Zooming in to see the bigger picture: Microfluidic and nanofabrication tools to study bacteria

Background Nanotechnology and bacteriology at first sight may seem like two disparate worlds, but a rapidly moving field of research has formed at the interface of these disciplines in the past decade. Bacteria experience spatial structure at many scales: Individual bacteria interact with nanoscale surface features, whereas bacterial communities are shaped by landscape structure down to the microscale. Nanofabrication and microfluidics are ideally suited to define and control the environment at those scales, allowing us to zoom in on the peculiarities of individual cells and to broaden our understanding of the processes that shape multi-species communities. Recently developed nanotools provide unprecedented control over the bacterial microenvironment and have been key to the discovery of new phenomena in bacteriology. Studying bacteria using nanofabrication and microfluidics. (A) Escherichia coli bacteria use their flagella to exploit submicrometer crevices for surface attachment [Reprinted with permission from (5) (reference list of full paper online)]. (B) Biofilm streamers form in a meandering flow channel (Pseudomonas aeruginosa, red; extracellular polymeric substances, green) [Reprinted with permission from (93)]. (C) E. coli undergo a shape transition when squeezing into a nanofabricated channel as shallow as half their width [Reprinted with permission from (26)]. Scale bars, (A) 2 µm; (B) 200 µm; and (C) 5 µm. Advances Nanofabrication and microfluidics have expanded our view on a myriad of bacterial phenomena. Microfluidics provides ways to study individual bacteria in dynamic and well-defined environments and has been used to address long-standing questions concerning bacterial aging and antibiotic persistence. Biological insights have been gained by exploring bacterial growth and movement in nanofabricated constrictions and revealed that bacteria can penetrate constrictions as narrow as only half their width. Furthermore, nanofabrication has been used to discriminate between competing hypotheses regarding the mechanisms that underlie intercellular electron transport. Confinement of single bacteria in tiny volumes has provided an individualistic perspective on collective phenotypes and demonstrated that density-dependent behaviors can even be exhibited by individuals. Bacteria growing in nanofabricated chambers adopt predefined shapes and have been used to study the geometry dependence of intracellular processes. Microfluidics and nanofabrication have been combined to create synthetic ecosystems in which the spatial eco-evolutionary dynamics of bacterial communities can be explored. Various approaches to mimic the intricate spatial structure of natural bacterial habitats now contribute to our understanding of competition and cooperation within bacterial populations. Microfluidic platforms have boosted research on unculturable environmental species by eliminating the need for pre-analysis culturing. On-chip whole-genome amplification of environmental isolates has recently provided a first genotypic glimpse on this “dark matter of biology.” Outlook Looking ahead, it is clear that the doors that nanofabrication and microfluidics have opened will continue to make important contributions to basic bacteriology research. A comprehensive investigation of the uncultured majority with microfluidic technologies, for instance, may uncover the vast potential of currently unknown species. Practical applications such as microbial fuel cells or antibacterial surfaces will benefit from the understanding of bacterial behavior at the nanoscale. Microfluidic devices are now beginning to be commonly used in microbiology labs because of a demand for precise measurements in complex environments that can be controlled at the microscale. This trend will undoubtedly continue as scientists delve deeper into the complex lives of bacteria. Experimental platforms for probing bacteria Bacteria respond to a host of changing cues provided by their environment. Hol and Dekker review how microfluidic and nanofabricated devices can provide a platform to deliver different stimuli in a variety of environments. Bacterial quorum sensing and electron transport are among the problems that can be studied in this way. Science, this issue 10.1126/science.1251821 The spatial structure of natural habitats strongly affects bacterial life, ranging from nanoscale structural features that individual cells exploit for surface attachment, to micro- and millimeter-scale chemical gradients that drive population-level processes. Nanofabrication and microfluidics are ideally suited to manipulate the environment at those scales and have emerged as powerful tools with which to study bacteria. Here, we review the new scientific insights gained by using a diverse set of nanofabrication and microfluidic techniques to study individual bacteria and multispecies communities. This toolbox is beginning to elucidate disparate bacterial phenomena—including aging, electron transport, and quorum sensing—and enables the dissection of environmental communities through single-cell genomics. A more intimate integration of microfluidics, nanofabrication, and microbiology will enable further exploration of bacterial life at the smallest scales.