Up-Regulation of the Nrf2/HO-1 Antioxidant Pathway in Macrophages by an Extract from a New Halophilic Archaea Isolated in Odiel Saltworks.

The production of reactive oxygen species (ROS) plays an important role in the progression of many inflammatory diseases. The search for antioxidants with the ability for scavenging free radicals from the body cells that reduce oxidative damage is essential to prevent and treat these pathologies. Haloarchaea are extremely halophilic microorganisms that inhabit hypersaline environments, such as saltworks or salt lakes, where they have to tolerate high salinity, and elevated ultraviolet (UV) and infrared radiations. To cope with these extreme conditions, haloarchaea have developed singular mechanisms to maintain an osmotic balance with the medium, and are endowed with unique compounds, not found in other species, with bioactive properties that have not been fully explored. This study aims to assess the potential of haloarchaea as a new source of natural antioxidant and anti-inflammatory agents. A carotenoid-producing haloarchaea was isolated from Odiel Saltworks (OS) and identified on the basis of its 16S rRNA coding gene sequence as a new strain belonging to the genus Haloarcula. The Haloarcula sp. OS acetone extract (HAE) obtained from the biomass contained bacterioruberin and mainly C18 fatty acids, and showed potent antioxidant capacity using ABTS assay. This study further demonstrates, for the first time, that pretreatment with HAE of lipopolysaccharide (LPS)-stimulated macrophages results in a reduction in ROS production, a decrease in the pro-inflammatory cytokines TNF-α and IL-6 levels, and up-regulation of the factor Nrf2 and its target gene heme oxygenase-1 (HO-1), supporting the potential of the HAE as a therapeutic agent in the treatment of oxidative stress-related inflammatory diseases.

Enhanced wastewater bioremediation by a sulfur-based copolymer as scaffold for microalgae immobilization (AlgaPol).

In recent years, there has been an increasing concern related to the contamination of aqueous ecosystems by heavy metals, highlighting the need to improve the current techniques for remediation. This work intends to address the problem of removing heavy metals from waterbodies by combining two complementary methodologies: adsorption to a copolymer synthesized by inverse vulcanization of sulfur and vegetable oils and phytoremediation by the microalga Chlorella sorokiniana to enhance the metal adsorption. After studying the tolerance and growth of Chlorella sorokiniana in the presence of the copolymer, the adsorption of highly concentrated Cd2+ (50 mg L-1) by the copolymer and microalgae on their own and the combined immobilized system (AlgaPol) was compared. Additionally, adsorption studies have been performed on mixtures of the heavy metals Cd2+ and Cu2+ at a concentration of 8 mg L-1 each. AlgaPol biofilm is able to remove these metals from the growth medium by more than 90%. The excellent metal adsorption capacity of this biofilm can be kinetically described by a pseudo-second-order model.

Exploring Nordic microalgae as a potential novel source of antioxidant and bioactive compounds.

Nordic microalgae are a group of photosynthetic organisms acclimated to growth at low temperature and in varying light conditions; the subarctic climate offers bright days with moderate temperatures during summer and cold and dark winter months. The robustness to these natural stress conditions makes the species interesting for large-scale cultivation in harsh environments and for the production of high-value compounds. The aim of this study was to explore the ability of nineteen species of Nordic microalgae to produce different bioactive compounds, such as carotenoids or polyphenols. The results showed that some of these strains are able to produce high amounts of carotenoids (over 12 mg·g-1 dry weight) and phenolic compounds (over 20 mg GAE·g-1 dry weight). Based on these profiles, six species were selected for cultivation under high light and cold stress (500 µmol·m-2·s-1 and 10 ˚C). The strains Chlorococcum sp. (MC1) and Scenedesmus sp. (B2-2) exhibited similar values of biomass productivity under standard or stress conditions, but produced higher concentrations of carotenoids (an increase of 40% and 25%, respectively), phenolic compounds (an increase of 40% and 30%, respectively), and showed higher antioxidant capacity (an increase of 15% and 20%, respectively) during stress. The results highlight the ability of these Nordic microalgae as outstanding producers of bioactive compounds, justifying their cultivation at large scale in Nordic environments.

Metabarcoding data of prokaryotes and eukaryotes inhabiting the phosphogypsum stockpiles on the salt marshes of Huelva (SW Spain).

