Microfluidics for adaptation of microorganisms to stress: design and application

Abstract

Microfluidic systems have fundamentally transformed the realm of adaptive laboratory evolution (ALE) for microorganisms by offering unparalleled control over environmental conditions, thereby optimizing mutant generation and desired trait selection. This review summarizes the substantial influence of microfluidic technologies and their design paradigms on microbial adaptation, with a primary focus on leveraging spatial stressor concentration gradients to enhance microbial growth in challenging environments. Specifically, microfluidic platforms tailored for scaled-down ALE processes not only enable highly autonomous and precise setups but also incorporate novel functionalities. These capabilities encompass fostering the growth of biofilms alongside planktonic cells, refining selection gradient profiles, and simulating adaptation dynamics akin to natural habitats. The integration of these aspects enables shaping phenotypes under pressure, presenting an unprecedented avenue for developing robust, stress-resistant strains, a feat not easily attainable using conventional ALE setups. The versatility of these microfluidic systems is not limited to fundamental research but also offers promising applications in various areas of stress resistance. As microfluidic technologies continue to evolve and merge with cutting-edge methodologies, they possess the potential not only to redefine the landscape of microbial adaptation studies but also to expedite advancements in various biotechnological areas. KEY POINTS: • Microfluidics enable precise microbial adaptation in controlled gradients. • Microfluidic ALE offers insights into stress resistance and distinguishes between resistance and persistence. • Integration of adaptation-influencing factors in microfluidic setups facilitates efficient generation of stress-resistant strains.

Keywords: Adaptive laboratory evolution; Gradient systems; Microbial adaptation; Microfluidics; Strain improvement; Stress resistance.

Fig. 1

General concept of main adaptive laboratory evolution (ALE) approaches. a ALE through batch culture. Aliquots are serially transferred at regular time intervals (Δt) to new cultures with a gradually increased stressor concentration. b In this system, population size is dynamically changing in every batch; however, the overall fitness is enhanced over time (t) as a result of the increasing stressor concentration. c ALE through continuous culture. Fresh media combined with a gradually increasing stressor concentration are fed continuously, and a proportional volume is removed to the waste. d Because of the optimized cultivation conditions, the population size mostly stays constant, while the overall fitness increases over time (t)

Fig. 2

Commonly existing and alternative flow gradient creation strategies. The gradient chamber for cultivation may be positioned in two distinct configurations: external to the fluid flow, referred to as “ex-flow” (a), or internal to the fluid flow, denoted as “in-flow” (b, c) gradients. In the conventional ex-flow (a) and in-flow (b) systems, the gradient and screening processes are typically aligned perpendicularly with respect to the flow direction. In contrast, the alternative model introduced in (c) implements a parallel orientation of both the gradient and screening relative to the flow direction. Low-stress conditions are indicated in blue at point X, while high-stress conditions are depicted in red at point Y. The goal of adaptation is to move cells from the low-stress conditions (point X) for selection and to enrich cells that become adapted to stress at areas of high stress (point Y). Adapted with permission from Zoheir et al. (2021) and the Authors (2021) Small

Fig. 3

Microfluidic systems for adaptive laboratory evolution (ALE). a–d Hexagonal gradient chamber for ciprofloxacin adaptation (Zhang et al. 2011b). a This system comprises a network of interconnected microwells, designed for the adaptation of microorganisms to ciprofloxacin (Cipro) within LB medium. b A gradient of ciprofloxacin is meticulously established within the chamber, as depicted. c Remarkably, resistant E. coli cells emerge within a mere 5 h, particularly at the steepest gradient point, aptly referred to as “Goldilocks” (highlighted by the orange arrow). d Subsequently, the adapted cells manifest their growth, visualized through green fluorescence after 30 h. e, f Gradient system for stress-directed growth (Deng et al. 2019). e In this system, a unique gradient strategy is employed, focusing on directing microbial growth toward regions with high-stress levels. On one side of the chip, a M9 minimal medium with limited nutrients coexists with LB medium containing not only rich nutrients but also the antibiotic ciprofloxacin (Cipro). f E. coli growth is clearly marked by green fluorescence in this innovative gradient system. a Adapted with permission from Zhang et al. (2011b), copyright (2011) American Chemical Society. b Adapted with permission from Bos and Austin (2018), copyright (2018) Elsevier. c, d Adapted with permission from Zhang et al. (2011a), copyright (2011) The American Association for the Advancement of Science. e, f Adapted with permission from Deng et al. (2019), copyright (2019) American Chemical Society

Fig. 4

Structure of the evo.S microfluidic chip. a The evo.S chip is fabricated using polydimethylsiloxane (PDMS), a biocompatible and gas-permeable material that ensures an ideal environment for microbial growth. b The chip accommodates microbial cultures within wells that serve as versatile microenvironments for hosting mixed populations of planktonic cells and biofilms. The dimensions of the wells are carefully tailored, and the wells are designed to be deeper than the channels. c The evo.S chip’s distinctive feature lies in its capability to cumulatively create chemical gradients within interconnected wells. This is achieved through the stepwise supplementation of the main flow with defined stressor concentrations at precise flow rates. The controlled formation of gradients is a pivotal aspect of the chip’s functionality, contributing to the success of ALE experiments conducted within this system. Reprinted with permission from Zoheir et al. (2021) and the Authors (2021) Small

Fig. 5

Impact of gradient profiles on the adaptability of E. coli to nalidixic acid (NA) within the evo.S chip. a The study employed two distinct concentration profile modes: a smooth gradient denoted as “chip-A” and a steep gradient designated as “chip-B.” Remarkably, both profiles yielded similar E. coli growth on the chip after approximately 6 days (b). c To assess the adaptability of E. coli populations, screening was conducted on selective agar plates containing 40 μg/mL NA. Notably, the colonies observed on these plates indicated successful evolution, primarily within the context of the steep gradient (chip-B) profiles, thus underscoring the role of gradient steepness in facilitating adaptation. d The influence of the antibiotic gradient on adaptation phenotypes within the evo.S chip. In the case of chip-A (smooth gradient), bacteria experience a sequential transition through phases: (1) a phase devoid of antibiotics, (2) a phase containing sub-inhibitory concentrations inducing persister cell formation, and (3) a phase featuring super-inhibitory concentrations. In this scenario, persister cells emerge and can endure the subsequent super-inhibitory concentrations, leading to persistence. In contrast, chip-B (steep gradient) directly exposes sensitive cells from phase (1) to super-inhibitory concentrations in phase (2), inducing and selecting for resistant mutants. Adapted with permission from Zoheir et al. (2021) and the Authors (2021) Small