Distillation Daydreams

I daydream about recreating human technology on my own. I've thought about it basically every damn day for years. I don't know how to stop. Maybe writing about some of it will help? It never has before. Maybe I'm just spreading bad thoughts by writing this. Consider not reading this if you don't want to daydream about recreating human technology on your own, every damn day for the rest of your natural life.

Lots of wonderful things can be made or purified through distillation. Some examples:

* Ethanol. If you let yeast ferment sugars into alcohol, you can concentrate the alcohol. Whiskey, brandy, rum, mezcal, vodka, et cetera. Also, you can let your fermented sugars spoil to get acetic acid and distill that out if you like.

* Essential oils. If you boil water and there are a bunch of flowers or pine chips or other plant material in the way of the steam, the steam can absorb some terpenoid oils, and condensing it down lets you make perfumes. Or home remedies for autism or something? I don't know what foolish people do with essential oils. Just use it for perfume. Or use it to flavor your vodka to make gin or limoncello or vanilla extract. But a little bit goes a really long way.

* Mineral acids. A bunch of sulfate minerals can be heated/roasted to produce sulfur trioxide vapors, which can be condensed in water to produce sulfuric acid. The traditionally used sulfate minerals (vitriols) aren't globally uncommon, but it's not like you're going to find them in quantity in your backyard or a river bed or a roadcut. The vitriol minerals are all sulfate salts of heavy transition metals (copper, iron, cobalt, zinc). The more common sulfate minerals on the earth's crust are evaporite deposits of sulfate salts with light alkaline metal cations. I think gypsum (hydrated calcium sulfate) is the most common sulfate mineral. Alchemists didn't use it to make sulfuric acid, but I've heard of it being done in modern times, so maybe it's just difficult? In the 1970s, the Marchon Chemical Works in Whitehaven, UK made sulfuric acid and calcium silicate by roasting gypsum with siliceous rocks. I think it required very high temperatures., since calcium sulfate and sodium sulfate both have thermal decomposition temperatures around 1100 deg C. Eventually, Marchon gave up on gypsum and switched to roasting elemental sulfur, but still, gypsum is some kind of an option, if not necessarily a good one. Other alkaline sulfate minerals found in evaporite deposits include epsomite (magnesium sulfate), baryte (barium sulfate), and thenardite/mirabilitie (sodium sulfate). Alum minerals (double sulfate salts that have both aluminum and an alkaline metal as cations) are also sulfates, and I think they're intermediately common between alkaline sulfates and transition metal sulfates. I don't know how hard it is to make sulfuric acid out of alum by heating. It's probably easier to make sulfuric acid from most of these by electrolysis than by distillation, but that's not an option in the first stages of my fantasy. Whatever the source, once you have made sulfuric acid, you can mix it with other minerals and then distill the mixture to make new exciting mineral acids .Sulfuric acid plus table salt, distilled, gives hydrochloric acid. Sulfuric acid plus nitrate minerals, distilled, give nitric acid. Glauber figured both of those recipes out. Thanks, Glauber. Sulfuric acid combined with with phosphate minerals gives phosphoric acid. Et cetera. It's a very useful chemical, that sulfuric acid. Also you can make sulfuric acid by roasting elemental sulfur or metal sulfide ores, instead of roasting sulfate minerals, but there are some extra steps. It's called the Lead Chamber Process and you have to control the mixing of three gasses and a sprayed liquid. It's not the easiest way to start out your technology recreation fantasy.

* Wood liquors. If you heat up wood in a low oxygen environment (pyrolysis, pyrolytic decomposition, destructive distillation), then gasses and vapors come off and you're left with charcoal. If you condense the vapors, you first get pyroligneous acid, which, besides water, is mostly made of acetic acid and methanol, and a decent amount of acetone. I've read that the distillation of cellulose gives acetic acid and the distillation of lignin gives methanol. Super interesting if true, right? But maybe a little misleading in its simplicity; cellulose is a polymer made of glucose monomers. When you heat it up to thermal degradation in a low-oxygen environment, acetic acid might be the first volatile to distill over, but lots of caramel-like stuff with a higher boiling point is being made in the pot. Likewise, lignin is an irregular polymer made of aromatic alcohols, and methanol might be the first and the main volatile given off by the pyrolysis of lignin, but it's not the only one. Anyway, acetic acid, methanol, and acetone all have some uses, and you can separate them out by fractional distillation (i.e. redistill the mixture with more precise temperature control and with a taller column in the still to encourage reflux). This whole post came about because I was looking into low-tech ways to get methanol for use in biodiesel transesterification. In addition to those three components, your pyroligneous acid will contain a million other random smoky caramelly tar-like organic contaminants in small amounts: formic acid, furfural, pyridine, methyl ethyl ketone, the list just goes on and on. As you keep heating up the wood, tar will start to come over with and then after the pyroligneous acid. Or I guess it could all come over at the same time, if you heat things up quickly or if your still doesn't have any real height for refluxing. If you let the distillation products cool and settle, the tar will be the viscous part at the bottom and the pyroligneous acid will be the runny part at the top. Tar is another wood liquor with some uses - like preserving ship timbers from rot -  although I mostly think of it as contaminant when trying to distill more valuable things. Similarly to distilling wood, if you distill pine resin, you can get turpentine.

