ࡱ > 4 '`
f bjbj$$ F F 5 5 5 8 5 j6 d | 4 6 D 9 49 49 49 c> . > > { { { { { { { $ C~ h 6{ Q gJ = c> gJ gJ 6{ 49 49 w { @ a a a gJ 49 49 Dv a gJ { a a jr s 49 6 P&$l=S 5 ] s $ Xu { H | 2s / ` / , s / s \ > oB a E $ ?G ( > > > 6{ 6{ _a d > > > | gJ gJ gJ gJ D # # Development and usage of an etched transparent flowcellfor visualisation of biofilm induced heterogeneities in porous media using Particle Image Velocimetry.
Geert M. van der Kraan , Floris Buijzen , Maarten de Ridder , Barbara Thuss , Mario Laros , Christian Poelma, Bart P. Lomans , Gerard Muyzer, Mark C.M. van Loosdrecht and Johannes Bruining, Development and usage of an etched transparent flowcell for visualisation of biofilm induced heterogeneities in porous media using Particle Image Velocimetry, E-poster: SPE 134895, SPE Annual Technical Conference and Exhibition, Florence, Florence Italy Sept. 20-22
Geert M. van der Kraana,f*, Floris Buijzena, Maarten de Riddera, Barbara Thussb, Mario Larosc, Christian Poelmad, Bart P. Lomanse, Gerard Muyzera, Mark C.M. van Loosdrechta and Johannes Bruiningf
aDepartment of Biotechnology, Delft University of Technology, Delft, 2628 BC, The Netherlands
bOTM Consulting Ltd, 44 Quarry Street, Guildford, GU1 3XQ, United Kingdom
cDelft Institute of Microelectronics and Submicron Technology, Delft, 2628 CD, The Netherlands
dDepartment of Mechanical Engineering, Delft University of Technology, Delft, 2628CD, The Netherlands
eShell International Exploration & Production, Rijswijk, 2288 GS, The Netherlands
fDepartment of Geotechnology, Delft University of Technology, Delft, 2628 CN, The Netherlands
Correspondence: Geert M. van der Kraan, Department of Biotechnology, Delft University of Technology, Julianalaan 67, Nl-2628 BC Delft, The Netherlands. Tel: +31-15-2789175; Fax: +31-15-2782355; e-mail: HYPERLINK "mailto:g.m.vanderkraan@tudelft.nl" g.m.vanderkraan@tudelft.nl
Short title: Development of a flowcell, to visualise biofilms in porous media.
Key words: Micro model, flowcell, biofilm, Particle Image Velocimetry, porous media, Pseudomonas
Abstract
Flow cells (or micromodels) are widely used to observe complex processes in two dimensional (2D) porous media. The objective of this study is to design, develop, and apply a wet-etched glass micromodel suitable for the observation of biofilm formation, transport of microorganisms and flow measurements using Particle Image Velocimetry (PIV). PIV in this case can be used for the visualisation and quantification of flow diversion. The wet-etching technique applied here is relatively straightforward. The chosen etching depth of around 25 m proved to be well suited for transport experiments with microorganisms. We show that the micromodel containing a wet-etched cell (wafer) provides a high quality image of transport and growth of microorganisms in porous media. The microorganism Pseudomonas chlororaphis was used as a model strain to perform the experiments on biofilm formation and transport. In the transparent cell, we could clearly observe biofilm formation. Moreover, we showed that PIV techniques can indeed visualize the change in flow pattern caused by biofilm presence.
1. Introduction
Biofilm formation and transport of microorganisms play an important role in numerous biological-mediated processes in the subsurface. Much of the research regarding transport of microorganisms is done for safe drinking water and bioremediation ADDIN EN.CITE Murphy20003317Murphy, Ellyn. M., Ginn, Timothy. R.Modeling microbial processes in porous mediaHydrogeological JournalHydrogeological Journal142-158812000(Murphy, 2000). There is renewed interest in biological aspects in the petroleum industry; not only for Microbial Enhanced Oil Recovery (MEOR) applications ADDIN EN.CITE Bryant20024417Bryant, S.L., Lockhart, T.P.Reservoir engineering analyses of microbial enhanced oil recoverySociety of Petroleum Engineers, Reservoir Evaluation & EngineeringSociety of Petroleum Engineers, Reservoir Evaluation & Engineering797192002(Bryant, 2002), but also to reduce H2S emissions or for the analysis of microorganisms found in wells as possible additional information source for reservoir performance ADDIN EN.CITE Pronk2009222217Pronk, M., Goldscheider, N., Zopfi, JMicrobial communities in karst groundwater and their potential use for biomonitoringHydrogeology JournalHydrogeology Journal37-48172009(Pronk, 2009) ADDIN EN.CITE van der Kraan2009343417van der Kraan, G. M.Bruining, J.Lomans, B. P.van Loosdrecht, M. C.Muyzer, G.Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.Microbial diversity of an oil-water processing site and its associated oil field: the possible role of microorganisms as information carriers from oil-associated environmentsFEMS Microbiol EcolFEMS microbiology ecologyFEMS Microbiol EcolFEMS microbiology ecologyFEMS Microbiol EcolFEMS microbiology ecology2009Nov 111574-6941 (Electronic)
1574-6941 (Linking)19958386http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19958386 Eng(van der Kraan, et al., 2009). As opposed to more conventional environments, oil reservoirs have more extreme conditions like high temperatures and salinities, but still hold diverse microbial communities. Transport of microorganisms and biofilm formation in the subsurface are important aspects if microorganisms are to be used in MEOR applications or as information carriers from oil reservoirs ADDIN EN.CITE Foppen2005232317Foppen, J. W.Schijven, J. F.UNESCO-IHE Institute for Water Education, P.O. Box 3015, 2601 DA Delft, The Netherlands. j.foppen@unesco-ihe.orgTransport of E. coli in columns of geochemically heterogeneous sedimentWater ResWater researchWater ResWater researchWater ResWater research3082-83913*Bacterial AdhesionCalcium Carbonate/chemistryCarbon/chemistryColloidsEnvironmental Monitoring*Escherichia coliGeologic Sediments/chemistry/*microbiologyIron Compounds/chemistryPorosityQuartzWater MicrobiologyWater Movements2005Aug0043-1354 (Print)15996706http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15996706 eng(Foppen & Schijven, 2005), ADDIN EN.CITE Zandvliet20085517Zandvliet, M., Handels, M., Van Essen, G., Brouwer, R. and Jansen, J.D.Adjoint-Based Well-Placement Optimization Under Production ConstraintsSPE JournalSPE Journal392-3991342008(Zandvliet, 2008), a process commonly known as biomonitoring ADDIN EN.CITE Rling2002212117Rling, Wilfred. F. M., van Verseveld, Henk. W.Natural attenuation: What does the subsurface have in store?BiodegradationBiodegradation53-64132002(Rling, 2002). In these environments microorganisms can occur in many forms; examples are: suspended cells, aggregates, or biofilms. Biofilms offer a protective environment for the bacteria to e.g. oxygen stress or biocides ADDIN EN.CITE Tolker-Nielsen2000292917Tolker-Nielsen, T.Molin, S.The Molecular Microbial Ecology Group, Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, DenmarkSpatial Organization of Microbial Biofilm CommunitiesMicrob EcolMicrobial ecologyMicrob EcolMicrobial ecologyMicrob EcolMicrobial