Search Results (87)

Archaeal lipids are a source of new biomaterials for pharmaceutical and nanomedical applications; however, their classical extraction method relies on chloroform and methanol, toxic solvents that conflict with green chemistry principles. In this paper, we explore the performance of an eco-friendly method for the extraction of total lipids from the haloarchaea Halorubrum tebenquichense. Using the bio-solvents ethyl acetate and ethanol in a two-step procedure, a fraction of total lipids (135 ± 41 mg phospholipids and 1.1 ± 0.4 mg bacterioruberin (BR)/100 g cell paste) was obtained containing the same composition as that resulting from extraction with the classical solvents, as confirmed by electrospray ionization mass spectrometry, although with lower phospholipid (PL) content, thus with a higher proportion of bacterioruberin (BR/PL ratio 9.0 vs. 6.8 µg/mg). The extracted lipids were subsequently utilized for the preparation of archaeosomes, which were characterized by uniform size distribution (406 ± 137 nm, 0.63 ± 0.13 polydispersity index), colloidal stability, and negative ζ potential (−38.2 ± 5.4 mV). The photoprotective potential of these archaeosomes was determined for the first time in human keratinocyte (HaCaT) cells exposed to UVB irradiation (270 mJ/cm²). Treatment with archaeosomes significantly (p < 0.05) enhanced cell viability (from ~43 to ~80%), reduced intracellular ROS generation and proinflammatory cytokine release (TNF-α), and mitigated UVB-induced apoptosis compared to untreated controls, indicating effective cytoprotection. This study demonstrates that ethyl acetate–ethanol-based extraction offers an alternative for archaeal lipid recovery and highlights the potential of archaeosomes as natural photoprotective agents for skincare applications. Full article

This study investigates the valorisation of lemon industry by-products as carbon sources to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using the halophilic archaeon Haloferax mediterranei. The resulting polymer (HFX PHBV) was supplemented with nucleating agents (orotic acid, boron nitride, and theobromine) and compared with a commercial PHBV grade (Enmat Y1000) under identical conditions. Fermentation strategies were optimised by varying the lemon by-product concentration, inoculum size, and nutrient stoichiometry (C:N:P ratios), followed by scaleup in a 2 L bioreactor. A 11% (v/v) lemon by-product combined with a 5% (v/v) inoculum yielded the highest productivity under minimal medium conditions (2.127 g/L PHBV), while enriched media further enhanced the polymer accumulation (up to 3.250 g/L PHBV). A comparative characterisation of HFX PHBV and Enmat Y1000, using NMR, TGA, MFR, DSC, Raman spectroscopy, XRD, and DMA, revealed that HFX PHBV exhibited lower crystallinity, increased flexibility, and a high hydroxyvalerate content (27.4%), which conferred improved ductility. Investigation of nucleating agents demonstrated that orotic acid was the most effective at enhancing the crystallisation kinetics. Overall, this study demonstrates an efficient PHBV production process based on waste valorisation, yielding a biopolymer with competitive physicochemical properties relative to a commercial standard, and provides integrated solutions to the global challenges of plastic pollution and food waste. Full article

Haloarchaea are moderate and extreme halophilic microorganisms inhabiting hypersaline environments characterised by high ionic and oxidative stress due to extremely high salt concentrations and high incidence of UV radiation (mainly in spring and summer). To be alive and metabolically active under these harsh conditions, haloarchaeal strains have developed molecular adaptations, like hyperpigmentation. Among the carotenoids produced by haloarchaeal species, the C₅₀ carotenoid called bacterioruberin (BR) and its derivatives, monoanhydrobacterioruberin and bisanhydrobacterioruberin, are the predominant natural pigments produced. This review aims to highlight the most significant characteristics of BR and their derivatives, as well as a description of the biological activities already reported that could provide benefits for human health, including antitumoral, immunomodulatory, antioxidant, skin protectant, antilipidemic, antiglycemic, and anti-atrophic effects, in addition to showing potential positive effects on sperm cells cryopreservation. Overall, C₅₀ carotenoids are fascinating natural biomolecules that could be utilised in processed food and nutraceuticals or as tools in the context of new strategies and/or pharmaceutical formulations to combat various human diseases or metabolic disorders. Full article

