Optimization of microorganism preservation conditions for the development of an acute toxicity bioassay for biocides

Biocides, also referred to as 'microbicides' or 'inhibitors', are widely used in industrial processes (e.g. utility water in cooling towers) to control and/or eliminate the growth of microorganisms. Because of their inherent toxicity, their presence in various sources (e.g. river sediments, potable water) can negatively affect ecosystems. Currently available biocide detection techniques are not suitable for 'point-of-use' applications since they are tedious, complicated, and often require experienced personnel to operate. To address this concern, we sought to develop a simple-to-use toxicity bioassay based on a model microorganism (E. coli) after short (<30 min) exposure to known biocides that can be stored at room temperature (preferably) or in the fridge. Based on recent work and our expertise in polymer-based preservation of biomolecules, we leveraged this knowledge to improve E. coli preservation for biocide detection purposes. A design-of-experiments strategy was used to evaluate 16 different preservation conditions from 5 process parameters (i.e. 2^5-1 fractional factorial). It was found that pullulan, a sugar-based polymer, improved E. coli culturability by an order of magnitude after three months of storage. Also, it was found that storing E. coli in the fridge in Milli-Q water was favorable for maintaining a high level of culturability. Finally, the toxicity of three common biocides (Cetyltrimethylammonium bromide (CTAB), ProClin™ 300, and Grotan^® BK) was evaluated using a fluorescence-based assay across all 16 preservation conditions. The response of the preserved E. coli was biocide specific and at certain conditions did not vary during the entire three-month storage period.