Hypersaline microbial mats develop through seasonal and diel fluctuations, as well as under several physicochemical variables. Hence, resident microorganisms commonly employ strategies such as the synthesis of polyhydroxyalkanoates (PHAs) in order to resist changing and stressful conditions. However, the knowledge of bacterial PHA production in hypersaline microbial mats has been limited to date, particularly in regard to medium-chain length PHAs (mcl-PHAs), which have biotechnological applications due to their plastic properties. The aim of this study was to obtain evidence for PHA production in two hypersaline microbial mats of Guerrero Negro, Mexico by searching for PHA granules and PHA synthase genes in isolated bacterial strains and environmental samples. Six PHA-producing strains were identified by 16S rRNA gene sequencing; three of them corresponded to a Halomonas sp. In addition, Paracoccus sp., Planomicrobium sp. and Staphylococcus sp. were also identified as PHA producers. Presumptive PHA granules and PHA synthases genes were detected in both sampling sites. Moreover, phylogenetic analysis showed that most of the phylotypes were distantly related to putative PhaC synthases class I sequences belonging to members of the classes Alphaproteobacteria and Gammaproteobacteria distributed within eight families, with higher abundances corresponding mainly to Rhodobacteraceae and Rhodospirillaceae. This analysis also showed that PhaC synthases class II sequences were closely related to those of Pseudomonas putida, suggesting the presence of this group, which is probably involved in the production of mcl-PHA in the mats. According to our state of knowledge, this study reports for the first time the occurrence of phaC and phaC1 sequences in hypersaline microbial mats, suggesting that these ecosystems may be a novel source for the isolation of short- and medium-chain length PHA producers.

Polyhydroxyalakanote (PHA) was produced by the marine bacteria Paracoccus seriniphilus Strain E71. Three methods were used for screening PHA in this strain:(1) microscopic analysis, (2) specifically designed primers for amplify fragments of phaC gene from Gram negative bacteria, and (3) measurements using spectroscopy, calorimetry, thermogravimetry, and rheology. The polyhydroxyalkanoic acid synthase gene (phaC) sequence had 77% identity with the phaC gene of P. denitrificans PD1222 strain. Additionally, the translated sequence showed an 86% similarity with the amino acid sequence of the phaC gene N-terminal portion of the P. denitrificans PD1222 strain. Our phaC sequence was closely related to two phaC sequences that correspond to P. denitrificans; therefore, this is the first report of a sequence of phaC that codifies a poly-(3-hydroxyalkanoate) synthase class I, specifically a polybeta-hydroxybutyrate polymerase from the marine bacteria Paracoccus seriniphilus. The polymer PHA of E71 melts at 167.86°C (T m ), which corresponded to the fusion of the crystalline polymer and thermally degrades at 296.52°C, indicating that the biopolymer has good thermal stability. Rheology showed that this polymer behaves as a nonNewtonian fluid. All these characteristics suggest that the E71 strain produces a PHA that corresponds to the crystalline thermoplastic polymer PHB type.

Knowledge regarding the diversity of methanogenic archaeal communities in hypersaline environments is limited because of the lack of efficient cultivation efforts as well as their low abundance and metabolic activities. In this study, we explored the microbial communities in hypersaline microbial mats. Bioinformatic analyses showed significant differences among the archaeal community structures for each studied site. Taxonomic assignment based on 16S rRNA and methyl coenzyme-M reductase (mcrA) gene sequences, as well as metagenomic analysis, corroborated the presence of Methanosarcinales. Furthermore, this study also provided evidence for the presence of Methanobacteriales, Methanomicrobiales, Methanomassiliicoccales, Candidatus Methanofastidiosales, Methanocellales, Methanococcales and Methanopyrales, although some of these were found in extremely low relative abundances. Several mcrA environmental sequences were significantly different from those previously reported and did not match with any known methanogenic archaea, suggesting the presence of specific environmental clusters of methanogenic archaea in Guerrero Negro. Based on functional inference and the detection of specific genes in the metagenome, we hypothesised that all four methanogenic pathways were able to occur in these environments. This study allowed the detection of extremely low-abundance methanogenic archaea, which were highly diverse and with unknown physiology, evidencing the presence of all methanogenic metabolic pathways rather than the sheer existence of exclusively methylotrophic methanogenic archaea in hypersaline environments.

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