| 1. |
- Franzén, Carl Johan, 1966, et al.
(författare)
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High gravity lignocellulose bioprocess development for ethanol and lactic acid production by multi-feed simultaneous saccharification and fermentation
- 2017
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Ingår i: Oral presentation at: Recent Advances in Fermentation Technology, RAFT12. Oct 29 - Nov 1, Bonita Springs, FL, USA..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Second generation bioethanol production is becoming established in production plants across the world. The process can also be viewed as a model biorefinery concept for biotechnological conversion of recalcitrant lignocellulosic raw materials to chemicals and other products. We have developed a Multi-Feed SSCF process: a systematic, model-driven design of fed-batch simultaneous saccharification and co-fermentation of steam-pretreated lignocellulosic materials in standard stirred tank reactors. The design includes feeding of solid substrate, enzymes, and active, robust cell factories adapted to the present substrate. The concept has been applied not only to ethanol production with S. cerevisiae, but also to lactic acid production from wheat straw by the thermophilic, cellulolytic strain Bacillus coagulans MA-13, isolated from bean processing waste. High Gravity operation, i.e. fermentation at high concentrations of water insoluble solids (WIS), pushes the process towards higher product concentrations and productivities, and improved energy and water economy. By using the multi-feed SSCF approach, the ethanol process was pushed towards final product concentrations above 60 g/L, at about 90% of the theoretical yields on consumed substrate, using 22% w/w accumulated WIS additions of acid- and steam explosion-pretreated wheat straw. Bacillus coagulans MA-13 was found to secrete cellulolytic enzymes and ferment lignocellulose-derived sugars to lactic acid; thus, it may be a potential platform for consolidated bioprocessing of lactic acid. We investigated its performance in multi-feed SSF and found that pre-adaptation of cells to the liquid fraction of the steam-pretreated lignocellulosic material improves lactate productivity and reduces the SSF time from 33 to 12 hours.
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| 2. |
- Wang, Ruifei, 1985, et al.
(författare)
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Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Cell mass and viability are tightly linked to the productivity of fermentation processes. In 2 generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-based viability tests had very limited applicability in such samples. In contrast, manual cell enumeration in a hemocytometer, plating and enumeration of colony forming units, qPCR, and in situ dielectric spectroscopy were further investigated. Parameter optimization to measurements in synthetic lignocellulosic media, which mimicked typical lignocellulosic fermentation conditions, resulted in statistically significant calibration models with good predictive capacity for these four methods. Manual enumeration of cells in a hemocytometer and of CFU were further validated for quantitative assessment of cell numbers in simultaneous saccharification and fermentation experiments on steam-exploded wheat straw. Furthermore, quantitative correlations could be established between these variables and in situ permittivity. In contrast, qPCR quantification suffered from inconsistent DNA extraction from the lignocellulosic slurries. Development of reliable and validated cell quantification methods and understanding their strengths and limitations in lignocellulosic contexts, will enable further development, optimization, and control of lignocellulose-based fermentation processes.
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| 3. |
- Wang, Ruifei, 1985, et al.
