Maintenance and assessment of cell viability in formulation of non-sporulating bacterial inoculants

Abstract

The application of beneficial, plant-associated microorganisms is a sustainable approach to improving crop performance in agriculture. However, microbial inoculants are often susceptible to prolonged periods of storage and deleterious environmental factors, which negatively impact their viability and ultimately limit efficacy in the field. This particularly concerns non-sporulating bacteria. To overcome this challenge, the availability of protective formulations is crucial. Numerous parameters influence the viability of microbial cells, with drying procedures generally being among the most critical ones. Thus, technological advances to attenuate the desiccation stress imposed on living cells are key to successful formulation development. In this review, we discuss the core aspects important to consider when aiming at high cell viability of non-sporulating bacteria to be applied as microbial inoculants in agriculture. We elaborate the suitability of commonly applied drying methods (freeze-drying, vacuum-drying, spray-drying, fluidized bed-drying, air-drying) and potential measures to prevent cell damage from desiccation (externally applied protectants, stress pre-conditioning, triggering of exopolysaccharide secretion, 'helper' strains). Furthermore, we point out methods for assessing bacterial viability, such as colony counting, spectrophotometry, microcalorimetry, flow cytometry and viability qPCR. Choosing appropriate technologies for maintenance of cell viability and evaluation thereof will render formulation development more efficient. This in turn will aid in utilizing the vast potential of promising, plant beneficial bacteria as sustainable alternatives to standard agrochemicals.

Figure 1

Formulation possibilities and inherent application techniques. Solids and slurries may be applied as seed treatment or soil amendment; liquids as seed treatments, soil amendment or foliar spray. The possibility of controlling the desiccation process depends thus on the form of the inoculant and application technique.

Figure 2

Strategies of improving desiccation tolerance in non‐sporulating bacteria. Externally added protectants stabilize the cell membrane from outside or can be accumulated in the cell (during cultivation). Stress pre‐conditioning results in intracellular adaptation (e.g. accumulation of protective agents) or secretion of EPS. Co‐cultivation with protectant‐excreting ‘helper’ strains provides external protection (e.g. by EPS).