half a cup of granular erythritol, glycerol, a whole mess of glass bottles and bottlecaps, a variety of high-caffeine black tea bags, miscellaneous kitchen implements, a couple of copper-coated scouring pads, a selection of two-liter PETE bottles, iodophor concentrate, a hydrometer, a somewhat overstressed and twisted mind, a wife, four cats, and a dog. What shall I make?

I’m thinking I should aim for a mildly sweet brew with a ginger bite, perhaps adding a bit of tea or pepper if the sweetness needs balancing – but of course part of the goal of the exercise here will be to try to adapt to whatever I’m getting along the way…

Suggestions?

�ber alkoholische G�rung ohne Hefezellen

In my last submission to “The Giants’ Shoulders” blog carnival, we saw how the famous surgeon Dr. Joseph Lister deftly demonstrated definitively that fermentation processes were caused by live microbes rather than some sort of mysterious soluble substance that just happened to be associated with microbes. In today’s episode, we will see how Eduard Buchner definitely demonstrated that fermentation was caused by a soluble substance that was associated with microbes, and no microbes are actually needed.

Better still…they’re both right. “Wait…what?” Read on, O Seeker of Microbiological Knowledge, and be enlightened by this month’s entry: Continue reading �ber alkoholische G�rung ohne Hefezellen

Saccharomyces cerevisiae – Shameless Libertine!

I’ve been wondering about starting my own little yeast-breeding operation. I haven’t yet figured out where you can by the necessary teeny, tiny yeast-sized versions of the Implements of Extremely Impolite Probing that breeders of other species use, but even before that, I need to understand yeast reproduction better in the first place.

I had gotten an impression from some of the stuff that I’d read in a Genetics textbook and online that yeasts were normally haploid, and only became diploid briefly during mating (you see, when an “?” haploid yeast cell and an “a” haploid yeast cell fall in love, sometimes they’ll…). On the other hand, while reading a review of yeast virology[1], the author explicitly wrote that yeast cells are normally diploid. How to resolve this issue? Plus, I was wondering how, if I happened to have a pure culture of a haploid yeast, how could I tell if it was “a” or “?”?

I recently realized that there was one Dr. Bryk in the department where I work who specifically researches yeast chromosomes…so I asked her…

Continue reading Saccharomyces cerevisiae – Shameless Libertine!

“Top Ten Favorite Microbes” proto-meme…

Dr. Joseph over on the “(It’s a…) Micro World (…after all)” blog posted a list of his ten favorite microbes. After showing up in the comments of his post and being a wiseass about E.coli and Gram staining, the least I can do is participate here. Besides, it’s a great idea. Therefore here are ten of my current favorite microbes:

Continue reading “Top Ten Favorite Microbes” proto-meme…

Benzoic Acid Part 2: “Sour Stuff”

Okay, now that the boring review is over with…

Consider the cell. It doesn’t matter what kind of cell – bacterial, archael, fungal, animal, whatever. It’s still a tiny droplet of slightly salty water, thickened by a bunch of enzymes, other proteins, and various other substances floating around in the water. There’s also one other component that makes this a “cell” rather than soup: a bubble made of fatty material that the droplet is wrapped in, called the cell membrane. Depending on what kind of cell you’re thinking of, there may or may not be a “cell wall” made of some sort of rigid material, with the cell membrane inside of it. There may also be more than one membrane as is the case with the classic “Gram negative” style of bacterium, which has a second “outer” membrane wrapped around its cell wall. If it’s a eukaryotic cell, it’ll even have tiny little “organelles” inside itself wrapped in their own little membranes…but whatever. It’s the innermost one, inside of whatever cell wall may be there but wrapped around the cell’s guts, that we’re concerned with here.

Since stuff that will dissolve readily in water doesn’t tend to dissolve well into fats, and vice-versa, the cell membrane not only prevents stuff dissolved in the water inside the cell from leaking out, it also prevents stuff in the water outside from getting in. This lets a cell maintain itself at near neutral pH even if it happens to live in a very acidic environment, or an appropriate level of, say, sodium salts even if it lives in the Great Salt Lake.

