Microwell arrays are important tools for studying single cell behavior and cell−cell interactions, both in microbial and mammalian systems. However, retrieval of cells from microwell arrays with high spatial precision remains a major technical hurdle that prevents follow-up genetic and phenotypic characterization of cells within observed microwells. This work describes a new, material-based approach to grow and retrieve live bacterial cells from small (≥20 μm diameter) microwells in an array using the plant pathogen Agrobacterium tumefaciens as a model bacterium. Our approach uses a light-responsive, steppolymerized poly(ethylene glycol) hydrogel interface as a membrane that confines motile cells within microwells while allowing nutrient exchange and cell growth. The key design feature is the photodegradability of the membrane, as it enables individual wells of interest to be opened using patterned UV light for selective release and retrieval of cells. Extraction can occur in parallel from any number and combination of wells defined by the user. These advancements represent a new use for light-responsive hydrogels and the ability to retrieve cells from microwells with high spatial precision enables several applications that require the isolation and characterization of cells with rare phenotypes from heterogeneous populations.

Understanding the consequences of microbe-microbe interactions is critical in efforts to predict the function of microbiomes and to manipulate or construct communities to achieve desired outcomes. The investigation of these interactions poses a significant challenge -in part due to the lack of suitable experimental tools. We present the Microwell Recovery Array, a high throughput approach designed to rapidly screen interactions across a microbiome and uncover higher-order combinations of strains that either inhibit or promote the function of a GFP-producing focal species. One experiment generates 10 4 unique microbial communities that contain a focal species combined with a unique combination of previously uncharacterized cells from plant rhizosphere. Cells are then sequentially extracted from individual co-culture wells that display highest or lowest levels of focal species function using a novel high-resolution photopolymer extraction system. Microbes present are subsequently identified and the putative interactions are validated. Using this approach, we screen the Populus trichocarpa rhizosphere for bacterial strains affecting the survival and growth of Pantoea sp. YR343, a plant growth promoting strain isolated from the P. trichocarpa rhizosphere. We were able to simultaneously isolate and validate multiple Stenotrophomonas strains that antagonize strain YR343 growth and a set of Enterobacter strains that promote strain YR343 growth. The latter demonstrates the unique ability of the platform to uncover multimembered consortia that generate emergent phenotypes. This knowledge will inform the development of beneficial consortia that promote the production of Populus biofuel feedstock, while the platform is adaptable to screening higher-order interactions in any microbiome of interest. Significance StatementAchieving a fundamental understanding of microbe-microbe interactions that occur within microbial communities is a grand challenge in microbiology due to the limited experimental tools available. In this report, we describe a new tool that enables one to screen microbial interactions across thousands of compositionally unique communities to discover collections of bacteria that antagonize or promote the survival and growth of bacteria with important functions. This approach has the unique ability to uncover higher-order combinations of bacteria that generate emergent

Lectin-functional interfaces are useful for isolation of bacteria from solution because they are low-cost and allow nondestructive, reversible capture. This study provides a systematic investigation of physical and chemical surface parameters that influence bacteria capture over lectin-functionalized polymer interfaces and then applies these findings to construct surfaces with significantly enhanced bacteria capture. The designer block copolymer poly(glycidyl methacrylate)-block-poly(vinyldimethyl azlactone) was used as a lectin attachment layer, and lectin coupling into the polymer film through azlactone−lectin coupling reactions was first characterized. Here, experimental parameters including polymer areal chain density, lectin molecular weight, and lectin coupling buffer were systematically varied to identify parameters driving highest azlactone conversions and corresponding lectin surface densities. To introduce physical nanostructures into the attachment layer, nanopillar arrays (NPAs) of varied heights (300 and 2100 nm) were then used to provide an underlying surface template for the functional polymer layer. Capture of Escherichia coli on lectin−polymer surfaces coated over both flat and NPA surfaces was then investigated. For flat polymer interfaces, bacteria were detected on the surface after incubation at a solution concentration of 10 3 cfu/mL, and a corresponding detection limit of 1.7 × 10 3 cfu/mL was quantified. This detection limit was 1 order of magnitude lower than control lectin surfaces functionalized with standard, carbodiimide coupling chemistry. NPA surfaces containing 300 nm tall pillars further improved the detection limit to 2.1 × 10 2 cfu/mL, but also reduced the viability of captured cells. Finally, to investigate the impact of cell surface parameters on capture, we used Agrobacterium tumefaciens cells genetically modified to allow manipulation of exopolysaccharide adhesin production levels. Statistical analysis of surface capture levels revealed that lectin surface density was the primary factor driving capture, as opposed to exopolysaccharide adhesin expression. These findings emphasize the critical importance of the synthetic interface and the development of surfaces that combine high lectin densities with tailored physical features to drive high levels of capture. These insights will aid in design of biofunctional interfaces with physicochemical surface properties favorable for capture and isolation of bacteria cells from solutions.

Understanding microbe-microbe interactions is critical to predict microbiome function and to construct communities for desired outcomes. Investigation of these interactions poses a significant challenge due to the lack of suitable experimental tools available. Here we present the microwell recovery array (MRA), a new technology platform that screens interactions across a microbiome to uncover higher-order strain combinations that inhibit or promote the function of a focal species. One experimental trial generates 104 microbial communities that contain the focal species and a distinct random sample of uncharacterized cells from plant rhizosphere. Cells are sequentially recovered from individual wells that display highest or lowest levels of focal species growth using a high-resolution photopolymer extraction system. Interacting species are then identified and putative interactions are validated. Using this approach, we screen the poplar rhizosphere for strains affecting the growth of Pantoea sp. YR343, a plant growth promoting bacteria isolated from Populus deltoides rhizosphere. In one screen, we montiored 3,600 microwells within the array to uncover multiple antagonistic Stenotrophomonas strains and a set of Enterobacter strains that promoted YR343 growth. The later demonstrates the unique ability of the platform to discover multi-membered consortia that generate emergent outcomes, thereby expanding the range of phenotypes that can be characterized from microbiomes. This knowledge will aid in the development of consortia for Populus production, while the platform offers a new approach for screening and discovery of microbial interactions, applicable to any microbiome.

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