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.

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.

Monitoring polymer degradation under different conditions in the marine environment.

The perdurability of plastics in the environment is one of the major concerns of plastic pollution and, as a consequence, oceans are accumulating large amounts of plastic. The degradation of conventional and biobased materials was evaluated through a laboratory experiment for a year simulating four different conditions in the marine environment. The water column environmental compartment was simulated under euphotic and aphotic (with and without light availability) conditions. The seafloor environmental compartment was simulated with sediment under non-polluted and polluted conditions. By combining weight loss (%), spectroscopic and thermal analyses, the degradation patterns regarding the polymer structure were assessed. The studied biobased materials were polylactic acid (PLA) based materials and showed higher degradability than conventional ones. The weight loss of conventional materials was not influenced by the water column or sediment, while in PLA-based materials, the degradation rates were ca. 5 times greater in the sediment than in the water column. The absorbance (Abs) value at 3400 cm-1 for polyethylene terephthalate (PET), and carbonyl (CO) index for PET and PLA could be useful to detect early signs of degradation. The crystallization index could be a useful parameter to discriminate degradation stages. The obtained results highlight the different degradability rates of materials depending on the specific environmental marine conditions.