Winogradsky Column

[Hookahhead]

Forewarning, this post has a little bit of discussion about what lead me to this post. If you care to skip it, the real content will be in the 2nd post.

So, I don’t know where the rabbit is that makes these holes all over the internet, but I ended up falling into another one today. After a good bit of reading and thinking, I did a search on here and RIU and didn’t see any mention of this topic. To tell the truth I’m very excited to bring this “new” topic to everyone’s attention here.

Like many of us here, I am a probiotic farmer. I try to get a healthy, diverse population living and thriving in my soil. I am successfully growing using only local materials, and making my own brews based on these materials. I have posted some of my use of these inputs in this thread (Local materials). I brewed random aerated teas for a while. Then I discovered LABs. I enjoy my experience growing them and they have given me a greater appreciation for knowing the microbes.

This lead me to seek out other microbes we can easily cultivate from the wild. If we look at a EM-1 recipe it contains

• LABs
• Yeasts
• Photosynthetic Bacteria

The Yeast I have experience with from wine brewing and baking. I haven’t really figure out how having a bunch of yeast is a good thing yet, but I haven’t looked to much into it either. If anyone has some good information on this please share it.

The Photosynthetic Bacteria were completely new to me though. I knew they existed, but I didn’t recognize how beneficial they are to the soil. Further research lead me to Rhodopseudomonas palustris. This organism is AMAZING! It has 4 different modes of metabolism. Meaning it can live off a wide variety of materials, and in any environment. It even generates an electric charge. My mind was blown for a second there! After settling back down, I stated to look into how to get this thing into my soil. It’s remarkable adaptivity means we’re likely surrounded by it.

Onto the next step, how do we isolate and cultivate this organism? I have a background in biology, I have worked with agar numerous times for cultivating different species of bacteria and fungi. However, aside from taking a few swipes from my phone, or hand, or whatever I have always started with known inoculants. I know that would be the proper way to approach this. Unfortunately, I don’t know where to find those materials where I’m living, and I’m looking for something less involved. I am also not an expert in microbiology, especially identification.

Down this rabbit hole I came across Sergei Winogradsky. This is one cool dude, and I’m really surprised his name hasn’t been mentioned in all of this Probiotic/KNF buzz. He discovered lithographic bacteria way back in 1887. These guys chew up rocks and other minerals, and excrete them in more plant friendly forms. Of course we need some of those! He also discovered nitrogen fixing bacteria, photoautotrophs, and chemoautotrophs. If my boy Sergei could do it with what he had back then, surely we can do it today? And for real, why aren’t we giving this guy more props!

 

[Hookahhead]

Winogradsky Column

Winogradsky worked his magic with a tool he invented called a Winogradsky column. This man was brilliant, he figured out how to make what I see as “column chromatography for useful bacteria”. This video does a great job explaining the concept.[YOUTUBEIF]https://m.youtube.com/watch?v=5TP0CrzPVZ0[/YOUTUBEIF]

It seems logical, simple, cheap, and efficient. What more could you ask for? Hopefully it works as advertised!

I happen to have a small stream in my back yard, so off I went to make my own Winogradsky column.

1. First you need to find somewhere water has saturated the ground for a while. This can be from the edge of a pond, lake, small stream, long standing mud puddles, and even brackish and salt water. This will be your inoculant source.
   
   
   
   
   
2. Next dig up some mud. Everything I saw said to omit as much rock and organic matter as possible, you want mud! Unfortunately my river is pretty sandy, and the only thing I consider mud was in the middle. I waded in and gathered some mud. Just under the sediment there was a rich black layer. Every shovelful sent a load of bubbles rising to the surface. There was a slight stench of sulfur. We have life!
   
