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

Abstract

SummaryThe 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.

The Great Five—an artificial bacterial consortium with antagonistic activity towards Pectobacterium spp. and Dickeya spp.: formulation, shelf life, and the ability to prevent soft rot of potato in storage

“…Although a number of attempts have been made to control SRP on potato using biological control agents, so far, none of them have resulted in development and commercialization of a microbial-based biocontrol product (Charkowski 2018;Czajkowski et al 2011). Likewise, there are only few examples of the use of such bioproducts being successful in other bacterial pathogen-crop systems (Azizbekyan 2019;Nega 2014;Sheppard et al 2003) The main reason for this is that for commercialization, the microorganisms need to be effectively formulated in order to remain viable and retain their properties throughout the storage period, in transport and upon application under environmental settings (Berninger et al 2018).…”

“…This most often involves drying of the product and its storage in low humidity (Rhodes 1993). Desiccation of microorganisms can be carried out in several ways, e. g., freeze-drying, vacuum-drying, spray-drying, fluidized bed-drying, or air-drying (Broeckx et al 2016); however, freeze-drying (lyophilization) is considered a method of choice as it offers good survival rate, it is applicable both on large and small scale, and results in viable cells that can be rehydrated directly prior to use (Powell 1992;Berninger et al 2018). The majority of microorganisms when freeze-dried without supplementation of lyoprotectants survive the process poorly (Heckly 1961).Viability rates as low as 0.1% have been frequently reported (Miyamoto-Shinohara et al 2006).…”

“…To reliably determine the shelf life of the formulations, defined as the viability of bacteria over time (Berninger et al 2018), two individual lots were prepared for each formulation, with a postponement between them of 8 months. For powder formulations, each lot comprised of cells freeze-dried in independent procedures.…”

“…Microbial formulations can be prepared as solids or liquids (Berninger et al 2018). Solid-state formulations include powders and granules, whereas liquid formulations comprise microbial cells suspended in water-based or oil-based carriers (Stephens and Rask 2000).…”

Predicted ecological niches and environmental resilience of different formulations of the biocontrol yeast Candida sake CPA-1 using the Bioscreen C

“…For example, at 25ºC and 0.96 aw at pH 3 and 7. The vital functions of microorganisms can often reach a dormant state during the drying process (Berninger et al 2018) and they only become active again during rehydration (García 2011). This may give liquid formulations an advantage allowing slightly faster recovery and growth when favourable environmental conditions occur.…”