Croda: The Power of Combined Knowledge in Delivering Microbial Formulations

Carola Peters and Edisa Garcia Hernandez, Research Scientists at Croda and Incotec explore the challenges in delivering microbial agricultural solutions and discuss why combining knowledge, expertise and capabilities across different agricultural markets helps overcome some of these challenges.

Microbial products are a key component in sustainable agriculture. Microbes which are able to protect plants from pests or stimulate plant growth are increasingly being used by growers worldwide. They have many advantages such as minimum risk of harmful exposure to non-target insects and humans.
 
Bringing a microbial product to market is a challenge. A different type of knowledge, capability and understanding is required compared to what is needed to produce chemical products. Developing formulations that provide the right environment for a microbe to survive but also maintain the performance requirements that a formulation has (such as no separation or sedimentation in storage) is difficult. Additionally, you need to be able to test initial compatibility and long-term viability of a microbe with the formulation components. Moreover, you also have to think about the end application (e.g. seed, foliar or soil) as this will affect the formulation design due to the different challenges associated with each of these applications.

At Croda we are in a unique position as we have three Ag businesses under the brands, Incotec (seed enhancement), Plant Impact (biostimulants) and Croda Crop Protection (agrochemical formulation design). The advantage of these businesses is the power of combining their knowledge, especially when it comes to delivering microbial solutions.

For example, across Croda’s Ag businesses, the process of formulation and evaluation involves multiple areas of expertise and capabilities worldwide. Experts on formulation (Croda Crop Protection) are responsible for carefully selecting components to provide a suitable delivery system that maintains the viability of the microbe until it is delivered to its end application. In seed enhancement technologies (Incotec), researchers focus on adherence of the components to the seed, flowability of seeds through equipment and prevention of unnecessary waste and dust emissions. The compatibility of microbes in seed and foliar solutions needs to be evaluated so our microbiologists ensure that the microbe is viable over time in the product (Incotec and Crop Protection). Moreover, the microbe needs to be efficacious in the end use and sometimes the addition of other compounds could further boost the performance of the microbe. Our experts on biostimulants (Plant Impact) have a significant role in the discovery and evaluation of synergistic effects of biostimulants on plant growth.

Let’s look at the different steps in the development process of a microbial product separately with some examples from different expertise’s within Croda. 

Formulating for foliar sprays
 
For foliar application, formulations can be water-free systems like wettable powder (WP), or oil dispersions (OD), but other systems like suspension concentrates (SC) can also be used, all with their own benefits and limitations. In OD formulation technology, the microbes are dispersed in an oil phase, meaning the formulation benefits from being water and preservative-free. Additionally, the oil phase can act as a built-in adjuvant which can improve characteristics of the final application, including penetration enhancement and improved spray retention. The formulation is stabilised using emulsifiers, dispersants and rheology modifiers, specifically selected for the type of oil and the microbe. Any component used for stabilisation can impact microbe viability and so should be tested for compatibility.  

To support the process of formulating ODs, we have designed three recipes that can be used as an initial starting point, with only the microbe required to complete the formulation. These OD formulation systems have been evaluated for compatibility with two commonly used microbes, Bacillus sp and Trichoderma sp. An easy way to test short-term compatibility is using a disk diffusion method (Figure 1). The test showed that the disks with the OD formulations did not affect the microbial growth around them, indicating a certain degree of compatibility. Since each microbe can react differently, making general product recommendations is difficult. However, these OD systems can be used as an initial recommendation, and we can work together to provide more tailored advice to your specific microbe if necessary. 

Figure 1. Compatibility of Trichoderma sp. and Bacillus sp.  with microbial OD systems 1, 2 and 3

Even though short-term compatibility shows good results, it is important to conduct longer-term viability studies to ensure the final product has the desired shelf-life. Therefore, the OD systems were evaluated using colony forming units (CFU) method. Table 1 displays the results of 20% w/w Bacillus sp. (a mixture of mixture of B.subtilis and B.amyloliquefaciens) incorporated into OD system 2. After 154 days stored at 20 °C minimal effect was seen which is an excellent result.