This brings us back again to benzoic acid, which you should recall from the previous post alternates between a dissociated hydrogen-ion-and-benzoate-ion form and a combined, netural form in water. You may have noticed that foods preserved with benzoates tend to be sour, like fruit juices or soda. That’s because “sour” is the flavor of acid, and benzoic acid’s ability to be a preservative is only good in acidic environments

Useless Knowledge Break: the German word for acid is “Saurstoff”. Yes, that is pronounced like “sour stuff”, and no, that is not a coincidence.

An acidic environment means lots of extra hydrogen ions (“protons”) floating around. That also means that when a molecule of benzoic acid splits into a hydrogen ion and benzoate ion, it takes less time before another hydrogen ion comes by and the molecule can recombine again and therefore a bigger majority of the benzoate floating around at any moment is in the combined, somewhat fat-soluble neutral form. In that form, it can soak into a cell membrane if it encounters one.

If that molecule drifts through the membrane and gets to the inside of the cell, it may touch the less acidic watery environment there and dissociate into ions again and be unable to return through the membrane. The released hydrogen ions mean the inside of the cell becomes more acidic. As of today (20080806), the Wikipedia entry for Sodium Benzoate cites a single paper from the early 1980’s saying that when the inside of a yeast cell gets acidic enough, it prevents a specific step in the energy-generating process from working. This may be true, but there’s more to the story than this.

Obviously the membrane can’t totally seal the cell off from the outside, or the cell would be unable to excrete wastes or take in food molecules, so there are numerous specialized “transport” proteins that stick through the membrane to allow specific kinds of molecules in and out. Lots of biochemical reactions release hydrogen ions, so there are transport proteins that can shove hydrogen ions out of the cell and into the cell’s surroundings. The problem is that all substances naturally diffuse from areas of higher concentration to areas of lower concentration, so in an acidic environment the natural direction that hydrogen ions “want” to flow is into the more neutral cell. These transport proteins can shove the hydrogen ions in the opposite direction, but like pushing a boulder uphill it costs energy. This seems to be the primary reason that benzoic acid prevents bacteria and yeasts from growing – it makes them waste energy that they would be using for growth just to keep taking the hydrogen ions that the benzoic acid helps leak in through the cell membrane and shoving them back outside. The figure above is linked to a page at Helsinki university that discusses this type of preservative action in more detail.

Simple and elegant, and this seems to have been assumed to be the whole explanation for some time. But what happens to the benzoate ion when its hydrogen ion gets pumped away? Does it do anything?

Coming up next: Endocannibalism!

SHENANIGANS! Caffeine is our FRIEND!

Our new Asylum has real internet finally now and we’re getting settled in. The Houston area here is one of the most hot and humid areas of the US. All hot and sweaty. So of course I’ve been advised that my favorite psychotropic substance – 1,3,7-trimethylxanthine [“caffeine” for party-poopers who aren’t into the fancier names] – is no longer my friend, because it’s a diuretic that’ll dehydrate me, right?

NO! Shenanigans! Caffeine is our FRIEND! And that stuff about it being a diuretic? CRAP! LIES AND SLANDER!

But don’t just take my word for it. After all, humans are a bunch of freakish multicellular soft-celled eukaryotes, and I normally focus on normal organisms like bacteria, archaea, and yeasts. So, let’s ask some real human-physiology type scientists and check out their official peer-reviewed findings:

Armstrong LE, Pumerantz AC, Roti MW, Judelson DA, Watson G, Dias JC, Sokmen B, Casa DJ, Maresh CM, Lieberman H, Kellogg M: “Fluid, electrolyte, and renal indices of hydration during 11 days of controlled caffeine consumption.”; Int J Sport Nutr Exerc Metab. 2005 Jun;15(3):252-65.

