Freakish Eukaryotes – Page 3 – The Big Room (and the little things in it)

“A simplified method of staining endospores”

One more for the “classic papers” challenge:

Schaeffer AB, Fulton MD: “A Simplified Method of Staining Endospores”; 1933; Science; 77; pg 194

If you take a microbiology lab, this is the endospore staining technique (or “technic” as they used to spell it) that you’ll practice. This is a nice, simple, one-page paper. Alice B. Schaeffer and co-author Mac Donald Fulton describe a few of the other variations on endospore staining techniques, then describe how they’ve further simplified what they felt was previously the simplest one, described by a Mr. Wirtz in 1908.

“Endospores” are a sort of “escape pod” for certain specific kinds of bacteria. Unlike spores formed by yeasts and molds, these are not reproductive – each bacterium only produces one thick-coated spore, into which it shoves it’s genetic material and a few vital enzymes to get itself going again later when the spore finds itself in favorable conditions.

Since only a few kinds of bacteria produce these endospores, if you see endospores in your unknown bacterial culture it goes a long way towards helping to identify the bacterial species, so having a simple method for staining your bacteria so that endospores are obvious under a microscope is helpful. (Of course, these days most of us would rather just get a 16s rDNA sequence with PCR, but never mind that for now…)

Evidently, Wirtz’s original method involved using Osmium Tetraoxide (“osmic acid”) to stick the bacteria to the slide before staining. Not only is that stuff poisonous, it’s also expensive. I found a site selling sealed glass ampoules containing 1 gram each of this stuff for $35.00 each. Schaeffer and Fulton’s method does away with this in favor of much cheaper and easier heat-fixing (just as is done with the Gram stain and others). They use the dye “Malachite Green” for the initial stain, and steam-heat the dye-covered bacterial slide a few times to sort of “cook” the dye into the thick-walled endospores if they are there. Rinsing then washes the dye out of everything but the endospores, and a light red dye (safranin) is added as a counterstain. The end result is that under the microscope you’ll see light-red bacteria. If any of them form endospores, you’ll be able to see them as smaller green dots – sometimes still bulging inside of bacterial cells, sometimes floating around freely having escaped from the now-empty bacterial cell.

The “Schaeffer-Fulton Endospore Stain” is pretty easy to do, though the occasionally messy steambath part can be annoying. The method is pretty resistant to errors, so it’s not too hard to get good results even if you’ve never done it before.

Incidentally, you can buy Malachite Green at many pet stores – it’s still used as a treatment for “ick” (Ichthyophthirius infestation) in tropical fish.

Hmmm…still a couple of hours before it’s not longer May – Perhaps I can throw in one last post before time’s up…

“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…]

“They laughed at me! But I’ll show them all! AH, HAHAHAHA!”

Another T-shirt to add to my list of T-shirts I want.

I’m spending more hours shoveling my way through the books and papers and crap we’ve got up here at House v1.0, since if all goes well I’ll be making a brief run back down to Southeast Texas so we can sign the papers for House v2.0 down there, at which point we’ll be able to start actually moving. I sure hope this one goes through. Not only is it our third attempt to buy a house down there, but I’ve already identified a convenient location to build my “Intentional Food Microbiology” brewlab in it.

Since there’s no way I can afford to buy a -80�F freezer, I have an obvious interest in alternate means of preserving the yeast, mold, and bacterial cultures that I want to keep. To me, drying seems like the most desirable method when it’s feasible, since dried cultures should require the least amount of maintenance. After a several-month delay, I’ve finally gotten around to getting back in touch with the archivist at Brewer’s Digest to see about getting an old article on the viability of dried yeast cultures[1].

Speaking of old but useful scientific papers, there’s an extremely nifty challenge going on through the month of May (deadline: May 31st) over at “Skulls in the Stars” blog: find a classic scientific paper, read it, and blog about it.

“My �challenge�, for those sciencebloggers who choose to accept it, is this: read and research an old, classic scientific paper and write a blog post about it. I recommend choosing something pre- World War II, as that was the era of hand-crafted, �in your basement�-style science. There�s a lot to learn not only about the ingenuity of researchers in an era when materials were not readily available, but also about the problems and concerns of scientists of that era, often things we take for granted now!”

I think this is a brilliant idea – the classic papers often seem to be forgotten and often explain things that people seem to take for granted these days. I already mentioned my post about the Gram Stain (original paper published in 1884), though that post really talks more about what has happened with the Gram Stain over the last 125 years rather than only being about the original paper. There are a couple of other classic microbiology papers that I’m going to try to get to if I have time before the May 31st deadline arrives.

