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As the biological agriculture market continues to grow, so does the demand for high-performing, compliant microbial products. However, the path from lab bench to market is rarely straightforward, mainly when it comes to improving microbial yield during bioprocesses and maintaining microbial viability and stability over time. Here we will explore how the presence of byproducts and impurities within surfactants can affect microbial viability during both upstream bioprocesses and within formulation of the final microbial product. Quality of ingredients can be a true differentiator shaping product stability and bioefficacy, thus improving overall market competitiveness.
The use of surfactants in upstream processes to optimise fermentation and enhance microbial yield has become an increasingly common strategy among biologicals manufacturers. At these early stages of bioprocessing, microorganisms are particularly sensitive to byproducts and impurities, making the selection of appropriate components critical for overcoming process challenges and maximising productivity.
Croda offers a specialised portfolio of biocompatible ingredients designed for use in upstream and fermentation processes. Our solutions include components that enhance membrane permeability and oxygen transfer in fermentation media, high-performance emulsifiers that improve the bioavailability of hydrophobic substrates, agents that stabilise cell membranes and protect against shear stress, and components to promote cell lysis maintaining the stability of the metabolites. Additionally, our ingredients support nutrient uptake, promote effective cell dispersion, and improve the solubility of oils and lipophilic substrates, delivering measurable benefits across the entire bioprocessing workflow. Recognising that every microbial strain and bioprocess is unique, Croda’s specialists offer bespoke solutions, leveraging deep expertise and advanced capabilities to address specific bioprocessing challenges. With optimised manufacturing processes, Croda’s products are high-quality with low levels of impurities, which make them ideal for use in upstream bioprocesses.
After microbial fermentation, a well-designed formulation can delay the loss of viability in microorganisms, effectively extending the shelf life of the final product. However, it’s important to highlight that formulation cannot enhance the inherent viability of a microorganism, this is an intrinsic property of the microbe itself. What a formulation can do, when composed of long-term compatible components, is create conditions that minimise harmful interactions between the microbes and extracellular metabolites or residual upstream inputs. This protective environment helps preserve cell viability over time.
While performance is key, the success of a biological formulation often depends on what is not necessarily visible as trace impurities from formulation components. These low-level contaminants (e.g. reaction byproducts, free monomers, formaldehyde, hydrogen peroxide, free radicals) can have a significant impact on microbial viability and long-term stability, particularly in sensitive biological products. One critical vulnerability lies in the microbial cell wall, which can be susceptible to adverse interactions with reaction byproducts, preservatives, and highly oxidative molecules. Even in small concentrations, these substances can compromise cell integrity, reduce viability, and accelerate degradation over time.
The data in Figure 1 illustrate how selecting high-purity, well-characterised ingredients can mitigate these risks, enhance formulation robustness, and support better overall product performance throughout shelf life. Viability was assessed using the conidial germination method applied to solid compositions of Trichoderma harzianum, aiming to evaluate the performance of the same dispersant chemical structure (same CAS number) at two different purity grades. After 180 days, the higher-purity dispersant maintained conidial viability at twice the level observed with the lower-purity version. This enhanced viability may translate into improved efficacy and more consistent biological performance.
Figure 1. Comparison of solid formulations containing regular and purified dispersants (same CAS number) on the conidial viability (%) of Trichoderma harzianum over 180 days at room temperature. Notably, byproducts and impurities from the production process negatively impact microbial viability throughout the shelf life.
It is critical to distinguish that when we refer to viability loss, we are talking about a decrease in the significand, not the exponent (Figure 2). For example, a formulation intended to ensure long shelf life must maintain the exponent for as long as possible (1) – typically 12–24 months, depending on the microorganism. A reduction in the significand is normal and expected. However, the formulation should not cause a drop in the exponent, either immediately after exposure to its components (which would indicate incompatibility) or during the shelf life. Indeed, an exponent drop in viability from 10¹¹ to 10⁷ over the shelf life is not acceptable for a product considered to have long shelf life. Such a drop suggests the possible presence of process byproducts or residues that are compromising cell viability, either immediately upon exposure or gradually during storage.