* Ammonia. Lots of organic matter has amino acids. Animal tissues in particular have a lot. Amino acids can be distilled to make ammonia gas. Condense it in water and you've got ammonium hydroxide. It's good for cleaning, it's good as fertilizer, it's a decent refrigerant, and it can be used to make hydrazine rocket fuel. And ammonium hydroxide is a convenient base in chemistry and it's in hair dye and other stuff. Lots of uses.

* Low boiling point metals. Some metal can be boiled/vaporized pretty easily and separated from their ores that way. Mercury and lead are pretty easy to distill off of their ores, for example. And at higher temperatures,  you can do more metals. It's pretty cool.

* Petrochemicals. If you have crude oil, which some places in the world do, you can distill it to separate out hydrocarbons by boiling point. Now you can use hexane for acid-base extractions or make candles out of paraffin wax or other things. I hear gasoline has some uses. You can make stuff that like. Petrochemicals can also be made by the Fischer-Tropsch process, although that's not something we do till a much later stage in the tech fantasy.

So there are all these amazing things you can get by heating up plants and rocks and animal tissue in a vessel and condensing the vapors. But how do you do actually distill those things?

"You buy a laboratory glassware distillation apparatus online." No. We're recreating technology on Mars or in the distant past or something. You don't get a glassware supplier. 

"You buy a... copper still online?" Still not quire there.

"You make your own copper still!" Much better! But I'm not very good at metal working, and also, where are we getting all this copper from? Have you ever mined copper? Do you know where to mine  copper? That's not a real plan. 

Honestly, it is a start of a plan. We could make aqueducts to hush the loam from the countryside, revealing bedrock, and then melt ores by fire-setting, maybe. Or we could crush rocks down by a river with a waterwheel and a hammer on a cam follower and then separate out the crushed rock according to density by gravity methods, and in that way find some heavy metals. I don't mean to say that mining isn't possible. But it's not easy and you don't have any guarantee of what you'll find in what places, and it's not usually one of the early steps in my primitive technology daydreams.

The low-tech way to distill things on your own, without a global economy that can extract copper from distant lands and transport it to you, is to make a still from clay. Clay is easy to find, in the distant past, or in your backyard, or even on Mars - all the usual daydream locations - and it's moderately easy to work in a primitive setting, especially compared to metal or glass.

It's a little dangerous to distill alcohol in clay, because the clay could shatter and now you're suddenly throwing alcohol on a fire in an enclosed space, but people in Mexico still make mezcal with clay plots; it's not an insurmountable risk. And it's not like distilling alcohol in metal or glass is without risk. 

The old-timey names for a still are an alembic or a retort, depending on whether it's made from multiple pieces or just one. If you use clay to make a still, and your still has more then one piece to it, with the pieces just stuck together through friction fitting - like a lid on a pot - then the still probably won't be airtight, and your vapors will come out the cracks instead of condensing where you want them. One ancient solution for this is to use dough to plug up holes. It cooks into a horrible little papery hardtack biscuit whish stops gases and vapors from escaping for a while. If your dough-plugs shrink too much as they cook and dry, you can just put on another rolled up snake of dough to plug the new gap. I've done that before with a metal still that was venting steam and I don't really ever want to do it again. What are some other options?

One option is to use a still that only has one piece: a retort. It's a kind of teardrop shape, and the tip bends over and away from the bowl at the bottom to form a condenser tube. Retorts are cool. Hard to clean out though. Kind of hard to fill with solids like animal tissue or woodchips too. I think there's another option.

What we have illustrated here above is a two-piece alembic. The only joint at which we need to stop gasses from venting out is the one where the conical upper piece meets the groove of the lower bowl. Here, the groove is filled with water to help with sealing; I think that's important. You could also put more things in the groove, if you wanted. Like sand, maybe. Or dough snakes. Pine pitch. I don't know. But I think a two-piece alembic makes cleaning and charging the vessel a lot easier than in the case of a retort, and maybe its single joint is a manageable challenge to engineer around, especially if you have a groove full of water, rather than just a friction fit between a pot and a lid. I suppose you could also put grooves on the upper piece and thereby make a little serpentine maze of traps. I don't thing that would help much, but maybe.

Some of the cool distillation products listed above are gasses at standard temperature and pressure and they need to be dissolved in water to make them easily storable and usable, like SO3 becoming sulfuric acid in water or NH3 becoming ammonium hydroxide in water. But if the condenser tube is leading into a container of water, and the seals of the groove also contain water, is there any reason to think that the valuable gas will go in the water that we want, instead of out the side? I don't really know. My hope is that, for example, water can only hold so much ammonia gas, and so the little bit of water in the groove will quickly become saturated, and then the larger amount of water under the condenser tube (not pictured) could keep collecting ammonia gas, and the gas wouldn't start escaping from the system until both reservoirs of water are saturated. I don't know if that's how gas actually works. If that doesn't work, I guess dough snakes are fine. I'd still probably try water and sand first. Or maybe use some wet unfired clay as a plug, in addition to water.