I also need to get some yeast activated and get my must processed – I’m hoping a brief boil will reduce the amount of a yeast-inhibiting substance in it. I’ll post more detail after I get it going.

[1] Wickerham LJ, AND Flickinger MH:”Viability of yeast preserved two years by
the lyophile process.” 1946; Brewers Digest, 21, 55-59; 65.

I finally got to Bristol Brewing Company…

Signage in front of the Bristol Brewing Company After spotting the company’s mention in Jeff Sparrow’s “Wildbrews“, I’ve been wanting to visit Bristol Brewing Company, particularly since they may be the only such brewery using local yeasts and bacteria that they isolated themselves from their local environment.

Since we’re moving from Southeastern Idaho to Southeastern Texas, I’ve been going back and forth between the two location. It just so happens that along with New Belgium Brewing Company, Bristol Brewing is actually right along a convenient route between the two locations. (Let’s see if I can figure out how to work the Google Maps plugin):

How’s that look? How does it work? (Those of you reading this on the RSS feed: The interactive map only appears on the website, it would seem. Please check it out here.) I made the KML file myself…

Bristol Brewing is a cozy little brewpub, and the people there are encouragingly helpful. They were in the middle of bottling, so there were no tours. Also, there were not currently any of the skull-‘n-bones beers available. However, I did get an educational series of tastes of their current brews on tap (thank you to the employee I spent most of the time talking to whose name I’ve embarrassingly forgotten, but who I believe was Tad Davis judging from the photos on the web site). I also lucked out and their microbiologist, Ken Andrews, happened to be there. I asked about their native Colorado brewing flora. Turns out Bristol Brewing isn’t quite as bold as I originally thought. They’re still doing their primary fermentation with “normal” brewing yeasts. What they’ve done is inoculated some wine barrels with the locally-isolated yeasts and bacteria, and they use the barrels for a secondary fermentation and aging instead. Much safer if you have to worry about having a drinkable product at the end, and of course it makes me feel like more of a crazed rebel for wanting to isolate my own local bugs for the main fermentation. So, a great visit overall.

Incidentally, it seems they’ll be tapping a new Skull-‘n-Bones brew in a couple of weeks, at 5pm on Tuesday, May 27th (2008). It sounds like they’ll probably have some available for a few days before it all disappears, so I’m hoping my return to Texas can be timed such that I can swing by and at least get a taste.

WANT: “Teamaker” hops

Just a brief “aw, crap, has it really been over two weeks since my last post?” post, really, but I thought this was interesting.

It would seem that there’s a variety of hops that’s been registered recently known as the “Teamaker” variety. It’s got all the magic bacteria-stopping power of a hops plant, but composed of almost entirely the non-bitter component. I’m not sure how hard it would be for me to get them to send me a plant or two to evaluate it’s usefulness for yeast cultivation (as an anti-firmicutes antibiotic) and for controlling the growth of bacteria in fermented foods and drinks…

House-hunting (“Yep, these are house droppings all right. Fresh ones too…” [everybody’s seen that Monty Python bit, right?]) in southeast Texas and the related travel (both in the area here and between here and the other abode in southeastern Idaho) is eating my life at the moment, but I’ll try not to neglect the blog so much.

More to follow.

Boosting fermentation with science

All right then – I’ve got five pounds of honey, a pound of frozen cherries, packets of a couple of different dried yeasts, miscellaneous other potential additives, two 2-gallon polyethylene terphthalate fermentation containers with screw-top lids and spigots, several feet of aquarium airline tubing and connectors, silicone sealant, and miscellaneous kitchen gadgets (including a hydrometer). Now it’s time to discuss what I’m about to do and fish for comments and criticisms before I jump into it.

My goal here with this brewing experiment is a quick primary fermentation. And to compare the results from two different yeast strains, uh, TWO goals, quick fermentation, yeast strain comparison, and fermentation container design. THREE goals. Quick fermentation, comparing yeast strains, fermentation container design, and to try to keep the yeast cultures from dying off too quickly during the fermentation. FOUR. Four goals…

In this post, I’ll stick to talking about what I’m putting into the brew and how I hypothesize my additives and process with speed the fermentation along and help keep a large portion of the yeast viable during the primary fermentation.

