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Old September 8, 2012   #31
greentiger87
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Well, it would basically be a diy version of submerged culture, which is an established way of culturing many useful organisms. A nutrient broth is inoculated with the microorganism in a flask, and then placed on a shaker table (with or without incubation). The nutrient broth is tailored precisely to fit whatever the goal is - producing a natural secondary metabolite or a transgenic product, producing spores for drying, or just producing more of the organism. It's scaled up to large tanks with air blowers for things like antibiotic production (and is likely how Actinovate and Serenade are made to begin with). The normal time period before a batch would be "ready", or full of spores and secondary metabolites, is usually around 48 hours.

I've investigated submerged culture of both bacillus subtilis and streptomyces lydicus... despite being very different animals, the actual requirements, especially for a rough/imprecise culture like this, are very similar. A carbon/carbohydrate source, a protein/nitrogen source, aeration, and trace elements (covered by tap water and whole food nutrient sources). In fact, other than being cultured at room temperature, this doesn't differ much from the submerged culture of E. coli, which I'm very familiar with.
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Old September 9, 2012   #32
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Thanks, It's good to have someone around here with your professional knowledge of microbiology. I'm an amateur But I do understand what you are saying from what I've read.
In the case of Bacillus subtilis which reproduces by both binary fission and by producing endospores, if you want spore production, you would lower the nutrient inputs, basically starving Bacillus subtilis. Bacillus subtilis is triggered into producing endospores when under nutrient stress, kind of insurance for species survival if things get hostile. Is that basically the same reaction as would happen with Streptomyces lydicus though it only reproduces by spores?
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Old September 9, 2012   #33
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Lol, just as a caveat. I'm not actually a microbiologist by profession. Most of my undergrad training was in biochemistry and microbiology, but I ended up getting a job as a materials chemist.

Streptomyces spp. are not nearly as well understood as Bacillus subtilis, but the basic triggers for spore formation are the same - nutrient depletion and environmental stress. Additionally, some aspects of primary metabolism may be triggers as well - e.g., accumulation of glycogen or other energy storage molecules.

You're right that Streptomyces only truly reproduces by spores (called conidiospores in this case). Conidiospores are much easier to kill than endospores, but they're omnipresent in the air and quite successful. Strepomyces is weird because it has some characteristics of a multicellular organism. When it first germinates, it goes through a phase of vegetative growth where it grows a filamentous, branched "primary mycelium" that's really similar to fungal mycelium. Each strand grows only from the tip, and is essentially one cell with many, many copies of the organism's genetic material. Rarely, the filament does actually go through "cell division" by partitioning the strand with a cell wall, but the new cell is not a new organism - it remains integrated with the rest of the mycelium.

When sporulation is triggered, Streptomyces produces an "aerial" mycelium that rises above the substrate and partitions into individual compartments, each with a copy of the genetic material. These compartments eventually become spores, and drift off in the air to find new and exciting adventures.

On a solid substrate, like agar, perlite, wheat bran, etc., sporulation is extremely obvious - a big fuzzy, hairy mass appears. It's usually a darker color than the primary mycelium as well.

It seems that the time to sporulation varies a lot, from 3 days to 14 days. Some of that has to do with the sheer size of the culture involved, but it may take a certain amount of time for the bacteria to develop regardless of other factors.

It's not yet clear to me how you can tell if sporulation has occurred in a liquid (submerged) culture. I'll have to do some more research there.

Truthfully though, it really doesn't matter. Even fragmented pieces of primary mycelium remain viable, and are a perfectly good way of delivering the organism when you're not worried about preserving, packaging, storing, and delivering a standardized product to a customer.

Multiple references do say that producing Streptomyces spores on a sterilized solid substrate is easier and more economical. But it's not clear to me whether or not mechanical aeration is necessary, and how it could be accomplished in a DIY context.

Btw, all of this is a piece of cake for Bacillus subtilis. Just throw it in with some nutrients and air, and let it multiply. Production of endospores, again, really isn't necessary.
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Old March 23, 2013   #34
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This is a good thread to bump with my questions. I have been trying to figure out a beneficial bacteria/fungi innoculant.

