Perspective Cite This: Biochemistry XXXX, XXX, XXX−XXX
pubs.acs.org/biochemistry
New Techniques for the Generation and Analysis of Tailored Microbial Systems on Surfaces Ariel L. Furst,† Matthew J. Smith,† and Matthew B. Francis*,†,‡ †
Department of Chemistry, University of California, Berkeley, Berkeley, California 94720-1460, United States Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460, United States
‡
ABSTRACT: The interactions between microbes and surfaces provide critically important cues that control the behavior and growth of the cells. As our understanding of complex microbial communities improves, there is a growing need for experimental tools that can establish and control the spatial arrangements of these cells in a range of contexts. Recent improvements in methods to attach bacteria and yeast to nonbiological substrates, combined with an expanding set of techniques available to study these cells, position this field for many new discoveries. Improving methods for controlling the immobilization of bacteria provides powerful experimental tools for testing hypotheses regarding microbiome interactions, studying the transfer of nutrients between bacterial species, and developing microbial communities for green energy production and pollution remediation.
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patterns. In addition, the new characterization techniques that are being used to understand the responses of microbes to external stimuli are discussed.
he specific interactions that occur when microorganisms bind to surfaces can profoundly influence their growth, gene expression, and pathogenicity. For mammalian cells, signaling events between interacting cells have been wellstudied in the contexts of immune synapse formation,1 stem cell differentiation,2,3 and cancer invasion,4 and the physiochemical properties of the extracellular matrix5 have also been observed to have profound effects on cell behavior. It is becoming increasingly appreciated that context-specific stimuli are similarly influential for microbial organisms as they engage different types of cells6 and surfaces.7 However, these interactions are difficult to study with precise control over the spacing of microbes and the ratio of individual microbes in microbial communities. At the center of efforts to improve control over the placement of microbes on surfaces are a growing number of techniques that can be used to establish and maintain specific interactions between microbial cells and other surfaces. As improvements are made to control bacterial immobilization, the complexity of biological questions that can be probed continues to increase. As we learn more about the microbial interactions involved in the human biome, these experimental tools will play an increasingly important role in testing hypotheses that are central to nutrient processing and the progression of many diseases. In addition, the ability to control the surface interactions of microbial organisms is expanding their roles as biosynthetic producers,8,9 biosensors,10 logic components,11 and even sources of electrical current.12,13 In this Perspective, we initially discuss current analytical techniques to evaluate biofilms and microbial communities. We then describe the methods that are currently available for the attachment of microorganisms to synthetic and biologically derived surfaces. The utility of each technique is evaluated for the generation of controlled biofilms, the tethering of microbes to electrodes, and the organization of multiple types of organisms into two- and three-dimensional multicellular © XXXX American Chemical Society
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CHARACTERIZATION TECHNIQUES FOR STUDYING CELLS ON SURFACES Recent years have seen an expansion of the biophysical techniques that can be used to characterize cell behavior. The best technique for a given application varies depending on the feature sizes to be observed, the transparency of the sample, and the time scale that needs to be evaluated. In many cases, multiple techniques are used in concert to provide complementary information. Brief summaries of the most common techniques in use today follow. Optical Microscopy. Optical microscopy is used extensively to study bacterial biofilms, especially in combination with the other imaging techniques described below. Imaging without staining can be used to determine the total biomass in a biofilm based on the transmittance of light through the film,14,15 and both Gram staining and live/dead cell staining can provide especially useful information about the distribution of cells.15 The use of chemically specific stains can reveal the distributions of particular biomolecules,16 including nucleotides, glycopolymers, and proteins, and immunofluorescence techniques can report the locations of specific molecules. For a comprehensive review of optical techniques, see the 2002 review by Dunne.17 Atomic Force Microscopy. Atomic force microscopy (AFM) is an especially powerful tool for imaging biofilms by raster-scanning a tip across a sample surface. This method can be performed under vacuum, at ambient atmosphere, or in Received: March 16, 2018 Revised: April 24, 2018
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DOI: 10.1021/acs.biochem.8b00324 Biochemistry XXXX, XXX, XXX−XXX
Perspective
Biochemistry
formation of biofilms. An electrochemical quartz crystal microbalance (EQCM) has been used to monitor the total biomass in biofilms throughout their development; voltammetric techniques are often used to monitor electroactive components within biofilms, and electrochemical impedance spectroscopy (EIS) can be used to monitor the development of biofilms and the density of microbes on an electrode surface. Reviews of several of these techniques are available, including EQCM,26 EIS,27 and voltammetry.28 Often, these techniques are used in combination with the other techniques described above to garner the most information possible about microbial adhesion and biofilm maturation.