“[…]The following variables were unaffected (P > 0.05) by different caffeine doses on days 1, 3, 6, 9, and 11 and were within normal clinical ranges: body mass, urine osmolality, urine specific gravity, urine color, 24-h urine volume, 24-h Na+ and K+ excretion, 24-h creatinine, blood urea nitrogen, serum Na+ and K+, serum osmolality, hematocrit, and total plasma protein. Therefore, C0, C3, and C6 exhibited no evidence of hypohydration.[…]”

Abstract on Pubmed

Armstrong LE, Casa DJ, Maresh CM, Ganio MS: “Caffeine, fluid-electrolyte balance, temperature regulation, and exercise-heat tolerance.” Exerc Sport Sci Rev. 2007 Jul;35(3):135-40.

“[…]This review, contrary to popular beliefs, proposes that caffeine consumption does not result in the following: (a) water-electrolyte imbalances or hyperthermia and (b) reduced exercise-heat tolerance.”

(Review article, apparently – Abstract on Pubmed)

Del Coso J, Estevez E, Mora-Rodriguez R: “Caffeine effects on short-term performance during prolonged exercise in the heat.” Med Sci Sports Exerc. 2008 Apr;40(4):744-51.

“[…]RESULTS: Without fluid replacement (NF and NF + CAFF), subjects were dehydrated by 3.8 +/- 0.3%[…]CONCLUSION: During prolonged exercise in the heat, caffeine ingestion (6 mg.kg body weight) maintains MVC and increases PMAX despite dehydration and hyperthermia. When combined with water and carbohydrate, caffeine ingestion increases maximal leg force by increasing VA (i.e., reducing central fatigue).”

(“NF” = “No Fluid replacement” – the “dehydration” mentioned here is due to exercising in the heat, and doesn’t appear to be related to whether the test subjects consumed caffeine or not)

Abstract on Pubmed

Scott D, Rycroft JA, Aspen J, Chapman C, Brown B:”The effect of drinking tea at high altitude on hydration status and mood.” Eur J Appl Physiol. 2004 Apr;91(4):493-8. Epub 2004 Feb 11.

“[…]Several markers of hydration status were also taken immediately pre and post each condition, including measures of urine specific gravity, urine electrolyte balance (K+, Na+), and urine colour. None of these measures indicated a difference in hydration status as a result of the dietary intervention in either the control or tea condition.[…]”

(In this study, the tea was the only caffeine-containing substance involved. The study group’s caffeine came solely from the tea. The control group got no caffeine at all.)

Abstract on pubmed

Paluska SA: “Caffeine and exercise.” Curr Sports Med Rep. 2003 Aug;2(4):213-9.

“[…]It[caffeine] is relatively safe and has no known negative performance effects, nor does it cause significant dehydration or electrolyte imbalance during exercise.[…]”

Abstract on Pubmed

Grandjean AC, Reimers KJ, Bannick KE, Haven MC.: “The effect of caffeinated, non-caffeinated, caloric and non-caloric beverages on hydration.” J Am Coll Nutr. 2000 Oct;19(5):591-600.

“[…]This preliminary study found no significant differences in the effect of various combinations of beverages on hydration status of healthy adult males.[…]”

Pubmed entry – full text available

See? Oh, I know what you’re going to say next – “But, like, dude! When I drink my Venti Mocha Crappucino [note: Link goes to “Foamy the Squirrel”, who is a bit of a pottymouth, ranting about the “Tall/Grande/Venti” nonsense.� It amused me.] or a can of Jolt Ultra I have to take a major whiz a little while later! Isn’t that ‘cuz of the caffeine?” Well, no, it isn’t. It’s because you just drank a bunch of liquid. Duh.

