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This project was supported by the U.S. National Science Foundation Center for Bio-mediated and Bio-inspired Geotechnics (ERC-1449501), the Swiss National Science Foundation (200021_188546), and the U.S National Science Foundation (MCA-2322428, EAR-1847689, DMR-2003849). Part&#160;of this study was carried out at the UC Davis Center for Nano and Micro&#160;Manufacturing (CNM2). We thank Steven Lucero and the UC Davis Tech Foundry for the design and fabrication of the microscope incubator and UVC irradiation system. We also thank Dr. Yuze Wang and Dr. Jason DeJong for providing the porous medium geometry used for microfluidic reactor fabrication, and Dr. Arunima&#160;Bhattacharjee, Dr. Noah Goshi, Dr. Gregory Girardi, and Dr. Hyehyun Kim for useful discussions and/or providing training on photolithography and soft lithography. Finally, we thank Dr. Gaitan Gehin, Dr. Tonya Kuhl, Dr. Filippo Miele, and Dr. Charles Graddy for valuable discussions on experimental design and image analysis.

The authors declare no conflicts of interest.

This experimental platform offers unique opportunities for investigating spatiotemporal dynamics of biogeochemical processes in porous environments free of artifacts that prevent the acquisition of high-fidelity data. This approach is particularly well-suited for Mn and Fe biomineralization, which produce mineral precipitates with characteristic ochre, brown, and black hues that can be readily segmented with optical microscopy and image analysis. Using Mn biomineralization as a case study, it is possible to evaluate patterns and rates of mineral precipitation in situ&#160;with high spatial and temporal resolution.
Several aspects of this protocol enabled the collection of reproducible measurements of biogeochemical transformations. First, using a sequential injection valve with low dead volume facilitated experiments with multiple fluids without perturbing the biofilms within the microfluidic pore space. This setup preserved the initial biofilm spatial distribution by preventing pressure surges that can lead to sloughing or displacement of biofilms when switching injected fluids, which may occur with other instruments such as syringe pumps. This component further provides the user with the flexibility to customize the geochemical conditions imposed on the resident microorganisms. Finally, because it preserves initial biofilm distribution, this setup allows the user to systematically test how different biofilm morphologies or biomass amounts influence the distribution and rate of Mn oxide precipitation.
Second, mitigating the formation and intrusion of bubbles in the microfluidic pore space allowed for multi-day experiments that captured the environmentally relevant timescales of Mn biomineralization. Bubbles, a common and often inevitable issue in long experiments above room temperature62,63, can fragment and block individual pores, reroute fluid flow, and disrupt reaction progress. To prevent bubble trapping in the pore space, the reactor was fully saturated prior to inoculation. To minimize the introduction of bubbles through the flow control system, the microscope incubator was set to 30 &#176;C well in advance of the experiment, and multiple volumes of influent solution were flushed prior to attaching the inoculated reactor. This step removed trapped air from the tubing and fittings and minimized the risk of fluid outgassing during the experiment. A bubble trap was also integrated into the flow control system to capture any residual bubbles, with a length of 200-&#181;m ID PTFE tubing attached to the trap outlet to increase resistance and enhance bubble removal efficiency. Finally, to prevent air trapping or leaking at the interface between the influent tubing and reactor inlet hole, all connections were made with droplets of fluid on both ends of the tubing and the punched hole, the hydrophobic PTFE tubing was coated with 1% PEG-DA, and the end of the inserted tubing was kept perpendicular to the PDMS surface throughout the experiment.
Third, incorporating a custom-built UVC irradiation system into the experimental platform maintained a defined geochemical boundary condition and reproducibly constrained Mn biomineralization within the microfluidic pore space. Pilot experiments showed that extraneous biofilms in the reactor inlet region precipitated Mn oxides upstream of the pore space, restricting the downstream availability of Mn(II). Briefly irradiating the reactor inlet and outlet regions with proper alignment&#160;following the no-flow settling period, prevented biofilm development from any initially deposited bacteria. Continuous unidirectional fluid flow prevented recolonization of the reactor inlet region following this initial UVC treatment. Irradiating the reactor outlet region at t = 17.5 h prior to the onset of the stationary phase and visible Mn oxide precipitation minimized the amount of mineral accumulation downstream of the pore space. This facilitated direct comparison of color brightfield image data with Mn mass digested and measured with ICP-MS.
The combination of image subtraction and endpoint mass measurements provided a semi-quantitative method for evaluating the kinetics of Mn oxide precipitation in situ. However, it is important to note that pixel intensity ceases to be directly proportional to Mn oxide mass at very high mineral density due to limited camera sensitivity to dark colors61. Evaluation of this non-linear behavior could be improved by acid-digesting samples at defined timepoints and comparing the digested mass to the original mass scaled by the final experimental timepoint. If exact quantification of mineral mass is required, users can develop a calibration curve using mineral samples of known intensity and mass, which has been demonstrated previously in Fadely et al.61, or make direct measurements with synchrotron-based X-ray fluorescence.
The microfluidic platform presented here can be applied to investigate biomineralization processes under a wide range of environmental conditions. While representative experiments were optimized for P. putida&#160;GB-1, this setup can be used to systematically test a range of influent solution chemistries (e.g., Mn or Fe concentration, carbon source, presence of co-occurring metals), flow rates, temperatures, or the presence of a microbial consortium. Specific acidic or suboxic conditions, such as those favorable to Fe-oxidizing bacteria, can be simulated by decreasing influent pH or coating the PDMS with an oxygen-impermeable polymer. Insights into the microbial controls on biomineralization can be obtained by implementing reporter gene fusion strains and tracking the resulting optical fluorescence59,64. Microfluidic reactors can be redesigned to simulate a range of porous environments&#160;by changing the particle size distribution, particle shape, or porosity, or by functionalizing the PDMS to mimic the surface charge of clay particles or natural organic matter. With additional modifications (e.g., replacing the glass reactor substrate with plastic), reactors can support direct spectroscopic measurements of metal oxidation state, mineralogy, organo-mineral associations, or the sorption or co-precipitation of secondary metals. Finally, this experimental platform can be expanded to investigate precipitation of other metal biominerals (e.g., phosphates, carbonates, sulfides), provided their optical properties are sufficiently distinct to allow for image segmentation. This protocol therefore offers a framework to comprehensively investigate biomineralization and microbe-mineral-fluid interactions in relevant porous environments.

