Actinobacteria from arctic and atlantic deep-sea sediments—biodiversity and bioactive potential

The deep-sea covers over 70% of the Earth’s surface and harbors predominantly uncharacterized bacterial communities. Actinobacteria are the major prokaryotic source of bioactive natural products that find their way into drug discovery programs, and the deep-sea is a promising source of biotechnologically relevant actinobacteria. Previous studies on actinobacteria in deep-sea sediments were either regionally restricted or did not combine a community characterization with the analysis of their bioactive potential. Here we characterized the actinobacterial communities of upper layers of deep-sea sediments from the Arctic and the Atlantic (Azores and Madeira) ocean basins, employing 16S rRNA metabarcoding, and studied the biosynthetic potential of cultivable actinobacteria retrieved from those samples. Metabarcoding analysis showed that the actinobacterial composition varied between the sampled regions, with higher abundance in the Arctic samples but higher diversity in the Atlantic ones. Twenty actinobacterial genera were detected using metabarcoding, as a culture-independent method, while culture-dependent methods only allowed the identification of nine genera. Isolation of actinobacteria resulted on the retrieval of 44 isolates, mainly associated with Brachybacterium, Microbacterium, and Brevibacterium genera. Some of these isolates were only identified on a specific sampled region. Chemical extracts of the actinobacterial isolates were subsequently screened for their antimicrobial, anticancer and anti-inflammatory activities. Extracts from two Streptomyces strains demonstrated activity against Candida albicans. Additionally, eight extracts (obtained from Brachybacterium, Brevibacterium, Microbacterium, Rhodococcus, and Streptomyces isolates) showed significant activity against at least one of the tested cancer cell lines (HepG2 and T-47D). Furthermore, 15 actinobacterial extracts showed anti-inflammatory potential in the RAW 264.4 cell model assay, with no concomitant cytotoxic response. Dereplication and molecular networking analysis of the bioactive actinobacterial extracts showed the presence of some metabolites associated with known natural products, but one of the analyzed clusters did not show any match with the natural products described as responsible for these bioactivities. Overall, we were able to recover taxonomically diverse actinobacteria with different bioactivities from the studied deep-sea samples. The conjugation of culture-dependent and -independent methods allows a better understanding of the actinobacterial diversity of deep-sea environments, which is important for the optimization of approaches to obtain novel chemically-rich isolates.info:eu-repo/semantics/publishedVersio

Environmental DNA as a complementary tool for biodiversity monitoring: A multi-technique and multi-trophic approach to investigate cetacean distribution and feeding ecology

38 pages, 4 figuresThe use of environmental DNA (eDNA) to assess the presence of biological communities has emerged as a promising monitoring tool in the marine conservation landscape. Moreover, advances in Next-Generation Sequencing techniques, such as DNA metabarcoding, enable multi-species detection in mixed samples, allowing the study of complex ecosystems such as oceanic ones. We aimed at using these molecular-based techniques to characterise cetacean communities, as well as potential prey in the northern coast of Mainland Portugal. During seasonal campaigns, we collected seawater samples, along with visual records of cetacean occurrence. The eDNA extracted from 64 environmental samples was sequenced in an Illumina platform, with universal primers targeting marine vertebrates. Five cetacean species were identified by molecular detection: common dolphin (Delphinus delphis), bottlenose dolphin (Tursiops truncatus), Risso’s dolphin (Grampus griseus), harbour porpoise (Phocoena phocoena) and fin whale (Balaenoptera physalus). Overall, except for the fin whale (not sighted during the campaigns), this cetacean community composition was similar to that obtained through visual monitoring, and the complementary results suggest their presence in the region all year round. In addition, the positive molecular detections of B. physalus are of special relevance since there are no visual records reported in the area. The detection of multiple known preys of the identified dolphins indicates they use these coastal areas for feeding purposes. While this methodological approach remains in a development stage, the present work highlights the benefits of using eDNA to study marine communities, with specific applications for research on cetacean distribution and feeding ecology, ultimately serving as the baseline of a methodological approach for biodiversity monitoring and marine conservationPh.D. fellowships for authors RV (SFRH/BD/144786/2019) and AG (PD/BD/150603/2020) were granted by Fundação para a Ciência e Tecnologia (FCT, Portugal) under the auspices of Programa Operacional Regional Norte (PORN), supported by the European Social Fund (ESF) and Portuguese funds (MECTES). This work is a result of the project ATLANTIDA (ref. NORTE-01-0145-FEDER-000040) supported by the Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement and through the European Regional Development Fund (ERDF); and of the project EMPHATIC funded by Biodiversa+, the European Biodiversity Partnership, under the joint call 2022 – 2023 BiodivMon for research proposals, co-funded by the European Commission and with the following funding organisations: Fundación Biodiversidad (FB, Spain), Fundação para a Ciência e Tecnologia (FCT, Portugal), Agence Nationale de la Recherche (ANR, France), and the Ministry of Universities and Research (MUR, Italy).Peer reviewe

