The factors leading to changes in the organization of microbial assemblages at fine spatial scales are not well characterized or understood. However, they are expected to guide the succession of community development and function toward specific outcomes that could impact human health and the environment. In this study, we put forward a combined experimental and agent-based modeling framework and use it to interpret unique spatial organization patterns of H1-Type VI secretion system (T6SS) mutants of P. aeruginosa under spatial confinement. We find that key parameters, such as T6SS-mediated cell contact and lysis, spatial localization, relative species abundance, cell density and local concentrations of growth substrates and metabolites are influenced by spatial confinement. The model, written in the accessible programming language NetLogo, can be adapted to a variety of biological systems of interest and used to simulate experiments across a broad parameter space. It was implemented and run in a high-throughput mode by deploying it across multiple CPUs, with each simulation representing an individual well within a high-throughput microwell array experimental platform. The microfluidics and agent-based modeling framework we present in this paper provides an effective means by which to connect experimental studies in microbiology to model development. The work demonstrates progress in coupling experimental results to simulation while also highlighting potential sources of discrepancies between real-world experiments and idealized models.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

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Results reveal that for an electrode-grown Geobacter sulfurreducens biofilm, as much as 70% of cytochrome hemes residing within hundreds of nanometers from the electrode surface store electrons even as extracellular electron transport is occurring across the biofilm/electrode interface.

Microbial communities form biofilms on material surfaces in a multitude of ecosystems, from the root hairs of a plant to the human gut. The hallmarks of an established biofilm include (1) the attachment of microbial cells to a surface, (2) production of extracellular polymeric substance, (EPS) (3) a complex structure or "architecture," and (4) the ability to exchange genetic information between cells. [1] Geobacter sulfurreducens forms unique, electrically conductive biofilms, a property that can be exploited in production and design of microbial fuel cells. In this work, examine biofilm formation, and biofilm properties of Geobacter sulfurreducens using a Scanning Electron Microscope (SEM) as well as a Helium Ion Microscope (HIM). SEM is a high-resolution imaging technique used for characterization of a broad variety of materials. However, in order to image highly insulating, soft biological materials, the samples must be coated for charge compensation. These (typically) metallic coatings create a homogenous surface and may cloak true biological behavior and material contrast in the micrograph. In the case of Geobacter sulfurreducens, metal coating precludes detailed investigation of microbial attachment, presence of EPS, and fine surface details that may elucidate the mechanisms behind architecture formation and genetic material exchange. Recently introduced HIM, offers more flexibility in investigating biological samples, as highly insulating sample can be imaged sui generis, without the use of a conductive coating. [2] This opens new pathways to capturing high resolution spatial details of biofilm formation and biofilm properties. Furthermore, high-resolution HIM imaging reveals true surface details of Geobacter sulfurreducens, such as flagella or pilin typically inaccessible by SEM. Finally, the effects of different sample preparation strategies for SEM and HIM will be illustrated and discussed [3].