ecology75-844022000Aug0095-3628 (Print)11029076http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11029076 Eng(Tolker-Nielsen & Molin, 2000). Important for all of the mentioned applications are the attachment and detachment processes of micro-organisms and the formation of biofilm structures in porous media. Examples of processes that trigger biofilm formation are, biobridging (formation of chains of microorganisms that stretch from sand grain to sand grain or in our case from pillar to pillar) , clogging of pores by released pieces of biofilm and adsorption due to interaction of bacteria with the pore-wall, ADDIN EN.CITE Rijnaarts19996617Rijnaarts, H.M., Norde, W., Lyklema, J. and Zehnder, J.B. DLVO and steric contributions to bacterial deposition in media of different ionic strengthsColloids and Surfaces B: BiointerfacesColloids and Surfaces B: Biointerfaces179-195141999(Rijnaarts, 1999), ADDIN EN.CITE van Loosdrecht19907717van Loosdrecht, M. C.Norde, W.Zehnder, A. J.Dept. of Bioprocess Engineering, Delft University of Technology.Physical chemical description of bacterial adhesionJ Biomater ApplJournal of biomaterials applicationsJ Biomater ApplJournal of biomaterials applicationsJ Biomater ApplJournal of biomaterials applications91-10652Bacterial Adhesion/*physiologyCell Wall/physiology*Models, Biological1990Oct0885-3282 (Print)2266489http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2266489 eng(van Loosdrecht, et al., 1990). It is asserted that these mechanisms can be visualized in two dimensional transparent flow cells (also known as micromodels). Visualization is an indispensable tool for a better understanding of the phenomena associated with the presence of biofilm and its effect on flow properties and vice versa (see references and text below).
Chatenever and Calhoun ADDIN EN.CITE Chatenever19528817Chatenever, A., and Calhoun Jr., J.C. Visual examinations of fluid behavior in porous media: Part 1. Petroleum Transactions, AIME Petroleum Transactions, AIME149-1561951952(1952) were the first to use a visual approach for the study of fluid flow in porous media. They used micromodels in which a single layer of glass beads was placed between two flat glass plates. Mattax and Kyte ADDIN EN.CITE Mattax19619917Mattax, C.C., and Kyte, J.R.Ever see a water flood?Oil and Gas Journal Oil and Gas Journal115-128591961(1961), were the first to use glass etching in order to create networks They used the then innovative etching techniques for the construction of a micromodel. In their study, a wax covered glass plate in which a pattern was drawn was etched chemically with hydrofluoric acid. Their purpose was to study multiphase flow in porous media. Davis and Jones ADDIN EN.CITE Davis1968101017Davis, J.A., and Jones, S.C.,Displacement mechanisms of residual solutions.Journal of Petroleum Technology Journal of Petroleum Technology1415 - 1428201968(1968) superseded the wax with photo resist, thus introducing photo etching techniques, which greatly improved the versatility of this approach
Micromodels differ from an ideal 2D shape, which is not always explicitly stated in micromodel studies. Chemical (wet) etching, as described in this manuscript expands spherically from the points exposed to the etching agent. Consequently the pillars in between, which constitute the porous skeleton, obtain a bell shape. The spherical expansion in combination with sufficiently deep pores to prevent spurious attachment to bottom and top plate excludes the creation of pores with a high aspect ratio. Therefore, the depth of the micromodel must exceed the diameter of the microorganism several times (in our case in the order of 10 m). Consequently the width of the pores in the micromodel equals roughly twice the depth of the pores. Wet-etched micromodel pores are therefore larger than pores in a real porous medium. The features described above may play an important role in the interpretation of the microbial trapping mechanisms and are therefore mentioned.
A number of studies has been devoted to the study of microbes in transparent micromodels. Paulsen and Oppen used glass micromodels to visualize oil degradation and mobilisation with pore throats ranging from 120-600 microns and a depth of 200 micron ADDIN EN.CITE Paulsen1999111117Paulsen, J.E., Ekrann S. and Oppen, E.,Visualisation of bacterial degradation and mobilisation of oil in a porous mediumEnvironmental Geology Environmental Geology204-2083831999(Paulsen, 1999). The microorganisms used were obtained from a marine oil-waste biotreatment plant. Steward and Fogler ADDIN EN.CITE Stewart2001131317Stewart, T. L.Fogler, H. S.Department of Chemical Engineering, University of Michigan, 3168 H. H. Dow Building, Ann Arbor, Michigan 48109-2136, USA.Biomass plug development and propagation in porous mediaBiotechnol BioengBiotechnology and bioengineeringBiotechnol BioengBiotechnology and bioengineeringBiotechnol BioengBiotechnology and bioengineering353-63723Biodegradation, Environmental*BiomassBiopolymers/metabolism/secretionCell DivisionColony Count, MicrobialDextrans/metabolism/*secretionDiffusionGlassKineticsLeuconostoc/cytology/*metabolismMicrospheres*Models, BiologicalPermeabilityPetroleumPorosityPressure*Soil MicrobiologyWater/metabolism2001Feb 50006-3592 (Print)11135206http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11135206 eng(2001) studied the formation of pore plugs in porous media by applying exopolymer producing Bacteria. They used the technique developed by Wan et al. to create a micromodel with a triangular pore structure arranged in a hexagonal pattern ADDIN EN.CITE Wan1996141417Wan, J., Togunaga, T.K., Tsang, C.F., and Bodvarsson, G.S.Improved glass micromodel methods for studies of flow and transport in fractured porous media.Water Resources Research Water Resources Research19641995321996(Wan, 1996). The pore throats had a width between 30 and 300 micron. The applied bacterial strain was Leuconostoc mesenteroides, a facultative anaerobe that grows under mesophilic conditions. They continued their investigation by focussing on pore scale level clogging development in porous media. A short paper on biofilm accumulation and transport of microorganisms was published by Dunsmore and Lappin-Scott ADDIN EN.CITE Dunsmore2004151517Dunsmore, B. C.Bass, C. J.Lappin-Scott, H. M.Oil Plus Ltd, Hambridge Road, Newbury, Berkshire, RG14 5TR, UK. B.Dunsmore@oilplusA novel approach to investigate biofilm accumulation and bacterial transport in porous matricesEnviron MicrobiolEnvironmental microbiologyEnviron MicrobiolEnvironmental microbiologyEnviron MicrobiolEnvironmental microbiology183-762*Bacterial Physiological PhenomenaBiofilms/*growth & developmentBiological TransportDesulfovibrio/physiologyModels, BiologicalPermeability2004Feb1462-2912 (Print)14756882http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14756882 eng(Dunsmore, et al., 2004). They created a micromodel in which an image of a thin slice of sandstone rock was etched chemically in a glass plate. They used