Light and drought: bioprospecting the microbiota of highly-irradiated and dry environments

La bioprospección es la búsqueda de microorganismos, o de sus partes, para el desarrollo de herramientas biotecnológicas. Los ambientes extremos, como aquellos altamente irradiados y secos, albergan microorganismos interesantes desde el punto de vista biotecnológico. Las condiciones duras a las que están sometidos en estos ambientes constituyen una presión de selección de mecanismos que les permiten lidiar con altas dosis de radiación y desecación. El trabajo descrito en esta tesis tiene como objetivo caracterizar el perfil de microorganismos asociado a superficies naturales y artificiales sometidas a irradiación (la zona intermareal mediterránea, el desierto de Tabernas y las cabinas UV de fototerapia de un hospital) mediante una estrategia holística que incluye técnicas dependientes e independientes de cultivo. Hemos puesto énfasis en recuperar la máxima diversidad microbiana y la descripción de nuevos taxones mediante la combinación de técnicas de cultivo sencillas, como el uso al mismo tiempo de diferentes condiciones de cultivo y medios. En paralelo, se han investigado las aplicaciones biotecnológicas y biomédicas de estos microorganismos, especialmente su potencial como antioxidantes. Además, se ha estudiado el uso de condiciones de cultivo alternativas de fermentación en fase sólida mediante el uso de matrices 3D en el modelo Saccharomyces cerevisiae. La combinación de medios de cultivo y condiciones de incubación ha permitido aislar un elevado número de géneros diferentes, potenciales nuevos taxones y cepas biotecnológicamente relevantes, tal y como evidencia la descripción de tres nuevas especies del género Kineoccoccus y dos nuevas del género Belnapia. Por otro lado, las técnicas de NGS permitieron determinar diferencias y similitudes entre los ambientes estudiados y las colecciones microbianas establecidas. En concreto, las comunidades de los tres ambientes estaban dominadas por Pseudomonadota, Actinomycetota y Bacteroidota. Además, la NGS reveló que la baja diversidad encontrada en la colección del litoral rocoso mediterráneo estaba influenciada por los métodos de cultivo y el rendimiento de la PCR. Finalmente, géneros como Hymenobacter, Rubellimicrobium, Paracoccus y Corynebacteriumeran, abundantes en NGS, no estaban en las colecciones. El diseño de matrices sólidas impresas en 3D pretendía estudiar la influencia que estas estructuras pueden tener en el crecimiento de levaduras y en la biotecnología. El crecimiento de Saccharomyces cerevisiae reveló que las matrices de manera drástica favorecen crecimiento celular comparado con los cultivos equivalentes estáticos y la fermentación alcohólica, tal y como confirma el perfil proteómico obtenido para los cultivos en diferentes condiciones de crecimiento. Además, los resultados diferenciales de consumo de glucosa y síntesis de etanol abren la puerta a posibles aplicaciones para la industria. Finalmente, el potencial antioxidante de aislados de la zona intermareal de la costa mediterránea se evaluó mediante ensayos in vitro e in vivo. Además, se estudió el potencial antioxidante del aislado CR10 (Micrococcus luteus), una bacteria productora de carotenoides, en un modelo de enfermedad mitocondrial (el Síndrome de Leigh), en colaboración con el grupo de Neuropatología Mitocondrial de la Universitat Autònoma de Barcelona. Este síndrome fatal se caracteriza por la neurodegeneración, alteraciones motoras y respiratorias, neuroinflamación y niveles incrementados de estrés oxidativo. Por ello se han explorado las terapias basadas en antioxidantes. Pese a que la administración de un preparado microbiano no tuvo efecto beneficioso sobre los animales enfermos, la serendipia nos llevó a identificar que la maltodextrina era responsable de una significativa