Why Does It Work? – Page 2 – The Big Room (and the little things in it)

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!

Why Benzoic Acid Works: Part 1 – “Some Boring Review Material”

It’s about time I got to the long-promised post about benzoic acid. The thing is, I don’t want to assume everybody reading this is well-versed in chemistry or anything, so after much thought I’m going to split this into three posts. This first one is a bit of chemistry review for some topics that are important to how benzoic acid acts as a preservative. People who are bored by this or know more about it than I do are welcome to either wait for the next post or leave corrections or questions in the comments as you see fit. (Brief note to people reading this from the RSS feed – I’ve noticed that the stylesheet information doesn’t transfer with the RSS, so you won’t see where the web page view would indicate that there is additional information available for some of the terms here. Try hovering over various words and phrases in this post, though, and the information should pop up if it’s there…or just pop in at the main site and post questions if you have any.)

There are several ways people separate types of molecules into opposites. For example, ionic vs. covalent, polar vs. non-polar, or hydrophilic vs. hydrophobic. Although these three categories are each a little different from each other, they all relate to the same thing. As with all other chemistry, it all has to do with what the electrons are doing.

When atoms react with each other, they have a big fight over each other’s electrons. The reaction “finishes” (reaches equilibrium) when this custody battle is concluded. Each of the three categories above relate to how equitable the electron-sharing arrangment ends up being. Once the molecule’s atoms arrange themselves, if the custody of the electrons is distributed fairly evenly around the entire molecule, the molecule is considered “non-polar”. On the other hand, if the atoms at some corner of the molecule end up with more custody of the electrons than the other areas, the molecule ends up having an end that’s slightly more negatively charged (remember electrons are arbitrarily defined as being “negative”) than the others, and the molecule is “polar”. If you dissolve that polar molecule in water and the atoms remain together stubbornly clinging to the shared electrons, the molecule is considered “covalent” (“valence” refers to the area around atoms that electrons “orbit”), whereas if one or more of the atoms readily gains or gives up complete custody of one or more electrons and drifts away from the rest of the molecule, the molecule is considered “ionic”. (It amuses me to think of these latter two terms as “homoelectrical” and “heteroelectrical”. Yes, I am easily amused, why do you ask?) Plain old table salt is what you get when atoms of Sodium (“Na“) and Chlorine (“Cl”) get into one of these electron fights. If you were to look at a Periodic Table of Elements, take a look at the column way over on the left, with Sodium (Na) and Potassium (K) and so on. All of these have one electron that they just don’t really give a crap about. Way over on the other side of the table, one column over from the far right, you’ll see Fluorine (F), Chlorine(Cl) and so on. All of THOSE desperately want an extra electron (Chlorine is the third most electron-greedy – “electronegative” – atom, behind Fluorine and Oxygen). Stick Sodium Chloride in water, and Chlorine says “MINE!”, and Sodium says “Ah, whatever, who needs it?” and the gentle pull of the water molecules around them easily overcome the electric charge based attraction of the now positively charged sodium ion and the now negatively charged chlorine ion, and the two atoms drift apart.

This brings us to “hydrophobic” and “hydrophilic”. There’s a truism in chemistry that “like dissolves like”. Polar substances tend to dissolve well in other polar substances, and non-polar substances tend to dissolve well in other non-polar substances, but polar and non-polar substances don’t mix well at all. Water is a polar substance – it’s got an electron-greedy oxygen atom in between two comparatively electron-apathetic hydrogen atoms. What’s more, the two hydrogen atoms aren’t on exactly opposite sides of the oxygen atom. The “H-O-H” arrangement is actually bent (at just over 104�, if you care), so a water molecule ends up being slightly triangular, with one corner being a little bit negative (where the oxygen atom clings more to the electrons) and two corners with the hydrogens being a little bit positive. Any other molecule with a slightly-positive or slightly-negative part will find that part attracted to one side or the other of water molecules, and as a result will tend to be pulled out into the water as the molecules bounce around [i.e. it will dissolve]. On the opposite end of the scale, molecules with their electrons relatively evenly spread over them tend not to be soluble in water. Large molecules like fats are in this category, which is why fat floats on top of water rather than dissolving in it.

There are two other random facts that I need to wedge in here somewhere. First, the line between “covalent” and “ionic” is actually kind of arbitrary. Water is considered “covalent”, but a very small fraction of the times that two water molecules run into each other, they’ll hit just right so that the slightly-negative oxygen atom on one of them manages to attract one of the slightly-positive hydrogen atoms enough to make it leave an electron behind and jump over. When that happens, you end up briefly with a positively-charged “hydronium” ion (“H₃O⁺“) and a negatively-charged “hydroxide” (OH^–) ion. It doesn’t take too long for a “hydronium” to find a “hydroxide” again and rearrange back into two water molecules, but in pure water at “standard temperature and pressure” (defined as 25�C and one atmosphere of pressure) at any time there are about 620,000,000,000,000,000 hydroniums and hydroxides floating around in a liter of water – assuming I didn’t screw up my math there.

