Defluorination as the key trait to gauge the biodegradability of fluorinated pollutants in environmental microbial communities.

Research on microbial defluorination is largely centred on controlled experiments using axenic or well defined microbial inocula. These approaches serve a relevant purpose in the field, offering fundamental biochemical and mechanistic insights on the intricacies of biological defluorination. However, they fail to account for the effective contribution of environmental microbial communities in the recycling of fluoroorganic pollutants, a highly relevant perspective from an environmental risk assessment standpoint, while also missing an important outlook on how community-wide dynamics can leverage the breakdown of C─F bonds in these recalcitrant compounds. With that in mind, this chapter provides experimental and methodological insights on the study of microbial defluorination in wild environmental communities, using this critical catabolic step as the de facto endpoint to evolve, select and cultivate microorganisms with improved defluorination performances.

Unveiling the culturable and non-culturable actinobacterial diversity in two macroalgae species from the northern Portuguese coast.

Actinomycetota, 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.

Corrigendum: Actinobacteria from Arctic and Atlantic deep-sea sediments-Biodiversity and bioactive potential.

[This corrects the article DOI: 10.3389/fmicb.2023.1158441.].

Impact of per- and polyfluorinated alkyl substances (PFAS) on the marine environment: Raising awareness, challenges, legislation, and mitigation approaches under the One Health concept.

Per- 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.

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.

Combined Effects of Temperature and Dietary Lipid Level on Body Composition, Growth, and Freshness Profile in European Seabass, Dicentrarchus labrax.

A fish trial was carried out to evaluate the combined effects of temperature and dietary lipid level on the body composition, growth performance, and freshness profile of the European seabass (Dicentrarchus labrax). Fish were kept for 56 days at 20 °C and 24 °C and fed on two diets, with 16% and 20% lipid. At the end of the trial, fish were euthanized at two temperature conditions (0.6 °C or -0.6 °C) and kept on ice for 10 days at 4 °C to evaluate their freshness condition. Findings demonstrated that fish reared at 24 °C presented a lower lipid level and a higher daily growth index than those at 20 °C. Additionally, sensory analysis (Quality Index Method-QIM) and microbiological analysis revealed that fish reared at 24 °C showed better freshness conditions than those at 20 °C. However, the 16S rRNA metabarcoding analyses revealed a higher proliferation of genera associated with fish-spoiling bacteria in the skin microbiome of fish reared at 24 °C, i.e., Vibrio and Acinetobacter, which was not observed in the skin microbiome of fish reared at 20 °C. Nevertheless, the dietary lipid level did not have any influence on fish freshness. Therefore, our data suggest that the increase in temperature to 24 °C is beneficial for the growth and freshness profile (lower QIM and lower CFUs/cm2) of this particular species. Additionally, the lower euthanasia temperature (-0.6 °C) seems to lead to higher fish freshness than the normal temperature (0.6 °C).

Bioleaching of Heavy Metals from Printed Circuit Boards with an Acidophilic Iron-Oxidizing Microbial Consortium in Stirred Tank Reactors.

In this study, bioleaching was carried out for the recovery of metals (copper, zinc, tin, lead, gold and silver) from printed circuit boards residues (PCBs), one of the most important wastes from electrical and electronic equipment, using an acidophilic iron-oxidizing bacterial consortium enriched with minerals from a gold mine in the Arequipa region, Peru. High-throughput sequencing and analysis of the 16S rRNA biomarker revealed that this consortium was predominantly composed of Tissierella, Acidiphilium and Leptospirillum bacteria, from which the latter is known to grow by chemolithotrophy through iron oxidation. After the enrichment process, the acidophilic iron-oxidizing consortium was first tested for its tolerance to different PCBs concentrations, showing best growth up to 10 g/L of PCBs and a tolerance index of 0.383. Based on these results, the bioleaching efficiency of the consortium was investigated for 10 g/L of PCBs in stirred tank reactors coupled to an aeration system, for 18 days. High bioleaching efficiencies were achieved for copper and zinc (69% and 91%, respectively), indicating that these two metals can be easily extracted in this leaching system. Lower extraction efficiencies were achieved for tin (16%) and gold (28%), while for lead and silver only a residual recovery (<0.25%) was detected. These results indicate that the enriched bacterial consortium originating from the Arequipa region, Peru, has a high capacity to recover different metals of economic importance.

