Microbial consortia are groups of diverse microorganisms that have the ability to act together in a community. Such consortia are common in nature and are known to play important roles in many ecosystems but are not always well understood. Soil management and nutrient mobilization are one area where complex communities of microbes are known to be important, whether it be a naturally occurring consortium, or a man-made consortium.
In this conversation, we talk with Dr. Ray Ketchum from Agrinos about the microbial consortia he and his team cultivate and produce to improve plant health and increase crop yields. We learn about the challenges of fermenting mixtures of more than 20 diverse microorganisms to reproducibly make products that improve plant health and mobilize nutrients in a completely organic way. His team uses dPCR to titer each of the species within their consortia for quality and regulatory purposes, a task that cannot be done by cell culture methods given the range of bacteria involved.
To learn more, listen to the Absolute Gene-ius dPCR Podcast Episode at www.thermofisher.com/absolutegeneius
Transcript from Absolute Gene-ius episode four “Shrimp-ly amazing science”. This transcript has been edited for clarity and brevity.
Ray Ketchum, PhD: What we do here is, we produce microbial consortia to use in agriculture. These are basically microbiomes that we manufacture and sell to growers, and those are applied to the field. What I do is, I lead the group that does much of the research and development as well as the technical production for the bacteria that we grow and also for doing the analysis of those bacteria. My group coordinates the quality control tests that our quality manager and our quality technicians actually use.
Interviewer: That’s awesome. So you’re talking about consortia. Can you elaborate a little bit on what that actually means and how that’s important?
Ray Ketchum, PhD: Agrinos manufactures three main agricultural products at this time. We have others that are in development but those are our commercial products.
One of them is a microbial consortium, which contains 22 different bacteria. These are all soil bacteria, they’re nothing genetically modified, all of our products are organic certified, and OMRI certified. These are just naturally occurring soil bacteria that we’ve taken. We’ve grown them up separately, at least to isolate them, but then as we manufacture them, we group them together.
We grew up this large fermentation which contains a collection of anaerobes in one fermentation and aerobes in another fermentation and we blend those together. These are all bacteria which are intended to improve plant health, mostly by increasing the availability of nutrients in the soil. Some of the bacteria actually fix nitrogen. Some of them help to solubilize inorganic nutrients that are in the soil, like phosphorus or certain types of inorganic potassium, and make those available to the plant. All these bacteria have been isolated from the rhizosphere, which is the part of the earth that surround the roots of the of each individual plant.
If you pull up a plant and you’ve got that big bundle of dirt, there are a lot of bacteria that are in that soil that are very closely associated with the roots and there’s sort of this beneficial interaction between those bacteria in the plants. The plants actually provide the bacteria with certain nutrients and in exchange, the bacteria also help to solubilize some of the materials or actually produce nitrogen that gets absorbed by the root. There’s this interaction and we’re trying to exploit that by making up these groups of bacteria that you can actually add to the soil. “Consortium” refers to just that large collection of bacteria in a single sample or a single product.
Now, I mentioned also that there are two other products that we manufacture. We’ve taken this and we’ve gone one more step. What we’ve done is, we have a production facility in Mexico that uses these same bacteria to help break down shrimp bio-waste in Sonora, Mexico, along the Gulf, or the Sea of California, the Sea of Cortez.
There’s a lot of shrimp farming that’s done down there and after the shrimp are deveined, the shells are removed, and they’re basically cleaned up, there’s all this waste material that’s left over. We take that waste material and we ferment that with these same bacteria.
From that, we produce two additional products. One is a bio-stimulant product, which is the liquid that’s left over from that fermentation. The other is all the solids that are left over are dried and milled down, and that’s our third product. We’ve been trying to be this green company where we’re helping to reduce waste by actually using that waste in different ways and applying that to agriculture, and it’s actually worked quite well.
Interviewer: Question about the shrimp. I’d love to talk about that. How did you even find out that the, that shrimp waste can be something that’s impactful for plants and for the soil?
Ray Ketchum, PhD: Shrimp, and almost any other type of shellfish or that type of waste, if they contain protein, they contain a source of nitrogen.
Nitrogen tends to be the nutrient that plants need the most because they can’t make it themselves. It’s returned to the atmosphere fairly readily. Any source of nitrogen is usually a good thing.