And, finally: a classical definition of an “acid” is something that “donates protons” (that is, hydrogen ions). In water, that means a molecule that provides extra available hydrogen atoms that water can pull off to form “hydronium” ions more often that water alone does.

And now, at last, we reach the subject of the preservative known as “benzoic acid”. If you read the ingredients lists of the food and drink you buy, you’ll probably never actually see “benzoic acid” on the label. Instead, you’ll see “sodium benzoate” or “potassium benzoate”. If you remember, sodium and potassium don’t really care about one of their electrons, so when you dump “sodium benzoate” in water, the sodium goes floating off to play with the water, leaving behind a negatively-charged benzoate ion with its electron. The extra electron hangs out around the part of the benzoate ion where the electron-greedy oxygen atoms are, making the molecule quite polar. Along comes a new “hydronium” ion, carrying a hydrogen that decides it misses its electron after all, and it jumps over to take over partial custody of the electron that the sodium left behind. In short, you’re going from Sodium + Benzoate + Hydronium + Hydroxide to Sodium + Hydroxide +…Benzoic Acid. (Plus a molecule of water, which is traditionally left out of these kinds of equations, which used to be the “hydronium”.) With the hydrogen attached and sharing the electron, benzoic acid no longer has so much of a charge imbalance and is a lot less polar. Being an acid, Benzoic Acid can also give that hydrogen ion back up again to a molecule of water – exactly the reverse of the reaction that formed it.

That’s the punchline to this: in water, a molecule of benzoic acid might at any one time be without it’s hydrogen and therefore charged/polar and hydrophilic, or it might have the attached hydrogen and be uncharged, relatively non-polar, and be comparatively hydrophobic…or “fat-soluble”.

Next post: So what?

“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…

“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.

Asterisk® is our Friend

I suppose this is slightly off the usual topics for this blog, but what the heck.

Asterisk� software is an open-source system for computer telephone stuff. Yes, I did just say “telephone stuff” instead of “PBX, VoIP, and Telephony”. Cope. Anyway, it’s an entirely legally-free (aside from the cost of a computer and any desired additional equipment) replacement for the kinds of many-thousands-of-dollars proprietary software systems that your cable TV and telephone companies use to prevent you from talking to human beings on the phone (so they can fire most of them, and outsource the rest to India or the Philipines or Florida or China or whatever other “developing” area has cheap low-grade labor). In other words, they seem to use their telephony system mainly for telephony prevention. The fact that “The Man�” uses the power of a PBX for evil in this way shouldn’t trick you into thinking that having your own is a bad thing, though. For example, my Minister of Domestic Affairs was recently in Australia for work. Since calls back to the US on the cellphone cost $1.50/minute, I set up a voice-over-IP client on her computer before she left. She could then use her computer’s internet connection to connect to the Asterisk box at no extra cost. The Asterisk box could then forward her Voice-Over-IP call out our residential phone line to my cellphone – a local call for the Asterisk box. No $1.50/minute for “The Man�”! Take that, The Man!

(Oh, “PBX”? That’s Private Branch eXchange. It’s a fancy way of saying it’s your own personal robotic Ernestine the Operator for your house or office.)

I discovered Asterisk a few years ago and have been puttering with it off and on. I figured if I wanted to learn how to use it, it’d be a good, simple start to replace my answering machine with it. It was a little trickier than I thought. I got my hands on a working “X100P“-type card, which is really just a specific variety of cheap voice-modem that was used originally for early development of Asterisk prior to the fancier hardware being developed. This connects my Asterisk box to the phone line. Like my old answering machine, it shares the line with an ordinary telephone that it doesn’t control.

Googling turned up all kinds of information on getting Asterisk to answer the phone and do all kinds of amazing tricks, but not a lot about controlling the answering in the first place. I wanted it to act like my old answering machine. It wasn’t answering the phone and taking messages that was hard to figure out, it was getting it to not answer the phone if someone in the house beat Asterisk to it.

I couldn’t find any references to this anywhere online at the time (and still can’t, actually, though they may be out there). Asterisk doesn’t seem to have any way – at least not with the X100P – to explicitly detect when another device picks up the shared line, but I came up with a workaround.

Now, when the lines starts ringing, I have Asterisk wait 11 seconds (which works out to about 3 rings) before doing anything. Then, I have it explicitly check for one more second to see if the line is still ringing. If not, the assumption is that someone picked up the phone and Asterisk leaves it alone. If it DOES detect one more ring, it picks and and carries on with whatever incoming-call magic I feel like programming into it – like detecting and saving incoming faxes. A copy of the relevant portion of my dialplan for any other Asterisk users out there who care may be found at the end of the post.