   
   
   
   
3. The inputs required are minimal. You want to add a calcium source. I used powdered egg shells (1/2 tsp), but you could also use other sources or calcium carbonate, calcium sulfate or calmag. You also need a source of sulfur, I used 1/4 tsp magnesium sulfate (Epsom salt), but you could use calcium sulfate and I even saw an egg yolk was suggested. None of my research listed any proportions, and the YouTube examples just tossed in small amounts. I looked up how much sulphur is in a yolk, and a non scientific source said 25mg in the yolk and 50 in the white. Personally I don’t like the idea of using a raw egg, but I used it to kind of rough gauge of how much sulfur to add. You also need a carbon source. Many of the tutorials used shredded paper, I used shredded paper eggcrate. It’s something I had on hand, and I prefer to use this material since it’s already been recycled at least once. I imagine you could also use bio char, which is something I may experiment with if all goes well. Finally, you need a bit of water from your source, so make sure you collect some of that too. Mix all of the materials together EXCEPT THE WATER
   
   
   
   
   
   
4. Fill up most of a clear container with mud. You can use plastic, but I think glass is preferable for me. A lot of things suggested 1L, but many examples used something smaller. My gut tells me bigger (taller) is better. I don’t currently have any 1L glass jars to devote to this, so I improvised with some pasta jars I had in the recycling. I made the effort to sterilize them by boiling hot water in the microwave. I was high, and it probably wasn’t necessary. Luckily I had let them cool before I collected any mud. The rest of the space I filled with water. I don’t know if it would be beneficial to leave a layer of air above the water. I could see how this could be beneficial as it’s another zone for microbes to grow. Again that’s something for further research.
   
   
   
   
   
5. 5. Set your jar somewhere with sufficient indirect sunlight. Direct sunlight could cook your jar. Now like everything in this hobby, you just need to wait. The internet says you should see changes in 3-6 weeks. Changes that are visible, easily identifiable, and consistent. I sure hope everything on the internet is true!

Follow along with me over the next couple weeks, I’ll post how our jar is progressing. In the mean time, let’s discuss the different organisms I may or may not be cultivating. We should also consider how I may extract and use these organisms after this step. If you enjoyed this post, please click the “this post was helpful” button or leave me some rating. I know it doesn’t mean anything on the board or in real life, but I enjoy seeing that people are following along and appreciate my contributions to this website.

Thanks!

 

[Hookahhead]

The video I posted above shows an idealized Winogradsky column. Looking at this image I realize an air space is a good idea. Also it’s not good to pressurize glass bottles, duh. I went out and removed the lids when I realized my mistake. However, my jars still lack the traditional airspace.

So let’s talk about the organisms found in the various layers.

• Sulfur Bacteria
• Purple Photosynthetic Bacteria
• Green Nonsulfur Bacteria
• Cyanobacteria and Algae

 

[troutman]

I built one last year. Didn't let it age long enough. Take months to mature.

They do sell photosynthetic bacteria.

Sadly, the product I really wanted is impossible to get in Canada and it has 4 species in it.

It's called AZOO PSB SUPER RED PHOTOSYNTHETIC BACTERIA and it has these 4 bacteria in it.

Rhodobacter sphaerorides, Rhodobacter capsulatus, Rhodosprillium rubrum and Rhodospeudomonas palustris.

I just emailed somebody who may be able to send it to me. We'll see if I can finally get it.

 

[Hookahhead]

Thanks for stopping by troutman. I’d be happy to hear any of your ideas, experiences or criticisms on this project.

The wiki has better info on construction than some of the other sources. I think I’m going to get a few more of these together in the next couple of days/weeks to test different inputs. It looks like we can swing the conditions to favor more specific groups/organisms. I like the idea of a small layer of sand over top of the mud, and a larger water area. I think as troutman pointed out the red/purple photosynthetic bacteria look like a good group to target.

The column is a rough mixture of ingredients – exact measurements are not critical. A tall glass (30 cm long, >5 cm wide) is filled one third full of pond mud, omitting any sticks, debris, and air bubbles. Supplementation of ~0.25% w/w calcium carbonate and ~0.50% w/w calcium sulfate or sodium sulfate is required (ground eggshell and egg yolk respectively are rich in these minerals), mixed in with some shredded newspaper, filter paper or hay (for cellulose). An additional anaerobic layer, this time of unsupplemented mud, brings the container to two thirds full. Alternatively, some procedures call for sand to be used for the layer above the enriched sediment as to allow for easier observation and sampling of resulting populations. This is followed by water from the pond to saturate the mud (or sand) and occupy half the remaining volume. The column is sealed tightly to prevent evaporation of water and incubated for several months in strong natural light.