Metal oxide minerals play a critical role in biogeochemical cycling1. Manganese (Mn) and iron (Fe) oxides are negatively charged at circumneutral pH and readily sorb cationic metals2,3,4,5,6,7,8, regulating micronutrient availability and contaminant mobility in the rhizosphere and hyporheic zone. These minerals influence the stability and degradation of organic matter9,10,11,12, dictating carbon uptake by plants and soil microbes. In engineered systems (e.g., water treatment filters, constructed wetlands), metal oxide precipitation is directly leveraged for Mn or Fe removal and remediation of co-occurring contaminants13,14,15,16,17. These metal oxides are identifiable by their characteristic colors, which range from ochre to dark brown to black18,19.
The precipitation of metal oxides is commonly facilitated by microorganisms in a process known as biomineralization20,21,22. Numerous Mn-oxidizing bacteria and fungi synthesize multicopper oxidases and peroxidases that catalyze electron transfer from Mn(II) to Mn(III) and Mn(IV) under aerobic conditions, resulting in the precipitation of birnessite-like Mn(III, IV) oxide minerals2,23,24. Additionally, diverse Fe-oxidizing bacteria enzymatically oxidize Fe(II) to Fe(III) in circumneutral or acidic environments limited in oxygen, producing Fe(II, III) (oxyhydr)oxides, including ferrihydrite and magnetite25,26,27,28. These biogenic minerals precipitate adjacent to cell surfaces and accumulate on extracellular polymers, sheaths, and other cellular structures, forming microbe-mineral assemblages2,23,25,27,29,30.
Despite the ubiquity of Mn and Fe biomineralization in complex environments, pore-scale insights are lacking. Pore spaces are structurally heterogeneous, exhibiting preferential fluid flow and nonuniform reactant transport that dictate the distribution of biogeochemical reactions31,32,33,34,35. Further, microbial activity may generate local geochemical gradients that promote or limit biomineralization, while fluctuations in pore fluid chemistry (e.g., pH, dissolved oxygen, redox potential) may enhance mineral precipitation or facilitate dissolution34,35. Studies of Mn and Fe biomineralization typically rely either on well-mixed batch conditions30,36,37,38,39,40 or columns41,42,43,44 to quantify oxidation kinetics or characterize biomineral precipitates. However, batch experiments lack the spatial heterogeneity inherent to porous systems. Columns containing packed granular media provide relevant physical architecture but are limited in their utility because they require destructive sampling at static timepoints, which halts the experiment progression and prevents unperturbed pore-scale observations. Therefore, new experimental approaches are needed to investigate the microbial precipitation of metal oxides in porous media.
Microfluidic systems are a tool to study biogeochemical reactions in situ&#160;and in real time. Microfluidic reactors can be fabricated rapidly and at low cost with reproducible and customizable geometries that simulate natural or idealized porous media45. The resulting devices are optically transparent, facilitating non-destructive visualization and quantification of pore-scale processes, including biofilm formation46,47,48,49, hotspot development50,51, abiotic metal precipitation52,53, and calcite biomineralization34,54,55,56. Additionally, microfluidic systems allow users to control and systematically vary boundary conditions, including metal concentration, presence or absence of dissolved oxygen, and fluid flow rate. Finally, depending on the reactor substrate material, direct measurements of microbe-mineral-fluid interactions can be acquired using X-ray, Raman, or infrared spectroscopy57. The development of such a microfluidics-based platform to study the microbial precipitation of metal oxides&#160;thus provides an avenue for novel research on the formation and reactivity of Mn or Fe mineral phases that drive carbon, nutrient, and contaminant cycling in porous environments.
This work uses Mn biomineralization as a case study to quantify the microbial precipitation of metal&#160;oxides in a dynamic porous environment. The following protocol demonstrates how to fabricate and inoculate a microfluidic reactor with Pseudomonas putida GB-1, a model bacterium known to oxidize Mn(II) to Mn(III, IV) at its stationary phase of growth58,59. Using a pressure-based flow control system, nutrient-rich growth medium and minimal salt solution are sequentially injected to promote biofilm formation and induce Mn oxide precipitation, respectively. High-resolution color brightfield images of the microfluidic pore space are collected at regular time intervals, capturing the color contrast between P. putida&#160;GB-1 biofilms and accumulating Mn oxides. At the end of each experiment, all Mn oxides contained within the reactor are digested, and the total mass is measured with inductively coupled plasma mass spectrometry (ICP-MS). The rate of Mn oxide precipitation is estimated as a function of time by integrating these endpoint mass measurements with an image subtraction algorithm. This workflow can be readily adapted to investigate other organisms or metal oxide precipitation processes under a variety of environmental conditions.