Environmental DNA as a cetacean monitoring tool in the Northern Coast of Continental Portugal

34th European Cetacean Society Conference, O Grove, 16-20 April 2023In recent decades, environmental DNA (eDNA) has emerged as a utopian monitoring tool in the marine conservation landscape, for its potential to collect data on presence and abundance of biological communities with insufficient knowledge and/or difficult access. In the ATLANTIDA Project, this tool is being optimized with the ultimate goal of detecting and identifying cetacean species on the northern coast of continental Portugal without relying on visual monitoring. To this end, a molecular biology-based methodology is being developed and tested in positive control samples, consisting of a mixture of DNA extracted directly from muscle tissue or gums of cetaceans with eDNA samples collected in ATLANTIDA dedicated at-sea surveys. Firstly, in silico analysis using metagenomic data was performed to assess the pair of primers directed to mitochondrial DNA presenting the highest variability and, consequently, a higher probability of distinction among the target species. For that purpose, different sets of primers previously described in the bibliography were analysed and unique single nucleotide polymorphisms between the species of interest were counted. After the selection of the most suitable primer set, several optimization tests have been conducted, through conventional PCR, in order to identify the most efficient DNA amplification protocol for the gene of interest. From these tests, we were able to identify the ideal annealing temperature, the best TAQ polymerase enzyme, and the detection of cetacean DNA up to very low concentrations (~5ng/μL) was achieved, with salt water not inhibiting the reaction. In environmental samples, we were still not able to detect cetacean DNA, probably because of its concentration below our detection limit in the samples tested. In conclusion, although the effectiveness of resorting to eDNA for cetacean monitoring programs remains unclear, these results represent a step forward towards that goalN

Blow microbiota of free-ranging short-finned pilot whales: comparison of two 16S rRNA primers for studies of blow prokaryotic communities

34th European Cetacean Society Conference, O Grove, 16-20 April 2023Respiratory disease is one of the main causes of death in cetaceans. Characterizing the microbial communities harbored in the exhaled breath condensate (EBC), or blow, has been proposed as a suitable methodology to assess cetacean health. However, still few studies in the literature focus on the respiratory microbiota of free-ranging animals. In the present study the short-finned pilot whales (Globicephala macrorhynchus) were used as a model species to: 1) develop a sampling/analysis protocol for the characterization of the microbial diversity in the EBC of free-ranging cetaceans; 2) perform a comprehensive comparison of two 16S rRNA gene primers set (regarding amplification and taxa coverage) able to describe the microbial communities of their respiratory tracts; 3) highlight possible potential pathogens in the animal‟s airway microbiota. For this purpose, a total of 12 pilot whale EBC samples were collected during at-sea campaigns in Madeiran waters, in the autumn 2018. Environmental DNA was extracted from the samples and then sequenced using the Illumina MiSeq platform to amplify V3-V4 and V4-V5 hypervariable regions of the 16S rRNA gene. DADA2 bioinformatic pipeline was used to process sequences and analyze the diversity and taxonomic profiles of blow prokaryotic communities. Results showed that, independently of the primer set used, all the sampled animals share Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla in their blow composition. The V4-V5 dataset showed higher microbial richness (rare biosphere and potentially pathogenic taxa), whereas V3-V4 captured more diversity. This research contributes to bringing new knowledge on the characterization of the respiratory-associated microbial communities, towards a non-invasive tool for monitoring the physiological state of the airways in cetaceans. However, the methodology still requires research effort and development / optimization of techniques to further advance the implementation of such monitoring toolN

Actinobacteria from Arctic and Atlantic deep-sea sediments—Biodiversity and bioactive potential

