Visualizing and Isolating Iron-Reducing Microorganisms at the Single-Cell Level

Abstract

Iron-reducing microorganisms (FeRM) play key roles in many natural and engineering processes. Visualizing and isolating FeRM from multispecies samples are essential to understand the in-situ location and geochemical role of FeRM. Here, we visualized FeRM by a “turn-on” Fe2+-specific fluorescent chemodosimeter (FSFC) with high sensitivity, selectivity and stability. This FSFC could selectively identify and locate active FeRM from either pure culture, co-culture of different bacteria or sediment-containing samples. Fluorescent intensity of the FSFC could be used as an indicator of Fe2+ concentration in bacterial cultures. By integrating FSFC with a single cell sorter, we obtained three FSFC-labeled cells from an enriched consortia and all of them were subsequently evidenced to be capable of iron-reduction and two unlabeled cells were evidenced to have no iron-reducing capability, further confirming the feasibility of the FSFC. IMPORTANCE Visualization and isolation of FeRM from samples containing multispecies are commonly needed by researchers from different disciplines, such as environmental microbiology, environmental sciences and geochemistry. However, no available method has been reported. In this study, we provide a solution to visualize FeRM and evaluate their activity even at single cell level. Integrating with single cell sorter, FeRM can also be isolated from samples containing multispecies. This method can be used as a powerful tool to uncover the in-situ or ex-situ role of FeRM and their interactions with ambient microbes or chemicals.

Multi-heme cytochrome-mediated extracellular electron transfer by the anaerobic methanotroph ‘Candidatus Methanoperedens nitroreducens’

“…M. nitroreducens’ cells exhibited bright FeRhoNox fluorescence (Fig. 1b, c ), indicating that Fe 2+ was absorbed to their outer surface 24 . FeRhoNox positive cells almost entirely overlapped with ‘ Ca .…”

“…M. nitroreducens’ via EET directly to iron, or to iron-reducing syntrophic partners 21 , 23 , FeRhoNox fluorescence staining was employed to identify the microorganisms directly responsible for the observed iron reduction. The method is based on Fe 2+ -selective chemosensory fluorescence on the cell surface of active iron-reducing microorganisms 24 . Under iron-reducing incubations, ‘ Ca .…”

Coupled magnetic nanoparticle‐mediated isolation and single‐cell image recognition to detect Bacillus' cell size in soil

“…Here, the combination of MMI and SCIR approach in our study makes it possible to focus on the single-cell level. This improvement is beneficial for the integration of downstream analysis with microbial morphology (Chen et al, 2021;Gan et al, 2021). Therefore, the established experimental and analytical methods are based on MMI.…”

“…Furthermore, single-cell image recognition (SCIR) was developed as a prospective technology to investigate the morphology of the active microbes isolated by MMI. Gan et al (2021) successfully identified and located active iron-reducing microorganisms from both pure culture and sediment-containing samples by coupling fluorescent and chemosensor confocal laser scanning microscopy at the single-cell level. However, coupling MMI and SCIR for visualisation of active functional microorganisms and evaluation of their size even at the single-cell level is still lacking in studies of the soil ecosystem.…”

Precision isolation and cultivation of single cells by vortex and flat-top laser ejection

“…Laser-induced forward transfer (LIFT), based on the interaction principles between laser and materials ( Serra and Piqué, 2019 ), possesses the capability to precisely transfer minute substances and has been applied in efficient isolation and cultivation of single cells ( Kattamis et al, 2007 ; Marquez et al, 2020 ). Compared to other sorting techniques, LIFT cell sorting technology has a higher microscopic resolution, and can be used to sort target microbial cells based on a variety of characteristics such as morphology, size, fluorescence, and Raman molecular fingerprints ( Song et al, 2017 ; Gan et al, 2021 ). Additionally, due to its capability to transfer cells from both liquid and solid samples, LIFT sorting technology enables single-cell isolation in a state closer to their original environment, making it more suitable for the separation of microorganisms in complex environments ( Ringeisen et al, 2015 ).…”