<table><tbody><tr><td>Adapter fitting (Elveflow, 6-40 to 1/4-28)</td><td>Darwin Microfluidics</td><td>LVF-KFI-08</td><td>Used to interface between flow sensor and flangeless fittings.</td></tr><tr><td>Agar (Fisher BioReagents)</td><td>Fisher Scientific</td><td>BP9744</td><td>Used to pour plates for streaking frozen stocks.</td></tr><tr><td>Air compressor (Jun-Air)</td><td>Elveflow</td><td>KCP-110</td><td>Used to pressurize flow controller.</td></tr><tr><td>Ammonium sulfate ((NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;, CAS: 7783-20-2)</td><td>Sigma-Aldrich</td><td>A4915</td><td>Used for experimental media and solutions.</td></tr><tr><td>Angled tweezers</td><td>TDI International</td><td>7-SA</td><td>Used to attach tubing to microfluidic reactor.</td></tr><tr><td>AutoCAD software</td><td>Autodesk&amp;nbsp;</td><td>--</td><td>Used to design microfluidic reactor geometry.</td></tr><tr><td>Autoclave</td><td>Steris</td><td>--</td><td>Used to sterilize materials and liquids.&amp;nbsp;</td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>ME2002T</td><td>Used to weigh PDMS elastomer base and curing agent.</td></tr><tr><td>Barbed adapter (1/4-28 swivel to 3/32-in barbed)</td><td>Elveflow</td><td>KFI-06</td><td>Used to connect polyether polyurethane tubing to manifold and fluid reservoir rack.</td></tr><tr><td>Biopsy punch (Rapid-Core, 1.2-mm diameter)</td><td>Darwin Microfluidics</td><td>PT-T983-12-25</td><td>Used to punch inlet/outlet holes in PDMS microfluidic channels.&amp;nbsp;</td></tr><tr><td>Bleach (Chlorox)</td><td>ULINE</td><td>S-19719</td><td>Used to sterilize flow control system. Dilute to 1% in ultrapure water.</td></tr><tr><td>Blunt-tip needle (Jensen Global, 21-gauge)</td><td>QSource</td><td>JG21-10HPX-J004</td><td>Used to connect tubing to syringes. Sterilized by autoclaving for 30 min.</td></tr><tr><td>Bunsen burner</td><td>Humboldt</td><td>H-5970</td><td>Used to maintain a sterile experimental workspace.</td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O, Fisher Chemical, CAS: 10035-04-8)</td><td>Fisher Scientific</td><td>C79</td><td>Used for experimental media and solutions.</td></tr><tr><td>Casamino acid (Fisher BioReagents, acid casein peptone)</td><td>Fisher Scientific</td><td>BP1424</td><td>Used for experimental media.</td></tr><tr><td>Cell density meter (WPA Biowave CO8000)</td><td>Avantor</td><td>490005-906</td><td>Used to measure OD&lt;sub&gt;600&lt;/sub&gt; of bacterial cultures.&amp;nbsp;</td></tr><tr><td>Clear tape (Scotch Magic)</td><td>ULINE</td><td>S-10223</td><td>Used to keep dust from sticking to PDMS microfluidic channels.</td></tr><tr><td>Cobalt(II) chloride hexahydrate (CoCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O, CAS: 7791-13-1)</td><td>Sigma-Aldrich</td><td>255599</td><td>Used for experimental media and solutions.</td></tr><tr><td>Compressed nitrogen</td><td>Airgas</td><td>NIUHP300</td><td>Used to dry microfluidic reactor components.</td></tr><tr><td>Conical tube (Falcon, 15-mL volume, polypropylene)</td><td>Fisher Scientific</td><td>14-959-49B</td><td>Used for influent solutions and ICP-MS samples. Pre-sterilized.</td></tr><tr><td>Conical tube (Falcon, 50-mL volume, polypropylene)</td><td>Fisher Scientific</td><td>14-432-22</td><td>Used for LB preculture. Pre-sterilized.</td></tr><tr><td>Copper(II) sulfate pentahydrate (CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O, CAS: 7758-99-8)</td><td>Sigma-Aldrich</td><td>C8027</td><td>Used for experimental media and solutions.</td></tr><tr><td>Cutting mat</td><td>ULINE</td><td>S-18543</td><td>Used to cut apart PDMS microfluidic channels and punch inlet/outlet holes.</td></tr><tr><td>Cuvette (Fisherbrand, 1.5-mL volume, polystyrene)</td><td>Fisher Scientific&amp;nbsp;</td><td>14-955-127</td><td>Used to measure OD&lt;sub&gt;600&lt;/sub&gt; of bacterial cultures.&amp;nbsp;</td></tr><tr><td>Digital camera (Hamamatsu ORCA-Fusion)</td><td>Nikon</td><td>77054115</td><td>Used for time-lapse color brightfield imaging of microfluidic experiments.</td></tr><tr><td>Distribution valve (MUX)</td><td>Elveflow</td><td>MUX-D-12</td><td>Used to sequentially inject multiple fluids.