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Applied and Environmental Microbiology, December 2005, p. 8721-8728, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8721-8728.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Application of Stereolithographic Custom Models for Studying the Impact of Biofilms and Mineral Precipitation on Fluid Flow

D. L. Stoner,^1* S. M. Watson,² R. D. Stedtfeld,¹ P. Meakin,³ L. K. Griffel,¹ T. L. Tyler,^1, L. M. Pegram,¹ J. M. Barnes,¹ and V. A. Deason²

Biological Sciences Department,¹ Physics Department,² Center for Advanced Modeling and Simulation, Idaho National Laboratory, Idaho Falls, Idaho³

Received 17 September 2003/ Accepted 1 August 2005

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Here we introduce the use of transparent experimental models fabricated by stereolithography for studying the impacts of biomass accumulation, minerals precipitation, and physical configuration of flow paths on liquid flow in fracture apertures. The internal configuration of the models ranged in complexity from simple geometric shapes to those that incorporate replicated surfaces of natural fractures and computationally derived fracture surfaces. High-resolution digital time-lapse imaging was employed to qualitatively observe the migration of colloidal and soluble dyes through the flow models. In this study, a Sphingomonas sp. and Sporosarcina (Bacillus) pasteurii influenced the fluid dynamics by physically altering flow paths. Microbial colonization and calcite deposition enhanced the stagnant regions adjacent to solid boundaries. Microbial growth and calcite precipitation occurred to a greater extent in areas behind the fabricated obstacles and less in high-velocity orifices.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Understanding the mechanisms by which microorganisms affect fluid flow in groundwater and subsurface environments is significant because of the importance of natural geohydrological processes (41), understanding the transport of microorganisms in the subsurface (12, 23), mitigating contaminant transport in the subsurface (12), and utilizing microorganisms for in situ processes such as mineral dissolution and recovery (3, 4, 7), enhanced oil recovery (15, 48), and contaminant remediation (11, 13, 39, 40). Microorganisms are only one of the interacting physical, chemical, and biological variables that can affect the behavior of fluid flow in subsurface systems. One of the greatest challenges in understanding and computationally simulating subsurface hydrogeological processes is the influence of microbial exudates and biofilm accumulation on liquid flow in fractures and porous geomatrices.

Experimental models have been utilized extensively to study multiphase flow phenomena (16, 19, 26, 47) in porous media and their effects on colloids (34, 47) and to study the mechanisms responsible for biomass plugging in porous media (17, 36, 37). Models are frequently used to examine the impact of biofilm formation on surface chemistry, materials degradation, fluid flow, and surface corrosion (25, 42). The impacts of surface topography, substratum composition, and fluid properties on bacterial adhesion, colonization, detachment, activity, and interactions among community members (24, 32, 35, 43) have also been examined using experimental models.

In many studies, experimental models with relatively simple geometries were fabricated by machining or glass etching (6, 32). However, these approaches cannot easily be applied to the fabrication of complex three-dimensional (3-D) models designed to examine the impact of microbial processes on fluid flow in subsurface fractures. Machining of complex cavities that bear a resemblance to natural fractures and porous media is difficult. Because of limited machine tolerances, it is often not possible to obtain reproducible results from a number of models fabricated with the same nominal geometry. Etching through a mask of inert material can be used to construct complex quasi-two-dimensional channel networks (6), but the construction of truly three-dimensional structures requires the use of a series of masks, and the variety of three-dimensional structures that can be fabricated in this manner is very limited. For both machining and etching, enclosed models must be fabricated from two pieces of material, which must be sealed to form an enclosed cavity.

