Engineered Microbial Habitats for Health, Fuel, and Food

Microorganisms are of vital importance in medicine, in industry, and in the natural environment. In agricultural systems, bacteria fix nitrogen, protect crop roots from pathogens, and promote water retention in soils. In the biotech industry, microbes are harnessed to produce food, pharmaceuticals and biofuels. Microbes are essential for health, yet microbial pathogens continue to cause more human deaths worldwide than all forms of cancer combined. In each case, whether microbes are found in soil, in industry, or in vivo, micron-scale habitat conditions critically impact the structure and function of microbial communities. In our work, we design and build artificial microbial habitats that emulate selected micron-scale features of real microbial habitats. In this talk I will survey ongoing research projects including (i) a microfluidic biofilm array for screening antimicrobial combinations and measuring the effect of antimicrobial delivery rate on biofilm inhibition; (ii) a growth chamber with appropriately-scaled oxygen gradients for sustaining the complex hindgut microbiome of cellulose-degrading termites; and (iii) a testing device for seed coating technology aimed at enhancing crop yields in the developing world

Bacterial Extracellular Polymeric Substances Amplify Water Content Variability at the Pore Scale

The function of microbial communities in soil is inextricably linked with the complex physical, chemical, and biological structure of the soil itself. Pore-scale water content controls the hydraulic connectivity of microbial communities and microbes' access to aqueous and gaseous substrates. In turn, soil bacteria directly influence local moisture conditions through the secretion of extracellular polymeric substances (EPS). However, the effect of a soil's physical geometry on EPS-mediated water retention is not well understood. In this study, we systematically measured the rate and extent of water evaporation from pore structures as a function of both EPS concentration and pore size. Three different chamber types were employed: (i) glass capillary tubes (1.2 mm pore diameter) to represent a uniform macropore geometry; (ii) emulated soil micromodels (pore widths ~10 to >300 μm) to represent an aggregated sandy loam pore geometry; and (iii) microfluidic capillary arrays (uniform channels 20 μm wide) to represent a uniform micropore geometry. All chambers were initially saturated with dilute EPS solutions collected from stationary-phase Sinorhizobium meliloti cultures and then the infiltration of air was tracked over time. In the largest chambers, EPS concentration had no effect on the extent of evaporation or on the magnitude or variability of the evaporation rate. However, in the chambers with micropore-sized physical features, EPS concentration strongly influenced rate, extent, and variability of pore water evaporation. In micropores, higher EPS concentrations enhanced water retention and led to greater variability in pore-scale water distributions. In real soil, these phenomena could act together to promote the intermediate water contents associated with productive soil systems, and more variable pore-scale water distributions could increase microbial community diversity and the resiliency of soil systems

Optical control of exopolysaccharide production in Sinorhizobium meliloti for studying biofilm formation and water retention

The rhizosphere contains many types of microbes interacting with plant roots, creating a complex symbiotic system. Microbial processes occurring in the rhizosphere are essential to the productivity of terrestrial ecosystems. In particular, exopolysaccharide produced by soil microbes allows dynamic regulation of soil moisture by modulating water transport. We have demonstrated that purified microbial exopolysaccharide (EPS) impacts soil water retention through enhancing the variability of water distributions in the soil microstructure. However, the impact of EPS on water transport in soil is not understood due to complex interaction of microbial EPS with soil microstructure and particle surface properties. To decipher the causal role of EPS in soil microstructures, we set out to develop engineered soil bacteria with spatially regulated EPS biosynthesis capabilities. Here we report genetic engineering of soil bacterium Sinorhizobium meliloti to enable in situ spatial control of EPS production. We show that the photo-sensitive transcription factor EL222, derived from Erythrobacter litoralis, allows robust control of gene expression in S. meliloti. Essential genes in the type II EPS (a major component of EPS from S. meliloti in the soil) production pathway were identified, and deletion strains were generated. Complementation of the essential gene using a synthetic promoter controlled by EL222 led to robust light-activated production of EPS. Optimization of the engineered genetic construct was performed by varying promoters, ribosome binding sites, and using alternative start codons. Using the engineered EPS production strain, we observed rapid settlement of EPS producing S. meliloti in liquid culture, and selective biofilm formation quantified by a crystal violet staining assay. This approach enables spatially regulated EPS production and biofilm formation. We will demonstrate control of gene expression in a synthetic soil microsystem that emulates aggregated sandy loam soil. We will also report our current progress on using these new strains of soil bacteria to study the impact of EPS production on water drying rate in the synthetic soil microsystem. We anticipate that the engineered genetic constructs will be broadly applicable for dissecting gene function in a defined population of microbes in the rhizosphere

Expanding Molecular Coverage in Mass Spectrometry Imaging of Microbial Systems Using Metal-Assisted Laser Desorption/Ionization

Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial ( approximately 67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels

Pore-scale monitoring of the effect of microarchitecture on fungal growth in a two-dimensional soil-like micromodel

In spite of the very significant role that fungi are called to play in agricultural production and climate change over the next two decades, very little is known at this point about the parameters that control the spread of fungal hyphae in the pore space of soils. Monitoring of this process in 3 dimensions is not technically feasible at the moment. The use of transparent micromodels simulating the internal geometry of real soils affords an opportunity to approach the problem in 2 dimensions, provided it is confirmed that fungi would actually want to propagate in such artificial systems. In this context, the key objectives of the research described in this article are to ascertain, first, that the fungus Rhizoctonia solani can indeed grow in a micromodel of a sandy loam soil, and, second, to identify and analyze in detail the pattern by which it spreads in the tortuous pores of the micromodel. Experimental observations show that hyphae penetrate easily inside the micromodel, where they bend frequently to adapt to the confinement to which they are subjected, and branch at irregular intervals, unlike in current computer models of the growth of hyphae, which tend to describe them as series of straight tubular segments. A portion of the time, hyphae in the micromodels also exhibit thigmotropism, i.e., tend to follow solid surfaces closely. Sub-apical branching, which in unconfined situations seems to be controlled by the fungus, appears to be closely connected with the bending of the hyphae, resulting from their interactions with surfaces. These different observations not only indicate different directions to follow to modify current mesoscopic models of fungal growth, so they can apply to soils, but they also suggest a wealth of further experiments using the same set-up, involving for example competing fungal hyphae, or the coexistence of fungi and bacteria in the same pore space

Application of microfluidic systems in modelling impacts of environmental structure on stress-sensing by individual microbial cells

Environmental structure describes physical structure that can determine heterogenous spatial distribution of biotic and abiotic (nutrients, stressors etc.) components of a microorganism's microenvironment. This study investigated the impact of micrometre-scale structure on microbial stress sensing, using yeast cells exposed to copper in microfluidic devices comprising either complex soil-like architectures or simplified environmental structures. In the soil micromodels, the responses of individual cells to inflowing medium supplemented with high copper (using cells expressing a copper-responsive pCUP1-reporter fusion) could be described neither by spatial metrics developed to quantify proximity to environmental structures and surrounding space, nor by computational modelling of fluid flow in the systems. In contrast, the proximities of cells to structures did correlate with their responses to elevated copper in microfluidic chambers that contained simplified environmental structure. Here, cells within more open spaces showed the stronger responses to the copper-supplemented inflow. These insights highlight not only the importance of structure for microbial responses to their chemical environment, but also how predictive modelling of these interactions can depend on complexity of the system, even when deploying controlled laboratory conditions and microfluidics

Applications of biosynthesized metallic nanoparticles - A review

We present a comprehensive review of the applications of biosynthesized metallic nanoparticles (NPs). The biosynthesis of metallic NPs is the subject of a number of recent reviews, which focus on the various “bottom-up” biofabrication methods and characterization of the final products. Numerous applications exploit the advantages of biosynthesis over chemical or physical NP syntheses, including lower capital and operating expenses, reduced environmental impacts, and superior biocompatibility and stability of the NP products. The key applications reviewed here include biomedical applications, especially antimicrobial applications, but also imaging applications, catalytic applications such as reduction of environmental contaminants, and electrochemical applications including sensing. The discussion of each application is augmented with a critical review of the potential for continued development.Web of Science10104042402

Mobility of Protozoa through Narrow Channels

Microbes in the environment are profoundly affected by chemical and physical heterogeneities occurring on a spatial scale of millimeters to micrometers. Physical refuges are critical for maintaining stable bacterial populations in the presence of high predation pressure by protozoa. The effects of microscale heterogeneity, however, are difficult to replicate and observe using conventional experimental techniques. The objective of this research was to investigate the effect of spatial constraints on the mobility of six species of marine protozoa. Microfluidic devices were created with small channels similar in size to pore spaces in soil or sediment systems. Individuals from each species of protozoa tested were able to rapidly discover and move within these channels. The time required for locating the channel entrance from the source well increased with protozoan size and decreased with channel height. Protozoa of every species were able to pass constrictions with dimensions equal to or smaller than the individual's unconstrained cross-sectional area. Channel geometry was also an important factor affecting protozoan mobility. Linear rates of motion for various species of protozoa varied by channel size. In relatively wide channels, typical rates of motion were 300 to 500 μm s(−1) (or about 1 m per hour). As the channel dimensions decreased, however, motilities slowed more than an order of magnitude to 20 μm s(−1). Protozoa were consistently observed to exhibit several strategies for successfully traversing channel reductions. The empirical results and qualitative observations resulting from this research help define the physical limitations on protozoan grazing, a critical process affecting microbes in the environment