Around 100 Mt of phosphogypsum (PG) of extreme acidity and with high concentrations of heavy metals and radionuclides have been deposited on the salt marshes of the Tinto River estuary in Huelva (SW Spain) for more than forty years. The microbial community able to thrive in these adverse conditions remains totally unknown, despite the fact that it can highly influence the biogeochemical cycle of the phosphogypsum components and include new species with biotechnological interest. High throughput sequencing of 16S/18S rRNA encoding genes is a potent tool to uncover the microbial diversity of extreme environments. This data article describes for the first time the prokaryotic and eukaryotic diversity of two water samples collected in the Huelva phosphogypsum stacks. The raw amplicons of the 16S/18S rRNA maker genes for the two phosphogypsum samples and two reference samples (seawater and the Tinto River water) obtained after sequencing on MiSeq platform are provided. The operational taxonomic units (OTUs) obtained after the treatment and clustering of the obtained reads with the QIIME2 pipeline and their taxonomic assignation performed by comparison with the SILVA database are also presented to complete the information of the article "Exploring the microbial community inhabiting the phosphogypsum stacks of Huelva (SW, Spain) by a high throughput 16S/18S rDNA Sequencing approach".

Simultaneous production of carotenoids and chemical building blocks precursors from chlorophyta microalgae.

Replacement of fossil fuels has to be accompanied by the incorporation of bio-based procedures for the production of fine chemicals. With this aim, the microalga Chlamydomonas reinhardtii was selected for its ability to accumulate starch, an environmentally-friendly alternative source of chemical building blocks, such as 5'-hydroxymethylfurfural or levulinic acid. The content of appreciated lipophilic coproducts was assessed in the selected microalga cultured at different nutritional conditions; and the parameters for the acidic hydrolysis of the algal biomass, obtained after pigments extraction, were optimized using a Central Composite Design. Response Surface Methodology predicted that the optimal hydrolysis conditions were elevated temperature, high DMSO % and short hydrolysis time for glucose. LA was favored at long times and high acid % and 5'-HMF at lower acid % and high DMSO %. Chlamydomonas can therefore be used as a sustainable feedstock for the simultaneous production of high-added value lipophilic compounds and platform chemicals.

Exploring the microbial community inhabiting the phosphogypsum stacks of Huelva (SW SPAIN) by a high throughput 16S/18S rDNA sequencing approach.

Around 100 Mt of phosphogypsum (PG) have been deposited in large stacks on the salt marshes of the Tinto River estuary in Huelva (SW Spain), covering about 1000 ha. These stacks contain extremely acidic water (pH < 2) with high concentrations of pollutants which can cause emissions into their surroundings, generating important environmental concerns. Despite many chemical, geological or hydrological studies have been conducted to characterize the PG stacks of Huelva, the microbial community inhabiting this extreme environment remains unexplored. Using a 16S/18S-rRNA-high throughput sequencing approach, we have uncovered the main taxonomic groups able to live in the acidic metal-contaminated water, which is in direct contact with the PG, demonstrating for the first time the existence of a huge diversity of microbial species in these extreme conditions. In addition, the physicochemical characteristics of the water sampled have been analyzed. These studies have revealed that the most abundant bacteria found in two different leachate samples of the PG stacks belong to the genera Acidiphilium, Pseudomonas, Leptosprillum, Acidithrix, or Acidithiobacillus, typically found in acid mine drainage (AMD) environments, which in total represent around 50% of the total bacterial community. Biodiversity of eukaryotes in PG water is lower than that of prokaryotes, especially in the water collected from the perimeter channel that surrounds the PG stacks, where the pH reaches a value of 1.5 and the activity concentrations exceed 300 Bq L-1 for 238U or 20 Bq L-1 for 210Po, values which are from four to five orders of magnitude higher than those usually found in unperturbed surface waters. Even so, an unexpected diversity of algae, fungi, and ciliates have been found in the PG stacks of Huelva, where chlorophyte microalgae and basidiomycetes fungi are the most abundant eukaryotes. Additional bioinformatics tools have been used to perform a functional analysis and predict the most common metabolic pathways in the PG microbiota. The obtained data indicate that the extreme conditions of these PG stacks hide an unexpected microbial diversity, which can play an important role in the dynamics of the contaminating compounds of the PG and provide new strains with unique biotechnological applications.

Impact of heavy metals in the microalga Chlorella sorokiniana and assessment of its potential use in cadmium bioremediation.