Figure 2. Assessment of microbial viability over the shelf life of the compositions. The control (fermentation broth) and formulations containing Bacillus velezensis maintain similar viability, with some differentiation over time. Note that the decreases in viability refer to changes in the significand, not in the logarithmic exponent, which is normal and expected. These results indeed indicate an extended shelf life of the compositions. DAF: days after fermentation.
When optimising formulation components, it’s essential to clarify what is measured in standard compatibility tests and how additional parameters can enhance the overall quality and potency of microbial-based products. Spore viability remains a widely used indicator of fungal quality in biological control, typically assessed through microscopic observation after a 24-hour incubation. However, this method can introduce bias, as early germinating spores may obscure non-viable ones on the agar surface. Despite this limitation, compatibility tests offer a practical and rapid way to screen large numbers of treatments. More advanced and precise methods are also available in the literature, offering improved accuracy and efficiency. In parallel, the concept of spore vigour, which reflects the strength and speed of germination and germ tube development, has gained attention as a more nuanced quality indicator. Vigour is strongly influenced by upstream factors such as fermentation conditions and nutrient availability, as well as downstream processing. Notably, germination speed is one of the most reliable indicators of vigour. Studies (1) have shown that weakened but viable spores tend to germinate slowly and exhibit reduced virulence, supporting the idea that relying solely on viability can lead to overestimating product quality.
Indeed, Croda’s internal data generated using PrecisionBio™ software (Figure 3) highlight the complementary nature and interpretive accuracy achieved when traditional CFU (colony-forming unit) measurements are analysed alongside conidial vigour assessments on solid fermentations containing Trichoderma harzianium conidia spores obtained from solid fermentation. This study presents a viability study comparing four formulations, each with varying proportions of different co-formulants, designed to identify the optimal balance of physicochemical and microbiological performance of the fermentation broth (control). Viability over time was assessed using CFU counts, all formulations demonstrated stability after 180 days, with results consistently in the order of 10⁸ CFU/ml.
Interestingly, when PrecisionBio™ was applied to evaluate germination tube development, a proxy for conidia vigour, Formulation 3 emerged as the top performer promoting a higher conidia vigour comparing to the control even after 180 days of shelf-life. Formulations 1, 2, and 4, on the other hand, did not compromise vigour relative to the control, but also did not provide any improvement after the same period.
Figure 3. Analysis of colony forming units and conidia vigor during shelf life. Assessment of the control (spores from solid fermentation) and solid formulation prototypes containing Trichoderma harzianium. All formulation prototypes maintained the viability and vigour of the conidia spores throughout the shelf life, presenting appropriate values. Notably, Formulation 3 enhanced conidial vigour even after 180 days of storage at room temperature, indicating a beneficial effect promoted by the formulation. DAI: days after incubation.
This case illustrates how integrating assessments of different microbial developmental stages can support more informed and strategic decisions in formulation design, especially when the goal is to optimise both shelf life and product performance. Additionally, this data highlights that the commonly used short-term microbial compatibility tests for co-formulant selection may not accurately predict the long-term stability of microbial compositions. For biological product developers, this implies the need for extended evaluations to validate long-term performance, often requiring a return to earlier development stages. As a result, the overall product development timeline may be significantly impacted.
High-quality ingredients with low levels of byproducts and impurities are preferential when formulating microbial product to maintain viability. This is important for both upstream bioprocesses as well as formulation development, resulting in microbial products that have good shelf life stability and bioefficacy. As well as Croda’s standard surfactants and co-formulants which are low in impurities, we are also able to refine and tailor products to meet the needs of microbe. Our commitment goes beyond supplying high-quality ingredients – we act as a strategic partner, working alongside formulators to design robust, compliant, and high-performing solutions from the ground up. By aligning certified, sustainable ingredients with deep formulation expertise, we are helping shape the future sustainable innovation, making the journey from lab to field more efficient, compliant, and competitive.
(1) Faria et al. 2015. Conidial vigor vs. Viability as predictors of virulence of entomopathogenic fungi. J Invertebr Pathol 125: 68-72.
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