One approach for overcoming the difficulties associated with the fabrication of models with complex internal geometries is to use stereolithography. In stereolithography, a three-dimensional solid structure is manufactured layer by layer, using a laser to convert a liquid or powder into a solid (1, 31). At each stage in the process, the laser polymerizes a thin layer of monomer containing a photoinitiator of the polymerization reaction or sinters a thin layer of powder. The newly formed layer is then again covered with a thin layer of liquid or powder, and the process is repeated many times until a solid body with the required shape is formed. Here we report the application of stereolithographic models and the integration of three-dimensional lattice Boltzmann simulations to microbiological studies. In support of this objective, models with a variety of internal configurations were designed, fabricated, and used to qualitatively examine the impact of biomass accumulation and microbially induced calcite precipitation on fluid flow.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cultures and cultivation.
Two cultures were utilized: a Sphingomonas sp. and Sporosarcina (Bacillus) pasteurii (ATCC 11859). The Sphingomonas sp. strain, designated R02-4, was isolated from a groundwater sample collected from a well near Mud Lake, Idaho, in June 2001. The culture was isolated by the spread plate method on R2A heterotrophic total bacterial counting medium (Difco medium; Becton, Dickinson and Company, Franklin Lakes, NJ) after incubation for 3 weeks at room temperature. The Sphingomonas strain was identified by fatty acid methyl ester analysis and by partial 16S rRNA gene sequencing and alignment with sequences deposited in GenBank (National Center for Biotechnology Information) by using the BLAST algorithm. Purified stocks of R02-4 were preserved at –20°C in LB broth containing 15% glycerol. Frozen cultures of R02-4 were revived in Trypticase soy broth (Difco) containing 2% urea (Acros Organics/Fisher Chemicals, Fairlawn, NJ). For the calcite precipitation experiments, the Sphingomonas strain was cultivated at 23°C in medium containing 5 mg KNO₃, 50 mg MgSO₄, 4 mg NaNO₃, 92 mg NaHCO₃, 6 mg KHCO₃, 1.8 g urea (Bio-Rad Laboratories, Hercules, CA), 0.5 g KH₂PO₄, 0.8 g Na₂HPO₄, and 1.0 g Karo syrup (Bestfoods, Englewood Cliffs, NJ) per liter of deionized water. For calcite precipitation, the medium was amended with 3 g CaCl_2. The medium was adjusted to a pH of 7 and sterilized by filtration (0.22-µm-pore-size filter; polyethersulfone; Corning Inc., Corning, NY). Sporosarcina pasteurii was cultivated at 23°C in the medium described by Stocks-Fisher et al. (38), containing 3 g nutrient broth (Difco), 20 g urea, 10 g NH₄Cl, and 2.12 g NaHCO₃ per liter of deionized water. The nominal pH of the medium was 7.8 to 8.0. Unless otherwise noted, reagents were acquired from Fisher Chemicals (Fairlawn, NJ).

Design and fabrication approach.
Stereolithographic prototyping permits the repeatable fabrication of hollowed, three-dimensional solids in a range of dimensions suitable for a variety of measuring systems (1). A standard model format was developed to support a variety of internal configurations. Once a design for a model was decided upon, three-dimensional design programs were used to create a fully rendered structural model (31). Final model designs were exported in the STereoLithography (*.stl) file format, which is compatible with commercial stereolithographic fabrication systems. Generation of the *.stl files converted the design into a triangulated representation of the boundaries, which is referred to as a mesh. The *.stl file format contains a description of the fracture aperture or open model and surrounding matrix, which were rendered as a collection of polygons. The greater the number of polygons, the more refined the models. However, files with a large number of polygons are computationally more intensive. Thus, the number of polygons is chosen such that the resulting faceting of the model is acceptable for the intended use.

Design of stylized models.
Stylized models (Fig. 1) were designed using I-deas version 9 (EDS, Inc., Plano, TX) and Argon version 5 (Ashlar Inc., Austin, TX). The computer-aided design (CAD) software packages employed nonuniform rational B-spline geometry, which is a mathematical representation that accurately characterizes complex 3-D surfaces and solids. The final design was exported in *.stl file format for fabrication.

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FIG. 1. Design and fabrication of stylized flow models. (A) CAD drawing for an open model with a triangle obstacle; (B) the design "meshing" step; (C) completed model with tapped threads to accept the fittings; (D) CAD drawing for an enclosed model with two layers of interconnected voids; (E) fabricated bilayer model with tapped threads; (F) side view of the bilayer model.

