Live and Let Die: The Importance of Active, Healthy Microbes

We talked to Becky Williams-Wagner, PhD in microbiology, to better understand microbial responses to stress, and we asked her about the importance of healthy microbes for biological products. 

What is the importance of active, healthy microbes versus inactive ones? 

To benefit plants, microbes must be alive and viable when applied. Biological products experience microbe die off in the supply chain and/or as a result of contamination introduced during large batch production.

Once applied, microbes must navigate factors in the soil including texture, pH, temperature, moisture, salinity, nutrients, competition with other soil microbiota, predation by higher organisms, and infection by bacteriophages. Colonization, or the necessary spread of a microbe on the root surface, aerial tissues, or internal plant tissues, is an active process that requires a living microbe that can adapt to a dynamic environment that may not be microbe-friendly (Knights et al., 2021). For microbes that colonize inside plant tissues (known as endophytes), some of these environmental factors are lessened  and may make these microbes more successful.  

One of the biggest challenges for the microbial biostimulant market is the inconsistent reporting of results in the field. The newly released 2023 Ag Biologicals Landscape clearly demonstrates the confidence that industry has in biological products. Undoubtedly, each of these companies believes their product will provide benefit to the targeted plant. These benefits may be in the form of nutrient use efficiency, direct biofertilizer action (e.g. biological nitrogen fixation), biocontrol, or plant growth promotion via phytohormones. 

However, if the microbes are not alive and active at application, they cannot provide those benefits.

Knights HE, Jorrin B, Haskett TL, Poole PS. Deciphering bacterial mechanisms of root colonization. Environ Microbiol Rep. 2021 Aug;13(4):428-444. doi: 10.1111/1758-2229.12934. Epub 2021 Feb 15. PMID: 33538402; PMCID: PMC8651005.

How do we know if microbes are active? 

Outside of the laboratory, there is not an easy way to assess microbe viability. Each microbe is different and will have different biological capabilities, and so a one size-fits-all method of evaluating viability is challenging to identify. 

One example, 3BarBio’s LiveMicrobeTM fermentation system allows the farmer to see that the microbes have grown. Initially, the liquid contained in the product will be translucent. After activation and exponential growth of the microbes, the liquid will become turbid, meaning that the light no longer passes through the media unobstructed. The product will look cloudy, and you may see “swirls” in the liquid. These swirls are bacterial cells that have grown to a sufficient density that they are visible to humans.

What is the typical die-off rate of microbes, and how does that affect product efficacy?

For traditionally produced microbe-based products, which are manufactured in large steel bioreactors, the culture has already experienced exponential growth. Following growth, the culture is formulated, which is the process of stabilizing the cells for storage prior to application. 

Current industry standards recommend that products have a two-year shelf-life, meaning the product maintains the viable cell counts indicated on the label for two years. However, reports suggest that many products do not meet this label guarantee (Herrmann et al 2015). Precisely when these products lose viability is unknown; for some products, this may occur after several months. 

The impact on efficacy of microbial die-off within the supply chain depends on the mode of action for the product. Some products contain secondary metabolites or enzymes that are produced by microbes, which provide the benefit in the field and not living bacteria themselves. Because of this, it is possible to purify the metabolites or enzymes, or deactivate the bacteria and sell these as standalone products. For these products, the active ingredient is 

applied to the plant or field, and is used or degraded. Live bacteria are also capable of producing secondary metabolites and enzymes, and may potentially continue to produce these products on the plant root, or within plant tissue for endophytic bacteria, as opposed to a single application for standalone products. 

However, other modes of action, such as biological nitrogen-fixation, require live bacteria to function. For biological nitrogen fixation to be effective, the bacterial cell must actively colonize the rhizosphere, such that a close (in proximity) relationship is established. The bacteria are then able to fix atmospheric nitrogen into a form that is readily available to the plant, and in exchange, the plant supplies the bacterial cells with a carbon source. Dead bacteria are unable to fix atmospheric nitrogen, and products with this mode of action, in which the bacteria have died, will not be effective in the field. 