After the column is sealed tightly the anaerobic bacteria will develop first, including Clostridium spp. These anaerobic bacteria will consume the cellulose as an energy source. Once this commences they create CO2 that is used by other bacteria and thus the cycle begins. Eventually colour layers of different bacteria will appear in the column. At the bottom of the column will be black anaerobic H2S dominated zone with sulfur reducing bacteria, the layer above will be green sulfur photosynthetic anaerobic bacteria, then the layer will be purple which is sulphur anaerobic bacteria, followed by another column of purple anaerobic non-sulfur bacteria and at the top will be a layer of Cyanobacteria which is sulphur oxidising bacteria. This top layer of aerobic bacteria, produces CO2 which feeds back into the column creating further reaction.

While the Winogradsky column is an excellent tool to see whole communities of bacteria, it does not allow one to see the densities or individual bacterial colonies. It also takes a long time to complete its cycle. However its importance in environmental microbiology should not be overlooked and it is still an excellent tool to determine the major bacterial communities in a sample.[2]

 

[Grapefruitroop]

Just Awesome!!

Just Awesome!!


Awesome Post!!!Very interesting! Thanks for sharing!!
Keep up the good work!!

 

[GOT_BUD?]

Tag

 

[Microbeman]

Hey Hooka; Great exercise for folks. You will find that I have recommended and referred to this process many times in the forum. Spurr did an exercise displayed with photos for growing PNSBs in similar fashion using fish [paste] as I recall.

Here is my most recent reference and a link to one of my heroes sites; https://www.icmag.com/ic/showpost.php?p=8474233&postcount=30

Pitcher plants are another good source for PNSBs and fish scales.

EDIT; It appears that Jimmy Deacon's site is not available. He did say he no longer monitors it.

 

[Hookahhead]

Hi MM thanks for stopping by, I was secretly hoping you would chime in! Wow, the post you linked is from the local materials thread I’ve been posting in, and somehow I still missed this info. I saw you reference PSB, but didn’t understand the acronym at the time. Also for some reason, a search for “Winogradsky” doesn’t turn up that post, maybe the basic search only covers thread titles? Anyhow, thanks for the info I’ll be sure to read up some more later today. If you have any insights, known issues, whatever please feel free to share them here.

Edit: It seems like the link you posted is down now. Here is a web archive of the link. https://web.archive.org/web/20190324163617/https://archive.bio.ed.ac.uk/jdeacon/microbes/winograd.htm

If anyone else wants to set up a column and share their progress, this is the tread to do it!

 

[Lost in a SOG]

EM1 has palustris in but not its relatives.

Edit: just noticed you basically mentioned that already

Great thread btw..

 

[Microbeman]

Here is a discussion with a lot of good info;
https://logicalgardener.org/viewtopic.php?f=24&t=393&hilit=Deacon

and try this link to Jimmy's take
https://archive.bio.ed.ac.uk/jdeacon/microbes/winograd.htm

EDIT: I do not know why the 2nd link can be reached from the other website but not from here.

 

[Fred Sanford]

Thank you for sharing this Mr. Head, I really appreciate it.

 

[Hookahhead]

From the link Microbeman posted...

A better image of an idealized column:

And this interesting bit of info:

Most of the water column above the photosynthetic bacteria is coloured bright red by a large population of purple non-sulphur bacteria. These include species of Rhodopseudomonas, Rhodospirillum and Rhodomicrobium. A mixed culture of them is shown in the bottle in Figure E (below).
These bacteria grow in anaerobic conditions, gaining their energy from light reactions but using organic acids as their carbon source for cellular synthesis. So they are termed photoheterotrophs. The organic acids that they use are the fermentation products of other anaerobic bacteria (e.g. Clostridium species), but the purple non-sulphur bacteria are intolerant of high H2S concentrations, so they occur above the zone where the green and purple sulphur bacteria are found.

The columns shown at the top of this page (Fig. A) have passed the stage where these organisms are common because the water columns became oxygenated by cyanobacteria (Fig. D).

I think the mixture of purple non-sulfur is where I am going to focus my efforts first. As already discussed these microbes seem very beneficial for soil health and nutrient cycling. They are found in the water column, so hopefully they will be easier to extract. I am planning on doing a “revised” version of my columns based on this idea. I’m thinking a lot less mud (1/4 or 1/3 of the column), covered by a layer of sand, then the rest water.