</td></tr><tr><td>Elveflow Smart Interface (ESI) software</td><td>Elveflow&amp;nbsp;</td><td>--</td><td>Used to control flow rate/pressure feedback between flow controller and flow sensor and perform distribution valve switching.</td></tr><tr><td>Erlenmeyer flask (Fisherbrand, 50-mL volume)</td><td>Fisher Scientific</td><td>FB50050</td><td>Used for mid-exponential culture. Sterilized by autoclaving for 30 min.</td></tr><tr><td>Ethanol (Fisher Chemical, reagent alcohol, 90% purity, CAS: 64-17-5)</td><td>Fisher Scientific</td><td>A962P-4</td><td>Used to clean PDMS and cover slips for reactor fabrication. Diluted to 70% in ultrapure water.</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA, CAS: 60-00-4)</td><td>Sigma-Aldrich</td><td>EDS</td><td>Used for experimental media and solutions.</td></tr><tr><td>Flangeless fitting (1/4-28 for 1/16-in OD tubing)</td><td>Elveflow</td><td>KFI-17</td><td>Used to connect tubing to Elveflow hardware components. Kit includes ferrules and threaded nuts.</td></tr><tr><td>Flow controller (OB1 MK3+)</td><td>Elveflow</td><td>OB1-2000</td><td>Used to provide flow rate/pressure feedback for controlled fluid injection.</td></tr><tr><td>Flow sensor (MFS2)</td><td>Elveflow</td><td>MFS-D-2</td><td>Used to maintain stable flow for microfluidic experiments using pressure feedback from flow controller.</td></tr><tr><td>Fluid reservoir rack&amp;nbsp;</td><td>Elveflow</td><td>KPT-4S</td><td>Used to pressurize influent fluids. Compatible with 15-mL Falcon conical tubes.</td></tr><tr><td>Glass cover slip (1.42-in x 2.36-in, #1 thickness)</td><td>Ted Pella</td><td>260460-100</td><td>Plasma bonded to PDMS channel as a substrate for microfluidic reactors.&amp;nbsp;</td></tr><tr><td>Glass gas-tight syringe (5-mL volume, Luer-Lock)</td><td>Hamilton</td><td>81520</td><td>Used for reactor inoculation and acid digestion (separate syringes). Inoculation syringe sterilized by autoclaving for 30 min.</td></tr><tr><td>HEPES buffer (CAS: 7365-45-9)</td><td>Sigma-Aldrich</td><td>H3375</td><td>Used for experimental media and solutions.</td></tr><tr><td>Hot plate</td><td>Corning</td><td>6795-420D</td><td>Used to cure PDMS.</td></tr><tr><td>Hydrophobic filter (Elveflow, 0.2-&amp;micro;m)</td><td>Darwin Microfluidics</td><td>LVF-KXX-14</td><td>Used to prevent liquid backflow into the flow controller.</td></tr><tr><td>Inductively coupled plasma mass spectrometer (ICP-MS, 7900)</td><td>Agilent</td><td>--</td><td>Used to measure Mn concentration in microfluidic reactor digest.</td></tr><tr><td>Inverted epifluorescence microscope (Nikon Ti2 Eclipse)</td><td>Nikon</td><td>MEA54000, MEE59925, MEL51920, MEP59394, MEC56130</td><td>Used for time-lapse color brightfield imaging of microfluidic experiments. Basic setup includes body, diascopic pillar, condenser turret, nosepiece, and stage.</td></tr><tr><td>Iron(III) chloride hexahydrate (FeCl&lt;sub&gt;3&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O, CAS: 10025-77-1)</td><td>Sigma-Aldrich</td><td>44944</td><td>Used for experimental media and solutions.</td></tr><tr><td>Isopropanol (Fisher Chemical, 100% purity, CAS: 67-63-0)</td><td>Fisher Scientific</td><td>A416P-4</td><td>Used to clean flow control system.</td></tr><tr><td>Laminar flow cabinet (Air Science Purair FLOW Vertical, 48-in)</td><td>Fisher Scientific</td><td>NC0875829</td><td>Used to maintain a low-dust environment for soft lithography, reactor assembly, and reactor degassing.&amp;nbsp;</td></tr><tr><td>Light engine (LIDA)</td><td>Lumencor</td><td>77060081</td><td>Used for time-lapse color brightfield imaging of microfluidic experiments.</td></tr><tr><td>Luria Broth powder (LB, Invitrogen, Miller&#039;s formulation)</td><td>Fisher Scientific</td><td>12-795-027</td><td>Used to culture P. putida GB-1 from frozen stocks. Sterilized by autoclaving for 20 min.</td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt; 7H&lt;sub&gt;2&lt;/sub&gt;O; CAS: 10034-99-8)</td><td>Sigma-Aldrich</td><td>230391</td><td>Used for experimental media and solutions.</td></tr><tr><td>Manganese(II) chloride tetrahydrate (MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O, CAS: 13446-34-9)</td><td>Sigma-Aldrich</td><td>221279</td><td>Used for experimental media and solutions.</td></tr><tr><td>Manifold</td><td>Elveflow</td><td>KM9</td><td>Used to distribute pressure from air compressor into fluid reservoir rack.</td></tr><tr><td>MATLAB software</td><td>Mathworks</td><td>--</td><td>Used for image analysis.</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5418</td><td>Used to wash LB preculture.&amp;nbsp;</td></tr><tr><td>Microcentrifuge tube (Fisherbrand, 1.5-mL volume)</td><td>Fisher Scientific</td><td>02-681-5</td><td>Used to wash LB preculture. Sterilized by autoclaving for 30 min.</td></tr><tr><td>Microcentrifuge tube (Fisherbrand, 2-mL volume)</td><td>Fisher Scientific</td><td>02-681-271</td><td>Used to prepare reactor inoculum. Sterilized by autoclaving for 30 min.</td></tr><tr><td>Microcentrifuge tube (Labcon, 5-mL volume)</td><td>Sigma-Aldrich</td><td>LABC-3048-860-000-9</td><td>Used to collect reactor effluent during time-lapse experiments.</td></tr><tr><td>Microfluidic bubble trap (25-&amp;micro;L internal volume, EZMount)</td><td>PreciGenome</td><td>PG-BT-REC25UL</td><td>Used to remove bubbles upstream of microfluidic reactor. Sterilized by autoclaving for 30 min.</td></tr><tr><td>Microscope incubator</td><td>--</td><td>--</td><td>Designed and fabricated by the UC Davis Tech Foundry.&amp;nbsp;</td></tr><tr><td>Microscope stage insert (TI2-S-HU universal holder)</td><td>Nikon</td><td>MEC59140</td><td>Used to mount UVC irradiation system and hold microfluidic reactors during time-lapse experiments.</td></tr><tr><td>NIS Elements software</td><td>Nikon</td><td>--</td><td>Used to automate time-lapse image acquisition.</td></tr><tr><td>Nitric acid (Fisher Chemical, 70% purity, CAS: 7697-37-2)</td><td>Fisher Scientific</td><td>A200-212</td><td>Used for acid digestion of Mn oxides in microfluidic reactor and ICP-MS sample dilution.</td></tr><tr><td>Objective (Plan Apo &amp;lambda;D 4x/0.20)</td><td>Nikon</td><td>MRD70040</td><td>Used for time-lapse color brightfield imaging of microfluidic experiments.</td></tr><tr><td>Oxalic acid dihydrate (CAS: 6153-56-6)</td><td>Sigma-Aldrich</td><td>247537</td><td>Used for acid digestion of Mn oxides in microfluidic reactor.&amp;nbsp;</td></tr><tr><td>PEEK tubing (63.5-&amp;micro;m ID, 1/16-in OD)</td><td>IDEX</td><td>1560XL</td><td>Used to provide fluidic resistance in flow control system. Connected to flow sensor outlet.</td></tr><tr><td>Petri dish (Fisherbrand, 100-mm diameter)</td><td>Fisher Scientific</td><td>FB0875712</td><td>Used to pour agar plates and to store PDMS microfluidic channels/assembled microfluidic reactors.</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G, PDC-VPE, PDC-VCG</td><td>Used to bond PDMS microfluidic channels to glass cover slips. Basic setup includes cleaner, vacuum pump, and vacuum gauge.</td></tr><tr><td>Plastic cup</td><td>ULINE</td><td>S-22541</td><td>Used to mix PDMS elastomer base and curing agent.</td></tr><tr><td>Plastic knife</td><td>ULINE</td><td>S-7304B</td><td>Used to mix PDMS elastomer base and curing agent.</td></tr><tr><td>Plastic syringe (10-mL volume, Luer-Lock)</td><td>U.S. Plastic Corporation</td><td>87368</td><td>Used to recover acid solution from reactor and tubing. Pre-sterilized.</td></tr><tr><td>Plastic syringe (3-mL volume, Luer-Lock)</td><td>U.S. Plastic Corporation</td><td>87366</td><td>Used to saturate microfluidic reactor. Pre-sterilized.</td></tr><tr><td>Plate incubator (VWR)</td><td>Avantor</td><td>97025-630</td><td>Used to grow bacterial colonies on agar plates at a stable temperature.</td></tr><tr><td>Pneumatic polyurethane tubing (4-mm ID, 6-mm OD)</td><td>Legris-Pneumatics</td><td>1025U0601</td><td>Used to connect air compressor to flow controller.</td></tr><tr><td>Pneumatic polyether polyurethane tubing (2.5-mm ID, 4-mm OD)</td><td>Legris-Pneumatics</td><td>1025U04R08</td><td>Used to connect flow controller to manifold and fluid reservoir rack.&amp;nbsp;</td></tr><tr><td>Polydimethylsiloxane (PDMS, Dow Corning, Sylgard 184)</td><td>Ellsworth Adhesives</td><td>4019862</td><td>Used to fabricate microfluidic reactors via soft lithography. Kit contains elastomer base and curing agent.