water, and it provides images in three dimensions. The subnanometer to nanometer resolution of AFM makes it useful for imaging components of biofilms, from whole cells to extracellular polymeric substances (EPS).18,19 AFM has been extensively used for imaging the conformational changes that occur during the early stages of biofilm development, especially on industrial surfaces.18 A 2017 review by Wright and coworkers provides insight into this technique for applications on surfaces relevant to water treatment and construction.20 Because the force applied to the tip during imaging can be varied, AFM is also an efficient method for evaluating the strength of cellular adherence to a particular surface. This information is vital to understanding disinfection and the removal of biofilms from industrial and medical surfaces. In 2016, Ting and co-workers applied AFM to study the adhesion strength of secreted EPS in Pseudomonas aeruginosa and Bacillus subtilis biofilms. Using single-molecule force spectroscopy (SMFC), they found that polysaccharide components increased adhesion strength significantly more than protein components did.21 Additional examples of the results of force applied to biofilms through AFM can be found in work by Gordon and co-workers.22,23 While this technique is advantageous for its real-time, nondestructive imaging, AFM is often limited by the size of the area that can be imaged. Although translucence is not required, it should also be noted that AFM can provide information about only the top surface of a sample. Scanning Electron Microscopy. As with AFM, scanning electron microscopy (SEM) provides three-dimensional (3D) images of both the cells within the biofilm and the EPS. Because of the ease of obtaining 3D images of complete biofilms with this method, SEM has been used extensively to evaluate the effects of antibacterial agents on the conformation of EPS components. SEM can also be used to confirm the integrity of cells that are immobilized on surfaces. For example, in our 2017 paper describing a bacterial biosensor that relies on Escherichia coli binding to a gold surface, SEM was used to confirm that the cells maintained their morphology following lyophilization.10 SEM is normally limited to the qualitative analysis of bacterial films and typically relies on the use of sputtered metal coatings that can mask specific chemical information about the original cells. Though quantitative SEM methods have been developed for biofilm analysis,24 the instrumentation required for such techniques is especially costly. Surface Plasmon Resonance. The optical technique surface plasmon resonance (SPR) is based on changes in the refractive index of a metal surface upon the adsorption of analytes of interest. It is especially useful for the study of biofilms and the mobility of cells within those films because it allows extremely sensitive monitoring of adhesion of a small molecule to a surface over imaging windows of ≤1 cm2. This method has been used to study the kinetics of bacterial biofilm formation on a variety of substrate materials. Jarvis and coworkers’ 2004 review provides an excellent overview of this technique for P. aeruginosa biofilm formation.25 The speed and low cost of SPR make it especially attractive for the rapid screening of binding of bacteria to a variety of surfaces. Electrochemistry. Electrochemistry provides a single technique that can both position microbes on surfaces and monitor biofilm conditions with high sensitivity. Electrochemistry has the additional advantage of providing rapid readout with low-cost and portable instrumentation. Several electrochemical techniques are commonly used to evaluate the
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ATTACHMENT OF MICROBES TO SURFACES THROUGH NONSPECIFIC ADHESION The most facile method of forming bacterial layers is to allow them to generate natural biofilms, either through nonspecific adhesion or after direct capture. These strategies provide a general method for the attachment of bacteria to surfaces, but they do not allow one to control the cell densities or the ratios of multiple cell types. Controlling the density of cells and the ratio of multiple species of cells on a surface would better enable the precise study of microbes throughout the biofilm formation process.29 In addition, controlling the relative ratio of multiple microbial species on a surface would facilitate the study of cocultures for feedstock production30,31 and development of mimics of biologically relevant microbial colonies, such as the human gut32,33 or skin microbiome.34 Nonetheless, this remains the most common technique for bacterial immobilization. Unassisted Biofilm Formation. Biofilms are comprised of microorganisms within a complex matrix of extracellular components. In fact, microorganisms are