Most of the text says the column should be ready anywhere from 3 weeks - 6 months. This is a huge gap, and not really efficient. If successful with this step, I plan to use it to I inoculate a media/environment that is geared towards multiplying the purple non sulfur bacteria. If you want to use culturing LABs as an analogy, I see this step as fermenting the rice/starch wash. You collect a lot of organisms at first, and afterwards create conditions where only specific organisms survive/proliferate.

If troutman, Microbeman, or anyone else has any suggestions on how to proceed with this second step I’m listening. A Well lit area and anaerobic conditions seems to be the environmental factors. Feedstock is organic acids, so vinegar or lactic acid seem suitable.

Edit: I just found this in the other link from the page Microbeman linked. Thanks again for those sources, they were very useful!

https://www.splammo.net/bact102/102pnsb.html
III. Enrichment and Isolation of Purple Non-Sulfur Photosynthetic Bacteria.

In looking for the purple non-sulfur bacteria, we find it most advantageous to set up conditions for photoheterotrophic growth, utilizing a source of light, anaerobic conditions (needed for phototrophic growth by these organisms), no hydrogen sulfide, and an organic carbon source not generally used by other bacteria under these conditions such as sodium succinate or malate. Note the medium formulas below. Not only will most other types of organisms be restricted from growing, but the purple non-sulfur photosynthetic bacteria will be easily recognized by the presence of photosynthetic pigments. When substantial pigmented growth shows up in the liquid medium or is seen in the natural source, it is referred to as a "bloom."

One may expect these organisms in their most likely habitat – i.e., anaerobic mud in ponds and lakes where there is access to sunlight. Other successful sources where they can be found as easily-recoverable contaminants include surface water from streams, bogs and transient puddles – and even rain, snow, icicles and hailstones! High concentrations of these organisms have even been found in the water trapped by the leaves of bromeliads and pitcher plants. Soil and flat leaf surfaces are worth a try.

Isolation of purple non-sulfur bacteria is accomplished easily by adding the source material to a liquid enrichment medium in a stoppered bottle. The final volume attained can be approximately 5-25% inoculum (a lower amount if a solid sample is used) with the medium filling up the rest of the container such that no air bubbles are trapped. These enrichments are placed near a tungsten light (one or more common desk lamps) at room temperature to 30°C. Once the enrichment has achieved turbidity with the consequent bloom, it is streaked onto plates which are then incubated anaerobically near the light source.

From the same page:

IV. Formulas for the Mineral Salts-Succinate Broth and Agar Media.

Of the several we have used in teaching and research over the decades, the medium detailed below has probably given us the best luck in obtaining rapid and substantial growth (enrichments and colonies) of purple non-sulfur photosynthetic bacteria. It is probably based on a medium formulated originally by Norbert Pfennig who was a distinguished and well-remembered visitor to our department.

With water samples containing a substantial amount of oxygenic phototrophs (mainly cyanobacteria and green algae), it may be possible to have these organisms come up within a week and overtake the enrichments! An example of this happening is shown in these images; the photos were taken 3 days (left) and 6 days (right) after inoculation. (These images are from a study of samples from source streams of the Mississippi River.) Therefore, it is probably best to streak your plates from the enrichments as soon as a reddish "bloom" of the desired organisms is evident.

 

[Hookahhead]

EDIT: I do not know why the 2nd link can be reached from the other website but not from here.

I just tried looking at the links, the issue is that ICMag changes all links to “https” instead of “http” for increased security. Unfortunately this breaks a lot of links on the site, you just need to be aware of this if you click a link here and it doesn’t load. The page loads fine if you remove the “s” from “https”.

 

[ReikoX]

 

[Badfishy1]

Definitely following along, good luck my man

 

[GOT_BUD?]

Here is a discussion with a lot of good info;
https://logicalgardener.org/viewtopic.php?f=24&t=393&hilit=Deacon

and try this link to Jimmy's take
https://archive.bio.ed.ac.uk/jdeacon/microbes/winograd.htm

EDIT: I do not know why the 2nd link can be reached from the other website but not from here.

I'm guessing it's the s added to the end of https: that the forums adds.

ETA - Yup. Remove the s and it takes you to the site just fine.

I also see Hookahhead beat me to it.

 

[Hookahhead]

Hey there everyone, thanks for all of the comments! I'm happy to know you're following along.