The chlorophyte microalga Chlorella sorokiniana was tested for the bioremediation of heavy metals pollution. It was cultured with different concentrations of Cu2+, Cd2+, As (III) and As (V), showing a significant inhibition on its growth at concentrations of 500 µM Cu2+, 250 µM Cd2+, 750 µM AsO33- and 5 mM AsO43- or higher. Moreover, the consumption of ammonium was also studied, showing significant differences for concentrations higher than 1 mM of Cu2+ and As (III), and 5 mM of As (V). The determination of intracellular heavy metals concentration revealed that Chlorella sorokiniana is an outstanding Cd accumulator organism, able to accumulate 11,232 mg kg-1 of Cd, and removing 65% of initial concentration of this heavy metal. Finally, antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), and enzymes involved in the production of glutamate and cysteine, such as glutamine syntethase (GS), glutamate dehydrogenase (GDH), O-acetylserine (thiol) lyase (OASTL) and NAD-isocitrate dehydrogenase (NAD-IDH) were studied both at gene expression and enzymatic activity levels. These enzymes exhibited different grades of upregulation, especially in response to Cd and As stress. However, GS expression was downregulated when Chlorella sorokiniana was cultured in the presence of these heavy metals.

Biochemical Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. HS Isolated in the Odiel Marshlands.

Alpha-amylases are a large family of α,1-4-endo-glycosyl hydrolases distributed in all kingdoms of life. The need for poly-extremotolerant amylases encouraged their search in extreme environments, where archaea become ideal candidates to provide new enzymes that are able to work in the harsh conditions demanded in many industrial applications. In this study, a collection of haloarchaea isolated from Odiel saltern ponds in the southwest of Spain was screened for their amylase activity. The strain that exhibited the highest activity was selected and identified as Haloarcula sp. HS. We demonstrated the existence in both, cellular and extracellular extracts of the new strain, of functional α-amylase activities, which showed to be moderately thermotolerant (optimum around 60 °C), extremely halotolerant (optimum over 25% NaCl), and calcium-dependent. The tryptic digestion followed by HPLC-MS/MS analysis of the partially purified cellular and extracellular extracts allowed to identify the sequence of three alpha-amylases, which despite sharing a low sequence identity, exhibited high three-dimensional structure homology, conserving the typical domains and most of the key consensus residues of α-amylases. Moreover, we proved the potential of the extracellular α-amylase from Haloarcula sp. HS to treat bakery wastes under high salinity conditions.

Effect of cadmium in the microalga Chlorella sorokiniana: A proteomic study.

Cadmium is one of the most common heavy metals in contaminated aquatic environments and one of the most toxic contaminants for phytoplankton. Nevertheless, there are not enough studies focused on the effect of this metal in algae. Through a proteomic approach, this work shows how Cd can alter the growth, cell morphology and metabolism of the microalga Chlorella sorokiniana. Using the sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS), we concluded that exposure of Chlorella sorokiniana to 250 µM Cd2+ for 40 h caused downregulation of different metabolic pathways, such as photosynthesis, oxidative phosphorylation, glycolysis, TCA cycle and ribosomal proteins biosynthesis. However, photorespiration, antioxidant enzymes, gluconeogenesis, starch catabolism, and biosynthesis of glutamate, cysteine, glycine and serine were upregulated, under the same conditions. Finally, exposure to Cd also led to changes in the metabolism of carotenoids and lipids. In addition, the high tolerance of Chlorella sorokiniana to Cd points to this microalga as a potential microorganism to be used in bioremediation processes.

Dataset for proteomic analysis of Chlorella sorokiniana cells under cadmium stress.

Cadmium is one of the most hazardous heavy metal for aquatic environments and one of the most toxic contaminants for phytoplankton. This work provides the dataset associated with the research publication "Effect of cadmium in the microalga Chlorella sorokiniana: a proteomic study" [1]. This dataset describes a proteomic approach, based on the sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS), derived from exposure of Chlorella sorokiniana to 250 µM Cd2+ for 40 h, showing the proteins that are up- or downregulated. The processing of data included the identification of the Chlamydomonas reinhardtii protein sequences equivalent to the corresponding of Chlorella sorokiniana sequences obtained, which made possible to use KEGG Database. MS and MS/MS information, and quantitative data were deposited PRIDE public repository under accession number PXD015932.

Antioxidant, Antimicrobial, and Bioactive Potential of Two New Haloarchaeal Strains Isolated from Odiel Salterns (Southwest Spain).

