Photodegradable Hydrogel Materials for Encapsulation, Isolation and Recovery of Functionally Rare Bacteria from Complex Microbial Communities

Photodegradable hydrogel materials can be used for cellular encapsulation and for spatiotemporally-controlled cell release, and therefore have been explored as materials for use in cell isolation applications. While most studies have focused on mammalian cells, here we extend the use of photodegradable polyethylene glycol (PEG) hydrogels towards isolation of bacteria with rare or unique function from complex microbial communities, which often contain thousands of unique species. Hydrogels are formed using the photocleavable macromer, PEG <i>o</i>-nitrobenzyl diacrylate, which is crosslinked into a hydrogel through thiol-acrylate Michael-type addition reactions with PEG-tetrathiol macromers. PEG molecular weights are selected to form hydrogels with a 10 nm mesh size, enabling physical encapsulation and immobilization of bacteria cells while also allowing for the transport of nutrients, chemical cues, and waste products throughout the material for culture. A patterned illumination tool is used to expose the hydrogel to user-defined 365 nm light patterns at 5-mm spatial resolution to selectively release desired cells from the hydrogel interface after phenotypic observations for recovery, genomic characterization, and application.<br/><br/>With this foundational approach, three applications will be highlighted. The first involves screening bacteria mutant libraries for phenotypically rare cells. Cells from mutant libraries are directly encapsulated into the hydrogels for high-throughput, microscopic observation. After identification of the desired cellular phenotype, rare cells are released with patterned light and recovered for whole genome sequencing, enabling one to link phenotype to genotype. Second, these materials are integrated into microdevices containing microwell arrays for bacteria co-cultures. These devices are designed to screen thousands of different co-cultures to uncover microbe-microbe interactions that impact beneficial or pathogenic bacteria. Finally, photodegradable hydrogels have been functionalized with lectin proteins, enabling high-affinity capture and removal of bacteria from young biofilms on host surfaces. After capture, bacteria can be sectioned from the hydrogel using patterned light for sequencing and characterization. We have applied this approach to study membrane biofouling in wastewater treatment systems, and specifically to identify early-colonizing bacteria from wastewater that accelerate the membrane biofouling process. Current and future work involves investigating the use of upconversion nanoparticles in these hydrogel systems for release of cells using near infrared light.