Novel Focused Ion Beam Liftouts for Spatial Characterization of Spherical Biominerals With Transmission Electron Microscopy.

Focused ion beam (FIB) is frequently used to prepare electron- and X-ray-beam-transparent thin sections of samples, called lamellae. Typically, lamellae are prepared from only a subregion of a sample. In this paper, we present a novel approach for FIB lamella preparation of microscopic samples, wherein the entire cross-section of the whole sample can be investigated. The approach was demonstrated using spherical, porous, and often hollow microprecipitates of biologically precipitated calcium carbonate. The microprecipitate morphology made these biogenic samples more fragile and challenging than materials commonly investigated using FIB lamellae. Our method enables the appropriate orientation of the lamellae required for further electron/X-ray analyses after attachment to the transmission electron microscopy (TEM) grid post and facilitates more secure adhesion onto the grid post. We present evidence of autofluorescence in bacterially precipitated vaterite using this lamella preparation method coupled with TEM selected area diffraction. This innovative approach allows studying biomineralization at the micro to nano scales, which can provide novel insights into bacterial responses to microenvironmental conditions.

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.

Mineralogy of microbially induced calcium carbonate precipitates formed using single cell drop-based microfluidics.

Microbe-mineral interactions are ubiquitous and can facilitate major biogeochemical reactions that drive dynamic Earth processes such as rock formation. One example is microbially induced calcium carbonate precipitation (MICP) in which microbial activity leads to the formation of calcium carbonate precipitates. A majority of MICP studies have been conducted at the mesoscale but fundamental questions persist regarding the mechanisms of cell encapsulation and mineral polymorphism. Here, we are the first to investigate and characterize precipitates on the microscale formed by MICP starting from single ureolytic E. coli MJK2 cells in 25 µm diameter drops. Mineral precipitation was observed over time and cells surrounded by calcium carbonate precipitates were observed under hydrated conditions. Using Raman microspectroscopy, amorphous calcium carbonate (ACC) was observed first in the drops, followed by vaterite formation. ACC and vaterite remained stable for up to 4 days, possibly due to the presence of organics. The vaterite precipitates exhibited a dense interior structure with a grainy exterior when examined using electron microscopy. Autofluorescence of these precipitates was observed possibly indicating the development of a calcite phase. The developed approach provides an avenue for future investigations surrounding fundamental processes such as precipitate nucleation on bacteria, microbe-mineral interactions, and polymorph transitions.

Controlling the Distribution of Microbially Precipitated Calcium Carbonate in Radial Flow Environments.

Bacterially driven reactions such as ureolysis can induce calcium carbonate precipitation, a well-studied process called microbially induced calcium carbonate precipitation (MICP). MICP is of interest in subsurface applications such as sealing leaks around wells. For effective field deployment, it is important to study MICP under radial flow conditions, which are relevant to near-well environments. In this study, a laboratory-scale radial flow reactor of 23 cm diameter, with a 1 mm glass bead monolayer serving as a porous medium, was used to investigate the effects of fluid flow rates and calcium concentrations on the mass and distribution of MICP by the ureolytic bacterium Sporosarcina pasteurii. Experiments were performed at hydraulic residence times of 14, 7, and 3.5 min and calcium to urea molar ratios of 0.5:1, 1:1, and 2:1. The total amount of CaCO3 precipitated in the reactor increased with increasing residence time and with decreasing Ca2+ to urea molar ratios. Increased bacterial attachment and increased CaCO3 precipitation were observed with distance from the center inlet of the reactor in all experiments. More uniform calcium distribution was achieved at lower flow rates. The relationship between reaction and transport rate (i.e., the Damköhler number) is identified as a useful parameter for the prediction of MICP in radial flow environments.