Croda: Taking Microbes from Production to Effective Delivery

Introduction    

The biologicals market has seen a CAGR of 9.2% since 2014, this growth rate has been mainly driven by biostimulants and microbials markets and significantly surpasses the CAGR of the overall crop protection market. Application of biopesticides is still, however, limited to approximately 2% of all plant protection products despite the growing requirement for more sustainable solutions (Agbioinvestor, 2021). There are many contributing factors limiting uptake to market such as expensive production methods, poor storage stability, susceptibility to environmental conditions and efficacy problems.

Croda is a key collaboration partner for those wishing to develop their offering in the biologicals market, supporting design and delivery of biological solutions. Herein we will discuss formulating with microbes, optimising formulation for maximum biological efficacy and the robust product solutions that are available at Croda.

Understanding your microbe

Within the agricultural market the two main types of microbes used are fungi and bacteria. Both can be used to promote crop growth and for disease control in combination with, or in the place of, their synthetic pesticide counterparts. Whilst fungi and bacteria are similar in the way they are utilised in application, they are different in terms of their cell structure and therefore behave uniquely within a formulation and in the field. Both the chemical composition and the physical form of a microbe will dictate which delivery system is most suitable. Having a good understanding of these parameters is important for successful formulation development, allowing you to optimise efficacy and delivery in the field.

Bacteria are single-celled microorganisms, broadly categorised as Gram-negative or Gram-positive depending on the thickness of their cell walls. A gram-negative bacterium, such as Pseudomonas sp., has a thin peptidoglycan cell wall surrounded by an outer membrane whereas Bacillus sp., a Gram-positive bacterium, has a much thicker peptidoglycan cell wall and no outer membrane. Some bacteria can form spores (generally the Gram-positive bacteria), and their ability to do this further enhances their robustness and therefore ease of formulating. Spores hold all the genetic information that is present in the vegetative form but in a dormant state. This essentially protects the genetic information from external stresses for example, extreme temperatures. As soon as a favourable environment is achieved, the spores can germinate allowing the genetic information to be expressed and the benefits of the microbe to be observed. Non-spore forming bacteria are more sensitive to external conditions as their genetic information is not protected in the same way.

Fungi can be single-celled or multicellular microorganisms; their cell walls are primarily made up of chitin and glucans. Fungi exhibit characteristics most like Gram-positive bacteria and are generally less sensitive than Gram-negative bacteria due to their more resistant structures, such as conidia and spores.

When considering formulation development of microbes, the characteristic of the microbial strain can present challenges, for example heightened sensitivity to surfactant impurities, moisture, or catalytic residues. Therefore, the more knowledge we have around a specific strain of a microbe, the easier it is to select an appropriate formulation type and combination of surfactants to produce an effective delivery system. Whether that be a super refined product with catalytic residues removed, or a low moisture variant where an anhydrous stabilising system can be guaranteed.

Selecting a Suitable Formulation Type

The morphological structure of microbes can be extremely hydrophobic. When dealing with a microbe in a solid powder form, for example Trichoderma sp., the dilution in water is often ineffective and can result in unevenly dispersed material resulting in suboptimal field application. Therefore, formulation of the microbe is recommended.

The presence or introduction of water in the formulation can encourage dormant spores to begin to germinate prematurely which can reduce the viability of the microbe at the time of spraying, excluding the choice of many liquid formulations such as suspension concentrates (SCs). Oil dispersions (ODs) and wettable powders (WPs) can provide water-free formulation environments, removing the likelihood of spores germinating during storage. OD formulations offer unique advantages for the formulation chemist, such as ease of use and the ability to incorporate adjuvants easily in the continuous phase of the formulation. However, the design of ODs can be complex, often comprising of several formulation aids. Selecting the right formulation aids for development is important, surfactant selection must:

• Be non-toxic to the microbe

• Have effective performance in application
  

• Ensure biological and physical stability
  

• Meet the demands of the microbe – low moisture, highly refined

• Meet the demands of the market – 100 % biobased, suitable for organic registration

In the concentrate:	On dilution in water:

Figure 1: Idealised surfactant configuration within an OD in the concentrate and upon dilution.

Compatibility and Viability Testing

Once the formulation type has been selected, formulation aids are chosen and their impact on the viability of the microbe is assessed. Assessing for viability is an essential step to refine the formulation development process. Croda recommends the use of a wide suite of testing to ensure the stability of the microbe has been optimised. These tests include:

• Inhibition zone method or Kirby-Bauer test – In these test methods it is possible to analyse the effects of different components on microorganisms in a quick and easy way, by visually assessing the suppression of growth of a microbe on an agar plate.

Figure 2: Results using the inhibition zone test method, highlighting a control where no growth inhibition zone has been created and a treatment where a significant growth inhibition zone has been created.

• Colony forming unit count (CFU) – This method is used to determine the concentration of microbes in a formulation and therefore how many microbes can produce colonies after being in contact with the formulation. This method allows for understanding around shelf-life of a product under different environmental conditions.
  

• Conidia germination test (CGT) (direct viability) – This method is specifically used for fungi conidia. When compared with the two previous analyses, this method provides an accurate value regarding the number of viable conidia in a sample.
  

Croda regularly supplement these methods with complementary ‘in vivo’ efficacy tests at our Product Validation Centre in Brazil.

Making OD Development More Efficient

To facilitate the development process, Croda has designed three emulsion chassis systems for microbial delivery in a range of oils, selected to capture the unique requirements of different microbes. These chassis are an excellent starting point for developments with microbes, which allow further optimisation after initial compatibility testing has been completed.

The components of these chassis have been screened for compatibility with different microbes to ensure the versatility of their use. Trichoderma sp. and Bacillus sp. were amongst the microbes screened and positive results from compatibility testing (inhibition zone method) can be observed in Figures 3 and 4.

Figure 3: Bacillus sp. with (left to right) Atlox™ 4916, Atlas™ G-1086, Tween™ L-1505, Tween™ 22, Atlox Rheostrux™ 200. Filter paper was saturated with a 10 % w/w dilution of surfactant and placed on an agar plate inoculated with the microbe.

Figure 4: Trichoderma sp. with (left to right) Atlox 4916, Atlas G-1086, Tween L-1505, Tween 22, Atlox Rheostrux 200. Filter paper was saturated with a 10 % w/w dilution of surfactant and placed on an agar plate inoculated with the microbe.

The main benefit of these OD chassis recipes is that they significantly decrease the formulation development time and can be used even with limited formulation experience. This is because:

• There is a high confidence in compatibility with common microbes
  

• Surfactant choice and ratios have already been identified for each oil
  

• The formulation method is provided with best practice advice

To explore these chassis formulations in more detail, get in touch with Croda for more detailed support and guidance or visit our Crop Care website.

Concluding remarks

Croda is the supplier of specialist ingredients; expertise within molecule design, manufacture and application allows Croda to offer a toolbox of materials designed with biologicals in mind, enabling the formulator to open doors in the design of new and innovative solutions. Cross functional collaboration across biology and chemistry teams have enabled the design of fully formulated delivery systems which formulators can apply to their needs and develop further. Croda are willing to support and collaborate to push the boundaries of microbial solutions to market.

This article will be published in AgroPages '2022 Formulation & Adjuvant Technology ' magazine to be published this May.

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