Here's an update on the Winogradsky columns I started 4 days ago. The water on top settled over night. No real remarkable growth is visible. I can see little bits of things floating in the water portion. The mud looks darker to me, almost black. I read that this is a sign of iron being oxidized. Again, I think this is way too much mud to be very useful, but I'll still let it go to see what happens.

 

[Hookahhead]

So I have been doing more reading, learning, pondering on all off this. Again, the links Microbeman posted helped bring me up to speed pretty quick. The page on Isolation and Enrichment of a sample is a good read. Basically, it tells you if you're aware of every step you take along the way; you're much more likely to succeed at successfully cultivating your targeted species.

One useful tool is to use an "selective enrichment", which limits the nutrients to only those required by your targeted organisms. It's pretty simple, if the proper food isn't there the other organisms can't compete. So if we look back at my last post, we see a vital piece of info. To set up conditions for photoheterotrophic growth, utilizing a source of light, anaerobic conditions (needed for phototrophic growth by these organisms), no hydrogen sulfide, and an organic carbon source not generally used by other bacteria under these conditions such as sodium succinate or malate. Unfortunately I do not have immediate access to succinate or malate, though malate should be easily found in stores catering to home brewing alcohol. I also don't have immediate access to some of the other chemicals listed in the recipe. They are all very cheap and easy chemicals to come by; you should be able to source them locally or online with little effort.

I already had the Epsom salt and table salt on hand. For the mean time, I had to improvise the rest. I did a little bit of math on the recipe. Here is what the breakdown looks like by nutrient per liter.

K = 0.09g
PO4 = 0.23g
Mg = 0.06g
SO4 = 0.26g
Na = 0.13g
Cl = 0.56g
NH4 = 0.17g
Ca= 0.02g
*With trace amounts of Fe, Zn, Mn, Bo, Co, Ni, Mo

I'm an organic guy, but looking at the NPK alone, it looks pretty close to a 10-20-10 fertilizer. I decided it was OK for me to step into synthetics for this. I would rather learn to effectively cultivate the organisms now, and try experimenting with organic inputs later on. I ended up going with a 20-20-20 because I couldn't find a 10-20-10 (I didn't check to see if this actually exists).

I still needed an "exclusionary" carbon source. Sugar fermenting organisms like LABs or Yeasts use various sugars as their carbon source. However, purple non-sulfur photosynthesizing bacteria (PNSPB) bacteria, can utilize organic acids as well. In the recipe I posted, Sodium Succinate or Malate is suggested, which are just the sodium salts of Succinic Acid and Malic Acid. Let's look at their structure. (Carbon is every point that is not labeled as another element)

Succinic Acid (4 carbon chain)

Malic Acid (4 carbon chain)

I do have 2 organic acids on hand, that might serve as feedstock. However, you can see these molecules have smaller carbon chains, and therefore may not be as suitable?

Acetic Acid (Vinegar - 2 carbon chain)

Lactic Acid (3 carbon chain)

After reading, pondering, and exploring a little more, I came across a ton of YT videos on growing purple sulfur bacteria. The Process looks pretty strait forward and simple, you can check it out here for now. I plan to try this and discuss it more later in this thread. The carbon source they are using is cheap and readily available... Monosodium Glutamate (MSG). Oh man, please don't kick me out of the Organics section for talking about synthetic nutes and MSG in the same post! MSG is just the Sodium salt of Glutamic Acid. I also learned that almost entirely all of the production of MSG comes from a fermentation with Corynebacterium species, how cool is that! So even though it's sold as a salt, the Glutamic Acid is organic in origin.


Glutamic Acid (5 carbon chain)

 

[Hookahhead]

Off to the store I went looking for my supplies. Not shown is the table salt. I am not able to match the suggested recipe exactly, but I improvised the best I could based on the materials that I had. Here is my recipe for 1L of water. With a slight bit of swirling, everything dissolved within a minute or 2. No precipitation was observed.

1g 20-20-20 fertalizer [0.2g each of N, P2O5, K2O]
.33g MgSO4 (Epsom Salt) [0.06g Mg, 0.26g SO4]
.33g NaCl (Table Salt) [0.13 Na, 0.20g Cl)
1g Monosodium Glutamate (MSG)

As for the minor elements, the fertilizer does contain trace amounts of many of them. However, as you will see I decided to use the inoculation sample as my media, and I hope that there is enough micronutrients in there to get the job done.