Desulfovibrio spp. as model organism. Research towards microbial improved oil recovery was performed by Soudmand-asli et al. ADDIN EN.CITE Soudmand-asli2007161617Soudmand-asli, A., Ayatollahi, S.S., Mohabatkar H., Zareie M., Shariatpanahi S.F. The in situ microbial enhanced oil recovery in fractured porous mediaJournal of Petroleum Science and EngineeringJournal of Petroleum Science and Engineering161-172581-22007(Soudmand-asli, 2007). A micromodel has been applied to study enhanced oil sweep induced by bacterial activity. They also address the situation found in fractured reservoirs. Their study uses 2 bacterial strains, i.e., Bacillus subtilis and Leuconostoc mesenteroides. The latter is able to produce large amounts of dextran under anaerobic conditions. The Bacillus subtilis strain is known for its interfacial tension reduction capabilities ADDIN EN.CITE Abtahi2003171717Abtahi, N., Roostaazad R., Ghadiri F.,Biosurfactant Production in MEOR for Improvement of Iran's Oil Reservoirs' Production Experimental ApproachInternational Improved Oil Recovery Conference in Asia Pacific. Kuala Lumpur, Malaysia International Improved Oil Recovery Conference in Asia Pacific. Kuala Lumpur, MalaysiaSPE Paper 573212003(Abtahi, 2003). Only a few papers can be found on studies where micromodel observations of microbes were combined with flow quantification, an example is ADDIN EN.CITE Yarwood2006282817Yarwood, R. R., Rockhold, M. L., Niemet, M. R., Selker, J. S., Bottomley, P. J. Impact of microbial growth on water flow and solute transport in
unsaturated porous mediaWater resources researchWater Resources Research42W104052006(Yarwood, 2006).
Objective
The objective of the research reported in this paper is to give a full description of the design, construction and operation of a conventional micromodel that allows observation of transport of microorganisms and biofilm growth in porous media, combined with flow field visualization/quantification. Visualization of flow diversion is performed using PIV. For the achievement of this objective this paper describes a combination of two methods.
1) The construction of a glass transparent micromodel that allows transport of microbes.
2) A method in which Particle Image Velocimetry is used to track particles passing through a micromodel elucidating heterogeneities created by biofilms.
Included is the procedure to grow the strain Pseudomonas chlororaphis, which is known for its biofilm formation capabilities. This strain was used as a model strain to grow the biofilm; subsequently flow experiments were performed in the presence of this biofilm.
Outline
First the construction of the etched glass cell (wafer) will be explained, giving a description of the techniques applied in the etching of the wafers. This also involved pattern and mask plate development and the procedures that are applied to transfer the developed patterns to the wafers. This is followed by a detailed description of the construction of the holder for the wet-etched glass wafers constituting the 2D micromodel. Then a description of the usage of the complete micromodel is given including the microscope and camera. The bacterial strain Pseudomonas chlororaphis, used for the transport and biofim experiments, is shortly described. Methods to grow the strain, including nutrient media, and induce biofilm formation in the micromodel are given. The usage of the PIV technique is elucidated subsequently. We end with providing results, demonstrating a proof of principle, and conclusions.
2. Development and description of the micromodel
2.1 Materials
Glass Borofloat 33 wafers were purchased from Plan Optik (Elsoff, Germany), which contain 81.3 % SiO2, 12.75% B2O3, 2.4% Al2O3 and 3.55% Na2O. The rings for the micromodel holder were constructed from a Perspex plate (Polymethyl methacrylate or acrylic glass, (C5O2H8)n). Perspex is naturally transparent, which allows optical visualization of the fluid flow through the micromodel holder. Standard equipment, tools and chemicals were obtained from standard local resources.
2.2 Micromodel mask design
2.2.1. Lithography (mask plate development)
A 2 mm thick mask plate was created in order to transfer the created micromodel pattern on the wafers. The designed pattern was created in the mask design program L-Edit. (L-Edit version 12.61, Tanner EDA, California, USA). The mask plate is made of glass on which a thin chromium (Cr) layer is deposited. This chromium layer has been coated with AZ 1518 photo-resist by the manufacturer. The pattern of the micromodel was then written to the photo-resist by using a UV Laser Beam Pattern generator, (LBPG, Heidelberg instruments, Germany). The laser causes changes in the photo-resist so that it can be dissolved later with the development liquid MF322 (Micro-posit, Shipley, MA, USA). After this step, the mask plate is exposed to an acidic bath etching away the Chromium, which is exposed to the acid. Subsequently the photo-resist is removed by dissolving it in an organic liquid, leaving the desired pattern on the mask plate. In our case, the mask contained a grid of circles (400 ( 253) with a diameter of 150 mm and at a minimum distance of 3 (m between the edge of the circles. Of the total area of 6 ( 6 cm2, the area containing the circles has a size of 6 ( 3.8 cm2, which is the medium of interest. The circles are placed in an equidistant staggered grid. The remainder consists of two rectangles (1.1 ( 6 cm2) on both sides of the medium of interest, designed to create highly permeable zones for uniform inflow, with 2 inflow channels, which are 3 mm wide. The patterndatawas then transferred to a Laser beam pattern generator. The pattern includes all the areas that are created by the etching process, viz., the inlet channels, the high permeable areas and the area that holds the pillars constituting the porous skeleton. The inlet channels are the connections from the porous medium to the micromodel holder. This mask plate can now be used as a template to be copied (transferred) into the wafers used in the micromodel.
2.2.2 Protection layer deposition
All Borofloat 33 glass wafers were first coated with an 800 nm poly-silicon (poly-Si) layer by means of Low Pressure Chemical Vapour Deposition (LPCVD). The PolySi was deposited during a 9 hour procedure using SiH4 as a gas. The deposition was performed at a temperature of 570 C, and simultaneously takes place on both sides of the wafers. The second coating consists of silicon carbide (SiC). A 500 nm thick layer was deposited through Plasma Enhanced Chemical Vapour Deposition (PECVD). As a basis, a mixture of SiH4 and CH4 was used. The procedure was performed in a Novellus concept one deposition system (Novellus, Ca, USA), and takes 8 minutes for one wafer and an additional 1.15 minutes for each extra wafer. The deposition was performed at a temperature of 400 C, and only takes place at the front of the wafer. To deposit the lower side, the wafers need to be flipped over and the process has to be repeated.
