Propagate your own Rhizobia bacteria for seed inoculation?

Rather than being dependent upon outside sources of legume seed inoculant, I'd prefer to close the loop in the classic permie manner. Once you have nitrogen-fixing Rhizobia nodules growing on your plants' roots, is there any way to harvest the nodules and propagate the bacteria in a culture, for subsequent inoculation of other seeds?

I've been trawling Google for an answer, but haven't found anything just yet. If you guys have any ideas or sources of information, I'd love to hear more!

That's a great question Patrick. I'm thinking once you've established the bacteria in your soil there would be no need to save or add it again. From a permie point of view I would say, the bacteria will show up on its own, just plant the host. The benefits of inoculation (instant population) the first year are undeniable though.

It appears to be a very intensive process! You need to get some outside chemicals, so the loop could never be closed on this unfortunately. It would be easier simply to buy the inoculant than to track down mannitol or dipotassium phosphate most likely, but if you're curious I've done the research and written up a how-to:

The bacteria nodules in the roots are harvested from successful nitrogen-fixing plants of the same species about to be planted, especially if you know those plants were inoculated beforehand. Harvest the nodules during flower. The nodules take the shape of rounded bumps forming on the roots.

The bacteria is extracted from the nodules, and placed in a sterile test-tube culture of “yeast mannitol broth.” The broth is made by combining 1 gram of brewer’s or baker’s yeast-water culture, 10 grams of mannitol (also known as “baby laxative”), 0.5 grams of dipotassium phosphate, 0.2 grams of magnesium sulfate, 0.1 grams of sodium chloride, and 1 gram of calcium carbonate to give the broth a pH of 6.8. The ingredients are combined in 1,000ml of distilled water, and heated just to boiling, then mixed well. The yeast mannitol broth is stored at below 86 degrees Fahrenheit in a tightly-sealed container.

The Rhizobia bacteria is introduced to the yeast mannitol broth, and incubated at 77 degrees Fahrenheit for 3-5 days, and then introduced to a larger 10-gallon fermenter with the same type of yeast extract medium at a temperature of 80 degrees Fahrenheit. An air hose is run down to the bottom of the fermenter, introducing heat-sterilized air bubbles. The bacteria is allowed to ferment in this medium for 72 hours.

Directly applying this bacteria liquid culture to the seeds or soil is ineffectual, and instead it needs to be inoculated into peat, which serves as the “carrier” for inoculating the seeds. Peat is superior to all other carrier candidates, everything from biochar to compost to plant meal. The harvested peat is dried at temperatures never exceeding 200 degrees Fahrenheit, and then ground into small bits. The peat’s high acidity needs to be neutralized to a pH of 6-7 with use of calcium carbonate.

Before adding the bacteria liquid, the peat should have a moisture level of 7%, and after the liquid is added it should have a moisture level of 40-50%, as anything higher or lower than this means bacteria will probably not survive. The liquid culture is blended with the dried peat carrier, and then spread in shallow layers to cure in open air at room temperature for several hours. The inoculant should be stored in a glass bottle plugged with sterilized cotton or wool. The bacteria is sensitive to heat and sunlight, and should be kept in a dark, cool place.

It’s impossible to overuse inoculant with seeds, and you may want to use an extra-large dose of inoculant if planting the seeds in soil where the species has never grown before, or the soil is poor, or there is less rainfall than usual. To inoculate the legume seeds, first coat the seeds in a sticky mixture of 1 quart milk and 2 tablespoons of molasses, and then sprinkle the seeds with the peat inoculant. The inoculated seed should be planted immediately, and if not planted in 4 hours, they should be inoculated again. Sowed seed should be covered with soil or mulch immediately to prevent the sunlight from killing the bacteria. The soil should be watered immediately if it is dry, to improve the bacteria survival rate.

My apologies, I misunderstood your question causing me to respond ignorantly.

Try this.
buy the innoculant ONCE, innoculate host plant and place in a pot indoors, under grow lights, feed the plant Fermented Plant Extracts(liquid nutrient derived from organic matter), and fish hydrolysate. When you need to innoculate new area's Prune the roots of your indoor host plant, toss trimmings where needed, then cover with some soil and plat seeds of the host species. repot your indoor host. this method works for mycorrhizae so it might work for rhizobia.

I am currently attempting to do this with Azospirillum brasilense on peppers, tomatoes, and going to be starting it on a lettuce mix.

Yes, I know this thread is 6 years old.

One of the loops I see on a farm, is the set of sugar loops.  Sugar can be fermented by yeast (fungi) to produce EtOH, EtOH can be fermented by bacteria to produce acetic acid.  The yeast, leftover fruit and what not often make good feed for all kinds of animals, and if not it can probably be composted.  If you are going to put things like spent fruit to other purposes, it is probably best to dry that product in such a way, that the exhaust of the drying process can serve as combustion air (with ethanol) for some kind of furnace.

My feeling is that while ethanol is most definitely volatile, it is not the pollutant that most people seem to think it is.  Because it has been part of the biosystem for so long.  Heck, ask any drunk bird that flies into your window during fruit season if ethanol is a natural part of the environment.  I have not been having much luck tracking down a proper life cycle analysis that is freely available.

To me, it makes more sense to pipe that small amount of ethanol into a burner, instead of just venting it to the atmosphere.  But even then, I don't think that EtOH emitted to the atmosphere is as big a problem as other volatile organics.