Design of a replicated natural surface model.
Models with a replicated surface of a limestone fracture were created by scanning the face of a broken limestone block and converting the vectorized scan data into a digital surface with a bounded box, which was used to create the model design for fabrication (Fig. 2). The block surface was scanned using a 3-D scanning system (the unique prototype system and associated software system were developed in the laboratory of W. R. Lloyd, Idaho National Laboratory) that allowed for submicrometer height (elevation) resolution with an elevation repeatability of 3 µm (20). The horizontal spatial resolution in the x,y plane was approximately 20 µm, which is roughly the spot size of the laser used in the scanning system, and the horizontal spatial repeatability is 5 µm. Vector (x,y,z) data were collected every 100 µm along the spatial plane. To generate a manageable file size for further manipulation, the data density was reduced to 16% (one-third of the original data points along a 40-mm axis and one-half of the original data points along a 25-mm axis). Finally, the average slope of the scanned surface was minimized by digitally tilting the fracture surface to decrease the z component by approximately 50%, using MATLAB (version 5.3.1.29215a [R11.1]; The MathWorks, Inc., Natick, MA). Manipulated vector data from the fracture surface were converted into "true solid" or digital surface format by using the R2V software package (Able Software Corp., Lexington, MA). The three-dimensional grid of the surface was exported as a triangle-based polygon surface bounded by a square polygon and incorporated into the basic model design and exported as a *.stl file for fabrication.

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FIG. 2. Design and fabrication of a flow models containing a replicated fracture surface. (A) Limestone block that was broken on one edge to form a fracture surface; (B) topographical scan of the fracture surface; (C) solid static rendering of fracture with minimized elevation in *.stl format; (D) meshed design that was imported into the model design; (E) transparent CAD drawing of completed flow model design; (F) fabricated natural fracture model.

Design of synthetic fracture models.
A model of a synthetic fracture was designed in a manner similar to that for a natural fracture (Fig. 3). The computer generation of a fracture aperture was based on the idea that the fracture of brittle materials forms rough fracture surfaces that have a self-affine fractal geometry (2, 18, 21, 22, 27, 33). The synthetic fracture generated for the flow model consisted of the gap between the two mathematically derived surfaces (5). A self-affine fractal surface consisting of a 128-by-128 array of heights was constructed using a random midpoint displacement algorithm, with successive random additions to ensure statistical homogeneity of the rough surface (46). Periodic boundary conditions were used, and one surface was displaced laterally by 12 units in the x direction and 20 units in the y direction. A Hurst exponent of 0.7, corresponding to a fractal dimension of 2.3 was used. The digital fractal pattern was converted into the same digital surface format as described above by using the R2V software package and imported into the model design, and the final model was exported as a *.stl file for fabrication.

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FIG. 3. Design and fabrication of a flow model containing a fractal-based surrogate fracture surface. (A) Mathematically derived fractal surface; (B) meshed design of a single fracture surface; (C) fracture aperture that was imported into the model design; (D) completed design of the model with the enclosed fracture aperture; (E) fabricated flow model with enclosed fractal fracture; (F) side view showing fractal fracture plane.

Model fabrication.
The open-diamond models with the triangle obstacle were fabricated by 3-D Systems, Inc. (Valencia, CA) in their demonstration facility, using AccuGen 100 epoxy resin and a Viper fabrication system. Other models were fabricated by Fine-Line Prototyping with colorless, transparent, polymer WaterShed 11120 (DSM Somos, New Castle, DE), using a Viper si2 SLA fabrication system (3-D Systems, Valencia, CA). After fabrication, the models were washed with solvent to remove excess liquid monomer. The external surfaces of the models, except for the surface of the replicated fracture, were polished and clear coated to improve clarity and visual resolution. Models were drilled and tapped in our laboratory to accept threaded fittings for the liquid flow system. The single-sided, open-cavity models were fitted with no. 1 glass coverslips held in place by machined aluminum plates and silicone rubber gaskets.

