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A microfluidic gradient chamber (MGC) and a homogeneous batch culturing system were used to evaluate whether spatial concentration gradients of the antibiotic ciprofloxacin allow development of greater antibiotic resistance in Escherichia coli strain 307 (E. coli 307) compared to exclusively temporal concentration gradients, as indicated in an earlier study. A linear spatial gradient of ciprofloxacin and Luria–Bertani broth (LB) medium was established and maintained by diffusion over 5 days across a well array in the MGC, with relative concentrations along the gradient of 1.7–7.7× the original minimum inhibitory concentration (MICoriginal). The E. coli biomass increased in wells with lower ciprofloxacin concentrations, and only a low level of resistance to ciprofloxacin was detected in the recovered cells (∼2× MICoriginal). Homogeneous batch culture experiments were performed with the same temporal exposure history to ciprofloxacin concentration, the same and higher initial cell densities, and the same and higher nutrient (i.e., LB) concentrations as in the MGC. In all batch experiments, E. coli 307 developed higher ciprofloxacin resistance after exposure, ranging from 4 to 24× MICoriginal in all replicates. Hence, these results suggest that the presence of spatial gradients appears to reduce the driving force for E. coli 307 adaptation to ciprofloxacin, which suggests that results from batch experiments may over predict the development of antibiotic resistance in natural environments.

Bacterial biofilms are a metabolically heterogeneous community of bacteria distributed in an extracellular matrix comprised primarily of hydrated polysaccharides. Effective inhibitory concentrations measured under planktonic conditions are not applicable to biofilms, and inhibition concentrations measured for biofilms vary widely. Here, we introduce a novel microfluidic approach for screening respiration inhibition of bacteria in a biofilm array morphology. The device geometry and operating conditions allow antimicrobial concentration and flux to vary systematically and predictably with space and time. One experiment can screen biofilm respiratory responses to many different antimicrobial concentrations and dosing rates in parallel. To validate the assay, onset of respiration inhibition following NaN₃ exposure is determined optically using an O₂-sensing thin film. Onset of respiration inhibition obeys a clear and reproducible pattern based on time for diffusive transport of the respiration inhibitor to each biofilm in the array. This approach can be used for high-throughput screening of antimicrobial effectiveness as a function of microbial characteristics, antimicrobial properties, or antimicrobial dosing rates. The approach may also be useful in better understanding acquired antimicrobial resistance or for screening antimicrobial combinations.

Subsurface environments often contain mixtures of contaminants in which the microbial degradation of one pollutant may be inhibited by the toxicity of another. Agricultural settings exemplify these complex environments, where antimicrobial leachates may inhibit nitrate bioreduction, and are the motivation to address this fundamental ecological response. In this study, a microfluidic reactor was fabricated to create diffusioncontrolled concentration gradients of nitrate and ciprofloxacin under anoxic conditions in order to evaluate the ability of Shewanella oneidenisis MR-1 to reduce the former in the presence of the latter. Results show a surprising ecological response, where swimming motility allow S. oneidensis MR-1 to accumulate and maintain metabolic activity for nitrate reduction in regions with toxic ciprofloxacin concentrations (i.e., 50× minimum inhibitory concentration, MIC), despite the lack of observed antibiotic resistance. Controls with limited nutrient flux and a nonmotile mutant (Δf lag) show that cells cannot colonize antibiotic rich microenvironments, and this results in minimal metabolic activity for nitrate reduction. These results demonstrate that under anoxic, nitrate-reducing conditions, motility can control microbial habitability and metabolic activity in spatially heterogeneous toxic environments.

Since some antifreeze proteins and glycoproteins (AF(G)Ps) cannot directly bind to all crystal planes, they change ice crystal morphology by minimizing the area of the crystal planes to which they cannot bind until crystal growth is halted. Previous studies found that growth along the c-axis (perpendicular to the basal plane, the crystal plane to which these AF(G)Ps cannot bind) is accelerated by some AF(G)Ps, while growth of other planes is inhibited. The effects of this growth acceleration on crystal morphology and on the thermal hysteresis activity are unknown to date. Understanding these effects will elucidate the mechanism of ice growth inhibition by AF(G)Ps. Using cold stages and an Infrared laser, ice growth velocities and crystal morphologies in AF(G)P solutions were measured. Three types of effects on growth velocity were found: concentration-dependent acceleration, concentration-independent acceleration, and concentrationdependent deceleration. Quantitative crystal morphology measurements in AF(G)P solutions demonstrated that adsorption rate of the proteins to ice plays a major role in determining the morphology of the bipyramidal crystal. These results demonstrate that faster adsorption rates generate bipyramidal crystals with diminished basal surfaces at higher temperatures compared to slower adsorption rates. The acceleration of growth along the c-axis generates crystals with smaller basal surfaces at higher temperatures leading to increased growth inhibition of the entire crystal.

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Microbial sulfate reduction (biosouring) is ubiquitous in natural and engineered environments. It can be especially problematic during oil recovery from unconventional reservoirs, where the growth of sulfate-reducing bacteria (SRB) biofilms can clog fracture pathways, decrease hydrocarbon production, and produce hydrogen sulfide in the produced oil and water that corrodes pipelines and threatens both human health and the environment. Relevant experimental data on shale fractures, assessing the conditions that affect SRB growth, distribution, and activity for sulfate reduction, are sparse, and this has limited our ability to evaluate predictive models for assessing the impacts. To address this limitation, a 250 μm wide, 170 μm deep, and 2 cm long microchannel was constructed within an actual sample of subsurface core from the Devonian age New Albany Shale of the Illinois Basin to simulate a hydraulically induced fracture (fracked shale). This real rock flow-through microfluidic system, called the GeoBioCell (GBC), was designed with two inlets and one outlet. At flow rates of 3.4−80 μL/h, an SRB enrichment culture with hydrocarbon degradation products (i.e., fatty acids) was fed into one inlet, while dissolved sulfate (30 mM) was introduced into the other inlet. These were not allowed to mix until they entered the microchannel, where rapid biomass growth and sulfate reduction took place. SRB biomass was quantified as a function of both influent sulfate flux using brightfield microscopy and SRB metabolic sulfide production using an effluent zinc trap. Results indicate that biomass grows on shale fracture walls and in the channel and increases with the influent organic acid and sulfate flux, and that the downflow SRB biofilm growth on microchannel walls is limited by the upstream metabolic consumption of electron donors. An advection-diffusion-reaction model was developed to interpret these results, and a specific sulfate reduction rate of 1.0 ± 0.4 × 10 −3 mmol SO 4 2− /(mmol biomass•s) or 9 ± 4 × 10 −6 mmol SO 4 2− /(mg biomass•s) was obtained. This rate, among the highest reported to date, suggests that nearly optimal conditions for SRB sulfate reduction were attained within the microchannel that were independent of mass transfer limitations. Future tests using similar real rock microfluidic experimentation will be useful for assessing worst case scenarios for subsurface reservoir biosouring models and establishing new mitigation strategies to prevent adverse human health and environmental impacts.