Wait a second, that's not how gas woks at all. You can bubble carbon dioxide though water for a long time and barely increase the carbonic acid content.

This leaves me with a few things I need to figure out to advance the narrative of my primitive technology fantasy, as it pertains to distillation, in the early stages of the fantasy before I've built up resources I need for metal- and glass-working. 

1) I need to prove to my satisfaction that I can make and fire a two-piece clay alembic with a groove for water sealing.

2)  I need to learn more about making sulfuric acid by distillation, either from common evaporite minerals like gypsum, or from elemental sulfur or sulfide minerals. I wouldn't be surprised if there was a good technique that I'm just not seeing in the historical literature or patent literature - maybe something that's too expensive for industry but it works at lower temperatures than the Marchon system.

3) I need to learn more about how gasses work, with respect to, like, partial vapor pressures and solubility and things. I've tried before and it's so boring it just passes between my ears, but maybe having a project on which to use the knowledge will help.

.

Another option that is basically materially equivalent to a clay alembic sealed with uncured clay, but on a larger scale, is to make a kiln out of fired clay bricks and seal it up with mud and straw. You'd still have to figure out a condenser design though. Maybe you could have a side chamber a ways away from the heated portion for liquids to condense or for gasses to dissolve into water. Or, I've heard that if you have a sloping hole out from the bottom of a a kiln where you distill wood, you can get pyroligneous acid and tar to just flow out on the floor. Maybe that works for other some things too. Probably not ammonia and SO3, because they have to be dissolved in water. But maybe some things. Or just make a ceramic tube and put it at the top of the kiln instead of the bottom. Lots of mud to seal, a tube leading into water, done.

...

Isolating Chlorella

I like microalgae. I've been culturing them for more than two years now, but not any fresh-water varieties yet. One of the coolest of the fresh water microalgae is Chlorella, and I just learned that it grows wild basically everywhere that has fresh water, and even in topsoil. So I'm going to try harvesting it from the wild! We've talked about the prokaryotic cyanobacterium Spirulina a lot on this blog. Chlorella isn't like that. It's a unicellular eukaryote with organelles such as a nucleus, a chloroplast, and a mitochondrion. It's what Melvin Calvin, of the Calvin cycle, and Otto Warburg (another towering Nobel laureate, more famous in oncology) experimented on to understand photosynthesis. It's a cool alga.

Actually, harvesting should be the easy part; it's not hard to grow green goo in a jar. What I really want to do is isolate it from other green goo. That means I need to be able to tell one kind of green goo apart from another. So I'm going to get a nicer microscope, because at low to medium resolution a lot of microalgae just look like green circles (they're "coccoid"); some genera can be identified based on larger features like colony shape, but I don't want to rely on just that. I'm going to learn about the distinguishing morphological features of freshwater microalgae, which might be really tiny features like the structure of the chloroplasts. And I'm going to do my best to find Chlorella and grow a sample of only Chlorella. Join me, won't you?

First up, microalgae come from multiple biological kingdoms. Blue-green algae are another name for cyanobacteria. They're prokaryotes with a very simple cell structure and no organelles. Green algae in contrast are eukaryotes, and in particular they're in the plant kingdom, although the single celled ones aren't much like garden plants. A bunch of other microalgae are diatoms, which are unicellular things with silica membranes. Diatoms are are often categorized as protists, which is barely a kingdom. 

The whole protist category is a junk drawer of weird stuff that isn't quite plant, fungus, or animal, but different protists might be more closely related to one of those three groups than to another protist. I'm planning to mostly avoid talking about diatom microalgae in this post, but maybe they'll be an important part of freshwater aquatic systems and I won't have the choice. From what I've read so far, the most common genera that include freshwater diatom species are probably Achnanthes, Amphora, Cymbella, Encyonema, Eunotia, Fragilaria, Gomphonema, Hantzschia, Navicula, Nitzschia, Pinnularia, Planothidium, Sellaphora, Stauroneis, and Surirella.

There are endless freshwater microalgae, but I think the most common genera that I'll have to be able to distinguish from Chlorella (plant) are:

* Microcystis - Cyanobacterium

* Scenedesmus - Plant

* Anabaena - Cyanobacterium

* Oscillatoria/Planktothrix - Cyanobacterium

* Aphanizomenon - Cyanobacterium

Those are the microalgae most commonly referenced in the literature on algae blooms in lakes. I think Nostoc (cyanobacterium) is also pretty common, and that one can grow aquatically or terrestrially, but maybe it doesn't form algal blooms, so it gets mentioned in different literature. Nostoc can also be a photosynthetic symbiont (a photobiont) of lichen colonies, and maybe I should be on the lookout for other lichen photobionts, like Trebouxia, Trentepohlia, Asterochloris, Rhizonema. I think some of them aren't really found as free-living organisms outside of lichen colonies, but if Nostoc is, then maybe some of the others are too. And probably I'll have to learn dozens more microalgae. As the great Delftish microbiologist Lourens Baas-Becking said, but in Dutch, "everything is everywhere"; I'm not going to be able to avoid also learning about all the slightly less common things like Chlamydomonas (plant), Cylindrospermopsis (cyanobacterium), Micrasterias (plant), Monoraphidium (plant), Dolichospermum (cyanobacterium), Lyngbya (cyanobacterium), Synechococcus (cyanobacterium), Raphidocelis (plant), and Pediastrum (plant). I'll have to learn it all. 

