Microbiological study of the Añana Salt Valley. Diversity analysis, isolation and screening of bioactivecompounds producers

REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Immunology, Microbiology, and Parasitology Department Immunologia, Mikrobiologia eta Parasitologia Saila Doctoral thesis/Doktorego tesia Maia Azpiazu Muniozguren 2025 Microbiological study of the Añana Salt Valley. Diversity analysis, isolation and screening of bioactive compounds producers Añanako Gatz Haranaren azterketa mikrobiologikoa. Dibertsitatearen analisia, isolamendua eta konposatu bioaktiboen baheketa Supervisors/Zuzendariak: Dr. Ilargi Martínez Ballesteros Dr. Javier Garaizar Candina REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 (cc) 2025 Maia Azpiazu Muniozguren (cc by-nc-nd 4.0) REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 FUNDING / FINANTZIAZIOA The present Doctoral thesis has been carried out thanks to the following funding: Doktorego tesi hau ondorengo laguntzei esker egin da: Agreement/Hitzarmena. Seguimiento espacio-temporal de variables ambientales en el espacio del Valle Salado. Funding Entity/ Erakunde finantzatzailea: Fundación Valle Salado. IP: Iñaki Antiguedad. 01/01/201731/12/2025. Project/ Proiektua. Estudio de la diversidad microbiana en el seguimiento espacio-temporal de variables ambientales en el espacio del Valle Salado de Añana (US19/01). Funding Entity/ Erakunde finantzatzailea: University of the Basque Country UPV/EHU and Biogenetics. IP: Ilargi Martinez Ballesteros. 29/11/2019-28/11/2021. Project/ Proiektua. MikroIker: Microbial genomics and characterisation (GIU21/021). Funding Entity/ Erakunde finantzatzailea: University of the Basque Country UPV/EHU. IPs: Irati Martínez Malax-etxebarria, Ilargi Martínez Ballesteros. 01/01/2022-31/12/2025. Project/ Proiektua. Producción y caracterización de biosurfactantes por procariotas halófilas del Valle Salado de Añana (PIBA23/06). Funding Entity/ Erakunde finantzatzailea: Basque Government/Eusko Jaurlaritza. IP: Irati Martínez Malax-etxebarria. 26/07/2023-30/06/2025. PhD fellowship from the University of The Basque Country UPV/EHU / UPV/EHUn ikertzaileak prestatzeko kontratazio deialdia (PIF22/12). A research stay at the Fundación MEDINA (Granada, Spain) was supported by a complementary grant from the University of the Basque Country UPV/EHU / UPV/EHUn mugikortasuna sustatzeko eta ikerketaren emaitzak hedatzeko laguntza deialdia. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 This research project has been approved by the Ethics Committee for Research on Biological Agents and Genetically Modified Organisms of the UPV/EHU (ABIEB-UPV/EHU) (M30/2020/181). Ikerketa proiektu honek UPV/EHUko Agente biologikoen eta genetikoki eraldatutako organismoen ikerketetarako Etika Batzodearen (ABIEBUPV/EHU) aldeko txostena dauka (M30/2020/181). The authors would like to thank the editors for permission to include the published articles in this Doctoral thesis. Egileek eskerrak eman nahi dizkiete editoreei argitaratutako artikuluak Doktorego tesi honetan sartzeko baimena emateagatik. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 SCIENTIFIC CONTRIBUTIONS / EKARPEN ZIENTIFIKOAK Publications / Argitalpenak  Azpiazu-Muniozguren M, Valgañón-Pérez E, García‑Martínez M, Rodriguez-Paniagua A, Poppy Clark H, Justicia C, Martín J, de la Cruz Moreno M, Reyes F, Laorden L, Martinez-Malaxetxebarria I, MartinezBallesteros I. Isolation and screening of bioactive compounds produced by halophiles and characterizing compounds produced by Pseudoalteromonas spp. ASV78. Under review.  Azpiazu-Muniozguren M, García-Martínez M, Zabaleta A, Antiguedad I, Garaizar J, Laorden L, Martinez-Malaxetxebarria I, Martinez-Ballesteros I. Prokaryotic Diversity and Community Distribution in the Complex Hydrogeological System of the Añana Continental Saltern. Microbial Ecology. 2024;87(1):171. DOI: https://doi.org/10.1007/s00248-02502488-2.  Azpiazu-Muniozguren M, García M, Laorden L, Martinez-Malaxetxebarria I, Seoane S, Bikandi J, Garaizar J, Martínez-Ballesteros I. Anianabacter salinae gen. nov., sp. nov. ASV31T, a facultative alkaliphilic and extremely halotolerant bacterium isolated from brine of a millennial continental saltern. Diversity. 2022;14(11):1009. DOI:https://doi.org/10.3390/d14111009.  Martinez-Ballesteros I, Azpiazu-Muniozguren M, Martínez Malaxetxebarria I, Laorden L, Garaizar J. Caracterización de la diversidad microbiana del agua de Salinas de Añana mediante cultivo y genómica (Spain). Book / Liburua: Seven millennia of saltmaking. Editor: Alberto Plata. Fundación Valle Salado. ISBN: 978-84-7821-936-0. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 La persona ha de tener la suficiente fe en sí misma para emprender aventuras, y dudar de sí misma lo suficiente para disfrutarlas. G. K. Chesterton REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 ESKER ONAK / AGRADECIMIENTOS Guztiak dauka hasiera bat eta amaiera bat. Hitz hauek idazterakoan atzeratz begiratu eta pertsona, momentu zein bizipen mordo batekit topo egiten dut, eta nola ez, posez betetzen naiz. Ezinezkoa bada ere lerro gutxi hauetan nire esker ona demostratzea, jakin ezazue momentu honetara iristeko zuek guztiok izan zaretela garrantzitsuak. Beraz, eskerrik asko denoi. Lehenik eta behin eskerrak eman nahi dizkiet nire tesi zuzendariei, Ilargi eta Javi, lan hau aurrera eramateko nigan jarri zenuten konfiantza eta bide honetan zehar eskaini didazuen laguntzagatik. Bereziki, eskerrik asko Ilargi, denbora guzti honetan zehar gertu egotearren, mila oztopori aurre egiten eta persona zein zientzialari eredu izaten. Eta nola ez Javi, eskerrik asko hasieratik talde giroa sustatzearren, ilusioa emanez ikerlari gazteoi “buru belarri equipo” behin eta berriro esanez. Eskerrik asko baita MikroIker ikerketa taldea osoari ere, lanean eta esfortsuan oinarrituta ilusioz beteriko ikerketa talde baten sorreraren parte izatea ahalbidetu duzuelako. Beti izango zaituztet gogoan laborategian momenturik politenak psatzea posible egin duzuen guztiok. Asko bazarete ere eskerrik asko benetan zuetako bakoitza ezagutzeko eta zuek gaindik ikasteko eman didazuen aukeragatik. Modu berean, Immunologia, Mikrobiologia eta Parasitologiako sail osoari, emandako baliabide, tratu eta gertukotasunagatik. Azkenik, nola ez, Añanako Gatz Harana Fundazioari, parte hartu dudan ikerketa proiektuan egindako apustuagatik eta zuekgandik jasotako prestutasunagatik. Por otro lado, cómo no dedicarles unas palabras de total agradecimiento a todo el equipo de la Fundación MEDINA. En particular muchas gracias Fernando, por haberme dado la oportunidad ir y de aprender de vosotros. Gracias a todo el equipo, por haberme acogido tan bien, empezando por micro, pasando por química hasta screening me habéis ayudado en todo momento y de verdad que guardo un muy buen recuerdo de todos vosotros. Mercedes, cómo no REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 agradecerte por esas charlas y risas, momentos fuera del laboratorio y bueno simplemente por ser asi como eres. ¡Muchísimas gracias a todos! Mila esker, ama eta aita proiektu honi aurre egiteko aukera, medioak eta animoak emateagatik. Baita Txikiri ere, hitzik gabe konektatzeko eta beti animoa altxatzeko gaitasuna izatearren. Eta nola ez, Jon, zu gabe ez nintzen proiektu honen hasierara iritsiko, beraz eskerrik asko bide honetan zehar ikasle-irakasle rolak partekatuz beti gertu egotearren. Eskerrik asko neskak, betiko lagunak, denpora honetan zehar izan duzuen ulerpena eta pazientziagatik, deskonektatzeko eman didazuen aukera bakoitzagatik eta zuekgandik jaso dudan berotasunagatik. Amaitzeko, guztion gainetik dagoen Jaun Goikoari, eskerrik asko, denbora eta pertsonez baliatuz bidea markatu eta beti sostengu izateagatik. Mila esker. Muchas gracias. Maia Azpiazu Muniozguren REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 LIST OF ABBREVIATIONS / LABURDUREN ZERRENDA English Euskera AAI Average Amino Acid Identity Aminoazidoen batez besteko identitatea ANI Average Nucleotide Identity Nukleotidoaren batez besteko identitatea ASV Amplicon Sequence Variant Anplikoien sekuentzien aldaera BGCs Biosynthetic gene clusters Gene-multzo biosintetikoak BPM Biosurfactant/bioemulsifier production medium Biosurfaktantea/bioemultsifikatzailea ekoizteko medioa CDSs Protein coding genes Proteinak kodetzen dituzten geneak CECT Spanish Type Culture Collection Espainiako mikroorganismo tipo-en bilduma CFS Cell-free supernatant Zelularik gabeko gainjalkina dDDH Digital DNA-DNA hybridization DNA-DNA hibridazio digitala DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen Alemaniako mikroorganismoen eta zelula-kulturen bilduma EI24 Emulsification index (%) Emultsifikazio indizea (%) EPS Exopolysaccharides Exopolisakaridoak EUCAST European Committee on Antimicrobial Susceptibility Testing Mikrobioen aurkako sentikortasun-proben Europako Batzordea FAO Food and Agriculture Organization Elikadura eta Nekazaritza Erakundea FDA Food and Drug Administration Elikagaien eta medikamentuen adminiztrazioa GIAHS Globally Important Agricultural Heritage System Mundu mailan garrantzitsua den Nekazaritza Ondare Sistema HPLC High-Performance Liquid Chromatography Bereizmen altuko likido kromatografia ICNP International Code of Nomenclature of Prokaryotes Prokariotoen Nomenklaturaren Nazioarteko Kodea REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 IR Infrared spectroscopy Espektroskopia infragorria iTOL Interactive Tree Of Life Bizitzaren zuhaitz interaktiboa ITS Ribosomal internal transcribed spacer Transkribatutako barne-espaziatzaile erribosomikoa LB Luria-Bertani culture media Luria-Bertani hazkuntza medioa LC-HRMS Liquid chromatography high resolution mass spectrometry Kromatografia likidoa bereizmen handiko masa-espektrometria MA Marine agar culture media Itsas agarra hazkuntza medioa MB Marine broth culture media Itsas salda hazkuntza medioa MDM "Microbial Dark Matter" Mikrobio-materia iluna EOR Enhanced oil recovery Lagundutako olioaren berreskurapena MH Müller-Hinton culture media Müller-Hinton hazkuntza medioa MIC Minimum Inhibitory Concentration Kontzentrazio minimo inhibitzailea MRSA Methicillin-resistant Staphylococcus aureus Staphylococcus aureus metilzilina erresistentea MS Mass spectra Masa espektroa NCBI National Center for Biotechnology Information Bioteknologiako Informazio Zentro Nazionala NGS Next Generation Sequencing Hurrengo belaunaldiko sekuentziazioa NMR Nuclear Magnetic Resonance Erresonantzia magnetiko nuklearra OGRI Overall Genomic Relatedness Index Erlazio-indize genomiko orokorra PCR Polymerase chain reaction Polimerasaren kate-erreakzioa PHA Polyhydroxyalkanoate Polihidroxialkanoatoa PICRUSt2 Phylogenetic Investigation of Communities by Reconstruction of Unobserved States Komunitateen ikerketa filogenetikoa behatu gabeko egoerak berreraikiz REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 POCP Percentage Of Conserved Proteins Kontserbatutako proteinen ehunekoa QIIME2 Quantitative Insights Into Microbial Ecology Mikrobio-ekologiari buruzko ikuspegi kuantitatiboak RAST Rapid Annotation with Subsystems Technology Anotazio azkarra azpisistemen teknologiarekin Rt Retention time Erretentzio denbora SDA Sabouraud dextrose agar Sabouraud dextrosa agarra SDS Sodium dodecyl sulphate Sodio dodezil sulfatoa SGIker Advanced Research Facilities Ikerkuntzarako Zerbitzu Orokorrak TLC Thin Layer Chromatography Geruza fineko kromatografia TYGS Type Strain Genome Server Andui tipoen genoma zerbitzaria UV Ultraviolet Ultramorea WGS Whole genome sequencing Genoma osoaren sekuentziazioa REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 TABLE OF CONTENTS / EDUKIEN TAULA ENGLISH VERSION List of figures ................................................................................ I List of tables ................................................................................. V Abstract ..................................................................................... VII Section 1 - Overview .................................................................................... 1 GENERAL INTRODUCTION ................................................................ 3 THEORETICAL FRAMEWORK ........................................................... 5 HYPOTHESES AND AIM OF THE STUDY ....................................... 26 METHODOLOGY ................................................................................. 28 SUMMARY OF THE RESULTS AND DISCUSSION ........................ 51 REFERENCES ..................................................................................... 105 Section 2 - Conclusions ........................................................................... 127 EUSKARAZKO BERTSIOA Irudien zerrenda .......................................................................... XI Taulen zerrenda ......................................................................... XV Laburpena ............................................................................... XVII 1. atala - Ikuspegi orokorra .................................................................... 131 SARRERA OROKORRA .................................................................... 133 ESPARRU TEORIKOA ...................................................................... 135 HIPOTESIAK ETA HELBURUA ....................................................... 157 METODOLOGIA ................................................................................ 159 EMAITZEN LABURPENA ETA EZTABAIDA ................................ 183 ERREFERENTZIA ITURRIAK .......................................................... 238 2. atala - Ondorioak ................................................................................ 239 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Abstract VII Abstract The Añana Salt Valley, in northern Spain, is a continental saltern located on a diapiric structure and fed by natural hypersaline springs that have been producing salt for more than 7,000 years. Despite its recognised historical and geological importance, the microbial inhabitants of this habitat have remained largely understudied. In this Doctoral thesis, 16S rRNA gene and ITS amplicon sequencing was conducted to study the prokaryotic and fungal communities present in different water sources in the valley. In parallel, halophilic and halotolerant bacteria were isolated and identified to establish a strain collection. A polyphasic characterisation was conducted on a number of the isolates that showed potential to be new taxa, with the aim of providing a description of them. Finally, the antimicrobial activity, the reduction of surface tension and the emulsifying capacity of the isolates were screened in order to identify those that were capable of producing the bioactive compounds of interest. In addition, a more detailed characterisation of the compounds produced by the ASV78 strain was conducted. The genomic approach revealed the existence of two distinct microbial communities, one from salty waters (∼200 g/L) and one from brackish waters (≤20 g/L). Salinity was shown to be the key environmental factor determining microbial composition, although niche-specific variations were also observed. Archaeal taxa, in particular the phylum Methanobacteriota, largely represented by the genus Halorubrum, predominated in salty waters, whereas bacterial taxa, including Pseudomonadota, were widespread, especially in brackish waters. Fungal diversity, studied for the first time in this continental saltern, revealed the ubiquity of the genus Saccharomyces, linked to environmental factors such as proximity to agricultural land. In addition, a collection of 150 halophilic and halotolerant isolates was generated, with Pseudomonadota being the phylum with the highest number of REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Abstract VIII isolates. Among these, two potential new bacterial isolates were finally found to have different genomic, phenotypic and chemotaxonomic characteristics compared to previously described taxa. One new species, Alteripontixanthobacter muriae sp. nov. (type strain SALINAS58T), was found to be a new member of the Erythrobacteraceae family. On the other hand, a new genus, Anianabacter salinae gen. nov. sp. nov. (type strain ASV31T), was found to be a new member of the family Paracoccaceae. Finally, bioassays identified 20 antimicrobial-producing isolates, 26 effective biosurfactant-producing isolates and 19 bioemulsifier-producing isolates. Members of the genus Pseudoalteromonas were found to be mainly responsible for the antibacterial activity, and members of the genus Halomonas for surface tension reduction and emulsifying activities. One isolate, Pseudoalteromonas sp. ASV78, was found to synthesizing both, antimicrobial and surface-active compounds. Further analysis of the strain ASV78 showed that the antibacterial activity against Gram-positive bacteria was related to the production of pentabromopseudilin and bromophene, and that the antibacterial activity against Gram-negative bacteria was exclusively related to the production of pentabromopseudilin. Of particular interest was low concentration of pentabromopseudilin at which it is effective against Staphylococcus aureus ATCC 29213. Regarding the surface-active compounds produced by strain ASV78, it was found that reduction in surface tension was associated with glycolipid-type biosurfactant production, and the ability to emulsify hydrophobic substrates was associated with lipopeptide-type bioemulsifier production. Taken together, these findings emphasise the role of the Añana Salt Valley as a reservoir of microbial diversity of extremophiles, as well as a promising source of new taxa and bioactive compounds of interest. This underscores the necessity for further study in this area. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - Overview REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 – GENERAL INTRODUCTION 3 GENERAL INTRODUCTION Hypersaline ecosystems are environments where the concentration of salt in water or soil is about 10 times that of seawater. These ecosystems can be coastal or continental systems and can include salt lakes or lagoons, salterns (coastal or continental), salterns and salt diapirs, among others (1). The salterns represent an environment of great ecological importance. So much so that in 2017, the Añana Salt Valley (Álava, Spain) was recognised by the Food and Agriculture Organization of the United Nations (FAO) as the first European Globally Important Agricultural Heritage System (GIAHS) (2), for being a saline habitat with exceptional biodiversity, typical of wetlands associated with saline environments. The Añana Salt Valley is one of the most emblematic examples of the valorisation of continental salterns. The salt present in this saltern is due to a large salt diapir, the result of the drying up of an ancient sea 200 million years ago. Fresh rainwater seeps through and dissolves the deeper layers of halite or rock salt and emerges in the valley as hypersaline springs, providing a habitat for a wide variety of halophilic and halotolerant microorganisms (3). The study of microbial biodiversity in hypersaline ecosystems is an area of great interest worldwide. This interest is mainly based on the fact that many of the microorganisms that inhabit these environments have special characteristics that allow them to live in the presence of high salt concentrations. Their metabolism and certain molecules they synthesise are therefore of great interest for industrial processes and biotechnological applications (production of bioplastics, pigments, antibiotics, elimination of pollutants, etc.) (4). During the 20th century, studies of halophilic microorganisms relied on culture-based approaches until the advent of new molecular and genomic sequencing techniques that allowed the development of genetic and culture-independent methods. Innovative genome-based techniques opened up a new way of understanding nature and showed that microbial diversity was REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 – GENERAL INTRODUCTION 4 much greater than originally thought (5). Hence, halophiles have been found in all three domains of life: Archaea, Bacteria and Eukarya, although the salt tolerance and strategies to cope with salt stress are quite different in each group. While recently developed genome-resolved metagenomics and single-cell genomics techniques have revealed the immense genetic diversity and metabolic potential of uncultivated halophiles, culture of these microorganisms is required to describe new taxa, study their physiology, confirm predicted biochemical pathways and exploit their biotechnological potential (6). Even though continental salterns that are still actively producing salt are exceptional (7) and despite the fact that archaeological, hydrogeological and salt studies have been carried out in the Añana Salt Valley, there is little microbiological data available. Furthermore, its distinctive salt-related architecture, encompassing springs, wells and pans built of stone, wood and clay, renders it a valuable subject for study, particularly with regard to the variations in the microbial community at different sites within the saltern. Conversely, no culture-dependent studies have yet been carried out on this saltern. This, in turn, increases the likelihood of discovering previously undescribed taxa, as well as halophiles that produce biomolecules with potential applications in current health needs. The knowledge gained could, in the future, contribute to the recovery and valorisation of the Añana Salt Valley, as well as to the knowledge of the microbial population living in hypersaline environments or the potential biotechnological use of halophilic and halotolerant microorganisms for the benefit of human and environmental health. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 5 THEORETICAL FRAMEWORK Hypersaline environments: the Añana Salt Valley Hypersaline environments are considered extreme environments, whether terrestrial or aquatic and are defined as those with a high concentration of salts. These environments are also characterised by elevated temperatures and/or low oxygen levels (5). Aquatic hypersaline environments are those where the salt concentration is higher than that of seawater (around 3.5% (w/v) in seawater vs. up to 35% (w/v) in brines). This type of environment includes ecosystems such as continental lakes, lagoons, salterns and coastal or continental salterns. All these environments, from seawater salinity to NaCl-saturated brines, are potential habitats for microbial life. Representative natural examples include the Dead Sea on the border between Israel and Jordan (346 g/L dissolved salts), the Great Salt Lake in Utah (270-300 g/L dissolved salts) and many others (4,5). In general, hypersaline aquatic environments can be divided into two groups according to the ionic composition of the water: thalassohaline and athalassohaline (5). The brines produced by the evaporation of seawater are called thalassohaline and reflect the ionic composition of the sea. Thalassohaline brines are low in calcium and, to a lesser extent, sulphate. As NaCl precipitates as halite, the ionic composition changes and the relative concentrations of potassium and magnesium increase. The Great Salt Lake of Utah, although long separated from the world's oceans, still reflects in its ionic composition the seawater that provided its salt, so its waters can still be classified as thalassohaline. Thalassohaline brines are characterised by neutral or slightly alkaline pH values. In other hypersaline environments, the ionic composition can vary significantly from that of seawater; these are called athalassohaline environments. The Dead Sea is a good example of an athalassohaline lake. In this environment, divalent cations predominate, with magnesium and calcium concentrations exceeding those of sodium and potassium. The pH of the Dead Sea brine is relatively low at around 6.0 (5). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 6 On the other hand, salterns are very diverse and can be classified according to various criteria, such as the location of the salt deposit, the hydrogeological origin of the deposit, the method of salt production or the present state of the salterns (8). Based on a classification according to the location of the resource, three main groups of salterns can be described, with some examples in Spain: coastal salterns (Sanlúcar de Barrameda, Cádiz or Fuencaliente Saltern, La Palma), salt mines (Remolinos, Zaragoza or Léniz, Guipúzcoa) and continental saltens (La Malahá, Granada or the Añana Salt Valley, Álava (Figure 1)). Figure 1. View of part of the Salt Valley with the village of Salinas de Añana in the background. The threshing floors for salt production can be seen. Photograph taken in this study. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 7 The variety of continental saltens in the Iberian Peninsula is very large, due to the variety of orographies, climatological conditions and geological components that make them up (8). The bulk of them date from the Late Triassic, more than 200 million years ago. This event is based on the theory that a large part of the eastern end of Spain was at that time submerged in the present-day Mediterranean Sea, which was then known as the Tethys Sea, as shown in Figure 2A. Repeated cycles of evaporation and flooding created a salty layer, which was fractured by tectonic plate movements and deposited on the surface or, in other cases, buried under other more modern edaphic layers, forming underground brine deposits. These unique conditions make the continental salterns of the Iberian Peninsula an exclusively Iberian phenomenon within the European continent. However, less than 10% of these salterns remain active. The rest are in danger of disappearing for a variety of reasons, including economic abandonment, pollution, changes in land use and, above all, ignorance of their historical and biological heritage (7,9). The Añana Salt Valley (Álava, Basque Country, Spain) is one of the best preserved continental saltern in Spain. The underground evaporitic rocks were formed during the early stages of the fragmentation of the supercontinent Pangaea (about 200 million years ago) and are composed of salt, gypsum, clay and others. These rocks are now part of a geologically complex diapiric structure (Figure 2B) that creates an ellipsoidal structure of approximately 19 km2 (10). The hydrogeological system around the Añana salt diapir is extremely complex and compartmentalised. According to the hydrochemical information, the different waters that emerge in the valley are the result of mixtures, in different proportions and depths, of at least two types of water. On the one hand, those from the eastern sector of the diapir, which are clearly sulphated and shallower. On the other hand, those from the southern sector of the diapir, clearly chlorinated and deeper, which condition the salty springs (11). The existence of these salty springs has enabled salt to be actively produced for at least 7,000 years. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 14 Studies of the phylogeny of halophiles have shown that the best-known group of extreme halophiles is represented by the archaeal phylum Methanobacteriota, which includes the widespread genera Halobacterium and Haloquadratum. The phylum Methanobacteriota also includes methane-producing anaerobic halophiles belonging to the genera Methanohalophilus and Methanonatronarchaeum. It is also worth mentioning the archaea halophiles in the “Candidatus” phylum Nanohalarchaeota. The analysis of the highly conserved ATP synthase subunits indicates an affiliation with the Methanobacteriota. However, at this time, no members of “Candidatus Nanohalarchaeota” have been cultivated in pure culture (21,25). On the other hand, the largest and best-known group of moderately halophilic bacteria is the family Halomonadaceae (phylum Pseudomonadota) (21). Halophilic bacteria are a diverse group which include Gram-positive and Gram-negative, phototrophic and heterotrophic and aerobic and anaerobic bacteria. Most halophilic bacterial species are included in the following phyla: Actinomycetota, Bacteroidota, Cyanobacteriota, Bacillota, Pseudomonadota and Mycoplasmatota (26,27). Some of the representative halophilic and halotolerant genera of these phyla are shown in the Figure 4. Halophilic members of the phylum Actinomycetota belong to the order Actinomycetales, while halophilic bacterial species of the phylum Bacteroidota are distributed among the three classes. The phylum Cyanobacteriota, also known as bluegreen algae, dominates the planktonic biomass and plays an ecological role as a major contributor to photosynthesis in many hypersaline lakes. Numerous halophilic species have been reported as members of the phyla Bacillota and Pseudomonadota, distributed in different families, while there are few moderately halophilic species belonging to the phyla Mycoplasmatota (26,27). Although it is considered that most of the halophilic bacteria are moderate halophiles, there are examples of extremely halophilic bacteria, such as Salinibacter ruber (phylum Rhodothermota), which is the major bacteria of hypersaline aquatic ecosystems worldwide, the photosynthetic purple bacteria REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 15 belonging to the genus Halorhodospira (phylum Pseudomonadota) or Actinopolyspora halophile (phylum Actinomycetota) (21,28). Within the Eukarya domain, halophiles are uncommon. The best knowns are the unicellular green algal genus Dunaliella, which is found in most high salt aquatic systems, the fungi Hortaea werneckii (phylum Ascomycota) and Wallemia ichthyophaga (phylum Basidiomycota) and the macroorganism brine shrimp Artemia (1,21). In general, yeasts and filamentous fungi tolerate hypersaline environments, growing better under aerobic conditions at moderate temperatures and acidic or neutral pH. Among the protozoa in hypersaline environments, the more widely studied is Fabrea salina, although other representatives are also present (19,29). Concerning the halotolerant microorganisms, the vast majority of them belong to the Bacteria and Eukarya domains. Examples include the bacteria of the genera Alteromonas, Lactobacillus, Bacillus, Myxococcus and Pediococcus, as well as many cyanobacteria and fungi such as Debaromyces, Hansenula, Cladosporium and Saccharomyces (22). Hypersaline environments as a source of novel microbial taxa Even though there has been great progress in the isolation and culture of extremophiles, most of these microorganisms cannot be cultured and do not grow in conventional laboratory conditions. Recent studies have estimated that 80% of microbial taxa remain uncultured and that the 85% of the phylogenetic diversity of prokaryotes consists of uncultured taxa (30). The term used to name the vast diversity of uncultivated microorganisms that can only be identified and studied using non-culture-based methods is "Microbial Dark Matter" (MDM). It is estimated that MDM dominate the vast majority of environments on Earth (31). In particular, the extremobiosphere that includes environments where the physicochemical conditions represent the known limits of life, is recognised as a hotspot of these microbes. These microorganisms possess special characteristics acquired during their adaptation to the environmental REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 16 niches in which they live. A significant number of previously unidentified taxa belonging to diverse phylogenetic lineages that inhabit hypersaline environments across the globe have been examined through the utilisation of cutting-edge metagenomics and other culture-independent methodologies. In fact, it was reported that among halophiles, the MDM includes 42 “Candidatus” phyla including “Zixibacteriota”, “Parcubacteriota”, “Asgardarchaeota”, “Bathyarchaeota” and members of the Nanobdellati kingdom. Albeit these studies yield substantial information, the ultimate objective should be to cultivate these novel species in a laboratory setting (21). The success of previous studies with Spiribacter (32), Haloquadratum (33) or Salinibacter (34) provides a positive outlook for the future isolation of new halophilic taxa, including members classified as “Candidatus” who have not yet been isolated. Metagenomic data allow research teams to develop tailored strategies to isolate members of uncharacterised dominant populations, with varying degrees of success. “Culturomics" methods can be useful, as demonstrated in the isolation of the archaeon Halorutilus (35). Others were serendipitous discoveries, such as the fascinating Actinarchaeum halophilum, which grew during a study to isolate halophiles of Actinomycetes class from a Chinese salt lake (36). Isolation in pure culture may not always be possible when different halophile species are not interdependent due to mutualism. An example of this is the metabolic crossfeeding discovered between a Halorubrum and a Marinococcus strain from the Cuatro Cienegas Basin (Mexico) growing together at 250 g/L salt (37). In this context, it is noteworthy that, despite the growing global trend towards an over-reliance on molecular approaches to study microbial communities, efforts continue to be made to conduct culture-based studies targeting novel halophilic taxa (38). Current prokaryotic taxonomy is based on phylogenetics and follows what is known as polyphasic characterisation. Phylogeny uses evolutionary associations of conserved gene sequences, mainly 16S rRNA gene in the case of prokaryotes, to determine the phylogenetic relationships of an isolate. Polyphasic characterisation combines genomic, chemotaxonomic, REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 17 physiological and cultural characteristics. Polyphasic characterisation is recognised as the standard method for the comprehensive classification of new taxa based on phenotypic, genotypic and chemotaxonomic characters (39). As a result, the number of taxa effectively recognised, for example in the class Halobacteria, has increased significantly in recent years, by around 50% between 2017 and 2023 (21). Biotechnological relevance of halophilic and halotolerant microorganisms The use of extremophilic microorganisms as cellular factories for the production of biomolecules has become of particular interest for the discovery of new biomolecules with multiple applications, from biomedical to biotechnological (40). Halophiles are extremophiles that have adapted to the high osmotic pressures of their environment. They are also considered polyextremophiles because they have adapted not only to high salinity but also to other environmental extremes such as extremely high or low pH or temperature (4). The genetic adaptation of halophilic and halotolerant microorganisms to extreme conditions have led to the production of a variety of compounds of great interest to industrial applications. Some of the most important are listed in Table 1. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Table 1. Some current and potential biotechnological uses of compounds produced by halophilic and halotolerant microorganisms. Product Application Examples of representative producers Bacteriorhodopsin Photochemical (optical memories) and photoelectric (motion biosensors, X-ray sensors and photovoltaic cells) applications. Commercialised by a German company (MIB, Munich Innovative Biomaterials). Halobacterium halobium Halobacterium salinarum Carotenoid pigments Bacterioruberin Antioxidant and food colouring agent. Halorubrum sp. β-Carotene Additive in cosmetics and food products. Dunaliella sp. Compatible solutes Ectoine Additive in dermatological moisturizers. Halomonas sp. Chromohalobacter israelensis Hydroxyectoine Protection of proteins against misfolding degradation and freezing. Halomonas elongata Marinococcus M52 Betaine In biomedicine, as a potential treatment for adipose infiltration of the liver at the onset of cirrhosis and anticoagulant. Halorhodopira halochloris Thioalkalovibrio versutus Halophilic enzymes Amylases In the food industry, in baking processes, brewing and fruit juices, where they are added to degrade the starch. Halomonas sp. Halobacterium salinarum Haloferax mediterranei Proteases Additives in pharmaceuticals and laundry detergents. Bacillus sp. Halobacillus sp. Halobacterium halobium Xylanases and cellulases Biobleaching and to accelerate the process of hydrolysis in plants for the extraction of fruit juices. Haloferax sulfurifontis Streptomonospora sp. Continued Section 1 - THEORETICAL FRAMEWORK 18 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Product Application Examples of representative producers Polyhydroxyalkanoates (PHAs) Polyhydroxybutyrate Biodegradable plastics and medical materials. Halomonas boliviensis Haloferax mediterranei Biosurfactants and bioemulsifiers Rhamnolipids Removal of crude oil, treatment of soil and wastewater, antiviral, antifungal, and antibacterial agents and cosmetics. Marketed by a number of US companies: GlycoSurf, AGAE Technologies LLC and Logos Technologies. Pseudomonas aeruginosa Pseudomonas putida Marinobacter sp. Surfactin Heavy metals removal. Bacillus subtilis Trehalose lipids Bioremediation of cold marine environments. Ectothiorhodospira sp. Exopolysaccharides Active hydrocarbon emulsifiers. Halomonas sp. Haloferax volcanii Halobacterium salinarum Emulsan Emulsion forming and stabilising properties especially in relation to hydrocarbon biodegradation. Acinetobacter venetianus Antimicrobial compounds Actinomycin C2 and pyrrole (1,2-A (pyrazine-1,4‑dione, hexahydro-3- (2methylpropyl)-) Antimicrobial activity against Escherichia coli, Sthapylococcus aureus and Pseudomonas aeruginosa. Nocardiopsis sp. Pyrrolo[1,2-a]pyrazine-1,4dione,hexahydro Antimicrobial activity against multidrug resistant S. aureus. Bacillus tequilensis Chaxamycins A–D Antimicrobial activity against S. aureus. Streptomyces sp. Detailed information in references (4,40–47). Section 1 - THEORETICAL FRAMEWORK 19 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 20 Among them, new antimicrobial compounds, as well as biosurfactant and bioemulsifier compounds, are in high demand due to the need to address today's pressing issues such as antimicrobial resistance and environmental contamination, which threaten both human and environmental health. In this context, bioactive products from microbes in extreme environments, including halophile and halotolerant microorganisms, have demonstrated a great capacity to produce valuable compounds. Thus, the upward trend of scientific research in the field of halophiles and halotolerant bioactivity is evident from the increase in publications on the subject year on year (Figure 5). Figure 5. Number of publications found in the ScienceDirect database after searching for studies on antimicrobials (blue) (a total of 819) and surface-active compounds (green) (a total of 388) related to halophilic and halotolerant microorganisms between 2000 and 2025 (accessed March 2025). The continuing rise of antibiotic resistance among many types of bacteria is one of the most serious health problems we face today. According to the data reported by the Lancet, nearly 5 million people died from drug-resistant 0 20 40 60 80 100 120 Number of publications Publication year Search query: (biosurfactant OR bioemulsifier) AND (halophil OR halotolerant) Search query: ("anti-infective agent" OR antimicrobial) AND (halophil OR halotolerant) REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 21 bacterial infections in 2019 and the crisis is expected to worsen in the near future (48). Finding new antimicrobial therapies is therefore one of the fundamental goals of science. In this sense, antagonistic interactions and antimicrobial compounds of halophilic bacteria and archaea have been studied. It has been shown that extreme conditions can increase competition and promote the evolution of biosynthesis of antimicrobial compounds with a narrow spectrum of activity but high stability and potency (49). Among halophilic and halotolerant bacteria, Actinomycetota are a predominant phylum of antimicrobial producing strains. Some examples of antimicrobial compounds produced by them are chaxamycins A–D (Streptomyces sp. from Salar de Atacama, Chile) (Figure 6A), anthracimycin (Streptomyces sp. from Santa Barbara, California) and abyssomicins B–D (Verrucosispora sp. from Sea of Japan) (50). In addition to Actinomycetes class members, other halophilic and halotolerant genera, including Bacillus, Kocuria, Vibrio and Halomonas, have been shown to produce molecules such as indole derivatives, alkaloids, trypenoids and peptides that are bioactive against certain pathogens. Interestingly, the efficacy of these halophilic biomolecules against drug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), fluconazole-resistant Candida albicans, norfloxacin and ciprofloxacin-resistant "Klebsiella quasivariicola" and penicillin-resistant Streptococcus pneumoniae has already been demonstrated (44). Recent advances suggest that halogenated antimicrobials may enhanced activity against drug-resistant bacteria. Indeed, studies have shown that the incorporation of halogenated compounds into peptides enhances antimicrobial activity against multi-resistant pathogens, with brominated analogues showing 32-fold increased activity against MRSA and 16-64-fold increased activity against Pseudomonas aeruginosa and Escherichia coli (51,52). In this context, species of the halophilic genus Pseudoalteromonas, often isolated from salterns, have been described as producers of halogenated secondary metabolites (Figure 6B). Most of them have strong antibacterial and antifouling REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 22 activity (53,54). In fact, genome mining approach to secondary metabolite discovery in Pseudoalteromonas genomes (42 publicly available) revealed that the pigmented strains had an average density of 10 biosynthetic gene clusters (BGCs) per genome, suggesting an untapped source of potentially interesting metabolites in this genus (55). Figure 6. Examples of the chemical structures of some antimicrobial compounds produced by A, Streptomyces and B, Pseudoalteromonas, both halophilic and halotolerant bacteria. On the other hand, surface-active biomolecules are another interesting group of compounds synthesised by various microorganisms as secondary metabolites. These biomolecules are essential for the bioavailability of substrates and thus for the survival of the microorganisms. Surface-active biomolecules have a hydrophilic (a sugar or peptide) and a hydrophobic (fatty acid chain) domain in the same molecule and are produced extracellularly or as part of the cell membrane. In general, surface-active biomolecules can be divided into two types according to their molecular weight (Figure 7). On the one hand, low molecular weight compounds, called biosurfactants, which reduce the surface and/or interfacial tension between two immiscible phases of liquid, liquid and solid or liquid and gas. On the other hand, high molecular weight compounds, termed bioemulsifiers, often referred to as exopolysaccharides (EPS), which allow the REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - THEORETICAL FRAMEWORK 23 formation of oil-in-water or water-in-oil emulsions (43,56). Conversely, based on their molecular structure, surface-active biomolecules are further classified as, lipoproteins, lipopeptides, glycolipids, fatty acids and polymers (Figure 7) (57). Figure 7. Classification of microbial surface-active compounds, grouped into biosurfactants and bioemulsifiers. Picture from Sánchez (58). Surface-active biomolecules are a suitable alternative to synthetic surfactants, as many of them are synthesised mainly from petroleum. They have been widely used in numerous industrial sectors, including hygiene, pharmaceuticals, medicine, cosmetics and agrochemicals (59). A wide range of surface activities including emulsifying, dispersing, solubilising, wetting and foaming have been demonstrated in surface-active biomolecules (60). As a result, biosurfactants and bioemulsifiers have demonstrated potential application in various fields (Figure 8) (57,61). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 30 Water samples from each site were collected directly in sterile labelled glass bottles. A Niskin bottle (Aquatic BioTechnology, El Puerto de Santa María, Spain) was used to collect water from the Santa Engracia and El Cautivo springs, both at 2 m depth. The groundwater from the piezometer S8 (at a depth of 60 m) was collected using a manual bailer system (Eijkelkamp, Giesbeek, The Netherlands). At all sampling sites, water temperature, pH and electrical conductivity were measured with a Combo tester (Hanna Instruments, Eibar, Spain) and salinity with a density hydrometer (Hartwig Instruments, The Netherlands), all parameters measured in situ. All of the samples were then transported to the laboratory for further processing and analysis. Of the 12 sampling sites, eight (the six springs, the stream and the piezometer) are part of a hydrogeological monitoring network of surface and groundwater started in 2017, so data on the ionic composition of these water samples were available (Section 3, APPENDIX I, Table S1). This ionic composition is periodically analysed by ion chromatography at the Advanced Research Facilities (SGIker) of the University of the Basque Country UPV/EHU. Assessment of microbial diversity and community distribution using amplicon sequencing technique Most recent studies assessing the diversity and composition of the microbial community in hypersaline environments have used culture-independent methods, as these provide a much more accurate representation of the microbial composition, as many of them are challenging to cultivate and therefore their presence is unknown (21). The use of Next Generation Sequencing (NGS) technology and techniques such as amplicon sequencing (sequencing of amplified genes of interest) has allowed the analysis of microbial communities and their potential ecological function by examining DNA extracted from water samples containing mixed assemblages of organisms. The relatively low cost of amplicon sequencing compared to other technologies (such as shotgun REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 31 sequencing, which involves sequencing fragments of all the genetic material present) led to its widespread use (68). Water samples collected from the Añana Salt Valley were processed (enriched and sequenced) at Biogenetics, S.L. (Álava, Spain). Briefly, 5 L of water were filtered under aseptic conditions using a custom-built vacuum/compression pumping device (Labbox, Premia de Dalt, Spain), ending with a 0.22 μm filter. Prokaryotic DNA was extracted using the Genomic Mini AX Bacteria Kit (A&A Biotechnology, Gdansk, Poland) and eukaryotic DNA was extracted using the Genomic Mini AX Yeast Kit (A&A Biotechnology, Gdansk, Poland). The DNA obtained was quantified using the QuantiFluor dsDNA System (Promega, United States). The V3-V4 hypervariable region of the 16S rRNA gene was amplified for simultaneous detection of bacteria and archaea. On the other hand, the nuclear ribosomal internal transcribed spacer 1 (ITS1) region was amplified for the molecular identification of eukaryotes. Libraries were prepared using a Nextera DNA Library Prep Kit (Illumina, United States) and sequencing was performed on an Illumina MiSeq platform, generating pairedend sequences in FASTQ format. The nucleotide sequence data from the prokaryotic and fungal studies are available in the DDBJ/EMBL/GenBank databases under accession numbers PRJNA1115049 and PRJNA749727 respectively. The amplicon sequencing data sets were analysed using the Quantitative Insights Into Microbial Ecology (QIIME2) bioinformatics pipeline (version 2021.4) (69). This pipeline coordinates the inputs and outputs of different bioinformatics tools to facilitate the analysis of samples that require many different analytical steps. The implementation of the denoising tool, DADA2, allows quality control of sequencing by removing sequencing errors and generating Amplicon Sequence Variants (ASVs) that retain all the observed biological variation. This data was used for taxonomic composition studies, differential abundance analysis and diversity analyses. Taxonomic assignment of ASVs based on similarity to sequences in 16S rRNA gene database SILVA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 32 database v.138 (70), allowed the construction of the prokaryotic taxonomic community. Similarly, ASVs derived from ITS1 region sequences were compared to the UNITE reference database (71), which allowed identification of fungal community. Diversity analysis was assessed at different scales, from within-sample (alpha) to between-sample (beta), using the q2‐diversity plugin in QIIME2 program. Alpha diversity metrics include observed ASVs, Chao1, Pielou's evenness, Shannon's and Simpson's diversity indexes. Diversity estimator like observed ASVs and Chao1 index, establish the microbial community richness. Quantitative community richness or diversity was assessed by Shannon's and Simpson's indexes and community equality or evenness by Pielou's evenness. On the other hand, beta diversity metrics include Bray‐Curtis dissimilarity, which identifies dissimilarities in ASV composition between the samples. The functional potential of the prokaryotic community was predicted using 16S rRNA gene abundance data via Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2). Similarly, the FUNGuild database was used to assign ecological guilds to all fungal ASVs. Figure 11 shows the general workflow employed for amplicon sequencing of the water samples. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 33 Figure 11. Scheme of the workflow performed for the amplicon sequencing analysis of the water samples. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 34 Isolation and identification of prokaryotic strains Water samples collected at the different sites mentioned above were also used for the isolation of halophilic and halotolerant bacteria. To carry out this approach, water samples were subjected to a series of filtration stages (Whatman® Grade 113V and Millipore 5 µm and 0.22 µm filters), followed by cultivation on marine agar (MA; Condalab, Spain) and MA supplemented with 23% NaCl (Sigma-Aldrich, Spain), with duplicate cultures of each sample. The media were then incubated at 25 °C. Colony formation was monitored periodically for up to 2 months. Subsequent sub-culturing of the isolates was carried out on the same medium until a pure culture was obtained. Pure cultures were maintained at -80 °C in marine broth (MB; Condalab, Spain) in 25% (v/v) glycerol. The diameter, edge, pigmentation, texture, height, surface, hardness and emulsifiability of the colonies were studied (72). At the same time, Gram staining and orange-acridine staining were performed to determine the microscopic characteristics of the colony cells according to the protocol proposed by Vázquez et al. (73). To identify the isolates, chromosome DNA was extracted using the PrepMan® UltraReactive (Applied Biosystems, United States) according to the manufacturer's instructions. In case of failure of this technique, extraction was performed by the use of glass beads and boiling. The concentration of the DNA was measured using a NanoDrop® 2000 spectrophotometer (Thermo Scientific, United States). The 16S rRNA gene was amplified by PCR using specific primers: 27F (5′-GAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3') for bacteria (74) and 21F (5′-TCCGGTTGATCCYGCCGG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3') for archaea (75). In cases where no amplification was obtained, universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were tried for fungal identification REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 35 (76). Amplification was performed on a T100 thermal cycler (Bio-Rad, United States) using the appropriate amplification programme. Visualisation of the amplified fragment was performed by 1% agarose gel electrophoresis (Condalab, Spain) in 1X TAE buffer (Thermo Scientific, United States) at 100 V for 50 min. GelRed 0.5X (Biotium, United States) was used to visualise nucleic acids and the molecular weight marker HyperLadder I (Bioline, Spain). Amplified fragments were visualised using a ChemiDoc XRS+ System Ultraviolet (UV) transilluminator (Bio-Rad, United States). The resulting amplicon was purified using the NucleoSpin Gel and PCR Clean-Up Kit (Macherey-Nagel, Germany). Purified PCR product was sequenced by the Sanger method (Stab Vida, Portugal) and the resulting sequences were analysed using Chromas 2.6.6 software and CLUSTAL W webserver version 2.0.12 (77). The EzBioCloud website (www.ezbiocloud.net/identify) (78) was used to identify closely related species and their 16S rRNA gene sequence similarity. For isolates for which it was not possible to obtain a complete or sufficiently long 16S rRNA gene sequence, a fragment similarity search was carried out using the BLAST web server (79) for their approximate identification. A 16S rRNA gene-based phylogenetic tree was constructed using the Neighbour-joining method in MEGA X software (80) to establish the phylogenetic relationships of all the isolated strains. Polyphasic characterisation of potential novel taxa Taxonomy provides the information necessary to identify and characterise a species in its ecological, clinical and industrial niche. The polyphasic taxonomic approach involves a combination of genotypic, phenotypic and chemotaxonomic data for the nomenclature and systematics of new taxa (39). Figure 12 summarised the polyphasic strategy used in this study for the taxonomic characterisation of potential new isolates. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 36 Genotypic methods provide information derived primarily from nucleic acids, DNA or RNA, of the isolated organism. 16S rRNA gene sequencing is a rapid and accurate identification method for bacterial and archaeal isolates. In this study, isolates with 16S rRNA gene sequence similarities between the strain under study and the closest related type species ≤ 98.7% (the threshold proposed by Chun et al. (81) for distinguishing two species) and ≤ 95.0% (the threshold proposed by Hördt et al. (82) for distinguishing two genera) were considered potential new taxa. The 16S rRNA gene sequences of closely related species were retrieved from the GenBank database to perform a comparative phylogenetic analysis based on that gene. The sequences were aligned by ClustalW 2.0.12. Phylogenetic trees were constructed using Neighbour-joining, Minimum-evolution and Maximum-likelihood methods in MEGA X software. Evolutionary distances for Neighbour-joining analysis were calculated using Kimura's two-parameter model algorithm with bootstrap values based on 1,000 replicates. The phylogenetic tree shows the position of the strain studied relative to its nearest neighbours by comparison with other sequences in the database, from which further genotypic, chemotaxonomic and phenotypic analyses are designed (39). The DNA for whole genome sequencing (WGS) was obtained by using the Nucleo Spin Tissue DNA extraction kit (Macherey-Nagel, Germany) according to the manufacturer's protocol. De novo assembly of contigs from raw pairedend reads obtained using the Illumina Miseq platform was performed using SPAdes (v3.13.0) (83) or Velvet (1.1.04) (84). Gene annotation was performed using the NCBI (National Center for Biotechnology Information) Prokaryotic Genome Annotation pipeline and the Rapid Annotation with Subsystems Technology (RAST) pipeline (85). A basic genotypic method for bacterial classification is the measurement of the mol% of guanosine and cytosine bases. Within a well-defined species and genus, the variation in G+C % content should not exceed 3% and 10%, respectively (39). In addition, the Overall Genomic Relatedness Index (OGRI) REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 37 reflects the similarity between two genomic sequences. There are many algorithms for calculating OGRI values, but the most widely used algorithm for taxonomic studies is Average Nucleotide Identity (ANI), which was computed in this study using the OrthoANI Calculator tool available at EzBiocloud (86). In addition, Average Amino Acid Identity (AAI) values were calculated using the Kostas Lab AAI calculator (http:// enveomics. ce. gatech. edu/ aai/) (87) and Digital DNA-DNA hybridization (dDDH) values were computed using the Genome-to-Genome Distance Calculator (http://ggdc. dsmz. de/ ggdc. php) (88). The AAI cut-off of 95% correlates well with 95% for ANI and 70% for dDDH when comparing similar microbial species, making them reliable tools for microbial taxonomy of prokaryotes (39). In addition, whole genome sequence-based tree was inferred using FastME 2.1.6.1 (89) from the GBDP distances calculated from the genome sequences using the Type Strain Genome Server (https://tygs.dsmz.de) (90). In order to assess genus affiliations, amino acid-level comparisons were performed using the AAI and the Percentage Of Conserved Proteins (POCP) for every pairwise combination of sequences. The POCP values were calculated using a Python script with the formula [(C1 + C2)/(T1 + T2)] x 100%, where C1 and C2 represent the conserved number of proteins in the two genomes being compared and T1 and T2 represent the total number of proteins in the two genomes being compared (91). Phenotypic tests, including chemotaxonomic techniques, are used to describe taxa, species and subspecies up to genus and family level. The morphology of the cells, their size and the presence of flagella were observed by transmission electron microscopy (1400 Plus, Jeol). The motility of the cells was checked by the hanging drop method and on semi-solid agar containing MB and 0.7% bacteriological agar (Scharlau, Spain). Colony morphology, size and colour were observed on MA after 3 days at 25 °C. The cellular pigments from the cultures grown on MB were analysed by means of High-Performance Liquid Chromatography (HPLC). Growth at different temperatures (4, 10, 15, 20, 25, REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 38 30, 37 and 42 °C), pH range (pH 4.5-10.5 in increments of 0.5 pH units) and NaCl concentration (0, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0, 15.0, 20.0 and 23.0 g/L) were determined. Anaerobic growth was tested on MA using GENbox anaer (bioMérieux, France) in an anaerobic chamber at 25 °C for 15 days. Oxidase activity was assessed using Bactident oxidase strips (Merck, United States) and catalase activity using ID colour catalase (bioMérieux, France). Hydrolytic capacity was determined on MA plates supplemented with 1% (w/v) skim milk, 1% (w/v) Tween 20 and 1% (w/v) Tween 80. API ZYM and API 20NE strips (bioMérieux, France) were used to determine other physiological and biochemical characteristics according to the manufacturer's instructions. Antibiotic susceptibility was determined on MA plates. Whole cell fatty acid composition was determined at the Spanish Type Culture Collection (CECT) according to the protocol recommended by the MIDI Microbial Identification System (92). Sherlock MIDI version 6.1 was used for identification and TSBA6 library for analysis of cellular fatty acid content. Analyses of polar lipids and respiratory quinones were performed by the identification service of the Leibniz Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 39 Figure 12. Schematic workflow for describing novel bacterial taxa using a polyphasic taxonomic approach. PCR, Polymerase chain reaction; WGS, Whole genome sequencing; OGRI, Overall Genomic Relatedness Index; ANI, Average Nucleotide Identity; AAI, Average Amino Acid Identity; dDDH, Digital DNA-DNA hybridization. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 46 Characterisation of bioactive compounds produced by the ASV78 isolate Among all the isolates tested the isolate ASV78 was selected for further analysis as it showed positive results for both the surface-active activity and the antibacterial activity. To optimise the production of the compounds of interest, MB medium supplemented with two carbon sources (1% w/v glycerol or glucose) was tested at two temperatures (25 or 30 °C) and two conditions (125 rpm or static) for a period of 5 days. The presence of the activities of interest was determined by the well diffusion assay (METHODOLOGY, Figure 14) and the Parafilm M, oil spreading and emulsification assays (METHODOLOGY, Figure 15). The most favourable conditions for production conditions were established and used for the subsequent extraction and characterisation analyses, summarised in Figure 16. Three different extractions were performed to separately obtain crude antibacterial extract, crude biosurfactant extract and crude bioemulsifier extract. The reason why two different methods are used to extract surfactant compounds is due to the different chemical nature of biosurfactants (low molecular weight molecules) and bioemulsifiers (high molecular weight molecules). The crude antibacterial extract was obtained using the CFS from 1 L culture and extracted twice with an equal volumes of ethyl acetate by vigorous shaking in a separating funnel. For the crude biosurfactant extract, the CFS from 1 L culture was first acidified to pH 2.0 with 1 M HCl and then extracted twice with equal volumes of ethyl acetate. In both cases, the solvent of the organic phase was then evaporated under vacuum using a Rotavapor R100 (Buchi, Switzerland). Finally, to obtain the crude bioemulsifier extract, the CFS was mixed with cold ethanol (1:1) and incubated overnight at -20 ºC. The bioemulsifier was then pelleted by centrifugation at 9,000 rpm for 15 min at 4 °C and dissolved in distilled water (1% w/v) before lyophilisation. The three crude extracts were weighed and stored at - 20 ºC until further analysis. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 47 Each crude extract was then tested for the presence of the corresponding activities as described in the METHODOLOGY subsection in the Figure 14 and Figure 15. Additionally, the crude antibacterial extract was separated by Thin Layer Chromatography (TLC) (Merck, Germany) and a contact bioautography analysis was used to determine which of the bands separated by TLC had antibacterial activity (98). For this purpose, the TLC plate was placed on MH agar inoculated with a pathogen strain at a concentration of 0.5 McFarland and it was left for 30 minutes to allow the compounds to diffuse from the silica plate to the agar medium. After incubation at 37 °C for 24 hours, the appearance of a growth inhibition zone on the agar indicated which band was active against the tested microbe. Dereplication of the active crude extracts was performed using liquid chromatography high resolution mass spectrometry (LC–HRMS) in an Agilent 1200RR HPLC system coupled with a Bruker maXis Q-TOF mass analyzer, under analytical conditions previously described (99–101). Dereplication is defined as the analytical method used to rapidly identify already known natural products in samples from natural sources (102). In order to purify the compounds of interest present in the crude antibacterial extract, a first fractionation was performed on an automatic flash chromatography system (CombiFlash Rf, Teledyne Isco) using a linear gradient from 5% to 100% acetonitrile in water (in 35 min) with a final step of 100% acetonitrile (for 10 min), collecting 47 fractions. The fractions were concentrated to dryness in a centrifugal evaporator. Fractions 27, 28 and 29 on the one hand and fractions 37, 38 and 39 on the other hand were pooled as they contained the two compounds of interest. These fractions were further purified separately by semi-preparative reversed-phase HPLC (XBridge® Prep C18, 10 × 150 mm, 5 μm, 3.8 mL/min, UV detection at 210 and 280 nm, 1.8 mL/fraction). A linear gradient of water-acetonitrile from 50% to 100% acetonitrile over 30 min was established. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 48 Nuclear Magnetic Resonance (NMR) spectra were recorded in CDCl3 on a Bruker Avance III spectrometer (500 and 125 MHz for 1H and 13C NMR, respectively) equipped with a 1.7 mm TCI MicroCryoProbe. The antibacterial activity of the two purified compounds was tested using the broth microdilution method in a 96-well plate format against Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Enterococcus faecalis ATCC 29212 and vancomycin-resistant Enterococcus faecium vanA 15167 (clinical isolate) (103,104). Vancomycin, ciprofloxacin and aztreonam were used as reference antibiotics (positive controls). A concentration of 80 μg/mL of the pure compounds was used for the initial assay. To evaluate any synergistic activity an initial concentration of 40 μg/mL was used for each of the pure compounds. Blank and growth controls were included as internal plate controls. To test the growth of the pathogens tubidity (absorbance [Abs] at 600 nm) was measured at baseline (T0) and after 24 hours of incubation (Tf) at 37 °C, using an EnVision™ Microplate Reader (PerkinElmer, United States). The Minimum Inhibitory Concentration (MIC) is defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) as the lowest concentration of antimicrobial agent that inhibits visible growth of a microorganism, expressed in mg/L or μg/mL (105). Three independent biological experiments (n = 3) were performed to determine the MIC values. The Genedata Screener software, version 18.0.4-Standard (Genedata, Switzerland) was used to process and analyze the data. On the other hand, infrared (IR) spectroscopy was used to analyse the bioactive compounds of interest in the crude extracts of biosurfactants and bioemulsifiers. The elucidation of functional groups was obtained from IR data spectra using a JASCO FT/IR-4100 spectrometer equipped with a PIKE MIRacle single reflection ATR accessory (JASCO Corp., Japan). All measurements were made at room temperature. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 49 Figure 16. Overview of the extraction and characterisation process of the bioactive compounds present in the extracts of this study. TLC, Thin Layer Chromatography; HPLC, High-Performance Liquid Chromatography; UV, Ultraviolet spectra; MS, Mass spectra; NMR, Nuclear Magnetic Resonance; IR, Infrared spectroscopy. Created in https://BioRender.com. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - METHODOLOGY 50 Finally, to identify any potential biosynthetic clusters in the ASV78 genome, WGS was performed on the Illumina NextSeq platform at the UPV/EHU Advanced Research Facilities (SGIker), combined with long-read sequencing on the MinION sequencer (Oxford Nanopore Technologies) at the University Hospital of Álava. After filtering for sequence quality, the raw Illumina reads and MinION long reads were assembled de novo using the Unicycler pipeline (Galaxy version 0.5.0+galaxy1). Gene annotation was performed using the Rapid Annotation with Subsystems Technology (RAST) pipeline (85) and Prokka v1.14.5 (106). Biosynthetic gene cluster (BGC) analysis was performed using antiSMASH v7.0 (107). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 51 SUMMARY OF THE RESULTS AND DISCUSSION The first part of this section consists of a summary of the results on the microbial diversity and community composition in the Añana Salt Valley, which are presented in APPENDIX I and APPENDIX II. The second part summarises the results obtained with regard to the characterisation of new taxa isolated from the Santa Engracia spring, which are included in APPENDIX III and APPENDIX IV. Finally, the results obtained with regard to the collection of prokaryotic isolates and the screening of halophilic and halotolerant bacteria for their ability to produce antimicrobial and/or surface-active compounds, with further characterisation of the compounds of interest produced by the strain ASV78, are presented in the Section 4, APPENDIX V. Microbial diversity and community composition in the Añana Salt Valley The study of the microbial community by Illumina-based 16S rRNA gene and ITS1 region sequencing in hypersaline environments reveals the possibilities and limits of life in the most extreme conditions (108), noting that it is based on the putative association of an amplicon with a taxon, defined as an Amplicon Sequence Variant (ASV). These studies, together with the analysis of physicochemical parameters of water samples, could be used to identify water physicochemical variables that predict community structure. In the Añana Salt Valley, physicochemical monitoring of the main watercourses that feed the valley has revealed the presence of salty and brackish waters of different origins. This can be attributed to the existence of multiple pathways for unsaturated water to infiltrate the salt deposits in the subsoil, where salt dissolution takes place (109). This process gives rise to springs with varying salinities, caused by the saltiness of underground water. Consequently, it can be assumed that the different water flows in the Añana diapiric structure are responsible for the differences in the spring water's hydrochemical characteristics. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 52 The dissolution of evaporite (halite) hundreds of meters deep in the diapir structure is characterised by a high and stable ionic composition such as Cl−, Na+ and K+, and relatively high temperature (16-20 °C), observed in salty water springs (Table 3). On the other hand, the dissolution of gypsum and anhydrite and of NO3 − ions, are the result of the mixing of deep flows with shallower flows, characterizing the brackish waters, with comparatively higher presence of SO4 2−, Ca2+ and Mg2+ ions (Table 3). The water from the S8 piezometer has a higher SO4 2−content than the other brackish waters (Table 3), due to its particular position in the flow scheme (Figure 17). While brackish water is close to neutral pH (7.2 to 7.4), salty water is slightly acidic (6.2 to 6.6) (Table 3). The low variability of the salt facies is a peculiarity of this valley and indicates a stable ionic environment, contrary to what has been described in other diapiric environments (e.g., Zechstein 2, Germany) (109). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 53 Table 3. Physicochemical parameters of the water at the different sampling sites analysed. Sampling sites Physicochemical parameters Measured in situ Measured by ionic chromatography* (mg/L) Salinity (g/L) T (°C) pH Cl𝐒𝐎𝟒 𝟐− 𝐍𝐎𝟑 − Na+ Ca2+ Mg2+ K+ Salty [1] 200 16 6.6 153,813 4,697 nd 106,513 1,860 286 519 [2] 200 17 6.6 ND ND ND ND ND ND ND [4] 220 17 6.6 142,721 4,475 nd 100,710 1,933 283 476 [6] 205 16 7.5 168,361 5,493 nd 118,100 1,823 300 567 [8] 210 16 6.2 148,983 4,778 nd 99,312 1,751 266 464 [9] 200 20 6.5 152,187 4,669 nd 102,345 1,845 281 580 [10] 220 29 7.6 ND ND ND ND ND ND ND [11] 210 27 7.5 ND ND ND ND ND ND ND [12] 200 18 7.8 ND ND ND ND ND ND ND Brackish [3] 20 13 7.4 5,515 921 17 3,853 391 65 19 [5] 10 13 7.3 5,460 939 17 3,812 396 62 19 [7] 4 13 7.2 3,060 2,374 6 2,380 673 193 21 Sampling sites: [1], Santa Engracia spring; [2], Santa Engracia channel; [3], San Juan stream; [4], El Cautivo spring; [5], El Pico Dulce spring; [6], El Pico spring; [7], S8 piezometer; [8], Hontana spring; [9], Fuenterriba spring; [10], Pond III; [11], Pond II and [12], Pond I. ND, not determined; nd, not detected; *, these data are the medium of the data obtained from the monitoring network during 2017-1022. Conductivity was measured in situ at [1], [6], [10], [11] and [12] sampling sitea and was saturated in all cases. Figure 17. The Añana Salt Valley map showing the 12 sampling locations included in the representation of the different water flows in the valley. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 54 Illumina sequencing was used to analyse prokaryotic communities at 11 sampling sites and eukaryotic communities at seven sampling sites. The microbial community analysis revealed a total of 583,454 prokaryotic sequences and 704,874 fungal sequences, corresponding to 1,801 and 2,204 different ASVs, respectively. The main factor influencing the microbial distribution in this valley was found to be salinity, as shown by the beta diversity indices. In fact, significant differences were found between sampling sites (ANOSIM test, p value < 0.05) for both prokaryotic and fungal diversity when sampling sites were grouped according to salinity (salty and brackish). However, no statistically significant differences were observed when comparing samples according to their sampling location (spring, pond, etc.). This was also reported related with the prokaryotes in the saltern of Margherita di Savoia (Italy) (110) or fungi in Rhode Island´s salterns (United States) (111). Alpha diversity metrics revealed that prokaryotic and fungal community richness (Observed ASVs and Chao1), quantitative community richness or diversity (Shannon and Simpson indexes) and community equality or evenness (Pielou’s evenness) varied widely among the samples (Table 4). The lowest prokaryotic and fungal richness and diversity was found in two salty springs, the Pico spring [6] and the Santa Engracia spring [1], respectively (Table 4). On the contrary, the brackish spring El Pico Dulce [5] is characterised by the highest prokaryotic richness and diversity, as well as by the high diversity of the fungi, although in the case of fungi, this can be equalled or exceeded in salty waters (Table 4). This phenomenon could be partly explained by the higher salinity stress encountered by microorganisms living in high salinity environments, which may have limited diversity due to the energetically costly lifestyle (112). However, other aspects, such as the availability of nutrients and oxygen, which were not determined in this study, must be considered among the main limiting factors of microbial diversity in waters originating from very deep flows (113), which is the case of the salty water spring in this valley. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 55 The dominance of some prokaryotic taxa in the San Juan stream [3] (members of the genus Arcobacter, according to subsequent analysis) and of fungal taxa in the Santa Engracia spring [1] (members of the genus Saccharomyces, according to subsequent analysis), could be observed (Table 4). This could be related to the ability of certain microorganisms in certain habitats to significantly increase their population and become dominant. In the case of Arcobacter dominance, two possible scenarios could be speculated. The first is related to its ability to adhere and form biofilms (114), since in this location, a stream, there could be prolonged contact with vegetation and rocks. The second is related to the ability of members of the genus Arcobacter to oxidise sulphide and reduce nitrate. This suggests that sulphide may act as an electron donor in denitrification and inhibit nitrous oxide reduction, making this niche specific and reducing species richness (115). Table 4. Alpha diversity indexes calculated for the locations of the saltern analysed in the study. Sampling sites Observed ASVs Chao1 Simpson’s index Shannon’s index Pielou’s evenness Prokaryotes Salty [1] 104 104 0.95 5.07 0.76 [2] 196 197 0.96 5.56 0.73 [4] 169 169 0.96 5.50 0.74 [6] 69 69 0.83 3.68 0.60 [9] 294 301 0.93 5.48 0.67 [10] 147 147 0.96 5.59 0.78 [11] 74 74 0.86 3.88 0.62 [12] 120 120 0.89 4.49 0.65 Brackish [3] 155 158 0.83 3.50 0.48 [5] 541 541 1.00 8.76 0.97 [7] 284 284 0.99 7.17 0.88 Fungi Salty [1] 71 116 3.59 0.77 0.58 [6] 354 633 6.27 0.95 0.74 [10] 263 337 6.07 0.94 0.75 [11] 396 699 6.47 0.94 0.75 [12] 264 452 5.44 0.93 0.67 Brackis h [5] 262 430 6.15 0.96 0.76 [7] 93 93 4.89 0.91 0.74 Sampling sites: [1], Santa Engracia spring; [2], Santa Engracia channel; [3], San Juan stream; [4], El Cautivo spring; [5], El Pico Dulce spring; [6], El Pico spring; [7], S8 piezometer; [9], Fuenterriba spring; [10], Pond III; [11], Pond II and [12], Pond I. ASVs, Amplicon Sequence Variants; Chao1, Confidence interval for richness estimator. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 62 In consideration of the taxonomic-inferred metabolic prediction derived from 16S rRNA gene amplicon sequencing, it was determined that the microbiome engaged in chemoheterotrophy (also referred to as aerobic) and fermentation was the most prevalent and extensive along the valley. It is known that most halophilic prokaryotes are aerobic chemoorganotrophs capable of decomposing organic compounds up to NaCl saturation (5). However, oxygen is poorly soluble in brines, allowing anaerobic halophilic heterotrophs to thrive (5) and actively coexist in the same niche. According to Wang and Bao (118), the main bacterial function is chemoheterotrophy, a process which also predominates among archaea; conversely, fermentation is widespread among the bacterial domain. On the other hand, ITS1 region sequencing-based prediction of fungal ecological functions revealed the ubiquity of saprotrophs throughout the salty waters, reaching up to 95.5% in the Santa Engracia spring, probably associated to the large presence of the genus Saccharomyces, a well-known saprotroph. In contrast, brackish waters presented a high abundance of prokaryotic and fungal microbiota involved in animal/plant parasites or symbionts. Finally, the high percentage of prokaryotic and fungal ASVs assigned to Unclassified taxa, together with the large number of taxa grouped as Uncultivated (12.6%) in the Santa Engracia spring, was striking in this study. This could imply that they correspond to sequences of taxa that have not yet been identified. At the same time, this might mean that dominant or abundant taxa could be overtaken by new or unidentified taxa, as noted by Oren (129). This premise highlights the need to carry out culture-based studies in this hypersaline environment, as they could provide relevant scientific knowledge. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 63 Isolation and identification of prokaryotic isolates Culture-based approaches, carried out for the first time in the Añana Salt Valley, identified a total of 150 halophilic and halotolerant isolates with different colony and cell morphologies. All isolates were bacteria, except for two fungi and two archaea. 89,3% of the strains (134/150) were isolated in MA medium and 10,7% (16/150) in MA containing 23% NaCl (Section 4, APPENDIX V, Table S1). The Pseudomonadota phylum predominated with 106 isolates (70.7%), in agreement with what has been described in the Rambla Salada, where the Pseudomonadota phylum represents 72.5% of the total isolates isolated there (130). Moreover, the outcomes are deemed to be logical in view of the culture media employed in the present study, as these media are designed for the cultivation of heterotrophic marine bacteria (131). The rest of the isolates were divided into the phylum Actinomycetota (21 isolates, 14.0%), Bacillota (12 isolates, 8.0%), Bacteroidota (seven isolates, 4.7%), Methanobacteriota (two isolates, 1.3%) and Basidiomycota (two isolates, 1.3%). At the genus level, the isolates were distributed across 48 different genera. In addition, a total of 95 different species were identified. The genus Halomonas was the genus with the highest number of isolates (36 isolates; 24.0%), as reported in the study of Rambla Salada (130), followed by Pseudoalteromonas (15 isolates; 10.0%). In particular, Halomonas sabkhae, Halomonas taeanensis and Pseudoalteromonas neustonica were the most frequently isolated species, 7 isolates each. The phylogenetic relationship between the isolates is shown in Section 4, APPENDIX V, Figure S1. Conversely, 24.7% of the identified isolates (37/150) exhibited values of less than 98.7% in the 16S rRNA gene sequence similarity to taxa previously described. These values are considered below the threshold proposed by Chun et al. for designating such a taxon to a known species (81). Consequently, it can be deduced that 37 of the isolates of the culture collection obtained in this study may represent new taxa. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 64 Polyphasic characterisation of new taxa isolated from Santa Engracia spring A polyphasic study was conducted to characterise the SALINAS58 (also named ASV58, in this study) and ASV31 strains isolated from the Santa Engracia spring, which is the main source of brine for the saltern. The polyphasic study consisted on genotypic, phenotypic and chemotaxonomic characterisation of the isolated strains. Characterisation of strain SALINAS58 Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain SALINAS58 belonged to the genus Altererythrobacter. The 16S rRNA gene sequence of the potentially novel isolate was deposited in GenBank under accession number MN918268. Nevertheless, in order to enhance the confidence of the results, the 16S rRNA gene sequence obtained from the WGS of the selected strain was utilised for subsequent phylogenetic analysis, due to its greater length. WGS data of strain SALINAS58 was deposited at DDBJ/ENA/GenBank under the accession numbers JAAFZT000000000. The 16S rRNA gene sequence (1,436 bp) analysis of strain SALINAS58 showed the highest similarity to Altererythrobacter marensis MSW-14T (96.6%), followed by Altererythrobacter aquaemixtae JSSK-8T (96.5%), Pontixanthobacter luteolus SW-109T (96.5%) and Altererythrobacter atlanticus 26DY36T (96.4%). Taking into account the reclassification of the family Erythrobacteraceae proposed by Xu et al. (132), at the time of writing Altererythrobacter marensis, Altererythrobacter aquaemixtae and Altererythrobacter atlanticus are homotypic synonyms, validly published under the International Code of Nomenclature of Prokaryotes (ICNP), but their correct names are Pelagerythrobacter marensis, Pontixanthobacter aquaemixtae and Croceibacterium atlanticum, respectively. The sequence similarity of the 16S rRNA gene between the strain SALINAS58 and other related species was lower than 98.7%, the threshold value proposed by Chun et REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 65 al. (81) for distinguishing two species. Neighbour-joining phylogenetic analysis based on the 16S rRNA gene sequence showed that strain SALINAS58 formed a distinct branch within the genus Altererythrobacter, despite not being supported by a high bootstrap value (Figure 21). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 66 Figure 21. Neighbour-joining phylogenetic tree from 16S rRNA gene sequences showing the relationships between strain SALINAS58, the type strains of Altererythrobacter species and representatives of several other related taxa. Only bootstrap values ≥ 50% (expressed as percentage of 1,000 replicates) are shown at divergence points. Filled circles indicate that the corresponding nodes were also found in both the minimum-evolution and maximum-likelihood trees (Section 3, APPENDIX III, Fig. S2 and Fig. S3, respectively). Paracoccus pacificus F14T (GenBank accession number KF924610) was used as an outgroup. Bar, 0.020 substitutions per nucleotide position. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 67 The genome size of strain SALINAS58 was 2.8 Mbp and was assembled into 56 contigs, with a N50 value of 1.9 Mbp. The DNA G+C content of strain SALINAS58 was 61.4%, within the range of the genus Altererythrobacter (54.5–69.0%) (133). The use of OGRI values or the construction of phylogenomic trees must necessarily be taken into account for the effective determination of the taxonomic position of a strain (39). Among OGRIs, the ANI values relative to strain SALINAS58 ranged from 71.5 to 73.1% compared to P. aquaemixtae JSSK-8T and P. marensis MSW-14T, respectively. The dDDH values (results obtained from the recommended formula 2) ranged from 18.3 to 18.9% with P. aquaemixtae JSSK-8T and P. luteolus SW-109T, respectively. AAI values ranged from 66.6 to 68.1% compared to A. epoxidivorans JCS350T and P. marensis MSW-14T, respectively. All of these values were below the thresholds specified for bacterial species identification (39,81). The whole genome phylogenetic tree confirmed that strain SALINAS58 forms a separate branch between relative type species (Section 3, APPENDIX III, Fig. S4). In addition, genome annotation predicted genes related to trehalose biosynthesis, N-cetylneuraminate synthase and the multidrug efflux pump component MtrF in strain SALINAS58, which were not found in related taxa (Section 3, APPENDIX III, Table S1). Phenotypic and chemotaxonomic characterisation analyses were also carried out to distinguish the SALINAS58 strain from the recognised species. For this purpose, all these analyses were carried out simultaneously with the purchased type strains P. marensis MSW-14T and P. aquaemixtae SSK-8T. The main phenotypic characteristics that distinguished strains SALINAS58 from its closest relatives are summarised in Table 5. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Table 5. Differential phenotypic characteristics of strain SALINAS58 and phylogenetically related type strains. Strains: 1, SALINAS58 (this study); 2, P. marensis MSW-14T (this study); 3, P. aquaemixtae SSK-8T (this study); 4, P. luteolus SW-109T (134,135); 5, Altererythrobacter epoxidivorans JCS350T (134,135). +, positive; −, negative; w, weakly positive; NA, no data available; ND, not determined. None of the strains were able to grow in anaerobic conditions or utilize acetate. In API ZYM strips, all the strains were positive for esterase (C4) and esterase lipase (C8) activities and negative for β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase activities. Characteristic 1 2 3 4 5 Colony colour Orange Beige Yellow Yellow Yellow Cell shape Rod Rod Rod Rod Ovoid–rod Oxidase activity - + w + + Nitrate reduction - - + - - Growth range (optimum): NaCl (%, w/v) 0-5 (1) 0-10 (2) 0-5 (1) 0.5-9 (2) 0.5-9 (2) Temperature (°C) 15-30 (30) 4-42 (30) 15-37 (30) 4-36 (30) 20-40 (35) pH 5.5-9 (6-6.5) 7-10 (7) 7-7.5 (7) 5.5-8 (7-8) 6-8.5 (6.5) Hydrolisis of: Casein + - - + + Starch - - - w - Tween 80 + - - + + Tween 20 + + - + NA Gelatin - - + - - Utilization of: Inulin + - ND NA NA Succinate - - + - - D-Xylose - + - + - Melibiose + + - - NA Sorbitol + + - - NA D-Fructose + + - + + Continued Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 68 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Characteristic 1 2 3 4 5 Inositol + + - - NA D-Galactose + + - + + Glycerol - + - NA NA Assimilation of: Malate - w - - - Mannitol - - - - + N-Acetylglucosamine - - - + + Maltose - - - + + Adipate - - - NA + Enzyme activity: Lipase (C 14) w - - + - Valine arylamidase w + + - + Cystine arylamidase - w w w + Trypsin w + + w + α-Chymotrypsin + + - + - Acid phosphatase w w - - + Naphthol-AS-BI-phosphofohydrolase + w w + - α-Glukosidase w - - w - β-Glucosidase w + + + + β-Galactosidase - - - + + Pigment (nm) 465 307, 340, 381, 415*a 449, 476, 477*b 340, 417, 449, 447 340, 381, 417, 541, 477 *Data from: a, Seo and Lee (136) and b, Park et al. (135). Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 69 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 70 A chemotaxonomic approach showed that the fatty acid profile of strain SALINAS58 was similar to that of related species, although differences in the proportions of some fatty acids were observed. The presence of C14:0, C18:0, C18:0 2OH and C16:0 iso 3OH only in strain SALINAS58 and the absence of C12:0 2OH, distinguish this strain from the other two strains analysed in this study. The predominant respiratory quinone identified in strain SALINAS58 was ubiquinone Q-10, which is characteristic of the genus Altererythrobacter (132). In addition, the major polar lipids detected in strain SALINAS58 were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidilglycerol, four unidentified glycolipids and one unidentified phospholipid (Section 3, APPENDIX III, Fig. S6). Overall, the analyses revealed differences in genomic, phenotypic and chemotaxonomic characteristics, indicating that strain SALINAS58 was a new species, for which the name Altererythrobacter muriae sp. nov. was proposed, being the type strain SALINAS58T. At the time of writing, Altererythrobacter muriae is a homotypic synonym, validly published under the ICNP, considering Alteripontixanthobacter muriae to be the correct name since the last reclassification in 2021 by Kim et al. (137). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 71 Description of Alteripontixanthobacter muriae sp. nov. Table 6. Description of Alteripontixanthobacter muriae strain SALINAS58T. Etymology muriae (mu’ri.ae. L. gen. fem. n. muriae, of brine, substance from which the strain was isolated). Type strain SALINAS58T (=CECT 30029T=LMG 31726T). Taxonomy Phylum Pseudomonadota, class Alphaproteobacteria, order Sphingomonadales, family Erythrobacteraceae and genus Alteripontixanthobacter. Cells Gram-negative, aerobic, nonmotile, rods 0.40.9 μm wide and 1.83.0 μm long and non flagellar. Colonies Orange, pigmented, translucent, smooth and they form a convex elevation after 3 days at 25 °C, with 1 mm of diameter. Pigments Carotenoid pigments, it does not contain bacteriochlorophyll a. Growth At pH 5.5-9 (optimum, pH 6-6.5), between 15 and 30 °C (optimum, 30 °C) and in the presence of 0-5% of NaCl (optimum 1%). Enzymatic activities Catalase-positive and oxidase-negative. It hydrolyses casein, Tween 20 and Tween 80, but not starch. It can use inulin, melibiose, sorbitol, D-fructose, inositol and D-galactose as carbon and energy sources, but not succinate, D-xylose, acetate or glycerol. In API 20NE tests, it is only positive for aesculin hydrolysis. In API ZYM tests, it shows strong alkaline phosphatase activity; intermediate esterase (C4), esterase lipase (C8), leucine arylamidase, α-chymotrypsin and naphthol-AS-BI-phosphohydrolase activity; and weak lipase (C14), valine arylamidase, trypsin, acid phosphatase, β-glucosidase activity. Continued Figure 23. Alteripontixanthobacter muriae strain SALINAS58T growth on MA. Colony morphology and colour are shown. Figure 22. Transmission electron micrograph of a cell of SALINAS58T. Bar 500.0 nm. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Characteristics 1 2 3 4 5 Enzymatic activities: Esterase (C 4) + w w NA + Esterase lipase (C 8) + w - NA + Valine arylamidase + - w NA - Acid phosphatase w w + NA + Naphthol-AS-BI-phosphohydrolase w w + NA w Cystine arylamidase w - - NA - α-glucosidase + + - NA - β-glucosidase - w - NA - Pigments Spheroidenone, spheroidene and BChl a Not detected*a Spheroidenone and BChl a*b ND ND *Data from: a, Sorokin et al. (143); b, Ramaprasad et al. (144). BChl a, bacteriochlorophyll a. Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 78 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 79 A chemotaxonomic approach showed that the main fatty acid, C18:0 cyclo ω8c, present in strain ASV31 was not present in T. pacifica DSM 10166T or R. algae LMG 29228T. In addition, ASV31 was the only strain without hydroxy fatty acids and the only one with unsaturated fatty acids. Moreover, summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) was the major fatty acid found in T. pacifica DSM 10166T (83.08%) and R. algae LMG 29228T (70.12%), while in ASV31 it was detected at a lower percentage (37.34%). Ubiquinone Q-10 was identified in strain ASV31, which is typical for most genera of the Paracoccaceae family (138). In addition, the polar lipid profile of strain ASV31 comprised phosphatidylglycerol, aminolipid, two unidentified glycolipids, two unidentified phospholipids and two unidentified lipids (Section 3, APPENDIX IV, Figure S4). It was observed that the phosphatidylethanolamine, which is one of the most important polar lipid in T. dalianensis and the members of Rhodovulum (138,145), did not match with the profile of strain ASV31. Overall, the analyses revealed differences in genomic, phenotypic and chemotaxonomic characteristics, indicating that strain ASV31 was the first species of a new genus within the family Paracoccaceae, for which the name Anianabacter salinae gen. nov. sp. nov. was proposed, being the type strain ASV31T. At the time of writing, Anianabacter salinae is validly published in accordance with the ICNP but the designation is addressed to Anianibacter salinarum due to other spelling of name or inaccurate spelling (146). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 80 Description of Anianibacter salinarum gen. nov. sp. nov. Table 8. Description of Anianibacter salinarum strain ASV31T. Etymology (A.ni.a.ni.bac’ter. N.L. masc. n. bacter, a rod; N.L. masc. n. Anianibacter, a rod from the Añana solar saltern) and (sa.li.na’rum. L. gen. pl. n. salinarum, of salt works). Type strain ASV31T (=CECT 30309T = LMG 32242T) Taxonomy Phylum Pseudomonadota, class Alphaproteobacteria, order Rhodobacterales and family Paracoccaceae. Cells Non-motile Gram-negative rods that occur singly or in aggregates with a mucous polysaccharide capsule around them. PHA granules are present inside the cells. Colonies Beige-to-pink coloured. The rod cells are 2.4-2.5 µm in length and 0.4-0.5 µm wide. Pigments Spheroidenone, spheroidene and bacteriochlorophyll a are present. Growth The species grows at pH 6.5-9.5 (optimum 7-9.5) and at temperatures between 1837 ºC (optimum 30 ºC). It has a high NaCl tolerance of 023% (w/v) (optimum 35%). Enzymatic activities Catalase and oxidase positive and urease negative. It hydrolyses aesculin and Tween 20. Nitrate reduction occurs in this species. In API ZYM tests, it shows strong alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase, valine arylamidase and α-glucosidase activity; and weak cysteine arylamidase, acid phosphatase and naphthol-AS-BIphosphohydrolase activity. Continued Figure 23. Transmission electron micrograph of a cell of ASV31T. Bar 500.0 nm. Figure 26. Anianibacter salinarum stain ASV31T growth on MA. Colony morphology and colour are shown. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 81 Antibiotic susceptibility Resistant to polymyxin B (300 IU), vancomycin (30 µg) and streptomycin (10 µg). Chemotaxonomy The unique ubiquinone is Q-10 and the major fatty acids are C18:0 cyclo ω8c and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The major polar lipids are phosphatidylglycerol, aminolipid, an unidentified glycolipid, an unidentified phospholipid and an unidentified lipid. WGS The genome size is 3.6 Mbp and the DNA G+C content of the type strain is 65.7 mol%. CECT, Spanish Type Culture Collection; LMG, Laboratorium voor Microbiologie Gent (Belgium); WGS, Whole genome sequencing. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 82 Screening of antimicrobial and/or surface-active compound producers With the exception of two isolates that proved unrecoverable, the remaining 148 isolates were subjected to a screening for antimicrobial production potential. This was based on their ability to inhibit the growth of pathogen strains using the deferred antagonism assay. Twenty isolates showed clear activity against at least one of the pathogen strains (Table 9). These antimicrobial producing isolates were then subjected to the well diffusion assay where nine of them were found to be positive for antimicrobial production (Table 9). Eight of these halofilic or halotolerant isolates produced compounds that inhibited the growth of both Gram-positive and Gram-negative bacterial strains, while antifungal activity was only observed for isolate ASV55 in the well diffusion assay (Table 9). Illustrations of some of the results obtained in both antagonism and well diffusion assays are shown in Figure 27A and Figure 27B, respectively. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 83 Figure 27. Example of the detection of antimicrobial compounds produced by some halophilic and halotolerant isolates. A, pathogen growth inhibition halo in the antagonism assay produced by isolates ASV12, ASV10, ASV14, ASV25, ASV89 and ASV106 and B, pathogen growth inhibition halo in the diffusion assay produced by isolates ASV12, ASV13, ASV55, ASV128, ASV106, ASV129 and ASV136. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 84 In addition, the diffusion assay suggested that the antimicrobial compounds were produced by the third day of incubation and that degradation was evident after the tenth day (Table 9). On the other hand, isolates for which activity was only observed in the delayed antagonism assay may imply that the production of the antimicrobial compound was based on nutrient competition with the pathogen strain, as described in other studies (147). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Table 9. Isolates showing antimicrobial activity against at least one of the pathogenic strains used. The diameter (mm) of the inhibition zone in the delayed antagonism assay (DAA) and the well diffusion assay (WDA) is given. Pathogen strains P. aeruginosa ATCC 27853T E. coli ATCC25922T B. subtillis CECT 356T S. aureus ATCC 29213T E. faecalis ATCC 29212T C. albicans ATCC 90029T Mean of the inhibition halo (mm) DAA WDA DAA WDA DAA WDA DAA WDA DAA WDA DAA WDA day day day day day day Isolate ID 3 10 17 3 10 17 3 10 17 3 10 17 3 10 17 3 10 17 ASV1 8 - - - - - - - - - - - - - - - - - - - - - - - ASV3 11 - - - - - - - - - - - - - - - - - - - - - - - ASV7 20 - - - - - - - 12 - - - 25 - - - - - - - - - - - ASV10 10 - - - 15 - - - - - - - 12 - - - - - - - - - - - ASV11 10 - - - - - - - - - - - - - - - - - - - - - - - ASV12 27 - - - 27 14 12 - 25 - - - 30 - - - 30 - - - - - - - ASV13 - - - - 9 15 11 - - - - - - - - - 31 - - - - - - - ASV14 12 - 15 - - - - - - - - - - - - - - - - - - - - - ASV25 12 - 12 - 15 - - - 16 - - - 10 - - - 13 - - - - - - - ASV55 - 15 13 - - 16 15 10 13 20 16 13 - 12 18 13 - 7 6 - - 15 13 - ASV78 12 - - - - - - - 20 - - - 17 - - - - - - - - - - - ASV89 9 - - - - - - - - - - - - - - - - - - - - - - - ASV106 - - - - - - - - 21 18 - - - - - - - - - - - - - - ASV117 9 - - - - - - - - - - - - - - - - - - - - - - - ASV118 9 - - - - - - - - - - - - - - - - - - - - - - - ASV120 - - - - - - - - 6 - - - - - - - - - - - - - - - ASV121 - - - - - - - - 6 - - - - - - - - - - - - - - - ASV128 - - - - - - - - 20 15 - - - - - - - - - - - - - - ASV129 - - - - - - - - 19 17 - - - - - - - - - - - - - - ASV136 6 - - - - - - - 14 9 - - 10 - - - - - - - - - - - -, negative Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 85 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 86 The genus Pseudoalteromonas (isolates ASV1, ASV3, ASV10, ASV11, ASV12, ASV13, ASV14, ASV25, ASV78, ASV89, ASV117, ASV118 and ASV136) was the main contributor to antibacterial activity in this study. It is noteworthy that the majority of the isolates, with the exception of ASV13, inhibited the growth of P. aeruginosa. In addition to the possibility of an inhibitory effect due to nutrient competition, another hypothesis for this occurrence is related to the ability of Pseudoalteromonas species to produce antibiofilm agents such as alterocin, thereby suppressing the growth of P. aeruginosa, a recognised biofilm-forming bacterium (148). The remaining of the isolates with antimicrobial capacity belonged to the genera Salinivibrio (ASV7), Pseudomonas (ASV55), Kocuria (ASV106 and ASV128), Halomonas (ASV120 and ASV121) and Streptomyces (ASV129). These genera have previously been documented to possess antimicrobial activity (149–152), with the exception of the genus Salinivibrio. Despite some reports of antifungal (153) and insecticidal (154) activity of members of the genus Salinivibrio, their antibacterial capacity, as seen in strain ASV7 in this study, has not been described. Conversely, all 148 isolates were also screened for biosurfactant production potential, based on their ability to reduce surface tension using oil spreading and Parafilm-M tests, and for bioemulsifier potential, based on their ability to emulsify oil and hydrocarbon compounds by EI24. Illustrations of some of the results are shown in Figure 28, while the detailed results are summarised in Section 3, APPENDIX III, Table S2. The mean results obtained for each isolate in the Parafilm-M test and the oil spreading test are shown in Figure 29 and Figure 30, respectively, grouped by genus. Isolates were designated as positive for biosurfactant production if the drop diameter in the Parafilm-M test and the clearing zone they produced in the oil spreading test were greater than the negative control (BPM) (≥ 3.4 mm and ≥ 7.0 mm, respectively). Similar cut-off values have been used in other studies to test positive for biosurfactant production (95,155). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 87 Figure 248. Examples of some of the halophilic and halotolerant strains that produce surface-active compounds in A, Parafilm-M test; B, oil spreading test; C, emulsification test, compared with the negative control. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 94 indicated that the ASV78 strain was able to synthesize biomolecules of different activities simultaneously and under the same conditions and that they could be extracted by the different techniques used. Figure 33. TLC-based bioauthography of the antimicrobial crude extract of ASV78 exhibiting antibacterial activity against S. aureus ATCC 29213. To identify the components of the antimicrobial extract, LC-HRMS dereplication was performed. The dereplication process yielded three peaks, which were identified as lumichrome, pentabromopseudilin and bromophene. Lumichrome was dereplicated as a match against the Fundación MEDINA library by matching the retention time, UV spectrum and molecular formula of the analysed peak with a standard stored in the spectral database (Section 4, APPENDIX V, Figure S5). Lumichrome is a known degradation product of riboflavin, by photodegradation in neutral or acidic solutions and by enzymes in bacteria, with plant growth promoting activity (163). On the other hand, pentabromopseudilin was dereplicated by interpreting the molecular formula of a UV peak that was very clearly defined. The MS REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 95 spectrum (Section 3, APPENDIX III, Figure S5) showed a characteristic isotopic pattern with five bromine atoms, thus suggesting C10H4Br5NO as the molecular formula, with pentabromopseudilin being the unique match in the Dictionary of Natural Products. The compound was purified from a bioactive extract using reversed phase chromatography and preparative/semi-preparative HPLC. At Rt 22 min, a total of 0.9 mg of pure compound was obtained. Then, (-)-MS spectrum and 1H-NMR analysis (Figure 34) confirmed its identity. Finally, the peak identified as bromophene did not give an interpretable (+)-HRMS spectrum. Consequently, purification of the compound was undertaken from the bioactive extract by reversed phase chromatography, followed by preparative/semi-preparative HPLC, thereby yielding a fraction that was deemed suitable for NMR analysis. Negative mode LC-HRMS produced a (-)-MS spectrum that was interpreted, based on its isotopic distribution, to correspond to a molecular formula of C12H6Br4O2 with eight coincidences in the Dictionary of Natural Products. At Rt 20 min, a total of 0.5 mg of pure compound was obtained. Then, (-)-MS spectrum and 1H-NMR analysis (Figure 35) confirmed the presence of bromophene by comparison with literature data (164). As described in this study, highly brominated metabolites from Pseudoalteromonas have also been documented. Specifically, pentabromopseudilin was previously isolated from P. phenolica and P. luteoviolacea and bromophene from P. phenolica (165). However, this study describes the ability to simultaneously produce both compounds by the strain ASV78, for which the phylogenetically closest species is Pseudoalteromonas neustonica (16S rRNA gene sequence similarity of 98.57%). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 96 Figure 34. (-) MS spectrum (top) and 1H-NMR spectra with the structure (bottom) of pentabromopseudilin produced by strain ASV78. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 97 Figure 35. (-) MS spectrum (top) and 1H-NMR spectra with the structure (bottom) of bromophene produced by strain ASV78. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 98 Despite the reported antibacterial activity of both pentabromopseudilin and bromophene against methicillin-resistant S. aureus, little is known about their broad spectrum activity (165,166). Furthermore, the simultaneous production of both compounds may not be common. To the best of our knowledge, this has only been documented in one strain, Pseudoalteromonas sp. CMMED 290 (165). Despite the same metabolic pathway for synthesising both compounds (167). In this respect, it would be worthwhile to investigate the potential synergistic effects of these compounds, as this has not been demonstrated to date. Such an investigation could prove beneficial for their practical application. In this regard, the assay hold with the pure compounds showed that both compounds exhibited antimicrobial activity against Gram-positive bacteria, with pentabromopseudilin additionally exhibiting activity against Gram-negative bacteria (Table 10). In particular, pentabromopseudilin exhibited MIC values ranging from 0.02 to 0.04 µg/mL against S. aureus ATCC 29213 and MIC values ranging from 10 to 20 µg/mL against P. aeruginosa ATCC 27853 (Table 10, top). Conversely, bromophene showed MIC values ranging from 2.5 to 5 µg/mL against S. aureus ATCC 29213 (Table 10, top). Table 10. Antibacterial activity of pure compounds and controls. MIC values at top and antibacterial activity (MIC values obtained from one replicate) at bottom. Compounds MIC (µg/mL) S.aureus ATCC 29213 P.aeruginosa ATCC 27853 Pentabromopseudilin 0.02-0.04 10-20 Bromophene 2.5-5 > 80 Synergy (1:1) 0.04-0.08 20-40 Vancomycin 0.5-1 ND Ciprofloxacin ND 0.25-0.5 Continued REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 99 Compounds Activity (µg/mL) A.baumannii ATCC 19606 E.faecalis ATCC 29212 E.faecium vanA 15167 Pentabromopseudilin 10-20 10-20 1.25-2.5 Bromophene > 80 40-80 20-40 Synergy (1:1) 20-40 20-40 2.5-5 Vancomycin ND 4-8 > 32 Aztreonam 80-160 ND ND ND; not determined MIC values against E. faecalis ATCC 29212, vancomycin-resistant E. faecium vanA 15167 and A. baumannii ATCC 19606 were determined using a single replicate assay. This was due to the paucity of samples and may require further validation in future studies. Pentabromopseudilin inhibited growth of E. faecalis ATCC 29212 at 10-20 µg/mL, vancomycin-resistant E. faecium vanA 15167 at 1.25-2.5 µg/mL and A. baumannii ATCC 19606 at 10-20 µg/mL (Table 10, buttom). On the other hand, it was observed that bromophene inhibited the growth of E. faecalis ATCC 29212 at 40-80 µg/mL and the growth of vancomycin-resistant E. faecium vanA 15167 at 20-40 µg/mL (Table 10, buttom). For none of the pathogens tested, synergistic activity between the two compounds was observed. Pentabromopseudilin's antibacterial activity against clinically relevant species including A. baumannii, E. faecium and S. aureus should be considered. The low concentration of pentabromopseudilin (MIC values ranging from 0.02 to 0.04 µg/mL) at which it is effective against S. aureus ATCC 29213 is striking, especially when compared to the vancomycin concentrations (MIC values of 0.5 to 1 µg/mL in this study, and 0.52 to 0.59 µg/mL according to Lepe et al. (168). It would therefore be very interesting to evaluate the spectrum of pentabromopseudilin against a wider range of pathogens and to further investigate its mechanism of action, which may be different from that of vancomycin, as evidenced by its activity against vancomycin-resistant E. faecium and Gram-negative bacteria. Indeed, multi-target inhibitors REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 100 represent the next frontier in the world of antibiotics, offering significant advantages over the development of drug resistance (169). With regard to the biosurfactant and bioemulsifier components of the bioactive extracts, IR spectroscopy was used to identify the chemical nature of these components. On the one hand, the IR spectrum of the biosurfactant extract revealed the presence of aliphatic hydrocarbon chains along with a polysaccharide moiety, thereby suggesting a glycolipid nature of the biosurfactant (Figure 36A). The presence of absorption bands at 3,311 cm−1 indicated the presence of -OH stretching of the hydroxy groups. The presence of bands at 2,919 and 2,847 cm−1 corresponded to the C-H stretching mode of an aliphatic chain. The peak at 1,652 cm−1 could be correlated with the presence of C=O groups. A peak at 1,408 cm−1 indicated that presence of C-H bending vibrations of CH3 and CH2 that may be attributed to polysaccharides. The peaks between 1,317-1,013 cm−1 indicated the presence of C-O stretching corresponding to the sugar moiety. The IR spectrum of the biosurfactant extract produced by strain ASV78 was similar to that of other glycolipid biosurfactants reported in previous studies (170–172). On the other hand, the IR spectrum of the bioemulsifier extract is shown in Figure 36B. Broad absorption bands indicating the presence of carboxylic groups were observed (3,369 cm-1, hydroxy stretching; 1,630 cm-1, carboxyl C=O stretching; 1,114 cm-1, carboxyl -C-O stretching). In addition, stretching vibrations at the wavelength of 1,556 cm-1 were observed for the amide group. They are associated with C-N and N-H groups, indicating the presence of acylated amino sugars and proteins/peptides in the bioemulsifier structure, forming glycoproteins. The emulsifying activity of a glycoprotein exopolymer produced by Pseudoalteromonas spp. has already been described (173,174). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Figure 36. IR spectra of A, crude biosurfactant extract and B, crude bioemulsifier extract obtained from the fermentation broth of strain ASV78. Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 101 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 102 It is well known that surface-active compounds are involved in the "pseudosolubilisation" strategy, which aims to improve bioavailability and facilitate access to hydrophobic compounds. In addition, the genus Pseudoalteromonas is typical among hydrocarbon-degrading microorganisms, so it is not surprising that a significant proportion of biosurfactant or bioemulsifier producers are found in this genus (60). Consequently, further characterisation and investigation of the potential applicability of the active compounds produced by strain ASV78 could be promising. Finally, the WGS of strain ASV78, obtained using a combination of MinION and Illumina approaches, yielded 19 contigs (the data is accessible at the NCBI under the BioProject accession number PRJNA1218939). The genome had a G+C DNA content of 41.5% and 5,215,181 bp. The assembled contigs were analysed using Prokka and a total of 4,674 genes were detected. Of these, 4,548 were identified as protein coding genes (CDSs) and their functions were assigned. A function was assigned to only 55.5% of the CDSs, while the rest were annotated as hypothetical protein CDSs. This result highlights the necessity to carry out further research in order to investigate the functions of these genes. The analysis of the genome using antiSMASH revealed a total of seven regions that are thought to encode biosynthetic gene clusters (BGCs) (Table 11). The total length of the predicted BGCs is approximately 250 Kb, representing ~4.8% of the ASV78 genome. The number of predicted BGCs in relation to the size of the genome is in the average range found in prokaryotes (175). It is interesting to note that only one of the predicted BGCs showed 100% similarity to the already described BGC of pentabromopseudilin (167). In this regard, Agarwal et al. emphasised that the bmp biosynthetic gene cluster is responsible for the production of a limited number of polybrominated diphenyl ether (PBDE) compounds, including bromophene and pentabromopseudilin. (167). In this sense, our results are consistent with the confirmed presence of both compounds in the extract via LC–HRMS. However, a phylogeny of the genus REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 1 - SUMMARY OF THE RESULTS AND DISCUSSION 103 revealed that numerous clades do not contain these genes (176). In fact, the detection of the bioactive compounds was mostly restricted to the clades containing the P. luteoviolacea and P. phenolica type strains (176), and not to P. neustonica as was the case for strain ASV78. Two other BGSs showed 75% and 45% similarity with the desferrioxamine E BGC (177) and APE Vf BGC (175), respectively. Desferrioxamine E and its analogues are siderophores widely produced by Streptomyces and related bacteria (178). However, in the Pseudoalteromonas genera where desferrioxamine is found (such as P. spongiae, P. ruthenica, P. piratica and P. tunicata), it is desferrioxamine B (179). Finally, aryl polyenes are yellow pigments, widespread among Gramnegative pathogens, such as, APE Ec from uropathogenic E. coli (180). They are structurally and functionally related to carotenoids and confer protection against photo-oxidative damage and lipid peroxidation (181). The yellowish colony color was observed when ASV78 was cultured on agar plates and is probably related to its genomic ability to synthesize aryl polyenes. The rest of the BGCs showed low (8% with N-myristoyl-D-asparagine) or no similarity to known BGCs, suggesting the potential to encode novel molecules. 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Arch Microbiol. 2020;202(8):2093-103. https://doi.org/10.1007/s00203-020-01927-7. 126. Alsammar H, Delneri D. An update on the diversity, ecology and biogeography of the Saccharomyces genus. FEMS Yeast Res. 2020;20(3):foaa013. https://doi.org/10.1093/femsyr/foaa013. 127. Blomberg A. Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 212 Analisi kimiootaxonomikoek erakutsi zuen ASV31 anduian nagusia zen gantz-azidoa, C18:0 cyclo ω8c, ez zegoela T. pacifica DSM 10166T eta R. algae LMG 29228T anduietan. Gainera, ASV31 hidroxi gantz-azidorik gabeko andui bakarra zen, eta gantz-azido asegabeak zituen bakarra. Are gehiago, summed feature 8 (C18:1 ω7c edota C18:1 ω6c) T. pacifica DSM 10166T (% 83,08) eta R. algae LMG 29228T (% 70,12) anduien gantz-azido nagusia izan zen, aldiz ASV31 anduian portzentaje baxuagoan (% 37,34) detektatu zen. Ubikinona Q-10 ASV31 anduian identifikatu zen, Paracoccaceae familiako genero gehienetan ohikoa den arnas kinona izanik (138). Gainera, ASV31 anduiaren lipido polarren perfilak honako hauek zituen: fosfatidilglizerola, aminolipidoa, identifikatu gabeko bi glikolipido, identifikatu gabeko bi fosfolipido eta identifikatu gabeko bi lipido (3. atala, IV. ERANSKINA, S4 irudia). Fosfatidiletanolamina ez detektatzeak, T. dalianensis-en eta Rhodovulum generoko kideen lipido polar garrantzitsuenetariko bat izanik (138,145), ASV31 anduia desberdintzen zuen. Oro har, analisiek ezaugarri genomiko, fenotipiko eta kimiotaxonomikoen desberdintasunak agerian utzi zituzten, eta horrek adierazten zuen ASV31 anduia genero berri baten lehen espeziea zela Paracoccaceae familiaren barruan, eta horregatik Anianabacter salinae gen. nov. sp. nov. izena proposatu zen, ASV31T andui tipoa izanik. Idazteko unean, Anianabacter salinae ICNPren arabera balio osoz argitaratua dago baina izendapena Anianibacter salinarum-era zuzentzen da, beste izen ortografia edo ortografia zehaztugabe batzuk direla eta (146). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 213 Deskribapena: Anianibacter salinarum gen. nov. sp. nov. 8. taula. Anianibacter salinarum ASV31T anduiaren deskribapena. Etimologia (A.ni.a.ni.bac’ter. N.L. masc. n. bacter, a rod; N.L. masc. n. Anianibacter, Añanako gatzagako bazilo bat) eta (sa.li.na’rum. L. gen. pl. n. salinarum, gatzagakoa). Andui tipoa ASV31T (=CECT 30309T = LMG 32242T) Taxonomia Pseudomonadota filuma, Alphaproteobacteria klasea, Rhodobacterales ordena eta Paracoccaceae familia. Zelulak Ez-mugikorrak, Gramnegatiboa diren baziloak, banaka edo inguruan polisakarido mukosozko kapsula bat duten agregatuetan agertzen direnak. PHA pikorrak biltzen dituzte zelulen barruan. Koloniak Beix-arrosa kolorekoak. Zelulek 2,4-2,5 µm-ko luzera eta 0,4-0,5 µm-ko zabalera dute. Pigmentuak Esferoidenona, esferoidenoa eta bakterioklorofila a agertzen dira. Hazkuntza pH 6,5-9,5 (optimoa 7-9,5) eta 18-37 ºC (optimoa 30 ºC) artean hazten da. NaCl tolerantzia handia du, % 0-23 (p/b) (optimoa % 35). Aktibitate entzimatikoa Katalasa eta oxidasa positiboa eta ureasa negatiboa. Esculina eta Tween 20 hidrolizatzen ditu. Nitratoen erredukzioa burutzen du. API ZYM probetan fosfatasa alkalino, esterasa (C 4), esterasa lipasa (C 8), leuzina arilamidasa, balina arilamidasa eta α-glukosidasa aktibitate handia erakusten ditu; eta aktibitate ahulak zisteina arilasa, fosfatasa azidoa eta naftola-AS-BIfosfohidrolasarentzako. Jarraitzen du 25. irudia. ASV31T anduiaren zelula baten transmisioko mikroskopia elektronikoa erabiliz egindako mikrografia. 500,0 nm barra. 26. irudia. Anianibacter salinarum ASV31T anduia MA hazkuntza medioan. Kolonien morfologia eta kolorea erakusten dira. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 214 Antibiotikoekiko suszeptibilitatea B polimixina (300 IU), bankomizina (30 µg) eta estreptomizinarekiko (10 µg) erresistentea. Kimiotaxonomia Ubikinona bakarra Q-10 da eta gantz-azido nagusiak C18:0 cyclo ω8c eta summed feature 8 (C18:1 ω7c edota C18:1 ω6c). Lipido polar nagusiak fosfatidilglizerola, aminolipida, identifikatu gabeko glikolipido bat, identifikatu gabeko fosfolipido bat eta identifikatu gabeko lipido bat dira. WGS Genomaren tamaina 3.6 Mbp-takoa da eta andui tipoaren DNAren G+C edukia % 65.7 mol-ekoa da. CECT, Espainiako mikroorganismo tipo-en bilduma; LMG, Ganteko Mikrobiologiako Laborategia (Belgika); WGS, Genoma osoaren sekuentziazioa. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 215 Konposatu antimikrobianoen edota tentsioaktiboen ekoizleak diren isolatuen baheketa Berreskuraezinak suertatu ziren bi isolaturen salbuespenarekin, gainontzeko 148 isolatuei antimikrobianoen ekoizpen ahalmenaren baheketa egin zitzaien. Hau, antagonismo entsegua erabiliz, andui patogenoen hazkuntza inhibitzeko zuten gaitasunean oinarritzen da. Hogei isolatuk jarduera argia erakutsi zuten gutxienez patogeno anduietako baten aurka (4. taula). Ondoren, antimikrobianoak ekoizten zituzten 20 isolatu horiekin difusio entsegua burutu zen, eta horietatik bederatzik antimikrobianoak ekoizteko gaitasuna azaldu zuten (4. taula). Isolatu halofilo edo halotolerante horietatik zortzik bakterio andui Gram-positiboen zein Gram-negatiboen hazkuntza inhibitzen zuten konposatuak ekoiztu zituzten; aldiz, ASV55 isolatuak difusio entseguan erakutsi zuen soilik aktibitate antifungikoa (4. taula). Antagonismo zein difusio entseguetan lortutako emaitz batzuen ilustrazioak 27. irudian agertzen dira. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 216 27. irudia. Isolatu halofilo eta halotolerante batzuek ekoztutako konposatu antimikrobianoen detekzioaren adibidea. A, antagonismo entseguan patogenoen hazkuntzaren inhibizio haloa, ASV12, ASV10, ASV14, ASV25, ASV89 eta ASV106 isolatuek sortua eta B, difusio entseguan patogenoen hazkuntzaren inhibizio haloa, ASV12, ASV13, ASV55, ASV128, ASV106, ASV129 eta ASV136 isolatuek sortua. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 217 Gainera, difusio entseguak iradoki zuen konposatu antimikrobianoak inkubazioaren hirugarren egunean ekoiztuak zeudela eta degradazioa nabaria zela hamargarren egunaren ondoren (4. taula). Bestalde, soilik antagonismo entseguan aktibitatea erakutsi zuten isolatuek, konposatu antimikrobianoaren ekoizpena patogenoarekiko mantenugaien lehian oinarritu zela suposa dezakete, beste ikerketa batzuetan deskribatu den bezala (147). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 9. taula. Aztertutako andui patogenoetako baten aurka, gutxienez, aktibitate antimikrobianoa erakutsi zuten isolatuak. Antagonismo entseguan (DAA) eta difusio entseguan (WDA) jasotako inhibizio haloaren diametroak (mm) ematen dira. Andui patogenoak P. aeruginosa ATCC 27853T E. coli ATCC25922T B. subtillis CECT 356T S. aureus ATCC 29213T E. faecalis ATCC 29212T C. albicans ATCC 90029T Inhibizio haloaren batezbestekoa (mm) DAA WDA DAA WDA DAA WDA DAA WDA DAA WDA DAA WDA eguna eguna eguna eguna eguna eguna Isolatuak 3 10 17 3 10 17 3 10 17 3 10 17 3 10 17 3 10 17 ASV1 8 - - - - - - - - - - - - - - - - - - - - - - - ASV3 11 - - - - - - - - - - - - - - - - - - - - - - - ASV7 20 - - - - - - - 12 - - - 25 - - - - - - - - - - - ASV10 10 - - - 15 - - - - - - - 12 - - - - - - - - - - - ASV11 10 - - - - - - - - - - - - - - - - - - - - - - - ASV12 27 - - - 27 14 12 - 25 - - - 30 - - - 30 - - - - - - - ASV13 - - - - 9 15 11 - - - - - - - - - 31 - - - - - - - ASV14 12 - 15 - - - - - - - - - - - - - - - - - - - - - ASV25 12 - 12 - 15 - - - 16 - - - 10 - - - 13 - - - - - - - ASV55 - 15 13 - - 16 15 10 13 20 16 13 - 12 18 13 - 7 6 - - 15 13 - ASV78 12 - - - - - - - 20 - - - 17 - - - - - - - - - - - ASV89 9 - - - - - - - - - - - - - - - - - - - - - - - ASV106 - - - - - - - - 21 18 - - - - - - - - - - - - - - ASV117 9 - - - - - - - - - - - - - - - - - - - - - - - ASV118 9 - - - - - - - - - - - - - - - - - - - - - - - ASV120 - - - - - - - - 6 - - - - - - - - - - - - - - - ASV121 - - - - - - - - 6 - - - - - - - - - - - - - - - ASV128 - - - - - - - - 20 15 - - - - - - - - - - - - - - ASV129 - - - - - - - - 19 17 - - - - - - - - - - - - - - ASV136 6 - - - - - - - 14 9 - - 10 - - - - - - - - - - - -, negatibo 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 218 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 219 Pseudoalteromonas generoa (ASV1, ASV3, ASV10, ASV11, ASV12, ASV13, ASV14, ASV25, ASV78, ASV89, ASV117, ASV118 eta ASV136 isolatuak) aktibitate antimikrobianoa erakutsi zuten isolatuen genero nagusia izan zen ikerketa honetan. Aipagarria da hauetako isolatu gehienek, ASV13a salbu, P. aeruginosa-ren hazkuntza inhibizio gaitasuna agertu zutela. Mantenugaien konpetentziak eragindako efektu inhibitzaile bat izateko aukeraz gain, beste hipotesi bat ere badago: Pseudoalteromonas espezieek gaitasun handia dute alterozina bezalako pelikulen aurkako agenteak ekoizteko eta modu horretan, P. aeruginosa-ren (biofilmak sortzen dituen bakterio ezaguna) inhibizioa eragiteko (148). Gaitasun antimikrobianoa erakutsi zuten gainerako isolatuak Salinivibrio (ASV7), Pseudomonas (ASV55), Kocuria (ASV106 eta ASV128), Halomonas (ASV120 eta ASV121) eta Streptomyces (ASV129) generoetan biltzen ziren. Genero hauetako kide batzuk aktibitate antibakterianoa dutela jasota dago (149–152), Salinivibrio generoko kideak izan ezik. Hauetan, aktibitate antifungikoa (153) eta intsektizidaz (154) gain, ez da deskribatu bakterioen aurkako aktibitaterik, ikerketa honetan ASV7 anduian ikusi den bezala. Aitzitik, 148 isolaturen biosurfaktante ekoizpen ahalmena ere aztertu zen, olioaren dispertsio testa eta Parafilm-M testa erabiliz gainazaleko tentsioa murrizteko duten gaitasuna determinatuz. Baita bioemultsifikatzaile ekoizpen ahalmena ere, emultsifikazio testaren bidez olio zein hidrokarburoak eraginkortasunez emultsionatzeko duten ahalmena ebaluatuz. 28. irudian, lortutako emaitz batzuen argazkiak ageri dira; xehatasun gehiago, berriz, 4. atala, V. ERANKINA, S2 taulan aurkitu daitezke. Parafilm-M eta olioaren dispertsio testetan isolatu bakoitzarentzat lortutako batez besteko emaitzak ageri dira, 29. irudian eta 30. irudian hurrenez hurren, generoaren arabera multzokatuta. Biosurfaktanteak ekoizteko gaitasuna zutela kontsideratu zen, baldin eta Parafilm-M testean tantaren diametroa eta olioaren dispertsio testean halo garbiaren diamtroa kontrol negatiboarenak (BPM) baino handiagoak baziren (≥ 3,4 mm eta ≥ 7,0 mm, hurrenez hurren). Beste ikerketa batzuetan REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 220 antzeko mozketa puntuak erabili dira biosurfaktanteen ekoizpena determinatzeko (95,155). 28. irudia. Konposatu tentsioaktiboak ekoizten dituzten zenbait andui halofiloren eta halotoleranteren adibideak A, Parafilm-M testean; B, olioaren dispertsio testean eta C, emultsifikazio testean, kasu guztietan kontrol negatiboarekin alderatuta. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 221 29. irudia. Biosurfaktanteak ekoizteko isolatuen baheketaren emaitzak Parafilm-M testean. Isolatuak generoaren arabera taldekatutako puntuen bidez irudikatuta daude, tantaren batezbesteko diametroa erakutsiz. Kontrol negatiboaren (BPM, gorriz) eta SDS kontrol positiboaren (% 1, p/b) (urdinez) emaitzak ere ageri dira. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 228 Bestalde, pentabromopseudilin konposatuaren identifikazioa oso argia izan zen bere UV espektroa eta formula molekularra interpretatuz. Izan ere, MS espektroak (4. atala, V. ERANSKINA, S6 irudia) bost bromo atomo dituen patroi isotopiko bereizgarri bat erakutsi zuen, modu horretan formula molekularrar bakarra, C10H4Br5NO, iradokiz, zeinak pentabromopseudilin konposatuarekin soilik kointziditzen zuen Produktu Naturalen Hiztegiaren arabehera. Jakineko aktibitatea medio, konposatu hau antimikrobiano estraktu gordinetik purifikatu zen alderantzizko fasean HPLC semi-preparative teknikaren bidez. Rt 22 minututan, 0,9 mg konposatu puru lortu ziren. Ondoren, (-) MS espektroak eta 1H-NMR analisiak (34. irudia) konfirmatu zuen formula eta purutasuna. Azkenik, bromophene bezala identifikatutako pikoak ez zuen (+) MS espektro interpretagarririk eman. Ondorioz, antimikrobiano estraktu gordinetik abiatuta, konposatuaren purifikazioari ekin zitzaion alderantzizko fasean HPLC semipreparative teknikaren bidez, eta, horrela, RMN analisirako egokitzat jo zen frakzio bat lortu zen. Frakzio honetan egindako (-) MS espektroan agertutako banaketa isotopikoan oinarrituta, Produktu Naturalen Hiztegian zortzi kointzidentzia zutuen C12H6Br4O2 formula molekularrarekin bat zetorrela ondorioztatu zen. Rt 20 minututan, 0,5 mg konposatu puru lortu ziren. Ondorengo (-) MS espektroak eta 1H-NMR analisiak (35. irudia) bromophene konposatuaren presentzia baieztatu zuten, literaturaren datuekin alderatuz (164). Ikerketa honetan deskribatu den bezala, Pseudoalteromonas gereroan metabolito bromatuen ekoizpena dokumentatua izan da. Zehazki, pentabromopseudilin, P. phenolica eta P. luteoviolacea espezieetatik isolatua izan da eta bromophene aldiz P. phenolica espezietik (165). Deigarria dena da, ikerketa honetan ASV78 anduiak bi konposatuak aldi berean ekoizteko gaitasuna azaldu duela, eta ASV78 anduitik filogenetikoki gertuen dagoen espeziea Pseudoalteromonas neustonica dela (% 98,57ko 16S rRNA genesekuentziaren antzekotasunarekin). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 229 34. irudia. ASV78 anduiak ekoiztutako pentabromopseudilin konposatuaren (-) MS espektroa (goian) eta 1H-NMR espektroa bere egiturarekin (behean). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 230 35. irudia. ASV78 anduiak ekoiztutako bromophene konposatuaren (-) MS espektroa (goian) eta 1H-NMR espektroa bere egiturarekin (behean). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 231 Metizilinarekiko erresistentea den S. aureus anduiaren aurkako aktibitatea pentabromopseudilin zein bromophene konposatuetan deskribatu bada ere, ez da askorik ezagutzen hauen aktibitate espektroaren inguruan (165,166). Gainera, bi konposatuak aldi berean ekoiztea ez da ohikoena; izan ere, oraingoz, Pseudoalteromonas sp. CMMED 290 andui bakarrean baino ez da dokumentatu (165), nahiz eta bide metabolikoa partekatua izan bi konposatuak ekoizteko (167). Hori horrela izanda, baloratzekoa da konposatu horiek izan dezaketen efektu sinergikoa ikertzea, orain arte ez baita halakorik frogatu. Ikerketa horren emaitzak onuragarria izan daitezke konposatu hauek praktikan aplikatzeko. Testuinguru honetan, konposatu puruekin egindako entseguek erakutsi zuen bi konposatuek bakterio Gram-positiboen aurkako aktibitate antimikrobianoa erakusten zutela; pentabromopseudilin-ak gainera bakterio Gram-negatiboen aurkako aktibitatea ere erakusten zuelarik (10. taula). Zehazki, pentabromopseudilin-ak 0,02 eta 0,04 µg/mL bitarteko MIC balioak agertu zituen S. aureus ATCC 29213 anduiaren aurka eta 10 eta 20 µg/mL bitarteko MIC balioak P. aeruginosa ATCC 27853 anduiaren aurka (10. taula, goiko aldean). Aldiz, bromophene-k 2,5 eta 5 µg/mL bitarteko MIC balioak erakutsi zituen S. aureus ATCC 29213 anduiaren aurka (10. taula, goiko aldean). 10. taula. Konposatu puruen eta kontrolen aktibitate antibakterianoa. MIC balioak goiko aldean eta bakterioen aurkako aktibitatea (erreplika batetik lortutako MIC balioak) behekaldean. Konposatuak MIC (µg/mL) S. aureus ATCC 29213 P. aeruginosa ATCC 27853 Pentabromopseudilin 0.02-0.04 10-20 Bromophene 2.5-5 > 80 Sinergia (1:1) 0.04-0.08 20-40 Bankomizina 0.5-1 ND Ziprofloxazinoa ND 0.25-0.5 Jarraitzen du REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 232 Konposatuak Aktibitatea (µg/mL) A.baumannii ATCC 19606 E.faecalis ATCC 29212 E.faecium vanA 15167 Pentabromopseudilin 10-20 10-20 1.25-2.5 Bromophene > 80 40-80 20-40 Sinergia (1:1) 20-40 20-40 2.5-5 Bankomizina ND 4-8 > 32 Aztreonama 80-160 ND ND ND; zehaztu gabe. MIC balioak E. faecalis ATCC 29212, bankomizinarekiko erresistentea den E. faecium vanA 15167 eta A. baumannii ATCC 19606 andui patogenoen aurka determinatu ziren baina soilik erreplika bakarra erabiliz. Hori konposatu puruen kantitate eskasiagatik gertatu zen, eta hortaz, baliteke etorkizuneko ikerketetan balioztatze handiagoa behar izatea. Pentabromopseudilin konposatuak E. faecalis ATCC 29212 anduiaren hazkuntza inhibitu zuen 10-20 µg/mL-tan, bankomizinarekiko erresistentea den E. faecium vanA 15167 anduiarena 1,25-2,5 µg/mL-tan eta A. baumannii ATCC 19606 anduiarena 10-20 µg/mLtan (10. taula, behekaldean). Bestalde, ikusi zen bromophene-k E. faecalis ATCC 29212 anduiaren hazkuntza 40-80 µg/mL-tan inhibitzen zuela eta bankomizinarekiko erresistentea zen E. faecium vanA 15167 anduiarena 20-40 µg/mL-tan (5. taula, behekaldean). Aztertutako patogeno bakar batentzat ere ez zen ikusi bi konposatuen arteko aktibitate sinergikorik. Pentabromopseudilin konposatuak erakutsi zuen aktibitate antibakterianoa kontuan hartzekoa da klinikoki garrantzitsuak diren espezieen aurka, besteak beste, A. baumannii, E. faecium eta S. aureus. Deigarria da pentabromopseudilin-aren kontzentrazio baxua (0,02 eta 0,04 μg/mL bitarteko MIC balioak) zeinetan S. aureus ATCC 29213 anduiaren aurka eraginkorra den, batez ere bankomizinaren kontzentrazioekin alderatuz (0,5 eta 1 μg/mL bitarteko MIC balioak ikerketa honetan, eta 0,52 eta 0,59 μg/mL bitartekoak Lepe et al.-en ikerketan (168)). Beraz, oso interesgarria izango litzateke pentabromopseudilin-aren aktibitate espektroa ebaluatzea patogeno sorta REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 233 zabalago baten aurka, eta haren ekintza mekanismoa ikertzen jarraitzea, zeina bankomizinarenarekiko desberdina izan daitekeen, bankomizinarekiko erresistentea den E. faecium eta bakterio Gram-negatiboen aurkako jarduerak agerian uzten duen bezala. Izan ere, diana anitzeko inhibitzaileek antibiotikoen munduko hurrengo belaunaldia agertzen dute, antimikrobianoekiko erresistentziaren garapenarekiko abantaila nabarmenak eskainiz (169). Biosurfaktante eta bioemultsifikatzaile estraktu gordinei dagokienez, IR erabili zen osagai horien izaera kimikoa identifikatzeko. Alde batetik, biosurfaktante estraktuaren IR espektroak erakutsi zuen hidrokarburo alifatikoen kateak zeudela polisakarido frakzio batekin batera, horrek biosurfaktantearen izaera glikolipidikoa iradokiz (36A irudia). Izan ere, 3.311 cm−1-tan xurgapen banda zabala egoteak hidroxitaldeen -OH luzaketak zeudela adierazten zuen. Gainera, 2.919 eta 2.847 cm–1-ko banden presentzia kate alifatikoen C-H luzaketa moduari zegokion. 1.652 cm−1-ko banda C=O taldeen presentziarekin elazionatuta egon liteke eta 1.408 cm–1-ko bandak polisakaridoei egotz zekizkiekeen CH3 eta CH2-aren C-H taldeei. Azkenik, 1.317-1,013 cm−1 arteko bandek, azukrearen hondarrari dagokion C-O luzaketaren presentzia adierazten zuten. Oro har, ASV78 anduiak ekoiztutako biosurfaktantearen estraktu gordinaren IR espektroa beste ikerketeta batzuetan (170–172) adierazitako biosurfaktante glikolipidikoen antzekoa izan zen. Bestalde, bioemultsifikatzaile estraktu gordinaren IR espektroa 36B irudian erakusten da. Talde karboxilikoen presentzia adierazten duten xurgapen banda zabalak ikusi ziren (3.369 cm-1, hidroxi-luzaketa; 1.630 cm-1, karboxilo C=O luzaketa; 1.114 cm-1, karboxilo -C-O luzaketa). Gainera, amida taldearentzat 1.556 cm-1-ko uhin luzeran luzatze bibrazioak ikusi ziren. Hauek C-N eta N-H taldeekin lotzen dira, bioemultsifikatzailearen egituran aminoazukre eta proteina/peptido azilatuak daudela adieraziz, glikoproteinak eratuz. Aurkikuntza hau bat dator beste Pseudoalteromonas spp. isolatu batzuetan deskribatu den glikoproteina izaeradun exopolimeroekin, zeintzuek gainera jarduera emultsionatzailea erakutsi duten (173,174). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 36. irudia. ASV78 anduiaren hartzidura saldatik lortutako A, biosurfaktante estraktu gordinaren eta B, bioemultsifikatzaile estraktu gordinaren IR espektroak. 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 234 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 235 Ezaguna da konposatu tentsioaktiboek "pseudosolubilizazio" estrategian parte hartzen dutela; estrategia horren helburua da bioerabilgarritasuna hobetzea eta konposatu hidrofoboen eskuragarritasuna erraztea. Gainera, Pseudoalteromonas generoa ohikoa da hidrokarburoak degradatzen dituzten mikroorganismoen artean, eta, beraz, ez da harritzekoa genero horretan biosurfaktante edo bioemultsifikatzaile ekoizleak diren isolatuen proportzio esanguratsu bat aurkitzea (60). Ondorioz, ASV78 anduiak ekoiztutako konposatu tentsioaktiboen aplikagarritasun potentzialaren karakterizazio eta ikerketa gehiago etorkizun handikoak izan litezke. Azkenik, ASV78 anduiaren genoma aztertu zen MinION eta Illumina tekniken konbinazioa erabiliz. Genomak 5.215.181 bp zituen eta 19 contig-etan mihiztatu zen (NCBIn eskuragarri PRJNA1218939 atxikitze zenbakiarekin), bere DNA G+C edukia % 41,5-ekoa izanik. Contig-ak Prokka erabiliz aztertu ziren eta guztira 4.674 gene detektatu ziren. Horietatik 4.548 proteinak kodetzen dituzten gene (CDS) gisa identifikatu ziren, zeintzuen artean soilik % 55,5ari esleitu zitzaien funtzio bat, gainerakoak proteina hipotetiko gisa sailkatu baitziren. Emaitza horrek agerian uzten du beharrezkoa dela ikerketa gehiago egitea gene horien funtzioak zehazteko. ASV78 anduiaren genoma antiSMASH bidez aztertu zen eta gene-multzo biosintetikoak (BGC) kodetzen zituzten zazpi eskualde aurkitu ziren (11. taula). Detektatutako BGCen luzera osoa 250 Kb ingurukoa izan zen, ASV78ren genomaren ~ % 4,8koa alegia. Genomaren tamainarekiko aurresandako BGCen kopurua prokariotoetan aurkitutako batez besteko tartean dago (175). Iragarritako BGCen artean bakar batek erakutsi zuen % 100eko antzekotasuna pentabromopseudilin konposatuaren sintsiarekin erlazionatuta (167). Horren harira, Agarwal et al.-ek azpimarratu zuen bpm gene biosintetikoen kluster hori, difenil eter polibromatu (PBDE) konposatuen kopuru mugatu baten ekoizpenaz arduratzen dela, hauen artean, pentabromopseudilin eta bromophene konposatuen sintesiaz (167). Lan honek hori konfirmatzen du, izan ere LC-HRMS teknikaren bidez estraktuan bi konposatuen ekoizpena REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 236 baieztatu zen. Dena den, Pseudoalteromonas generoaren filogeniak agerian utzi zuen klado ugarik ez dituztela gene horiek (176). Izan ere, konposatu bioaktiboen detekzioa P. luteoviolacea eta P. phenolica espezieetako anduiak zituzten kladoetara mugatu zen gehienbat (176), eta ez P. neustonica-ra ASV78 anduiaren kasuan gertatzen zen bezala. Beste bi BGC E desferrioxaminaren (177) eta APE Vf-ren (175) BGCekin antzekotasuna erakutsi zuten. E desferrioxamina eta bere analogoak Streptomyces eta erlazionatutako bakterioek maiz ekoizten dituzten sideroforoak dira (178). Hala ere, desferrioxamina detektatu den Pseudoalteromonas generoetan (P. spongiae, P. ruthenica, P. piratica eta P. tunicata kasu), B desferrioxamina izan da (179). Azkenik, aril polienoak Gramnegatibo patogenoen artean hedatutako pigmentu horiak dira, hala nola E. coli uropatogenikoaren APE Ec (180). Estrukturalki eta funtzionalki karotenoideekin erlazionatuta daude eta kalte fotooxidatiboen eta lipidoen peroxidazioaren aurkako babesa ematen dute (181). Litekeena da ASV78 anduiaren koloniek agar plaketan kultibatzerakoan erakusten zuten kolore horixka aril polienoak sintetizatzeko duen gaitasun genomikoarekin erlazionatuta egotea. Gainerako BGC antzekotasun baxua erakutsi zuten (% 8 N-myristoyl-D-asparagine-rekin) edo ez zuten antzekotasunik erakutsi BGC ezagunekin, hauei molekula berriak kodetzeko ahalmena iradokiz. Zentzu honetan, identifikatu gabeko BGCen artean ASV78 anduiak sortutako biosurfaktanteak egon litezke, izan ere beste ikerketa batzuetan erribosomikoak ez diren peptido sintetasak (NRPS) identifikatu dira, surfactin edo fengycin bezalako biosurfaktante aitortuekin erlazionatuta (182). REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1. atala - EMAITZEN LABURPENA ETA EZTABAIDA 237 11. taula. AntiSMASH erabilita ASV78 anduiaren genoman detektatutako ustezko gene-multzo biosintetikoak (BGC). Eremua Mota Hasiera (bp) Bukaera (bp) Ezagutzen den talderik antzekoena (MIBiG Erref.) Antzekotasuna Eremua 1 PBDE 865.555 889.181 Pentabromopseudilin (BGC0000890) 100% Eremua 2 NRPS, Type I PKS 1.224.295 1.296.460 Eremua 3 RiPP-like 1.498.707 1.509.561 N-myristoyl-Dasparagine (BGC0000972) 8% Eremua 4 NRPS 2.574.952 2.643.824 Eremua 5 NIsideroforoa 3.032.695 3.063.100 Desferrioxamine E (BGC0001572) 75% Eremua 6 Aril polienoak 478.747 522.306 APE Vf (BGC0000837) 45% Eremua 7 RiPP-like 804.360 816.540 PBDE, difenil eter polibromatuak; NRPS, erribosomikoak ez diren peptido sintetasak; PKS, polyketide sintasak; RiPP, erribosomikoki sintetizatutako eta itzulpen ostean aldatutako peptido produktua; NI-sideroforoa, NRPS-sideroforoa independentea; APE Vf, Aliivibrio fischeri ES114 anduian detektatutako aril polienoaren gene-multzo biosintetikoa. REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 3. atala - APPENDIX I. ERANSKINA 245 APPENDIX I. ERANSKINA Prokaryotic Diversity and Community Distribution in the Complex Hydrogeological System of the Añana Continental Saltern Reference: Azpiazu-Muniozguren M, García-Martínez M, Zabaleta A, Antiguedad I, Garaizar J, Laorden L, Martinez-Malaxetxebarria, I, and Martinez-Ballesteros, I. Prokaryotic Diversity and Community Distribution in the Complex Hydrogeological System of the Añana Continental Saltern. Microb Ecol. 2024; 87(1):171. Microbial Ecology (2025); 87(1):171 DOI: 10.1007/s00248-025-02488-2 Impact factor 2023: 3.3 Relative position: 70/161, Q2 Microbiology REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Vol.:(0123456789) Microbial Ecology (2024) 87:171 https://doi.org/10.1007/s00248-025-02488-2 RESEARCH Prokaryotic Diversity andCommunity Distribution intheComplex Hydrogeological System oftheAñana Continental Saltern MaiaAzpiazu‑Muniozguren1· MinervaGarcía‑Martínez1· AneZabaleta2· IñakiAntiguedad2· JavierGaraizar1,3· LorenaLaorden1,3· IratiMartinez‑Malaxetxebarria1,3· IlargiMartinez‑Ballesteros1,3 Received: 10 October 2024 / Accepted: 2 January 2025 © The Author(s) 2025 Abstract The Añana Salt Valley (northern Spain) is a continental saltern consisting of a series of natural springs that have been used for salt production for at least 7000years. This habitat has been relatively understudied; therefore, prokaryotic diversity was investigated through Illumina-based 16S rRNA gene sequencing to determine if the waters within the valley exhibit distinctive microbiological characteristics. Two main types of water were found in the valley: salty (approximately 200g/L salinity) from the diapiric structure and brackish (≤ 20g/L salinity) from shallow streams. The beta diversity indices showed that salinity was the primary factor influencing the prokaryotic distribution. However, a niche-specific influence was observed between waters of the same origin, with significant differences in the relative abundance of the ASVs. The microbiome of the saltern revealed that the archaeal domain was mainly restricted to salty waters, while the bacterial domain was ubiquitous throughout the saltern, with a notable prevalence in brackish waters. The main bacterial and archaeal phyla identified were Pseudomonadota and Halobacterota, respectively. The genus Halorubrum was abundant and widespread in salty waters, while Pseudomonas was a significant part of the prokaryote community, mainly in brackish waters. The relative abundance of the genera Haloplanus and Salinibacter increased in the salt ponds used for salt production. The taxa involved in chemoheterotrophy and fermentation were widespread, sharing the same niche. Overall, the location of this saltern on a diapiric structure favors the occurrence of waters with different origins that affect the prokaryotic distribution beyond the niche location in the valley. Keywords Salt diaper· Continental saltern· 16S rRNA gene sequencing· Prokaryotic diversity Introduction Hypersaline environments represent highly bioproductive habitats with a large microbial diversity adapted to these conditions [1]. To better understand these environments, it is essential to characterize the microbial communities due to their contribution to nutrient cycling and ecosystem functioning [2]. Therefore, the study of hypersaline environments such as solar salterns [3], salt mines [4], and salt lakes [5] has received considerable interest. Many different types of halophilic and halotolerant microorganisms including archaea of the order Halobacteriales and bacterial species of the order Halanaerobiales and Halomonadaceae, Desulfohalobiaceae, and Salinibacteriaceae families, among others [6], are found in these environments. While halophiles are less common within the Eucarya domain, the green alga Dunaliella is typically present in aerobic environments with high salt content [5]. As microbial communities are vital to ecosystem functions and are influenced by the physical and geological characteristics of the site, parallel studies in these other areas are also necessary to gain a comprehensive understanding of these ecosystems [1, 7]. *Ilargi Martinez-Ballesteros [email protected] 1 MikroIker Research Group, Immunology, Microbiology andParasitology Department, Faculty ofPharmacy, University oftheBasque Country UPV/EHU, Paseo de La Universidad 7, 01006Vitoria-Gasteiz, Spain 2 Hydro-Environment Processes Research Group. Geology Department, Faculty ofScience andTechnology, University oftheBasque Country UPV/EHU, Barrio Sarriena S/N, 48940Leioa, Spain 3 Bioaraba, Microbiology, Infectious Diseases, Antimicrobial Agents, andGene Therapy, 01006Vitoria-Gasteiz, Spain Section 3. atala - APPENDIX I. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 M.Azpiazu-Muniozguren et al. 171 Page 2 of 15 Among the different types of salterns that exist, continental ones are less well known, and not many are still in use today. The Añana Salt Valley (Álava, Basque Country, Spain) is placed in one of the best-preserved continental salterns in Europe. Salt production is active in the valley since at least 7000years, and it has been recognized by Food and Agriculture Organization (FAO) as being Europe’s first Globally Important Agricultural Heritage System (GIAHS). The underground evaporitic rocks, comprising salt, gypsum, clay and others, were formed during the initial stages of the fragmentation of Pangaea supercontinent (approximately 200 million years ago). These rocks now form a geologically complex diapiric structure where the salts are actively rising [8]. The geological complexity of the area leads to hydrogeological complexity, with very slow deep flows mixing with shallower flows in some areas of the valley. This results in the emergence of springs with varying salinity levels, dependent on the route taken by the water through the subsoil. This gives rise to the presence of springs with salty (≈ 200g/L salinity) or brackish (≤ 20g/L salinity) water that are very close to each other. Despite the singularity of this environment, and although studies have been carried out in this saltern on archeology, hydrogeology, or the salt itself, there is limited microbiological data available. There is only one publication on microbial and viral changes from groundwater to surface water [9] and one on fungal community diversity [10], so further research is needed to fully understand this ecosystem. Therefore, in this context, this work present an extended study along this ancient saltern in which the prokaryotic diversity was studied by Illumina-based 16S rRNA gene sequencing to determine whether the waters within the valley have distinctive microbiological characteristics. Taking into account the physico-chemical parameters of the main waters supplying the valley (six springs, a stream that cross the saltern, and groundwater) and water involved in salt production (water from a brine distribution channel and three brine resting ponds), prokaryotic community structure and composition was studied. The integration of all data is not only important for expanding scientific understanding but also for facilitating the recovery and enhancement of this active saltern. Material andMethods Sampling Site Features andSampling Procedure Añana Salt Valley (42°47′59.82″ N, 2°59′3.23″ W; altitude 570–598m) is in western Álava, Basque Country, Spain. For this study, 12 sampling sites (Fig.1a) were selected based on their accessibility and minimal human intervention. The Santa Engracia spring is situated at the highest point of the valley, and it is the main brine supplier to the saltern. Immediately, its water is channeled (called in this study Santa Engracia channel) and distributed throughout the valley to the temporary accumulation or resting ponds (i.e., Pond I, Pond II, and Pond III) from which the brine is distributed to the crystallization pans at the end of the salt production system. Santa Engracia spring is very close to the brackish water source of the San Juan stream, which runs all along the saltern, but it is not part of the salt production system. A little further north are the other springs, some salty (El Cautivo, El Pico, Fuenterriba, and Hontana) and some brackish (El Pico Dulce, which is only two meters from El Pico). To the east in the valley, there is a piezometer (called S8) from which brackish groundwater can be sampled at a depth of 60m. Most of the springs are in direct contact with the natural surrounding environment, except for the Santa Engracia spring and the El Cautivo spring, which have a wooden structure around them (Fig.1a). More information about the complex geological and hydrogeological context of the valley is presented in the Supplementary Material. Given the importance of salt production in the economic and social development of the area and its relevance as a natural, cultural and heritage site, there is since 2017 a surfaceand ground-water monitoring network throughout the valley. Of the 12 sampling sites, eight (the six springs, the stream, and the piezometer) are part of this monitoring network; therefore, the ionic composition of the watersamples was available. This composition is periodically analyzed by ion chromatography at the SGIker Advanced Research Facilities of the University of the Basque Country UPV/EHU. In a first phase of this study (spring 2018), water samples from three springs (Santa Engracia spring, El Pico spring, and El Pico Dulce spring), groundwater from S8 piezometer, and water from the ponds (pond I, II, and III) were collected. In a second phase of the study (spring 2021), the sampling was extended to the rest of the springs (Fuenterriba spring, El Cautivo spring, and Hontana spring), the San Juan stream, and the brine distribution channel (Santa Engracia channel). Water samples were collected directly using sterile glass bottles. Water from the Santa Engracia and El Cautivo springs was collected using a Niskin bottle (Aquatic BioTechnology, El Puerto de Santa María, Spain) both at 2m depth. Groundwater from the S8 piezometer (60m depth) was collected using a manual bailer system (Eijkelkamp, Giesbeek, The Netherlands). At all sampling sites, water temperature, pH, and electrical conductivity were measured with a Combo tester (Hanna Instruments, Eibar, Spain) and salinity with a density hydrometer (Hartwig Instruments, Netherlands), all parameters measured insitu. The samples were then transported to the laboratory in Section 3. atala - APPENDIX I. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Prokaryotic Diversity andCommunity Distribution intheComplex Hydrogeological System of… Page 3 of 15 171 properly labeled containers at 4°C and within 1h for further processing. Prokaryotic Community andDiversity Study by16S rRNA Gene Deep Sequencing DNA Extraction andSequencing Fig. 1 The Añana Salt Valley; a map showing the 12 sampling locations and their pictures (A-L) and b the representation of the different water flows in the valley Section 3. atala - APPENDIX I. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 M.Azpiazu-Muniozguren et al. 171 Page 4 of 15 Water samples were enriched by filtering 5 L of water under aseptic conditions through a custom-made device driven by vacuum/compression pumps (Labbox, Premia de Dalt, Spain) and ending up in a 0.22-µm pore size filter. DNA was extracted using the Genomic Mini AX Bacteria Kit (A&A Biotechnology, Gdansk, Poland) and quantified using the QuantiFluor dsDNA System (Promega, Madison, WI, USA). The V3-V4 hypervariable region of the 16S rRNA gene was amplified [11], and the amplicons were sequenced using the paired-end method on a MiSeq Illumina platform at Biogenetics (Álava, Spain). Libraries were prepared from isolated DNA using the Nextera DNA Library prep kit (Ilumina, San Diego, CA, USA). Sequencing data from the study are available under the accession number PRJNA1115049. Prokaryote Diversity andDistribution Sequence analysis was performed using the packages and pipelines of the Quantitative Insights Into Microbial Ecology (QIIME2) program (version 2021.4) [12]. Joining of paired-end reads, sequence quality control, and feature table construction were performed by denoising with DADA2 plugin (q2-dada2) with standard parameters [13]. Taxonomic assignment of DADA2-generated amplicon sequence variants (ASVs) was performed using the Bayesian taxonomy classifier classify-sklearn with the q2-feature-classifier addon [14] with taxonomy.py using the SILVA database v.138 [15], clustered at 99% sequence similarity. Alpha diversity metrics (observed ASVs, Faith’s Phylogenetic Diversity, Pielou’s evenness, Shannon’s and Simpson’s diversity indexes) and beta diversity metric (Bray‐Curtis dissimilarity) were estimated using q2‐diversity after samples were rarefied to the smallest number of non-chimeric sequences of all samples tested. Statistical significance of the alpha diversity values between sampling sites was assessed using the Kruskal–Wallis H test. To assess the relationship of the water properties with α-diversity indices, Pearson’s coefficient test was carried out. For comparing the microbial community structure among samples, Bray–Curtis dissimilarity coefficient was assessed by analysis of similarities (ANOSIM) with 999 permutations. In all analyses, it was assumed a p < 0.05 for statistically significant differences. An analysis of composition of microbiome (ANCOM) test was applied to assess differentially abundance genera using the q2-composition QIIME2 plugin [16]. A heatmap was performed by ggplot2 R package to visualize differences at genus-level composition between samples. Association between samples were established by the Bray–Curtis dissimilarity. Venn analysis was performed by Venn diagram software (available online at: http:// bioin forma tics. psb. ugent. be/ webto ols/ Venn/, accessed on 13 October 2023) to determine common and unique prokaryotic taxa on samples. A Canonical Correspondence Analysis (CCA) was performed to correlate environmental variables with prokaryotic taxa and samples. To make the CCA, PAST 4 software package [17] was used and a Monte Carlo test with 999 unrestricted permutations was carried. Functional Prediction ofProkaryotic Communities The functional potential of the microbial community was predicted using 16S rRNA gene abundance data via Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) [18]. The recommended maximum NSTI cut-off point of two was implemented by default in PICRUSt2, which excluded 1.3% of ASVs. Predicted pathways were categorized according to KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology groups and compared between samples. In addition, the functional annotation of prokaryotic taxa via Functional Annotation of Prokaryotic Taxa (FAPROTAX) was carried out (http:// www. ehbio. com/ Image GP/ index. php/ Home/ Index/ FAPRO TAX. html) [19]. Results Physico‑chemical Characteristics ofWaters The physico-chemical analysis of the water samples revealed that they could be classified into two distinct categories: salty and brackish (Table1). The table presents two sets of data on which the classification of the type of water is based: the first set comprises data obtained insitu at all sampling sites, while the second set comprises ion concentration data for sites belonging to the monitoring network. The values displayed for the latter dataset represent the mean value obtained from measurements conducted between 2017 and 2022. Detailed information is given in Supplementary TableS1. Monitoring showed that there is very little change in ion concentration over the years, thus adequately reflecting the stable character of the physico-chemical characteristics of the waters of the valley, which are considered stable over the years. They also maintain their flow rates (a total of about 3 L/s for the salty and 5 L/s for the brackish) fairly constant over time. Both salty and brackish water show a clear sodium chloride facies, a direct result of the dissolution of halite in the diapiric structure. However, it is important to note the high presence of sulfate and calcium, due to the dissolution of gypsum-anhydrite, which is comparatively higher in the brackish waters (Table1). The water taken from the S8 piezometer sampling site has a higher sulfate content than the other brackish water (Table1), due to its particular position in the flow scheme (Fig.1b). It is also worth noting the high concentration of magnesium and potassium in the salty Section 3. atala - APPENDIX I. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Prokaryotic Diversity andCommunity Distribution intheComplex Hydrogeological System of… Page 5 of 15 171 Table 1 Physico-chemical parameters of the water at the different sampling sites analyzed NA not analyzed, nd not detected *These data are obtained from the monitoring network in the valley that measures these parameters periodically. The data shown are the media from the data obtained during 2017–2022 Sampling sites Physico-chemical parameters Measured insitu Measured by ionic chromatography* Salinity (g/L) Conductivity T (°C) pH Cl− (mg/L) SO2− 4 (mg/L) NO− 3 (mg/L) Na+ (mg/L) Ca2+ (mg/L) Mg2+ (mg/L) K+ (mg/L) Salty Santa Engracia spring 200 Saturated 16 6.6 153,813 4697 nd 106,513 1860 286 519 Santa Engracia channel 200 NA 17 6.6 NA NA NA NA NA NA NA El Pico spring 205 Saturated 16 7.5 168,361 5493 nd 118,100 1823 300 567 El Cautivo spring 220 NA 17 6.6 142,721 4475 nd 100,710 1933 283 476 Hontana spring 210 NA 16 6.2 148,983 4778 nd 99,312 1751 266 464 Fuenterriba spring 200 NA 20 6.5 152,187 4669 nd 102,345 1845 281 580 Pond I 200 Saturated 18 7.8 NA NA NA NA NA NA NA Pond II 210 Saturated 27 7.5 NA NA NA NA NA NA NA Pond III 220 Saturated 29 7.6 NA NA NA NA NA NA NA Brackish San Juan stream 20 NA 13 7.4 5515 921 17 3853 391 65 19 El Pico Dulce spring 10 NA 13 7.3 5460 939 17 3812 396 62 19 S8 piezometer 4 NA 13 7.2 3060 2374 6 2380 673 193 21 Section 3. atala - APPENDIX I. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 M.Azpiazu-Muniozguren et al. 171 Page 6 of 15 waters (Table1). In addition, as expected, nitrates are only present in the shallower brackish waters (Table1). The waters of the five salty springs originate from very slow deep flows (hundreds of meters) and have a very high salinity (220–240g/L) and relatively high temperature (16–20°C). The two brackish waters springs have a much lower salinity (15–30g/L) and correspond to the mixture of deep waters with shallower currents. The pH of the brackish water is close to neutral (7.2 to 7.4) while the pH of the salty water is slightly lower (6.2 to 6.6). Prokaryotic Diversity andCommunity Composition A total of 583,454 sequences corresponding to 1801 different ASVs were obtained from 11 sampling sites. The data from the Hontana spring had to be discarded due to the low number of sequences obtained. Rarefaction curves were computed by rarefying each sample to the minimum frequency of sequences, which was 14,641 reads (Fig.S1). Diversity was calculated after normalization of the samples. Prokaryotic Diversity Alpha diversity analysis revealed that prokaryotic community richness (Observed ASVs and Chao1), quantitative community richness or diversity (Shannon and Simpson indexes), and community equality or evenness (Pielou’s evenness) varied widely among the samples (Table2). In particular, the highest prokaryotic richness, diversity, and evenness were observed in the El Pico Dulce spring, while the lowest richness was found in the El Pico spring. In contrast, the lowest diversity and evenness were found in the San Juan stream, with values similar to those of the El Pico spring. Pearson’s coefficient test showed a statistically significant negative correlation between salinity and ASVs, and also between salinity and Chao1 diversity indexes (p value < 0.05). Beta diversity identifies dissimilarities between the sampling sites. Dissimilarity in ASV composition was represented by the Principal Coordinate Analysis (PCoA) plot. The first three principal components explained 57.61% (PC1 + PC2 + PC3) of the total variation (Fig.2). For the analysis of multivariate homogeneity among groups, the analysis of similarities (ANOSIM) test showed significant differences in the prokaryotic diversity between groups based on their salinity (salty and brackish water) (p-value < 0.05). However, no statistically significant differences were observed when comparing samples according to their sampling site (spring, pond, stream, or groundwater). Assessment oftheProkaryotic Community Composition andIts Distribution The 1801 different ASVs were subsequently assigned to different taxonomic levels. Fifty-nine of the ASVs (3.3%) could not be assigned to any known phylum. The taxonomic assignment of each ASV is shown in Supplementary TableS2. The taxonomic assignment showed that the archaeal domain was essentially restricted to salty waters, whereas bacteria were present at all sampling sites, with the majority in brackish water (99.4 to 99.8%) (Fig.3a). The bacterial and archaeal domains were distributed in 31 phyla, 57 classes, 120 orders, 182 families, and 258 genera. The distribution of genera whose relative abundance was greater than 3% in at least one of the analyzed sites is shown in Fig.3b. It can be observed that the 60.7% of the ASVs were classified at genus level (6.3% of them as uncultured organisms), while 39.3% remained Unclassified. The major bacterial and archaeal phyla were Pseudomonadota and Halobacterota, respectively. At the genus level, Table 2 Alpha diversity indexes calculated for the locations of the saltern analyzed in the study ASVs amplicon sequence variants, Chao1 confidence interval for richness estimator Sampling site Observed ASVs Chao1 Simpson’s index Shannon’s index Pielou’s evenness Santa Engracia spring 104 104 0.95 5.07 0.76 Santa Engracia channel 196 197 0.96 5.56 0.73 El Pico spring 69 69 0.83 3.68 0.60 El Cautivo spring 169 169 0.96 5.50 0.74 Fuenterriba spring 294 301 0.93 5.48 0.67 Pond I 120 120 0.89 4.49 0.65 Pond II 74 74 0.86 3.88 0.62 Pond III 147 147 0.96 5.59 0.78 San Juan stream 155 158 0.83 3.50 0.48 El Pico Dulce spring 541 541 1.00 8.76 0.97 S8 piezometer 284 284 0.99 7.17 0.88 Section 3. atala - APPENDIX I. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Prokaryotic Diversity andCommunity Distribution intheComplex Hydrogeological System of… Page 7 of 15 171 the most representative bacterial genera (relative abundance > 1%) were Pseudomonas, Undibacterium, Salinibacter, Marinomonas, and Bacteroides. Similarly, the most abundant archaeal genera (relative abundance > 1%) were Halorubrum, Halonotius, Haloplanus, Halomicroarcula, and Natronomonas. Each of these genera varied greatly in abundance at different sampling sites (Fig.3b). Although some genera were found in more than one location, different compositional profiles can be defined at the genus level. Clustering using Bray–Curtis dissimilarity coefficient based on genus abundance confirmed that brackish waters showed greater similarity to one another, as did salty waters (Fig.4). ANCOM analysis identified the genera Natronomonas and Halorubrum as significantly more abundant genera in the salty water samples. Furthermore, when the analysis was performed according to the water salinity of the samples, only 25 out of 258 genera were shared (Fig.5a). However, 64 and 169 genera were unique to salty and brackish water, respectively. The distribution of genera between salty springs revealed 12 genera shared among these samples (Fig.5b); including Halomarina, Halonotius, Owenweeksia, Halohasta, Natronomonas, Flexistipes, Salinibacter, Halorubellus, Haloplanus, Halorubrum, Halobaculum, and Halomicroarcula. Halorubrum was the most abundant (8.6 to 61.2%) among them, except in the Santa Engracia spring, where Halohasta was the most abundant one. In the Fuenterriba spring, 22 genera were identified not present in the other saline springs. However, the genera found there were in the minority (Acinetobacter, 0.8%; Marinobacter, 0.4%; Enterococcus, 0.2%). The investigation of the possibility of groundwater contact between the ElPico and the ElPico Dulce springs, which are only two meters apart but have different physicochemical parameters (Fig.1 and Table1), revealed a different taxonomic composition. Of the 132 genera detected, the majority (n = 111) were only present at the ElPico Dulce spring and only two (Halomonas and Cellulosimicrobium) were shared (Fig.5c). Moreover, the relative abundance of both genera based on ASV taxonomic assignment was very low in both sampling sites, 0.05% and 0.71% and 0.19% and 0.54%, respectively. Fig. 2 UniFrac distance-based Jackknife clustering based on the ASVs data, with the first three principal coordinates (PCs) shown: unweighted UniFrac with PC1 (26.90%), PC2 (19.21%), and PC3 (11.50%) Section 3. atala - APPENDIX I. 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Turroni F, Ribbera A, Foroni E etal (2008) Human gut microbiota and bifidobacteria: from composition to functionality. Antonie Van Leeuwenhoek 94:35–50. https:// doi. org/ 10. 1007/ s104820089232-4 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Section 3. atala - APPENDIX I. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 APPENDIX I. ERANSKINA The supplementary figures and tables in APPENDIX I can be downloaded by scanning this QR code. I. ERANSKINeko irudi eta taula osagarriak QR kode hau eskaneatuta deskarga daitezke. Section 3. atala - APPENDIX I. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 3. atala - APPENDIX I. ERANSKINA APPENDIX II. ERANSKINA Fungal Diversity and Composition of the Continental Solar Saltern in Añana Salt Valley (Spain) Reference: Azpiazu-Muniozguren M, Perez A, Rementeria A, MartinezMalaxetxebarria I, Alonso R, Laorden L, Gamboa J, Bikandi J, Garaizar J, and Martinez-Ballesteros I. Fungal diversity and composition of the continental solar saltern in Añana Salt Valley (Spain). Journal of Fungi. 2021; 7(12):1074. Journal of Fungi (Basel) (2021); 7(12):1074 DOI: 10.3390/jof7121074 Impact factor 2021: 5.724 Relative position: 40/137, Q2 Microbiology 263 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Fungi Journal of Article Fungal Diversity and Composition of the Continental Solar Saltern in Añana Salt Valley (Spain) Maia Azpiazu-Muniozguren 1, Alba Perez 1, Aitor Rementeria 2, Irati Martinez-Malaxetxebarria 1, Rodrigo Alonso 1, Lorena Laorden 1, Javier Gamboa 3, Joseba Bikandi 1, Javier Garaizar 1 and Ilargi Martinez-Ballesteros 1,*   Citation: Azpiazu-Muniozguren, M.; Perez, A.; Rementeria, A.; Martinez-Malaxetxebarria, I.; Alonso, R.; Laorden, L.; Gamboa, J.; Bikandi, J.; Garaizar, J.; Martinez-Ballesteros, I. Fungal Diversity and Composition of the Continental Solar Saltern in Añana Salt Valley (Spain). J. Fungi 2021,7, 1074. https://doi.org/ 10.3390/jof7121074 Academic Editors: Nalin Wijayawardene and Nattawut Boonyuen Received: 26 October 2021 Accepted: 12 December 2021 Published: 14 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 MikroIker Research Group, Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain; [email protected] (M.A.-M.); [email protected] (A.P.); [email protected] (I.M.-M.); [email protected] (R.A.); lor[email protected] (L.L.); [email protected] (J.B.); javier[email protected] (J.G.) 2 Department of Immunology, Microbiology and Parasitology, Faculty of Science and Technology, University of the Basque Country UPV/EHU, Barrio Sarriena s/n, 48940 Leioa, Spain; aitor[email protected] 3Biogenetics, Portal de Zurbano 3, 6-B, 01013 Vitoria-Gasteiz, Spain; [email protected] *Correspondence: ilar[email protected]; Tel.: +34-945-013904 Abstract: The Añana Salt Valley in Spain is an active continental solar saltern formed 220 million years ago. To date, no fungal genomic studies of continental salterns have been published, although DNA metabarcoding has recently expanded researchers’ ability to study microbial community structures. Accordingly, the aim of this present study was to evaluate fungal diversity using the internal transcribed spacer (ITS) metabarcoding at different locations along the saltern (springs, ponds, and groundwater) to describe the fungal community of this saline environment. A total of 380 fungal genera were detected. The ubiquity of Saccharomyces was observed in the saltern, although other halotolerant and halophilic fungi like Wallemia,Cladosporium, and Trimmatostroma were also detected. Most of the fungi observed in the saltern were saprotrophs. The fungal distribution appeared to be influenced by surrounding conditions, such as the plant and soil contact, cereal fields, and vineyards of this agricultural region. Keywords: ITS; metabarcoding; fungi; biodiversity; continental saltern 1. Introduction Saline and hypersaline environments are widely distributed around the world. The study of these habitats is becoming more common, not only as a means to increase knowledge regarding the structure of their microbial communities but also to gain insights into the adaptations of these organisms to these unique locations. Among hypersaline environments, solar salterns are of particular interest. Different types of salterns exist that may be classified as littoral salterns and continental or inland salterns. Continental salterns are those that use brine obtained from spring sources, for the production of salt via evaporation and solar activity [1]. The Añana Salt Valley (30 km southwest of Vitoria-Gasteiz, Álava, Northern Spain; 42.80 N 2.98 W) is an active continental solar saltern formed about 220 million years ago following the evaporation of water from the great ocean (Tethys Ocean) that covered most of the Earth. This process led to the deposit of extensive layers of evaporites (salts, anhydrite, and gypsum), which, emanating from highly plastic stratigraphic levels and subjected to great pressure, rose through the sedimentary layers of the Earth’s crust, crossing and deforming these layers and resulting in the formation of a diapir. Rainwater crosses the upper stratum of the diapir rock, and then layers of salt appear on the surface decades later in the form of hypersaline or brackish springs [ 2 ]. Depending on the path the filtrated J. Fungi 2021,7, 1074. https://doi.org/10.3390/jof7121074 https://www.mdpi.com/journal/jof Section 3. atala - APPENDIX II. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 J. Fungi 2021,7, 1074 2 of 14 water takes through the rocks, it encounters areas of higher or lower salt content. For this reason, springs that are next to each other contain water with different levels of salinity. The hypersaline springs located in the Añana Salt Valley are water sources that supply brine at the surface level in a natural and continuous way without any need for drilling or for the use of pumps. Archaeological remains show that these water sources have been used by locals for 7000 years, for the extraction of salt [ 3 , 4 ]. Consequently, the physical, chemical, and anthropogenic characteristics of the Añana Salt Valley environment make it an exceptional setting for study and research. What is more, these salt flats contain deposits with important paleo-environmental and paleo-climatic information, in addition to the biodiversity of their extreme environments [3,4]. For many years, saline and hypersaline environments were thought to be populated almost exclusively by prokaryotic microorganisms. Recently, it has been shown that eukaryotic microorganisms are present too [ 5 – 7 ]. Studies regarding prokaryotic diversity and distribution have been conducted at different salterns using both culture-dependent and culture-independent methods [ 8 – 12 ]. Fewer studies have been conducted on the examination of fungal communities [ 13 – 15 ]. In the last few years, DNA metabarcoding has become an effective tool for the identification of yeast and other fungal species. Different studies have been conducted on soil, marine, and saline ecosystems; vineyards and wineproduction systems; and in glaciers [ 16 – 23 ], among other ecosystems. Although a study on prokaryotic and virus diversity in the Añana Salt Valley has been published [ 24 ], no fungal metabarcoding survey in this ecosystem has been published nor of other continental or inland salterns worldwide. The aim of this present study, therefore, was to evaluate fungal diversity in the extreme ecosystem of the Añana Salt Valley using metabarcoding to describe the composition of the fungi present in this location. 2. Materials and Methods 2.1. Sampling Sites and Proceedings Water samples from diverse sites around the Añana Salt Valley were collected to perform the fungal diversity study (Figure 1). Individual water samples from each of the seven sites were collected during the survey and studied for fungal diversity. Two of these sites, Santa Engracia (SE) Spring and Pico Spring, were springs containing high-salinity water. SE Spring is the main brine supplier for salt production in the saltern, with an average flow of 3 L/s and 200 g of salt/L. Pico Spring provides a lower brine flow to the system. The salt production brine is distributed by an interconnected wooden canal system, channeling the brine to a series of distribution ponds and crystallizing pans. Three of these ponds (Pond I, II, and III), containing high-salinity water and located along the salt-production system, were also selected for this study. In the valley, other springs with a lower-salinity water content arise naturally. From among these, brackish water from the Pico Dulce Spring was analyzed. Finally, brackish groundwater from an aquifer at 60 m depth, accessible through a piezometer called S8, was also analyzed. Water samples were collected in October 2017 (SE, Pico Spring, and ponds) and in June 2018 (S8 and Pico Dulce Spring). Section 3. atala - APPENDIX II. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 J. Fungi 2021,7, 1074 3 of 14 Figure 1. Locations along the Añana Salt Valley studied for fungal diversity and composition determination (photograph obtained from Visor geoEuskadi at https://www.geo.euskadi.eus/s69bisorea/es/x72aGeoeuskadiWAR/index.jsp, accessed on 26 July 2021). The exact coordinates of each location are as follows: Pond I, 42.8014 N 2.9860 W; Pond II, 42.8009 N 2.9852 W; Pond III, 42.8006 N 2.9851 W; Pico Dulce, 42.7989 N 2.9858 W; Pico, 42.7986 N 2.9856 W; Santa Engracia, 42.7965 N 2.9863 W; S8 piezometer, 42.7970 N 2.9838 W. Water samples were directly obtained using sterile glass bottles or a Niskin bottle (Aquatic BioTechnology, El Puerto de Santa María, Spain). Groundwater from the S8 piezometer was taken at a 60 m depth using a manual bailer system (Eijkelkamp, Giesbeek, The Nederlands). Physicochemical parameters, such as water temperature, pH, NaCl concentration, and conductivity (related to salt content), were determined in situ (Combo tester, Hanna Instruments, Eibar, Spain). Water samples were taken directly to the laboratory for processing. 