Custom-engineered micro-habitats for characterizing rhizosphere interactions

The interactions amongst plants and microorganisms within the rhizosphere have a profound influence on global biogeochemical cycles, and a better understanding of these interactions will benefit society through improved climate change prediction, increased food security, and enhanced bioenergy production. However, the rhizosphere is one of the most complex and bio-diverse ecosystems on earth, making it difficult to parse apart specific interactions between species. This difficulty is compounded by the inability to directly visualize rhizosphere interactions through the soil. Additionally, conventional laboratory techniques do not offer real-time, high-resolution visualization or the proper environmental control to isolate and probe these interactions. A knowledge gap persists in how to design appropriate culturing platforms that allow researchers to collect spatially and temporally sensitive information about physical and chemical interactions in the rhizosphere. This dissertation addresses that gap by demonstrating the design and use of several custom-engineered micro-habitats in characterizing plant-microbe interactions. Specifically this thesis introduces novel protocols for culturing plants and microorganisms together in microfluidic platforms, pairing platforms to multi-modal imaging techniques with organelle scale resolution, and recreating the structural complexity of the rhizosphere in a microfluidic habitat. Not only does this thesis introduce novel engineered systems, but the work contained herein also goes beyond proof-of-concept experiments and demonstrates the ability of these platforms to generate hypotheses and answer outstanding biological questions

Accessing microfluidics through feature-based design software for 3D printing

<div><p>Additive manufacturing has been a cornerstone of the product development pipeline for decades, playing an essential role in the creation of both functional and cosmetic prototypes. In recent years, the prospects for distributed and open source manufacturing have grown tremendously. This growth has been enabled by an expanding library of printable materials, low-cost printers, and communities dedicated to platform development. The microfluidics community has embraced this opportunity to integrate 3D printing into the suite of manufacturing strategies used to create novel fluidic architectures. The rapid turnaround time and low cost to implement these strategies in the lab makes 3D printing an attractive alternative to conventional micro- and nanofabrication techniques. In this work, the production of multiple microfluidic architectures using a hybrid 3D printing-soft lithography approach is demonstrated and shown to enable rapid device fabrication with channel dimensions that take advantage of laminar flow characteristics. The fabrication process outlined here is underpinned by the implementation of custom design software with an integrated slicer program that replaces less intuitive computer aided design and slicer software tools. Devices are designed in the program by assembling parameterized microfluidic building blocks. The fabrication process and flow control within 3D printed devices were demonstrated with a gradient generator and two droplet generator designs. Precise control over the printing process allowed 3D microfluidics to be printed in a single step by extruding bridge structures to ‘jump-over’ channels in the same plane. This strategy was shown to integrate with conventional nanofabrication strategies to simplify the operation of a platform that incorporates both nanoscale features and 3D printed microfluidics.</p></div

Droplet generators.

<p>(a) The T-junction device was operated with the fluorescein flow rate at 1μl/min and the oil at (a1) 5μl/min and (a2) 20μl/min. (b) The flow-focusing device operates with the same flow rates. (b1-2) show the oil channels pinching off a droplet from the fluorescein channel. (c) Formed droplets are highly replicable and can be controlled by altering the oil flow rate from 2μl/min to 20μl/min.</p

3D microfluidics.

<p>Using 3D capabilities of the feature-based software, bridges were printed to create an overlapping design with three channels from an offset (a) and side (b) view. (d)Top view—overlapping channels remain separate from one another. (c) Side view—the bridging structure raises off the plane of the glass slide. The expanded view shows the printing direction for the bridging structures. (e) The microfabricated structure along with an inset of the chambers with each channel independent of one another. (f) Shows 3D printed structures connecting channels and overlapping to simplify the device control.</p

Fabrication process diagram.

<p>(a) The device was printed on a heated print bed. (b) Acetone was applied to the surface of the device to anneal the ABS surface. (c) PDMS was cast over the mold, (d) a vacuum degassed the PDMS, and (e) the heated print bed cured the device. (f) The device was removed from the bed and ABS mold and (g) bound to a glass slide.</p

Fabrication process.

<p>(a) The device was designed by combining fluidic parts into a custom fluidic network. (b) The design was sent to an FDM 3D printer. (c) The ABS mold is removed from the PDMS device after being cast on the heated print bed. The device was cut into individual devices, and (d) bonded to a substrate for use.</p

Acetone annealing gradient generators.

<p>(a) SEM images show the surface of the ABS mold annealed by applying acetone. (b) A microfluidic gradient mixer produced using our ABS mold printing process. (c-d) Images of the device show dilution channels recombining. (c) Annealing smooths the surface for more even imaging. (d) Non-annealed device shows rough surfaces from the printing process. (e) The maximum fluorescent intensity from the individual channels prior to rejoining shows greater variability in non-annealed devices. (f) The fluorescent intensity profile in the channels after recombining show the gradient forming. Variation in the chamber height from 3D printing causes variation across the profile deviating from the expected linear gradient.</p

Feature parameters and program GUI.

<p>(a) Table of features available for the design process. The GUI consists of 3 sections (b) the printer and feature parameters are given, (c) the design is represented graphically, and (d) the parameter list of all the parts in the current design for editing.</p

Synthetic Soil Aggregates: Bioprinted Habitats for High-Throughput Microbial Metaphenomics

The dynamics of microbial processes are difficult to study in natural soil, owing to the small spatial scales on which microorganisms operate and to the opacity and chemical complexity of the soil habitat. To circumvent these challenges, we have created a 3D-bioprinted habitat that mimics aspects of natural soil aggregates while providing a chemically defined and translucent alternative culturing method for soil microorganisms. Our Synthetic Soil Aggregates (SSAs) retain the porosity, permeability, and patchy resource distribution of natural soil aggregates—parameters that are expected to influence emergent microbial community interactions. We demonstrate the printability and viability of several different microorganisms within SSAs and show how the SSAs can be integrated into a multi-omics workflow for single SSA resolution genomics, metabolomics, proteomics, lipidomics, and biogeochemical assays. We study the impact of the structured habitat on the distribution of a model co-culture microbial community and find that it is significantly different from the spatial organization of the same community in liquid culture, indicating a potential for SSAs to reproduce naturally occurring emergent community phenotypes. The SSAs have the potential as a tool to help researchers quantify microbial scale processes in situ and achieve high-resolution data from the interplay between environmental properties and microbial ecology

Pore-scale hydrodynamics influence the spatial evolution of bacterial biofilms in a microfluidic porous network.

Bacteria occupy heterogeneous environments, attaching and growing within pores in materials, living hosts, and matrices like soil. Systems that permit high-resolution visualization of dynamic bacterial processes within the physical confines of a realistic and tractable porous media environment are rare. Here we use microfluidics to replicate the grain shape and packing density of natural sands in a 2D platform to study the flow-induced spatial evolution of bacterial biofilms underground. We discover that initial bacterial dispersal and grain attachment is influenced by bacterial transport across pore space velocity gradients, a phenomenon otherwise known as rheotaxis. We find that gravity-driven flow conditions activate different bacterial cell-clustering phenotypes depending on the strain's ability to product extracellular polymeric substances (EPS). A wildtype, biofilm-producing bacteria formed compact, multicellular patches while an EPS-defective mutant displayed a linked-cell phenotype in the presence of flow. These phenotypes subsequently influenced the overall spatial distribution of cells across the porous media network as colonies grew and altered the fluid dynamics of their microenvironment