Liquid flow control and continuous cultivation in models.
A syringe pump (model 33 dual syringe pump; Harvard Apparatus, Holliston, MA) was used to control flow into the micromodels. The syringe pump was interfaced to a computer via an RS-232 port and controlled by a software module written in the LabView (National Instruments, Inc., Austin TX) graphical programming language.

The models were sterilized by filling the models with a 10% household bleach solution, sealing the ports with end caps, and letting them sit for approximately half an hour. Afterwards, models were flushed thoroughly with 60 ml of filter-sterilized deionized water. For inoculation with Sporosarcina pasteurii, experimental models were filled with a culture, the ports were capped, and the models were incubated for 2 weeks at 37°C. During incubation, the air in the headspace and the liquid medium were replaced weekly. For calcite deposition experiments, the models were connected via polyether ether ketone (PEEK) tubing to sterile 60-ml syringes, and Stock-Fisher medium (38) amended with 1.4 g CaCl₂/liter was introduced into the model at a rate of 40 µl/min. Models were left on the bench top and incubated at room temperature (23°C). Dye tests were initiated 6 days later, when visible amounts of calcite had accumulated within the model. The Sphingomonas sp. culture was handled in a similar manner except that cultivation and calcite precipitation occurred at room temperature.

System imaging.
Automated time-lapse photography (Fig. 4) was used to monitor the progression of 5 µl soluble (food coloring) or colloidal (India ink) dye through the model. Dyes were manually injected after the data acquisition was initiated. Digital, single-lens reflex cameras (Kodak DSC 760; Eastman-Kodak Co., Rochester, NY) equipped with zoom-micro lenses (Nikkor ED 70 to 180 mm; Nikon, Inc., Melville, NY) and operated in manual mode were used to image models. A single camera was used for imaging single-sided models, whereas two cameras were used for simultaneous imaging of the double-sided models with a bilayer internal structure. Camera lens configurations allowed an area of approximately 40 mm by 25 mm to be imaged at a distance of 165 mm from the lens to the model. The cameras utilized 6-million-pixel imagers (2:3 aspect ratio) and were operated at a virtual film speed of 80 ISO. Camera control and file manipulation were accomplished via the computer, using proprietary software packages (Kodak Camera Manager and Kodak Digital Camera System Photodesk). A virtual intervalometer in the Camera Manager software was used for the timing of image acquisition (one picture taken every 10 to 15 seconds) as the dyes propagated through the model. Image data were acquired in Digital Camera System format and were converted to Joint Photographic Experts Group (JPEG) format, assembled as a JPEG video, and then saved in an Audio Video Interleaved (AVI) movie format using four JPEG frames per second.

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FIG. 4. Time-lapse imaging system. Dual cameras enable the simultaneous imaging of both sides of a bilayer model. Dashed lines represent the bidirectional communication interfaces between the computers and the equipment. Solid lines represent the tubing.

A custom-fabricated frame within a cabinet was used to support the model and cameras and to eliminate stray light. A custom-machined aluminum block with setscrews was used to hold the flow model securely, align the model for imaging, and provide mechanical strain relief for the fluid lines affixed to the inlet and outlet ports of the flow model. Continuous illumination (400 to 650 nm) for this holder assembly was provided by thin-film (0.3-mm thickness) electroluminescent material, which was fixed to the inner surface of the holder. The electroluminescent film was powered by three magnetic resonating transformer-type inverters (9 VDC to 40 to 80 Vrms at frequencies of 400 to 800 Hz and current densities of 0.33 to 1.5 mA/in²) to provide five stepped light levels.

