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- Combined effects of temperature and dietary lipid level on body composition, growth, and freshness profile in european seabass, dicentrarchus labraxPublication . Cardoso, Patrícia G.; Gonçalves, Odete; Cavalheri, Thais; Amorim, Vânia E.; Cao, Weiwei; Alexandrino, Diogo A. M.; Jia, Zhongjun; Carvalho, Maria F.; Vaz-Pires, Paulo; Ozório, Rodrigo O. A.The effects of increasing temperature and dietary lipid level on the body composition, growth performance, and freshness profile of the European seabass (Dicentrarchus labrax) were evaluated through a fish trial lasting 56 days. Findings demonstrated that fish reared at 24 °C presented a lower lipid level and a higher daily growth index than those reared at 20 °C. On the other hand, the sea bass condition index did not change among treatments. Additionally, sensory analysis (the Quality Index Method) and microbiological analysis revealed that fish reared at 24 °C showed better freshness conditions than those at 20 °C. Nevertheless, the dietary lipid level did not have any influence on fish freshness conditions. Therefore, our data suggest that the increase in temperature to 24 °C is beneficial for the growth and freshness profile of this particular species in aquaculture.
- Actinobacteria from arctic and atlantic deep-sea sediments—biodiversity and bioactive potentialPublication . Ribeiro, Inês ; Antunes, Jorge T. ; Alexandrino, Diogo A. M.; Tomasino, Maria Paola ; Almeida, Eduarda ; Hilário, Ana ; Urbatzka, Ralph ; Leão, Pedro N. ; Mucha, Ana P. ; Carvalho, Maria F.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.
- Exploring the possible link between fluoride sensitivity and bacterial defluorinationPublication . Maia, Tiago; Carvalho, Maria F.; Alexandrino, Diogo A. M.; Alexandrino, DiogoFluoorganic compounds are ubiquitous environmental pollutants due to their widespread use and high environmental persistence, mostly attributed to the stability of their carbon-fluoride bonds. The biotransformation of these compounds has been observed in some microorganisms, but defluorination (cleavage of carbon-fluorine bonds) remains the limiting step. Intracellular accumulation of fluoride occurs during microbial defluorination, which can cause several toxic effects. This work hypothesizes that intracellular fluoride stress may potentially affect the defluorination process in bacteria, limiting this critical catabolic step for the eventual mineralization of fluoroorganic pollutants. Fluoride sensitivity was first ascertained in defluorinating bacteria Labrys portucalensis F11 and Delftia acidovorans MFA5 (known degraders of fluorobenze1 and fluoroacetate2, respectively), with an Escherichia coli strain as the non-defluorinating control. Sensitivity was tested for increasing concentrations of f luoride (0-0.6 mM NaF) both in oligotrophic (minimal salts medium with acetate) and mesotrophic media (Nutrient Broth), based on bacterial growth inhibition for 48 hours at 28 ºC. After ascertaining their sensitivity thresholds, these strains are now being tested for their defluorination ability, against their preferred fluorinated substrates, when exposed to the NaF concentration with the highest observed growth inhibition. Results showed that fluoride stress was more severe in oligotrophic media, with 0.4 mM NaF presenting the highest growth inhibition among tested strains. Strain MFA5 was also shown to be the least sensitive to fluoride, while F11 was the most affected. Conclusions: Fluoride can exert cytostatic effects even in bacterial strains with proven ability to biodegrade fluorinated compounds. These results will allow to enlighten the ties between fluoride sensitivity and bacterial defluorination, thus broadening the knowledge on influencing factors of a critical catabolic reaction.
- Microcystin-degrading bacteria reduce bioaccumulation in Fragaria vulgaris and enhance fruit yield and qualityPublication . Haida, Mohammed; Khalloufi, Fatima El; Essadki, Yasser; Alexandrino, Diogo A. M.; Mugani, Richard; Hejjaj, Abdessamad; Campos, Alexandre; Vasconcelos, Vitor; Carvalho, Maria F.; Díez‑Quijada, Leticia; Cameán, Ana M.; Oudra, Brahim; Alexandrino, DiogoIn Morocco, red fruit production has thrived, primarily utilizing hydroponic methods to control crops, increase fruit yield and quality, and avoid soil-related problems. However, the irrigation of these expansive hydroponic farms relies heavily on water sourced from dams, many of which are contaminated with Microcystins (MCs). To address this contamination issue, ongoing research is focused on discovering effective and cost-efficient biological solutions for eliminating MCs. In this study, we isolate and identify bacterial strains capable of degrading MCs, evaluate the rate of degradation, and investigate how soil inoculated with these bacteria affects the accumulation of MCs in plant tissue. The partial 16S rRNA analyses of three bacterial sequences were conducted, identifying them through NCBI as follows: Ensifer sp. (B1) isolated from soil, Shinella sp. (B2) from a cyanobacterial bloom, and Stutzerimonas sp. (B3) from water. These bacteria exhibited the ability to degrade MCs, with approximately 34.75%, 73.75%, and 30.1% of the initial concentration (20 µg/L) being removed after a 6-day period for B1, B2, and B3, respectively. Moreover, strawberry plants were cultivated hydroponically in a greenhouse for a duration of 90 days. These plants were subjected to extracts of cyanobacteria containing 10 and 20 µg/L of Microcystins (MC), as well as water from an artificial lake contaminated with MC, both with and without the presence of isolated bacterial strains. Among these strains, Shinella sp. exhibited the highest efficacy in mitigating MC accumulation. Specifically, it resulted in a reduction of approximately 1.159 µg of MC per kilogram of root dry weight, leading to complete elimination in the leaves and fruits. The findings also indicated that the inoculation of perlite with the three MC-degrading bacterial strains significantly enhanced growth, photosynthetic pigments, yield, biochemical constituents, and quality attributes of strawberries (p≤0.05). These promising outcomes suggest the potential of this approach for addressing the adverse impacts of crops irrigated with MC-contaminated water in future agricultural practices.
