Borregaard Lignin Biopolymers in Biological Formulation

Sumit Ganguly, PhD

Researcher Borregaard AS

Introduction:

The global agrochemical market is shifting steadily towards more sustainable and greener products as a replacement of synthetic products. In crop protection, there is a fast-growing interest in formulating with biological substance as the active ingredient. The global bio market revenue is estimated to reach $25 Billion by 2030; with the global biocontrol market likely to exceed $15 billion by 2029 (source: DunhamTrimmer). One of the key motivations to use a biological substance is its sustainable ecological footprint. At the same time, they are economically efficient, safe to handle, and can control a wide area of crop after application. The biocontrol market is divided into three subcategories – 1) Macroorganisms (insects, mites etc.), 2) Biochemicals (plant extracts, organic acids etc.) and 3) Microbials (bacteria, fungi, virus etc.). Formulating with a microbial can be challenging as to maintain its efficacy after application, it is important that it is compatible with the co-formulants. Additional stress factors like UV-radiation can also play an important role. Thus, special care needs to be taken when choosing a proper co-formulant to achieve a stable formulated product.

Borregaard's lignin-based biopolymers serve as a sustainable alternative to fossil based co-formulants. The lignin biopolymers are robust, bio-based dispersants and have excellent compatibility with a variety of biologicals. In addition, Borregaard’s unique product Activance UV can act as protector to shield the microbe from harmful UV-radiation. 

Sustainability: 

Borregaard produces sustainable lignin-based biopolymers derived from renewable, non-agricultural biomass sources. Through life cycle analyses, we've found that our products reduce CO₂ emissions by 70% compared to synthetic alternatives, offering a significant environmental advantage. Additionally, our lignin-based biopolymers maintain a negative CO₂ footprint throughout their service life.

Figure 1: Life Cycle Analysis of Borregaard's lignin biopolymers vs. synthetic dispersant (polycarboxylate)

Microbial Compatibility:

Maintaining the viability of microorganism is undoubtedly the most challenging aspect in any biological formulation. For the biocontrol agent to maintain its shelf life and efficacy after application, the co-formulants used in the formulation should be compatible with the microbial. 

Borregaard’s lignin biopolymers were tested against four different microorganisms – two bacteria Bacillus thuringiensis (gram +ve) and Pseudomonas fluorescens (gram –ve); and fungi Beauveria bassiana and Metarhizium anisopliae. For the qualitative screening, a disk diffusion assay was performed where filter paper disks soaked in 10% (w/w) lignin biopolymer solution were placed in agar plates containing microbial suspension in appropriate dilution. The results of the compatibility test are shown in Table 1 with lignin biopolymers (BP 2-4) including Activance UV with varying pH, molecular weight, sulfonation degree etc.

Table 1: Microbial compatibility test results with Borregaard's lignin biopolymers

An illustrative example is shown in Figure 2 where culture of Bacillus thuringiensis was grown on nutrient agar plates in presence of three Borregaard lignin biopolymers (BP 1-3) and four equivalent competitor products (CP 1-4). The bacteria grew around the filter paper discs soaked with Borregaard's lignin biopolymers and no inhibition zone was observed around them, whereas prominent inhibition ring was observed for all the competitor products. This clearly showcases the superior microbial compatibility of lignin biopolymers over the equivalent competitor products (CP 1-4). 

Figure 2: Compatibility test with bacteria (Bacillus thuringiensis) and Borregaard's lignin biopolymers – no inhibition was observed for Borregaard products as was seen for the competitor products.

To further establish our claim for microbial compatibility, a quantitative study was performed where bacteria Bacillus thuringiensis (Bt) was grown up to stationary phase and incubated with Activance UV; the bacterial suspension was later assessed for viability. 

As presented in Figure 3, Bt mixed with Activance UV for 24 hr, showcased a viability comparable to the control (i.e., with 0% Activance UV added), suggesting that there is no drop in viability after mixing with Activance UV. This proves that Activance UV is highly compatible with the bacteria.

Figure 3: Viability assay performed with Bt mixed with Activance UV after 24 hr.

UV-protection:

One of the biggest challenges for any biological formulation is to maintain the efficacy of the active after application from external stress factors like UV-radiation or heat. Borregaard’s lignin biopolymers, owing to their unique chemical structure, can absorb UV-radiation and thereby provide UV-protection to the microbes. Borregaard’s special product Activance UV has a high UV-absorption potential over a wide range of wavelengths (Figure 4), making it a perfect candidate as an UV-protectant in biological formulations.

Figure 4: UV-absorbance spectra of Activance UV and synthetic benzotriazole

To test the hypothesis, bacteria Bacillus thuringiensis mixed with Activance UV, was tested in an artificial sunlight simulation chamber, the ATLAS SUNTEST XLS+ (Figure 5). The Bt suspension was subjected to simulated sunlight of irradiance value 27 W/m² for 4 hours (wavelength 300-400 nm) and was later checked for viability assays. 

Figure 5: The ATLAS SUNTEST XLS+ sun simulation chamber used in this study.

As can be seen in Figure 6, the bacteria that was premixed with Activance UV showed growth after UV exposure whereas in the control (without any Activance UV) no growth was observed. This shows that Activance UV helps the bacteria to stay alive by shielding them from the harmful UV-radiation, and in absence of Activance UV, the bacteria were completely degraded by sun exposure. Thus, it can conclusively be stated that Activance UV provides UV-protection to the microbial, and thus can play an important role in a biological formulation.

Figure 7: Bacterial suspension on agar plates after UV-exposure – In presence of Activance UV (left), and Without Activance UV (right)

Formulation Examples:

Borregaard’s lignin biopolymers are an excellent choice as a bio-based dispersant for any biological formulation, aiding in the suspension ability of the microbial spores for application in the field. In addition, owing to the microbial compatibility, the lignin biopolymer provides extra stability to the microbial species during storage. Given below are few examples showcasing the advantages of including Borregaard’s lignin biopolymer when formulating with a biological active.

1. A wettable powder (WP) formulation of fungi Beauveria bassiana and lignin biopolymers were attempted by spray drying at low temperature setting (60/30 ºC); the recipe for the formulation is provided in Figure 8. The formulated product showed good dispersion of conidia; also, viability of fungi spores was maintained after drying.

   
   

   
   

   Figure 8: Recipe for the WP formulation (left), Agar-plates of fungi before and after drying (middle), and the suspensibility data of the formulated product (right).

   
   

2. A granulated product was prepared with peptide, a microbial, and Activance UV as binder, using the fluidised bed. The recipe is provided in Figure 9. The resulted granules exhibit high suspensibility in water (85%).

   
   

   
   

   Figure 9: Recipe for the formulation (left), the granulated powder (centre), and the granulated powder dispersed in water (right)

   
   

3. A wettable formulation formed by spray drying, with Bt and Activance UV shows excellent dispersibility of bacterial spores, and Activance UV was proven to exhibit UV-protection.

   
   

   
   

   Figure 10: Viability assays of the formulated Bt after UV exposure (left), and dispersibility of Bt spores with and without Activance UV (right).

Conclusion:

Borregaard’s lignin-based biopolymers can serve as multipurpose co-formulants for a wide variety of biocontrol formulations. In addition to dispersing and binding abilities, the lignin biopolymers have excellent microbial compatibility and the unique Activance UV can offer UV-protection to the microbial active. Moreover, Borregaard’s lignin biopolymers are derived from 100% natural sources, are OMRI-listed and approved for use in organic farming.

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