Simulation.
The fluid flow in enclosed cavities was computationally simulated using three-dimensional lattice Boltzmann simulations (29, 30, 44). The lattice Boltzmann model is based on the ideas that the flow of all simple fluids at low velocities can be described by the Navier Stokes equation and that any system of particles in which the interparticle interactions are isotropic and conserve momentum will generate hydrodynamic behavior on sufficiently large length scales. A simple particle-tracking approach was used to simulate the transport of the dye. In most of the simulations, the diffusion of the dye was not included, because advection was dominant in the experiments that were carried out under large-Peclet-number conditions. Some simulations were carried out using the lattice Boltzmann approach of Flekkoy (8, 9), which allowed the effects of diffusion to be simulated in a manner which is fully consistent with the flow simulation after the flow velocity field has been calculated. We used the D3Q19 model, which is based on a simple cubic lattice with 19 velocity vectors, including a zero-velocity vector and six degenerate vectors (28). The particles travel with relative velocities of 0, 1, and the square root of 2. Some simulations were also carried out using the D3Q15 model (28).

The simulations were carried out with constant-pressure (constant-particle-density) boundary conditions at the inlet and outlet of the model. Nonslip boundary conditions were represented using "bounce-back" boundary conditions (30). Flow was induced by maintaining a small density difference, (/ = 0.0001, where is the fluid density), between the entrance and exit of the model, and the simulation was run until a steady state was reached. After the three-dimensional flow field was calculated, the advection of the dye was simulated by tracking the motion of a swarm of tracer particles, which were released at the entrance of the flow.

Contact angle measurement.
Contact angles between deionized water and solid surfaces were determined using the sessile drop method with a contact angle and surface tension instrument (FTÅ200; First Ten Angstroms, Portsmouth VA). Results are reported as the average and standard deviation of three separate measurements.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Stereolithography allows the fabrication of models with complex internal configurations that are not possible with other fabrication techniques. Models can be designed and fabricated in a few days, and because there are a number of fabrication companies, there is no need for an individual investigator to invest in expensive fabrication equipment. Most high-end CAD programs support 3-D modeling and can output the *.stl file format used by the stereolithographic fabrication community. In this study, flow models ranged in design complexity from simple geometric shapes to those that incorporated replicated surfaces of natural fractures. Of the two types of polymers that were used to create the models, the Watershed polymer was more durable. The models fabricated with AccuGen polymer degraded with use. For example, when flow models were subjected to continuous-flow conditions for periods of up to a month, the tapped threads began to leak. The Watershed and AccuGen polymers were relatively hydrophobic. The Watershed polymer had a contact angle of 81.6 ± 1.3°, which was greater than that of the epoxy models (57 to 65°) used by Geller et al. (11). The AccuGen polymer had a contact angle of 45.3 ± 9.9°. In comparison to the polymers, a glass microscope slide (Fisher brand, precleaned) had a water contact angle of 9.8 ± 1.5°.

The use of stereolithographically fabricated models to examine the impact of microbial growth and mineral precipitation was demonstrated using a microbial-mineral system in which calcite was precipitated from the medium. Sphingomonas spp. and Sporosarcina pasteurii mediate the hydrolysis of urea, which results in the formation of carbonate and ammonium ions and an increase in alkalinity (10, 14, 45). When they are grown on media containing urea and calcium ions, the change in geochemical conditions resulting from urea hydrolysis results in the precipitation of calcium carbonate.

Stylized complex models.
Calcite was deposited within the flow models via urea hydrolysis by Sphingomonas sp. strain R02-4. For the dye test at a flow rate of 200 µl/min, the soluble dye followed a relatively direct path to the outlet, rushed over fabricated obstacles, and pooled behind the large obstacle in the middle (Fig. 5B). Calcite was deposited unevenly throughout the flow model, with more material depositing in the lower-flow-velocity regions in the corners and along the far edges of the flow model (Fig. 5C). The calcite is visible as a white material in the image of this flow model and for other models described below. Flow paths (right to left) over fabricated obstacles, as delineated by the soluble dye, decreased (Fig. 5D) relative to that of the control flow model. The flow path from the inlet to the outlet was less direct. By the time the leading edge of the soluble dye reached the outlet, the dye dispersed throughout the flow model to a greater extent than it did in the control without calcite precipitation. Compared to the case for the control flow model, the leading edge of the dye took an extra 10 s to travel across the flow model with calcite precipitate. There was also a noticeable lack of dye of in the stagnant region, which was located downstream of the large obstacle in the middle of the void.