reducción en la neuroinflamación, un aumento de la longevidad y un incremento de la variabilidad de Akkermansia spp. en el intestino.Bioprospecting is the search for microorganisms, or their parts, to develop biotechnological tools. Extremophilic environments, such as highly irradiated and dry environments host interesting microbes from the biotechnological perspective. The harsh conditions to which microorganisms are subjected to in these environments constitute a selection pressure of mechanisms that allow them to cope with high doses of radiation and desiccation. The work described in this thesis aims at characterizing the microbial profiles associated to natural and artificial surfaces subjected to irradiation (the Mediterranean rocky-shore, the Tabernas Desert and hospital phototherapy UV cabins), through a holistic strategy including culture-independent and -dependent techniques. We have emphasized improving the recovery of the maximum microbial diversity and the description of novel taxa through the combination of simple culturing strategies, such as combining culture conditions and media. In parallel, the biotechnological and biomedical applications of these microorganisms have been investigated, specifically regarding the potential as antioxidants. Furthermore, the use of alternative culture configurations of solid state fermentation through the use of 3D matrixes has been studied in the model Saccharomyces cerevisiae. The combination of culture media and incubation conditions has resulted in the isolation of a high number of different genera, potentially new taxa and biotechnologically-relevant strains, as evidenced by the description of three new Kineococcus and two Belnapia species. Moreover, NGS allowed us to determine differences and similarities among the studied sites, and with the microbial collections. Specifically, the communities of the three highly-irradiated environments were dominated by Pseudomonadota, Actinomycetota and Bacteroidota. Moreover, NGS revealed that the low diversity found in the collection from the Mediterranean rocky shore was influenced by culturing methods and PCR performance. Finally, genera such as Hymenobacter, Rubellimicrobium, Paracoccus and Corynebacterium were abundant in NGS and completely absent in the collections. The design of 3D-printed solid matrixes aimed at studying whether these structures may influence yeast growth and their impact on biotechnology. The growth of Saccharomyces cerevisiae revealed that our matrixes dramatically fostered cell-yields compared to the equivalent static cultures and appeared to favour alcoholic fermentation, as revealed by the proteomic profiles obtained for cultures in different growth conditions. Moreover, differential results were also obtained in terms of glucose consumption and ethanol biosynthesis, which opens the door for further applications in industry. Finally, the antioxidant potential strains isolated from the Mediterranean supratidal zone was assessed through in vitro and in vivo assays. Moreover, we further tested the antioxidant potential of strain CR10 (Micrococcus luteus), a carotenoid-synthetizing bacteria, in a model of a human mitochondrial disease (Leigh Syndrome), as a collaboration with the group of Mitochondrial Neuropathology in the Universitat Autònoma de Barcelona. This fatal syndrome is characterized by neurodegeneration, motor and respiratory alterations, neuroinflammation and increased levels of oxidative stress. Thus, therapies based in antioxidant compounds have been explored. Although the administration of a microbial preparation had no beneficial effects on the diseased mice, serendipity led us to identify that maltodextrin was responsible of a significant reduction in neuroinflammation, increased lifespan and variability in the abundance of Akkermansia spp. in the gut