Finally, a sufficient number of live cells are required for colonization to occur, which is necessary for most plant growth promoting properties. If the culture is dead before application in the field, it is not surprising that the products are inefficacious. 

Finkel, S. Long-term survival during stationary phase: evolution and the GASP phenotype. Nat Rev Microbiol 4, 113–120 (2006).

Does the level of activity or sensitivity to die off vary by type of microbe? 

Microbial products may be composed of fungi or bacteria. For the purposes of this question, the focus will be on bacteria. Some species of bacteria used in agriculture, primarily Bacillus, are able to differentiate into endospores when nutrients become depleted. 

Endospores are dormant cells, which are also protected by proteinaceous coat and cortex layers that make them resistant to many stresses such as heat, desiccation, and UV radiation (McKenney et al., 2013). Because of this resistance to stress, endospores are capable of surviving for years, which is why industry has focused on developing products using endospore-forming Bacillus species. 

Once conditions become favorable for growth, the endospores may germinate into vegetative cells, which are metabolically active and can reproduce. However, conditions used to induce sporulation can impact revival of spores. Finally, the endospore must be able to detect favorable growth conditions in order to germinate and potentially colonize a root (Setlow, 2013). If favorable conditions are not detected, then the endospore will remain dormant. Therefore, the endospore must be deposited in close proximity to a root to detect nutrients secreted by the plant, which provide favorable growth conditions for germination to occur. 

Non-endospore-forming bacteria do not naturally enter this resistant dormant state, and therefore, they must be manufactured in a way that induces dormancy. This is most easily achieved by drying the bacteria cells (Garcia, 2011), but this process can be stressful to the cells and can increase manufacturing costs. The shelf-life of the cells that survive the drying process will likely be shorter than that of an endospore. 

However, the shelf-life of dried bacterial cells will be longer than that of a liquid culture. Furthermore, a dried culture will be less prone to contamination effects because the water activity is too low to permit microbial growth.

McKenney, P., Driks, A. & Eichenberger, P. The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nat Rev Microbiol 11, 33–44 (2013).

Setlow, P. (2013), Summer meeting 2013 – when the sleepers wake: the germination of spores of Bacillus species. J Appl Microbiol, 115: 1251-1268.

García A. H. (2011). Anhydrobiosis in bacteria: from physiology to applications. Journal of biosciences, 36(5), 939–950.

Does the formulation method affect microbe activity and die off? How does that happen? 

Formulation is the process of preparing and stabilizing microbes prior to their application in the field. There are several steps involved in this process, all of which can be varied and can affect the final product: 

1. The first step in this process is the growth of the bacterial cells, which involves selecting the proper growth media for the bacterial cells and can be strain-dependent, although there are some general-purpose growth media that can be used for multiple species. 

2. Next, physical properties for bacterial growth must be considered such as temperature, pH, and agitation. For growth in a bioreactor, the option to oxygenate the media is available. 

3. Once the growth conditions have been set, the optimal harvest time is the next consideration. There are five distinct phases of bacterial growth, which are lag phase, exponential growth phase, stationary phase, death phase and extended-stationary phase. 

During lag phase, the cells adapt to their environment such that exponential, or rapid, growth can begin. Finally, as one or more nutrients are exhausted, growth slows, and the rate of growth equals the rate of death. 

This phase is called stationary phase, and the viable cell counts stabilize during this phase. Furthermore, the cells activate expression of genes that facilitate survival during stress and repress expression of genes that favor rapid growth. 

Death phase occurs when the cells become too depleted of nutrients, or a toxic waste product accumulates. 

Ultimately the culture enters the extended stationary phase. During this phase the total viable cell counts are stable, but at a significantly lower level; nearly 99% of the cells originally present in the culture have died. The remaining cells are a conglomerate of sub-populations that emerge as the cells acquire mutations that permit growth on nutrients released by dead cells in the culture. This population can diverge from the original parental strain due to genetic drift. 

4. In general, it is best to harvest cells during the stationary phase before significant cell death occurs. However, some reports indicate success when cells are harvested during exponential growth phase. 