Table 1. Viability data of 20% w/w Bacillus sp in OD system 2 at a dilution factor of 10^-7 per plate, based on a starting material concentration of 3.7 x 10¹⁰

Formulation and application on seed
 
When microbes are applied on seed, again formulation compatibility is an important factor, as well as the microbe remaining efficacious after application. Application of microbes on seed can be achieved with several different technologies such as, film coating, encrustment or pelleting. The choice will depend on the specific crop-microbe combination. Film coating is a very thin layer of only 1-2 µm around the seed. Encrustment and pelleting of seeds involves powders and binders and creates a much thicker layer around the seed (Figure 2). 

Figure 2. Different seed treatment technologies, from left to right: naked, film coated, encrusted and pelleted carrot seed

A film coating is an intricate formulation that can contain up to 15 different components that all can have an impact on microbe viability. Research has shown that microbes from the same family can respond completely different to a formulation.  In the example in Figure 3, three different Trichoderma strains were tested for compatibility with one specific film coating. Trichoderma 1 remains viable in the film coating for 24 hours, Trichoderma 3 significantly decreases in viability over time and the viability of Trichoderma 2 has decreased below the detection limit after 24 hours. This demonstrates the need for tailor-made solutions to be developed based on the microbe characteristics.

Figure 3. Differences in the viability of three strains of Trichoderma sp. in a film coating for 0, 4 and 24 hours

Governments, consumer pressure and climate change are calling for a reduction of artificial fertilisers. A microbe that can assist with this is Rhizobium, a bacterium that can provide the needed nitrogen to legumes, through nitrogen fixation. To do this, the bacteria need to colonise the root system and form nodules. In these nodules the nitrogen is fixated and in exchange, the bacteria receive sugars from the plant. Rhizobium is typically delivered via treating a seed before planting. Therefore, on seed viability and long-term stability needs to be determined, but it is most important to analyse how efficacious the microbe is at the point the seeds are sown. To prove Rhizobium can still colonise the root system and form nodules, after being applied to a seed, we perform grow out tests. In these tests, we observe that Rhizobium can grow out of the film coat layer and are still able to form nodules in the root system (Figure 4). 

Figure 4. Example of Rhizobium sp. nodules on a soybean root (left) and dry nodules to calculate dry mass nodules per plant (right). 

Enhancing microbe performance

To enhance the beneficial functions of these microbes, additional components can be added to the seed. For instance, in the process of nitrogen fixation by Bradyrhizobium, molybdenum (Mo) and cobalt (Co) are required. The concentration of these elements can be quite low in some soils, limiting the beneficial effect of the bacterial symbiosis on the plant. As a result, these elements may need to be added, however, many Co and Mo salts can be toxic to Rhizobium when applied together on seed. Plant Impact’s InSync™ CoMo is designed for soybean seeds and is compatible with symbiotic Bradyrhizobium, ensuring that these two crucial elements are available in the surrounding soil during germination.

An example of biostimulant synergy between Plant Impact technologies is the InSync Plus seed treatment, that also contains a Plant Impact technology to support nodulation and stimulate plant growth, whilst providing cobalt and molybdenum (CoMo) in a rhizobia compatible formulation. This combination resulted in a significant yield increase in field trials (Figure 5).

Figure 5. Soybean yield increase after application of InSync Plus seed treatment

Closing remarks

As discussed, there are several challenges (and opportunities) when it comes to delivering microbial agricultural solutions. Combining knowledge, expertise and capabilities across different agricultural markets helps overcome some of these challenges meaning the Croda Ag businesses can:

• Provide troubleshooting and optimisation of microbial formulations  
  

• Tailor seed enhancement technologies to a certain microbe  
  

• Perform biostimulant synergy tests
  

• Advise on plant and crop trials
  

These are only some examples of what Croda can offer. The websites of Incotec, Croda Crop Care and Plant Impact contain resources related to biological formulations and how you can contact us.

Join us to show your expertise in the coming issue!

Contact: joyce@agropages.com