Polyethylene glycol hydrogel coatings for protection of electroactive bacteria against chemical shocks.

Loss of bioelectrochemical activity in low resource environments or from chemical toxin exposure is a significant limitation in microbial electrochemical cells (MxCs), necessitating the development of materials that can stabilize and protect electroactive biofilms. Here, polyethylene glycol (PEG) hydrogels were designed as protective coatings over anodic biofilms, and the effect of the hydrogel coatings on biofilm viability under oligotrophic conditions and ammonia-N (NH4+-N) shocks was investigated. Hydrogel deposition occurred through polymerization of PEG divinyl sulfone and PEG tetrathiol precursor molecules, generating crosslinked PEG coatings with long-term hydrolytic stability between pH values of 3 and 10. Simultaneous monitoring of coated and uncoated electrodes co-located within the same MxC anode chamber confirmed that the hydrogel did not compromise biofilm viability, while the coated anode sustained nearly a 4 × higher current density (0.44 A/m2) compared to the uncoated anode (0.12 A/m2) under oligotrophic conditions. Chemical interactions between NH4+-N and PEG hydrogels revealed that the hydrogels provided a diffusive barrier to NH4+-N transport. This enabled PEG-coated biofilms to generate higher current densities during NH4+-N shocks and faster recovery afterwards. These results indicate that PEG-based coatings can expand the non-ideal chemical environments that electroactive biofilms can reliably operate in.

Polymer Surface Dissection for Correlated Microscopic and Compositional Analysis of Bacterial Aggregates during Membrane Biofouling.

Multispecies biofilms are a common limitation in membrane bioreactors, causing membrane clogging, degradation, and failure. There is a poor understanding of biological fouling mechanisms in these systems due to the limited number of experimental techniques useful for probing microbial interactions at the membrane interface. Here, we develop a new experimental method, termed polymer surface dissection (PSD), to investigate multispecies assembly processes over membrane surfaces. The PSD method uses photodegradable polyethylene glycol hydrogels functionalized with bioaffinity ligands to bind and detach microscale, microbial aggregates from the membrane for microscopic observation. Subsequent exposure of the hydrogel to high resolution, patterned UV light allows for controlled release of any selected aggregate of desired size at high purity for DNA extraction. Follow-up 16S community analysis reveals aggregate composition, correlating microscopic images with the bacterial community structure. The optimized approach can isolate aggregates with microscale spatial precision and yields genomic DNA at sufficient quantity and quality for sequencing from aggregates with areas as low as 2000 µm2, without the need of culturing for sample enrichment. To demonstrate the value of the approach, PSD was used to reveal the composition of microscale aggregates of different sizes during early-stage biofouling of aerobic wastewater communities over PVDF membranes. Larger aggregates exhibited lower diversity of bacterial communities, and a shift in the community structure was found as aggregate size increased to areas between 25,000 and 45,000 µm2, below which aggregates were more enriched in Bacteroidetes and above which aggregates were more enriched with Proteobacteria. The findings demonstrate that community succession can be observed within microscale aggregates and that the PSD method is useful for identification and characterization of early colonizing bacteria that drive biofouling on membrane surfaces.

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation.

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

Photodegradable Hydrogels for Rapid Screening, Isolation, and Genetic Characterization of Bacteria with Rare Phenotypes.

Screening mutant libraries (MLs) of bacteria for strains with specific phenotypes is often a slow and laborious process that requires assessment of tens of thousands of individual cell colonies after plating and culturing on solid media. In this report, we develop a three-dimensional, photodegradable hydrogel interface designed to dramatically improve the throughput of ML screening by combining high-density cell culture with precision extraction and the recovery of individual, microscale colonies for follow-up genetic and phenotypic characterization. ML populations are first added to a hydrogel precursor solution consisting of polyethylene glycol (PEG) o-nitrobenzyl diacrylate and PEG-tetrathiol macromers, where they become encapsulated into 13 µm thick hydrogel layers at a density of 90 cells/mm2, enabling parallel monitoring of 2.8 × 104 mutants per hydrogel. Encapsulated cells remain confined within the elastic matrix during culture, allowing one to track individual cells that grow into small, stable microcolonies (45 ± 4 µm in diameter) over the course of 72 h. Colonies with rare growth profiles can then be identified, extracted, and recovered from the hydrogel in a sequential manner and with minimal damage using a high-resolution, 365 nm patterned light source. The light pattern can be varied to release motile cells, cellular aggregates, or microcolonies encapsulated in protective PEG coatings. To access the benefits of this approach for ML screening, an Agrobacterium tumefaciens C58 transposon ML was screened for rare, resistant mutants able to grow in the presence of cell free culture media from Rhizobium rhizogenes K84, a well-known inhibitor of C58 cell growth. Subsequent genomic analysis of rare cells (9/28,000) that developed into microcolonies identified that seven of the resistant strains had mutations in the acc locus of the Ti plasmid. These observations are consistent with past research demonstrating that the disruption of this locus confers resistance to agrocin 84, an inhibitory molecule produced by K84. The high-throughput nature of the screen allows the A. tumefaciens genome (approximately 5.6 Mbps) to be screened to saturation in a single experimental trial, compared to hundreds of platings required by conventional plating approaches. As a miniaturized version of the gold-standard plating assay, this materials-based approach offers a simple, inexpensive, and highly translational screening technique that does not require microfluidic devices or complex liquid handling steps. The approach is readily adaptable to other applications that require isolation and study of rare or phenotypically pure cell populations.

Identification of Critical Surface Parameters Driving Lectin-Mediated Capture of Bacteria from Solution.

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 103 cfu/mL, and a corresponding detection limit of 1.7 × 103 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 × 102 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.