Analysis of Polyhydroxyalkanoates Granules in Haloferax mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy

Haloferaxmediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.This work was funded by a research grant from Instituto de Fomento de la Region de Murcia (INFO), Spain (2020.08.CT01.0039) and the University of Alicante (VIGROB-309)

Haloarchaea as Cell Factories to Produce Bioplastics

Plastic pollution is a worldwide concern causing the death of animals (mainly aquatic fauna) and environmental deterioration. Plastic recycling is, in most cases, difficult or even impossible. For this reason, new research lines are emerging to identify highly biodegradable bioplastics or plastic formulations that are more environmentally friendly than current ones. In this context, microbes, capable of synthesizing bioplastics, were revealed to be good models to design strategies in which microorganisms can be used as cell factories. Recently, special interest has been paid to haloarchaea due to the capability of some species to produce significant concentrations of polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), and polyhydroxyvalerate (PHV) when growing under a specific nutritional status. The growth of those microorganisms at the pilot or industrial scale offers several advantages compared to that of other microbes that are bioplastic producers. This review summarizes the state of the art of bioplastic production and the most recent findings regarding the production of bioplastics by halophilic microorganisms with special emphasis on haloarchaea. Some protocols to produce/analyze bioplastics are highlighted here to shed light on the potential use of haloarchaea at the industrial scale to produce valuable products, thus minimizing environmental pollution by plastics made from petroleum.This work was funded by research grant from the MINECO Spain (RTI2018‐099860‐B‐I00), University of Alicante, Spain (VIGROB‐309), INFO Región de Murcia, Spain (2020.08.CT01.0039) and grant from Deanship of Scientific Research, University of Jeddah, Jeddah, Saudi Arabia, (UJ‐02‐015‐ICGR)