Enhanced manganese oxidation at the biofilm−fluid interface drives pore-scale patterns in mineral precipitation

Microbial oxidation of manganese (Mn) from aqueous Mn(II) to solid-phase
Mn(III, IV) minerals catalyzes Mn(II) removal in natural and engineered
porous systems. However, little is known about the spatiotemporal
evolution of Mn biomineralization in confined spaces that experience
simultaneous Mn(II) delivery and Mn oxide precipitation. In this work, we
combined time-lapse microscopy, image analysis, and mass spectrometry to
quantify the extent and rate of Mn biomineralization by Pseudomonas putida
GB-1 in an optically transparent two-dimensional porous medium. We
conducted microfluidic experiments in triplicate and collected both
optical images (color brightfield and mCherry fluorescence) and effluent
samples for each replicate. We also developed a calibration curve using
optical microscopy and micro-X-ray fluorescence to convert pixel intensity
to Mn oxide mass. Images were analyzed with custom MATLAB (2024b) scripts
to quantify total Mn oxide accumulation and the rate of mineral
precipitation as a function of time and space. Effluent samples were
analyzed with inductively coupled plasma mass spectrometry (ICP-MS) to
measure Mn(II) removal. From these data, we found that Mn(II) oxidation
initially occurred within biofilms but shifted over time towards the edges
of biofilms in contact with pore fluid. Minerals precipitated outside of
the initial biofilm footprint due to surface-mediated oxidation of Mn(II)
by nascent biogenic Mn oxides, reinforcing a gradient in mineral
accumulation from the Mn(II) source near the reactor inlet to the outlet.
The rate of mineral precipitation outside the biofilm footprint surpassed
the rate of mineral accumulation inside biofilms within six hours and
accounted for two-thirds of the total Mn oxide mass in the pore space at
the end of the experiment. This work advances a mechanistic understanding
of coupled biotic and abiotic Mn oxidation in porous environments, while
providing a novel platform to quantify microbe-mineral-fluid interactions.