The deep-sea covers over 70% of the Earth’s surface and harbors predominantly uncharacterized bacterial communities. Actinobacteria are the major prokaryotic source of bioactive natural products that find their way into drug discovery programs, and the deep-sea is a promising source of biotechnologically relevant actinobacteria. Previous studies on actinobacteria in deep-sea sediments were either regionally restricted or did not combine a community characterization with the analysis of their bioactive potential. Here we characterized the actinobacterial communities of upper layers of deep-sea sediments from the Arctic and the Atlantic (Azores and Madeira) ocean basins, employing 16S rRNA metabarcoding, and studied the biosynthetic potential of cultivable actinobacteria retrieved from those samples. Metabarcoding analysis showed that the actinobacterial composition varied between the sampled regions, with higher abundance in the Arctic samples but higher diversity in the Atlantic ones. Twenty actinobacterial genera were detected using metabarcoding, as a culture-independent method, while culture-dependent methods only allowed the identification of nine genera. Isolation of actinobacteria resulted on the retrieval of 44 isolates, mainly associated with Brachybacterium, Microbacterium, and Brevibacterium genera. Some of these isolates were only identified on a specific sampled region. Chemical extracts of the actinobacterial isolates were subsequently screened for their antimicrobial, anticancer and anti-inflammatory activities. Extracts from two Streptomyces strains demonstrated activity against Candida albicans. Additionally, eight extracts (obtained from Brachybacterium, Brevibacterium, Microbacterium, Rhodococcus, and Streptomyces isolates) showed significant activity against at least one of the tested cancer cell lines (HepG2 and T-47D). Furthermore, 15 actinobacterial extracts showed anti-inflammatory potential in the RAW 264.4 cell model assay, with no concomitant cytotoxic response. Dereplication and molecular networking analysis of the bioactive actinobacterial extracts showed the presence of some metabolites associated with known natural products, but one of the analyzed clusters did not show any match with the natural products described as responsible for these bioactivities. Overall, we were able to recover taxonomically diverse actinobacteria with different bioactivities from the studied deep-sea samples. The conjugation of culture-dependent and -independent methods allows a better understanding of the actinobacterial diversity of deep-sea environments, which is important for the optimization of approaches to obtain novel chemically-rich isolates

Removal of metals and emergent contaminants from liquid digestates in constructed wetlands for agricultural reuse

Given the increasing pressure on water bodies, it is imperative to explore sustainable methodologies for wastewater treatment and reuse. The simultaneous presence of multiples contaminants in complex wastewater, such as the liquid effluents from biogas plants, can compromise biological treatment effectiveness for reclaiming water. Vertical subsurface flow constructed wetlands were established as low-cost decentralized wastewater treatment technologies to treat the liquid fraction of digestate from municipal organic waste with metals, antibiotics, and antibiotic resistance genes, to allow its reuse in irrigation. Twelve lab-scale planted constructed wetlands were assembled with gravel, light expanded clay aggregate and sand, testing four different treating conditions (liquid digestate spiked with oxytetracycline, sulfadiazine, or ofloxacin, at 100 μg/ L, or without dosing) during 3 months. Physicochemical parameters (pH, chemical oxygen demand (COD), nutrients, metals, and antibiotics), the microbial communities dynamics (through 16S high-throughput sequencing) and antibiotic resistance genes removal (qPCR) were monitored in influents and effluents. Systems removed 85.8%–96.9% of organic matter (as COD), over 98.1% of ammonium and phosphate ions, and 69.3%–99.4% of nitrate and nitrite ions, with no significant differences between the presence or absence of antibiotics. Removal of Fe, Mn, Zn, Cu, Pb and Cr exceeded 82% in all treatment cycles. The treatment also removed oxytetracycline, sulfadiazine and ofloxacin over 99%, and decreased intl1, tetA, tetW, sul1 and qnrS gene copies. Nonetheless, after 3 months of ofloxacin dosing, qnrS gene started being detected. Removal processes relied on high HRT (14 days) and various mechanisms including sorption, biodegradation, and precipitation. Microbial community diversity in liquid digestate changed significantly after treatment in constructed wetlands with a decrease in the initial Firmicutes dominance, but with no clear effect of antibiotics on the microbial community structure. Removals above 85% and 94% were observed for Streptococcus and Clostridium, respectively. Results suggest that vertical subsurface flow constructed wetlands were a suitable technology for treating the liquid digestate to reuse it in irrigation agricultural systems, contributing to the circular bioeconomy concept. However, a more profound understanding of effective wastewater treatment strategies is needed to avoid antibiotic resistance genes dissemination

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Inter-comparison of marine microbiome sampling protocols

Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d\u27Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22–3 µm, 3–20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and “whole water” (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume