CITROFOL^® AI as a carrier fluid for microbials

Microbials in agriculture

Microbial biocontrol agents and biostimulants enhance the resilience of plants against biotic and abiotic stressors, and are thus important sustainable crop inputs in agriculture. However, maintaining cell viability and thus a long shelf life is a persistent challenge, even for currently available microbial products. Formulation plays a crucial role in addressing this challenge. Carrier fluids providing a low water activity environment can stabilise microbial cell viability, while aqueous carriers are often not suitable. CITROFOL^® AI (triethyl citrate) is an oily, water-free but partially water-soluble fluid and a suitable carrier for formulating dust-free and easy-to-dose microbial products with extended shelf lives.

CITROFOL^® AI is part of Jungbunzlauer’s globally recognised brand of citrate esters. It is a clear, colourless, and odourless oily liquid produced through the esterification of raw materials derived from fermentation, making it 100% bio-based and biodegradable. The physicochemical properties of CITROFOL^® AI offer distinct advantages in handling and formulation. It is non-flammable, non-volatile according to Directive 2004/42/EC, and non-toxic, ensuring safe storage and handling. With water solubility of up to 58 g/L, CITROFOL^® AI can homogeneously dilute into water at relevant use rates, eliminating the need for emulsifiers and simplifying formulations. It remains stable at cold temperatures and has good pourability across a wide temperature range. CITROFOL^® AI is listed in international chemical inventories and complies with the EU REACH Regulation. In the USA, triethyl citrate is approved as an inert ingredient for use in pesticide products applied to both food and non-food items, following the restrictions outlined in 40 CFR Part 180. Additionally, CITROFOL^® AI is USDA Certified Biobased under the USDA BioPreferred^® Program, making it suitable for bio-based applications in specific categories.

Microbial viability tests

To evaluate the impact of CITROFOL^® AI on microbial viability, commercially significant microbials were sourced as formulated products from the market. These included the Gram-positive bacterium Bacillus velezensis (available as wettable powder and aqueous suspension concentrate) and the fungi Trichoderma harzianum and Beauveria bassiana (both in wettable powder form). Cell suspensions were prepared by blending the wettable powder products with CITROFOL^® AI. All formulations and commercial products were stored at 40°C and/or room temperature for up to six months, with cell viability assessed periodically through plate counting.

Cell viability of B. velezensis

The viability of the spore-forming bacterium B. velezensis was monitored under challenging storage conditions at 40°C. Despite these harsh conditions, the B. velezensis wettable powder product demonstrated resilience and maintained full viability throughout the six-month testing period. In contrast, the commercial B. velezensis liquid product, an aqueous suspension concentrate, experienced a decline in cell viability from 1.5 x 10¹⁰ CFU/g to 4.3 x 10⁸ CFU/g over the same six-month period at 40°C. Although not as robust as the wettable powder formulation, suspending in CITROFOL^® AI resulted in improved viability and shelf life for liquid product formats. The viability of B. velezensis declined slightly from 1.5 x 10⁹ CFU/g to 4.8 x 10⁸ CFU/g, less than one order of magnitude, during six months at high temperatures (Figure 1).

Figure 1: Cell viability of B. velezensis stored at elevated temperatures for an accelerated storage test, comparing a commercial wettable powder with a commercial aqueous suspension concentrate and a non-aqueous suspension in CITROFOL^® AI. Differences in starting viability are due to different cell concentrations in the products.

Cell viability of T. harzianum

T. harzianum was found to be more susceptible to high storage temperatures, but monitoring its shelf life at 40°C for six months was still feasible. There was a pronounced drop in cell viability in the wettable powder product over this period, from 1.8 x 10⁹ CFU/g to 5.1 x 10⁴ CFU/g. However, suspending it in CITROFOL^® AI mitigated this loss: the CITROFOL^® AI suspension had a viability of 2.7 x 10⁶ CFU/g at the end of the measurement period, almost two orders of magnitude higher than the reference powder product (Figure 2).