So, you see, caffeine really is our friend. Be nice to caffeine. But don’t feed it to your yeast in the presence of benzoic acid because it’ll kill them. See? I managed to turn this into a segue back to the stuff I was talking about before the whole “buy a house in Texas” thing started interfering. Stay tuned…

Very brief post…

Sorry about having another brief bout of blogstipation. We’ve finally managed to close on a house and now we’re probationary Texans (y’all). I’ve been spending the last week+ driving back to SE Idaho (by way of Best Friends, since we now have room to hire a second dog to hopefully keep Cornelia the Laser Dog company. I’ve got to lug 3 cats (and one goldfish in a small fish tank) a distance of 1600 miles or so starting in about 6-8 hours. Wish me (good) luck.

After the weekend, I should have some time to get back to the real posts. The Mountain Dew Wine was virtually unfermented when I returned, even after sitting there 10 days, but since I’ve gotten back it’s started going. Still slowly – the bubbler spits out 3-5 bubbles ever 40 seconds or so – but it’s going. It’ll be interesting to see what I end up with. I wanted to do 2-3 posts on the effects of benzoic acid on yeasts (that’s the preservative in Mountain Dew®), and I would swear I had one or two others in mind. Oh, yes, and an update on getting a Magellan GPS replacement that can actually be used – they seem to have located a slightly lower-end model eXplorist that they can send me. I sent them back their “walled garden”-based Triton 500, so the replacement unit ought to show up next week, I think. Their service has been pretty good, at least.

“A small modification of Koch’s plating method.”

Only two more days for the Classic Papers Challenge, so if I’m going to get any more up, I’d better get my butt in gear.

Here’s a nice easy one:

Petri, R. J.:”Eine kleine Modification des Koch�schen Plattenverfahrens.” Centralblatt f�r Bacteriologie und Parasitenkunde; 1887; Vol. 1, pages 279-280.

The American Society for Microbiology has a translation available online. It’s only about a page-and-a-half of relatively large type – check it out.

There’s a trick we microbiologists use for growing bacteria. You make a solid (but wet) surface that contains whatever nutrients the microbe (bacteria, archaea, yeasts, mold spores…) you’re interested in need, and then you spread a diluted mixture of the microbe on it. The idea is that since the surface is solid the microbes can’t move around too much, and at any spot where a single cell starts initially, a whole pile of that cell and it’s genetically-identical (non-sexually-produced) clone-children will form until it gets big enough to see without a microscope. This cell-pile is called a “colony”, and you can poke or rub it with a sterile object, then stick the object into a sterile nutrient source. The end result is you have a “pure” culture of microbes that are effectively genetically identical. The solid material could be a lot of things – I’ve seen references to using slices of potato – though these days agar-agar gel mixed with nutrients is the preferred substance.

Koch (that is, Robert Koch of “Koch’s Postulates” fame, not Ed Koch the former mayor of New York City) used gelatin (so, hey, here’s another thing you can do with your expired Jell-O®). He apparently used to have a stack of shallow bowls, and had to use a special pouring device to carefully dump the gelatin into each stacked bowl in turn, then cover the works with a bell jar in order to keep stuff from falling into them from the air and contaminating them.

This was kind of a pain to work with, so some clever guy named Julius came up with a modification of this method in 1887, using pairs of shallow dishes, one slightly larger than the other so that it could be turned upside down to use as a lid. Then, you don’t necessarily need the bell jar, and you don’t need to stack them so they’re easier to pour.

Julius Robert Petri’s idea was so useful that we still use it today. Oh, yeah, and they named the dish-and-lid combination after him.

How’s that for a “classic” paper?

Meanwhile, my “Mountain Dew� Wine” project is turning out to be substantially more educational and fascinating than I’d hoped. There seems to be a decent amount of information available on how benzoic acid affects yeasts. I intend to turn that into a post later, but first I’ll try to find at least one more old paper to post before tomorrow is over…

Another cheatin’ “Open Thread” and random stuff

No single topics to dominate a post today. I’m in a hurry (as usual lately) and have very little time. Tomorrow morning I’m back on the 1600-mile route back to Southeast Texas, hopefully to sign the closing paperwork on the house we’re trying to buy.

