Evonik’s novel biobased system to incorporate Gram-negative bacteria into liquid formulations for agriculture applications to extend shelf-life and improve field efficacy

Dr. Ines Ochrombel Head of Innovation Domain Agriculture Solutions Creavis, Evonik Operations GmbH	Dr. Stefan Gilch Project Manager Agriculture Solutions Creavis, Evonik Operations GmbH

Increasing food demands, diminishing arable land, and changing regulatory expectations call for strategies to intensify agricultural systems and improve environmental stewardship. Therefore, it is important to develop formulations that are not only effective in promoting plant growth and reducing disease, but also have a longer shelf-life and are environmentally friendly. Gram-negative bacteria present a unique challenge for agriculture formulations, requiring innovative solutions to promote stability and field efficacy. Gram-negative bacteria, unlike Gram-positive bacteria and fungi, cannot form persistence spores, and only have a thin murein layer envelope to stabilize the cell membrane, which limits their survival in liquid agricultural formulations. 

Evonik’s products have been used for decades in agriculture for formulations and as tank side adjuvants in conventional agriculture and now Evonik has developed an innovative process for biobased liquid agriculture formulations with Gram-negative bacteria. New developments show the value of using Evonik’s biocompatible additives for physically stable solid and liquid formulations with a vast range of microbial species. Evonik’s new approach is to use encapsulation technology to protect bacteria from environmental stresses and provide a controlled release of the active ingredient. This can improve the stability and efficacy of the product and reduce the need for additional carbon sources. Evonik’s new composition uses OMRI (Organic Materials Review Institute) certified co-formulants. 

The use of beneficial bacteria in agriculture has great potential to improve crop productivity and sustainability. The development of effective and sustainable formulations is critical to the success of this approach. This requires a multidisciplinary approach that combines microbiology, formulation science, and sustainable chemistry to develop products that are effective, stable, and environmentally friendly.

Evonik’s production process for successful formulation with Gram-negative bacteria

Evonik’s production process is a two-step approach. The first step involves the conversion of a liquid culture of Gram-negative bacteria into a biopowder. The second step involves the suspension of the biopowder into a liquid carrier solution. To make the biopowder, the bacterial cell culture is spray dried on a silica carrier product which is Evonik’s SIPERNAT^® 50. During this spray drying process, a sugar is coated around the bacteria / silica particles (Figure 1A). In the second step, the biopowder is dispersed into a liquid product called BREAK-THRU^® SP 133 (Figure 1B). This product is a unique biobased carrier fluid which protects microbes during storage. BREAK-THRU^® SP 133 interrupts small amounts of water in the formulation from interacting with any cells. Thus, this keeps the Gram-negative bacteria in a dormant state during storage and prevents growth of contaminating microbes. This carrier liquid maintains microbial viability during storage. In addition, BREAK-THRU^® SP 133 is effective during the application process. Once the liquid composition is mixed with water by the grower, the sugar from the bacteria and silica particle dissolves and releases the bacteria into the solution. This sugar in the solution now serves as a nutrient source for the microbes to begin growing. Both SIPERNAT^® 50 and BREAK-THRU^® SP 133 will also dissolute into the spray solution. BREAK-THRU^® SP 133 is a beneficial adjuvant and enhances the delivery of the sprayed microbes to leaf surfaces. This adjuvant reduces surface tension to cause the spray droplets to stick to plant surfaces, spread over plant surfaces and encourage penetration into plants (Figure 1C).

Figure 1: Microplastic free carrier particle SIPERNAT^® 50 and a sugar layer protect Gram-negative bacteria from environmental influences (A). High storage stability of these microorganisms is achieved by the combination of SIPERNAT^® 50 and the carrier fluid BREAK-THRU^® SP 133 (B). Field efficacy of the Gram-negative bacteria is achieved due to even spreading and penetration of leaves through sticking characteristics and anti-drift properties of BREAK-THRU^® SP 133 in foliar application (C).

SIPERNAT^® 50

Evonik’s SIPERNAT^® 50 is OMRI certified (allows for organic agriculture in the USA) and microplastic free. It is a precipitated silica, comparable to amorphous silica found in plants and is naturally derived. It is used as a performance additive in various applications, such as a carrier (absorptive substrate for microbes and additives), free-flow agent, and anti-caking agent in powders. Evonik’s SIPERNAT^® 50 will enter the natural geological cycle in the same way as plant derived silica. In this new process Evonik has developed SIPERNAT^® 50 as a substrate in the drying process to create a free flowing and easy to handle biopowder (Figure 2). 

Figure 2: SIPERNAT^® 50 is an amorphous silica used as a substrate in the drying process to create a free-flowing and easy-to-handle biopowder with Gram-negative bacteria. The high absorption capacity and porous structure of SIPERNAT^® 50 makes it suitable for such applications, as it can absorb moisture and prevent clumping. This improves the handling and storage properties of powders.