- Impact of per- and polyfluorinated alkyl substances (PFAS) on the marine environment: Raising awareness, challenges, legislation, and mitigation approaches under the One Health conceptPublication . Bilela, Lada Lukić; Matijošytė, Inga; Krutkevičius, Jokūbas; Alexandrino, Diogo A.M.; Safarik, Ivo; Burlakovs, Juris; Gaudêncio, Susana P.; Carvalho, Maria F.; Alexandrino, DiogoPer- and polyfluorinated alkyl substances (PFAS) have long been known for their detrimental effects on the ecosystems and living organisms; however the long-term impact on the marine environment is still insufficiently recognized. Based on PFAS persistence and bioaccumulation in the complex marine food network, adverse effects will be exacerbated by global processes such as climate change and synergies with other pollutants, like microplastics. The range of fluorochemicals currently included in the PFAS umbrella has significantly expanded due to the updated OECD definition, raising new concerns about their poorly understood dynamics and negative effects on the ocean wildlife and human health. Mitigation challenges and approaches, including biodegradation and currently studied materials for PFAS environmental removal are proposed here, highlighting the importance of ongoing monitoring and bridging research gaps. The PFAS EU regulations, good practices and legal frameworks are discussed, with emphasis on recommendations for improving marine ecosystem management.
- Complementary field and laboratory batch studies to quantify generation rates of perfluoroalkyl acids in a contaminated agricultural topsoil with unknown precursorsPublication . Haluska, Alexander Arthur; Röhler, Klaus; Fabregat-Palau, Joel; Alexandrino, Diogo A. M.; Abramov, Sergey; Thompson, Katharine J.; Straub, Daniel; Kleindienst, Sara; Bugsel, Boris; Zweigle, Jonathan; Zwiener, Christian; Grathwohl, Peter; Alexandrino, DiogoSoil microbiome changes and generation rates of per- and polyfluoroalkyl substances (PFAS) precursors were studied in a contaminated agricultural field using a combination of field and laboratory batch microcosm studies. 16S rRNA gene amplicon sequencing was used to track how microbial community composition changed over time, while perfluoroalkyl acids (PFAA) generation rates were quantified using a combination of field and batch incubations combined with the direct total oxidizable precursor (dTOP) assay. The study site in Brilon-Scharfenberg, North Rhine-Westphalia, Germany, has PFAS contamination in the topsoil (0 to 30cm) originating from compost. Generation rate constants of these short-chain PFAA estimated from batch incubations (0.12 to 0.751/year) were higher but similar to rate constants from the fields (0.05 to 0.221/year). Long-term field mass discharge data (2009 to 2023) suggest that at least 60years are needed to remove 99.99% of short-chain PFAA and their precursors. 16S rRNA gene amplicon sequencing data revealed a major impact of PFAA on the biodiversity of soil microorganisms, with batch-incubated contaminated soils showing higher richness and diversity indexes than field control soils. However, most of these impacts occurred at lower taxonomical ranks and did not seem to have a prominent impact on the overall structure of the autochtonous microbial communities of the soils where PFAA were produced and accumulated. Overall, our findings demonstrate that well-controlled aerobic batch test combined with the results of dTOP assay are a suitable approach for estimating short-chain PFAA generation rates. Additionally, our research suggests that the complete removal of PFAA precursors from topsoil will take decades.
- Unveiling the culturable and non‐culturable actinobacterial diversity in two macroalgae species from the northern Portuguese coastPublication . Girão, Mariana; Alexandrino, Diogo A. M.; Cao, Weiwei; Costa, Isabel; Jia, Zhongjun; Carvalho, Maria F.; Alexandrino, DiogoActinomycetota, associated with macroalgae, remains one of the least explored marine niches. The secondary metabolism of Actinomycetota, the primary microbial source of compounds relevant to biotechnology, continues to drive research into the distribution, dynamics, and metabolome of these microorganisms. In this study, we employed a combination of traditional cultivation and metagenomic analysis to investigate the diversity of Actinomycetota in two native macroalgae species from the Portuguese coast. We obtained and taxonomically identified a collection of 380 strains, which were distributed across 12 orders, 15 families, and 25 genera affiliated with the Actinomycetia class, with Streptomyces making up approximately 60% of the composition. Metagenomic results revealed the presence of Actinomycetota in both Chondrus crispus and Codium tomentosum datasets, with relative abundances of 11% and 2%, respectively. This approach identified 12 orders, 16 families, and 17 genera affiliated with Actinomycetota, with minimal overlap with the cultivation results. Acidimicrobiales emerged as the dominant actinobacterial order in both macroalgae, although no strain affiliated with this taxonomic group was successfully isolated. Our findings suggest that macroalgae represent a hotspot for Actinomycetota. The synergistic use of both culture-dependent and independent approaches proved beneficial, enabling the identification and recovery of not only abundant but also rare taxonomic members.