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FIG. 5. Impact of calcite precipitation mediated by Sphingomonas sp. strain R02-4 on liquid flow through a stylized flow model. (A) Top view of flow model; (B) image of the flow model with soluble dye delineating the flow paths around the obstacles, taken 50 seconds after the leading edge of the dye entered the flow model; (C) model with white calcite precipitate; (D) dye path in flow model with calcite precipitate, with image taken 60 s after the leading edge of the dye entered the model. Flow tests were run at 200 µl/min, and flow direction is right to left.

The bilayer model was developed to simulate a complex fracture network with interconnecting channels and voids. Time-lapse photography with the dual camera system allowed the simultaneous visualization of the upper and lower layers (Fig. 6). The dye was diverted around air bubbles trapped within the model. In the colonized model with the calcite precipitate, the dye traveled through the model in less time (Fig. 6C and D) than in the abiotic control model (Fig. 6A and B). This suggested that there was a decreased liquid volume due to the accumulation of biomass and calcite and the presence of gas bubbles. In the figures presented, the dye tracked into the lower level of the model. However, in other tests dye flowed into the upper level, indicating potential variability in flow paths.

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FIG. 6. Impact of calcite precipitation and microbial colonization by Sporosarcina pasteurii on liquid flow through a bilayer flow model. (A) Image of upper layer of the uncolonized model; (B) image of lower layer of the uncolonized bilayer; (C) image of upper layer of the colonized model; (D) image of lower layer of the colonized model. The flow rate during the dye tests was 80 µl/min, and flow direction is right to left. Images were taken 2 min after the soluble dye first entered the flow model.

Replicated natural fracture model.
Well-characterized models that are representative of natural topographical features will enable the interactions among natural topological features, microbial colonization and mineral deposition patterns, and hydrodynamics to be examined. The natural fracture model (Fig. 7A) was colonized with Sporosarcina pasteurii, which, under continuous-flow conditions, induced the formation of calcite precipitation via urea hydrolysis. The image in Fig. 7B shows the colloidal dye path in the control model as the liquid flowed at 200 µl/min from the right to the left. After colonization and calcite precipitation, there was a vein-like appearance of the dye path (Fig. 7D), which was not observed in the abiotic control flow model. Furthermore, the dye traced a narrower flow channel during the later periods of the dye test.

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FIG. 7. Impact of calcite precipitation mediated by Sporosarcina pasteurii on liquid flow through the natural fracture flow model. (A) Top view of the entire model; (B) close-up image of the control model; (C) image of the path taken by the colloidal dye as the dye first reached the effluent ports of the flow model; (D) image of dye path through flow model with precipitate as the dye first reached the effluent port. Flow tests were run at 200 µl/min, and flow direction is right to left.

Synthetic fracture model.
Numerically generated "fracture surfaces" that were based on fractal mathematics were used to develop a fracture aperture which was entirely enclosed within the model (Fig. 3). Under natural conditions in the subsurface, the displacement of one fracture surface with respect to its partner may crush and fracture asperities, and some part of the fracture aperture may be occluded. A model was fabricated in one piece by using stereolithography in which a fracture was represented by partially overlapping self-affine surfaces, where the overlapping regions represent occlusions.

For the fractal fracture experimental model, the colloidal dye traveled through the fracture in two distinct paths (Fig. 8). After colonization and calcite precipitation by Sporosarcina pasteurii, the images of the flow paths were less distinct due to the decreased clarity of the model. Nevertheless, in the colonized-flow model, the colloidal dye diffused to a greater extent as it entered the fracture aperture and traveled to the effluent channel.

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FIG. 8. Impact of calcite precipitation mediated by Sporosarcina pasteurii on liquid flow through the fractal fracture flow model. (A) Top view of flow model; (B) image of colloidal dye path taken as the dye first reached the effluent ports of the flow model about 5 1/2 min after the dye was injected; (C) image of colloidal dye path taken later in the flow test; (D) top view of flow model with white calcite precipitate; (E) image of dye path through flow model with precipitate as the dye first reached the effluent port; (F) image of dye path through the flow model with precipitate, taken 15 min later. Dye tests were run at a flow rate of 200 µl/min, and flow direction is right to left.