What if I bought mycogrow: http://www.fungi.com/product-detail/...uble-1-oz.html

and tried to keep a constant culture of it in the water I used for my greenhouse plants? I have a 220 gallon horse trough I can fill with water. I would need a lightproof lid and an air or water pump for aeration, but that is no big deal. Last year I kept bluegill in the tank and used that water. It's a similar idea.

I have read that a lot of the US uses chloramine now in the tap water and not chlorine. Chloramine has a half-life of about three months, unlike Chlorine which is closer to three days. I am hesitant about using tap water at all. I could use pond water; it would be bacteria soup. I guess that would mess up my culture effort. Maybe pond water treated with chlorox that has sat for a week? It might be easier just to buy a reverse osmosis water filter.
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Old March 23, 2013   #35
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Originally Posted by Cole_Robbie View Post
This is a good thread to bump with my questions. I have been trying to figure out a beneficial bacteria/fungi innoculant.

What if I bought mycogrow: http://www.fungi.com/product-detail/...uble-1-oz.html

and tried to keep a constant culture of it in the water I used for my greenhouse plants? I have a 220 gallon horse trough I can fill with water. I would need a lightproof lid and an air or water pump for aeration, but that is no big deal. Last year I kept bluegill in the tank and used that water. It's a similar idea.

I have read that a lot of the US uses chloramine now in the tap water and not chlorine. Chloramine has a half-life of about three months, unlike Chlorine which is closer to three days. I am hesitant about using tap water at all. I could use pond water; it would be bacteria soup. I guess that would mess up my culture effort. Maybe pond water treated with chlorox that has sat for a week? It might be easier just to buy a reverse osmosis water filter.
Most Myco need a living plant in fertile healthy soil or compost to propagate. It is a symbiotic relationship. Most the bacteria can be grown in the "compost tea".
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Old March 24, 2013   #36
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Originally Posted by Cole_Robbie View Post
This is a good thread to bump with my questions. I have been trying to figure out a beneficial bacteria/fungi innoculant.

What if I bought mycogrow: http://www.fungi.com/product-detail/...uble-1-oz.html

and tried to keep a constant culture of it in the water I used for my greenhouse plants? I have a 220 gallon horse trough I can fill with water. I would need a lightproof lid and an air or water pump for aeration, but that is no big deal. Last year I kept bluegill in the tank and used that water. It's a similar idea.

I have read that a lot of the US uses chloramine now in the tap water and not chlorine. Chloramine has a half-life of about three months, unlike Chlorine which is closer to three days. I am hesitant about using tap water at all. I could use pond water; it would be bacteria soup. I guess that would mess up my culture effort. Maybe pond water treated with chlorox that has sat for a week? It might be easier just to buy a reverse osmosis water filter.
I don't think it's worth it to try to keep a constant water culture going, some species may not even be able to survive long term in such conditions. I've never seen any recommendations for keeping an AACT or inoculant tea going for more than 36 hours. A lot depends on the water temperature, oxygen level and food sources.
Treating your soil is not something that needs to be done regularly anyway. Get them in the soil and they'll take care of themselves.
Although many bacteria can be cultured in a liquid medium, Mycorrhizae need to be near living roots, they can't be cultured in a tea. Trichoderma fungi are cultured in a substrate.

If using tap water, find out whether Chloramine is being used or not in your city water. Chlorine is much easier to deal with, Chloramines would need to be treated, liquid humic acids are supposed to work well at neutralizing Chloramines.
I prefer whenever possible to use rainwater for preparing commercial inoculants or AACT, no worries there.
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Old March 24, 2013   #37
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I prefer whenever possible to use rainwater for preparing commercial inoculants or AACT, no worries there.
If you have Chloramines and don"t have a rainwater system set up to first get them in the ground you might could use distilled water
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Old March 24, 2013   #38
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If you have Chloramines and don"t have a rainwater system set up to first get them in the ground you might could use distilled water
Yep, RO Water would work as well, but they would get expensive if you need a large amount.
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Old March 24, 2013   #39
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Thanks guys. It's confusing, because there are liquid amendment products as well as soil mix products that advertise that they contain beneficial bacteria and fungi. I guess they contain spores? Do you think it would be accurate to say that everyone's healthy plants would ultimately grow roots containing the same bacteria/fungi makeup anyway, but the innoculant method just gets the plant to that point faster?

