(478c) Detection of Biocides in Industrial Wastewater Using Pullulan-Encapsulated Microorganisms

Biocides, also frequently referred to as ‘microbicides’ or ‘inhibitors’, are used in industry to control or eliminate microbial growth in environments which are prone to microbiological activity. An evaporative cooling tower is an example of where biocides such as cetyl trimethylammonium bromide (CTAB) are deliberately used to control microbial growth to optimize cooling efficiency when cooling a building or a piece of machinery [1] [2]. Recent changes to water effluent quality standards are making the treatment of industrial wastewater increasingly difficult as municipalities are updating their sewer use by-laws to include restrictions on the amount of biocides. Therefore, there is a need for specialized tests to identify the presence of biocides in any potential incoming streams to municipal wastewater treatment plants (WWTPs) because the biocides are non-specific and thus will kill the beneficial microorganisms used in biological treatment processes (e.g. removal of ammonia). Existing methods for biocide detection rely on specialized techniques (e.g. High-Performance Liquid Chromatography, Liquid Chromatography – Mass Spectrometry) that are both time consuming and require the attention of specialized operators. This motivation has compelled our team to develop a robust, easy-to-use, biocide screening technology capable of accurately determining the presence of any biocide in wastewater. We envision that such a technology would be extremely useful for achieving ‘point-of-use’ reductions in the amount of biocides used at industrial facilities.

Using our expertise in the areas of WW separation processes and applied molecular biology, we have developed a bacterial cell-based kit that is capable of quantifying the relative toxicity levels of several WW samples in parallel in just minutes. The kit works by analyzing the survival rate of a library of microorganisms upon exposure to WW. For example, the survival rate of DH5-α E. Coli decreased from 85 ± 3% to 15 ± 3% as the concentration of CTAB increased from 60 to 120 µM. In order to obviate the need for cold-temperature storage, we have drawn upon our team’s previous work and understanding in enzyme encapsulation using water soluble pullulan, and extended this to microbial cell preservation at room temperature. Pullulan is the polysaccharide used to make Listerine™ breath strips, and it has been reported as being extremely effective in protecting enzymes, RNA, DNA and small molecules against thermal and oxidative degradation [3]. In this new work, we performed an extensive optimization study that covered numerous experimental parameters (e.g. pullulan concentration, cell concentration, humidity) and storage conditions that resulted in a very robust and effective method of preserving microbial cells which are viable for long periods of time when maintained at room temperature conditions and higher (up to 40°C) - we have shown that DH5-α E. Colicells can survive without refrigeration for long periods of time (>3 months), while maintaining survival rates as high as 50%. This works paves the way for creating a new area in sensing that uses whole cells as stable reagents that can be readily used in the field. In this work, we demonstrate this by showing that whole cells encapsulated in pullulan films can be used to detect the presence of the biocide CTAB.

References

[1] Baltimore Aircoil Company, "What is Evaporative Cooling?," 2010. [Online]. Available: http://www.baltimoreaircoil.com/english/what-is-evaporative-cooling. [Accessed 11 April 2017].

[2] S. Maruthamuthu, S. Ponmariappan, K. Indiral, A. Subramanian and N. Rengaswamy, "Interference Between Biocides and Inhibitors in Cooling Water Systems," Bulletin of Electrochemistry, vol. 16, no. 5, pp. 209-213, 2000.

[3] S. Jahanshahi-Anbuhi, B. Kannan, V. Leung, K. Pennings, M. Liu, C. Carrasquilla, D. White, Y. Li, H. R. Pelton, D. J. Brennan and D. C. Filipe, "Simple and ultrastable all-inclusive pullulan tablets for challenging bioassays," Chem. Sci, vol. 7, pp. 2342-2346, 2016.