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

Abstract

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.

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

“…Five microorganisms were used to validate the bioprinting capability, including Escherichia coli Dch1A: mScarlet RFP and Pseudomonas fluorescens SBW25 : GFP [ 17 ], Escherichia coli MJK2: GFP [ 18 , 19 ], Paenibacillus polymyxa DSM 36 [ 20 ], and Neurospora crassa : tdTomato [ 21 ]. These organisms represent a diverse array of microorganisms including Gram-negative bacteria, Gram-positive bacteria, and fungi, and all, except P. polymyxa , express constitutive production of a fluorescent protein.…”

“…We also examined the potential for SSAs to demonstrate biogeochemical processes via biosensors. The E. coli GFP used in this study was specifically engineered to produce urease for urea hydrolysis [ 18 , 19 ]. Urease activity of this organism changes its growth media’s pH from 6.8 to 8+ in the presence of urea.…”

Sub-Micron Scale Chemical and Mineralogical Analyses on Microbially Induced Calcium Carbonate Precipitates

“…Our previous work that utilized a droplet-based microfluidics platform for MICP enables us to produce single micro-precipitates inside droplets [3]. After collecting and drying the micro-precipitates, we image and analyze them using a suite of correlative microscopy and microanalysis techniques.…”

“…Our current work includes characterizing the distribution of CaCO 3 polymorphs within a single microprecipitate based on our earlier finding of potential co-presence of two polymorphs. 3 To determine whether the precipitates contained calcite or vaterite, Selected Area Electron Diffraction (SAED) was performed using a Themis Scanning Transmission Electron Microscope. Our studies apply a correlative microscopic and microanalytical approach with a unique micro-scale system to study biologically precipitated minerals.…”

Micro-scale Mineralogy and Imaging Reveal Polymorph Variance in Microbially Precipitated Calcium Carbonate

“…Our data show that bacterial cells (gfp -green in Fig 1b ) were often encapsulated in a sheath of precipitate (calcite autofluorescence -red in Fig 1b). 4 Fig 2 shows a single precipitate collected from a droplet and visualized using TEM (a,c) and SEM (b,d), where tubular structures resembling bacteria are seen covered with precipitate. This material showed an atomic EDS signal indicative of calcium carbonate.…”