BREAK-THRU^® SP 133

BREAK-THRU^® SP 133 is OMRI certified (allows for organic agriculture in the USA). It is manufactured with plant derived raw materials (Figure 3), is readily biodegradable (OECD 301 F) and is toxicologically benign. BREAK-THRU^® SP 133 is a hydrophobic polyglycerolester based adjuvant, which is emulsifiable in water as well as soluble in many solvents. BREAK-THRU^® SP 133 is suitable for organic crop production, and it improves the efficacy of biologicals (fungicides, insecticides, herbicides, desiccants, biostimulants) as a tank side adjuvant. This adjuvant is water-free and can coordinate small amounts of water. This is how BREAK-THRU^® SP 133 when used as a carrier fluid prevents the growth of Gram-negative bacteria and also any potential contaminating microbes and keeps bacteria in a dormant state. Once the formulation is diluted in water, BREAK-THRU^® SP 133 no longer coordinates all the water and the microbes can begin to grow. The function of  BREAK-THRU^® SP 133 once diluted in water is as an adjuvant to improve the application of the bacterial suspension.  

Figure 3: BREAK-THRU^® SP 133 is a hydrophobic polyglycerolester based adjuvant.

Testing Evonik’s novel liquid formulation with Gram-negative bacteria

Evonik has tested its new liquid formulation with the Gram-negative bacterial species of Rhizobium species. Rhizobium species are known to form an endosymbiotic nitrogen-fixing association with roots of legumes and other plant species. They are used in agriculture to increase productivity by increasing plant-available nitrogen with reduced fertilizer inputs. We have prepared a composition with these Gram-negative bacteria on the solid carrier SIPERNAT^® 50 with a sugar layer and then suspended these particles in the carrier liquid BREAK-THRU^® SP 133. 

Evonik’s DC (dispersible concentrate) guideline formulation for Gram-negative bacteria is shown in Table 1. This formulation not only contains the active ingredient, as well as the carrier liquid BREAK-THRU^® SP 133, but also the hydrophilic dispersant BREAK-THRU^® DA 647 which will be soluble in the DC-formulation and later in the spray tank. The additional use of AEROSIL^® R 202, a polydimethylsiloxane post-treated fumed silica, provides an excellent increase of the thixotropic effect in oils and in water.

Table 1: Evonik’s guideline formulation for Gram-negative bacteria DC (dispersible concentrate).

We then tested the microbe viability and physical stability after storage at ambient temperature. Our results demonstrate that the cell viability remains high for 540 days (see Figure 4) and the composition is physical stable and. Evonik’s novel liquid composition can be used for other liquid formulations with Gram-negative bacteria. 

Figure 4: Vertical axis shows the colony forming units (CFU) determination of a Gram-negative bacteria, Rhizobium metallidurans, and the horizontal axis the time in days. The results show the microbe viability remains high with SIPERNAT^® 50 and BREAK-THRU^® SP 133 and stored at room temperature over the days of the test. 

Conclusion:

Evonik's innovative composition process for stabilizing Gram-negative bacteria in agricultural applications is a significant advancement in the field of study for biofertilizers and biopesticides. This new two-step process creates an encapsulation of Gram-negative bacteria with silica (SIPERNAT^® 50) and with the adjuvant (BREAK-THRU^® SP 133) addresses the challenge of maintaining the viability of these bacteria outside their natural environment.

Key components and benefits of Evonik's composition process:

1. Encapsulation with SIPERNAT^® 50: This silica-based carrier helps to create a protective environment for the Gram-negative bacteria, keeping them dormant yet viable until they are ready to be used. The encapsulation process ensures that the bacteria are physically stable and protected from environmental stresses that could otherwise reduce their effectiveness.

2. BREAK-THRU^® SP 133: This adjuvant is crucial for maintaining a low moisture environment, which is essential for the longevity of the bacteria in storage. It does not have intentionally added water and causes low water activities. The adjuvant also serves as a delivery enhancer, improving the coverage of plant surfaces and adherence of the bacteria to plant surfaces during the application.

3. Sugar Carbon Source: The inclusion of a sugar has two functions:  1) Sugar glass layer to protect the microbes during storage; 2) Food source once the formulation is added to water during the application.  

4. OMRI Certification: Both SIPERNAT^® 50 and BREAK-THRU^® SP 133 are certified by the Organic Materials Review Institute (OMRI), indicating that they are suitable for use in organic agriculture. This certification is important for farmers who are looking to maintain organic status for their crops.

5. Sustainability and Biodegradability: The components of the composition are designed to be environmentally friendly. SIPERNAT^® 50 is microplastic-free and comparable to natural silica, while BREAK-THRU^® SP 133 is also microplastic-free, made from biobased building blocks, has no toxicology labeling, and is readily biodegradable according to OECD 301F standards.

6. Convenience and Compatibility: The liquid formulation offers ease of handling and is stable at ambient temperatures, making it convenient for farmers to store and use. It can be easily mixed with water without the need for additional emulsifiers and can be applied using standard agricultural sprayers. The quality of the product can be guaranteed, because contamination (even after opening the package) has a limited ability to grow.

7. Customizable Formulations: The process allows for the creation of hybrid solutions that can combine Gram-negative bacteria with other chemicals, offering flexibility to address specific agricultural needs.

Overall, Evonik's novel process represents a significant advancement in the practical application of beneficial Gram-negative bacteria in agriculture. Evonik’s products enhances the physical stability and microbial viability and enhance field efficacy. This is a good step forward for microbial formulations with Gram-negative bacteria and will allow growers to more effectively utilize products with this type of bacteria to promote plant health, improve soil quality, and potentially reduce the reliance on chemical fertilizers and pesticides.

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