The need to survive in extreme environments has furnished haloarchaea with a series of components specially adapted to work in such conditions. The possible application of these molecules in the pharmaceutical and industrial fields has received increasing attention; however, many potential bioactivities of haloarchaea are still poorly explored. In this paper, we describe the isolation and identification of two new haloarchaeal strains from the saltern ponds located in the marshlands of the Odiel River, in the southwest of Spain, as well as the in vitro assessment of their antioxidant, antimicrobial, and bioactive properties. The acetone extract obtained from the new isolated Haloarcula strain exhibited the highest antioxidant activity, while the acetone extracts from both isolated strains demonstrated a strong antimicrobial activity, especially against other halophilic microorganisms. Moreover, these extracts showed a remarkable ability to inhibit the enzyme cyclooxygenase-2 and to activate the melanogenic enzyme tyrosinase, indicating their potential against chronic inflammation and skin pigmentation disorders. Finally, the aqueous protein-rich extracts obtained from both haloarchaea exhibited an important inhibitory effect on the activity of the acetylcholinesterase enzyme, involved in the hydrolysis of cholinergic neurotransmitters and related to several neurological diseases.

Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae.

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3'-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae.

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg-1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

Using agro-industrial wastes for mixotrophic growth and lipids production by the green microalga Chlorella sorokiniana.

There has been growing interest in the use of microalgae for the production of biofuels, but production costs continue to be too high to compete with fossil fuel prices. One of the main limitations for photobioreactor productivity is light shielding, especially at high cell densities. The growth of the green microalga Chlorella sorokiniana, a robust industrial species, has been evaluated under different trophic conditions with traditional carbon sources, such as glucose and sucrose, and alternative low cost carbon sources, such as carob pod extract, industrial glycerol and acetate-rich oxidized wine waste lees. The mixotrophic cultivation of this microalga with wine waste lees alleviated the problems of light shielding observed in photoautotrophic cultures, improving specific growth rate (0.052 h-1) compared with the other organic sources. The fed-batch mixotrophic culture of Chlorella sorokiniana in a 2 L stirred tank reactor, with optimized nutritional conditions, 100 mM of acetate coming from the oxidized wine waste lees and 30 mM of ammonium, produced an algal biomass concentration of 11 g L-1 with a lipid content of 38 % (w/w). This fed-batch strategy has been found to be a very effective means to enhance the biomass and neutral lipid productivity.

Characterization of the Microbial Population Inhabiting a Solar Saltern Pond of the Odiel Marshlands (SW Spain).

The solar salterns located in the Odiel marshlands, in southwest Spain, are an excellent example of a hypersaline environment inhabited by microbial populations specialized in thriving under conditions of high salinity, which remains poorly explored. Traditional culture-dependent taxonomic studies have usually under-estimated the biodiversity in saline environments due to the difficulties that many of these species have to grow at laboratory conditions. Here we compare two molecular methods to profile the microbial population present in the Odiel saltern hypersaline water ponds (33% salinity). On the one hand, the construction and characterization of two clone PCR amplified-16S rRNA libraries, and on the other, a high throughput 16S rRNA sequencing approach based on the Illumina MiSeq platform. The results reveal that both methods are comparable for the estimation of major genera, although massive sequencing provides more information about the less abundant ones. The obtained data indicate that Salinibacter ruber is the most abundant genus, followed by the archaea genera, Halorubrum and Haloquadratum. However, more than 100 additional species can be detected by Next Generation Sequencing (NGS). In addition, a preliminary study to test the biotechnological applications of this microbial population, based on its ability to produce and excrete haloenzymes, is shown.

Effect of selenate on viability and selenomethionine accumulation of Chlorella sorokiniana grown in batch culture.

The aim of this work was to study the effect of Se(+VI) on viability, cell morphology, and selenomethionine accumulation of the green alga Chlorella sorokiniana grown in batch cultures. Culture exposed to sublethal Se concentrations of 40 mg · L(-1) (212 µM) decreased growth rates for about 25% compared to control. A selenate EC50 value of 45 mg · L(-1) (238.2 µM) was determined. Results showed that chlorophyll and carotenoids contents were not affected by Se exposure, while oxygen evolution decreased by half. Ultrastructural studies revealed granular stroma, fingerprint-like appearance of thylakoids which did not compromise cell activity. Unlike control cultures, SDS PAGE electrophoresis of crude extracts from selenate-exposed cell cultures revealed appearance of a protein band identified as 53 kDa Rubisco large subunit of Chlorella sorokiniana, suggesting that selenate affects expression of the corresponding chloroplast gene as this subunit is encoded in the chloroplast DNA. Results revealed that the microalga was able to accumulate up to 140 mg · kg(-1) of SeMet in 120 h of cultivation. This paper shows that Chlorella sorokiniana biomass can be enriched in the high value aminoacid SeMet in batch cultures, while keeping photochemical viability and carbon dioxide fixation activity intact, if exposed to suitable sublethal concentrations of Se.

Efficiency of different heterologous promoters in the unicellular microalga Chlamydomonas reinhardtii.