The biosynthesis of polyhydroxyalkanoate (PHA) biopolymer is highly dependent on the activity of a key enzyme, PHA synthase (PhaC). The halophilic archaeon Haloferax mediterranei can accumulate large amounts of PHAs from different carbon sources under non-sterilized conditions. In this study, a PhaC enzyme from H. mediterranei was produced and subsequently partially purified by ion exchange chromatography. The protein was visualized by SDS-PAGE, with a subunit molecular mass of 56.4 kDa. The purified enzyme converts hydroxybutyryl CoA molecules into PHA, being optimally active at pH 10.0 and pH 8.0. The PhaC was thermoactive in the range of 30 °C to 70 °C, with maximum activity registered at 50 °C. The enzyme was confirmed to be haloalkaliphilic (active at pH > 7.0 and high salt concentration) and exhibit a degree of stability at 25 °C for 24 h. Full article

Bioplastics such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are promising alternatives to conventional plastics. However, the high production cost limits their industrial application. In this study, PHBV production was optimized in Haloferax mediterranei by the homologous overexpression of the key enzyme PHA synthase (PhaEC), resulting in the OEphaEC strain. The growth and PHBV production of OEphaEC compared with the parental strain (HM26) were evaluated in three culture media with different nitrogen sources (KNO₃, NH₄Cl, and casamino acids). The OEphaEC strain exhibited a 20% increase in PHBV production and a 40% increase in 3-hydroxyvalerate monomer (3HV) content in a defined medium with nitrate as a nitrogen source, as determined by GC-MS. Moreover, enzyme activity, measured spectrophotometrically, increased from 2.3 to 3.9 U/mg. Soluble and insoluble protein fractions were analysed to assess the overexpression of PHA synthase. Only PhaE was found in the insoluble protein fraction, where PHBV granules accumulate. Transmission electron microscopy (TEM) images confirmed a higher PHBV content in OEphaEC compared to the parental strain. These results demonstrate that the homologous overexpression of the key enzyme implicated in PHBV biosynthesis can enhance PHBV content, making its production competitive for industrial applications. Full article

Haloarchaea, a group of extremophilic archaea, thrive in hypersaline environments characterized not only by high salinity but also by other extreme conditions, such as intense UV radiation, high osmotic pressure, heavy metal contamination, oxidative stress, and fluctuating temperatures. This study investigates the environmental adaptation strategies of species of two genera, Haloarcula and Natrinema, the second and third largest haloarchaeal genera, respectively, after Halorubrum. Comparative genomic analyses were conducted on 48 species from both genera to elucidate their genomic diversity, metabolic potential, and stress-tolerance mechanisms. The genomes revealed diverse metabolic pathways, including rhodopsin-mediated phototrophy, nitrogen assimilation, and thiamine biosynthesis, which support their survival and adaptation to extreme conditions. The analysis identified mechanisms for oxidative stress mitigation, DNA repair, “salt-in” and “salt-out” osmoregulatory strategies, adaptations to temperature shifts and heavy metal exposure, and immune defense. Experimental validation of four representative species, Haloarcula terrestris S1AR25-5A^T, Haloarcula saliterrae S1CR25-12^T, Haloarcula onubensis S3CR25-11^T, and Natrinema salsiterrestre S1CR25-10^T, isolated from the heavy-metal-rich hypersaline soils in the Odiel Saltmarshes (Huelva, Spain), demonstrated their tolerance, especially to arsenic, corroborating genomic predictions. This study advances our understanding of the resilience of haloarchaea under poly-extreme conditions and underscores their ecological significance and promise for biotechnological applications, such as the bioremediation of heavy-metal-polluted environments and the production of valuable biomolecules. Full article