</td></tr><tr><td>Polyethylene glycol diacrylate (PEG-DA, M.W. 700, CAS: 26570-48-9)</td><td>Sigma-Aldrich</td><td>455008</td><td>Used to wet PTFE tubing when attaching to reactor inlet. Diluted to 1% in ultrapure water and filter sterilized.</td></tr><tr><td>Potassum phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, CAS: 7778-77-0)</td><td>Sigma-Aldrich</td><td>P0662</td><td>Used for experimental media and solutions.</td></tr><tr><td>PTFE membrane filter for bubble trap</td><td>PreciGenome</td><td>PG-BT-FLT-Q5</td><td>Compatible with microfluidic bubble trap.</td></tr><tr><td>PTFE tubing (200-&amp;micro;m ID, 1/16-in OD)</td><td>Darwin Microfluidics</td><td>BL-PTFE-1602-20</td><td>Used to connect bubble trap outlet to microfluidic reactor.&amp;nbsp;</td></tr><tr><td>PTFE tubing (500-&amp;micro;m ID, 1/16-in OD)</td><td>Darwin Microfluidics</td><td>BL-PTFE-1605-20</td><td>Used to connect distribution valve to flow sensor and PEEK tubing to bubble trap inlet.</td></tr><tr><td>PTFE tubing (Elveflow, 1/32-in ID, 1/16-in OD)</td><td>Darwin Microfluidics</td><td>LVF-KTU-15</td><td>Used to connect fluid reservoir rack to distribution valve.</td></tr><tr><td>Razor blade</td><td>ULINE</td><td>H-595B</td><td>Used to cut apart PDMS microfluidic channels.</td></tr><tr><td>Sample frame (steel, outer: 35 x 60 mm, inner: 25 x 50 mm, 1 mm thickness)</td><td>--</td><td>--</td><td>Designed and fabricated by the UC Davis Tech Foundry.&amp;nbsp;</td></tr><tr><td>Scalpel (Integra Miltex)</td><td>Fisher Scientific</td><td>12-460-456</td><td>Used to cut cured PDMS out of microfluidic mold.</td></tr><tr><td>Shaker incubator (Excella)</td><td>New Brunswick</td><td>E24</td><td>Used to grow bacterial cultures at a constant temperature and speed.</td></tr><tr><td>Sodium hydroxide (NaOH, 1 M, CAS: 1310-73-2)</td><td>Sigma-Aldrich</td><td>109137</td><td>Used to adjust pH of Fe-EDTA and HEPES stock solutions.</td></tr><tr><td>Sodium molybdate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O, CAS: 10102-40-6)</td><td>Sigma-Aldrich</td><td>331058</td><td>Used for experimental media and solutions.</td></tr><tr><td>Sodium phosphate dibasic dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O, Fisher Chemical, CAS: 10028-24-7)</td><td>Fisher Scientific</td><td>S472</td><td>Used for experimental media and solutions.</td></tr><tr><td>Syringe filter (Fisherbrand, PES, 0.22-&amp;micro;m)</td><td>Fisher Scientific</td><td>09-720-511</td><td>Used to filter sterilize stock solutions. Pre-sterilized.</td></tr><tr><td>Syringe pump (New Era)</td><td>SyringePump</td><td>NE-4002X</td><td>Used for reactor inoculation.</td></tr><tr><td>Tape</td><td>Fisher Scientific</td><td>15-953</td><td>Used to secure tube lid and tubing.</td></tr><tr><td>Tygon tubing (Masterflex Microbore, ND-100-80, 0.02-in ID, 0.06-in OD)</td><td>Avantor</td><td>MFLX06419-01</td><td>Used for reactor inoculation and as outlet tubing during experiment. Sterilized by autoclaving for 30 min.</td></tr><tr><td>Ultrapure water (Milli-Q)</td><td>Millipore Sigma</td><td>--</td><td>Used to prepare experimental media/solutions and clean flow control system. Sterilized by autoclaving for 30 min.</td></tr><tr><td>Ultraviolet-C (UVC) irradiation system</td><td>--</td><td>--</td><td>Designed and fabricated by the UC Davis Tech Foundry.&amp;nbsp;</td></tr><tr><td>Union fitting (Elveflow, 1/4-28 to 1/4-28)</td><td>Darwin Microfluidics</td><td>LVF-KFI-10</td><td>Used to connect PEEK and PTFE tubing.&amp;nbsp;</td></tr><tr><td>Vacuum desiccator (Bel Art)</td><td>Fisher Scientific</td><td>08-594-16C</td><td>Used for PDMS and reactor degassing.&amp;nbsp;</td></tr><tr><td>Vacuum pump for desiccator (Welch DryFast)</td><td>Fisher Scientific</td><td>1257156</td><td>Used to maintain vacuum pressure in desiccator.</td></tr><tr><td>Yeast extract (Fisher BioReagents)</td><td>Fisher Scientific&amp;nbsp;</td><td>BP1422</td><td>Used for experimental media.</td></tr><tr><td>Zinc sulfate heptahydrate (ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O, CAS: 7446-20-0)</td><td>Sigma-Aldrich</td><td>221376</td><td>Used for experimental media and solutions.</td></tr></tbody></table>

