Dynamic nitrogen fixation in an aerobic endophyte of Populus.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

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.

Hotspots of root-exuded amino acids are created within a rhizosphere-on-a-chip.

The rhizosphere is a challenging ecosystem to study from a systems biology perspective due to its diverse chemical, physical, and biological characteristics. In the past decade, microfluidic platforms (e.g. plant-on-a-chip) have created an alternative way to study whole rhizosphere organisms, like plants and microorganisms, under reduced-complexity conditions. However, in reducing the complexity of the environment, it is possible to inadvertently alter organism phenotype, which biases laboratory data compared to in situ experiments. To build back some of the complexity of the rhizosphere in a fully-defined, parameterized approach we have developed a rhizosphere-on-a-chip platform that mimics the physical structure of soil. We demonstrate, through computational simulation, how this synthetic soil structure can influence the emergence of molecular "hotspots" and "hotmoments" that arise naturally from the plant's exudation of labile carbon compounds. We establish the amenability of the rhizosphere-on-a-chip for long-term culture of Brachypodium distachyon, and experimentally validate the presence of exudate hotspots within the rhizosphere-on-a-chip pore spaces using liquid microjunction surface sampling probe mass spectrometry.

Modeling root system growth around obstacles.

State-of-the-Art models of Root System Architecture (RSA) do not allow simulating root growth around rigid obstacles. Yet, the presence of obstacles can be highly disruptive to the root system. We grew wheat seedlings in sealed petri dishes without obstacle and in custom 3D-printed rhizoboxes containing obstacles. Time-lapse photography was used to reconstruct the wheat root morphology network. We used the reconstructed wheat root network without obstacle to calibrate an RSA model implemented in the R-SWMS software. The root network with obstacles allowed calibrating the parameters of a new function that models the influence of rigid obstacles on wheat root growth. Experimental results show that the presence of a rigid obstacle does not affect the growth rate of the wheat root axes, but that it does influence the root trajectory after the main axis has passed the obstacle. The growth recovery time, i.e. the time for the main root axis to recover its geotropism-driven growth, is proportional to the time during which the main axis grows along the obstacle. Qualitative and quantitative comparisons between experimental and numerical results show that the proposed model successfully simulates wheat RSA growth around obstacles. Our results suggest that wheat roots follow patterns that could inspire the design of adaptive engineering flow networks.

Microfluidics and Metabolomics Reveal Symbiotic Bacterial-Fungal Interactions Between Mortierella elongata and Burkholderia Include Metabolite Exchange.

We identified two poplar (Populus sp.)-associated microbes, the fungus, Mortierella elongata strain AG77, and the bacterium, Burkholderia strain BT03, that mutually promote each other's growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.

Label-free time- and space-resolved exometabolite sampling of growing plant roots through nanoporous interfaces.

Spatial and temporal profiling of metabolites within and between living systems is vital to understanding how chemical signaling shapes the composition and function of these complex systems. Measurement of metabolites is challenging because they are often not amenable to extrinsic tags, are diverse in nature, and are present with a broad range of concentrations. Moreover, direct imaging by chemically informative tools can significantly compromise viability of the system of interest or lack adequate resolution. Here, we present a nano-enabled and label-free imaging technology using a microfluidic sampling network to track production and distribution of chemical information in the microenvironment of a living organism. We describe the integration of a polyester track-etched (PETE) nanofluidic interface to physically confine the biological sample within the model environment, while allowing fluidic access via an underlying microfluidic network. The nanoporous interface enables sampling of the microenvironment above in a time-dependent and spatially-resolved manner. For demonstration, the diffusional flux through the PETE membrane was characterized to understand membrane performance, and exometabolites from a growing plant root were successfully profiled in a space- and time-resolved manner. This method and device provide a frame-by-frame description of the chemical environment that maps to the physical and biological characteristics of the sample.

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.

Increasing access to microfluidics for studying fungi and other branched biological structures.

