Percorrer por autor "Alexandrino, Diogo"
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- 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.
- Bioprospecting for cellulose-degrading microorganismsPublication . Almeida, Liliana; Alexandrino, Diogo; Oliveira, Rui S.; Carvalho, M. Fátima; Freitas, Marisa; Freitas, MarisaCellulose, a complex polysaccharide, is one of the main components of plant and algae biomass and therefore the most abundant polymer found in nature, serving as a significant carbon source in various ecosystems. Bioprospecting for cellulolytic microbes is a promising strategy for discovering potential biocatalysts for the hydrolysis of lignocellulosic materials, including residues. This work aims to assess the cellulolytic potential of microbial isolates (bacteria and fungi) sourced from diverse environments and to establish a synthetic microbial consortium capable to enhance the management of cellulosic waste. The experimental approach encompasses biodegradation assays coupled with optimized colorimetric methods, to estimate cellulose consumption (Congo Red assay) and the concomitant production of oligomeric/monomeric sugars (Dinitrosalicylic acid assay). For bacterial strains, these biodegradation assays involve batch incubations in minimal liquid medium supplied with cellulose as the sole carbon source, while solid medium is being used for fungi. Both assays are being run under mesophilic temperatures (28-30 ºC) and aerobic conditions. Upon achieving a final selection, strains exhibiting the best cellulose degradation performances will be assembled into a synthetic consortium that will be evaluated for their cellulose degradation capability. Future steps will also include phylogenetic identification of the cellulose degrading isolates through 16S and ITS rRNA gene sequence analysis. This study has the potential to identify novel cellulolytic microbes, highlighting the substantial role microorganisms play in ecosystem processes and their diverse applications in biotechnology.
- 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.
- 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.
- 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.
- 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.