研究多孔介质中生物堵塞的微流体平台
Summary
本协议描述了一个微流体平台，通过将高分辨率显微镜成像与同时压差测量相结合来研究 准二维多孔介质中的生物膜发展。该平台量化了多孔介质中孔径和流体流速对生物堵塞的影响。
Abstract
细菌生物膜存在于几种环境和工业多孔介质中，包括土壤和过滤膜。生物膜在某些流动条件下生长并会堵塞孔隙，从而改变局部流体流动的方向。生物膜堵塞孔隙的能力，即所谓的生物堵塞，会对多孔介质的局部渗透性产生巨大影响，在系统中产生压力积聚，并影响通过它的质量流。为了了解生物膜生长与流体流动在不同物理条件下（例如，在不同的流速和孔径下）之间的相互作用，在本研究中，开发了一个微流体平台，以在外部施加的受控物理条件下使用显微镜可视化生物膜的发展。可以使用压力传感器同时测量多孔介质中生物膜引起的压力积聚，然后与生物膜的表面覆盖率相关联。所提出的平台为研究流动条件下多孔介质中生物膜引起的生物堵塞的系统方法提供了基线，并且可以适用于研究环境分离物或多物种生物膜。
Introduction
生物膜 – 嵌入自分泌的超聚合物物质（EPS）基质中的细菌菌落 – 在天然多孔介质中无处不在，例如土壤和含水层1，以及技术和医疗应用，例如生物修复2，水过滤3和医疗设备4。生物膜基质由多糖、蛋白质纤维和细胞外 DNA5，6 组成，并且在很大程度上取决于微生物、营养物质的可用性以及环境条件7。然而，矩阵的功能是通用的;它形成生物膜结构的支架，保护微生物群落免受机械和化学应力的影响，并且主要负责生物膜的流变特性5。
在多孔介质中，生物膜的生长会堵塞毛孔，导致所谓的生物堵塞。生物膜的形成由流体流动和孔径控制，定义为多孔介质8，9，10的两根支柱之间的距离。孔径和流体流量都控制着养分的输送和局部剪切力。反过来，生长的生物膜堵塞孔隙，影响流体11，12，13的速度分布，质量传递和多孔介质14，15的导水率。水力传导率的变化通过密闭系统中压力的增加反映出来16，17，18，19。目前生物膜开发和生物堵塞的微流体研究集中在研究均匀几何形状16，20（即具有单一孔径）或异质多孔介质12，21，22中的流速的影响。然而，为了解开流速和孔径对生物膜发育的影响以及生物堵塞多孔介质中由此产生的压力变化，需要一个高度可控和多功能的实验平台，可以并行研究不同的多孔介质几何形状和环境条件。
本研究引入了一种微流体平台，该平台将压力测量与多孔介质内不断发展的生物膜同时成像相结合。由于其透气性、生物相容性和通道几何形状设计的灵活性，由聚二甲基硅氧烷（PDMS）制成的微流体装置是研究多孔介质中生物膜发育的合适工具。微流体允许高精度地控制物理和化学条件（例如，流体流动和营养浓度），以模拟微生物栖息地的环境23.此外，微流体装置可以使用光学显微镜以微米分辨率轻松成像，并与在线测量（例如，局部压力）相结合。
在这项工作中，实验的重点是研究在受控的强加流动条件下均匀多孔介质模拟中孔径的影响。使用注射泵施加培养基的流量，并通过压力传感器同时测量通过微流体通道的压差。通过在微流体通道中播种 枯草芽孢 杆菌的浮游培养物来启动生物膜开发。对不断发展的生物膜进行定期成像和图像分析，可以在各种实验条件下获得有关表面覆盖的孔隙尺度分辨信息。压力变化和生物堵塞程度的相关信息为生物堵塞多孔介质的渗透率估计提供了重要的输入。
Protocol
1. 硅片制备 在计算机辅助设计（CAD;见 材料表）软件中设计微流体通道的几何形状，并将其打印到透明薄膜上以创建光掩模（图1A）。 按照以下步骤通过软光刻（在洁净室条件下）制造母模。将硅片在200°C下烘烤2小时。 将晶圆放在旋涂机的中心，然后将SU8 3050光刻胶（见 材料表）倒在晶圆上。以 1，700 rpm ?…
Representative Results
在本研究中，使用具有三个具有不同孔径的平行微流控通道的微流控装置（图1）系统地研究多孔介质中生物膜的形成。使用明场显微镜可视化生物膜形成过程。细菌细胞和生物膜在图像中显示为较暗的像素（图2）。此外，观察到逐渐堵塞的过程;在24小时的实验中，最初随机生长的生物膜定植了几乎整个多孔介质。 在 Q = 1 mL…
Discussion
微流体多孔介质类似物与压力传感器相结合，为研究多孔介质中的生物膜发展提供了合适的工具。微流体多孔介质设计的多功能性，特别是柱子的排列，包括直径、不规则形状和孔径，允许研究许多几何形状。这些几何形状的范围从单个孔隙到高度复杂、不规则排列的障碍物，模仿不同的自然（例如土壤）和工业（例如膜和过滤器）多孔介质。在目前的微流体平台中，创建了三种多孔介质几何形?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢SNSF PRIMA赠款179834（给E.S.），ETH（给RS），苏黎世联邦理工学院研究基金（给R.S.和J.J.M.）的酌情资助，以及Eawag（给J.J.M.）的酌情资助。作者要感谢Roberto Pioli在 图1B 中说明实验设置，并感谢Ela Burmeister用于硅晶圆制备。
Materials
| Acrodisc 25 mm Syringe Filter, 1.2 µm Versapor Membrane | Pall Corporation | PN4190 | 1.2 µm filters |
| BD 10 mL Syringe (Luer-Lock) | BD | 300912 | used to fill the channel with deionised water |
| Box Incubator | Life Imaging Services | used to have a stable temperature during the biofilm growth experiment | |
| Cell density meter CO8000 | WPA biowave | OD meter | |
| Centrifuge vial | Eppendorf | 30120086 | 1.5 mL |
| CETONI Base 120 | CETONI GmbH | syringe pump | |
| CorelCAD | CorelDRAW | software used to design the microfluidic channel geometries | |
| Culture tubes (14 mL, sterile) | greiner bio-one | Culture tubes | |
| Drying oven, VENTI-Line | VWR | Oven to cure the PDMS | |
| Handy | Migros | Detergent solution | |
| Hot plate with temperature control | VRW | to cure the PDMS-glass bonding after plasma treatment | |