Another consideration is that of preconditioning the cells to survive the ultimate stress of formulation by application of sub-lethal stress during preparation of biomass. This could include heat-shock, cold-shock, osmotic stress, or pH stress, which can activate stress response pathways to protect the cell from damage. If these pathways are activated, they may protect the cell during subsequent formulation steps. 

After the cells are harvested, they are often combined with additives that are designed to protect the cells during any subsequent formulation steps or storage. These additives can include different sugars, polymers or amino acids. For liquid products, this may be the final step before packaging and distribution. This material should maintain the viability of the cells during storage. However, the shelf-life of liquid products is often relatively short, and these liquid products are prone to contamination due to the high water activity and the presence of nutrient components in the formulation left over from biomass production or released by dead cells. 

To improve the shelf life of these products, the formulation components are dried, which promotes dormancy of the cells and reduces the chance of contamination. Drying methods can include lyophilization, spray drying, fluidized bed drying, and other emerging technologies. 

During lyophilization the biomass is frozen and the frozen water is sublimed under vacuum. Spray drying involves atomization of the formulated biomass at high temperatures to promote drying. This is a cost-effective and scalable method, but is difficult to apply to non-endospore-forming bacteria because the temperatures are too high for the bacterial cells to survive. However, this method could be effective for endospore-forming bacteria. During fluidized bed drying, the formulated biomass is sprayed onto granules, which are dried by application of a stream of warm air. This method is cost-effective like spray-drying and requires cooler temperatures for drying, which makes this method attractive for non-endospore-forming bacteria. 

All of this is to say that there are many steps involved in the formulation of microbial cells for storage and distribution. The impacts of variation of each of these steps will be strain-dependent. The ultimate goal of formulation is to enhance the shelf life of the product. Viable cells are required for colonization of the plant, which is vital for efficacy in the field. Each of these formulation steps can impact the stability of the cells and, ultimately, the efficacy in the field.

Are there conditions that hasten die off, and can delivery methods and packaging have a positive effect? 

There are several factors that influence the stability of microbial cells once formulated and packaged, which include temperature, humidity/moisture, light, oxygen, and microbial contamination

Temperature is a major factor impacting the stability of microbial products. In general, viability declines at higher temperatures. One way to mitigate this is to refrigerate products, but this can be costly to implement during distribution and not every farmer has this capability for these products. Unfortunately, packaging alone is unable to control the temperature experienced by the contained products. 

Another factor to consider is moisture. For liquid products, this is not typically a problem, but for dry products, moisture is a major factor in determining the shelf-life of the product. As moisture or relative humidity exposure increases, the viability will decrease. This is because moisture permits reawakening of the microbes, which were previously formulated to promote dormancy and therefore stability during storage. 

Once the microbes have awakened, they require nutrients to survive. If they are awakened and applied to a plant, then the plant will provide this nutrition. But if they are awakened in the packaging by humidity with limited nutrients, they will eventually die. 

Fortunately, packaging and formulation are able to mitigate the impact of relative humidity on the stability of the product. Ideally, microbial products will be stored in packaging with a low moisture vapor transmission rate, which will reduce the amount of moisture able to permeate the packaging. In addition, formulation components may be added to absorb moisture before it can impact the microbial cells. 

Manufacturing and assembly of the product should occur under controlled humidity conditions to reduce humidity exposure. Relative humidity is temperature dependent, and so the humidity in the packaging can vary based on the external storage temperature. For dry products that are recommended to store at ambient temperature, a transition to cooler/refrigerated temperatures could trigger condensation of relative humidity in the packaging, which could damage the product. 

Other considerations include exposure to oxygen or UV light, which are both known to damage molecules within the cell, such as DNA (the microbial genetic material) and the cell membrane. Damage to the molecules can be lethal to cells, and so their exposure must be reduced. Packaging and formulation additives can ameliorate the effects of these factors. UV light exposure could be reduced by colored or opaque packaging. Antioxidants in the packaging or formulation can reduce oxygen exposure.

Finally, microbial contamination can impact the stability of the product during storage. This is especially true of liquid products, which may include formulation components that can serve as nutrients for contaminating species. As these contaminating species grow, they can actively kill the desired product or compete with these cells for nutrients. Once contamination is rampant in the product, it could be a health concern to people, animals, or plants. One way to mitigate this is to manufacture products under aseptic conditions, but this is costly at scale.