Supplementary Material: Maritalea mediterranea sp. nov., isolated from marine plastic residues from Valencia, Spain

The 16S rRNA gene sequence of strain P4.10XT have been deposited in GenBank under the accession number MZ994596. The genomic assembly of strain P4.10XT has been deposited under the GenBank accession number ASM2156869v1.Peer reviewe

Lessons from a survey on the public perception of synthetic biology and related disciplines

Financial support from the European CSA on biological standardization BIOROBOOST (EU grant number 820699, http://standardsinsynbio.eu) is acknowledged. EMM is funded with a Formación de Profesorado Universitario (FPU) grant from the Spanish Government (Ministerio de Ciencia, Innovación y Universidades), with reference FPU17/04184. ALP is a recipient of a Doctorado Industrial fellowship from the Ministerio de Ciencia, Innovación y Universidades (Spain), with reference DI‐17‐09613.Peer reviewe

Gillisia lutea sp. nov., isolated from marine aluminium residues from the Mediterranean sea

A novel Gram-reaction-negative, facultatively anaerobic, rod-shaped, non-motile, non-spore forming, orange-pigmented bacterium identified as M10.2AT, was isolated from marine residues submerged on the Malva-rosa beach (València, Spain), on the western coast of the Mediterranean Sea. This strain was catalase-positive and oxidase-negative and grew under mesophilic, neutrophilic and halophilic conditions. With respect to the 16S rRNA gene sequences, M10.2AT showed similarities with Gillisia mitskevichiae DSM 19839T and Gillisia hiemivida IC154T (97.57 and 97.50 % gene sequence similarity, respectively). The genome of M10.2AT was sequenced and has been deposited in the DDBJ/ENA/GenBank databases under the accession code JAKGTH000000000. The genomic DNA G+C content was 36.13 %. Its adscription to a novel species of the genus Gillisia was confirmed by the genomic indexes average nucleotide identity by blast (ANIb) and digital DNA–DNA hybridisation (dDDH). The major fatty acids were iso-C15 : 0, iso-C15 : 1G, iso-C16 : 0 3-OH, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c). According to the results of this polyphasic study, strain M10.2AT represents a novel species of the genus Gillisia, for which name Gillisia lutea sp. nov. (type strain M10.2AT = CECT 30308T = DSM 112385T) is proposed.Financial support from Spanish Government (Grant SETH with reference RTI2018-095584-B-C41-42-43-44 co-financed by FEDER funds and Ministerio de Ciencia e Innovación) and European Union (MICRO4BIOGAS project with reference ID101000470 funded by European Union’s Horizon 2020 research and innovation programme) is acknowledged. E.M.M. and À.V.V. are recipients of a Formación del Profesorado Universitario (FPU) grant with references FPU17/04184 and FPU18/02578, respectively, from the Spanish Government (Ministerio de Ciencia e Innovación, Spain).Peer reviewe

Microbial communities of the Mediterranean rocky shore: ecology and biotechnological potential of the sea‐land transition

Microbial communities from harsh environments hold great promise as sources of biotechnologically relevant strains and compounds. In the present work, we have characterized the microorganisms from the supralittoral and splash zone in three different rocky locations of the Western Mediterranean coast, a tough environment characterized by high levels of irradiation and large temperature and salinity fluctuations. We have retrieved a complete view of the ecology and functional aspects of these communities and assessed the biotechnological potential of the cultivable microorganisms. All three locations displayed very similar taxonomic profiles, with the genus Rubrobacter and the families Xenococcaceae, Flammeovirgaceae, Phyllobacteriaceae, Rhodobacteraceae and Trueperaceae being the most abundant taxa; and Ascomycota and halotolerant archaea as members of the eukaryotic and archaeal community respectively. In parallel, the culture‐dependent approach yielded a 100‐isolates collection, out of which 12 displayed high antioxidant activities, as evidenced by two in vitro (hydrogen peroxide and DPPH) and confirmed in vivo with Caenorhabditis elegans assays, in which two isolates, CR22 and CR24, resulted in extended survival rates of the nematodes. This work is the first complete characterization of the Mediterranean splash‐zone coastal microbiome, and our results indicate that this microbial niche is home of an extremophilic community that holds biotechnological potential.Financial support from the Spanish Government (Grant Helios, Reference: BIO2015‐66960‐C3‐1‐R co‐financed by FEDER funds and Ministerio de Ciencia, Innovación y Universidades) and from the European CSA on biological standardization BIOROBOOST (EU grant number 820699) is acknowledged. EMM is funded with a Formación de Profesorado Universitario (FPU) grant from the Spanish Government (Ministerio de Ciencia, Innovación y Universidades), with reference FPU17/04184. KT is a recipient of a Doctorado Industrial fellowship from the Ministerio de Ciencia, Innovación y Universidades (Spain), with reference DI‐16‐08976. ÀVV is funded with a Formación de Profesorado Universitario (FPU) grant from the Spanish Government (Ministerio de Ciencia, Innovación y Universidades), with reference FPU18/02578.Peer reviewe

Kineococcus vitellinus sp. nov., Kineococcus indalonis sp. nov. and Kineococcus siccus sp. nov., isolated nearby the Tabernas desert (Almería, Spain)