The other thing that shrimp, other types of shellfish, and crustaceans in general have is chitin in their exoskeletons. Chitin can also serve as another source of a more slowly degrading nitrogen, as long as you have bacteria that are in the soil that are able to break that down. They can break down that chitin into to nitrogen as well as some other carbohydrates, and again, those are all very beneficial for plants.
Interviewer: So, it was looking at the at the chitin in the material that the shellfish have and seeing how you could apply that into the consortia, and what you offer to try and get more nitrogen into the plants is how that stemmed is, is that a correct assumption?
Ray Ketchum, PhD: Right. I mean, that’s basically the end product. The solids that are leftover, that’s the material that contains the chitin.
The other interesting thing is, when you actually do this process, you separate the solids and the liquids. The liquid portion of this, the lipids actually get removed from that. The lipid, or the material that’s leftover is this liquid portion that’s, in many cases, what’s left over from the bacteria when they’re actually using this as a food source. It’s some of the metabolic products that the bacteria produce, enriched in this liquid.
What’s fascinating about that particular liquid is it’s a stress relief mechanism. It’s a way of helping plants to recover from certain types of stress, like water deficit, or in some cases herbicide or pesticide drift or anything like that. It seems to be very effective in in terms of relieving stress.
Interviewer: It’s like a seafood broth for when you’re sick. A nice broth to calm the throat, it does the same thing for the plants?
Ray Ketchum, PhD: Exactly. Instead of a chicken broth, it’s more like a shrimp broth.
Interviewer: Some might call it “shrimply amazing.” I’ll see myself out. You have fun. Bye now.
Interviewer: Talk a little bit about the R&D behind some of these products. Do you look at individual strains or species of bacteria and try to figure out how they might interact as a consortium and if it’s worthwhile to add them in? Or how does that R&D function?
Ray Ketchum, PhD: We’d have a large consortium, or a large collection I should say, of bacteria that have been collected through the years from a lot of these field sites and things that are in our stores or in our master cell bank. That gives us a large library to go back to and pick out certain types of function.
A lot of the bacteria had been screened for metabolic functions and characterizations. That’s one of the things that the R&D group and my group does. We screened different bacteria for different abilities, in terms of breaking down waste, or chitin, for instance nitrogen fixation, solubilization. But then we also will take individual strains and we will DNA sequence them. Those are some of the types of activities that we do, but probably the main thing that my group does and spends most of their time on is just figuring out how to grow these bacteria together.
What’s a little bit unusual about our company compared to other companies is a lot of companies will put a bacterial product together that may have four or five bacteria in it, in which case they’ve grown each of those individual bacteria separately. One of the things that we found, and it applies to this bio-stimulant product that I was telling you about, this liquid portion, is that there is a benefit to growing all of the bacteria together.
There seems to be communication between certain bacterial strains, almost interaction between those strains. And if you think of some of those ecosystem studies that you might have done in biology, where you had a fish tank and you threw some duckweed in there, maybe some algae, and you threw some fertilizer in there, and then you just kept taking samples throughout the year and you saw how that thing changed over time, that’s what you see with some of these bacteria. The whole population will change at certain times during the fermentation.
We want to get to a point where things are pretty much stabilized, we know the numbers of certain types of bacteria within there, and we know that it’s going to be an effective product once it’s applied to the field.
Interviewer: Interesting. So, you’re seeing a benefit to growing them all together. There’s not some kind of competition, in a sense, between the bacteria, or?
Ray Ketchum, PhD: There is. And again, that’s it, there absolutely is. That’s where the real fun starts, figuring out how do you get these things to grow together so that they’ll all come up, they’ll all reach a certain titer, a certain population within that product.
There’s competition in, if you don’t do it right there will be certain bacteria which will completely dominate and mess up the whole culture. That’s the proprietary work that we have in terms of figuring out, how do we get them all to grow together? And how do we get them all to get up to that particular titer?
That’s where the magic happens.
Interviewer: That’s very cool. Now, I would assume as you scale from R&D to production level for a product, there are some difficulties that arise in the scale-up. Is there a way that you need to QC or QA as you kind of scale up the product and make sure that you’re seeing the same kind of breakdown of bacteria?
Ray Ketchum, PhD: That is one of the main challenges: making sure that we’re hitting a certain titer.