Does the delivery method, system, or package affect microbe activity and die off? 

Microbial products are typically sold in two formats, liquid or dry. In general, liquid products will fit into the farmer’s standard practice. However, liquid products are less shelf-stable than dry products, the exception being liquid endospore products, which will be stabilized by the spore physiology. This is because, in the liquid format, microbes are metabolically active and require nutrients for this activity. Without sufficient nutrients, the microbes will eventually enter the death phase. Furthermore, liquid products are more susceptible to low-level contamination that may be introduced during manufacturing. 

Dry products are more shelf-stable because the microbes are stabilized and dormant due to desiccation. However, these do not typically fit easily into the farmer’s standard practice. Generally, these products must be combined with a liquid or another carrier material for application on the farm. By separating the microbial powder from carrier material in a single package, the viability can be protected and the step of combining the microbial powder with the liquid carrier can be easy for the farmer. 

Finally, farmers must use caution when combining microbial products with other agricultural inputs. Some fertilizers can induce lethal osmotic shock to the microbial cells when combined. In this case, the bacterial cells may still be alive in the package, but they are destroyed when added to the planter tank with certain fertilizers. In addition, certain pesticides may be harmful to microbial products. Compatibility should be tested at the tank-mix rate to ensure one product does not destroy the other. 


Live Microbe Fermentation System

In the LiveMicrobeTM  fermentation  system, the dried microbial powder is contained in one chamber, and a sterile liquid  is contained in a second chamber. By activating the first chamber (for example, by breaking the foil seal), the microbial powder is combined with the liquid. Once combined, the microbial cells will become metabolically active (either through fermentation or rehydration). This system combines the shelf-life benefits of the dried biomass with the ease of use of the liquid product for application.

Another example of this separated package is Meristem’s Bio-Capsule technology, in which the dried microbial biomass is contained in cartridges separate from the talc/graphite carrier material. The farmer combines the microbial biomass with the talc/graphite carrier material with the push of a button, and applies it to the seed before planting. This keeps the microbial biomass separate from the carrier material, which can be harmful to the microbes until application. 

What are the larger financial implications of microbe activity versus die off? 

There are two broad areas where microbe die off presents a financial concern: ecology and manufacturing. 

Ecology is often overlooked when considering application of microbial products. The whole system should be considered when applying these products and anticipating results. If, for example, a healthy and effective microbe is applied to a field which already has excellent soil and irrigation, sufficient nutrients present for the plants, and low disease pressure, then the microbe may not enhance plant growth. This is because the plant and its system already have everything they need. 

However, if a healthy microbe is applied to a stressed system, then it is more likely to improve the growth of the plant. For a farmer using these products and not seeing an effect, the system should be evaluated. Perhaps these products are not needed. Alternatively, the products may not be viable when applied to the field. 

In these examples, farmers may well have wasted their money.

For manufacturers, the cost of producing microbial products at a high density to account for the inevitable loss of viability is a waste of manufacturing dollars and resources. Stabilizing the microbe effectively is vitally important to reduce the waste of money, energy and resources. 

3Bar’s LiveMicrobe innovative approach shifts growth of the bacterial cells to a point closer to application and ensures that the population is viable and resources are not wasted. With increasing emphasis on more sustainable practices, the energy and water required to produce these products just for them to die in the supply chain is negligent and wasteful. 

Profile photo of Rebecca Williams-Wagner, Ph.D.

Becky Williams-Wagner earned her BS in Molecular Biology from Otterbein University, and a PhD in microbiology from The Ohio State University. During graduate school and as a postdoctoral researcher, she studied physiology and gene regulatory mechanisms of the soil bacterium, Bacillus subtilis, in an effort to understand microbial responses to stress. She mentored graduate and undergraduate students in the laboratory setting, and was an Adjunct Faculty of chemistry and biology at Otterbein and Ohio Dominican Universities. At OSU, she was a founding member of the RNA Biology Student Organization and a recipient of University and Center for RNA Biology Fellowships.