a microfluidic platform to investigate microbial precipitation of metal oxides in porous media

A Microfluidic Platform to Investigate Microbial Precipitation of Metal Oxides in Porous Media

Microorganisms commonly precipitate metal oxide nanoparticles in soils, sediments, and other environmentally relevant porous media. However, the formation and reactivity of manganese (Mn) and iron (Fe) oxides remain poorly characterized in physically complex environments. While laboratory systems can incorporate porous media as a substrate, they require destructive sampling and are not readily amenable to pore-scale analyses. The dynamic yet inaccessible nature of porous media, therefore, requires the ability to resolve microbially mediated mineral precipitation in situ and in real time. To close this gap, a microfluidic platform is developed to evaluate microbial precipitation of metal oxides in a model pore space. This platform is validated with the Mn-oxidizing bacterium Pseudomonas putida GB-1. Biofilm development and associated Mn oxide precipitation are visualized under continuous flow with time-lapse color brightfield microscopy. An image subtraction algorithm is then combined with endpoint mass measurements to estimate the rate of Mn oxide precipitation. This approach provides a tool to systematically test how microbial precipitation of metal oxides varies in response to changing physical and chemical conditions.

This protocol provides an experimental platform for spatiotemporally resolved imaging of biofilm formation and Mn biomineralization. Color brightfield images demonstrated that P. putida GB-1 biofilms were uniformly distributed throughout the microfluidic pore space at t = 18.5 h prior to the onset of Mn oxide precipitation (Figure 5A). Subsequently, Mn oxides preferentially accumulated on the inlet side of the pore space closest to the influent Mn(II) source (Figure 5B). The difference between the reference image and subsequent image timepoints revealed the isolated contribution of Mn oxide precipitates to the overall image intensity (Figure 5C) and enabled quantification of the observed reactor-scale gradient in mineral accumulation (Figure 5D). At the biofilm scale, this image subtraction approach captured the gradual precipitation of Mn oxides on biofilm surfaces and accumulation of minerals at the edges of biofilms in contact with pore fluid (Figure 6).
The protocol further provides a workflow to estimate the kinetics of Mn oxide precipitation from image subtraction and ICP-MS measurements. Triplicate microfluidic reactors contained 4.29 ± 0.22 µg Mn at the final experimental timepoint. Acid digestion of the reactor contents was validated previously using synchrotron-based measurements of Mn oxide mass and found to result in less than 18% error61. Therefore, the intensity-corrected matrices were rescaled to this final mass. The resulting estimated rate of Mn oxide precipitation was non-monotonic, increasing steeply between t = 19.5 h and t = 21 h, before peaking at 0.29 ± 0.04 µg·h-1 and decelerating gradually until t = 43.5 h (Figure 7). It should be noted that this rate calculation does not account for any nanoparticulate Mn oxides below the resolution of the optical microscope or for any oxide mass lost due to transport over the experiment duration, which is expected to be minimal. This approach therefore takes advantage of accessible microscopy techniques to semi-quantitatively evaluate temporal trends in biomineralization kinetics.
<img alt="Pore structure analysis via microscopy; pore image and corrected intensity data shown in graph form." class="xfigimg" src="/files/ftp_upload/71047/71047fig5v4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
Figure 5: Reactor-scale distribution of biofilms and Mn oxides. (A). Color brightfield image of P. putida GB-1 biofilms within the microfluidic pore space at the onset of the stationary phase (t = 18.5 h, tref). (B). Color brightfield image of Mn oxide precipitates on and around P. putida GB-1 biofilms at the final experimental timepoint (t = 43.5 h). (C). Intensity-corrected matrix of mineral accumulation at the final experimental timepoint (t = 43.5 h), with grains shown in gray and pores shown in white. Scale bar is 1 mm. (D). One-dimensional spatial distribution of total corrected intensity from the inlet (left) to the outlet (right) side of the pore space (t = 43.5 h). The solid line represents a 1-mm moving average along the pore space from inlet to outlet averaged over triplicate experiments, and the shading represents the standard deviation. <a href="https://www.jove.com/files/ftp_upload/71047/71047fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell growth time-lapse; microscopy images; contrast-enhanced analysis; experimental timeline." class="xfigimg" src="/files/ftp_upload/71047/71047fig6.jpg" data-altupdatedat="1787630408000" data-alt="Figure 6"> 
Figure 6: Biofilm-scale Mn oxide accumulation. (A). Select color brightfield images of representative P. putida GB-1 biofilms. Manganese oxides were detectable first as a color change from gray to brown on biofilm surfaces (shown at t = 20 h and t = 21 h) and later as an accumulation of dark brown precipitates at biofilm edges (shown at t = 33 h and t = 43 h). (B). Intensity-corrected matrices of mineral accumulation with grains in dark gray, pores in white, and underlying biofilms in light gray. Scale bar is 150 µm. <a href="https://www.jove.com/files/ftp_upload/71047/71047fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Precipitation rate vs. time graph showing data analysis for experimental results in μg·h⁻¹ over 45 hours." class="xfigimg" src="/files/ftp_upload/71047/71047fig7.jpg" data-altupdatedat="1787630408000" data-alt="Figure 7"> 
Figure 7: Estimation of Mn oxide precipitation rate over time. The rate (µg·h-1) was quantified by rescaling the total corrected intensity at each timepoint to the endpoint Mn mass measured with ICP-MS, then taking the derivative with respect to time. Markers represent the average of triplicate experiments, and the shading represents the standard deviation. <a href="https://www.jove.com/files/ftp_upload/71047/71047fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Step1_ImageRegistration.m.<a href="https://www.jove.com/files/ftp_upload/71047/Step1_ImageRegistration.m" target="_blank">Please click here to download this file.</a>
Supplementary File 2: Step2_ImageSubtraction.m.<a href="https://www.jove.com/files/ftp_upload/71047/Step2_ImageSubtraction.m" target="_blank">Please click here to download this file.</a>
Supplementary File 3: Step3_Figures.m.<a href="https://www.jove.com/files/ftp_upload/71047/Step3_Figures.m" target="_blank">Please click here to download this file.</a>

This protocol presents a microfluidic system for studying the microbial precipitation of manganese oxide minerals. Biomineral formation is measured in situ with optical microscopy and image analysis. This approach can be adapted to investigate the precipitation of other distinctively colored biogenic minerals in porous environments.

Microorganisms commonly precipitate metal oxide nanoparticles in soils, sediments, and other environmentally relevant porous media. However, the formation ...