As I acquire wild samples and microscope images, I'm going to post them here and discuss what I think they might be, and also chronicle my isolation procedures. Can I use gelatin instead of agar for immobilization? Can I dissolve salts from pulverized rocks to make an effective growth medium or do I need something more precisely formulated like Bold's basal medium? How terrible do these things smell? All this and more in the coming months.

:: The Microscope.

You know microscopes? Pioneered by the great Delftish microbiologist van Leeuwenhoek? We'll need one of those. People on microscope forums say that the popular models on Amazon, like AmScope, OMAX, and Swift are all kind of crummy, and that it's better to buy a used Olympus, Leica, Zeiss, or Nikon microscope. I thought it was a little weird that they were recommending used ones. Do those companies not make new microscopes anymore? They totally do, but the websites for Olympus, Leica, Zeiss, and Nikon do not make it easy to buy or even find a price for new scopes. Maybe they mainly cater to bulk purchasers like universities? So Ebay it is. I'm fine with that.

YouTube has some pretty nice microalgae videos taken through a Leica ATC 2000, with moderate organelle resolution, and those are only like $150 to $250 on eBay, which is a lot cheaper than some of the supposedly crummy Amazon brand models. But the YouTube videos of microalgae from the supposedly crummy Amazon models seem pretty comparable? Let's trust the forums for a moment and keep looking for Olympus, Leica, Zeiss, or Nikon.

I think Leica ATC 2000 uses phase contrast to make sharper images, which can make kind of ugly halos of light around things, but it's still better than not having it, and the videos are honestly pretty cool. If you go a couple hundred or thousand dollars more expensive, you can get microscopes that use differential interference contrast (DIC), which has no halos, and it gives really nice pseudo-3D relief images. Technically, the two contrast techniques are showing different information (path length magnitude versus path length gradient), so they're kind of complementary. I think you can use either one to see fine cellular details like gas vesicles. I've definitely seen some DIC images where you can see amazing organelle structure, but I've also seen ones that kind of just seem to show surface detail, while phase contrast looks more like a somewhat blurry X-ray right through. So I'm not sure if I want DIC or not. It's definitely going to look amazing, but it might be worse for identification if I can't see organelles inside. Most DIC scopes are thousands of dollars, which would make up my mind handily, but there's one on eBay right now for $500. *intense hand wringing* You know, what? I don't believe it. All the DICs are thousands of dollars. There's something wrong about that $500 listing. I'm just going to get a phase contrast microscope. Now I need to figure out why some Leica listings differ by $100 for what appears to be the same model. Ah, some of them don't turn on. They're being sold for parts. It's been a while since I did circuit repair, and I was never very good. I'll just buy a working one.

Hm, eBay is making this more difficult than it should be... Screw it, I don't believe the forums. I'm getting a new one. One with lots of features. AmScope T490B-DK. No regrets. Should be here next week. That gives me some time to get samples.

Oh, butt. Maybe some regrets. The AmScope I bought doesn't have phase contrast. Maybe I should do an interlude about .... histological staining? I feel like I might to need learn a lot about staining in order to resolve cytological features anywhere near as well as I would get with a phase contrast microscope. Hopefully the one I get can be upgraded? Eventually. No rush.

:: The Growth Medium

When growing things in a lab, it's nice to have a well defined food for your algae, but Chlorella really isn't too picky in where it lives and what it eats. From Adaptation to Aquatic and Terrestrial Environments in Chlorella vulgaris (Chlorophyta):

Microalgae in the genus Chlorella (Trebouxiophyceae) are found in almost all geographic regions. The genus comprises species in freshwater lakes, soil, marine, brackish and terrestrial habitats, and some species are also symbionts of lichens, protozoa and invertebrates (Luo et al., 2010; Bock et al., 2011; Darienko et al., 2015).

Two common laboratory growth media for cultivating microalgae are Bold's basal medium and BG-11 medium. Recipes like those mostly just have a bunch of mineral salts dissolved in distilled water. Among the cations of the salts, all of the common alkali metals are represented (Na, K, Ca, Mg) and the anions include nitrate, phosphate, sulfate, chloride, and usually some amount of borate. Then there are usually tiny amounts of some heavy metal salts with cations of iron, zinc, copper, manganese, cobalt, and molybdenum. Also a chelating agent called EDTA is commonly used. I don't know why.