(författare)
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Which methods for viable yeast cell quantification can be used in lignocellulosic fermentation processes
- 2016
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Ingår i: European Symposium of Biochemical Engineering Science (ESBES) 2016, 11-14 September, Dublin, Ireland.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Cell concentration is a primary characteristic of fermentation processes. The total cell concentration in aparticle-free liquid medium can be easily assessed by cell counts, optical density or dry weight. The quantification of viable cells is not as straightforward. Viable cells can be defined as culturable, metabolically active and intact cells. Culturable cells can be assessed by colony-forming unit (CFU) assay. Metabolically active and intact cells have been quantified by e.g. qPCR, dielectric spectroscopy probes, and flow cytometry using various dyes. All these methods work well for applications in clear liquid media, but have not been validated in 2nd generation bioprocesses using lignocellulosic materials.In this study we evaluate the applicability of several methods for quantitative assessment of both total and viable cell concentrations in lignocellulosic media. In order to mimic typical conditions of lignocellulosic fermentations, we used a central composite design of experiments with known cell numbers, water insoluble solids content (WIS) and osmolality as factors. For the osmolality, we used sorbitol and NaCl to differentiate hyperosmotic conditions at different ion strengths and conductivities. The cell concentrations were determined using cell enumeration in a hemocytometer (with and without methylene blue staining), plating and enumeration of CFU, qPCR on extracted DNA and RNA, and on-line permittivity using a capacitance probe. These methods have the potential to be less affected by impurities and water insoluble solids in lignocellulosic media than e.g. dry weight and turbidity. The number and viability of cells used to create the test conditions of the experimental design were first determined from the seed culture on defined mineral medium. Considering all experimental points and some validation points within the design space, all the selected methods were used for measuring total and viable number of cells. With these data we built a quantitative model to fit all interaction effects and curvature, and to calibrate the qPCR and permittivity results to the number of total and culturable cell counts. Data of qPCR on DNA were fitted to total cell numbers, WIS level and osmolality. The permittivity measured by the dielectric probe was fitted to CFUs, WIS level, osmolality and measured conductivity. Parameter optimization resulted in statistically significant models with good predictive capacity. The results showed that cell counts and CFU were not sensitive to WIS and osmolarity levels. Therefore they can be used asreference methods in lignocellulose-based media. Furthermore, using the selected methodologies in simultaneous saccharification and fermentation (SSF) process of pre-treated wheat straw showed consistent results in total and viable cell numbers.Development of reliable and validated total and viable cell quantification methods will contribute to wellmonitored lignocellulosic fermentation processes both for research and industry in bio-based production.
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| 4. |
- Aulitto, Martina, 1991, et al.
(författare)
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Bacillus coagulans MA-13: a promising thermophilic and cellulolytic strain for the production of lactic acid from lignocellulosic hydrolysate
- 2017
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 10:210
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Tidskriftsartikel (refereegranskat)abstract
- Background: The transition from a petroleum-based economy towards more sustainable bioprocesses for the production of fuels and chemicals (circular economy) is necessary to alleviate the impact of anthropic activities on the global ecosystem. Lignocellulosic biomass-derived sugars are suitable alternative feedstocks that can be fermented or biochemically converted to value-added products. An example is lactic acid, which is an essential chemical for the production of polylactic acid, a biodegradable bioplastic. However, lactic acid is still mainly produced by Lactobacillus species via fermentation of starch-containing materials, the use of which competes with the supply of food and feed. Results: A thermophilic and cellulolytic lactic acid producer was isolated from bean processing waste and was identified as a new strain of Bacillus coagulans, named MA-13. This bacterium fermented lignocellulose-derived sugars to lactic acid at 55 degrees C and pH 5.5. Moreover, it was found to be a robust strain able to tolerate high concentrations of hydrolysate obtained from wheat straw pre-treated by acid-catalysed (pre-) hydrolysis and steam explosion, especially when cultivated in controlled bioreactor conditions. Indeed, unlike what was observed in microscale cultivations (complete growth inhibition at hydrolysate concentrations above 50%), B. coagulans MA-13 was able to grow and ferment in 95% hydrolysate-containing bioreactor fermentations. This bacterium was also found to secrete soluble thermophilic cellulases, which could be produced at low temperature (37 degrees C), still retaining an optimal operational activity at 50 degrees C. Conclusions: The above-mentioned features make B. coagulans MA-13 an appealing starting point for future development of a consolidated bioprocess for production of lactic acid from lignocellulosic biomass, after further strain development by genetic and evolutionary engineering. Its optimal temperature and pH of growth match with the operational conditions of fungal enzymes hitherto employed for the depolymerisation of lignocellulosic biomasses to fermentable sugars. Moreover, the robustness of B. coagulans MA-13 is a desirable trait, given the presence of microbial growth inhibitors in the pre-treated biomass hydrolysate.
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| 5. |
- Aulitto, Martina, 1991, et al.
(författare)
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Draft genome sequence of bacillus coagulans ma-13, a thermophilic lactic acid producer from lignocellulose
- 2019
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Ingår i: Microbiology Resource Announcements. - 2576-098X. ; 8:23
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus coagulans MA-13 is an efficient lactic acid producer which withstands high concentrations of the growth inhibitors formed during the pretreatment of lignocellulosic feedstock. This draft genome sequence is expected to pave the way toward the understanding of mechanisms responsible for the robustness of MA-13 during simultaneous saccharification and fermentation.
