Browsing by Author "Alexandrino, Diogo A. M."
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- 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.
- Assembly of bacterial consortium for the biodegradation of PFAS and related subproductsPublication . Neves, David M. B.; Pinto, Ana Sofia; Mucha, Ana Paula; Almeida, C. Marisa R.; Alexandrino, Diogo A. M.; Carvalho, Maria F.; Alexandrino, DiogoPer- and polyfluoroalkyl substances (PFAS) are man-made chemicals with wide application in consumer products since the 1950s. A recent revision of the PFAS definition has also introduced several polyfluorinated pharmaceuticals and agrochemicals into this class, further exacerbating the urgency of any PFAS-related pollution scenario. Their many favourable properties, including improved persistence and lipophilicity, has caused PFAS to be considered mobile pollutants with the capacity to accumulate in the environment for various decades. In fact, their increased presence in the aquatic environment has negative effects on the environment and human health, so it is of great importance to develop and improve remediation techniques to remove PFAS and other related subproducts from aquatic matrices. This work aims to create a synthetic bacterial consortium and study its capacity to degrade different PFAS and/or their subproducts. To achieve this, different fluoroorganic-degrading bacterial strains are currently being screened. Among them, a fluoroaliphatic (Delftia acidovorans MFA5) and a fluoroaromatic-degrading strains (Labrys portucalensis F11) have already been preselected to be included in the consortium. Soon, when a final selection of prospective fluoroorganic-degrading strains is achieved, their co-cultivation compatibility will be investigated through growth inhibition tests (cross-streak and diffusion disc activity assays). Strains with favourable co-cultivation dynamics will then be assembled in a synthetic bacterial consortium and tested for its ability to degrade different PFAS (individually) and related subproducts, based on bacterial growth analysis and on defluorination efficiency. This work will contribute to the ongoing effort of designing an efficient PFAS bioremediation unit to outfit a novel hybrid water treatment technology that combines nanophotocatalysis and bioremediation for the mitigation of PFAS aquatic pollution.
- 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.
- 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.
- Development of a bioremediation system for the removal of PFAS from aquatic environmentsPublication . Pinto, Ana Sofia; Neves, David M. B.; Maia, Tiago; Mucha, Ana Paula; Almeida, C. Marisa R.; Martins, Pedro; Lanceros-Mendez, Senetxu; Alexandrino, Diogo A. M.; Alexandrino, DiogoAmong the many pollutants that afflict aquatic ecosystems, PFAS (per- and polyfluoroalkyl substances) stand out due to their distribution, environmental persistence and ecotoxicity. Currently, there are no suitable remediation technologies capable of mitigating PFAS-related pollution in these ecosystems. Yet, the combination of nanophotocatalysis (NPC) and bioremediation (BRMD) may prove useful in combating PFAS aquatic pollution. NPC has shown promising results for the breakdown of persistent pollutants, while BRMD processes can benefit from the high degree of redundancy and promiscuity of bacterial catabolism to efficiently degrade various pollutants and their sub-products. As such, this work aims to develop an efficient BRMD unit, based on a synthetic bacterial consortium with orthogonal defluorination capacity, and explore its potential to act as a secondary water treatment step in tandem with a TiO2-based NPC treatment, to remove PFAS from aquatic matrices. The first development step, currently ongoing, is set on screening different bacterial strains enriched with fluorinated pollutants, petroleum hydrocarbons or cyanotoxins. This sorting is being achieved by validating the degradative capabilities of the strains based on their bacterial growth and defluorination performances. So far, these preliminary tests led to the selection of two prospective consortium members, Delftia acidovorans MFA5 and Labrys portucalensis F11, based on their capacity to defluorinate 50 mgL-1 of fluoroacetate and fluorobenzene in 8 days, respectively. Once all strains are selected and their co-cultivation dynamics are ascertained, a synthetic bacterial consortium will be assembled and tested as a BRMD step coupled to a primary NPC treatment against two different PFAS in quasi-real aquatic matrices.
- 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.
- Isolation of Actinomycetes from marine sediments with potential to produce bioactive compoundsPublication . Ribeiro, Inês; Alexandrino, Diogo A. M.; Oliveira, Rui S.; Santos, Chiara; Pereira, Filipe; Mucha, Ana P.; Carvalho, Maria F.The various health problems experienced by the humanity, together with an increasing number of antibiotic-resistant microorganisms have been driven scientists to look for additional reserves of new bioactive substances. Actinomycetes are an important source of bioactive compounds with industrial and pharmaceutical interest. The distribution of this vast microbial group in the oceans is largely unexplored, making oceans an untapped and promising source of novel bioactive compounds.
- Microbial degradation of Sodium Trifluoroacetate under aerobic and anaerobic conditionsPublication . Alexandrino, Diogo A. M.; Oliveira, Rui S.; Carvalho, M. FátimaThe extensive use of hydrofluorocarbons (HFCs) and hydrochlorofluorocarbons (HCFCs) as environmental acceptable alternatives to chlorofluorocarbons (CFCs) has been responsible for the introduction in the environment of significant amounts of Trifluoroacetate (TFA). Specifically, TFA is produced by the atmospheric breakdown of HCFC-123, HCFC-124, HFC-134a and HFO-1234yf, which undergo oxidative, photolytic and hydrolytic reactions to generate the compound. TFA is a highly persistent, water soluble and extremely stable fluorinated compound, that tends to accumulate in low-streamed aquatic environments, with marine ecosystems as its ultimate environmental sink. Although it does not exert considerable toxicological effects neither in microbial communities, nor in aquatic organisms, it reveals a mild toxicity regarding plants (Boutonnetet al. , 1999). There are no reports on the aerobic biodegradation of TFA, but the mineralisation of the compound by anaerobic methanogenic bacteria was demonstrated in few studies (Visscher, et al ., 1994; Kim et al., 2000).
- 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.