This article belongs to the Section Environmental Microbiology.Three novel Gram-positive, aerobic, chemoheterotrophic, motile, non-endospore-forming, orange-pigmented bacteria designated strains T13T, T90T and R8T were isolated from the Tabernas Desert biocrust (Almería, Spain). Cells of the three strains were coccus-shaped and occurred singly, in pairs or clusters. The three strains were oxidase-negative and catalase-positive, and showed a mesophilic, neutrophilic and non-halophilic metabolism. Based on the 16S rRNA gene sequences, the closest neighbours of strains T13T, T90T and R8T were Kineococcus aurantiacus IFO 15268T, Kineococcus gypseus YIM 121300T and Kineococcus radiotolerans SRS 30216T (98.5%, 97.1% and 97.9% gene sequence similarity, respectively). The genomes were sequenced, and have been deposited in the GenBank/EMBL/DDBJ databases under the accession numbers JAAALL000000000, JAAALM000000000 and JAAALN000000000, respectively, for strains T13T, T90T and R8T. The average nucleotide identity (ANIb) and digital DNA-DNA hybridization (dDDH) values confirmed their adscription to three new species within the genus Kineococcus. The genomic G + C content of strains T13T, T90T and R8T ranged from 75.1% to 76.3%. The predominant fatty acid of all three strains was anteiso-C15:0. According to a polyphasic study, strains T13T, T90T and R8T are representatives of three new species in the genus Kineococcus, for which names Kineococcus vitellinus sp. nov. (type strain T13T = CECT 9936T = DSM 110024T), Kineococcus indalonis sp. nov. (type strain T90T = CECT 9938T = DSM 110026T) and Kineococcus siccus sp. nov. (type strain R8T = CECT 9937T = DSM 110025T) are proposed.Financial support from Spanish Government (Grant HELIOS with reference: BIO2015-66960-C3-1-R; and grant SETH with reference RTI2018-095584-B-C41-42-43-44 co-financed by FEDER funds and Ministerio de Ciencia, Innovación y Universidades) is acknowledged. EMM is a recipient of a Formación del Profesorado Universitario (FPU) grant with reference FPU17/04184, from the Ministerio de Ciencia, Innovación y Universidades (Spain).Peer reviewe

A 3D printed plastic frame deeply impacts yeast cell growth

Solid State Fermentation (SSF) processes have been explored for yeast growth and protein and metabolites production. However, most of these processes lack standardization. In this work, we present a polylactic acid (PLA) 3D printed matrix that dramatically enhances yeast growth when embedded in liquid media compared to equivalent static cultures, and changes yeast expression patterns at the proteome level (data are available via ProteomeXchange with identifier PXD043759). Moreover, differences in sugar assimilation and ethanol production, as the main product of alcoholic fermentation, are observed. Our results suggest that these matrixes may be useful for a vast range of biotechnological applications based on yeast fermentation.Financial support from the Spanish Government (Grant SETH, Reference: RTI2018-095584-B-C41-42-43-44 co-financed by FEDER funds and Ministerio de Ciencia, Innovación y Universidades), the European CSA on biological standardization BIOROBOOST (EU grant number 820699) and Micro4Biogas (European Commission H2020 Program Ref. Grant Agreement ID 101000470) is acknowledged. EM-M and ÀV-V are funded with a Formación de Profesorado Universitario (FPU) grant from the Spanish Government (Ministerio de Ciencia, Innovación y Universidades), with references FPU17/04184 and FPU18/02578, respectively.Peer reviewe

Maritalea mediterranea sp. nov., isolated from marine plastic residues

A novel Gram-reaction-negative, aerobic, motile, rod-shaped, grey bacterium, strain P4.10XT, was isolated from plastic debris sampled from shallow waters in the Mediterranean Sea (Valencia, Spain). P4.10XT was catalase- and oxidase-positive, and grew under mesophilic, neutrophilic and halophilic conditions. The 16S rRNA gene sequences revealed that P4.10XT was closely related to Maritalea myrionectae DSM 19524T and Maritalea mobilis E6T (98.25 and 98.03 % sequence similarity, respectively). The DNA G+C content of the genome sequence of P4.10XT was 53.66 %. The genomic indexes average nucleotide identity by blast (ANIb) and digital DNA–DNA hybridization (dDDH) confirmed its classification as representing a novel species of the genus Maritalea. The predominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c) and C18 : 1 ω7c 11-methyl. The results of this polyphasic study confirm that P4.10XT represents a novel species of the genus Maritalea, for which the name Maritalea mediterranea sp. nov. is proposed (type strain P4.10XT=CECT 30306T = DSM 112386T).Financial support from Spanish Government (SETH Project with reference RTI2018-095584-B-C41-42-43-44 co-financed by European Regional Development Fund - Ministry of Science and Innovation – State Research Agency; MIPLACE Project with reference PCI2019-111845-2 funded by Ministry of Science and Innovation – State Research Agency) and European Union (MICRO4BIOGAS project with reference ID101000470 funded by European Union's Horizon 2020 research and innovation programme) is acknowledged. E.M.M. and À.V.V. are recipients of a University Faculty Training Programme (FPU) grant with references FPU17/04184 and FPU18/02578 respectively, from the Spanish Government (Ministry of Science, Innovation and Universities).Peer reviewe