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| 6. |
- Aulitto, Martina, 1991, et al.
(författare)
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Seed culture pre-adaptation of Bacillus coagulans MA-13 improves lactic acid production in simultaneous saccharification and fermentation
- 2019
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Lignocellulosic biomass is an abundant and sustainable feedstock, which represents a promising raw material for the production of lactic acid via microbial fermentation. However, toxic compounds that affect microbial growth and metabolism are released from the biomass upon thermochemical pre-treatment. So far, susceptibility of bacterial strains to biomass-derived inhibitors still represents a major barrier to lactic acid production from lignocellulose. Detoxification of the pre-treated lignocellulosic material by water washing is commonly performed to alleviate growth inhibition of the production microorganism and achieve higher production rates. Results In this study, we assessed the feasibility of replacing the washing step with integrated cellular adaptation during pre-culture of Bacillus coagulans MA-13 prior to simultaneous saccharification and lactic acid fermentation of steam exploded wheat straw. Using a seed culture pre-exposed to 30% hydrolysate led to 50% shorter process time, 50% higher average volumetric and 115% higher average specific productivity than when using cells from a hydrolysate-free seed culture. Conclusions Pre-exposure of B. coagulans MA-13 to hydrolysate supports adaptation to the actual production medium. This strategy leads to lower process water requirements and combines cost-effective seed cultivation with physiological pre-adaptation of the production strain, resulting in reduced lactic acid production costs.
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| 7. |
- Aulitto, Martina, et al.
(författare)
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Thermus thermophilus as source of thermozymes for biotechnological applications: homologous expression and biochemical characterization of an alpha-galactosidase
- 2017
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The genus Thermus, which has been considered for a long time as a fruitful source of biotechnological relevant enzymes, has emerged more recently as suitable host to overproduce thermozymes. Among these, alpha-galactosidases are widely used in several industrial bioprocesses that require high working temperatures and for which thermostable variants offer considerable advantages over their thermolabile counterparts. Results: Thermus thermophilus HB27 strain was used for the homologous expression of the TTP0072 gene encoding for an a-galactosidase (TtGalA). Interestingly, a soluble and active histidine-tagged enzyme was produced in larger amounts (5 mg/L) in this thermophilic host than in Escherichia coli (0.5 mg/L). The purified recombinant enzyme showed an optimal activity at 90 degrees C and retained more than 40% of activity over a broad range of pH (from 5 to 8). Conclusions: TtGalA is among the most thermoactive and thermostable a-galactosidases discovered so far, thus pointing to T. thermophilus as cell factory for the recombinant production of biocatalysts active at temperature values over 90 degrees C.
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| 8. |
- Fusco, Salvatore, 1985
(författare)
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A standardized protocol for the UV induction of Sulfolobus spindle‑shaped virus 1
- 2015
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Ingår i: Extremophiles. - 1433-4909 .- 1431-0651. ; 19, s. 539-546
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Tidskriftsartikel (refereegranskat)abstract
- The Fuselloviridae prototype member Sulfolobusspindle-shaped virus 1 is a model of UV-inducibleviruses infecting Crenarchaeota. Previous works on SSV1UV induction were bases on empirically determined parametersthat have not yet been standardized. Thus, in manypeer reviewed literature, it is not clear how the fluence andirradiance have been determined. Here, we describe a protocolfor the UV induction of SSV1 replication, which isbased on the combination of the following instrumentallymonitored parameters: (1) the fluence; (2) the irradiance;(3) the exposure time, and (4) the exposure distance. Withthe aim of finding a good balance between the viral replicationinduction and the host cells viability, UV-irradiatedcultures were monitored for their ability to recover in theaftermath of the UV exposure. This UV irradiation procedurehas been set up using the well-characterized Sulfolobussolfataricus P2 strain as model system to study host–virus interaction.
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| 9. |
- Fusco, Salvatore, 1985
(författare)
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The identification of a novel Sulfolobus islandicus CAMP‑like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules
- 2015
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 14:126
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Tidskriftsartikel (refereegranskat)abstract
- Background: Pathogenic bacteria easily develop resistance to conventional antibiotics so that even relatively newmolecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for thetreatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with anamphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa.Results: A novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76,encoded by pSSVx, a hybrid plasmid–virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physicaland functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires adefined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogeniccapability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on thecell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findingssuggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets.Conclusions: VLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaealmicroorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, resultsfrom this work show that DNA/RNA-binding proteins could be used as sources of CAMPs.