My Mountain Dew� Wine appears to be still sitting there after several hours. Either the benzoic acid is still inhibiting the fermentation (in which case it’ll go REALLY slowly) or the yeast is just in shock or something. We’ll see how it looks in the morning. I’ll leave it for a week or so anyway to see how it does. Meanwhile I’ll refrigerate the other batch of yeast culture until I get back. If I have to develop my own strain of “Mountain Dew Yeast” I will, dagnabbit!

I did get a chance to go for a quick walk in the Big Room on the way back from some errands yesterday, so it gives me an excuse to play with the wordpress map plugin again (RSS feed readers: the map doesn’t get inserted there. Please check out the interactive map at the blog’s website here.) Comments on the map (or anything else, really – I DID say “Open Thread” after all) are encouraged – what do you think? I’d like to do some audio content for points on a map at some point, too. Maybe some video.

Lava Rock Walk [height=560;width=560]

If anyone’s bored enough to want to see how I get from Southeast Idaho to Southeast Texas, I can post a map of that tomorrow, too…

I’m not a real hillbilly, but I play one on the internet…

My first “Will It Ferment” project is now in process.

See, I’m stuck with a somewhat unpredictable schedule and a need to travel frequently, cramped spaces to work in at the moment, and a streak of gustatory perversion that I just can’t help an urge to rebel against purists at the moment.

Quick bit of background on that latter statement: I’ve noticed that people seem to think there are only two-and-a-half kinds of “real” non-commercial fermented beverages. There’s beer (which is apparently defined as a strong tisane of hops, flavored by mixing it into fermented malt), there’s wine (which is always made of grapes of course), and out on the fringes of respectability is Mead (“Honey wine”) which seems to be slowly gaining some acceptance as a mildly exotic brew. The attitude is that anything else you might want to brew (say, a beer flavored and preserved with something besides hops, or a wine made out of anything but grapes) is probably some quaint “country” (i.e. hillbilly) thing for people who either live too far from civilization or are too poor to just buy a “real” beverage, or are too ignorant to know the difference. Either that, or it’s just some desperate attempt to make something to get drunk on. Might as well be making pruno.

The attitude kind of annoys me, so I’m trying to make a sort of “free person’s pruno”. I figure if the end result is as palatable as it could be, it’ll probably resemble “Zima�”.� Uh, no, I don’t expect it to be great – this is merely an experiment.� My must has an Original Specific Gravity (“O.G.”) of approximately 1.054, about the same as most lagers start out. As I write this, my gallon of must is on the stove in a stainless steel pot. I’m going to try to heat it to boiling and let it boil for a few minutes in hopes of driving off much of the benzoic acid in it, so as not to inhibit the yeast fermentation.

To your right, you should see the ingredients used in this project. Yes, those two bottles in the background are there on purpose. I’m trying to make…Mountain Dew� Wine.

Here’s the plan:

