Winogradsky Column

Success!

Success!

They say seeing is believing. Today I slipped into the lab over my lunch break to use the microscope. This scope is equipped with a camera, but the laptop that hooks up to it was being used. So I took the pictures using my cellphone over the eyepiece. I used the first successful bottle (white cap @ 28 days) for my sample.

Here’s what we’re looking for

Source: https://www.mbio.ncsu.edu/mb452/purple_nonsulfurs/purples.html

What did I find? A ton of life!


Rhodospirillum - Live


Rhodospirillum - Dead

Rhodospirillum is the easiest to identify because they’re large and have a unique shape. I’m hoping Microbeman can chime in and confirm/deny these others. Again, microscopy is not my forte.


Rhodomicrobium?


Rhodobacter?

I have a 10 second video of various creatures zipping around the field of view. I’ll try to find somewhere to upload it.

Almost certainly rhodospirrilum considering the source, however on the others your estimate is as good as mine. After so much mis-identification has occurred in the science world the only true id is PCR.

Thanks for the input Mm, I should have known that proper identification would be difficult without other tools/methods. For the time being, I am content knowing that I am actually culturing PNSB, regardless of knowing the exact composition of my brew. I encourage Mm, and anyone else experimenting with this, to post your pictures, methods, observations on your own brews in this thread!

Everyday I read a little more about PNSB. I’ve covered a lot of the literature that isn’t over my head, and is pertinent to my application. There is a good bit of information out there, but most of it is repeating the same things. No doubt there are a number of benefits PNSB offer plants. I found a really nice review of the agricultural side I’d like to share.

Purple non‐sulphur bacteria and plant production: benefits for fertilization, stress resistance and the environment
https://onlinelibrary.wiley.com/doi/10.1111/1751-7915.13474

This quote is from about half way through the article, but I think it provides a good summary.

Critical analysis of the existing literature revealed the different functions of each PNSB product type, resulting in various effects and interactions with plants. More specifically, the dead cells mainly deliver nutrients, while living cells additionally convert nutrients into plant‐available forms and continuously supply PGPS. This results in the most prominent impact, promoting increased plant growth performance, while suppressing abiotic and biotic stress, as well as reduction of GHG emissions.

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It’s a pretty long article, and I realize not everybody’s cup of tea. Here are some things I found interesting...

Direct Fertilization

The use of dead PNSB biomass provides the benefit of a direct process where the only parameter to be considered is the nitrogen/phosphorus/potassium (N/P/K) content, determining the amount of biomass that should be dosed according to the plant's nutrient requirements. This type of microbial fertilizer presents a slow‐release pattern, as the dead microbial biomass is decomposed by the autochthonous soil microorganisms, and the gradually released nutrients are utilized by the plant...

...The same effect is presented when living PNSB cells are used, as the decaying cells can provide N/P/K to the plants. While no literature reports on the full nutrient content of PNSB were found, the N/P/K was measured as 8.5/2.4/0.5% in dry weight (DW) (own data for dried biomass of Rhodobacter sp.).

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Sometimes it’s easy to forget this benefit of adding microbes to your soil. When you add any sort of tea, a large number of those organisms are going to die off. Your taking an organism from their ideal environment and tossing them into the “wild” where they are going to have to compete with a number of other species. Earlier I wrote that I wasn’t sure how a bunch of yeast is beneficial to the soil environment. However, what I was over looking is the important role yeast plays in providing nutrients (vitamins, amino acids, organic molecules). Many agar and nutrient broth recipes, including those for PNSB, benefit from the addition of yeast extract.

Nitrogen Fixation

a roughly doubled agronomic nitrogen use efficiency (dry yield per unit of nitrogen supplied) was demonstrated by inoculation with R. palustris on pak choi (Wong et al., 2014). Similarly, inoculation with R. palustris PS3 increased the nitrogen efficiency of lettuce (17%) and pak choi (22–44%) (Hsu et al., 2015). Hence, the supply of PNSB can enhance the nitrogen efficiency, potentially contributing to a more sustainable agriculture.

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PNSB can fix nitrogen from the atmosphere similar to rhizobia bacteria in legumes. While they are most efficient at this in anaerobic and illuminated conditions, they have shown to be somewhat tolerant in other environments. Although they are not an incredible “fertilizer” on their own, they have a synergistic effect when used with other nitrogen inputs.