Sagittula salina sp. nov., isolated from marine waste

A novel Gram-stain-negative, non-motile, halophilic bacterium designated strain M10.9XT was isolated from the inner sediment of an aluminium can collected from the Mediterranean Sea (València, Spain). Cells of strain M10.9XT were rod-shaped and occasionally formed aggregates. The strain was oxidase-negative and catalase-positive, and showed a slightly psychrophilic, neutrophilic and slightly halophilic metabolism. The phylogenetic analyses revealed that strain M10.9XT was closely related to Sagittula stellata E-37T and Sagittula marina F028-2T. The genomic G+C content of strain M10.9XT was 65.2 mol%. The average nucleotide identity and digital DNA–DNA hybridization values were 76.6 and 20.9 %, respectively, confirming its adscription to a new species within the genus Sagittula . The major cellular fatty acids were C18 : 1  ω7c/C18 : 1  ω6c and C16 : 0. The polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, an unidentified glycolipid, an unidentified phospholipid and an unidentified lipid. According to the resuts of a polyphasic study, strain M10.9XT represents a novel species of the genus Sagittula for which the name Sagittula salina sp. nov. (type strain M10.9XT=DSM 112301T=CECT 30307T) is proposed.This manuscript was financially supported by the EU funded project Micro4Biogas (FNR-12-2020 (RIA), Project ID 101000470) and the Spanish Government on SETH Project (Reference: RTI2018-095584-B-C41-42-43-44 co-financed by FEDER funds and Ministerio de Ciencia, Innovación y Universidades). LS is funded by European project BIOROBOOST. EMM and ÀVV are recipients of a Formación del Profesorado Universitario (FPU) grant with references FPU17/04184 and FPU18/02578, respectively, from the Spanish Government (Ministerio de Ciencia, Innovación y Universidades, Spain).Peer reviewe

Living in a bottle: Bacteria from sediment-associated Mediterranean waste and potential growth on polyethylene terephthalate

Ocean pollution is a worldwide environmental challenge that could be partially tackled through microbial applications. To shed light on the diversity and applications of the bacterial communities that inhabit the sediments trapped in artificial containers, we analyzed residues (polyethylene terephthalate [PET] bottles and aluminum cans) collected from the Mediterranean Sea by scanning electron microscopy and next generation sequencing. Moreover, we set a collection of culturable bacteria from the plastisphere that were screened for their ability to use PET as a carbon source. Our results reveal that Proteobacteria are the predominant phylum in all the samples and that Rhodobacteraceae, Woeseia, Actinomarinales, or Vibrio are also abundant in these residues. Moreover, we identified marine isolates with enhanced growth in the presence of PET: Aquimarina intermedia, Citricoccus spp., and Micrococcus spp. Our results suggest that the marine environment is a source of biotechnologically promising bacterial isolates that may use PET or PET additives as carbon sources.Financial support by Ministerio de Ciencia e Innovación (grant SETH ref. RTI2018‐095584‐B‐C41‐42‐43‐44 co‐financed by FEDER), the European Union H2020 (BIOROBOOST project ID 210491758; Micro4Biogas project ref.101000470), Agencia Estatal de la Innovación AEI (MIPLACE projectref. PCI2019‐111845‐2), and Agència Valenciana de la Innovación AVI (ENTOMOPLAST project ref. INNEST/2021/334) are acknowl-edged. Àngela Vidal‐Verdú and Esther Molina‐Menor are funded with a Formación del Profesorado Universitario grant from the Spanish Ministerio de Ciencia, Innovación y Universidades with references FPU18/02578 and FPU17/04184, respectively. Adriel Latorre‐Pérezis a recipient of a Doctorado Industrial fellowship from the Spanish Ministerio de Ciencia, Innovación y Universidades (reference DI‐17‐09613).Peer reviewe