It seems like keeping a tank of fish is the best I'm going to do at culturing bacteria in water. I am yet to find a good explanation of why plants like fish tank water so much. I am thinking it is something at a bacterial level that might not be especially well understood. It could be as simple as the fish being a mechanism to get the water treatment chemicals out of my tap water.
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Old March 24, 2013   #40
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I agree, it can be confusing and there are more and more amendment products coming out all the time with bacterial and fungal inoculants included as technology and mass production techniques have made them very inexpensive.
Almost all inoculants are delivered in spore form since they can be microencapsulated and kept dormant for long periods of time in a dry or liquid carrier. All fungi reproduce by spores, many bacteria do but not all.
I think it's only accurate to say that bacteria and fungi are ubiquitous in the environment, what beneficial species as well as pathogenic species may be present in native soils will vary with climate, soil type and cultural practices. Cultivated soils for crop production are many times the most abused and damaging to soil life. Excessive tillage, chemical salt fertilizers, fungicides and pesticides can inhibit or kill off many beneficial species including higher forms of life like earthworms, protozoa, beneficial nematodes, mites, and insects that are all important to the Soil Food Web. Using inoculants are a good way to cheat a little and introduce known species that will be beneficial to the soil and plants faster. Composts help that too.

Your fish tank has is like soil too, it contains bacteria, fungi, algae and other critters also, maybe not all good ones. Being a closed system it takes steps to keep toxins from building up which can kill your fish. Fish waste contains ammonia and nitrites which can get to toxic levels for the fish. Your plants like that fish water in part because those compounds can be converted to ammonium and nitrates by soil bacteria. I found this, there is even a live culture of Nitrifying Bacteria you can buy that does just that in an aquarium.
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Old March 24, 2013   #41
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Robbie,
There are 10's of thousands of bacteria, fungi, and other microbiology in every square foot of soil. Most are beneficial, a few are not. But they vary widely in the degree to which they are beneficial.

When you inoculate you are simply making sure the best of the best (that we know of) get an advantage first. This helps insure that the few bad guys have a disadvantage in their quest for domination. It is not 100%, but it does help. Theoretically, in a perfect world, in perfect soil, you wouldn't have to inoculate at all.

As far as aquarium water goes, I use it too. I think it is a good use for the water I have to change anyway. But I doubt it is actually all that significant a difference. Sure there are a few trace nutrients in very trace amounts. However, I seriously doubt it is really all that important. The soil is the biggie.
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Old June 13, 2013   #42
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So.. I've been culturing a certain biological antifungal product that many of us have found extremely useful. And it's been really successful! It's a streptomycete. I'm using a submerged culture technique, inoculating directly from the product with each batch, and using the product immediately. So basically, this allows the product to go a lot further - but you still need to buy it.

I've cultured 5 times now successfully, with a few failures. I'm using a 5 gallon bucket filled to about 3.5 gallons and an aquarium air pump with two airstones (each a cube of 1 inch).

Success is incredibly obvious because of the sweet smell streptomycetes produce. The effects on plants are pretty unmistakable as well.

I've tried a few different "recipes". The important thing is to keep nitrogen relatively low, use no simple sugars, and as few simple carbohydrates as possible. This favors actinobacteria over other bacteria. The most successful recipe is below.

1/4 cup crab or shrimp shell (provides chitin along with other macro and micro nutrients)
1 tbsp vinegar (this brings my tap water to slightly below neutral pH)
2 quarts of tapwater (my tapwater is hard, high in sodium, and alkaline)
1/4 cup cornmeal (carbohydrate and other nutrients)
1 tsp brewers yeast powder (provides full spectrum of nutrients and possible minor growth factors)

Chitin as the primary nitrogen source promotes the production of antifungal enzymes/metabolites and favors actinobacteria. For actinobacteria, the brewers yeast powder may actually be counterproductive. Actinobacteria are well adapted to situations with very little nutrition, and perhaps no exogenous growth factors, unlike many bacteria. However, it is a standard component of many culture mediums. I haven't yet experimented with leaving it out, but I will.