| ImageJ | FIJI | Image analysis software | |
| Innova 42 Inc Shaker (New Brunswick) | Eppendorf | Incubator | |
| Isopropanol (> 99.8%) | Sigma Aldrich | 67-63-0 | |
| Masterflex transfer tubing | Masterflex | HV-06419-05 | 0.020'' ID, 0.06'' OD |
| Micro Slides, Plain, 75 x 60 mm | Corning | 2947-75X50 | Glass slides |
| Microfluidic pressure sensor (1 bar) | Elveflow | Pressure sensors | |
| Miltex Biopsy puncher, diameter 1.5 mm | Integra | Puncher to make the inlet and outlet holes of the microfluidic channel | |
| mrDev600 developer | Microresist | ||
| Nikon Eclipse Ti2 | Nikon Instruments | Microscope | |
| Nutrient broth n°3 | Sigma Aldrich | ||
| Omnifix Syringe with Luer-Lock | B.Braun | syringes of different volume | |
| Plasma chamber Zepto | Diener Electronic | ZEPTO-1 | used to plasma bond the PDMS and the glass slide |
| Precision wipes (Kimtech Science) | Kimberly Clark | KCP-7552 | to dry the glass slide |
| Scale | VWR-CH | 611-2605 | used to weigh the elastomer to crosslinking agent ratio |
| Silicon wafer (10 cm) | Silicon Materials Inc. | N//Phos <100> 1-10 Ω cm | |
| Spincoater, Spin module SM150 | Sawatec | ||
| SU8 3050 Photoresist | Kayakuam | ||
| Süss MA6 Mask aligner | SUSS MicroTec Group | used to align the chrome-glass mask | |
| Sylgard 184 | Dow Corning | silicone elastomer kit; curing agent | |
| Techni Etch Cr01 | Technic | Technic | |
| Tissue culture dish 150 | TPP | 93150 | |
| Trichloro (1H, 1H, 2H, 2H perfluorooctyl) silane | Sigma Aldrich | Sigma Aldrich | used to silanize the silicane wafer |
| Veeco Dektak 6 M | Veeco | Profilometer |
References
- Flemming, H. C., Wuertz, S. Bacteria and archaea on Earth and their abundance in biofilms. Nature Reviews Microbiology. 17 (4), 247-260 (2019).
- Cunningham, A. B., Sharp, R. R., Hiebert, R., James, G. Subsurface biofilm barriers for the containment and remediation of contaminated groundwater. Bioremediation Journal. 7 (3-4), 151-164 (2003).
- Pronk, W., et al. Gravity-driven membrane filtration for water and wastewater treatment: A review. Water Research. 149, 553-565 (2019).
- Caldara, M., Belgiovine, C., Secchi, E., Rusconi, R. Environmental, microbiological, and immunological features of bacterial biofilms associated with implanted medical devices. Clinical Microbiology and Infection. 35 (2), 00221 (2022).
- Flemming, H. C., Wingender, J. The biofilm matrix. Nature Reviews Microbiology. 8 (9), 623-633 (2010).
- Davey, M. E., O’Toole, G. A. Microbial biofilms: from ecology to molecular genetics. Microbiology and Molecular Biology Reviews. 64 (4), 847-867 (2000).
- Stoodley, P., Dodds, I., Boyle, J. D., Lappin-Scott, H. M. Influence of hydrodynamics and nutrients on biofilm structure. Journal of Applied Microbiology. 85 (1), 19-28 (1998).
- Thomen, P., et al. Bacterial biofilm under flow: First a physical struggle to stay, then a matter of breathing. PLoS ONE. 12 (4), 0175197 (2017).