Actually, the health of the yeast populations and the speed of fermentation are overlapping goals; more cells remaining alive and healthy means more cells simultaneously chewing up sugars and spitting out ethanol for me, resulting (hypothetically) in faster primary fermentation. In this experiment, I’m going to be focussing on nutrients and spices that are reported to benefit yeast activity. Here’s the process I am currently planning to follow, focussing primarily on the fermentation-boosting parts:

I’ll boil the 5 pounds of honey with enough tap-water to make about 2 gallons of must, adding the frozen cherries sometime after the boil gets underway.

Fermentation boost: we have water so hard that you have to wear a helmet to take a shower. (Joke stolen from my Environnmental Chemistry instructor, so you can blame Dr. Rosentreter for that one). It’s loaded with Mg²⁺ and Ca²⁺, which seem to be able to help the yeast to produce ethanol faster and survive higher ethanol concentrations better[1][2] as well as just being general nutrients[4].

Two approximately �-liter amounts of the must will be put into clean glass quart bottles and used to develop the initial yeast culture for pitching (each one for a different strain of yeast).

Fermentation Boost: Growing up a large population of yeast from the dried yeast packets before pitching will give me a faster start. In addition, the large headspace and the use of cloth rather than plastic or rubber covering of the top will allow oxygen to get into the starter culture, helping it to develop more quickly and in a more healthy fashion (i.e. a larger proportion of healthy, viable cells).

Nitrogen supplementation: Capsules of arginine picked up cheap at a certain big-box store will be added to the yeast starter.

Fermentation Boost: “Free Amino Nitrogen” is perhaps the most important bulk nutrient for yeast, and arginine seems to be the preferred amino acid source[3][4], presumably because it contains the most reduced nitrogen per molecule of the amino acids. I actually want to try to develop a process for using dry milk powder instead, but achieving sufficient hydrolysis of the milk proteins looks like it’s going to take some development on my part. For now I’ll “cheat” and use arginine instead.

Vitamin supplementation: A single well-crushed children’s “chewable vitamin” (“Flintstones�” or generic equivalent) will be added to each starter culture as well.

Fermentation Boost: Pantothenic Acid (Vitamin B5), Inositol, trace minerals, and small amounts of additional potassium and phosphate to supply vital nutrients to the yeast culture.[4]

Fermentation-enhancing spices: I will be adding ground ginger and cinnamon (actually cassia) to the must near the end of the boil.

Fermentation Boost: In addition to providing what I think will be excellent complementary flavors to the final product, it appears that even fairly large amounts of these two spices – among others – provide a boost to fermentation rate[5] (via Shirley O. Corriher’s “Cookwise”[6]) of Saccharomyces cerevisiae cultures. If I’m doing the conversions appropriately, the peak fermentation boost for ginger works out to something like 3 tbsp of ground ginger per liter, or something like (very roughly) 10 tablespoons per gallon. I don’t plan to add quite so much, but a couple of tablespoons of each spice in the two-gallon batch ought to provide some nice flavor while still hopefully providing a boost to the fermentation rate as well.

“Cinnamon”: In the US, the rust-colored stuff labelled “Cinnamon” is not, actually, cinnamon. True cinnamon (Cinnamomum zeylanicum)is actually tan in color. What you get in the US when you buy a bottle of “Ground Cinnamon” actually comes from Cassia (Cinnamomum aromaticum), a closely related plant. Realistically, as far as I have been able to find out so far, there’s not likely to be a huge difference in the active components or flavor. While I haven’t yet gotten my hands on a copy of the old article from Cereal Chemistry[5] mentioned above, I’d give good odds that the “cinnamon” used in the study was also actually cassia anyway.

There’s one more thing that I hypothesize would help promote my goals that could be added: small amounts of oxygen[7] (say, less than 13% O₂, or very roughly speaking, around half of the normal atmospheric concentration or less). However, I’m still trying to work out an easy way to achieve this automatically and am not yet ready to try it. Besides, this is already pretty poorly-designed for a “real” scientific experiment as it is, considering the number of variables that are really contained in this brewing process. Really, my hypothesis here boils down to a relatively vague “This mixture and process will allow me to finish the primary fermentation within a day or two of pitching”. If I ever have opportunity to do serious experimentation on this, it’ll require setting up a large number of separate fermentation reactions to assess the effects varying each individual set of hypothetically-fermentation-boosting additives. Hopefully one of these days things will settle down enough to let me try it.