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| 10. |
- Fusco, Salvatore, 1985
(författare)
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Transcriptome analysis of Sulfolobus solfataricus infected with two related fuselloviruses reveals novel insights into the regulation of CRISPR-Cas system
- 2015
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Ingår i: Biochimie. - 0300-9084. ; 118, s. 322-332
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Tidskriftsartikel (refereegranskat)abstract
- Fuselloviruses SSV1 and SSV2 are model systems to investigate virusehost relationships in stablyinfected cells thanks to their temperate nature. Although they are very similar in morphology, genomeorganization and gene synteny, their replication is induced by different stimuli, i.e.: by UV-light exposure(for SSV1) and by the growth progression of the host (for SSV2). In this study, we have analysed globalgene expression in SSV1- and SSV2-lysogens of Sulfolobus solfataricus P2 in the absence of any stimuli.Additionally, the interplay among SSV1, SSV2 and the host has been investigated in a double-infectedstrain to explore both virusehost and virusevirus interactions. Whereas SSV1 did not induce majorchanges of the host gene expression, SSV2 elicited a strong host response, which includes the transcriptionalactivation of CRISPR loci and cas genes. As a consequence, a significant decrease of the SSV2copy number has been observed, which in turn led to provirus-capture into the host chromosome. Resultsof this study have revealed novel aspects of the hosteviral interaction in the frame of the CRISPRresponse.
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| 11. |
- Fusco, Salvatore, 1985
(författare)
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Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1
- 2015
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 89:12, s. 6453-6461
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACTSulfolobus spindle-shaped virus 1 represents a model for studying virus-host interaction in harsh environments, and it is so farthe only member of the family Fuselloviridae that shows a UV-inducible life cycle. Although the virus has been extensively studied,mechanisms underpinning the maintenance of lysogeny as well as those regulating the UV induction have received little attention.Recently, a novel SSV1 transcription factor, F55, was identified. This factor was able to bind in vitro to several sequencesderived from the early and UV-inducible promoters of the SSV1 genome. The location of these binding sites together with thedifferential affinity of F55 for these sequences led to the hypothesis that this protein might be involved in the maintenance of theSSV1 lysogeny. Here, we report an in vivo survey of the molecular events occurring at the UV-inducible region of the SSV1 genome,with a focus on the binding profile of F55 before and after the UV irradiation. The binding of F55 to the target promoterscorrelates with transcription repression, whereas its dissociation is paralleled by transcription activation. Therefore, we proposethat F55 acts as a molecular switch for the transcriptional regulation of the early viral genes.IMPORTANCEFunctional genomic studies of SSV1 proteins have been hindered by the lack of similarity with other characterized proteins. As aresult, few insights into their in vivo roles have been gained throughout the last 3 decades. Here, we report the first in vivo investigationof an SSV1 transcription regulator, F55, that plays a key role in the transition from the lysogenic to the induced state ofSSV1. We show that F55 regulates the expression of the UV-inducible as well as the early genes. Moreover, the differential affinityof this transcription factor for these targets allows a fine-tuned and temporal coordinated regulation of transcription of viralgenes.
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| 12. |
- Gaglione, R., et al.
(författare)
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Insights into the anticancer properties of the first antimicrobial peptide from Archaea
- 2017
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Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 1872-8006 .- 0304-4165. ; 1861:9, s. 2155-2164
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Tidskriftsartikel (refereegranskat)abstract
- Background: The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. Methods: Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. Results: It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. Conclusions: VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP.
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| 13. |
- Strazzulli, A., et al.
(författare)
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Metagenomics of microbial and viral life in terrestrial geothermal environments
- 2017
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Ingår i: Reviews in Environmental Science and Biotechnology. - : Springer Science and Business Media LLC. - 1572-9826 .- 1569-1705. ; 16:3, s. 425-454
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Forskningsöversikt (refereegranskat)abstract
- Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme-and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes.
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