Apportioning bacterial carbon source utilization in soil using ¹⁴C isotope analysis of FISH‐targeted bacterial populations sorted by fluorescence activated cell sorting (FACS): ¹⁴C‐FISH‐FACS

Abstract

An unresolved need in microbial ecology is methodology to enable quantitative analysis of in situ microbial substrate carbon use at the population level. Here, we evaluated if a novel combination of radiocarbon-labelled substrate tracing, fluorescence in situ hybridisation (FISH) and fluorescence-activated cell sorting (FACS) to sort the FISH-targeted population for quantification of incorporated radioactivity ( C-FISH-FACS) can address this need. Our test scenario used FISH probe PSE1284 targeting Pseudomonas spp. (and some Burkholderia spp.) and salicylic acid added to rhizosphere soil. We examined salicylic acid- C fate (mineralized, cell-incorporated, extractable and non-extractable) and mass balance (0-24 h) and show that the PSE1284 population captured ∼ 50% of the Nycodenz extracted biomass C. Analysis of the taxonomic distribution of the salicylic acid biodegradation trait suggested that PSE1284 population success was not due to conservation of this trait but due to competitiveness for the added carbon. Adding 50KBq of C sample enabled detection of C in the sorted population at ∼ 60-600 times background; a sensitivity which demonstrates potential extension to analysis of rarer/less active populations. Given its sensitivity and compatibility with obtaining a C mass balance, C-FISH-FACS allows quantitative dissection of C flow within the microbial biomass that has hitherto not been achieved.

Agricultural management and plant selection interactively affect rhizosphere microbial community structure and nitrogen cycling

“…A highly useful starting point would be to delve into dynamic variation in microbial genes involved in carbon metabolism and nitrogen cycling in the rhizosphere, in combination with root exudate metabolomics and measurements of root N uptake. Stable isotope labeling and in situ visualization methods [69–73] could further complement our understanding of how management, plant roots, and their interactive effects shape rhizosphere processes.…”

Advances in automated real‐time flow cytometry for monitoring of bioreactor processes

“…Thus, stochastic average masked signals of heterogeneous populations can be avoided. Consequently, the level of biological noises could be controlled and clear correlations could be established [ 123 , 124 , 125 , 126 , 127 ].…”

Dynamic dielectric properties characterization of tapered dielectrophoresis microelectrodes for selective detection and rapid manipulation of cells

“…The processes of selective detection and rapid manipulation of particles can be done through direct contact and contactless manipulation of targeted particles. The former includes techniques such as the pressure-driven membrane filtration using micro membrane or nano membrane (Chung et al, 2017), micro-gripper (Bolopion et al, 2010), colour label techniques such as epithelial cell adhesion molecule (EpCAM) (Torrico et al, 2018), fluorescence activated cell sorting (Verma et al, 2018;(Gougoulias et al, 2018) and mixing of magnetic materials including magnetic activated cell sorting (MACS) (Suzuki et al, 2007). Overall, these techniques have negative impacts on particles and their medium because of the nature of direct contact and contaminating of materials owing to the dye marker colour and magnetic beads.…”

“…Therefore, in this work, a DEP MEMS consist of microelectrode arrays with tapered profile, have been studied as a tool for highly efficient detection and manipulation of particles and its performance has been compared with those of conventional designs (Chen et al, 2014;Srivastava et al, 2008;Han et al, 2011). The proposed DEP MEMS microelectrodes can selectively detect and manipulate according to the lateral and vertical forces and can be implemented for development of lab-on-a-chip application.…”