Once the house-hunting frenzy I’m in the middle of dies down, I’d like to start adding some nerdier features. For example, we’re moving to Southeast Texas, where there are occasional tornado warnings. Apparently, the National Weather Service’s warnings online contain embedded geographic information defining the boundaries of the warning area. I could have Asterisk watch the warnings page, and call my cellphone to tell me if I have to worry about tornadoes or not. (Kind of silly, I know…). It’d also be nice to finally test the fax reception that hypothetically is set up to work on my Asterisk box, too. (Dang crippled Motorola cellphones won’t let me fax despite supposedly supporting Class 1 fax mode, among other missing features…But that’s another post for another time.)

And now, the dialplan (or fragment thereof) (Update 20080523, fixed missing “]” after “[incoming”):

[incoming]
;give time to allow for someone to pick up 'regular' phone before asterisk does
exten => s,1,Wait(11)
;pause to check for one last ring, just in case someone picks up at the last second
exten => s,n,WaitForRing(1)
;So, you get 11 seconds - about 3 rings - to answer the phone.
;after that, Asterisk waits one more second for another ring.
;obviously if someone has picked up the phone before then,
;that last ring will never come and Asterisk will leave the call alone.
;otherwise, answer the phone:
exten => s,n,Answer()
;supposedly this will correctly jump to the fax extension if it's an incoming fax

;give announcement that ain't nobody here..
;(after waiting 3 seconds in case of fax tone detection)
exten => s,n,Wait(3)
exten => s,n,Background(nobody-but-chickens)
;...then go to 'leave a message' like a normal (if extremely powerful) answering machine
exten => s,n,Voicemail(9000)
exten => s,99,Hangup()
;end of line for now

Any questions?…

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

What really counts as a “microbe”?

Just a brief pre-post before the main one I’ve got brewing now (which will be posted either later today or tomorrow).

A tapeworm: Since when does 30-36 feet long count as 'micro'??? Microbiology is the dominating topic of this particular blog, but I don’t think I’ve ever addressed what I consider to really count as “micro”biology. This isn’t necessarily an obvious topic. My old “Microbiology” book from 8 years ago, plus the textbook from last year’s “Pathogenic Microbiology” class both contained large sections discussing organisms that are visible without a microscope. Heck, the “Pathogenic Microbiology” text even had a whole section on spider and insect bites. And, tapeworms? Since when is “over 30 feet long” considered “micro”? As I like to say: It’s time for Microbiology to grow up and move out of Medicine’s basement.

So: Here are the defining features of what I consider to be a “microbe”, at least for purposes of what I tend to discuss here on the blog:

Obvious: the organism cannot be effectively examined visually without a microscope and individual organisms can virtually never be observed by the “naked eye”.
In nature, a full normal population of a microbe can and will develop from a single live cell, and isolated individual cells are reasonably commonly observed.
Microbes do not “eat”.

It’s that last point that prompted me to write this post, mainly because it’s such an important part of why microbes work and how they affect their surroundings, especially when it comes to food microbes. What I mean by “do not eat” is that they are incapable of taking large (microbially speaking) chunks of material into themselves to use. Any cell nutrient for a microbe must be in the form of small molecules, like sugars, small peptides or individual amino acids, and so on that can be easily transported across the cell membranes and through the cell wall where applicable.

The importance of this is that for a microbe to grow on a complicated substance like meat or bread (for example), they have to excrete specialized enzymes that break down the substances out in the environment into simpler components like sugars or small peptides. If a microbe cannot secrete a protein-digesting “protease” enzyme, it can be surrounded by tasty, nutritious proteins and still starve to death. If a microbe can’t secrete an amylase (starch-digesting) enzyme, it doesn’t matter that starch is made of nice yummy glucose molecules because they’re all wadded up into long chains of starch that the microbe can’t get at.

And that, finally, is important because it brings up issues of growing multiple microbes together to accomplish something. Sake, for example, is made by fermenting rice, but rice is made primarily of starch. Saccharomyces yeasts don’t make amylases, so in order to make sake, you also have to add a kind of mold (Aspergillus oryzae, one of the types of white-mold-with-little-black-specks that you may see growing on the bread you’ve left sitting around for too long). A. oryzae is also a microbe and therefore can’t “eat”, but it does produce amylase. Since the amylase is breaking down the starches outside of the cells, this means the released glucose is also available for the yeast to use.

Admittedly, my definition above isn’t perfect. On the one hand, it leaves out protozoa (like amoebae and the well-known Paramecium, both of which actually do take in “chunks” of food, but both of which most people would normally consider to be “microbes”. It also leaves IN things like mushrooms, which are not usually thought of as being “microbes” by people who aren’t microbiologists. And, of course, it leaves me with no excuse not to go and learn something about eukaryotic (“plant”) algae (as opposed to bacteria-algae, a.k.a. cyanobacteria) and diatoms. Suggestions for updating my definition may be left in the comments…

Just something that came up while I was assembling what will be the next post. Stay tuned.