Buying, measuring, and combining all of those salts sounds like a lot of work when Chlorella grows wild in so many environments - including environments that probably have zero EDTA and much lower concentrations of heavy metals. I'm pretty sure you can just use, like, a handful of loamy soil in tap water and you'll have something suitable for Chlorella.

I want to try something a little in-between those two routes in terms of effort. A paper from 1987, "An inexpensive inorganic medium for the mass cultivation of freshwater microalgae", gives a growth medium called DS medium that starts with 90% distilled water and 10% ocean water. This provides all of the alkali metals (Na, K, Ca, Mg) and also chlorides, sulfates, and borates. All they have to add is phosphate, nitrate, and trace heavy elements. I don't live by the ocean, but I do live by rocks, and the salt in the ocean comes from the erosion of continental rocks. Strickler's 3rd Law of GeoFantasy: "The earth breaks what it makes and puts it in the ocean." So how about I try that? I'll pulverize some rocks into powder and maybe some small portion will be immediately dissolvable into water without any acidification (which would simulate weathering over long times scales by slightly acidic rain). Or if it does need acidification, I can use some phosphoric acid and some nitric acid, and now those anions are covered also. I think this is an excellent idea. It can be done inorganically, which limits sample contamination, it can be done far from oceans, which is convenient if you're in, say, Wisconsin or a Mars habitat, it doesn't require any EDTA chelating agent. There is a question in my mind of how much rock I'm going to have to pulverize to get appreciably quantities of growth medium with a salinity 10% that of ocean water, but that's a problem for near-future me. If you've followed my writing about growing Spirulina on Mars, you also know that there's an easy way to get phosphates and nitrates for growing algae, if you don't have the corresponding mineral acids handy.

Oh! After a little reflection, it seems to me that pulverizing rocks to extract soluble salts is perhaps a rather high effort option. It's still absolutely something I'm going to do; I need to know if it's viable. But first, I'm going to try something else. I've already got a nice abiological growth medium for my Spirulina with lots of mineral salts, including nitrates and phosphates and carbonates. Spirulina likes salty brine lakes, comparable in salinity to ocean water I think but with much lower acidity (much higher pH). So my first idea for a low effort abiological culture medium is start with spirulina medium, raise the acidity a little so that we're closer to ocean water, and then dilute with 9 parts distilled water to 1 part fake ocean water, just like the DS medium. That's probably not a great option for you kids following along at home, but it's what I'm going to do till I've had a day to crush rocks.

:: Our Beloved Protagonist

Chlorella was discovered in a pond in the Netherlands by the great Delftish microbiologist Martinus Willem Beijerinck, who also discovered viruses, nitrogen fixation, and sulfate reducing bacteria, among many other things. One of the things that makes Chlorella interesting is its potential use as a food source. It's a complete a protein, and it makes bioavailable vitamin B12 (but possibly only if it already has a source of non-bioavailable pseudo-B12), and its makes triglyceride oil rich in omega-3 fatty acids. I think it could be a very useful passenger on long-term space missions, where it is would also be useful for its ability to improve air through photosynthetic carbon capture, just as plants do.

What does it look like? What are its identifying morphological characteristics? It's green. It's round. Each cell has a size between 2 and 10 μm (mirometers, microns). Unfortunately, the common and hepatotoxic alga Microcystis is also green and round, and it has a size between 2 and 7 μm. Microcystis also happens to be a cyanobacterium, so no internal organelles. Chlorella doesn't have any flagella for moving. I'm pretty sure it's also non-motile, but some unicellular organisms without flagella can move a little bit anyways, and we're still figuring out how in some cases, and I'm not sure if Chlorella is one of them. One method used by bacteria is called twitching and it involves type IV pili, which are protein fibers on the surfaces of some bacteria. Another strange method of bacterial motion is gliding, which involves gliding motors in the cell membrane pushing against adhesion complexes? Chlorella isn't a bacteria, and as for as I know, Chlorella can't do either of those. In microscopy videos, it certainly looks pretty damn immotile, except that its contents expand and push against each other a little when it reproduces. I've heard that Microcystis can twitch or glide, but I haven't seen a video of it moving. Sources often remark on Chlorella's chloroplasts as distinguishing features. One source claims that each cell has a single cup-shaped chloroplast. Another says its chloroplasts are plate-like or cup-shaped, and doesn't give a count.. When they say cup-shaped, I think they mean that, looking down on the cell, you see a broken circle, like the letter "C". I'm going to be real with you: most high resolution images of Chlorella just look kind of bumpy. It's hard to tell what the bumps are. But even if we can positively say, "these bumps are a cup-shaped chloroplast", I don't know how diagnostic that is. If you google "cup-shaped chloroplast", you'll see that Chlamydomonas more famously has them. Chlamydomonas has a flagellum, so you're not going to confuse it for Chlorella, but the point is, a cup-shaped chloroplast isn't unique. Not super related, but Chlamydomonas with the flagellum, it can grow in snow! So cool. Oh hey, I just saw another source saying that Chlorella has only one chloroplast per cell. I believe it. I just don't know it when I see it. One Chlorella diagram on the web shows two chloropasts, each taking up about half of the cell volume, looking a lot like a yin-yang symbol. That's interesting in that the yin-yang components are kind of cup-shaped, but it doesn't match any images of Chlorella I've seen and also, two chloroplasts? Pretty suspicious.