Governments all over the world have made a lot of money off EtOH (often spelled booze).  They have more laws than you can shake a stick at.  This note is going to ignore said laws.

I may be skipping big chunks of things here.

If you want to harvest nitrogen fixing bacteria from legumes (but probably not honey locust, which seems to primitive to form the nodules in question, even if it still has a positive nitrogen balance), probably the best sterilizer for the surfaces of the roots is bench ethanol.  You can distill any EtOH containing solution which has been fermented by yeast multiple times, and get something approaching 96% EtOH.  If your farm is considering using acetic acid as a herbicide, you may want to play games involving ethyl acetate which probably gives you the ability of producing glacial acetic acid and 100% EtOH.

If you are serious about making wine/beer/mead (and this includes things like barley wine to make whiskey), you are probably interested in culturing multiple kinds of fungi (yeast) and storing them.  Probably on slants (nothing to do with Asians, the vial of (usually? agar gel) is kept at an angle so the gelled surface is on a slant).  As part of testing things like nitrogen fixing bacteria, you probably want to have a Chinese style greenhouse, and on the other side of the mass wall you could put a lab for working with the various bacteria and fungi.  You can do surface bonding of dry stacked concrete block with a mortar that contains "strings", but for this I think what makes a whole bunch more sense (and probably doubles the surface bonding cost) is "wet" out your dry concrete block with epoxy resin, apply a layer of glass fabric of reasonable strength, apply more epoxy resin to work in.  If you are working with an epoxy with a pot time of about 1 hour, I will suggest that the surface of the epoxy will be chemically active for about 1 week after it "sets up".  Apply a white polyurethane paint (zinc oxide or titanium dioxide white) with UV stabilizers to that epoxy within that 1 week period, the earlier the better (more active sites).  The paint will form a chemical bond with the epoxy.  Install regular lights in the room, but also install lights that emit a lot of UV.  You can get ceramic tiles with titanium dioxide or nano-silver, which when illuminated with UV light in the presence of water, generate hydrogen peroxide.  Self-bleaching surfaces.  You want to mist the surfaces before you turn on the UV.

Being white, it should be easy to see stains on the walls and floor.

Now you have a place where you can prepare, culture and scale-up bacteria and fungi.

I think you want (at least) 2 receiving rooms: one for bacteria and one for fungi.  You may be able to work with more than one bacteria or fungi at a time, but I don't think you should be working both at the same time.

If you are trying to collect a nitrogen fixing bacteria, you go to the bacteria receiving room.  Things like loose dirt should have probably been removed before.  The sample of root is nominally clean.  In the case of nitrogen fixing nodules on roots, I believe most (all?) of them are pinkish.  Sterilize your outer root surface with bench EtOH.  You can then probe the nodule, to try and extract the bacteria.  I think you then want to "scratch" an agar plate, and incubate.  There may be places on the incubated plates which seem to have a lot of bacteria. and places with little.  There are books on identifying the bacteria of interest.  You may need to scale up a possible and then test it (in the attached greenhouse).

Scale-up isn't that big a deal.  Anyone who has accidentally food poisoned others, has successfully scaled up a bacteria.  There are recipes, and use the right recipe and things should work out fine.  If your scale-up involves more than 1 kg of something (I am scaling up nitrogen bacteria to seed 1/4 section of land), there are lots of nit picking details which engineering typically does in stages and heat flow becomes an important problem.

But, I think you store things on slants, under the proper conditions.  So, maybe you extract a white clover bacteria one year, and a red clover bacteria another year.  Do you need to store bot?  You can test (scale ups) of the two slants.  The first thing, is to produce two new slants (which can get infected in the process).  So you can test the white bacteria on white clover and on red clover.  And likewise for the red bacteria.  And follow what happens in the attached greenhouse.

Using a living system to "store" the bacteria or fungi as Julian Kirby suggested sounds interesting.  It avoids problems like "the power was out, and all our slants were lost".  It may be that there is a best plant, a best soil, a best pH, and other variables which need to be found.

Patrick Winters wrote:Rather than being dependent upon outside sources of legume seed inoculant, I'd prefer to close the loop in the classic permie manner.  Once you have nitrogen-fixing Rhizobia nodules growing on your plants' roots, is there any way to harvest the nodules and propagate the bacteria in a culture, for subsequent inoculation of other seeds?

I've been trawling Google for an answer, but haven't found anything just yet.  If you guys have any ideas or sources of information, I'd love to hear more!

As you have discovered, it can be done but you need a laboratory to do it properly.
However, once you have Rhizobia present in your soil, you really don't have a need to inoculate successive plantings unless you just want to.
The Rhizobia will thrive in the soil as long as you build your soil and those soil present rhizobia will migrate to the developing roots and form nodules where they will do their feeding of both themselves and their host plant.
Having healthy soil that has had a legume crop or two grown in it, the rhizobia will be present from the previous crop, the only way to remove them would be to pull up the plants without leaving any of the nodules in the soil.

If you wanted to expand the growing areas, then producing rhizobia cultures would perhaps be worthwhile but you could alternately take a few spoons of soil from the previous growing site and use that to inoculate the soil in the new site.
Depending upon how large an area you want to grow legumes in would in the alternative proposal require about 2 gallons of primer soil per 100 sq. ft. to get complete inoculation over a two week period.

Redhawk