Haloarchaea are a group of moderate and extreme halophilic microorganisms, belonging to the Archaea domain, that constitute relevant microbial communities in salty environments like coastal and inland salted ponds, marshes, salty lagoons, etc. They can survive in stress conditions such as high salinity and, therefore, high ionic strength, high doses of ultraviolet radiation (UV), high temperature, and extreme pH values. Consequently, most of the species can be considered polyextremophiles owing to their ability to respond to the multiple extreme conditions characterizing their natural habitats. They cope with those stresses thanks to several molecular and metabolic adaptations. Thus, some of the molecules produced by haloarchaea show significantly different biological activities and physicochemical properties compared to their bacterial counterparts. Recent studies have revealed promising applications in biotechnology and medicine for these biomolecules. Among haloarchaeal biomolecules, rare natural pigments (C₅₀ carotenoids) and small peptides called halocins and microhalocins have attracted attention worldwide due to their effects on animal and human commercial tumoral cells, apart from the role as antibiotics described for halocins or the immunomodulatory activity reported from C₅₀ carotenoids like bacterioruberin. This review summarizes recent knowledge on these two types of biomolecules in connection with cancer to shed new light on the design of drugs and new therapies based on natural compounds. Full article

The haloarchaeon Haloferax mediterranei synthesizes poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under unfavorable nutritional conditions without the addition of any precursor to the culture, which is an advantage compared to other microbial counterparts able to synthesize polyhydroxyalkanoates (PHA). PHBV is a biodegradable polymer showing physiochemical properties of biotechnological and biomedical interest and can be used as an alternative to plastics made from chemical synthesis (which are not environmentally friendly). The versatile metabolism of H. mediterranei makes the use of waste as a carbon source for cellular growth and PHA synthesis possible. In this work, cellular growth and the production and characterization of PHBV using two different types of confectionery waste were analyzed and compared with cellular growth and PHBV synthesis in a standard culture media with glucose of analytical grade as a carbon source. The PHBV granules produced were analyzed by TEM and the biopolymer was isolated and characterized by GC-MS, FTIR NMR, and DSC. The results reveal that H. mediterranei can use these two residues (R1 and R2) for pure PHBV production, achieving 0.256 and 0.983 g PHBV/L, respectively, which are among the highest yields so far described using for the first-time waste from the candy industry. Thus, a circular economy-based process has been designed to optimize the upscaling of PHBV production by using haloarchaea as cell factories and valorizing confectionery waste. Full article

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide acquired heritable protection to bacteria and archaea against selfish DNA elements, such as viruses. These systems must be tightly regulated because they can capture DNA fragments from foreign selfish elements, and also occasionally from self-chromosomes, resulting in autoimmunity. Most known species from the halophilic archaeal genus Haloferax contain type I-B CRISPR-Cas systems, and the strongest hotspot for self-spacer acquisition by H. mediterranei was a locus that contained a putative transposable element, as well as the gene HFX_2341, which was a very frequent target for self-targeting spacers. To test whether this gene is CRISPR-associated, we investigated it using bioinformatics, deletion, over-expression, and comparative transcriptomics. We show that HFX_2341 is a global transcriptional regulator that can repress diverse genes, since its deletion results in significantly higher expression of multiple genes, especially those involved in nutrient transport. When over-expressed, HFX_2341 strongly repressed the transcript production of all cas genes tested, both those involved in spacer acquisition (cas1, 2 and 4) and those required for destroying selfish genetic elements (cas3 and 5–8). Considering that HFX_2341 is highly conserved in haloarchaea, with homologs that are present in species that do not encode the CRISPR-Cas system, we conclude that it is a global regulator that is also involved in cas gene regulation, either directly or indirectly. Full article

