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Stimuli-responsive hydrogels that provide controlled degradation can be used as bacteria delivery systems for advanced therapeutic applications. Here, we report the first use of photodegradable hydrogels as materials that can direct bacterial movement, tune mean bacteria speed, and control bacteria delivery through spatiotemporal control of degradation. Hydrogels were formed using base-catalyzed Michael addition reactions between photodegradable poly(ethylene glycol) (PEG) o-nitrobenzyl diacrylate macromers and PEG tetra-thiol cross-linkers within microfluidic channels. Nutrient gradients were generated across the channel, and micron-scale regions of the hydrogel were partially degraded by exposure to controlled doses (2.1–168 mJ/mm^2) of patterned 365 nm light. Hydrogel degradation was then characterized in situ using fluorescence visualization of fluorescein-labeled hydrogels. Following characterization, Bacillus subtilis expressing green fluorescent protein was introduced into the device, and its movement up the nutrient gradient was monitored using time-lapse fluorescence microscopy to enable a systematic study of bacteria chemotaxis through the hydrogels at varied levels of degradation. B. subtilis showed minimal adhesion to partially degraded PEG hydrogels, and bacteria mean speed and mean directional change were tunable according to the level of hydrogel photodegradation, with a 2.6-fold difference in mean cell speed measured across the partially degraded hydrogel regions. Finally, the ability to alter bacteria speed and directionality through tunable degradation and without significant adhesion was used to achieve controlled release profiles of bacteria to delivery sites. These findings advance the use of PEG-based hydrogel materials as delivery vehicles for bacterial therapeutic applications and other living material applications that require controlled bacteria transport. 

Abstract Hydrogel materials can be used to integrate bacteria cells into biohybrid systems. Here, we investigate the use of polyethylene glycol-based hydrogels that employ different Michael-type addition crosslinking chemistries, including thiol-acrylate, thiol-vinyl sulfone, and thiol-maleimide click reactions, for covalent hydrogel network formation and bacteria encapsulation. All crosslinking chemistries generated hydrogels that provided stable encapsulation and culture ofBacillus subtilis; however, significant differences in cell viability and cell morphology after encapsulation were identified. Thiol-acrylate hydrogels provided the highest cell viability and favored encapsulation of single cells, while thiol-maleimide hydrogels had the lowest cell viability and favored encapsulation of larger aggregates. These findings demonstrate the impact of crosslinking strategies for encapsulation of microorganisms into hydrogel networks and suggest that thiol-acrylate chemistries are favorable for many applications. Graphical abstract 

Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen Agrobacterium tumefaciens sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens. 

Plant growth-promoting bacteria (PGPB) are valuable for supporting sustainable food production and may alleviate the negative impacts of chemical fertilizers on human health and the environment. While single-strain inoculations have proven unreliable due to poor survival and colonization in the rhizosphere, application of PGPB in multispecies consortia has the potential to improve these outcomes. Here, we describe a new approach for screening and identifying bacterial consortia that improve the growth of corn relative to plants inoculated with a single strain. The method uses the microwell recovery array (MRA), a microfabricated high-throughput screening device, to rapidly explore the maize ( Zea mays L .) rhizobiome for higher-order combinations of bacteria that promote the growth and colonization of the nitrogen-fixing PGPB, Azospirillum brasilense . The device simultaneously generates thousands of random, unique combinations of bacteria that include A. brasilense and members of the maize rhizobiome, then tracks A. brasilense growth in each combination during co-culture. Bacteria that show the highest levels of A. brasilense growth promotion are then recovered from the device using a patterned light extraction technique and are identified. With this approach, the screen uncovered growth-promoting consortia consisting primarily of bacteria from the Acinetobacter - Enterobacter - Serratia genera, which were then co-inoculated with A. brasilense on axenic maize seedlings that were monitored inside a plant growth chamber. Compared to maize plants inoculated with A. brasilense alone, plants that were co-inoculated with these consortia showed accelerated growth after 15 days. Follow-up root colonization assays revealed that A. brasilense colonized at higher levels on roots from the co-inoculated seedlings. These findings demonstrate a new method for rapid bioprospecting of root and soil communities for complementary PGPB and for developing multispecies consortia with potential use as next-generation biofertilizers. 