So at present, to identify Chlorella, the best things we have to go on are 1) small, round and green with no flagellum (which rules out Chlamydomonas) and 2) does have at least one cup-shape chloroplast (which rules out Microcystis, if we can figure from microscope images where the chloroplast is, or isn't in the case of the cyanobacterium).

Some unicellular microalgae form interesting colonies. Small Mycrocystis colonies have individual cells grouped together loosely in a ball of clear mucous, not always directly touching. A larger colony sometimes elongates and looks more like a fuzzy caterpillar, or just a fuzzy ball if it doesn't elongate. What about Chlorella? One source says it can produce colonies up to 64 cells. Some cyanobacterial microalgae, when they form colonies, will have a few cells that fix atmospheric nitrogen for the colony instead of performing photosynthesis, and they look different (bigger usually, I think) and the nitrogen cells are called heterocysts. I don't think Chlorella can do that. If you see a colony with heterocysts, you haven't isolated Chlorella. So what's the point of forming a colony for Chlorella? It's not for division of labor with heterocysts, and it's not for mating, because Chlorella reproduces asexually. Does it excrete an extra-cellular polymeric substance that becomes more useful when all the colony chips in?

...

:: Unialgal And Axenic Isolation

The basic procedure for isolating an alga is to spread an environmental water sample very thinly on an immobilizing substrate like agar in a Petri dish, perhaps by dipping a wire loop or needle into the sample and then streaking it across the dish. Next, incubate for a few days and, finally, examine your handiwork. If you spread very small quantities thinly on the dish, then each little region on the dish will hopefully have just one species of alga. Now it's unialgal. Congrats. But what if there's one alga and also something else on there that isn't an alga? The easiest thing you can do repeat the process until you get just one species. If that works, then your culture is "axenic". Double congrats. What if, like, all of your chlorella cells have infections? Maybe each one contains an even tinier vampire bacterium called Vampirovibrio. What do you do now? I'm not sure. Maybe hope that you find a chemical or genomic treatment that attacks the parasite and not the host. Or maybe "every cell is infected" isn't a real problem. Like, when a Chlorella cell reproduces, I don't think the daughter cells would all have the infection. If you make a streak plate, one of your regions will eventually be pure, just by statistical coincidence. It's not a real problem. 

Or rather it wouldn't be a problem if I were trying to get an axenic culture. I'll do my best to limit contamination in my samples, but you need some high grade aseptic laboratory equipment and procedures to get rid of all contaminants and keep them away. Everything I just said can be done with great fastidiousness to avoid contamination, like by wearing surgical gloves, and disinfecting surfaces before working (including the gloves), and working in a laminar flow hood, and sterilizing your sampling wire under a spirit flame, and sealing your Petri dishes with thermoplastic parafilm, and incubating your dishes in a sealed chamber incubator, et cetera. But even with all of that, it's still really pretty hard to get and maintain an axenic culture. So I'll settle for a unialgal culture.

I'm going to try some other immobilizing agents besides agar to start, using materials at hand. I've heard gelatin works pretty well so long as the organisms in your sample don't digest it, but that this is a common problem. I've also heard of mixed success with either xanthan gum or pectin as an immobilizing agent for making streak plates. And as long as we're trying stuff, how about a gel made from polyethylene glycol and calcium acetate. That sounds interesting to me. Chlorella can eat acetate, but I still want to try. Oh! Silica gel! Absolutely yes, going to do silica gel.

How do you make silica gel? Start with silica, for example, silica beads from desiccant packets, or bulk silica beads that they sell in some craft stores for drying out flowers. If you use desiccant packets, probably don't use the color changing variety since they can have cobalt chloride as an indicator of hydration, which is a little cancerous. Combine your silica with water and sodium hydroxide to make a basic solution of sodium silicate. Now add in sulfuric acid dropwise. Lower the pH down around 6 and the let it stand; a fairly opaque gel will form throughout the whole container. Or maybe you can stir it a little bit to prevent localized coagulation, but don't add more acid. Use an inorganic nutrient medium instead of water and now you have a immobilizing nutrient gel which can support microbial life while halting diffusion and convection currents. And you can use other acids like hydrochloric acid if you want. Then your nutrient medium will be a little enriched with sodium chloride instead of sodium sulfate. I feel like phosphoric acid or nitric acid would be ever better, nutritionally. Or a mix? I'll try some stuff and let you know how it goes.

