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Advancing the Use of Microfluidic Models for Studying Microbial Communities: Integration of Microfluidic Model Experimentation, Multimodal Imaging, and Modeling

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The microbial breakdown of cellulose and related by-products is a key process in the global carbon (C) cycle. Cellulose, the most common organic compound on Earth, is a primary structural component of plants. It's estimated that annually, more than 1011 tons of cellulose are synthesized by plants and subsequently degraded by microorganisms.

In soil environments, this cellulosic material provides a carbon and energy source for many heterotrophic microorganisms, which in turn produce CO2 via respiration. While this CO2 production may seem insignificant on a local scale, it accounts for the release of between 75-100 billion metric tons of C globally each year. Therefore, a greater understanding of the factors and processes controlling the temporal and spatial dynamics of soil microbial respiration is necessary for addressing issues related to climate change, modeling Earth systems, and land use policy.

Pore scale studies are key. Within soil systems, the microbial processes that drive cellulose degradation and CO2 production occur within pore spaces, where a range of advective and diffusive transport processes supply biomass with nutrients, O2 (in the case of aerobic systems), and remove the products of microbial respiration and growth.

Micromodels
Micromodels consist of a series of pillars and channels etched into a substrate that simulate networks of pores and channels in soil environments. Enlarged View
Pore structures
A variety of pore structures will be used within these microfluidic models to obtain both advection-dominated and diffusion-dominated regions. On the left: Model design illustrating dead-end pores where diffusion-dominated processes will occur. Right: Flow velocity within a model design. Enlarged View

Micromodels to the rescue. Investigations using microcosms, mesocosms, or even field experiments do not allow for the study of these processes at the pore-scale. To achieve this level of resolution, this MCI project is using microfluidic models, or micromodels, as structures for studying pore-scale microbe-cellulose interactions via a range of novel non-destructive imaging-based technologies.

Challenges. A number of technical challenges exist within this project. Cellulose films must be created within the micromodel for cellulose-degrading bacteria to act upon. We are working on 1) techniques for depositing cellulose films in the pore spaces, and 2) non-destructive imaging to quantify cellulose degradation. Currently, microcrystalline Avicel has been successfully introduced into the micromodels.

As the cellulose film is degraded, a corresponding decrease in intensity of its natural fluorescence should be observed. We are also developing fluorescently tagged nanocrystalline cellulose thin films that, in combination with highly advanced imaging approaches, should enable more sensitive imaging of cellulose degradation. Cellulose imaging will be carried out in conjunction with a novel optode-based O2 sensor for determining O2 concentrations within the micromodel.

We anticipate that as cellulose breakdown proceeds in the pore spaces, certain regions (such as diffusion dominated regions) may become O2-limited. By coupling O2 readings to measurements of the remaining cellulose, we aim to determine the effects of geochemical heterogeneity on C cycling.

Initial experiments will aim to grow one or two cellulose-degrading bacterial strains in the micromodels. These initial proof-of-concept experiments will attempt to integrate the technologies being developed for detection of cellulose degradation and O2 use. In addition, a range of novel approaches will be deployed for monitoring biomass accumulation within the pore spaces of the micromodel, including the use of Raman spectroscopy, and fluorescently-labeled bacterial strains.

Following the successful demonstration of these technologies, more complex cellulose-degrading communities will be introduced into the models. Ultimately, the aim of this research is to interrogate ecological concepts associated with cellulose degradation in a heterogeneous pore-scale system.

The spatial distribution of cellulose films within these micromodels will allow resource island hypotheses to be investigated, while microbial niche concepts will be inferred from growth patterns. Data from these studies can then be integrated into modeling efforts being carried out as part of the MCI project "Multiscale Models for Microbial Communities."

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Extending Genomics & Proteomics to Quantitative Functional Analyses

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