Do not add molasses, honey, sugar, or anything remotely sweet. It's just not necessary for actinobacteria, and will likely result in other bacteria taking over the culture. I've tried. The smell was horrible.

Cornmeal is very, very cheap at feed stores.

I powder the ingredients in a spice grinder if necessary.

Option 1) Tyndallization - Mix the solids and liquids in a stockpot. Bring to a low boil for thirty minutes, replacing water as needed. Let it sit overnight, covered. Boil again for thirty minutes and it's ready to use.

Pressure cookers used to scare me, so this is what I would have done in the past and have tried twice with success on this organism. Allowing the liquid to sit overnight promotes the germination of dormant spores that weren't killed by the first boiling. The second boiling kills most organisms before they have a chance to sporulate again.

Option 2) Ghetto Autoclave - Stick everything in a pressure cooker and let it go to three whistles or whatever makes you happy. Add extra water to account for the lost steam. Can be ready to use within 30 minutes of mixing.

Now you just dump everything in a well cleaned 5 gallon bucket and dilute to about 3.5 gallons. Turn on the pump, and add a tsp of the product. Wait three days or so. Smell it when you get impatient to reassure yourself.

Airline tubing, bubble stones, and the 5 gallon bucket should be thoroughly cleaned and disinfected after every use. Note that the air you're blowing into the culture is obviously not disinfected or sterilized. Apparently the streptomycete has enough of a headstart that this is not a problem.

I haven't had to use any chemical fungicides so far this season!

Last edited by greentiger87; June 13, 2013 at 11:11 AM.
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Old June 13, 2013   #43
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I have asked a few places and I seem to get the same answer. Some version of- I have never thought of that. I currently pay a lot more than I would like to for these products. I started with the liquid. I then read the powder label and it seemed to have more types of beneficial bacteria in it. I mentioned it to my Wife and the next day I get home from work and she had bought me a big tub of it(I think she is awesome! I originally thought she did it because she loves me. I now think she just wants a gazillion cherry tomatoes...hmmm)I now have both. Great, I am excited and rotate between the two. Doesn't change the fact this stuff is pretty expensive for the amount you get. My question is can you increase the amount you get? I have been using compost or worm tea and I know the reason you let it bubble for hours on end is to let the bacteria and fungi grow. If I add mycorrhizae to a bucket with molasses or some other food source can I turn a few cc's into a few gallons?
No. You can grow your own Myco, but not by brewing it.
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Old June 13, 2013   #44
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I remember reading somewhere that Glomus mosseae was the most effective species of mycorrhizae at colonizing bean plants, other species didn't colonize as well or at all and didn't make much difference in growth or yield. It's a common workhorse species in most myco inocculants, but it might already be native to your soil for all we know. I don't know what variety of species are in Garden-ville's innoculant.
Other than mycorrhizae, Trichoderma fungi and the Nitrogen fixing Rhizobium bacteria species that colonize legume roots are highly beneficial to bean plants.
Some plants will benefit from the inoculation of both Mycorrhizae and Rhizobium. I used both this year.
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Old June 13, 2013   #45
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Originally Posted by greentiger87
Chitin as the primary nitrogen source promotes the production of antifungal enzymes/metabolites and favors actinobacteria. For actinobacteria, the brewers yeast powder may actually be counterproductive. Actinobacteria are well adapted to situations with very little nutrition, and perhaps no exogenous growth factors, unlike many bacteria. However, it is a standard component of many culture mediums. I haven't yet experimented with leaving it out, but I will.
Thanks for sharing your experiment in such detail.
Since yeast are a single celled fungi and their cell walls are held together by chitin, wouldn't the antifungal metabolites made by the streptomyces also kill off the saccharomyces cerevisiae yeast?

Did you have the opportunity to try your brew against an active fungal leaf infection? An active culture already rich in antifungal enzymes sounds likes it may be very potent against fungal pathogens.

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Some plants will benefit from the inoculation of both Mycorrhizae and Rhizobium. I used both this year.
Yes, I've done the same with peas and beans.
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