Another trick to help with isolation, besides the streak plate with an inorganic immobilizing agent and the aseptic work space, is serial dilution. Instead of taking straight from a sample of pond water when streaking, you can dilute your pond sample by putting just a little it into some growth medium. And then you dilute that by putting it into a bunch of batches of growth medium. And then you dilute that. And then you do streak plates for a bunch of your dilute samples, and hopefully one of them will end up free of whatever molds and non-photoautotrophic bacteria were in the original sample. Or if they're not gone, then at least you will have diluted the soil-like carbon sources that they feed on, so that the photosynthetic guys can dominant the sample, since they can feed on CO2 gas and whatever carbonate or bicarbonate anions if you have in your growth medium. Serial dilution is especially a good idea for soil samples, since they're full of organic carbon and molds and bacteria.

When you're all done isolating a sample, one easy test for the presence of fungi and non-photosynthetic bacteria is to take a little out and add a complex carbon source, like some sugar. It won't take long to spoil if they're in there.

:: Diagnostic Antibiotics

If more than half of the common microalgae are cyanobacteria, can we just use an antibacterial to double our chances of isolating Chlorella? Maybe. Table 2 on page 4 of the paper "Combined Effects of Sulfamethoxazole and Erythromycin on a Freshwater Microalga" (Zhang et al., 2021) shows that Chlorella is pretty resistant to sulfamethoxazole, an antibiotic commonly prescribed for humans, in comparison to some other cyanobacteria and microalgae plants. 

I'm absolutely going to get some aquarium grade antibiotics and do some tests. This sounds awesome. It might sounds less awesome to you if you've ever had to take antibiotics orally, and you know that they mostly smell and taste like sewage. I'm not worried about the smell. I think pond scum is more likely to smell bad if I don't treat it with antibiotics than if I do, and I was already prepared for that experience. I still think this will be a great.

What's even left after you get rid of the cyanobacteria and the diatoms?

The class of Chlorella is called Chlorophyceae. Its members seem to be fairly common. Famous genera include Botryococcus, Chlamydomonas, Chlorella, Dunaliella, Haematococcus, and Scenedesmus. Some other honorable mentions are (Tetraspora, Pediastrum, Hydrodictyon, Neochloris, Tetraselmis, Nannochloropsis).

There's another class of freshwater unicellular green algae called Charophyceae that's more closely related to land plants than Chlorophyceae, but also I think a lot rarer. It's most famous genera are probably (Closterium, Cosmarium, Desmidium, Micrasterias, Spirotaenia, Spirogyra, Staurastrum) with (Actinotaenium, Euastrum, Gonatozygon, Hyalotheca, Netrium, Pleurotaenium, Spondylosium, Xanthidium) coming in second tier.

I'm not sure if any of the others families matter. I'm still reading.

A lot of the genera above aren't coccoid, or they form filamentous colonies, or they have flagella, and none of those things are true of Chlorella. I think if I can filter rout cyanobacteria and diatoms and fungi and non-photosynthetic bacteria, and if Chlorella is as cosmopolitan as reports lead me to believe, then I have a good chance of identifying and isolating it.

I think, to avoid breeding antibiotic resistant bacteria, I'm going to use antibiotics diagnostically, but not for isolation. I'll isolate species with streak plates, and then rule out ones that are probably not Chlorella  by taking a little out and testing their susceptibility to antibiotics.

Most antibiotics are used as anti-bacterials, and if you want to get rid of yeasts and molds, then you use specialized antifungals. I don't anticipate needing to use antifungals diagnostically, but there are some green spherical yeasts and maybe I should be prepared for those.

Ooh, just found a paper called "The use of antibiotics to obtain axenic cultures of algae" which even talks about Chlorella. Things are looking up. And it even talks about common Chlorella contaminants, which is super important to me because I think Chlorella might need a source of pseudo-B12 (like a bacterial contaminant) in order to make B12 that's bioavailable for itself and for humans.

:: Vitamin B12 tho?

I think it's funny that almost all of the pioneering microbiologists I've mentioned have been Dutch dudes who lived or at least studied in Delft. There's one more I'd like to make a reference to, Albert Kluyver, and this will probably be the section.

Vitamin B12 is only made by archaea and bacteria, I believe. Chlorella cultures contain B12. Chlorella is a eukaryote, not an archaeon or bacterium. What gives?

The paper has the explanation, but the title completely skips the interesting bit. Bacteria make their own B12, then Chlorella converts it into a form of B12 that it and we can use. That paper talks about Chlorella combining bacterial B12 with a chemical called DMB on the lower ligand. Where does *that* cone from? We just found out in 2015.

So at present, my prediction is that a pure sample of Chlorella, a unialgal axenic culture, can't make B12, and all chlorella cultures that contain B12 have bacterial contamination. It would be cooler if Chlorella had absorbed a cyanobacterium and used it as a B12 organelle, or if it was making pseudo-B12 from scratch following cross-species gene transfer, but not this time.

What are the likely contaminants? The cool answer would be Chlorella's vampire. It's a cyanobacterium that gave up on photosynthesis in preference to predation. So cool. As a bacterium associated with Chlorella, might it be Chlorella's source of pseudo-cobalamin? The literature doesn't say anything about it, and it's the sort of thing we'd investigate and publish, so probably not.

