Imaging nutrient uptake in bacterial biofilms using quantum dots

Background/Question/Methods

The metabolic activities of soil microbes are the primary drivers of biogeochemical processes controlling the terrestrial carbon cycle, nutrient availability to plants, contaminant remediation, water quality, and other ecosystem services. However, we have a limited understanding of microbial metabolic processes such as nutrient uptake rates, substrate preferences, or how microbes and microbial metabolism are distributed throughout the three-dimensional complex of the soil. Here we use a novel imaging technique with quantum dots (QDs, engineered semiconductor nanoparticles that produce size or composition-dependent fluorescence) to measure bacterial uptake of substrates of varying complexity. Cultures of two organisms differing in cell wall structure -- Bacillus subtilis (a member of the Firmicutes) and Pseudomonas fluorescens (a member of the Proteobacteria) -- were grown in biofilms in one of four ecologically relevant experimental conditions: nitrogen limitation, phosphorus limitation, nitrogen and phosphorus limitation, or no nutrient limitation. The biofilms were then exposed to QDs with and without organic nutrients attached.

Results/Conclusions

We found that uptake of QDs conjugated to organic substrates varied depending on growth conditions and substrate, suggesting that they are a useful indicator of bacterial ecology. Cellular uptake was similar for the two bacterial species, ranging from 130 to 6,700 nanoparticles per cm³ of cell tissue. Uptake of QDs not conjugated to an organic molecule was negligible, indicating that bacteria actively consume the QD-labeled nutrient rather than QDs passively entering cells. On average, QD assimilation was six times greater when nitrogen or phosphorus was limiting (i.e., the substrate conjugated to the QD-provided nitrogen and/or phosphorus that was experimentally limited in the growth medium). Overall, cells took up about twice as much phosphoserine compared to other substrates, likely because it was the only compound providing both nitrogen and phosphorus.  These results showed that regardless of their cell wall structure, bacteria can selectively take up quantifiable levels of QDs based on substrate and environmental conditions. These findings offer a new way to experimentally investigate basic bacterial ecology such as metabolic activity and biofilm development and function.