Despite the biotechnological interest of microalgae, no robust and stable methods for genetic transformation of most microalgal strains exist. The scanty and disperse data about the efficiency of heterologous promoters in microalgae and the use of different transformation methods, DNA quantities and reporter genes in the existing studies makes very difficult a real comparison of their efficiency. Using Chlamydomonas reinhardtii as a host, we have evaluated the efficiency of the heterologous promoters of cauliflower mosaic virus 35S (CaMV 35S) and Agrobacterium nopaline synthase (NOS) genes. These promoters were fused to the paromomycin conferring-resistance aminoglycoside 3'-phosphotransferase encoding gene (APHVIII), and C. reinhardtii was transformed by the glass beads agitation method. The transformation efficiency and the APHVIII transcript and protein levels were evaluated in a series of transformants for each promoter. The chimeric promoter HSP70A/RBCS2 and the promoter-less APHVIII marker gene were used for comparison. We found significantly higher transformation efficiencies and higher level of APHVIII expression in those transformants harboring the NOS promoter than in those transformed with CaMV 35S promoter. The NOS promoter, widely used for genetic manipulation of higher plants, has been very rarely used for the transformation of microalgae. The results shown here suggest the possibilities of this heterologous promoter as an efficient system for the genetic manipulation of microalgae.

Microalgae mediated photoproduction of beta-carotene in aqueous-organic two phase systems.

Improving productivity is a usual requirement for most biotechnological processes, and the utilisation of two-phase aqueous organic systems has proved to be an effective way to improve the productivity of poorly water-soluble or toxic compounds. The high hydrophobicity of beta-carotene, which is highly demanded by the pharma and agrofood industry, makes it a good candidate for aqueous/organic biphasic photoproduction. In the present work we have investigated the viability of a two-phase system for the production of beta-carotene by the marine microalgae Dunaliella salina using decane as organic phase. Decane, with a logP(octanol) value of 5.6, showed no toxicity to Dunaliella cells for more than 72 h, and its ability for beta-carotene extraction is acceptable. Transferring Dunaliella cells from standard to carotenogenic conditions caused inhibition of chlorophyll production and induced a strong synthesis of beta-carotene. The two-phase aqueous/decane system was stable and beta-carotene content of the cells was increasing during 4-days. About 8% of the total carotenoids produced were excreted and extracted into the decane phase.

Metal toxicity in Chlamydomonas reinhardtii. Effect on sulfate and nitrate assimilation.

Cadmium (Cd(2+)) or copper (Cu(2+)) ions are toxic for Chlamydomonas reinhardtii growth, at 300 microM, and the alga may accumulate about 0.90+/-0.02 and 0.64+/-0.02% of its dry weight, respectively. Metal contamination changes the elemental composition of dried alga biomass, which indicates the possibility to use C. reinhardtii as biosensor and bioremediator of the aquatic contamination by heavy metals. Either, Cd(2+) or Cu(2+), inhibits about 20% of the nitrate consumption rate by the cells, while only Cd(2+) increases about 40% the sulfate consumption rate. The presence of 1 mM calcium (Ca(2+)) in the culture medium increases the C. reinhardtii productivity (about 50%), the nitrate uptake rate (about 20%) and the sulfate uptake rate (about 30%). In addition, Ca(2+) overcomes the Cd(2+) (300 microM) toxicity by decreasing (about 35%) the intracellular accumulation of metal. Sulfur-starvation induces in C. reinhardtii the expression of serine acetyltransferase and O-acetylserine(thiol)lyase activities, but decreases 50% the consumption rate of nitrate by the cells. Sulfate is also required for the full expression of the nitrate reductase (NR), nitrite reductase (NiR) and glutamate synthase activities.

Carotenoids as protective response against oxidative damage in Dunaliella bardawil.

In the present work the relation between carotenoids production and cell response mechanisms to oxidative damage was studied. High light intensity and nitrogen starvation, both conditions, which may increase the oxidative damage in microalgae, significantly increased total carotenoids content in Dunaliella bardawil, the effect of N-starvation being more noticeable when acting synergetically with light on carotenoid production. S-starvation stimulated carotenoids production as much as N-starvation. The use of norflurazon, inhibitor of phytoene desaturase that blocks formation of epsilon-carotene from phytoene, caused a decrease of carotenoid content down to 5% that of the control cells incubated without the inhibitor. The decrease in the oxygen consumption rate of D. bardawil cells exposed to norflurazon suggests a connection between carotenoids desaturation and chloroplastic oxygen species dissipation processes reported in the literature for other algae. It is an indication of the carotenoids involvement in chloroplastic response mechanisms to oxidative damage.