2.2.3. Transfer of the pattern
The pattern transfer is done by the same lithographic method as used for the patterning of the mask plate. The wafers are first coated with SPR3017M photo-res i s t ( 3 m t h i c k ) o b t a i n e d f r o m S h i p l e y C o m p a n y ( M a r l b o r o , M a s s a c h u s e t t s , U . S . A . ) . P h o t o - r e s i s t d e p o s i t i o n o n t h e w a f e r s w a s p e r f o r m e d o n a n E V G 1 2 0 s y s t e m ( E V g r o u p , A u s t r i a ) b y s p i n c o a t i n g . S u b s e q u e n t l y t h e w a f e r s a r e i l l u m i n a t e d w i t h U V l i g h t , w h i c h i s guided through the mask plate thereby copying the pattern onto the wafers. Illumination is performed on an EV420 contact aligner (EV group, Austria). The wafers are then developed on the same EVG 120 system with Microposit MF-322 developer, also obtained from Shipley Company.
2.2.4 Protection layer plasma etching
Prior to wet etching of the glass, the photo-resist pattern is transferred into the glass protection layers by plasma etching in an Alcatel Gir300 machine (Alcatel, Annecy, France) with a mixture of CF4, SF6 and O2 gasses. This procedure takes 12 minutes. After the glass etching the machine is also used for removing the residue of the protection layers on both sides of the wafers. In this case the procedure takes 12.5 minutes on average for the front and 15 minutes for the back of the wafer.
2.2.5 Chemical etching of the glass wafers
Wet-etching of the Borofloat 33 glass wafers is done in a heated mixture of HF and H3PO4. During this process the acid mixture will etch the glass on the unprotected areas, thus transferring the pattern into the glass. Deposition of the poly-Si and SiC masking layers on the flipside of the wafers was necessary to prevent this side from also being attacked. The wet-etching procedure took about 45 minutes to reach a depth of 25 m.
2.2.6 Cutting of the etched wafers (post etching modifications)
The etched wafer was positioned on a Wafer/frame tape applicator, were a double-layered Nitto foil layer was applied. This layer was required to keep the wafer in place and to protect it during the cutting procedure. The layered wafer was then positioned in a Disco DAD321 Automatic Dicing Saw (Disco Corporation, Tokyo, Japan) and aligned accordingly. A strip holding a width of 2.5 mm was cut out of the glass wafer. Subsequently, the cut wafer was cleaned in a GS Ultratech Model 2066 High pressure cleaning station (GS Ultratech equipment, USA) where DI-water was used at a pressure of 30 bars. CO2 is dissolved in the water to reduce its interfacial tension. This open end in the wafer was required to allow liquid to exit the model avoiding an increase in pressure in the constructed cell itself.
2.3 Construction and assembling of the holder including an etched wafer.
For the construction of the complete micromodel set-up the etched wafers were mounted correctly in the developed (wafer) holder. This holder consists of two Perspex rings with a thickness of 5 mm. Both rings hold an inner diameter of 90 mm and outer diameter of 130 mm. In the inside of the bottom ring, a secondary ring with a width of 10.4 mm and a depth of 1.2 mm was carved out allowing the correct placement (alignment) of both wafers (both have an outer diameter of 10 cm). These wafers are adhered to the Perspex using a Teflon elastomer (CAF 4 Silicone elastomer (Bluestar Silicones, Lyon, France)), an additional 0.4 mm deep area was carved to compensate for the thickness of this material. In order to create a leak free, waterproof micromodel the wafers were clamped and sealed between the two Perspex rings. To this purpose, plugholes were drilled through both rings (holders) where screws could be fitted in. The locations of these holes are shown in Figure 1.
To allow liquid flow from the Perspex holder to the created cell (consisting of 2 wafers), a reservoir (l ( w ( h = 30( 4( 4 mm3) and two identical connecting channels (l( w( h = 40mm ( 2mm ( 1.2 mm) were made in the bottom ring, to serve as the liquid inlet and bubble trap. Liquid is transferred from this reservoir via these channels to the wafers. As a liquid inlet from the infusion pump hose to the reservoir, a 3 mm steel tube was inserted along the long axis of the Perspex ring penetrating the reservoir from the side. The total volume of the reservoir including both channels is 0.672 ml. The created top ring has the same dimensions as the bottom ring, although it has no reservoir or channels, nor does it have a carved ring to position the wafers. In both rings, indicator marks were applied to be able to align them correctly later on.
Figure 1: Schematic overview of the micromodel. Four M6 and ten M3 plugholes were created in the holder.
3. Application of the micromodel and the observation of biofilm development.
3.1 Description of experimental set-up and utilization of the micromodel
The fully assembled micromodel was connected to a Cole-Parmer Single-Syringe Infusion Pump (Series EW-74900-00, Cole-Palmer, Illinois USA) with a silicon hose. The inflow velocity of the pump during the experiments was 0.100 ml/hour. The fluid traversed the micromodel in approximately 34 minutes, corresponding to a horizontal fluid velocity of 4.52*10-5 m/s or 3.9 m/day (interstitial velocity) corresponding to a Darcy velocity of 1.6 m/day. This value is somewhat larger than an average Darcy velocity in oil reservoirs (1 m/day).
Bacteria were grown on growth medium overnight. They were introduced in the micromodel by three hours of suspension pumping as an inoculum at about the same rate as used in the experiments. Flow was then stopped for one hour to give the bacteria time to adhere to the solid surface areas of the porous medium. Subsequently, fresh sterile medium was flushed through the micromodel. Biofilm formation was observed with the microscope at enlargements of 100( and 400(. Images were taken at different time frames, commonly every day.