For our main product, we have a specification on our label which we have to meet just from a regulatory standpoint; QC is very much involved in that with us. The other thing that we need to do, too, is depending on if it’s a new product, we need to figure out, once we’re able to grow things together, “Well, what is that titer that we can reliably hit?” So, there’s a lot of work done on saying, you know, “If we adjust this parameter, can we get everybody up here. or do we have to settle with everybody down here or what?”
To get those titers, if we’re working with individual bacteria, it’s super easy to do, just play them out. But if you have a collection of bacteria, that’s where things get a little bit more complicated. That’s where the digital PCR comes in.
Interviewer: How does digital PCR as a technology play a role in that in that process?
Ray Ketchum, PhD: We wouldn’t be able to do what we’re doing without digital PCR. The reason for that is because we have a product that has 22 different bacteria together. It’s impossible to plate these bacteria and count them all at once.
Some bacteria don’t plate very well. Some of them are anaerobes and some of them are aerobes, so they can’t grow under the same conditions. All of these variables make digital PCR really a keystone to the technology to our quality control.
What we do is, we do a whole genome sequence for each of our bacteria. We figure out within that genome, which genes are single copy and which ones are unique to that particular species, and then we design primers and probes for that portion of that particular gene. In some cases, it’s just a DNA sequence; it may not actually be a coding gene.
Once we have that, we can take a very complex mixture of bacteria, do total DNA extract on that, and then analyze that, with the assumption being that every signal that we get on digital PCR relates back to one single bacterium. By that, we can actually get some pretty good numbers.
Interviewer: Makes sense. Do you run into any inhibition problems as well, when it comes to bacteria or some of the samples? PCR inhibitors or any type of inhibitors that might impact, say, real-time PCR versus digital PCR?
Ray Ketchum, PhD: I would say that, at this point we haven’t actually done the real-time PCR versus the digital PCR comparison to see if there’s a difference there. I don’t suspect that there is, simply because the way the DNA is extracted, most of those inhibitors should be removed. But there is another issue that we have run into recently and I think it’s something that the industry as a whole is starting to notice a little bit more.
When you take this complex collection of bacteria and you’re doing a consortium on them, whether it’s from a gut, human gut microbiome, the soil microbiome or whatever, there has been this assumption that your DNA extraction is extracting the DNA from all of the bacteria equally well, and that’s simply not the case. In our case, what we found is that a lot of our bacteria are spore-forming. And if they’ve formed spores, we just have a heck of a time really getting the DNA out, representative of what’s actually in the in the product.
It turns out that we’ve been way under counting what our product actually has. Those are some of the issues that we’ve run into. We’re working on those. We don’t have a solution to those yet. We’re really not sure exactly how much of an impact this is going to have. We started working with some of those 22 species, but we haven’t done all 22 species yet. This is something that we’re working on right now.
Interviewer: How does this product impact the customer? Is there an impact on food production, per se, for farmers?
Ray Ketchum, PhD: What our product does is it improves the yield in the field. We’ve seen individual studies where, depending on the crop, it could be up to a 30% increase. That’s really unusual. More frequently, it’s in the 5 to 15% range.
But what the product does is, as I mentioned, it does fix nitrogen. It adds additional nitrogen to the soil. It solubilizes any sort of inorganic nutrients that may be in the soil already. Some of the bacteria helped to break down residual field waste, a lot of the straw and canes that are left over from the harvest. They help to break that down. There’s a lot of benefits that way.
I mentioned, also, the other two products. One is a stress relief product. That’s our “B Sure,” is what it’s called. “iNvigorate” is the consortium that we’re talking about. “Uplift” is the chitin containing product as well. So, all of those, yes, increase in crop yield. And mostly it’s for agricultural crops. Okay, some of the B Sure’s been used very successfully in turf.
Interviewer: So, any studies into potential impacts on, say, climate change?
Ray Ketchum, PhD: We haven’t done any studies specifically about these bacteria. Certainly, one of the things that they will provide is additional nutrients to the plants without additional artificial inputs such as chemical fertilizers.
At least in that regard, you know, that 5% increase in yield is coming not at the expense of using additional petrochemical-based products and gas and diesel that’s used in the field, but just from the bacteria themselves. But we haven’t really looked at any sort of carbon sequestration issues with the with the products yet.
To get more information and hear career advice, listen to the Absolute Gene-ius dPCR Podcast Episode at www.thermofisher.com/absolutegeneius
Learn more about digital PCR at www.thermofisher.com/absoluteq
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