- Horn, H., Reiff, H., Morgenroth, E. Simulation of growth and detachment in biofilm systems under defined hydrodynamic conditions. Biotechnology and Bioengineering. 81 (5), 607-617 (2003).
- Thullner, M., Mauclaire, L., Schroth, M. H., Kinzelbach, W., Zeyer, J. Interaction between water flow and spatial distribution of microbial growth in a two-dimensional flow field in saturated porous media. Journal of Contaminant Hydrology. 58 (3-4), 169-189 (2002).
- Bottero, S., et al. Biofilm development and the dynamics of preferential flow paths in porous media. Biofouling. 29 (9), 1069-1086 (2013).
- Durham, W. M., Tranzer, O., Leombruni, A., Stocker, R. Division by fluid incision: Biofilm patch development in porous media. Physics of Fluids. 24 (9), 091107 (2012).
- Coyte, K. Z., Tabuteau, H., Gaffney, E. A., Foster, K. R., Durham, W. M. Microbial competition in porous environments can select against rapid biofilm growth. Proceedings of the National Academy of Sciences. 114 (2), 161-170 (2017).
- Taylor, S. W., Jaffé, P. R. Biofilm growth and the related changes in the physical properties of a porous medium: 1. Experimental investigation. Water Resources Research. 26 (9), 2153-2159 (1990).
- Cunningham, A. B., Characklls, W. G., Abedeen, F., Crawford, D. Influence of biofilm accumulation on porous media hydrodynamics. Environmental Science and Technology. 25 (7), 1305-1311 (1991).
- Valiei, A., Kumar, A., Mukherjee, P. P., Liu, Y., Thundat, T. A web of streamers: Biofilm formation in a porous microfluidic device. Lab on a Chip. 12 (24), 5133-5137 (2012).
- Biswas, I., Sadrzadeh, M., Kumar, A. Impact of bacterial streamers on biofouling of microfluidic filtration systems. Biomicrofluidics. 12 (4), 044116 (2018).
- Hassanpourfard, M., Ghosh, R., Thundat, T., Kumar, A. Dynamics of bacterial streamers induced clogging in microfluidic devices. Lab on a Chip. 16 (21), 4091-4096 (2016).
- Stewart, T. L., Scott Fogler, H. Pore-scale investigation of biomass plug development and propagation in porous media. Biotechnology and Bioengineering. 77 (5), 577-588 (2002).
- Hassanpourfard, M., Ghosh, R., Thundat, T., Kumar, A. Dynamics of bacterial streamers induced clogging in microfluidic devices. Lab on a Chip. 16 (21), 4091-4096 (2016).
- Aufrecht, J. A., et al. Pore-scale hydrodynamics influence the spatial evolution of bacterial biofilms in a microfluidic porous network. PLoS ONE. 14 (6), 0218316 (2019).
- Karimifard, S., Li, X., Elowsky, C., Li, Y. Modeling the impact of evolving biofilms on flow in porous media inside a microfluidic channel. Water Research. 188, 116536 (2021).
- Yawata, Y., Nguyen, J., Stocker, R., Rusconi, R. Microfluidic studies of biofilm formation in dynamic environments. Journal of Bacteriology. 198 (19), 2589-2595 (2016).
- Preibisch, S., Saalfeld, S., Tomancak, P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics. 25 (11), 1463-1465 (2009).
- Ratkowsky, D. A., Olley, J., McMeekin, T. A., Ball, A. Relationship between temperature and growth rate of bacterial cultures. Journal of Bacteriology. 149 (1), 1-5 (1982).
- Ostvar, S., et al. Investigating the influence of flow rate on biofilm growth in three dimensions using microimaging. Advances in Water Resources. 117, 1-13 (2018).
- Carrel, M., et al. Biofilms in 3D porous media: Delineating the influence of the pore network geometry, flow and mass transfer on biofilm development. Water Research. 134, 280-291 (2018).
- Iglauer, S., Favretto, S., Spinelli, G., Schena, G., Blunt, M. J. X-ray tomography measurements of power-law cluster size distributions for the nonwetting phase in sandstones. Physical Review E. 82 (5), 10-12 (2010).
- Wu, C., Chu, J., Wu, S., Cheng, L., van Paassen, L. A. Microbially induced calcite precipitation along a circular flow channel under a constant flow condition. Acta Geotechnica. 14 (3), 673-683 (2019).
- Nassar, M. K., et al. Large-scale experiments in microbially induced calcite precipitation (MICP): reactive transport model development and prediction. Water Resources Research. 54 (1), 480-500 (2018).
- Jimenez-Martinez, J., Nguyen, J., Or, D. Controlling pore-scale processes to tame subsurface biomineralization. Reviews in Environmental Science and Biotechnology. 21 (1), 27-52 (2022).
Cite This Article
Kurz, D. L., Secchi, E., Stocker, R., Jimenez-Martinez, J. A Microfluidic Platform to Study Bioclogging in Porous Media. J. Vis. Exp. (188), e64689, doi:10.3791/64689 (2022).