3.2 Description of the used microbial strain applied for the testing of the set-up
The biofilm growth experiments were performed using the bacterial strain Pseudomonas chlororaphis (ATCC 55729). This strain was obtained from CBS (Centraal Bank Schimmelculturen, Utrecht, the Netherlands) on behalf of the Delft University of Technology. P. chlororaphis is a rod-shaped, motile, and facultative aerobe that is Gram negative. Strains of P. chlororaphis typically contain 4-8 polar flagella. It is able to grow at temperatures between 5 and 37 C, with an optimum at 30 C ADDIN EN.CITE Haynes196216916917Haynes, WilliamRhodes, LenoraComparative Taxonomy of Crystallogenic Strains of Pseudomonas Aeruginosa and Pseudomonas ChlororaphisJ. Bacteriol.1080-1084845micromodel1962citeulike-article-id:4198886http://jb.asm.org/cgi/content/abstract/84/5/1080(Haynes & Rhodes, 1962). P. chlororaphis is a level 1 terrestrial microorganism that is known not to be hazardous to any extent. It is, however, well known for its biofilm forming capabilities. The medium used to cultivate this microorganism is adapted from Stoodley et al. 2005 ADDIN EN.CITE Stoodley20054417P. StoodleyFlowing biofilms as transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor systemBiofouling161-168213/42005(Stoodley, 2005) and consists of the following compounds (mg/l): KH2PO4 70, K2HPO4 30, (NH4)2SO4 110, Glucose 1000, CaCl2 40, NaCl 585, trace elements/MgSO4 solution consisting of the following components (mg L-1): EDTA (Trilon B) 5, FeSO4 7H2O 2, ZnSO4 7H2O 0.1, MnCl2 0.03, H3BO3 0.3, CoCl2 6H2O 0.2, CuCl2 0.01, NiCl2 2H2O 0.02, Na2MoO4 0.02, MgSO4 7H2O 0.2. Buffer containing only KH2PO4 and K2HPO4 was autoclaved at 120 C. A 20% w/v (NH4)2SO4 was prepared separately and autoclaved at 120 C. A 20% w/v glucose stock solution was prepared and autoclaved at 110 C. Trace metals and MgSO4 were autoclaved at 120 C separately. All compounds were added together under sterile conditions. This was tested by incubating sterile medium bottles at 30 C overnight followed by a contamination check. Biofilm formation was induced by using a medium with a C:N ratio of 20. This ratio is believed to be favourable for biofilm formation and extracellular polysaccharide (EPS), production.
3.3 Microscopy study
Images were made on a Zeiss Axioplan 2 microscope (Carl Zeiss Imaging Solutions GmBH, Mnchen, Germany). The used camera is an AxioCam MRm (Carl Zeiss MicroImaging GmbH, Gttingen, Germany). The software package used to analyze the images is a Leica Qwin pro software package version 3.2.1 also from Leica microsystems. The SEM images of the etched wafers were made on a Philips XL electron microscope (FEI company/Philips, Eindhoven, the Netherlands).
3.4 Particle Image Velocimetry
During PIV experiments, the micromodel was placed under a combined stereo/mono epifluorescent microscope (Leica MZ 16 FA). The stereo mode is used for preparation of the experiments only (e.g., for tracer injection). The motion of these tracer particles is recorded on a digital camera (PCO Sensicam QE, 1376נ1040pixels using 22 binning; acquisition rate 5 Hz). Local cross-correlation is used to determine the local displacement and thus velocity (by dividing the displacement by the temporal separation between consecutive images). The set-up is controlled using a PC running DaVis 7 software (LaVision GmbH). This software is also used for data acquisition and storage. Polystyrene spheres with a diameter of 1.28 micron were used as tracer particles, containing a fluorescent dye Rhodamine 6G (Microparticles GmbH). The particles are bio-inert or stealth, because of a poly-ethylene glycol (PEG) coating. The particles are naturally buoyant and have a very small (Stokes) response time.
Typically 1500 images are recorded for each measurement. This corresponds with a 300 second time interval, at a frequency of 5 Hz. Background image subtraction was performed to circumvent dominant reflection originating from the biofilm itself. Hereby the first image is used as a reference image, and all subsequent images are mapped onto this image. To determine the required image transformation, the disparity between the images is determined by local cross-correlation using 96 ( 96 pixels interrogation windows with 50% overlap, covering the entire raw image. A second-order polynomial fit is performed using the disparity data. Although in general the corrections needed were small, this step is crucial in the PIV process. A more detailed description of this technique can be found in the article by Poelma ADDIN EN.CITE Poelma2008181817Poelma, C.; Vennemann, P., Lindken, R.; Westerweel, J.In vivo blood flow and wall shear stress measurements in the vitelline networkExperiments in fluidsExperiments in fluids703-7134542008(2008).
4. Results
4.1 Micromodel development and the wet-etching of the glass wafers
The wet-etching procedure used here, posed little difficulties with respect to the etching process. The obtained depth was around 25 m. This depth is well suited for transport experiments with microorganisms and did not cause operational problems while using the micromodel. It was observed that the pillars assumed a characteristic bell shape, as mentioned. It was also observed that the pillars do not obtain a perfect circular shape when observed perpendicularly from the top. The reached upper diameter of the pillars was 110 m. The average pore throats were 40 m holding a final aspect ratio throat width/depth of 1.6. As can be seen in Figure 2, the pattern was transferred correctly. In the mask plate the spherical etching (undercutting) was taken into account. The porosity of the pattern can be described as the pore volume divided by the total volume and is 41% in this case.
a b
Figure 2. SEM image showing a: wet etched pattern of the wafer (80xs magnification); b: effects of spherical etching (500xs magnification). Note the irregularities (lines) originating from the pillars in figure 2b, caused by the wet etching procedure. Note in addition the bell-shaped channels.
4.2 Biofilm development and the formation of specific structures in 2D porous media: a proof of principle.
After inoculation of the micromodel with the microorganism P. chlororaphis, biofilm growth was observed in the micromodel already after one day. The formation of biobridging was clearly observed throughout the model. These structures are described as a chain of attached microorganisms that form a bridge between soil particles. In the case of the micromodel, a biobridge is equivalent to the formation of a bridge between two adjacent pillars. This phenomenon is observed at numerous locations in the micromodel. In Figure 3, biobridge structures in progressing stages are visualized. When chains of microorganisms stretch from pillar to pillar, the bridge is complete (Figure 3a). Often, crossing bio-bridges merge together to form a web of biofilm (Figure 3b). Over time, these webs increase in size and become clusters, which can block a significant fraction of the pores (Figure 3c). It could be seen that the thick biofilm grew longer every day, following the direction of flow (Figure 3d). Individual microorganisms could be distinguished in these structures, surrounded by layers of EPS. During the initial microbial inoculation, the cell density was relatively low. With subsequent flushing with fresh medium, the cell density increased over time. Furthermore, transport of both single microorganisms and released pieces of biofilm was visible during pulses with fresh media. These released pieces showed similar transport behaviour as single organisms.
a
b
c
d
Figure 3. Close up of micromodel showing different stages of biofilm development. Figure 3a,b&c, the pillars show up as dark disks, where porespace is the lighter region in between. Magnification in (a,b,c) 400(s and in (d) 100(s.. Refraction and reflection cause the white and black circles surrounding the pillars, which are 110 m in diameter. a: Initial attachment observed as string of bacteria connecting one pillar to the other. Also we observe individual bacteria, as black or white dots and initial elongation of the string. b: Formation of biobridging and subsequent cluster forming. c: Formation of the mature biofilm. d: Overview of bioweb (The pillars now show up as white disks).