Australian isolates of Haloquadratum walsbyi, a square-shaped haloarchaeon, often harbor small cryptic plasmids of the pL6-family, approximately 6 kb in size, and five examples have been previously described. These plasmids exhibit a highly conserved gene arrangement and encode replicases similar to those of betapleolipoviruses. To assess their global distribution and recover more examples for analysis, fifteen additional plasmids were reconstructed from the metagenomes of seven hypersaline sites across four countries: Argentina, Australia, Puerto Rico, and Spain. Including the five previously described plasmids, the average plasmid size is 6002 bp, with an average G+C content of 52.5%. The tetramers GGCC and CTAG are either absent or significantly under-represented, except in the two plasmids with the highest %G+C. All plasmids share a similar arrangement of genes organized as outwardly facing replication and ATPase modules, but variations were observed in some core genes, such as F2, and some plasmids had acquired accessory genes. Two plasmids, pCOLO-c1 and pISLA-c6, shared 92.7% nt identity despite originating from Argentina and Spain, respectively. Numerous metagenomic CRISPR spacers matched sequences in the fifteen reconstructed plasmids, indicating frequent invasion of haloarchaea. Spacers could be assigned to haloarchaeal genera by mapping their associated direct repeats (DR), with half of these matching Haloquadratum. Finally, strand-specific metatranscriptome (RNA-seq) data could be used to demonstrate the active transcription of two pL6-family plasmids, including antisense transcripts. Full article

Climate change and the scarcity of primary resources are driving the development of new, more renewable and environmentally friendly industrial processes. As part of this green chemistry approach, extremozymes (extreme microbial enzymes) can be used to replace all or part of the chemical synthesis stages of traditional industrial processes. At present, the production of these enzymes is limited by the cellular chassis available. The production of a large number of extremozymes requires extremophilic cellular chassis, which are not available. This is particularly true of halophilic extremozymes. The aim of this review is to present the current potential and challenges associated with the development of a haloarchaea-based cellular chassis. By overcoming the major obstacle of the limited number of genetic tools, it will be possible to propose a robust cellular chassis for the production of functional halophilic enzymes that can participate in the industrial transition of many sectors. Full article

In recent years, interest in very small proteins (µ-proteins) has increased significantly, and they were found to fulfill important functions in all prokaryotic and eukaryotic species. The halophilic archaeon Haloferax volcanii encodes about 400 µ-proteins of less than 70 amino acids, 49 of which contain at least two C(P)XCG motifs and are, thus, predicted zinc finger proteins. The determination of the NMR solution structure of HVO_2753 revealed that only one of two predicted zinc fingers actually bound zinc, while a second one was metal-free. Therefore, the aim of the current study was the homologous production of additional C(P)XCG proteins and the quantification of their zinc content. Attempts to produce 31 proteins failed, underscoring the particular difficulties of working with µ-proteins. In total, 14 proteins could be produced and purified, and the zinc content was determined. Only nine proteins complexed zinc, while five proteins were zinc-free. Three of the latter could be analyzed using ESI-MS and were found to contain another metal, most likely cobalt or nickel. Therefore, at least in haloarchaea, the variability of predicted C(P)XCG zinc finger motifs is higher than anticipated, and they can be metal-free, bind zinc, or bind another metal. Notably, AlphaFold2 cannot correctly predict whether or not the four cysteines have the tetrahedral configuration that is a prerequisite for metal binding. Full article

