Microencapsulation of engineered bacteria towards whole-cell-based environmental biosensing.

INTRODUCTION: Legitimate concerns about the quality of environmental waters are on the rise. As a consequence, efforts are being made by both the scientific community and policymakers to develop proficient methods for the monitoring and detection of pollutants of emerging concerns (PECs). In line with the European Union's "Zero Pollution" action plan, there is a growing need to develop real-time, multiplexed, on-site monitoring systems. The study explores the encapsulation of engineered Pseudomonas putida cells designed to produce a luminescent response upon exposure to specific analytes. The aim was to develop a suitable, miniaturized, biocompatible, safe-and-sustainable-by-design, sensing element for future integration with optical-electrochemical transduction systems. METHODS: Genetically modified P. putida cells were encapsulated in sub-200 µm alginate microcapsules using a layer-by-layer method with poly-L-lysine (PLL) to avoid bacterial escape. Capsules morphology and structure were characterized using Laser Scanning Confocal and cryo-Scanning Electron microscopies. Effects of cell load, storage temperature, and storage medium were evaluated through encapsulated cells' fluorescent response to induction. Long-term fluorescent activity was evaluated over a 2-month period. Response-time of the encapsulated cells was investigated using both experimental and diffusion modelling approaches. RESULTS: Encapsulation strategy using alginate-PLL capsules revealed a good mechanical stability and resistance to saline water, with minimal cell leakage. About 75% of encapsulated cells remained viable after 45 days, and fluorescence increase upon induction was still observed after 2 months of storage at room temperature without nutrient supply. These findings suggest that a subpopulation of cells entered a dormant or low-metabolic state, enabling long-term sensing under non-ideal conditions. Higher cell loads correlated with stronger responses, likely due to an increased fraction of metabolically reactivable cells. Encapsulated cells were also reusable, showing measurable sensing response for five sensing cycles within 2 weeks. DISCUSSION: Results demonstrate that response dynamics are primarily governed by reduced metabolic activity and limited oxygen access, rather than by analytes diffusion through the hydrogel matrix.