4.3 Particle Image Velocimetry
The flow patterns in the micromodel are visualized by means of microscopic Particle Image Velocimetry ADDIN EN.CITE Santiago1998191917Santiago, J.G., Wereley, S.T., Meinhart, C.D., Beebe, D.J., Adrian, R.J. A particle image velocimetry system for microfluidicsExperiments in FluidsExperiments in fluids316-3192541998(Santiago, 1998). A recent review can be found in ADDIN EN.CITE Lindken2009242417Lindken, R.Rossi, M.Grosse, S.Westerweel, J.Laboratory for Aero- and Hydrodynamics, Delft University of Technology, Delft, The Netherlands. r.h.lindken@tudelft.nlMicro-Particle Image Velocimetry (microPIV): recent developments, applications, and guidelinesLab ChipLab on a chipLab ChipLab on a chipLab ChipLab on a chip2551-679172009Sep 71473-0197 (Print)19680579http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19680579 eng(Lindken, et al., 2009). The micromodel is perfused at a flow rate of 0.100 ml/hr with a m e d i u m c o n t a i n i n g 1 . 2 8 m f l u o r e s c e n t p a r t i c l e s , w h i c h a c t a s t r a c e r p a r t i c l e s f o r f l u i d m o t i o n . F i r s t t h e f l o w p a t t e r n i n t h e m i c r o m o d e l w i t h o u t m i c r o o r g a n i s m s o r a n y f o r m o f b i o f i l m i s d o c u m e n t e d ( d a t a n o t s h o w n ) . S u b s e q u e n t l y , P I V m e a s u r e m e n t s i n m i cromodels with biofilm are performed. An example is shown in Figure 4. The flow pattern is represented by a vector field, in which both the vector length and colors indicate the velocity magnitude. To obtain a better overview, the length is only an indication of the magnitude of the velocity but not proportional to it. The velocities typically range from 0.5e-5 ms-1 (dark blue) to 6e-5 ms-1 (red). The vector field has been superposed on a dark field image of the model, which shows the location of biofilm clusters (white patches).
In the figure, it can clearly be observed that the presence of biofilm alters the flow pattern, as can be seen in the red rectangle (Figure 4). Particles follow preferential pathways, on occasion also against the direction of the overall main flow direction. On a small scale, movement through narrow pathways led to an increase in velocity. On a larger scale, it could be seen (due to its size it could not be included in this paper) that in certain regions (wakes), lying behind thick biofilm formations, the flow rate as a whole was lower than in adjacent areas. To quantify the permeability reduction the parts of the flow pattern must be interpreted, using a flow simulation. This is, however, outside the scope of the present paper. However, with such an interpretation models of biofilm growth in porous media can be improved by incorporating a relation between permeability and biofilm concentration. Furthermore, the reproducibility of the PIV-measurements was determined by measuring the same region four times, as can be seen in Figure 5. This figure shows that even if the direction of flow is similar; the tracer particles show fluctuations in the flow rate.
Figure 4. Example of a PIV measurement in the micromodel. The colour of the arrow represents the particle velocity, ranging from blue (slow) to red (fast). The white structures are a web of biofilm. The white dot on the left hand side of each pillar is a reflection artefact. In this picture, two types of regions can be distinguished: one with biofilm formation and subsequent alteration of initial flow patterns (left) and one with little or no biofilm and a more regular flow pattern (right).
Figure 5. Recordings of the same area in the flowcell during four separate measurements using a time interval of 10 minutes. The direction of flow remains similar; however we see small variations in the flow rates (color of arrows) and intensity. The intensity changes are caused by small movements of the light source. Reasons for the changing color pattern are discussed in the text.
5. Discussion
The performed experiments are relevant for many applications in petroleum engineering and hydrology ADDIN EN.CITE Cunningham1991313117Cunningham, A. B., Characklis, W. G., Abedeen, F., Crawford, D. Influence of biofilm accumulation on porous media hydrodynamicsEnvironmental science & technologyEnvironmental science & technology1305-13112571991(Cunningham, 1991). Much attention is devoted to improving oil recovery using metabolic activity of micro-organisms. The most important envisioned application, however, is in bio-diversion or bio-sealing. Therefore, the interest here is on biofilm formation and pore clogging. Bio-diversion in this context implies that high permeable regions bounded by impermeable shale layers that are watered out are clogged such that for example the oil from lower permeable regions can be produced ADDIN EN.CITE Vermolen2004323217Vermolen, F.J., Bruining, J., van Duijn, C.J.Gel placement in porous media: constant injection rateTransport in Porous MediaTransport in Porous Media247-2664422004(Vermolen, 2004). A well is usually connected to a number of layers with different permeabilities. These layers may be separated by impermeable shale layers. In bull-headed injection in the production well, the placement of nutrients will be more effective in high permeable layers. Hence microbial growth will be more effective in the high permeable layers, in which a reduction of permeability and hence flow occurs. Consequently flow will be redirected via the low permeable layers from which oil will now be produced.
Indeed, microbes including EPS occupy the pore-space thus considerably reducing the permeability as observed in the experiments discussed in this paper. Furthermore observation of microbes in the wells can be used as a marker of processes occurring in the reservoir. All these applications require understanding of mechanisms of transport of microbes in the subsurface. In this, formation and destruction of biofilms play an important role. In biofilm growth nutrient supply, inhibitors and hydrodynamics influence its formation.
5.1 Proof of principle: The combination of a micromodel and the use of PIV techniques.
The combination of a 2D micromodel set-up and PIV techniques as demonstrated and applied in this study is a powerful method to study transport and attachment of microbes in porous media, e.g., in water management and oil recovery processes. Biofilm growth starts with attachment of single microbes, from where clusters of microbes develop. Sometimes clusters of microbes move in the porous medium ADDIN EN.CITE Vadas1973303017Vadas, E.B., Goldsmith, H.L., Mason, S.G.The Microrheology of Colloidal DispersionsJournal of Colloid and Interface ScienceJ Colloid Interface SciJournal of colloid and interface science630-6484331973(Vadas, 1973). Pore bridges originate and expand presumably by collector effects of bacteria and cell division. The origin of the observed biobridges can also be the effect of the wet-etching procedure in which a bacterium at a certain time is retained by the irregularities (lines caused by the wet etching procedure) as can be seen in figure 2b. This implies that bacterial attachment is enhanced by irregularities, which are naturally present at the porous medium surface ADDIN EN.CITE Mitik-Dineva2008333317Mitik-Dineva, N.Wang, J.Mocanasu, R. C.Stoddart, P. R.Crawford, R. J.Ivanova, E. P.Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, Australia.Impact of nano-topography on bacterial attachmentBiotechnol JBiotechnology journalBiotechnol JBiotechnology journalBiotechnol JBiotechnology journal536-4434Bacterial Adhesion/*physiologyBiocompatible Materials/*chemistryGlass/*chemistryMaterials TestingNanostructures/*chemistry/*ultrastructureParticle SizePseudomonas/*cytology/*physiologySurface Properties2008Apr1860-7314 (Electronic)18246568http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18246568 eng(Mitik-Dineva, et al., 2008). When the clusters develop biofilms are formed. It was observed that the biofilm formation affects both direction and velocity of the fluid. Large biofilm clusters cause the flow to divert into regions with less biofilm. Hereby, preferential flow pathways originate, as has been shown in this research. An important aspect of visual observations is the interpretation of the results. A clear observation is that flow avoids regions of high microbial concentrations. However, the flow rates flowing towards a node point and away from the node point should balance. From the observed rates, i.e. the color of the arrows, one would conclude that this balance is not satisfied at some node points. Below we list a number of artefacts that can lead to misinterpretation of the results.