One likely contaminant is Thaumarchaeota (aka Nitrososphaerota), a globally distributed archaeon phylum found widely in aquatic environments. It's everywhere, but especially prevalent relative to other B12 producers in colder and deeper water columns

Chlorella can also live terrestrially though. Where does it get its pseudo-B12 when it's on land? Maybe the same place:"Thaumarchaeota are the dominant archaea in most soil systems where they constitute up to 5% of all prokaryotes"(from Ammonia-Oxidising Archaea – Physiology, Ecology and Evolution).

So it seems likely that Chlorella associates with different Thaumarchaeota in the wild, not to rule out associations with cyanobacteria or proteobacteria or anything else. But wait, Thaumarchaeota might not be the contaminant in laboratory cultures, because... I think they're all huge. They're sometimes called giant archaea and their sizes can be like 13 to 28 microns wide, while Chlorella is like 2 to 10 micron. Hugeness means they're less likely to be a contaminant in lab culture, the same way we don't expect squirrels to be contaminants on account of their size. You'd see them on the streak plate when doing your isolation procedures. In comparison, Chlorella's vampire is ~ 0.6 micron.

But wait, someone found out the common laboratory contaminants in " "The use of antibiotics to obtain axenic cultures of algae"! 

First they reference an older paper:

"A typical large-scale continuous culture of Chlorella sorokiniana Shihira et Krauss for example, was found by Litchfield, Colwell & Prescott (1969) to be heavily infected with Pseudomonas, Acinetobacter, Flavobacterium and Bacillus bacteria, even when grown at the selective temperature of 39 ° C."

Very interesting. In their own experiments,

"The Chlorella cultures yielded an abundance of both yeasts and orange colonies of isolate Type No. 1, it was strange, however, that only from the most complex antibiotic mixture were bacterial colonies visible."

"Yeast-free healthy cultures of Chlorella were obtained with the streptomycinfree antibiotic mixture No. 2. It was surprising that the richer mixture, No. 1, did not eliminate the orange coryneform bacteria; it may have been due to antagonistic effects in the antibiotic mixtures."

So yeasts are common contaminants, but they don't make any form of B12. So let's look into the genera from the referenced older paper (Pseudomonas, Acinetobacter, Flavobacterium, Bacillus) and also the "orange coryneform bacteria" in the present paper.

"Coryneform" just means that the bacteria have a rod-like or club-like shape. I think the orange coryneform bacteria are all gram-positive, which means they take up crystal violet stain and appear purple in the Gram stain test. The reason they take up violet stain is that they have peptidoglycan (amino aid + sugar) polymers in their outer plasma membranes/cell walls. The orange coryneform bacteria include genera of Corynebacterium, Brevibacterium, Dermabacter, Microbacterium, and Cellulomonas. One of those, Brevibacterium, is responsible for the orange color on the rinds of traditionally-made Munster and Limburger cheese, though what you get in the grocery store is probably just colored with annatto made from achiote seeds.

So, now we have a list of bacterial genera that are common contaminants of laboratory Chlorella cultures. Which ones make some kind of cobalamin?

* Pseudomonas: This one can make B12. Pseudomonas denitrificans is even used industrially to produce B12 at scale.

* Acinetobacter: No B12 production that I can find.

* Flavobacterium: Definitely makes B12. The paper "Production of Vitamin B12 by Microorganisms and Its Occurrence in Plant Tissues" (1952) lists nine different species that make B12, but they all have different names now because biologists suck.

* Bacillus: Some species in this genus produce B12. Bacillus megaterium is used industrially to produce B12 at scale. Bacillus badius makes it, as reported in "Utilization of Hydrocarbons and Vitamin B12 Production by Bacillus badiust" (1976). The 1952 paper with the Flavobacteria also lists Bacillus subtilis, but the paper "Microbial production of vitamin B12" (2017) seems to say say no: "Bacillus subtilis lack[s] the genes involved in the cobalamin synthesis pathway after precorrin-2". 

So most of those can  make B12. But I can't find statements that any of the the orange coryneform ones make B12.

* Corynebacterium: I can only find references to  Corynebacterium species that do not produce B12. The genus Corynebacterium includes diphtheria, and also C. glutamicum, which is used to make massive quantities of the amino acids L-glutamate and L-lysine for animal feed, including human feed. 

No positive references yet for any species in the genera Brevibacterium, Dermabacter, Microbacterium, or Cellulomonas that can make B12, but while looking I found to fairly general statements about some things to do make it:

"Vitamin B12 can usually be synthesized by many bacterial species, especially heterotrophic bacteria, and nearly all of the oxygenic photosynthetic cyanobacteria,"

"Nearly all methylotrophic bacteria can produce vitamin B12 at concentrations ranging from 30 to 150 μg/gbiomass [81], with members of the Methylobacterium genus reported to produce B12 at higher concentrations compared to other genera"

So those are interesting.

:: Samples

:: Bioreactors

:: First Contact

...