BACKGROUND: Microfluidic systems are well-suited for studying mixed biological communities for improving industrial processes of fermentation, biofuel production, and pharmaceutical production. The results of which have the potential to resolve the underlying mechanisms of growth and transport in these complex branched living systems. Microfluidics provide controlled environments and improved optical access for real-time and high-resolution imaging studies that allow high-content and quantitative analyses. Studying growing branched structures and the dynamics of cellular interactions with both biotic and abiotic cues provides context for molecule production and genetic manipulations. To make progress in this arena, technical and logistical barriers must be overcome to more effectively deploy microfluidics in biological disciplines. A principle technical barrier is the process of assembling, sterilizing, and hydrating the microfluidic system; the lack of the necessary equipment for the preparatory process is a contributing factor to this barrier. To improve access to microfluidic systems, we present the development, characterization, and implementation of a microfluidics assembly and packaging process that builds on self-priming point-of-care principles to achieve "ready-to-use microfluidics." RESULTS: We present results from domestic and international collaborations using novel microfluidic architectures prepared with a unique packaging protocol. We implement this approach by focusing primarily on filamentous fungi; we also demonstrate the utility of this approach for collaborations on plants and neurons. In this work we (1) determine the shelf-life of ready-to-use microfluidics, (2) demonstrate biofilm-like colonization on fungi, (3) describe bacterial motility on fungal hyphae (fungal highway), (4) report material-dependent bacterial-fungal colonization, (5) demonstrate germination of vacuum-sealed Arabidopsis seeds in microfluidics stored for up to 2 weeks, and (6) observe bidirectional cytoplasmic streaming in fungi. CONCLUSIONS: This pre-packaging approach provides a simple, one step process to initiate microfluidics in any setting for fungal studies, bacteria-fungal interactions, and other biological inquiries. This process improves access to microfluidics for controlling biological microenvironments, and further enabling visual and quantitative analysis of fungal cultures.

Soil Aggregate Microbial Communities: Towards Understanding Microbiome Interactions at Biologically Relevant Scales.

Soils contain a tangle of minerals, water, nutrients, gases, plant roots, decaying organic matter, and microorganisms which work together to cycle nutrients and support terrestrial plant growth. Most soil microorganisms live in periodically interconnected communities closely associated with soil aggregates, i.e., small (<2 mm), strongly bound clusters of minerals and organic carbon that persist through mechanical disruptions and wetting events. Their spatial structure is important for biogeochemical cycling, and we cannot reliably predict soil biological activities and variability by studying bulk soils alone. To fully understand the biogeochemical processes at work in soils, it is necessary to understand the micrometer-scale interactions that occur between soil particles and their microbial inhabitants. Here, we review the current state of knowledge regarding soil aggregate microbial communities and identify areas of opportunity to study soil ecosystems at a scale relevant to individual cells. We present a framework for understanding aggregate communities as "microbial villages" that are periodically connected through wetting events, allowing for the transfer of genetic material, metabolites, and viruses. We describe both top-down (whole community) and bottom-up (reductionist) strategies for studying these communities. Understanding this requires combining "model system" approaches (e.g., developing mock community artificial aggregates), field observations of natural communities, and broader study of community interactions to include understudied community members, like viruses. Initial studies suggest that aggregate-based approaches are a critical next step for developing a predictive understanding of how geochemical and community interactions govern microbial community structure and nutrient cycling in soil.

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

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.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform.

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Two Poplar-Associated Bacterial Isolates Induce Additive Favorable Responses in a Constructed Plant-Microbiome System.

The biological function of the plant-microbiome system is the result of contributions from the host plant and microbiome members. The Populus root microbiome is a diverse community that has high abundance of ß- and γ-Proteobacteria, both classes which include multiple plant-growth promoting representatives. To understand the contribution of individual microbiome members in a community, we studied the function of a simplified community consisting of Pseudomonas and Burkholderia bacterial strains isolated from Populus hosts and inoculated on axenic Populus cutting in controlled laboratory conditions. Both strains increased lateral root formation and root hair production in Arabidopsis plate assays and are predicted to encode for different functions related to growth and plant growth promotion in Populus hosts. Inoculation individually, with either bacterial isolate, increased root growth relative to uninoculated controls, and while root area was increased in mixed inoculation, the interaction term was insignificant indicating additive effects of root phenotype. Complementary data including photosynthetic efficiency, whole-transcriptome gene expression and GC-MS metabolite expression data in individual and mixed inoculated treatments indicate that the effects of these bacterial strains are unique and additive. These results suggest that the function of a microbiome community may be predicted from the additive functions of the individual members.