Marine and extreme environments harbor a huge diversity of microorganisms able to produce new bioactive metabolites with beneficial health effects. In this study, ethanol, aqueous, methanol, and acetone extracts and protein hydrolysates were obtained from five different microalgae species and two haloarchaea. An in vitro study of cytotoxicity, migration, angiogenic effect, antioxidant capacity, and modulation of detoxifying enzyme expression was carried out using resistant (HCT-15) and non-resistant (T84) colon cancer tumor lines. Our results showed that the aqueous extract of the microalga Chlorella sorokiniana induced the greatest cytotoxic effect in both cell lines, while the ethanolic extracts of the archaea Haloarcula hispanica and Halobacterium salinarum caused the greatest inhibition on the migratory capacity. Meanwhile, the protein hydrolyzate and the aqueous extract of the microalga Chlorella sorokiniana significantly protected cells against hydrogen peroxide damage. Moreover, the aqueous extracts of Haloarcula hispanica and Halobacterium salinarum resulted in inducing the greatest increase in the activity of the detoxifying enzymes enzyme quinone oxidoreductase and glutathione S-transferase. These preliminary results suggest that aqueous extracts of some microalgae and haloarchaea may be promising candidates for an adjuvant therapy against colorectal cancer. However, additional research is required to identify the active principles and elucidate the mechanisms of action involved. Full article

Halophilic archaea, also termed haloarchaea, are a group of moderate and extreme halophilic microorganisms that constitute the major microbial populations in hypersaline environments. In these ecosystems, mainly aquatic, haloarchaea are constantly exposed to ionic and oxidative stress due to saturated salt concentrations and high incidences of UV radiation (mainly in summer). To survive under these harsh conditions, haloarchaea have developed molecular adaptations including hyperpigmentation. Regarding pigmentation, haloarchaeal species mainly synthesise the rare C₅₀ carotenoid called bacterioruberin (BR) and its derivatives, monoanhydrobacterioruberin and bisanhydrobacterioruberin. Due to their colours and extraordinary antioxidant properties, BR and its derivatives have been the aim of research in several research groups all over the world during the last decade. This review aims to summarise the most relevant characteristics of BR and its derivatives as well as describe their reported antitumoral, immunomodulatory, and antioxidant biological activities. Based on their biological activities, these carotenoids can be considered promising natural biomolecules that could be used as tools to design new strategies and/or pharmaceutical formulas to fight against cancer, promote immunomodulation, or preserve skin health, among other potential uses. Full article

The genus Halomicroarcula, classified within the family Haloarculaceae, presently comprises eight haloarchaeal species isolated from diverse saline habitats, such as solar salterns, hypersaline soils, marine salt, and marine algae. Here, a detailed taxogenomic study and comparative genomic analysis of the genus Halomicroarcula was carried out. In addition, two strains, designated S1CR25-12^T and S3CR25-11^T, that were isolated from hypersaline soils located in the Odiel Saltmarshes in Huelva (Spain) were included in this study. The 16S rRNA and rpoB’ gene sequence analyses affiliated the two strains to the genus Halomicroarcula. Typically, the species of the genus Halomicroarcula possess multiple heterogeneous copies of the 16S rRNA gene, which can lead to misclassification of the taxa and overestimation of the prokaryotic diversity. In contrast, the application of overall genome relatedness indexes (OGRIs) augments the capacity for the precise taxonomic classification and categorization of prokaryotic organisms. The relatedness indexes of the two new isolates, particularly digital DNA–DNA hybridization (dDDH), orthologous average nucleotide identity (OrthoANI), and average amino acid identity (AAI), confirmed that strains S1CR25-12^T (= CECT 30620^T = CCM 9252^T) and S3CR25-11^T (= CECT 30621^T = CCM 9254^T) constitute two novel species of the genus Halomicroarcula. The names Halomicroarcula saliterrae sp. nov. and Halomicroarcula onubensis sp. nov. are proposed for S1CR25-12^T and S3CR25-11^T, respectively. Metagenomic fragment recruitment analysis, conducted using seven shotgun metagenomic datasets, revealed that the species belonging to the genus Halomicroarcula were predominantly recruited from hypersaline soils found in the Odiel Saltmarshes and the ponds of salterns with high salt concentrations. This reinforces the understanding of the extreme halophilic characteristics associated with the genus Halomicroarcula. Finally, comparing pan-genomes across the twenty Halomicroarcula and Haloarcula species allowed for the identification of commonalities and differences between the species of these two related genera. Full article