5.2 Artefacts of the PIV measurement in the micromodel.
As can bee seen in Figure 4, at some places flow paths seem to start and end abruptly. This, however, is likely to be an artefact caused by the autofluorescence of the biofilm itself. If the biofilm is thick enough, the fluorescence of the biofilm can at some places overrule the signal of the tracer particles. This would lead to a distortion of the vector pattern. Another plausible possibility is that the particle moves out of the field of vision, due to 3D effects, e.g., a fluorescent particle passing below a piece of biofilm.
In Figure 5, it was illustrated that the flow direction in the different pictures was comparable in the region that was measured four times. It can be expected that the number of tracer particles fluctuates in the region under consideration. Therefore, the flow rate shows small fluctuations. This artefact will decrease when time interval of the measurement is increased, due to an averaging effect with the increased amount of captured tracer particles.
5.3 Advantages of the micromodel.
The application of wet etching in the construction of the etched cells is a relatively easy technique to create patterns in glass or SiO2. A suitable depth (20-40 m) to perform flow experiments, unlike, e.g., with plasma etching ADDIN EN.CITE Metwalli2003272717Metwalli, E., Pantano, C.G.Reactive ion etching of glasses: Composition dependenceNuclear Instruments and Methods in Physics Research BNuclear Instruments and Methods in Physics Research B21-272072003(Metwalli, 2003), is easily reached. We used a depth between 20-25 m and a width of 40-50 m. however, if a micromodel with small pores is required different etching techniques are needed.
The choice to use ordinary glass wafers for the construction of a micromodel has been proven fruitful. Glass allows a better quality of observation than an intransparent silicon (Si) wafer covered with a glass plate. Additionally, glass is hydrophilic resembling most subsurface environments; this in contrast to many other transparent materials like SU8, which are commonly hydrophobic. Since a mask plate has been designed, multiple wafers can be constructed allowing multiple flow experiments in cells that have the same etched pattern.
5.4 Limitations of the applied etching technique and the constructed micromodel.
In the current design we used two inlet channels followed by a high permeable area, before the area of interest is reached. However, it does not create a completely uniform waterfront as intended. All the same it did improve the uniform shape of the waterfront compared to earlier versions of the model in which the high permeable areas were not created. Furthermore, the use of conventional wafers with a thickness of 0.5 mm as commonly used in the electronic industry has its limitations. The use of chemical etching as mentioned earlier is rather straightforward and therefore frequently used in micromodel studies. It, however, puts a limit on the size of the pores. The fact that the pores used in our micromodel are relatively large excludes certain mechanisms in which microorganisms can clog pores, like size exclusion (filtration effects) and the observation of effects due to inaccessible and excluded pore volumes. The fact that this type of etching causes small irregularities in the pore structure bottom plate requires a critical interpretation of the observed bacterial attachment processes.
6. Conclusions
Chemical etching techniques can be used to construct transparent glass or silica micromodels, which have the advantage, as opposed to silicon models, that they are transparent and hence can provide clearer images of processes on the micro-scale. The wet etching technique limits the resolution of the pore sizes due to the undercutting artefact, i.e., minimal pore size is twice the depth. This limitation effectively rules out the construction of small pores, and hence the observation of size exclusion, inaccessible and excluded pore volume or filtration effects.
It is possible to successfully introduce Pseudomonas chlororaphis into the model. Biofilm formation was successfully induced. The designed holder functions properly with a wet-etched micromodel into place and it is shown that biofilm growth can be observed. Preliminary observations show that micromodels are a versatile method for the observations of microbial processes in porous media. They provide detailed insights in processes on the pore level. Experiments have shown that some of the mentioned processes, e.g., bio-bridging, that are related to biofilm formation can be observed. The biobridging, however, can also be a consequence of the created imperfections by the wet etching procedure. Therefore it is advised to describe the structure of the pore network in detail and to verify the experimental observations regarding bacterial attachment with the pore network structure.
The combination of 2D micromodels with PIV techniques allows the observation of flow irregularities caused by biofilm development at specific locations. On these locations, preferential flow pathways and wake zones were observed. The constructed micromodel has proven to be well suited for observations of these kinds of phenomena.
In principle the PIV measurments can interpreted in terms of permeablity modification. Such an interpretation would allow to incorporate a permeability-biofilm relation in the modelling.
This set-up provides an experimental tool for elucidating some of the transport mechanisms that determine the movement of microbes in oil reservoirs and aquifers. experimental results in which computer models regarding transport of microbes and biofilm formation in porous media can be compared and verified to real experiments. This is a welcome contribution towards a better understanding of these processes, also on a larger scale.
7. Acknowledgements
The authors would like to thank Bert Goudena for his contribution to this study. Special thanks are given to Cor Kuijvenhoven (Shell Exploration and Production) and Leon van Paassen (TU Delft) for fruitful discussions. Also we would like to thank Jan Etienne for his assistance during the assembly of the micromodel. Last but not least, special thanks to Astrid Kloosterman, for her assistance during the PIV measurements.
The research was carried out within the context of the ISAPP Knowledge Centre. ISAPP (Integrated Systems Approach to Petroleum Production) is a joint project of the Netherlands Organization for Applied Scientific Research TNO, Shell International Exploration and Production, and Delft University of Technology.
References
ADDIN EN.REFLIST [1] Abtahi N, Roostaazad R., Ghadiri F., (2003) Biosurfactant Production in MEOR for Improvement of Iran's Oil Reservoirs' Production Experimental Approach. International Improved Oil Recovery Conference in Asia Pacific. Kuala Lumpur, Malaysia
[2] Bryant SL, Lockhart, T.P. (2002) Reservoir engineering analyses of microbial enhanced oil recovery. Society of Petroleum Engineers, Reservoir Evaluation & Engineering.