Phosphate solubilization

There is evidence that only about 10–20% of the phosphorus applied to agricultural soils is taken up by plants (Schoumans et al., 2015). The remaining inorganic phosphorus is adsorbed to clay minerals, as well as iron (Fe3+) and aluminum (Al3+) ions (at pH < 5.5) or forms crystalline structures with calcium (Ca2+) and magnesium (Mg2+) ions (at pH > 6) (Schoumans et al., 2015). As a result, many agricultural soils are phosphorus‐saturated, creating a phosphorus reservoir (Tóth et al., 2014) that shall be exploited. An approach to valorize this reservoir is the application of phosphate‐solubilizing microorganisms (Qian et al., 2010). These microorganisms, typically including Pseudomonas sp. and Bacillus sp. (Sharma et al., 2013), can render soil‐bound phosphorus soluble and available to the plants, lowering the need for synthetic fertilizers. Alori et al. (2017) suggest that the principal mechanism of phosphate solubilization is the production of mineral dissolving compounds such as organic acids, siderophores, protons (H+), hydroxyl ions (OH−) and carbon dioxide (CO2); which result in pH changes or are active as chelating agents. The ability of PNSB for inorganic phosphate solubilization from soil has been demonstrated by several studies, yet the underlying mechanisms remain unknown (Koh and Song, 2007; Lee et al., 2008; Rana et al., 2016). Taking into account that the availability of phosphorus can be the limiting step in plant nutrient uptake (Rodríguez and Fraga, 1999), PNSB with phosphate‐solubilizing properties can significantly contribute to an improved plant growth.

Rana et al. (2016) tested the solubilization of Ca3(PO4)2, Mg3(PO4)2 and Zn3(PO4)2 by PNSB: Rhodospirillum rubrum was able to solubilize these inorganic phosphorus forms with an efficiency of 100%, 100% and 51% respectively. When a mixture of fly‐ash (P‐rich mineral residue of coal combustion) and R. rubrum was tested as a fertilizer mixture, the presence of resp. 20, 10 and 4.0 mg l−1 of Ca2+, Mg2+ and Zn2+ was observed, indicating the solubilization of phosphate without the release of toxic metal ions (Mn, V, Ni, Cd, As, Hg, B, Cu, Co, Cd, Se, Zn, Mo or Pb)

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A major benefit of having a living soil is the cycling of nutrients. Calcium and magnesium are known to combine with phosphorous and form insoluble precipitates. This is one of the reasons bottles nutrients use 2-part A-B system. Then we throw in more Cal-Mag and further contribute to the problem. In a living soil, the various organisms are able to solubilize, uptake, and release these elements back into plant available forms.

Plant Growth Promoting Substances

The most studied PNSB strains (Tables 1 and 2) in regard to their PGPS production potential are able to produce indole‐3‐acetic acid (IAA) and 5‐aminolevulinic acid (ALA). Melatonin, which is synthesized by some PNSB (e.g. R. rubrum) (Manchester et al., 1995), may also be a compound of interest as it is considered to be the first‐line defence against oxidative stress in plants (Tan et al., 2013). Even though the ability of plants to absorb exogenous melatonin through their roots is proven (Tan et al., 2007), its role as a PNSB‐derived PGPS is unexplored to our knowledge and therefore not further discussed in this review.

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This isn’t a subject I am very knowledgeable on. I do know IAA is found in rooting hormone powders and gels. Perhaps a PNSB broth would be just as effective. There are also potential benefits when PGPS are applied as a foliar spray. The article discusses how these hormones may increase resistance to stress and diseases.

The article contains a lot more information, but I find it unnecessary to direct quote all of it. Hopefully I have sparked your interest enough to read it for yourself. If you have any thoughts, questions, or comments please post so we can further the discussion.

It’s been a little bit since I updated. Sorry I haven’t posted more pictures of the Winogradsky columns. As we’ve already discussed, they are actually not as thrilling or useful as I first thought. It did send me down the PNSB road though, so I’m happy about that. Tomorrow I’ll try to get some photos to show how they are progressing.

Here’s an update on the colony expansion. The 3L is just starting to “bloom”. I’m very surprised it took this long, especially after going cloudy so quickly. They always bloom from the bottom up.


The other two haven’t changed much as far as I can tell. The red capped one MIGHT be a bit cloudy. As always I’ll ley them sit around for a while and see what happens.

Sorry I haven’t updated in a while, life got a little busy for me. I have some more experiments planned to start soon.

The Winogradsky column around day 55ish. Again we can see things definitely grew, but It doesn’t seem useful to extract them. Also, the layers definitely aren’t stratified like the idealized column. The dogs knocked over the other two, effectively ending that experiment.


The vinegar/LAB experiments still haven’t changed at all, even after inoculation. Personally I’m fairly surprised by this since the literature says they should be able italics various food sources and live in a wide variety of conditions.


The colony expansion is around 24 days I think. It worked, but it was a few days slower than I expected. Also it didn’t turn very dark. I’m not sure if this was because they ran out of food, or if the larger volume container actually inhibits light penetration?

Hey hey, it’s been a while since I’ve updated this thread. I gave up on the Winogradsky columns. As we already figured out they weren’t super practical for what I was trying to do.

I’m still making PNSB broth following my same recipe. It works every time, either from a new sample or an active culture. Today I finally got around to making a batch using sea water instead of river water. I expect things will proceed in a similar fashion, with a possibility of different species from the marine environment. I kept the recipe the same for this trial.

Day 1

Been real interesting to follow, ty