Figure 2: Cell viability of T. harzianum stored at elevated temperatures for an accelerated storage test, comparing a commercial wettable powder product with a suspension in CITROFOL^® AI. Data points at 12 weeks for the wettable powder were below the detection limit. 

Cell viability of B. bassiana

Among the microorganisms tested in the present study, B. bassiana was the most sensitive to elevated storage temperatures. To provide better resolution, shelf life was monitored both at room temperature and 40°C. After just two weeks at 40°C, cell viability in the wettable powder product had dropped from 5.3 x 10⁸ CFU/g to below the detection threshold of approx. 1.0 x 10³ CFU/g. Under the same conditions, the suspension in CITROFOL^® AI had only decreased slightly to 1.1 x 10⁸ CFU/g after two weeks, and remained as high as 1.0 x 10⁶ CFU/g after twelve weeks (Figure 3). As expected, viability declined more slowly when B. bassiana formulations were stored at room temperature. Under these conditions, cell counts in the wettable powder product dropped from 5.3 x 10⁸ CFU/g to 4.1 x 10⁷ CFU/g after 24 weeks. Shelf life was even better in CITROFOL^® AI, where cell counts were 2.5 x 10⁸ CFU/g at the start and 1.6 x 10⁸ CFU/g at the end of the test period (Figure 4). Our observations align with published results showing an increased tolerance of B. bassiana to elevated temperatures when formulated in an oily carrier.^[1]

Figure 3: Cell viability of B. bassiana stored at elevated temperatures for an accelerated storage test, comparing a commercial wettable powder product with a suspension in CITROFOL^® AI.

Figure 4: Cell viability of B. bassiana stored at room temperature, comparing a commercial wettable powder product with a suspension in CITROFOL^® AI.

Optimising rheology

The formulation-relevant physical characteristics of a carrier fluid are crucial considerations. Adding hydrophobic fumed silica at a low usage rate of 3.0 wt% was adequate to modify the rheological properties of CITROFOL^® AI as desired. The apparent viscosity of the thickened fluid was approx. 100,000 mPa·s at a low shear rate of 0.01 sec^-1, indicating effective stabilisation of dispersed cells against sedimentation. Simultaneously, the thickened fluid exhibited strong shear thinning properties, noticeable as the shear rate increased to 10 sec^-1, resulting in a 100-fold reduction in viscosity to around 1,000 mPa·s (Figure 5). These values demonstrate that formulations based on thickened CITROFOL^® AI have good pourability and dosability. 

Figure 5: Flow curves of CITROFOL^® AI thickened with varying amounts of hydrophobic fumed silicas. 

Conclusion

Our results confirm the suitability of the citrate ester CITROFOL^® AI as a carrier fluid for microbials. CITROFOL^® AI serves as a sustainable co-formulant, is highly safe, and exhibits favourable physicochemical characteristics. Notably, it combines the low water activity of an oily carrier with partial water solubility, eliminating the need for emulsifiers and preservatives and simplifying formulations. CITROFOL^® AI is fully compatible with plants and has substantial potential to enhance the shelf life of liquid microbial formulations, as demonstrated with all organisms tested in this study (B. velezensis, T. harzianum, B. bassiana). It aids in maintaining high cell viability even at elevated temperatures, which is crucial for farmers operating in warm climates or where continual cooling of the product during transport and storage is impractical. Ensuring high cell viability and prolonged shelf life is a significant advancement in overcoming obstacles in the commercialisation of microbials. Viable microbial cells are essential for achieving satisfactory results in the field, thereby increasing confidence in the benefits and economic viability of these sustainable crop inputs.  

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References

[1] Oliveira DGP, Lopes RB, Rezende JM, Delalibera Jr I. Increased tolerance of Beauveria bassiana and Metarhizium anisopliae conidia to high temperature provided by oil-based formulations. J. Invertebr. Pathol. 2018;151:151–157.

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