2.2. DNA Extraction and Sequencing Samples were enriched by filtering approximately 5 L of water in aseptic conditions through a tailored device, driven by vacuum/compression pumps, which concentrated the samples via a series of decreasing flow-rate polycarbonate membranes (Labbox, Premia de Dalt, Spain), ending in a 0.2 µ m pore size filter. DNA extraction and quantification of the samples were conducted according to the manufacturer’s instructions via the Genomic Mini AX Yeast Kit (A&A Biotechnology, Gdansk, Poland) and the QuantiFluor dsDNA System (Promega, Madison, WI, USA), respectively. The fungal internal transcribed spacer 1 (ITS1) region was amplified, as suggested in the ITS Metagenomics Protocol of Illumina by modified ITS1-F and ITS2 primers set [ 25 ]. The library was prepared by a Nextera DNA Library prep kit (Ilumina, San Diego, CA, USA) according to that protocol. Highthroughput sequencing (HTS) was performed on an Illumina MiSeq platform, which generates paired-end sequences in FASTQ format. The nucleotide sequence data from the study are available in the DDBJ/EMBL/GenBank databases under the accession number PRJNA749727. 2.3. Sequencing Data and Statistical Analysis Most of the analyses was carried out using QIIME2 version 2021.2 software tools and pipelines [ 26 ]. Joining of paired-end reads, sequence quality control, and feature table construction were performed by denoising with DADA2 plugin (q2-dada2) [ 27 ]. During this step, sequence denoising, dereplication, and chimera filtering were undertaken. Sequences were trimmed at a 200 bp length. Amplicon sequence variants (ASVs) [ 28 ] Section 3. atala - APPENDIX II. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 J. Fungi 2021,7, 1074 4 of 14 generated by DADA2 were assigned taxonomically using the q2-feature-classifier [ 29 ] classify-sklearn naïve Bayes taxonomy classifier with the UNITE 8.3 dynamic database [ 30 ] at 97% similarity level. Manually searching of unidentified ASVs was performed by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 6 October 2021). Alpha and beta diversity metrics were estimated using q2-diversity plunging after rarefaction of the samples to 2270 sequences per sample. Alpha diversity estimators, such as observed ASVs and Chao1 index, were determined to establish the species richness at each point. To study the diversity of the species in the samples, α estimators, such as Shannon and Simpson indexes, were used: the higher the Shannon index, the greater the diversity, and the closer the Simpson index is to 0, the lower the diversity. Pielou’s evenness, which quantifies how equal a community is, was also determined (the closer to 0, the higher the dominance of a species). Statistical significance of the alpha diversity values between sampling sites was assessed using the Kruskal–Wallis H test. A pvalue < 0.05 was considered statistically significant. To establish the correlation of diversity indexes with physicochemical characteristics, Pearson’s coefficient test was performed (pvalue < 0.05 was considered statistically significant). Dissimilarity between sample groups was also examined by the Bray–Curtis coefficient and differences among groups were assessed by ANOSIM with 999 permutations (p< 0.05). A heatmap was performed by ggplot2 R package to visualize differences at the genus level composition among samples. Associations between samples were established by the Bray–Curtis dissimilarity. Venn analysis was performed by Venn diagram software (available online at: http://bioinformatics.psb.ugent.be/webtools/Venn/, accessed on 21 July 2021) to determine common and unique fungal taxa on samples. The FUNGuild database was used to assign ecological guilds within three trophic modes (i.e., pathotroph, symbiotroph, and saprotroph) to all ASVs [ 31 ]. Only fungal ASVs with probable and highly probable confidence matches were considered for further analysis. 3. Results 3.1. Water Physicochemical Data The physicochemical characteristics of water samples measured in situ are shown in Table S1. Two different degrees of salinity were observed, which were used to establish groups for further analysis: SE Spring, Pico Spring, and the three brine ponds were classified as salty water, and Pico Dulce Spring and S8 piezometer were classified as brackish water due to their lower salt concentration. 3.2. Sequence Analysis and ASV Determination The fungal diversity and community structure in the water samples taken from representative sites in the Añana Salt Valley were investigated by HTS (Figure 1). After denoising and quality control of sequenced DNA, a mean of 132,520 reads per sample was obtained. A total of 704,874 sequences were obtained corresponding to 2204 different ASVs determined by DADA2. These ASVs were subsequently assigned at different taxonomy levels for fungal identification. Among the ASVs, 853 failed to be identified at any known phylum (38.7% of the total). 3.3. Fungal Diversity in the Saltern Rarefaction curves were computed by rarefying each sample to the minimum number of sequences (2270 reads). Rarefaction curves were not parallel for all the samples analyzed but started to flatten (Figure S1). The alpha diversity was calculated after normalization of the samples. Table 1shows the taxa richness and diversity values obtained by the different alpha diversity estimators. SE Spring had the lowest diversity. There, the smallest number of ASVs (71 ASVs) was detected and the Shannon index was 3.59 (ranging from 4.89–6.47 for the rest of the locations). The other six locations demonstrated a similar diversity, except for S8, which was less diverse. While 93 ASVs and a Shannon index of 4.89 were obtained at S8, the observed ASVs from the other five locations ranged from 262–396 and the Shannon index ranged from 5.44–6.47. Pielou’s evenness of SE was 0.58 (the closest value to 0 in the Section 3. atala - APPENDIX II. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 J. Fungi 2021,7, 1074 5 of 14 study), which indicated the presence of a dominant taxon (presumably Saccharomyces). No statistically significant differences were observed in the alpha diversity indexes between sample sites. Table 1. Alpha diversity indexes calculated for the locations of the saltern analyzed in the study. Sample Site Observed ASVs Chao1 Shannon Simpson Pielou’s Evenness Santa Engracia Spring 71 116 3.59 0.77 0.58 Pico Spring 354 633 6.27 0.95 0.74 Pond I 264 452 5.44 0.93 0.67 Pond II 396 699 6.47 0.94 0.75 Pond III 263 337 6.07 0.94 0.75 Pico Dulce Spring 262 430 6.15 0.96 0.76 S8 93 93 4.89 0.91 0.74 ASVs, amplicon sequence variants. The diversity and pH showed a statistically significant correlation based on Pearson’s coefficient test (Table 2), indicating that environmental diversity decreases as the pH of the water decreases too. No correlation was observed in regard to the NaCl concentration or temperature. Table 2. Correlation between water physicochemical parameters and fungal diversity. The analysis was carried out using Pearson’s coefficient test. Parameter Pearson’s Coefficient Test (pValues) Shannon Index Simpson Index NaCl 0.896 0.732 Temperature 0.314 0.262 pH 0.029 * 0.015 * * statistically significant correlation based on p-value (p< 0.05). The ANOSIM test based on Bray–Curtis dissimilarity identified differences in the fungal diversity among groups based on their salinity (salty and brackish water) ( p-value 0.046 ). No differences were observed regarding different water types (spring, pond, or groundwater). 3.4. Fungal Taxonomic Assignment and Community Composition The taxonomic assignment of ASVs showed that Basidiomycota and Ascomycota were the phyla present in the saltern (0.18–63.6% and 23–81.1%, respectively) (Figure 2). No other fungal phylum was detected in the samples. As referenced above, 38.7% of the ASVs were classified only as fungi. The manual identification of unidentified ASVs by BLAST search did not improve these results, so a low taxonomic assignment to the species level was obtained in the study. The taxonomic assignment of each ASV can be found in Table S2 . A total of 380 fungal genera were detected in the survey (Table S3). In Figure 2, genera whose relative abundance was more than 3% in at least one of the locations analyzed at the saltern are shown. Regarding SE Spring, 30 ASVs were taxonomically assigned to genus level; 184 with respect to Pico Spring; 147, 196, and 110 genera with respect to Pond I, II, and III, respectively; 81 at Pico Dulce Spring; and 28 genera at S8. Section 3. atala - APPENDIX II. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 J. Fungi 2021,7, 1074 12 of 14 References 1. Hueso, K.; Carrasco, J.F. Las salinas de interior, un patrimonio desconocido y amenazado. Def. Patrim. Geológico Min. 2006 ,6–7, 23–28. 2. Murelaga, X.; Zuluaga, M.C.; Alonso, A.; Baceta, J.I.; Ortega, L.O. 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ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 3. atala - APPENDIX III. ERANSKINA APPENDIX III. ERANSKINA Altererythrobacter muriae sp. nov., isolated from hypersaline Añana Salt Valley spring water, a continental thalassohaline-type solar saltern Reference: Azpiazu-Muniozguren M, Martinez-Ballesteros I, Gamboa J, Seoane S, Alonso R, Laorden L, Garaizar J, and Bikandi J. Altererythrobacter muriae sp. nov., isolated from hypersaline Añana Salt Valley spring water, a continental thalassohaline-type solar saltern. International Journal of Systematic and Evolutionary Microbiology. 2021; 71(3):004734. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY (2021); 71(3) DOI: 10.1099/ijsem.0.004734 Impact factor 2021: 2.689 Relative position: 106/137, Q4 Microbiology 281 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 1 Altererythrobacter muriae sp. nov., isolated from hypersaline Añana Salt Valley spring water, a continental thalassohalinetype solarsaltern MaiaAzpiazuMuniozguren1, IlargiMartinezBallesteros1,*, JavierGamboa2, SergioSeoane3, RodrigoAlonso1, LorenaLaorden1, JavierGaraizar1 and JosebaBikandi1 TAXONOMIC DESCRIPTION Azpiazu-Muniozguren etal., Int. J. Syst. Evol. Microbiol. 2021;71:004734 DOI 10.1099/ijsem.0.004734 Author affiliations: 1MikroIker Research Group. Immunology, Microbiology and Parasitology Department, Farmacy Faculty, University of the Basque Country UPV/EHU, VitoriaGasteiz, Álava, Spain; 2Biogenetics, VitoriaGasteiz, Álava, Spain; 3Plant Biology and Ecology Department, Faculty of Science and Technology, University of the Basque Country UPV/EHU, Leioa, Spain. *Correspondence: Ilargi MartinezBallesteros, ilargi. martinez@ ehu.eus Keywords: Altererythrobacter muriae; saltern; halophile; poliphasic taxonomy. Abbreviations: AAI, average aminoacid identity; ANI, average nucleotide identity; dDDH, digital DDH; DDH, DNA–DNA hybridization; MA, marine agar; MB, marine broth; OGRI, overall genome related index. The Genbank accession number for the 16S rRNA gene sequence of strain SALINAS58T is MN918268. The Whole Genome Shotgun sequence was deposited at DDBJ/ENA/GenBank under the accession JAAFZT000000000. Six supplementary figures and one supplementary table are available with the online version of this article. 004734 © 2021 The Authors Abstract A novel salttolerant alphaproteobacterium, designated SALINAS58T, was isolated from Santa Engracia hypersaline spring water in the Añana Salt Valley, Álava, Spain. The isolate was Gramnegative, aerobic, nonmotile, catalasepositive, oxidasenegative, rodshaped and formed orange colonies on marine agar. Optimal growth was observed at pH 6.0–6.5, at 30 °C and in the presence of 1% (w/v) NaCl. The main cellular fatty acids (>20%) were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The major respiratory quinone was ubiquinone Q-10 and the major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidilglycerol, four unidentified glycolipids and one unidentified phospholipid. Strain SALINAS58T had the highest 16S rRNA gene sequence similarity to Altererythrobacter marensis MSW-14T (96.6%), Altererythrobacter aquaemixtae JSSK-8T (96.5%) and Pontixanthobacter luteolus SW-109T (96.5%) followed by Altererythrobacter atlanticus 26DY36T (96.4%). Results of the phylogenetic analysis, based on 16S rRNA gene sequences, and phylogenetic approaches based on whole genome nucleotide differences, showed that strain SALINAS58T could be distinguished from recognized species of the genus Altererythrobacter. The genomic DNA G+C content was 61.4 mol%. Digital DNA–DNA hybridization, average nucleotide identity and average aminoacid identity values between the genome of strain SALINAS58T and A. marensis MSW-14T were 18.4, 73.1 and 68.1%, respectively. Based on data from this polyphasic characterization, strain SALINAS58T (=CECT 30029T=LMG 31726T) is considered to be classified as representing a novel species in the genus Altererythrobacter, for which the name Altererythrobacter muriae sp. nov. is proposed. Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Altererythrobacter falls within the family Erythrobacteraceae [1]. The recent genomicbased taxonomic classification of the family Erythrobacteraceae reclassifies all taxons into 16 genera, including 11 novel ones [2]. The genus Altererythrobacter was proposed in 2007 by Kwon et al. through the description of a single species, Altererythrobacter epoxidivorans (type species) [1, 3, 4]. The genus Altererythrobacter comprises, at the time of writing, 44 validly published species, including synonyms (https:// lpsn.dsmz. de/). The characteristic features of this genus include Gramnegative, aerobic growth, nonmotile, generally rodshaped cells, oxidaseand catalasepositive and Q-10 as dominant respiratory quinone with the absence of bacteriochlorophyll a (BChl a) [2, 3, 5]. Members of the genus require NaCl for growth and they can be characterized by yellow to orangered colony colours on agar plates. The temperature range for optimal growth is 15–35 °C [2–4]. The dominant fatty acid (>10%) is C18 : 1 ω7c and the major polar lipids are phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid. The DNA G+C content is 52.0–61.8 mol% [2]. The habitat range of the genus Altererythrobacter includes mainly aquatic environments, frequently seawater [6, 7]. However, there are also isolation reports from diverse Section 3. atala - APPENDIX III. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 2 Azpiazu-Muniozguren etal., Int. J. Syst. Evol. Microbiol. 2021;71:004734 environments such as coldseep sediment [1], deepsea sediment [8], lagoon sediments [9], mangrove sediment [10], estuary environment water [11, 12], the junction between the ocean and a freshwater spring [13], salt marsh plant [14], sea urchin [15], hot spring [16], air [5] and desert soil [17]. Altererythrobacter has also been recovered from polycyclic aromatic hydrocarbons and crude oil degrading consortia enriched from marine sediments [4]. In this work, we describe strain SALINAS58T isolated from hypersaline spring water collected in the Añana Salt Valley, Spain. The phenotipic analyses, the comparative 16S rRNA gene sequence analysis and the genomic analysis indicated that strain SALINAS58 T is affiliated phylogenetically to the genus Altererythrobacter. Therefore, according to the results, the species Altererythrobacter muriae sp. nov. is proposed. ISOLATION AND ECOLOGY Strain SALINAS58T was isolated from a hypersaline water sample collected from the Santa Engracia natural spring in the Añana Salt Valley (Spain; Fig. S1, available in the online version of this article), an active continental thalassohalinetype solar saltern declared as a Globally Important Agricultural Heritage System by the Food and Agriculture Organization of the United Nations (FAO). The hypersalinity of the water sources in this valley is explained by the phenomenon known as diapir, whose origin lies in marine water from the Triassic period. The Santa Engracia spring is located at 598 m above sea level and the approximate geographic coordinates are 42.79665° N 2.98602° W. Water samples were collected in May 2017. Water temperature was 16.0 °C, pH 6.6 and salinity 200 g l−1, and the conductivity was saturated. The strain was isolated by multiple filtration steps (Whatman Grade 113V, and Millipore 5 and 0.22 µm filters), then 100 µl of the filtered out water was cultivated at 25 °C on marine agar (MA; Conda). After 20 days of incubation, a visible orangecoloured colony was transferred and subcultured on MA medium until a pure culture was obtained and cultivated routinely at 25 °C on MA. Strain SALINAS58T was maintained at −80 °C in marine broth (MB; Conda) and 25% (v/v) glycerol. 16S rRNA GENE PHYLOGENY After incubation at 25 °C for 3 days, genomic DNA was extracted from strain SALINAS58T using PrepMan Ultra DNA extraction reagent (Applied Biosystems), according to the manufacturer’s instructions. The 16S rRNA gene was amplified by PCR with the primers 27F (5′- AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [18]. The amplicons were purified and Sanger sequencing was performed (Stab Vida). Sequence data were aligned and analysed by Chromas 2.6.6 software (Technelysium). The 16S rRNA gene sequence (1296 bp) was deposited at Genbank under accession number MN918268. Due to the short 16S rRNA gene sequence obtained, the 1436 bp 16S rRNA sequence from the genome sequencing was used for further phylogenetic analysis (accession number JAAFZT000000000; NODE 27). Identification of closely related species was carried out using the EzBioCloud website (www.ezbiocloud.net/identify) [19]. The 16S rRNA gene sequence indicated that strain SALINAS58T belongs to the genus Altererythrobacter. The 16S rRNA gene sequence analysis of strain SALINAS58T showed the highest similarity to Altererythrobacter marensis MSW-14T (96.6%), followed by Altererythrobacter aquaemixtae JSSK-8 T (96.5%), Pontixanthobacter luteolus SW-109T (96.5%) and Altererythrobacter atlanticus 26DY36T (96.4%). The 16S rRNA gene sequence similarities among strain SALINAS58 T and other related species were lower than 98.7%, the threshold proposed by Chun et al. [20] for differentiating two species. The 16S rRNA gene sequences of representatives of the genus Altererythrobacter were retrieved from the GenBank database and were aligned by ClustalW 2.0.12 (Kyoto University Bioinformatics Centre). Phylogenetic trees were reconstructed by mega X software [21] using the neighbourjoining [22], minimumevolution [23] and maximumlikelihood [24] methods. Evolutionary distances for the neighbourjoining analysis were calculated using the algorithm of Kimura’s twoparameter model with bootstrap values based on 1000 replications [25]. The neighbourjoining phylogenetic analysis based on 16S rRNA gene sequence, showed that strain SALINAS58 T formed a distinct branch within the genus Altererythrobacter, albeit not supported by a high bootstrap value (Fig.1). Similar results were observed in both the minimumevolution and the maximumlikelihood trees (Figs S2 and S3). GENOME FEATURES Whole genome sequencing of strain SALINAS58T was performed using the Illumina Miseq platform at SGIker General Services of the University of the Basque Country UPV/EHU (Leioa, Spain). The DNA for genome sequencing was obtained by using the Nucleo Spin Tissue DNA extraction kit (MachereyNagel) according to manufacturer’s protocol. De novo assembly from raw pairedend reads to contigs was performed using Velvet 1.1.04 software [26]. The authenticity was confirmed by the presence of the 16S rRNA gene sequence obtained by PCR on the assembled genome, and the contamination of the genome sequence was checked by GenomeQC platform (https:// genomeqc. maizegdb.org/) [27]. Genome sequences were annotated by the NCBI Prokaryotic Genome Annotation Pipeline and by the Rapid Annotation with Subsystems Technology (RAST) [28]. Whole genome sequencing data were deposited at DDBJ/ENA/GenBank under the accession number JAAFZT000000000. The genome size of strain SALINAS58T was 2.8 Mbp and was assembled into 56 contigs, with an N50 value of 1.9 Mbp. The DNA G+C content of strain SALINAS58T was 61.4%, within those of the genus Altererythrobacter (54.5–69.0%) [5, 17]. The combination of 16S rRNA sequence similarity and the overall genome related index (OGRI) can be used systematically to identify and recognize bacterial strains at the level of species. Among the OGRIs, the average nucleotide identity (ANI) value has been most widely used for species delineation. Section 3. atala - APPENDIX III. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 3 Azpiazu-Muniozguren etal., Int. J. Syst. Evol. Microbiol. 2021;71:004734 Altererythrobacter lutipelagi GH1-16T (LT797153) 88 Altererythrobacter aerophilus Ery1T (MG183676) Altererythrobacter rhizovicinus AY-3RT (MH611372) Altererythrobacter xinjiangensis S3-63T (HM028673) Altererythrobacter soli MN-1T (KT906300) 73 91 90 Altererythrobacter aerius 100921-2T (KU311004) Altererythrobacter rigui WW3T (KP997219) Altererythrobacter deserti THG-S3T (KY287245) Altererythrobacter fulvus S-54T (KY117470) Altererythrobacter amylolyticus NS1T (KX601069) 87 Altererythrobacter troitsensis KMM 6042T (AY676115) 86 Tsuneonella dongtanensis JM27T (GU166344) 80 Altererythrobacter flavus MS1-4T (KX099616) 57 Altererythrobacter mangrovi C9-11T (MF034045) Altererythrobacter oceanensis Y2T (KF924606) Altererythrobacter xiamenensis LY02T (KC520828) Altererythrobacter epoxidivorans JCS350T (DQ304436) Altererythrobacterinsulae BPTF-M16T (MH206217) Altererythrobacter ishigakiensis NITE-AP48T (AB363004) 89 Pelagerythrobacter marinus H32T (EU726272) 62 Altererythrobacter marensis MSW-14T (FM177586) Altererythrobacter muriae SALINAS58T (JAAFZT000000000) 87 Altererythrobacter aquaemixtae JSSK-8T (KY614064) Altererythrobacter aestiaquae HDW-31T (KJ658262) Altererythrobacter gangjinensis KJ7T (JF751048) 96 55 82 Altererythrobacter aquiaggeris KEM-3T (KX812543) Altererythrobacter confluentis KEM-4T (KX129915) 99 Altererythrobacter sediminis CAU 1172T (KP779619) Pontixanthobacter luteolus SW-109T (AY739662) 66 Altererythrobacter spongiae HN-Y73T (MG437235) Altericroceibacterium endophyticum BR-75T (KY310591) 84 Altericroceibacterium indicum MSSRF26T (DQ399262) 83 Altererythrobacter atlanticus 26DY36T (KC018454) Altererythrobacter xixiisoli S36T (KJ150597) 89 Altererythrobacter salegens XY-R17T (KT886062) Alteraurantiacibacter buctensis M0322T (KJ599648) 88 Alteraurantiacibacter aquimixticola SSKS-13T (MK194299) 83 51 Altererythrobacter lauratis YIM 75003T (KX808673) 100 Altererythrobacter palmitatis YIM 75004T (KX808674) 54 Altererythrobacter halimionae CPA-5T (KY310593) Alteraurantiacibacter aestuarii KYW147T (FJ997597) Paraurantiacibacter namhicola KYW48T (FJ935793) Parapontixanthobacter aurantiacus O30T (KF924607) 99 Alteripontixanthobacter maritimus HME9302T (KF385494) Erythrobacter spongiae HN-E23T (MG655147) 97 Erythrobacter atlanticuss 21-N3T (KP994305) Erythrobacter longus JCM 6170T (D12699) 91 Erythrobacter citreus RE35F/1T (AF118020) Paracoccus pacificus F14 T (KF924610) 0.020 Fig. 1. Neighbourjoining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between strain SALINAS58T, the type strains of Altererythrobacter species and representatives of some other related taxa. Only bootstrap values ≥50% (expressed as percentages of 1000 replications) are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in both the minimumevolution and maximumlikelihood trees. Paracoccus pacificus F14T (GenBank accession number KF924610) was used as the outgroup. Bar, 0.02 substitutions per nucleotide position. Section 3. atala - APPENDIX III. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 APPENDIX III. ERANSKINA The supplementary figures and tables in APPENDIX III can be downloaded by scanning this QR code. III. ERANSKINeko irudi eta taula osagarriak QR kode hau eskaneatuta deskarga daitezke. Section 3. atala - APPENDIX III. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 3. atala - APPENDIX VI. ERANSKINA APPENDIX IV. ERANSKINA Anianabacter salinae gen. nov., sp. nov. ASV31T, a Facultative Alkaliphilic and Extremely Halotolerant Bacterium Isolated from Brine of a Millennial Continental Saltern Reference: Azpiazu-Muniozguren M, García M, Laorden Muñoz L, Martínez Malax-Echevarría I, Seoane Parra S, Bikandi Bikandi J, Garaizar J, and Martínez-Ballesteros I. Anianabacter salinae gen. nov., sp. nov. ASV31T, a Facultative Alkaliphilic and Extremely Halotolerant Bacterium Isolated from Brine of a Millennial Continental Saltern. 2022; 14(11):1009. Diversity (2022); 14(11) DOI: 10.3390/d14111009 Impact factor 2022: 2.4 Relative position: 26/65, Q2 Biodiversity conservation 293 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Citation: Azpiazu-Muniozguren, M.; García, M.; Laorden, L.; Martinez-Malaxetxebarria, I.; Seoane, S.; Bikandi, J.; Garaizar, J.; Martínez-Ballesteros, I. Anianabacter salinae gen. nov., sp. nov. ASV31T, a Facultative Alkaliphilic and Extremely Halotolerant Bacterium Isolated from Brine of a Millennial Continental Saltern. Diversity 2022, 14, 1009. https://doi.org/10.3390/ d14111009 Academic Editors: Michael Wink and Stuart Donachie Received: 25 September 2022 Accepted: 18 November 2022 Published: 21 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). diversity Article Anianabacter salinae gen. nov., sp. nov. ASV31T, a Facultative Alkaliphilic and Extremely Halotolerant Bacterium Isolated from Brine of a Millennial Continental Saltern Maia Azpiazu-Muniozguren 1, Minerva García1, Lorena Laorden 1,2 , Irati Martinez-Malaxetxebarria 1,2 , Sergio Seoane 3, Joseba Bikandi 1, Javier Garaizar 1,2 and Ilargi Martínez-Ballesteros 1,2,* 1 MikroIker Research Group, Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Álava, Spain 2Bioaraba, Microbiology, Infectious Disease, Antimicrobial Agents and Gene Therapy, 01006 Vitoria-Gasteiz, Álava, Spain 3 Plant Biology and Ecology Department, Faculty of Science and Technology, University of the Basque Country UPV/EHU, Barrio Sarriena s/n, 48940 Leioa, Bizkaia, Spain *Correspondence: ilar[email protected] Abstract: During a prokaryotic diversity study in Añana Salt Valley, a new Rhodobacteraceae member, designated ASV31 T , was isolated from Santa Engracia spring water. It was extremely halotolerant, tolerating up to 23% NaCl, and facultatively alkaliphilic, growing at pH 6.5–9.5 (optimum at 7.0–9.5). The isolate was a Gram-negative, rod-shaped, aerobic and non-motile bacterium that formed beigeto-pink colonies on marine agar. According to a 16S rRNA gene-based phylogenetic analysis, strain ASV31 T forms a distinct branch of the family Rhodobacteraceae, with Thioclava pacifica DSM 10166 T being its closest type strain (95.3%). This was confirmed with a phylogenomic tree and the values of ANI (73.9%), dDDH (19.3%), AAI (63.5%) and POCP (56.0%), which were below the genus/species level boundary. Additionally, an ability to degrade aromatic compounds and biosynthesise secondary metabolites was suggested by the genome of strain ASV31 T . Distinguishing fatty acid profiles and polar lipid content were also observed. The genome size was 3.6 Mbp, with a DNA G+C content of 65.7%. Based on the data obtained, it was considered that strain ASV31 T ( =CECT 30309T= LMG 32242T ) represents a new species of a new genus in the family Rhodobacteraceae, for which the name Anianabacter salinae gen. nov., sp. nov. is proposed. Keywords: continental saltern; extremely halotolerant bacteria; alkaliphilic; Anianabacter salinae; biotechnological applications 1. Introduction Solar salterns are extreme habitats in which halophilic microorganisms with different adaptations to such environments are found. For this reason, the study of microbial communities of these environments is important, not only to increase knowledge around their diversity, but also because the metabolites produced by members of these communities could be interesting for different biotechnological applications. An active continental solar saltern, located in Añana Salt Valley (Álava, Northern Spain, 42.82 N 2.98 W), was recently declared a Globally Important Agricultural Heritage System by the Food and Agriculture Organization of the United Nations (FAO). Its origin lies in the marine water of the Triassic period (between 251 and 208 million years ago), when a diapir was formed by the deposit of evaporite layers that rose through the Earth’s surface, resulting in the hypersalinity of the water that is now found in this valley [ 1 ]. During a study of the microbial diversity of this saltern, a beigeto pink-pigmented bacterial strain from the Santa Engracia spring (the main brine support for the salt production in the saltern) was isolated. The isolate was designated ASV31 T , which represented a potential novel species of a novel genus in the Rhodobacteraceae family. Diversity 2022,14, 1009. https://doi.org/10.3390/d14111009 https://www.mdpi.com/journal/diversity Section 3. atala - APPENDIX VI. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Diversity 2022,14, 1009 2 of 17 The family Rhodobacteraceae, belonging to the order Rhodobacterales, class Alphaproteobacteria, was first proposed in 2005 by Garrity et al. [ 2 ] and was amended by Hördt et al. in 2020 [ 3 ]. This family is a large phenotypically, metabolically and ecologically diverse taxonomic group. Most members of the family originated from aquatic environments and many of them require sodium ions or combined salts for growth [ 4 ]. At the time of writing, the family Rhodobacteraceae consists of 227 validly published genera, including synonyms (https://lpsn.dsmz.de/family/rhodobacteraceae, accessed on 20 January 2022) which are divided into six different phylogenetic subgroups: Stappia,Amaricoccus,Paracoccus,Rhodobacter,Rhodovulum and Roseobacter [ 5 ]. The latter has been proposed to be split from the Rhodobacteraceae family to form a new one, the Roseobacteraceae family [ 6 ]. In general, members of the family Rhodobacteraceae are Gram-negative and multiply by binary fission or budding, following monopolar growth. While they are mainly aerobic photoand chemoheterotrophs, purple non-sulphur bacteria that perform photosynthesis in anaerobic environments are also found. They are characterised chemotaxonomically by the presence of ubiquinone-10 (Q-10) and C18:1 ω 7c is the predominant cellular fatty acid. They are considered to be the major group of marine heterotrophic bacteria and have different roles such as contributing to sulphur, nitrogen and carbon cycles, decomposing various compounds and generating secondary metabolites [7,8]. Members of the Rhodobacteraceae family could be utilised for industrial applications. Exopolysaccharide (EPS)-producing species, such as Palleronia marisminoris [ 7 ], can be found in this family. Microbial EPSs can be used in the food industry as viscosity agents, stabilisers, emulsifiers, gelling agents and water-binding agents. Moreover, in the medical industry, microbial EPSs are emerging as promising materials for drug-release systems, due to their ability to retain large amounts of water and remain insoluble, as well as drugtargeting carriers, based on their particular binding and penetrating features to cellular receptors [ 9 ]. Other genera, such as Methylarcula, are able to synthesise ectoine as a major compatible solute [ 5 ]. Ectoine is commercially available as a protectant of proteins, DNA and mammalian cells [ 10 ]. Others, such as Palleronia,Salipiger and Tranquillimonas, can synthesise polyhydroxyalkanoates (PHAs), or, more commonly, polyhydroxybutyrate (PHB), as a carbon reserve material [ 5 ]. PHAs are a family of biodegradable and biocompatible polyesters accumulated by many microorganisms and are developed for industrial usage (e.g., as bioplastics, biofuels and fine chemicals) or for medicinal use [ 10 ]. Furthermore, extremophilic carbohydrate-active enzymes (CAZymes) have recently garnered a lot of attention due to their advantages [ 11 ]. In this context, a genomic annotation allows for the identification of various metabolic pathways either directly involved or not in the synthesis of these metabolites of industrial interest. Using a polyphasic approach, the aim of this study was to characterise the strain ASV31 T (isolated from the brine of a saltern) and to determine its taxonomic position within the family Rhodobacteraceae. 2. Materials and Methods 2.1. Strain Isolation and Maintenance Strain ASV31 T was isolated from the Santa Engracia natural spring in the continental saltern at Añana Salt Valley, located 598 m above sea level (the approximate geographic coordinates are 42.79 N 2.98 W). Water samples were collected in June 2016. The water temperature was 16.0 ◦ C, pH 6.6 and salt content 20%. In order to isolate halophilic and halotolerant bacteria, the water samples were first processed through multiple filtration steps (Whatman ® Grade 113 V, Maidstone, UK, and Millipore 5 µ m and 0.22 µ m filters (Burlington, MA, USA)). Subsequently, 100 µ L of filtered water was cultivated on marine agar (MA; Conda) and incubated at 25 ◦ C. After 7 days of incubation, a visible beige-to-pink colony was observed and subcultured on MA, until pure culture was obtained. Strain ASV31Twas stored at −80 ◦C in marine broth (MB; Conda) and 25% (v/v) glycerol. Section 3. atala - APPENDIX VI. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Diversity 2022,14, 1009 3 of 17 2.2. 16S rRNA Gene-Based Identification and Phylogeny Strain ASV31 T chromosomal DNA was extracted from three-day colonies on MA using PrepMan Ultra Reactive (Applied Biosystems, Waltham, MA, USA) following the manufacturer’s specifications. The DNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). 16S rRNA gene sequencing was used for strain identification. The gene was amplified using the universal bacterial primers 27F (5 0 -AGAGTTTGATCMTGGCTCAG-3 0 ) and 1492R ( 50-GGTTACCTTGTTACGACTT-30 ) [ 12 ], and was, subsequently, purified by a NucleoSpin Gel and PCR clean-up kit (MachereyNagel, Düren, Germany). Purified PCR products were sequenced using the Sanger method (Stab Vida, Caparica, Portugal) and the sequencing data were analysed with Chromas 2.6.6 software (Technelysium, South Brisbane, Australia). Identification of closely related species and the measurement of their 16S rRNA sequence similarity were carried out using the EzBioCloud database (www.ezbiocloud.net/identify, accessed on 15 June 2021) [13]. The 16S rRNA gene sequences of representatives of the family Rhodobacteraceae were retrieved from the GenBank database and a phylogenetic tree was constructed with MEGA X software [14] using the neighbour-joining [15], minimum-evolution [16] and maximumlikelihood [ 17 ] methods. Evolutionary distance matrices were calculated using the algorithm of Kimura’s two-parameter model with bootstrap values based on 1000 replications [17]. 2.3. Phenotype and Chemotaxonomy Type strains Thioclava pacifica DSM 10166 T and Rhodovulum algae LMG 29228 T were tested alongside ASV31 T for comparison. The phenotypic characteristics of cells and colonies were determined after three days of growth at 30 ◦ C on MA. Cell morphology, size and presence of flagella were determined with transmission electron microscopy (1400 Plus, JEOL, Tokyo, Japan) at the UPV/EHU Advanced Research Facilities (SGIker, Leioa, Spain). Gram staining was performed using the method described by Smith et al. [ 18 ]. Cellular pigments were analysed by HPLC. Pigment extraction was performed with 90% acetone following the method described by Zapata et al. [ 19 ], with the modifications explained by Seoane et al. [ 20 ]. The absorbance chromatogram was extracted at 440 nm. PHA production was tested using Sudan black B staining according to the procedures of Smith et al. [ 21 ] and Santhanam et al. [ 22 ], inoculating 10 mL of MB supplemented with glucose (2% w/v) and yeast extract (2 g/L) as the carbon and nitrogen sources, respectively, and incubating this for 48 h at 28 ◦ C and 150 rpm. Cell motility was measured using the hanging drop method in semisolid agar including MB and 0.7% bacteriological agar (Condalab, Madrid, Spain). Anaerobic growth was tested at 30 ◦ C for 15 days on MA in an anaerobic chamber with GENbox anaer (bioMérieux, Marcy-l ' Étoile, France). Growth at different temperatures (4, 8, 15, 20, 25, 30, 37 and 42 ◦ C) was determined on MA after 3 to 15 days of incubation. Salt tolerance and requirement for growth were tested in MB supplemented with NaCl at final concentrations of 0, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0, 15.0, 20.0 and 23.0 g/L. These media were prepared according to the MB formula, but without NaCl. The pH range supporting growth was determined in MB at 30 ◦ C, with the pH adjusted (pH 4.5–10.5 in increments of 0.5 pH units) with different buffers: sodium acetate/acetic acid (for pH 4.5–6.0) and NaHCO 3 /Na 2 CO 3 (for pH 6.5–10.5). Energetic metabolism was characterised on a modified MA medium for photo- (anaerobic, light 2400 lx) and chemo- (aerobic, dark)organoheterotrophy (with pyruvate (0.03%, w/v) as the carbon source/electron donor), photolithoautotrophy (anaerobic, light (2400 lx), with Na 2 S 2 O 3 .5H 2 O (1 mM) as the electron donor and NaHCO 3 (0.1%, w/v) as the carbon source), chemolithoautotrophy (dark, aerobic, with Na 2 S 2 O 3 .5H 2 O (1 mM) as the electron donor and NaHCO 3 (0.1%, w/v) as the carbon source) and fermentative growth (dark, anaerobic, with pyruvate/glucose (0.3%, w/v) as the fermentable substrates). Oxidase activity was tested with Bactident Oxidase strips (Merck, Rahway, NJ, USA) and catalase activity with ID colour catalase (bioMérieux). Hydrolysis capacity was determined on MA plates supplemented with 1% (w/v) skimmed milk, 1% (w/v) Tween 20 and Section 3. atala - APPENDIX VI. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Diversity 2022,14, 1009 4 of 17 1% (w/v) Tween 80. Other physiological and biochemical features were examined using API ZYM and API 20NE strips (bioMérieux) according to the manufacturer’s instructions, with cells suspended in a saline solution. Susceptibility to antibiotics was tested on MA plates incubated at 30 ◦ C for three days using discs containing the following: ciprofloxacin (10 µ g), erythromycin (15 µ g), ofloxacin (5 µ g), penicillin G (5 IU), cephazolin (30 µ g), rifampicin (2 µ g), kanamycin (5 µ g), gentamicin (10 µ g), polymyxin B (300 IU), vancomycin (30 µg) and streptomycin (10 µg). Whole-cell fatty acid composition was determined following the protocol recommended by the MIDI Microbial Identification System [ 23 ] at the Spanish Type Culture Collection (CECT). Identification was performed with the Sherlock MIDI version 6.1 and cellular fatty acid content was analysed using the TSBA6 library [ 24 ]. Cells were grown for 72 h at 30 ◦ C on MA to obtain their biomass. Analyses of polar lipids and respiratory quinones were carried out using the Identification Service at the Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) following the protocol based on Bligh and Dyer [25] and Tindall et al. [26]. 2.4. Genome Sequencing and Analysis Whole-genome sequencing of the ASV31 T isolate was performed using the Illumina Miseq platform at the UPV/EHU Advanced Research Facilities (SGIker). DNA extraction was conducted using the NucleoSpin Tissue DNA extraction kit (Macherey-Nagel) according to the manufacturer’s protocol. Assembly from raw paired-end reads to contigs was performed using the SPAdes assembler v3.13.0 available in PATRIC 3.6.9 software [ 27 ]. Authenticity was confirmed with the presence of the 16S rRNA gene sequence obtained using PCR on the assembled genome, and the genome’s quality was confirmed using CheckM v1.0.18 [28]. Genome-based comparisons of strain ASV31 T with its closest species were performed with average nucleotide identity (ANI) values computed using the OrthoANI Calculator tool available at EzBiocloud (www.ezbiocloud.net/tools/ani, accessed on 20 January 2022 ) [ 29 ] and digital DNA–DNA hybridisation (dDDH) values computed using the genome-togenome distance calculator (http://ggdc.dsmz.de/ggdc.php, accessed on 20 January 2022 ) [ 30 ]. In order to assess genus affiliations, amino acid-level comparisons were performed using the average amino acid identity (AAI) and percentage of conserved proteins (POCP) for every pairwise combination of genomes. The AAI values were calculated using the Kostas Lab AAI calculator (http://enve-omics.ce.gatech.edu/aai/, accessed on 20 January 2022 ) [ 31 ]. The POCP values were calculated using a Python script with the formula [(C1 + C2)/(T1 + T2)] ×100% , where C1 and C2 represent the conserved number of proteins in the two genomes being compared, and T1 and T2 represent the total number of proteins in the two genomes being compared [32]. A whole-genome-based taxonomic analysis was performed on the Type Strain Genome Server (https://tygs.dsmz.de, accessed on 18 February 2022) [ 30 ] and a genome-based phylogenetic tree was inferred with FastME 2.1.6.1 [ 33 ] from the GBDP distances calculated from genome sequences. Branch lengths were scaled in terms of GBDP distance formula d5. In order to provide further information about the taxonomic position of strain ASV31 T , the amino acid sequences of RpoC, 2-oxoglutarate dehydrogenase and acyl-CoA synthetase proteins were obtained from the genome. These sequences were analysed with BLAST in order to find the most similar taxa. Phylogenetic trees based on these proteins were constructed with MEGA X using the maximum-likelihood method. Amino acid sequences were retrieved from the GenBank database. Gene annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline and the Rapid Annotation with Subsystems Technology (RAST) pipeline [ 34 ]. The detection of gene clusters related to secondary metabolite production was performed using the antiSMASH 6.0.1 webserver (https://antismash.secondarymetabolites.org, accessed on 11 May 2022) [ 35 ]. The dbCAN meta server (https://bcb.unl.edu/dbCAN2/, accessed on Section 3. atala - APPENDIX VI. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Diversity 2022,14, 1009 5 of 17 25 May 2022) [ 36 ] was used to identify the coding sequences (CDSs) encoding carbohydrateactive enzymes (CAZymes) using an HMMER annotation. 3. Results and Discussion A polyphasic study was carried out in order to characterise strain ASV31 T , which was isolated from a hypersaline water spring in an active continental saltern. This saltern was formed as a result of the deposit of evaporites millions of years ago during the desiccation of the great ocean that covered almost the entire Earth. 3.1. 16S rRNA Gene-Based Identification and Phylogeny Phylogenetic analysis showed that strain ASV31 T belonged to the Bacteria domain, Proteobacteria phylum, Alphaproteobacteria class, Rhodobacterales order and Rhodobacteraceae family. Due to the short 16S rRNA gene sequence (1337 bp) obtained by PCR (sequence deposited at GenBank under accession number MW691205), the 1468 bp 16S rRNA sequence from genome sequencing was used for further phylogenetic analysis (accession number JAHQZS010000014.1). A comparison of 16S rRNA sequences determined that Thioclava GU322906_s YC6923 was the most closely related strain (95.4% similarity between sequences), followed by Thioclava pacifica DSM 10166 T (95.3%) and Thioclava sediminum TAW-CT134 T (95.2%) type strains. Other related taxa were Tropicimonas AM176881_s SZB22 (95.1%), Haematobacter massiliensis CCUG 47968 T (95%) and Rhodovulum adriaticum DSM 2781 T (94.9%). Hördt et al. [ 3 ] proposed that when the 16S rRNA gene sequence similarity is below 95%, the taxa represent different genera. The similarity of the 16S rRNA sequences of Thioclava species and strain ASV31 T was just at this limit, which was not sufficient to support the placement of strain ASV31 T into the Thioclava genus. Furthermore, the phylogenetic tree, constructed with the neighbour-joining method based on the 16S rRNA gene sequence, showed that strain ASV31 T was not defined by any genus in the family Rhodobacteraceae, as it clustered in a differentiated branch between the Rhodobacter and Roseobacter groups (Figure 1). In fact, strain ASV31 T was in an independent branch that did not show a defined affiliation either to Thioclava spp. (Rhodobacter group) or Tropicimonas spp. (Roseobacter group). Similar results were observed in both the maximum-likelihood and the minimum-evolution trees (Figures S1 and S2). These results suggested that strain ASV31Tmay represent a novel genus of the Rhodobacteraceae family. 3.2. Phenotypic and Chemotaxonomic Characterisation Strain ASV31 T is a Gram-negative bacillus 2.4–2.5 µ m in length and 0.4–0.5 µ m in width, with a mucous polysaccharide capsule (slime) all around (Figure 2A). This feature was also seen genetically in the metabolic reconstruction (results shown below). Unlike Thioclava members, which are described as rarely motile by means of a polar flagellum [ 37 ], and Rhodovulum members, which also exhibit motility by a polar flagellum [ 5 ], no flagella were observed in ASV31 T using transmission electron microscopy. Motility was neither observed using the hanging drop method nor in semisolid agar. The presence of PHA as intracellular granules was confirmed through Sudan black B staining (Figure 2B). This capacity has also been reported in Rhodobacter capsulatus,Palleronia marisminoris,Salipiger spp., Tranquillimonas spp., Labrenzia spp. and Rhodovulum sulfidophilum in the Rhodobacteraceae family, with PHB being the main PHA class [5]. Section 3. atala - APPENDIX VI. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Diversity 2022,14, 1009 6 of 17 Diversity 2022, 14, x FOR PEER REVIEW 6 of 17 Figure 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences that shows the relationship between strain ASV31T and the type strains of Rhodobacter, Roseobacter and Rhodovulum groups of the family Rhodobacteraceae. The black circles indicate the clades that were also conserved in both the maximum-likelihood and minimum-evolutionary phylogenetic trees. Bootstrap values (expressed as percentages of 1000 replications) greater than 50% in the clade nodes are shown. Escherichia coli NCCB 54008T was used as outgroup. Scale: 0.02 substitutions per nucleotide position. 3.2. Phenotypic and Chemotaxonomic Characterisation Strain ASV31T is a Gram-negative bacillus 2.4–2.5 μm in length and 0.4–0.5 μm in width, with a mucous polysaccharide capsule (slime) all around (Figure 2A). This feature was also seen genetically in the metabolic reconstruction (results shown below). Unlike Thioclava members, which are described as rarely motile by means of a polar flagellum [37], and Rhodovulum members, which also exhibit motility by a polar flagellum [5], no flagella were observed in ASV31T using transmission electron microscopy. Motility was neither observed using the hanging drop method nor in semisolid agar. The presence of PHA as intracellular granules was confirmed through Sudan black B staining (Figure 2B). This capacity has also been reported in Rhodobacter capsulatus, Palleronia marisminoris, Salipiger spp., Tranquillimonas spp., Labrenzia spp. and Rhodovulum sulfidophilum in the Rhodobacteraceae family, with PHB being the main PHA class [5]. Figure 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences that shows the relationship between strain ASV31 T and the type strains of Rhodobacter,Roseobacter and Rhodovulum groups of the family Rhodobacteraceae. The black circles indicate the clades that were also conserved in both the maximum-likelihood and minimum-evolutionary phylogenetic trees. Bootstrap values (expressed as percentages of 1000 replications) greater than 50% in the clade nodes are shown. Escherichia coli NCCB 54008 T was used as outgroup. Scale: 0.02 substitutions per nucleotide position. Diversity 2022, 14, x FOR PEER REVIEW 7 of 17 (a) (b) Figure 2. Anianabacter salinae ASV31 T cell characteristics observed using microscopy: (a) Transmission electron micrograph of a cell. A polysaccharide mucous capsule was observed around the cell and darker spots in the cytoplasm. Bar 500.0 nm. (b) Sudan black B staining of PHA granules observed as black intracellular granules under 100× oil immersion objective. Colonies of strain ASV31 T were less than 1 mm in diameter and beige-to-pink coloured, with a circular shape, convex elevation, transparent smooth edge and they glistened on the MA medium (Figure S3). The colony morphology was different from the Thioclava pacifica DSM 10166 T and Rhodovulum algae LMG 29228 T strains that were studied alongside ASV31 T in this study. ASV31 T was not capable of growing under anaerobic conditions. Strain ASV31 T grew in a temperature range of 18–37 °C, with an optimum temperature of 30 °C, the same as its relatives. The pH range for growth was pH 6.5–9.5, with an optimum of pH 7.0–9.5, indicating it was slightly alcaliphilic compared with its close relatives. The NaCl tolerance for growth was 0.0–23.0%, with an optimum of 3.0–5.0%. In contrast with its relatives, strain ASV31 T could grow at higher salt concentrations and did not require NaCl to grow like most Thioclava and Rhodovulum members [5]. Members of Rhodobacteraceae are mainly aerobic photoand chemoheterotrophs [5]. In this sense, strain ASV31 T is an aerobic chemoorganoheterotroph. This capacity was also observed in Thioclava pacifica DSM 10166 T and Rhodovulum algae LMG 29228 T . Chemolitoautotrophic, photolithoautotrophic and photoorganoheterotrophic growth could not be demonstrated in ASV31 T . Moreover, Thioclava members are considered facultative sulphur chemolithotrophs, are included in the Rhodobacter group and are able to grow using thiosulfate oxidation and inorganic carbon fixation through the Calvin cycle [5]. This characteristic was observed in this study. Photorganoheterotrophy was also observed in Rhodovulum algae LMG 29228 T (as described by Ramaprasad et al. [38]). Fermentative growth was not seen in any of the strains analysed, as described in [5]. Spheroidenone was the main pigment in ASV31 T , while spheroidene and bacteriochlorophyll a (BChl a) were also detected in the strain. It is known that Rhodovulum members can also synthesise BChl a and carotenoids of the spheroidene series (spheroidene, spheroidenone, demethylspheroidene, hydroxyspheroidene and neurosporene, among others) in different proportions. However, Thioclava members do not synthetise BChl a or carotenoids [5,39]. Oxidase activity was positive in ASV31 T and its relatives, while catalase was present in ASV31 T and Rhodovulum algae LMG 29228 T . Urease was only present in Rhodovulum algae LMG 29228 T . A capacity for Tween 20 hydrolysis was only seen in strain ASV31 T and none of the strains had a casein or Tween 80 hydrolysis ability. In API 20NE tests, ASV31 T was positive for aesculin hydrolysis and nitrate reduction, but negative for indole production, glucose fermentation, gelatine hydrolysis, arginine dihydrolase, urease and β-galactosidase, and the assimilation of glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate. The API Figure 2. Anianabacter salinae ASV31 T cell characteristics observed using microscopy: ( a ) Transmission electron micrograph of a cell. A polysaccharide mucous capsule was observed around the cell and darker spots in the cytoplasm. Bar 500.0 nm. ( b ) Sudan black B staining of PHA granules observed as black intracellular granules under 100×oil immersion objective. Section 3. atala - APPENDIX VI. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Diversity 2022,14, 1009 7 of 17 Colonies of strain ASV31 T were less than 1 mm in diameter and beige-to-pink coloured, with a circular shape, convex elevation, transparent smooth edge and they glistened on the MA medium (Figure S3). The colony morphology was different from the Thioclava pacifica DSM 10166 T and Rhodovulum algae LMG 29228 T strains that were studied alongside ASV31 T in this study. ASV31 T was not capable of growing under anaerobic conditions. Strain ASV31 T grew in a temperature range of 18–37 ◦ C, with an optimum temperature of 30 ◦ C, the same as its relatives. The pH range for growth was pH 6.5–9.5, with an optimum of pH 7.0–9.5, indicating it was slightly alcaliphilic compared with its close relatives. The NaCl tolerance for growth was 0.0–23.0%, with an optimum of 3.0–5.0%. In contrast with its relatives, strain ASV31 T could grow at higher salt concentrations and did not require NaCl to grow like most Thioclava and Rhodovulum members [ 5 ]. Members of Rhodobacteraceae are mainly aerobic photoand chemoheterotrophs [ 5 ]. In this sense, strain ASV31 T is an aerobic chemoorganoheterotroph. This capacity was also observed in Thioclava pacifica DSM 10166 T and Rhodovulum algae LMG 29228 T . Chemolitoautotrophic, photolithoautotrophic and photoorganoheterotrophic growth could not be demonstrated in ASV31 T . Moreover, Thioclava members are considered facultative sulphur chemolithotrophs, are included in the Rhodobacter group and are able to grow using thiosulfate oxidation and inorganic carbon fixation through the Calvin cycle [ 5 ]. This characteristic was observed in this study. Photorganoheterotrophy was also observed in Rhodovulum algae LMG 29228 T (as described by Ramaprasad et al. [ 38 ]). Fermentative growth was not seen in any of the strains analysed, as described in [5]. Spheroidenone was the main pigment in ASV31 T , while spheroidene and bacteriochlorophyll a (BChl a) were also detected in the strain. It is known that Rhodovulum members can also synthesise BChl a and carotenoids of the spheroidene series (spheroidene, spheroidenone, demethylspheroidene, hydroxyspheroidene and neurosporene, among others) in different proportions. However, Thioclava members do not synthetise BChl a or carotenoids [5,39]. Oxidase activity was positive in ASV31 T and its relatives, while catalase was present in ASV31 T and Rhodovulum algae LMG 29228 T . Urease was only present in Rhodovulum algae LMG 29228 T . A capacity for Tween 20 hydrolysis was only seen in strain ASV31 T and none of the strains had a casein or Tween 80 hydrolysis ability. In API 20NE tests, ASV31 T was positive for aesculin hydrolysis and nitrate reduction, but negative for indole production, glucose fermentation, gelatine hydrolysis, arginine dihydrolase, urease and β -galactosidase, and the assimilation of glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate. The API ZYM tests showed alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase, valine arylamidase and α -glucosidase activity, weak activity of cysteine arylamidase, acid phosphatase and naphthol-AS-BIphosphohydrolase, and negative activity of lipase (C 14), α -chymotrypsin, trypsin, α - and β -galactosidases, β -glucuronidase, β -glucosidase, Nacetylβ -glucosaminidase, α -mannosidase and α -fucosidase. Strain ASV31 T was resistant to polymyxin B (300 IU), vancomycin (30 µ g) and streptomycin (10 µ g). The most relevant phenotypic characteristics that distinguished strain ASV31 T from its closest relatives are summarised in Table 1. Section 3. atala - APPENDIX VI. ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Diversity 2022,14, 1009 14 of 17 Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/d14111009/s1: Figure S1: Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences showing the relationships between Anianabacter salinae ASV31 T and other strains of Rhodobacteraceae family. Figure S2: Minimum evolution phylogenetic tree based on 16S rRNA gene sequences showing the relationships between Anianabacter salinae ASV31 T and other species of Rhodobacteraceae family. Figure S3: Anianabacter salinae ASV31 T growth on marine agar medium. Figure S4: Thin-layer chromatogram of polar lipids of Anianabacter salinae ASV31 T . Figure S5: Whole-genome-based taxonomic tree, showing the position of the strain ASV31 T and related strains of Rhodobacteraceae family. Figure S6: RpoC-based maximum-likelihood tree: A, performed with the most similar amino acidic sequences; B, performed with sequences from representatives of the Rhodobacteraceae family. Tree was generated using MEGA X. Only bootstrap values (expressed as percentages of 1000 replications) greater than 50% are shown at branching points. The scale bar indicates the number of substitutions per site. Figure S7: Acyl-CoA synthetase-based maximum-likelihood tree: A, performed with the most similar amino acidic sequences; B, performed with sequences from representatives of the Rhodobacteraceae family. Tree was generated using MEGA X. Only bootstrap values (expressed as percentages of 1000 replications) greater than 50% are shown at branching points. The scale bar indicates the number of substitutions per site. Figure S8: 2oxoglutarate dehydrogenase-based maximum likelihood tree; A, performed with the most similar amino acidic sequences, B, performed with sequences from representatives of the Rhodobacteraceae family. Tree was generated using MEGA X. Only bootstrap values (expressed as percentages of 1000 replications) greater than 50% are shown at branching points. The scale bar indicates the number of substitutions per site. Table S1: AAI and POCP matrix from pairwise whole-genome comparison. Table S2: Presence and absence of subsystems in strain ASV31 T compared to its closest relatives. Table S3: CAZyme types present in ASV31Tstrain. Author Contributions: Conceptualisation, I.M.-B., J.G. and J.B.; methodology, I.M.-B. and J.G.; formal analysis, M.A.-M., M.G., L.L., I.M.-M., S.S. and I.M.-B.; resources, J.B.; data curation, M.A.-M. , M.G., L.L., I.M.-M., S.S. and I.M.-B.; writing—original draft preparation, M.A.-M. and I.M.-B.; writing—review and editing, all authors; funding acquisition, J.G. and I.M.-B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the University of the Basque Country UPV/EHU (grant number US19/01) and the Añana Salt Valley Foundation (specific agreement between the Añana Salt Valley Foundation and the University of the Basque Country UPV/EHU). Institutional Review Board Statement: Not applicable. Acknowledgments: The authors are grateful for the technical and human support provided by the UPV/EHU Advanced Research Facilities (SGIker). Conflicts of Interest: The authors declare no conflict of interest. References 1. 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ERANSKINA REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 Section 4. atala – Work under review / Berrikuspenean dagoen lana REGISTRO TELEMÁTICO Sarreren Erregistro Orokorra / Registro General de Entradas 01/07/2025 13:49 EHU2025E029511 [Document text truncated for crawler view.]