The simple, accurate, and rapid detection of foodborne pathogens is essential for public health. Development of an immunomagnetic separation (IMS) multiplex touchdown PCR (IMS–multiplex TD–PCR) assay for simultaneous detection and distinguishing of C. jejuni and C. coli is reported herein. Polyclonal antibody (pAb) against multiepitope antigen (MEA) was conjugated to ferromagnetic nanoparticles (FMNs) to produce anti-MEA FMNs. Optimal anti-MEA FMNs loading yielded 26.7 μg of immunoglobulin G (IgG) molecules per mg of FMNs with an average size of 72 ± 9  nm, corresponding to an 83% rate of pAb conjugation. Anti-MEA FMNs (20 μg) for IMS captured culturable C. jejuni cells at 3.54 × 10 2 colony-forming unit (CFU)/mL in pure culture, while higher amounts (40 and 60 μg) reduced the recovery. The scanning electron microscope (SEM) analysis revealed the attachment of anti-MEA FMNs to target bacteria, forming aggregated cells and magnetic nanoparticles in ellipse-like shapes. The subsequent multiplex TD–PCR assay simultaneously detected and distinguished C. jejuni and C. coli at 104 CFU/mL in mixed culture and at 103 CFU/mL for each individual species. Furthermore, the limit of detection (LOD) of the IMS–multiplex TD–PCR assay was 104 CFU/g in spiked chicken breast samples. Specificity was 100% for both C. jejuni and C. coli as none of the amplicons were detected in control samples where Campylobacter was absent. This assay is able to detect and distinguish C. jejuni and C. coli simultaneously and is simple, accurate, and rapid with a time to result of 4 h without an enrichment step, making it a promising approach for rapid and culture-free detection of Campylobacter in chicken products. 

Semi-arid regions faced with increasingly scarce freshwater resources must manage competing demands in the food-energy-water nexus. A possible solution modifies soil hydrologic properties using biosurfactants to reduce evaporation and improve water retention. In this study, two different soil textures representative of agricultural soils in Kansas were treated with a direct application of the biosurfactant, Surfactin, and an indirect application via inoculation of Bacillus subtilis . Evaporation rates of the wetted soils were measured when exposed to artificial sunlight (1000 W/m 2 ) and compared to non-treated control soils. Experimental results indicate that both treatments alter soil moisture dynamics by increasing evaporation rates by when soil moisture is plentiful (i.e., constant rate period) and decreasing evaporation rates by when moisture is scarce (i.e., slower rate period). Furthermore, both treatments significantly reduced the soil moisture content at which the soil transitioned from constant rate to slower rate evaporation. Out of the two treatments, inoculation with B. subtilis generally produced greater changes in evaporation dynamics; for example, the treatment with B. subtilis in sandy loam soils increased constant rate periods of evaporation by 43% and decreased slower rate evaporation by 49%. In comparing the two soil textures, the sandy loam soil exhibited a larger treatment effect than the loam soil. To evaluate the potential significance of the treatment effects, a System Dynamics Model operationalized the evaporation rate results and simulated soil moisture dynamics under typical daily precipitation conditions. The results from this model indicate both treatment methods significantly altered soil moisture dynamics in the sandy loam soils and increased the probability of the soil exhibiting constant rate evaporation relative to the control soils. Overall, these findings suggest that the decrease in soil moisture threshold observed in the experimental setting could increase soil moisture availability by prolonging the constant rate stage of evaporation. As inoculation with B. subtilis in the sandy loam soil had the most pronounced effects in both the experimental and simulated contexts, future work should focus on testing this treatment in field trials with similar soil textures. 

This report evaluates the use of on-going, open-ended research problems taken from the instructor’s laboratory and assigned as projects in Transport Phenomena. Projects were structured following a hybrid active learning model and designed to engage student groups by providing them the opportunity to impact research in their department. The impact of these assignments on student comprehension and engagement is evaluated by comparing exam performance of student cohorts with and without projects and through student surveys. 

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 10 4 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.