If anybody sees anything stupid (or just interesting) up there, please say something…

[1] Dombek KM, Ingram LO: “Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation.”; Appl Environ Microbiol. 1986 Nov;52(5):975-81.
[2] Nabais RC, S�-Correia I, Viegas CA, Novais JM: “Influence of Calcium Ion on Ethanol Tolerance of Saccharomyces bayanus and Alcoholic Fermentation by Yeasts.”; Appl Environ Microbiol. 1988 Oct;54(10):2439-2446.
[3] Carter BL, Halvorson HO: “Periodic changes in rate of amino acid uptake during yeast cell cycle.”; J Cell Biol. 1973 Aug;58(2):401-9.
[4] Fugelsang KC, Edwards CG: “Wine Microbiology – Practical Applications and Procedures (2nd Ed.)”; 2007; Springer Science+Business Media LLC; pp 15-18
[5] Wright WJ, Bice CW, Fogelberg JM: “The Effect of Spices on Yeast Fermentation.”; Cereal Chemistry. 1954 Mar;Vol.31,100-112
[6] Corriher, SO: “Cookwise”; 1997; HarperCollins Publishers, inc; New York; pp 69-70
[7] Nagodawithana TW, Castellano C, Steinkraus KH: “Effect of dissolved oxygen, temperature, initial cell count, and sugar concentration on the viability of Saccharomyces cerevisiae in rapid fermentations.”; Appl Microbiol. 1974 Sep;28(3):383-91.

Fermentation: not just for alcohol

What does gluconic acid taste like, anyway?

Well, that was an interesting reminder. I’m tracking “fermentation” on Twitter, and caught a random reference to an interesting fermented beverage being made in Germany. The “reminder” I drew from this serendipitous reference was that “fermentation” doesn’t necessarily mean alcoholic fermentation.

“Fermentation” seems to be slightly tricky to define accurately. Most definitions seem to directly mention alcohol production from sugar, but this is only an example and not a definition. I’ve also seen the term used to mean simply “to grow a culture of microorganisms” (because the tank they are grown in can be referred to as a “fermentor”.)

Properly speaking, fermentation is what you get when you have microbes growing under conditions where the elelectrons that get sucked away from “food” molecules like sugars ends up on another, simpler carbon compound rather than something like oxygen, and therefore fermentation is implicitly anaerobic although that’s not the same as saying that fermentation cannot happen in the presence of oxygen (e.g. the Crabtree Effect, and of course fermentation of ethanol to vinegar requires oxygen). The end product is generally assumed to be organic acids (like acetic acid [vinegar]) or alcohols, and carbon dioxide. So, making beer and wine is fermentation. Making vinegar is fermentation. Making yogurt (lactic acid) is fermentation. Citric acid can be made by fermentation of glucose by Aspergillus molds, as can malic (apple) acid (see US Pat#3063910). You can make tartaric (grape) acid from glucose by fermentation as well (see US Pat#2314831).

I am familiar with the flavors of all of those products. One I’ve never directly tasted is gluconic acid, which is the main product of the fermentation process used to make “BIONADE�” (it seems to be written in all-caps everywhere).

According to their English-language page discussing their process – linked from the image at right, click to view – they are starting with malt, just as one would for beer, but instead of Saccharomyces yeasts, they are fermenting this wort-like liquid with “acid bacteria”. I’m going to hazard a guess that the bacterium in question is a strain of Gluconobacter oxydans or one of its close relatives. This group of bacteria is in the Acetobacteraceae family of bacteria which is involved in turning your wine into vinegar. It would appear that under the right conditions, the enzyme Glucose Oxidase (EC 1.1.3.4) produced by G.oxydans converts glucose to a compound which reacts with water to form gluconic acid. BIONADE� then adds flavor extracts and juices to the filtered fermentation product, carbonates it, and bottles it.

Not being familiar with the flavor of gluconic acid, I’m aching to get my hands on some of this stuff and try it.

For another example of a relatively non-alcoholic fermented beverage, see also Kombucha, which is essentially sweetened tea fermented by acetic-acid bacteria and non-Saccharomyces yeasts…which I also have yet to taste.

geostr:50.4600,10.2208:200804110105-06:geostr (at least if Google Maps interpretation of the address I could find at the moment is correct, and assuming the information I dug up and my interpretation of it is correct, this should be the approximate location of the brewery responsible for BIONADE� production.)

The care and feeding of Saccharomyces

Let me pause now for a moment to review what I’ve learned so far:

Yeast are filthy little jerks

No, seriously. I’ve previously reviewed their promiscuous sex lives,
their sexually-transmitted diseases, and their toiletry habits. Somehow, though I still want to do more brewing, so let’s continue.

Bag of 'Parodina Yeast Chow'. I am not affiliated with Purina Mills corporation! This image is PARODY!