We've developed a microfluidic platform to study the dynamic precipitation of metal oxides by bacteria in a porous environment. This protocol enables in situ and real-time measurement of mineral precipitation, unlike conventional methods such as batch or column systems. To begin, add 10 milliliters of LB medium to a 50 milliliter conical tube.Inoculate the medium using a single colony of Pseudomonas putida GB-1, obtained from a plate that was streaked 20 hours earlier. Incubate the LB pre-culture in a shaker incubator at 30 degrees Celsius and 180 revolutions per minute for 16 hours to reach the stationary phase. Then, pellet one milliliter of LB pre-culture by centrifugation at 8, 000 G for one minute.After washing the pellet three times, re-suspend the final pellet in one milliliter of salt solution without manganese. Then, dilute the washed cell suspension fivefold in salt solution without manganese in a cuvette, and measure the optical density at 600 nanometers using a cell density meter. Inoculate 20 milliliters of growth medium without manganese with the washed cell suspension to an optical density of 0.01 in a 50 milliliter Erlenmeyer flask.Incubate at 30 degrees Celsius and 180 revolutions per minute for six hours to reach mid-exponential phase. Before beginning the experiment, verify that the microscope incubator is maintained at 30 degrees Celsius and prime the flow control system. Pour 10 milliliters of growth medium and salt solution with manganese into a 15 milliliter conical tube, and connect both tubes to the fluid reservoir rack.Degas the prepared microfluidic reactor in a vacuum desiccator for 20 minutes. Draw one to two milliliters of salt solution with manganese into a three milliliter plastic syringe. Attach a blunt tip needle and 20 centimeters of 0.02 inch inner diameter Tygon tubing, press the syringe plunger until the tubing is fully saturated.After de-gassing, use angled tweezers to attach the tubing to the reactor outlet hole. Manually saturate the reactor with the syringe until a droplet forms at the reactor inlet hole. Next, attach 20 centimeters of 0.02 inch inner diameter Tygon tubing to the reactor inlet hole.Continue dispensing salt solution with manganese into the reactor until all air bubbles are removed. To prepare the reactor inoculum, remove the mid-exponential culture from the shaker incubator six hours after inoculation. Dilute fourfold in salt solution without manganese in a cuvette.And measure the optical density. Then, dilute the mid-exponential culture in two milliliters of salt solution with manganese to an initial optical density of 0.005 in a two milliliter microcentrifuge tube. Invert the tube several times to mix.Next, place the reactor inlet tubing in the microcentrifuge tube, ensuring that a droplet of fluid contacts the inoculum to prevent air introduction in the tubing. Partially close the microcentrifuge tube and secure the lid with tape to prevent contamination. Next, withdraw four to five milliliters of salt solution with manganese into a five milliliter glass gas tight syringe to wet the barrel.Dispense the solution until 0.5 milliliters remain in the syringe and load the syringe into a syringe pump. After disconnecting the plastic syringe from the needle, attach the needle to the glass syringe while avoiding trapping air in the needle hub. To inoculate the reactor, withdraw the inoculum into the reactor for 20 minutes at a flow rate of 8.33 microliters per minute.Turn off the syringe pump following inoculation and allow the bacteria to settle and attach for one hour with no flow. Then, remove the glass syringe from the syringe pump while leaving it connected to the outlet tubing. Next, move the reactor and syringe from the bench top to the microscope incubator.On the microscope stage, remove the inlet tubing from the reactor and push gently on the syringe plunger to create a droplet at the reactor inlet hole. Gently bend the polytetrafluoroethylene, or PTFE tubing, from the flow control system at a right angle. Dip the end of the PTFE tubing into a 1%polyethylene glycol diacrylate solution to wet the exterior of the hydrophobic tubing.Ensure a droplet of growth medium with manganese is present at the end of the PTFE tubing, and attach the tubing to the reactor inlet hole. After disconnecting the syringe, place the outlet tubing into a five milliliter microcentrifuge tube to collect the reactor effluent. Set the flow rate to 1.33 microliters per minute in the flow control system software.Program the system to supply growth medium with manganese for 15 hours to promote biofilm formation, followed by salt solution with manganese for 28.5 hours to induce manganese biomineralization. Next, place a sample frame underneath the reactor for stability. Rigidly mount the frame and reactor in the microscope stage insert to ensure full immobilization.Affix the PTFE tubing to the stage insert with a piece of tape to prevent its detachment from the reactor inlet hole. Align the ultraviolet C-arms two millimeters from the pore space edge on the outlet side and three millimeters on the inlet side to minimize UV light scattering and biofilm damage. Set up a program for reactor irradiation to treat the reactor inlet and outlet regions for five minutes at the experiment onset, then treat only the outlet region for five minutes, 17.5 hours after the experiment onset to inactivate remaining biofilms.Manually adjust the Kohler alignment, following the manufacturer's instructions to ensure even illumination. To prevent quilting artifacts, select Acquire and open the shading correction panel, then capture a shading image and set it as the current shading image. Select ND Acquisition and choose XY to set the center coordinate of the stitched image.Next, choose Large Image and set up the stitched image scan area as five by four fields of view, and apply a 1%overlap. Select Lambda and set up the acquisition of Color Brightfield images using the 4X objective. Select time and set the imaging frequency with intervals of one hour for 15 hours and 0.5 hours for 28.5 hours, then run the time lapse program.Brightfield images demonstrated that Pseudomonas putida GB-1 biofilms were uniformly distributed throughout the microfluidic pore space at 18.5 hours before the onset of manganese oxide precipitation. Subsequently, manganese oxides preferentially accumulated on the inlet side of the pore space closest to the influent manganese source. The difference between the reference image and subsequent image time points revealed the contribution of manganese oxide precipitates to the overall image intensity.Quantification of the observed mineral accumulation gradient was enabled at the reactor scale. At the biofilm scale, the image subtraction approach captured the gradual precipitation of manganese oxides on biofilm surfaces and accumulation of minerals at the edges of biofilms in contact with pore fluid. The resulting estimated rate of manganese oxide precipitation was non-monotonic, increasing steeply between 19.5 hours and 21 hours, peaking at 0.29 micrograms per hour and decelerating gradually until 43.5 hours.This protocol allows researchers to study the timing and location of mineral precipitation in a porous medium. It's critical to prevent biofilm growth in the reactor inlet region. This ensures a constant boundary condition and restricts precipitation to the pore space.Future studies can explore coupled microbe mineral fluid interactions under conditions relevant for environmental and engineering applications.

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JoVE Journal

Environment

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University of California, Davis.  This protocol presents a microfluidic system for studying the microbial precipitation of manganese oxide minerals. Biomineral formation is measured in situ with optical microscopy and image analysis. This approach can be adapted to investigate the precipitation of other distinctively colored biogenic minerals in porous environments.

A Microfluidic Platform to Investigate Microbial Precipitation of Metal Oxides in Porous Media

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