Description
Microbially induced calcium carbonate precipitation (MICP) often uses ureolytic bacteria, such as Sporosarcina pasteurii (S. pasteurii), which hydrolyze urea and, in the presence of sufficient Ca²⁺, promote calcium carbonate (CaCO₃) precipitation. MICP has been proposed or applied in engineering applications (e.g., soil strengthening, well sealing, hydraulic barriers), but controlling where and when ureolysis occurs remains challenging. MICP is a coupled process involving bacterial transport, attachment, growth, biofilm formation, nucleation, and CaCO₃ precipitation. Here, we use microfluidic flow cells with controlled surface chemistry to investigate the role surface properties, particularly charge and hydrophobicity, play in bacterial attachment, growth, and MICP. It is essential to have well-controlled conditions for microfluidics to be a valuable experimental method to determine system characteristics of MICP processes.
Two types of microfluidic devices were used to study the impact of surface properties, one made of stacked borosilicate glass/silicon/borosilicate glass and the other made using polydimethylsiloxane (PDMS). PDMS is considered hydrophobic and, at least initially, negatively charged while also being gas-permeable. Glass is generally hydrophilic, typically negatively charged, and gas impermeable. Surface modifications of glass flow cells established hydrophobic or positively charged surfaces through silanization with either 1H,1H,2H,2H-perfluorooctyltriethoxysilane (FOTS) to increase hydrophobicity or 3-aminopropyl-methyl-diethoxysilane (APMDES) to introduce terminal amine groups, yielding a net positive surface charge.
Bacterial attachment, growth and CaCO₃ precipitation in the flow cells were visualized using a combination of light and fluorescence microscopy involving SYBR Green® staining. Images were acquired before injection of the CaCl₂- and urea-containing cementation solution and again after ~80 pore volumes of cementation solution had passed through the microfluidic device.
Experiments conducted in PDMS flow cells with and without a nitrogen headspace demonstrated that oxygen had only a minor effect on bacterial attachment and CaCO₃ precipitation. In untreated glass flow cells, bacterial attachment and CaCO₃ precipitation were marginal. Cells remained mostly suspended and formed micrometer-sized, low-density flocs of cells, extracellular polymeric substances, and CaCO₃, which were largely flushed out of the flow cell. This likely reflects the net negative surface charge of both S. pasteurii cells and untreated glass under the prevailing pH (>8).
In surface-modified glass cells, bacterial attachment and CaCO₃ formation were significant and comparable to PDMS. Cells appeared immobilized and only a negligible number of cells were transported by the flow. In contrast to the micro-flocs in the untreated glass flow cells, cells attached readily, and dense, surface-attached biofilms formed in the APMDES-treated flow cells. Since APMDES-treated glass presents terminal amine groups and is net-positively charged, attractive bacteria–surface interactions are hypothesized to promote attachment, biofilm formation, CaCO₃ nucleation and aggregate growth. In FOTS-modified glass flow cells, hydrophobic interactions between the bacterial surface and the substrate may likewise enhance attachment and CaCO₃ precipitation.
In conclusion, the two glass surface modifications in these microfluidic models demonstrate that surface chemistry strongly influences bacterial attachment and, consequently, the characteristics of observable MICP behavior in microfluidics. These approaches support future microfluidic studies and emphasize the importance of controlling the surface properties, and when needed, strategies to enhance bacterial attachment in larger scale applications.