[3] Chatenever A, and Calhoun Jr., J.C. (1952) Visual examinations of fluid behavior in porous media: Part 1. . Petroleum Transactions, AIME 195: 149-156.
[4] Cunningham AB, Characklis, W. G., Abedeen, F., Crawford, D. (1991) Influence of biofilm accumulation on porous media hydrodynamics. Environmental science & technology 25: 1305-1311.
[5] Davis JA, and Jones, S.C., (1968) Displacement mechanisms of residual solutions. Journal of Petroleum Technology 20: 1415 - 1428.
[6] Dunsmore BC, Bass CJ & Lappin-Scott HM (2004) A novel approach to investigate biofilm accumulation and bacterial transport in porous matrices. Environ Microbiol 6: 183-187.
[7] Foppen JW & Schijven JF (2005) Transport of E. coli in columns of geochemically heterogeneous sediment. Water Res 39: 3082-3088.
[8] Haynes W & Rhodes L (1962) Comparative Taxonomy of Crystallogenic Strains of Pseudomonas Aeruginosa and Pseudomonas Chlororaphis. J. Bacteriol. 84: 1080-1084.
[9] Lindken R, Rossi M, Grosse S & Westerweel J (2009) Micro-Particle Image Velocimetry (microPIV): recent developments, applications, and guidelines. Lab Chip 9: 2551-2567.
[10] Mattax CC, and Kyte, J.R. (1961) Ever see a water flood? Oil and Gas Journal 59: 115-128.
[11] Metwalli E, Pantano, C.G. (2003) Reactive ion etching of glasses: Composition dependence. Nuclear Instruments and Methods in Physics Research B 207: 21-27.
[12] Mitik-Dineva N, Wang J, Mocanasu RC, Stoddart PR, Crawford RJ & Ivanova EP (2008) Impact of nano-topography on bacterial attachment. Biotechnol J 3: 536-544.
[13] Murphy EM, Ginn, Timothy. R. (2000) Modeling microbial processes in porous media. Hydrogeological Journal 8: 142-158.
[14] Paulsen JE, Ekrann S. and Oppen, E., (1999) Visualisation of bacterial degradation and mobilisation of oil in a porous medium. Environmental Geology 38: 204-208.
[15] Poelma CV, P., Lindken, R.; Westerweel, J. (2008) In vivo blood flow and wall shear stress measurements in the vitelline network. Experiments in fluids 45: 703-713.
[16] Pronk M, Goldscheider, N., Zopfi, J (2009) Microbial communities in karst groundwater and their potential use for biomonitoring. Hydrogeology Journal 17: 37-48.
[17] Rijnaarts HM, Norde, W., Lyklema, J. and Zehnder, J.B. (1999) DLVO and steric contributions to bacterial deposition in media of different ionic strengths. Colloids and Surfaces B: Biointerfaces 14: 179-195.
[18] Rling WFM, van Verseveld, Henk. W. (2002) Natural attenuation: What does the subsurface have in store? Biodegradation 13: 53-64.
[19] Santiago JG, Wereley, S.T., Meinhart, C.D., Beebe, D.J., Adrian, R.J. (1998) A particle image velocimetry system for microfluidics. Experiments in Fluids 25: 316-319.
[20] Soudmand-asli A, Ayatollahi, S.S., Mohabatkar H., Zareie M., Shariatpanahi S.F. (2007) The in situ microbial enhanced oil recovery in fractured porous media. Journal of Petroleum Science and Engineering 58: 161-172.
[21] Stewart TL & Fogler HS (2001) Biomass plug development and propagation in porous media. Biotechnol Bioeng 72: 353-363.
[22] Stoodley P (2005) Flowing biofilms as transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system. Biofouling 21: 161-168.
[23] Tolker-Nielsen T & Molin S (2000) Spatial Organization of Microbial Biofilm Communities. Microb Ecol 40: 75-84.
[24] Vadas EB, Goldsmith, H.L., Mason, S.G. (1973) The Microrheology of Colloidal Dispersions. Journal of Colloid and Interface Science 43: 630-648.
[25] van der Kraan GM, Bruining J, Lomans BP, van Loosdrecht MC & Muyzer G (2009) Microbial diversity of an oil-water processing site and its associated oil field: the possible role of microorganisms as information carriers from oil-associated environments. FEMS Microbiol Ecol.
[26] van Loosdrecht MC, Norde W & Zehnder AJ (1990) Physical chemical description of bacterial adhesion. J Biomater Appl 5: 91-106.
[27] Vermolen FJ, Bruining, J., van Duijn, C.J. (2004) Gel placement in porous media: constant injection rate. Transport in Porous Media 44: 247-266.
[28] Wan J, Togunaga, T.K., Tsang, C.F., and Bodvarsson, G.S. (1996) Improved glass micromodel methods / 8
A
g
h
j
, - . / 0 6 7 ʰʞʞʧ h{ h{ CJ aJ h?rK CJ aJ h'(2 h?rK CJ aJ h?rK CJ H*aJ h{ CJ H*aJ h{ CJ aJ hW CJ H*aJ hql6 CJ H*aJ hql6 CJ aJ hmB 5CJ aJ h|M hmB hmB hY hmB hw4 5CJ aJ hql6 5CJ aJ 2 h
i
j
. / 6 N
O
P
h i d I $d Eƀ چ a$
$d a$gd?rK
h*$^hgdmB $d a$ e f N
O
P
J K L f g i ' 8 B D * < T c i i ֹ֮敌wl hql6 6CJ ]aJ hql6 5CJ aJ hql6 6CJ \]aJ hql6 CJ \aJ hql6 5CJ aJ hiR
CJ aJ h2 CJ aJ h hql6 CJ aJ hql6 0J CJ aJ %j hql6 CJ UaJ mHsHj hql6 CJ UaJ mHsHhql6 CJ aJ h?rK CJ aJ h?rK CJ H*aJ &i ' i j z A dK ,O !r ,r s Dt t u u u y y y y
$d a$gdg
$d a$gd+o
$d a$gdiR
$d a$ d d gdiR
i z n o j k 5 6 , - : ; < = " " " " " " ^$ _$ * * + + + + - - - - - - *0 +0 90 :0
1 1 5 5 5 5 5 \7 ]7 7 7 : : ѼѼѰѼ٤٤
hql6 0J <hql6 CJ aJ mH sH h@!i h@!i 6CJ aJ j h@!i CJ UaJ hql6 CJ H*aJ h@!i CJ aJ j hql6 CJ UaJ h+o CJ aJ hql6 CJ aJ hql6 5CJ \aJ >: : : : : : @ @ )@ 1@ 8@ 9@ ;@ @ @ @ @ @ @ @ @ A A kA A A A D D D D D D G G G G G sH tH J J J J J M M O (O &P