Yeast need to be fed particular sugars

The three major elements needed by pretty much every living thing for “food” are Carbon, Nitrogen (as reduced “amino” nitrogen), and phosphorus (as oxidized phosphate) (Reduced sulfur is also needed in small amounts for proteins). Glucose (“dextrose” or “corn sugar”), fructose, or sucrose (“table sugar”, each molecule of which is made of a molecule of glucose attached to a molecule of fructose) are all used as carbon sources by Saccharomyces yeasts. Possibly also Galactose under certain conditions[1]. Saccharomyces yeasts don’t appear to be able to use lactose (“milk sugar”, each molecule of which is made of a molecule of glucose and a molecule of galactose), so some recipes include lactose in order to ensure there is some residual “sugar” in the mix at the end, for flavor and “body”.

Yeast need reduced nitrogen (amino nitrogen or ammonia…or urea)

Aside from sugars, this seems to be possibly the most important yeast nutrient. The most
“natural” source of this nutrient would seem to be amino acids or very short peptides (2-5 amino acids long). Apparently urea (carbamide) also makes a good yeast nutrient, but:

You don’t want TOO much nitrogen available to the yeast, or there’ll be excess urea dumped back into the brew

This could combine with the ethanol to make “ethyl carbamate”, which is considered
a probable carcinogen, at least if it’s present at a high enough level. Obviously if you use urea as a
yeast nutrient, that’s only going to increase the possibility of a problem.

Saccharomyces yeasts are effectively incapable of using proteins for nutrition.

Proteins can be a source of amino nitrogen (and carbon and sulfur), but like all real microbes, yeast cells cannot just “eat” chunks of protein. They have to be broken down into very small chains of amino acids or even as individual amino acid molecules before the yeast can suck them up and use them. Saccharomyces yeasts do not appear to normally excrete protein-digesting enzymes, so by themselves they cannot make any use of protein for nutrition[3].

Yeast need oxygen

Oxygen is necessary for making certain components of the cell membrane, in addition to it’s more obvious role in respiration. Without a way to replace used up membrane components, the yeast stop reproducing and eventually fall apart and die. There seems to be some suggestion that to a certain extent one can substitute some raw membrane material for oxygen here (either as “yeast hulls” or possibly even certain of the natural waxes on some fruits).

If you give yeast oxygen, though, they consume the sugars entirely instead of making alcohol…

…or do they? Between the “Crabtree effect” (when there are high concentrations of glucose, alcohol production continues even in the presence of oxygen) and indications in scientific papers[2], it seems SMALL amounts of oxygen may not be a problem, and might very well be beneficial.

Yeast need vitamins and minerals

B1 (“Thiamine”) is commonly mentioned, though apparently the need for it varies from strain to strain. Also potentially important are Pantothenic Acid (B5), Niacin (Nicotinic Acid, Vitamin B3), Biotin, Inositol, as well as Potassium, Magnesium, and trace amounts of calcium and a few other minerals[4].

Unhealthy yeasts are more prone to make (EEK!) Off-Flavors and Off-Odors (EEK again!)

For one thing, it seems to be a general rule that you don’t want your brew sitting on the corpses of dead yeast (the “lees” of wine, or “trub” of beer), because that is a potential source of (insert dramatic music and crash of thunder here)Off-Flavors and Off-Odors. Yeast dying and falling apart is also a major source of urea being dumped into the brew, too. Some strains of yeast under certain conditions, such as insufficient pantothenic acid, may be prone to producing nasty-smelling sulfides as well.

So, in most cases what we want to do when brewing is keep our yeast as alive and happy as possible, and get them to hurry up and finish our primary fermentation before they start dying off. Coming up: My (as yet untested) plot for accomplishing this – without specialized scientific equipment or materials.

[1] Wilkinson JF: “The pathway of adaptive fermentation of galactose by yeast” Biochem J. 1949; 44(4): 460�467
[2] Nagodawithana TW, Castellano C, Steinkraus KH: “Effect of dissolved oxygen, temperature, initial cell count, and sugar concentration on the viability of Saccharomyces cerevisiae in rapid fermentations.” Appl Microbiol. 1974 Sep;28(3):383-91.
[3] Bilinski CA, Russell I, Stewart GG: “Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects.” Appl Environ Microbiol. 1987 Mar;53(3):495-499.
[4] Fugelsang KG, Edwards CG: “Wine Microbiology: Practical Applications and Procedures” 2007; Springer Science+Business Media LLC, New York; pg 17