Last night, I took a couple of clean glass 1 Qt milk bottles, rinsed them with iodophor solution to sanitize, let them dry. Then, I put about 12 ounces of tap water (unchlorinated) into one and dumped the contents of both yeast packets you see in the picture into it to rehydrate.
The yeast packets were opened over a year ago – I don’t even recall what I did with the tiny amount of yeast I poured out. They’ve been sitting in the ‘fridge since then. What’s more, they had expired in December of 2006 to begin with.
The two yeasts (Red Star Montrachet and Red Star Flor Sherry) were both mixed into the same container in hopes that I could encourage a yeast orgy, giving me as much genetic diversity as possible from the two strains and maximizing the chance that I’d be able to get a culture which can grow in flat Mountain Dew and whatever benzoic acid (from Sodium Benzoate) might remain in it. I had read that V8 juice was actually an excellent medium for inducing yeast sporulation. Since sporulation occurs as a result of yeast cells mating, I made a huge leap of logic towards thinking the V8 might maximize my chances of getting some yeast mating going on.
After a few minutes to rehydrate, I dumped the 12-ounce can of V8 into the bottle and shook well. I also crushed up a children’s chewable vitamin (see bottle at lower-left) and a small portion (perhaps 1/5) of a capsule of the L-Arginine as a nitrogen source and added them as well. I then poured it back and forth between the two bottles a few times to aerate, then split the mixture between the two bottles, capped loosely, and went to bed.
As of this morning, fermentation was obviously occurring in the mixture, so sitting in my fridge open for over a year hadn’t killed off ALL of the cells. I opened the bottles and swirled to re-aerate, and added about a tablespoon of “corn sugar” (glucose a.k.a. dextrose) to wake the yeast cells back up. Since then, I’ve been pouring a bit of Mountain Dew right out of a third bottle into each of the yeast starter bottles every couple of hours. The amount of bubbling I see suggests to me that fermentation is still going on (and is not just from the carbonation of the Mountain Dew). Hopefully this will help the yeast culture acclimate a bit to the benzoic acid.
I dumped the other 2 2L bottles of Mountain Dew into a stainless steel 8-qt pot and heated to boiling, whisking with a steel whisk frequently to help get the dissolved gases to bubble out (hopefully along with some benzoic acid in the steam). At this moment, it’s up to 75�C (about 165�F) according to the thermometer I have stuck in it. It’s steaming a bit, and I can smell some of the citrusy aroma boiling off, unfortunately. I was afraid that’d happen. UPDATE: our ancient stove with one working burner seems to have trouble getting this much liquid above 200�F without cranking it up all the way and risking the bottom getting too hot. Since there’s a substantial amount of hot water vapor coming off, I’m going to hope that’s carrying away some benzoic acid and just let it start cooling down. I’ll stir it vigorously and frequently with the whisk until the temperature drops to about 160�F and then I’ll cover it for the night.
Meanwhile, before I go to bed, I’m going to recombine as much of the two yeast culture bottles as I can into one bottle, then rinse out the other. In that one, I’ll mix up about 12 ounces of tap water and enough corn sugar to reach a gravity of about 1.050-1.055. I’ll crush up another chewable vitamin and about half of the remaining arginine capsule into it, mix well, and warm it with a quick spin in the microwave (no exact measurements, just until it’s “obviously warm” to the touch after mixing). Then I’ll shake the V8 culture well to mix, and splash about a tablespoon’s worth into the new bottle.
In the morning, I’ll re-aerate the new culture and add a tablespoon of corn sugar to wake it back up, then once it’s going, I’ll start adding the now-cooled cooked flat Mountain Dew to it in small increments. Assuming it keeps going, I’ll dump in the remainder of the L-Arginine capsule, crush in one last chewable vitamin, and them combine the new culture with the rest of the cooked flat Mountain Dew in a nice plastic 2-gallon “water” container that I picked up (already rinsed with iodophor and dried). Cap it with a latex glove attached with a rubber band as described in the “Pruno” entry in Leon Kania’s “Alaskan Bootlegger’s Bible” (Click image for link) just because I thought it was the funniest airlock design I’d ever run into. Yes, I am easily amused.
If I’m lucky, there’ll be so much live and active yeast in the second culture at that point that the fermentation will finish quickly, because I’m driving to Texas the following day. Alternatively, I may be lucky and the remaining benzoic acid will slow the yeast down, so I can safely leave it fermenting (sitting in my sink in case of overflow while I’m gone) for the week that I’ll be gone.

If I’m UNlucky, either it won’t ferment at all (and I can then use it to try to develop a Mountain Dew Tolerant strain of yeast), or it’ll ferment but taste utterly disgusting (in which case I can use it to try to obtain some Gluconobacter strains and make Mountain Dew Vinegar), or it’ll be “infected” and will already BE Mountain Dew Vinegar, which would also be pretty funny anyway. So, other than completely unforseen results, this experiment can’t be a TOTAL waste.

[Update 20080727: Preliminary results of this perverse project may be read In this more recent post…]