Integration of experimental and computational activities.
To demonstrate integration of computational and experimental approaches, the dye tracer experiments were simulated for one of the flow models (Fig. 9). While there was good qualitative agreement between our dye tracer experiments and computer simulations, there were some quantitative differences. We attribute these differences to the way in which the tracer dyes were introduced into the model. In the simulations, the tracer particles were introduced uniformly across the inlet to the model. In the experiments, the dye is injected into the fluid before it reaches the model. Because fluid flows more rapidly in the center of the inlet tube than it does near the walls, the dye entering the model will not be uniformly distributed across the inlet. The incorporation of diffusion in the simulation may decrease the observed discrepancies between the simulation and experiment.

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FIG. 9. Comparison of experimental and computational results for a stylized flow model with a triangle obstacle. Time series images are ordered from top to bottom for (A) time-lapse images of the dye tracer test and (B) computational results. The red circles in the computational model are the inlet and outlet ports. Flow tests were run at 200 µl/min, and flow direction is left to right.

Experimental applications.
The flow models described here were developed to assess the interactive effects of physical configuration of a fracture, saturated liquid flow, and deposition of calcite. The application of transparent flow models will enable the study of the impact of aperture or porous medium configuration on microbial colonization and calcite deposition. Replicate models will allow the examination of strain-specific calcium carbonate crystallization patterns (14) on hydrodynamics. We are currently extending the technique to develop flow models comprised of interconnected vesicles such as would be found in a basalt geomatrix and those comprised of a simulated granular medium where flow paths would occur in the interstitial spaces. Flow models fabricated in this manner will enable the investigation of multiple models with identical particle or vesicle distributions, which would enable experimental replication or the comparison of effects of species or treatment variations on hydrodynamics.

To address issues relevant to biogeochemistry requires the development of flow models with geochemically relevant surfaces. Coating the surfaces of open models by sputter coating with metallic species, vapor deposition, or chemical/vapor deposition is one approach for achieving geochemical models. Another approach, one that we are currently investigating, is to fabricate the models from crushed geomatrices such as powdered basalt. One method for achieving such "natural" models involves the fabrication of thin sheets of micrometer-sized mineral particles bonded by a polymer matrix. A laser cuts this material into a series of horizontal slices of the 3-D model. These are stacked, heated to remove the organic components, and then fired to sinter the particles and fuse the layers into a cohesive form that conforms to the original model. Shrinkage is high, but it can be largely compensated for by proper design of the model. Complex internal and external shapes can be fabricated in this manner by using any of a variety of ceramic or natural materials. For completely opaque models fabricated out of crushed geological materials or from ceramic, alternative methods of imaging such as nuclear magnetic resonance imaging or high-resolution X-ray synchrotron tomography would be required.

Conclusion.
We have shown that stereolithography can be used to fabricate a variety of models with complex geometries that resemble natural fracture apertures and fracture networks. The ability to fabricate models with the required geometry from transparent materials will facilitate the study of the interactive effects of aperture geometry, biomass accumulation, and liquid flow. In follow-up work, we plan to investigate the use of surface treatments to impose different surface geochemistries and alter wetting properties, to investigate the fabrication of flow models from natural materials such as powdered rock, and to develop methods to quantitatively assess changes in flow paths and dye densities.

 
This work was supported through the INL Laboratory Directed Research and Development (LDRD) Program under DOE Idaho Operations Office contract DE-AC07-05ID14157.

 
* Corresponding author. Present address: Department of Chemistry, University of Idaho at Idaho Falls, 1776 Science Center Drive, Suite 306, Idaho Falls, ID 83402. Phone: (208) 282-7907. Fax: (208) 282-7950. E-mail: stondl{at}if.uidaho.edu.

Present address: Biology Department, Idaho State University, Pocatello, ID 83209.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, December 2005, p. 8721-8728, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8721-8728.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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