Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

Cell Adhesion and Biofilm Formation Analysis.

Cell adhesion to surfaces and ulterior biofilm formation are critical processes in microbial development since living in biofilms is the preferred way of life within microorganisms. These processes are known to influence not only microorganisms development in the environment, but also their participation in biotechnological processes and have been the focus of intense research that as a matter of fact, was mainly directed to the bacterial domain. Archaea also adhere to surfaces and have been shown forming biofilms, but studies performed until present did not exploit the diversity of methods probed to be useful along bacterial biofilm research.An experimental setup is described here with the aim of stimulating archaeal biofilm research. It can be used for studying cell adhesion and biofilm formation under controlled flow conditions and allows performing in situ optical microscopy (phase contrast, fluorescence, or confocal) and/or spectroscopic techniques (UV-Vis, IR, or Raman) to determine structural and functional biofilm features and their evolution in time. Variants are described with specific aims as working in anaerobiosis and allow sampling of biological material along time.

Biofilms of Halobacterium salinarum as a tool for phenanthrene bioremediation.

The use of hyperhalophilic microorganisms is emerging as a sustainable alternative to clean hydrocarbon-polluted hypersaline water bodies. In line with this practice, this work reports on the ability of the archaeon Halobacterium salinarum to develop biofilms on a solid surface conditioned by the presence of phenanthrene crystals, which results in the removal of the contaminating compound. The cell surface hydrophobicity does not change during the removal process and this organism is shown to constitutively produce a surfactant molecule with specific action on aromatic hydrocarbons, both indicating that phenanthrene removal might proceed through a non-contact mechanism. A new approach is presented to follow the process in situ through epifluorescence microscopy by monitoring phenanthrene auto-fluorescence.

Temporal Analysis of the Microbial Community from the Crystallizer Ponds in Cabo Rojo, Puerto Rico, Using Metagenomics.

The Cabo Rojo solar salterns are a hypersaline environment located in a tropical climate, where conditions remain stable throughout the year. These conditions can favor the establishment of steady microbial communities. Little is known about the microbial composition that thrives in hypersaline environments in the tropics. The main goal of this study was to assess the microbial diversity present in the crystallizer ponds of Cabo Rojo, in terms of structure and metabolic processes across time using metagenomic techniques. Three samplings (December 2014, March and July 2016) were carried out, where water samples (50 L each) were filtered through a Millipore pressurized filtering system. DNA was subsequently extracted using physical-chemical methods and sequenced using paired end Illumina technologies. The sequencing effort produced three paired end libraries with a total of 111,816,040 reads, that were subsequently assembled into three metagenomes. Out of the phyla detected, the microbial diversity was dominated in all three samples by Euryarchaeota, followed by Bacteroidetes and Proteobacteria. However, sample MFF1 (for Muestreo Final Fraternidad) exhibited a higher diversity, with 12 prokaryotic phyla detected at 34% NaCl (w/v), when compared to samples MFF2 and MFF3, which only exhibited three phyla. Precipitation events might be one of the contributing factors to the change in the microbial community composition through time. Diversity at genus level revealed a more stable community structure, with an overwhelming dominance of the square archaeon Haloquadratum in the three metagenomes. Furthermore, functional annotation was carried out in order to detect genes related to metabolic processes, such as carbon, nitrogen, and sulfur cycles. The presence of gene sequences related to nitrogen fixation, ammonia oxidation, sulfate reduction, sulfur oxidation, and phosphate solubilization were detected. Through binning methods, four putative novel genomes were obtained, including a possible novel genus belonging to the Bacteroidetes and possible new species for the genera Natronomonas, Halomicrobium, and Haloquadratum. Using a metagenomic approach, a 3-year study has been performed in a Caribbean hypersaline environment. When compared to other salterns around the world, the Cabo Rojo salterns harbor a similar community composition, which is stable through time. Moreover, an analysis of gene composition highlights the importance of the microbial community in the biogeochemical cycles at hypersaline environments.

Draft genome sequence of Halorubrum tropicale strain V5, a novel halophilic archaeon isolated from the solar salterns of Cabo Rojo, Puerto Rico.

The genus Halorubrum is a member of the family Halobacteriaceae which currently has the highest number of described species (31) of all the haloarchaea. Here we report the draft genome sequence of strain V5, a new species within this genus that was isolated from the solar salterns of Cabo Rojo, Puerto Rico. Assembly was performed and rendered the genome into 17 contigs (N50 = 515,834 bp), the largest of which contains 1,031,026 bp. The genome consists of 3.57 MB in length with G + C content of 67.6%. In general, the genome includes 4 rRNAs, 52 tRNAs, and 3246 protein-coding sequences. The NCBI accession number for this genome is LIST00000000 and the strain deposit number is CECT9000.

Draft genome of Haloarcula rubripromontorii strain SL3, a novel halophilic archaeon isolated from the solar salterns of Cabo Rojo, Puerto Rico.

The genus Haloarcula belongs to the family Halobacteriaceae which currently has 10 valid species. Here we report the draft genome sequence of strain SL3, a new species within this genus, isolated from the Solar Salterns of Cabo Rojo, Puerto Rico. Genome assembly performed using NGEN Assembler resulted in 18 contigs (N50 = 601,911 bp), the largest of which contains 1,023,775 bp. The genome consists of 3.97 MB and has a GC content of 61.97%. Like all species of Haloarcula, the genome encodes heterogeneous copies of the small subunit ribosomal RNA. In addition, the genome includes 6 rRNAs, 48 tRNAs, and 3797 protein coding sequences. Several carbohydrate-active enzymes genes were found, as well as enzymes involved in the dihydroxyacetone processing pathway which are not found in other Haloarcula species. The NCBI accession number for this genome is LIUF00000000 and the strain deposit number is CECT9001.

Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.