Rapid Enrichment and Isolation of Polyphosphate-Accumulating Organisms Through 4'6-Diamidino-2-Phenylindole (DAPI) Staining With Fluorescence-Activated Cell Sorting (FACS).

Mia Terashima,Souichiro Kato,Yoichi Kamagata

doi:10.3389/fmicb.2020.00793

Abstract

Screening for bacteria with abilities to accumulate valuable intracellular compounds from an environmental community is difficult and requires strategic methods. Combining the experimental procedure for phenotyping living cells in a microbial community with the cell recovery necessary for further cultivation will allow for an efficient initial screening process. In this study, we developed a strategy for the isolation of polyphosphate-accumulating organisms (PAOs) by combining (i) nontoxic fluorescence staining of polyphosphate granules in viable microbial cells and (ii) fluorescence-activated cell sorting (FACS) for the rapid detection and collection of target cells. To implement this screening approach, cells from wastewater sludge samples were stained with 4’6-diamidino-2-phenylindole (DAPI) to target cells with high polyphosphate (polyP) accumulation. We found a staining procedure (10 μg/ml of DAPI for 30 min) that can visualize polyP granules while maintaining viability for the majority of the cells (>60%). The polyP positive cells were recovered by FACS, purified by colony isolation and phylogenetically identified by 16S rRNA gene sequencing. Follow-up analysis confirmed that these isolates accumulate polyP, indicating that DAPI can be implemented in staining living cells and FACS can effectively and rapidly screen and isolate individual cells from a complex microbial community.

Highlights

The genetic and metabolic diversity of microbial species provide a wealth of possibilities for biotechnological applications
We propose a strategy for the isolation of polyphosphate-accumulating organisms (PAOs) by coupling (i) specific staining of an intracellular accumulate without compromising cell viability, and (ii) rapid detection and sorting of target cells by using fluorescence-activated cell sorting (FACS), for which wastewater sludge samples were utilized
We examined the viability of the cells after DAPI staining and determined a condition that allows for the majority of stained cells remain alive, enabling effective isolation of polyphosphateaccumulating organisms (PAOs) after FACS-based phenotype screening

Summary

Introduction

The genetic and metabolic diversity of microbial species provide a wealth of possibilities for biotechnological applications. Highly effective isolation methods are largely based on selection by growth capabilities. The isolation of microorganisms that accumulate valuable compounds is often a result of laborious individual strain screening, hypothesisdriven isolation methods, or by chance discovery. Persistent screenings of bacterial isolates from seaweed led to the identification of a strain capable of polyhydroxyalkanoates (PHA) production from starch, after investigation of numerous strains for PHA accumulation (Han et al, 2014). Identification of a dominant phylotype Dechloromonas in a wastewater treatment plant using 16S rRNA sequencing followed by screening for bacterial strains allowed the isolation of polyphosphateaccumulating strains (Terashima et al, 2016). In order to avoid laborious screening of single strains during targeted isolation endeavors, establishing a method to initially enrich for strains showing promising characteristics directly from a mixed population would vastly accelerate the downstream screening process. Methods to analyze accumulation of specific compounds in individual living cells from a community, followed by recovery and isolation, are still limited

Methods

Results

Conclusion