Revisiting pesticide pollution: The case of fluorinated pesticides.

Fluorinated pesticides acquired a significant market share in the agrochemical sector due to the surge of new fluoroorganic ingredients approved in the last two decades. This growing trend has not been accompanied by a comprehensive scientific and regulatory framework entailing all their potential negative impacts for the environment, especially when considering the hazardous properties that may result from the incorporation of fluorine into organic molecules. This review aims to address the safe/hazardous dichotomy associated with fluorinated pesticides by providing an updated outlook on their relevancy in the agrochemical sector and how it leads to their role as environmental pollutants. Specifically, the environmental fate and distribution of these pesticides in the ecosystems is discussed, while also analysing their potential to act as toxic substances for non-target organisms.

Atlas of the microbial degradation of fluorinated pesticides.

Fluorine-based agrochemicals have been benchmarked as the golden standard in pesticide development, prompting their widespread use in agriculture. As a result, fluorinated pesticides can now be found in the environment, entailing serious ecological implications due to their harmfulness and persistence. Microbial degradation might be an option to mitigate these impacts, though environmental microorganisms are not expected to easily cope with these fluoroaromatics due to their recalcitrance. Here, we provide an outlook on the microbial metabolism of fluorinated pesticides by analyzing the degradation pathways and biochemical processes involved, while also highlighting the central role of enzymatic defluorination in their productive metabolism. Finally, the potential contribution of these microbial processes for the dissipation of fluorinated pesticides from the environment is also discussed.

Combining Culture-Dependent and Independent Approaches for the Optimization of Epoxiconazole and Fludioxonil-Degrading Bacterial Consortia.

Epoxiconazole (EPO) and fludioxonil (FLU) are two widely used fluorinated pesticides known to be highly persistent and with high ecotoxicological potential, turning them into pollutants of concern. This work aimed to optimize two degrading bacterial consortia, previously obtained from an agricultural soil through enrichment with EPO and FLU, by characterizing the contribution of their corresponding bacterial isolates to the biodegradation of these pesticides using both culture-dependent and independent methodologies. Results showed that a co-culture of the strains Hydrogenophaga eletricum 5AE and Methylobacillus sp. 8AE was the most efficient in biodegrading EPO, being able to defluorinate ca. 80% of this pesticide in 28 days. This catabolic performance is likely the result of a commensalistic cooperation, in which H. eletricum may be the defluorinating strain and Methylobacillus sp. may assume an accessory, yet pivotal, catabolic role. Furthermore, 16S rRNA metabarcoding analysis revealed that these strains represent a minority in their original consortium, showing that the biodegradation of EPO can be driven by less abundant phylotypes in the community. On the other hand, none of the tested combinations of bacterial strains showed potential to biodegrade FLU, indicating that the key degrading strains were not successfully isolated from the original enrichment culture. Overall, this work shows, for the first time, the direct involvement of two bacterial species, namely H. eletricum and Methylobacillus sp., in the biodegradation of EPO, while also offering insight on how they might cooperate to accomplish this process. Moreover, the importance of adequate culture-dependent approaches in the engineering of microbial consortia for bioremediation purposes is also emphasized.

Harnessing the Potential of Native Microbial Communities for Bioremediation of Oil Spills in the Iberian Peninsula NW Coast.

Oil spills are among the most catastrophic events to marine ecosystems and current remediation techniques are not suitable for ecological restoration. Bioremediation approaches can take advantage of the activity of microorganisms with biodegradation capacity thus helping to accelerate the recovery of contaminated environments. The use of native microorganisms can increase the bioremediation efficiency since they have higher potential to survive in the natural environment while preventing unpredictable ecological impacts associated with the introduction of non-native organisms. In order to know the geographical scale to which a native bioremediation consortium can be applied, we need to understand the spatial heterogeneity of the natural microbial communities with potential for hydrocarbon degradation. In the present study, we aim to describe the genetic diversity and the potential of native microbial communities to degrade petroleum hydrocarbons, at an early stage of bioremediation, along the NW Iberian Peninsula coast, an area particularly susceptible to oil spills. Seawater samples collected in 47 sites were exposed to crude oil for 2 weeks, in enrichment experiments. Seawater samples collected in situ, and samples collected after the enrichment with crude oil, were characterized for prokaryotic communities by using 16S rRNA gene amplicon sequencing and predictive functional profiling. Results showed a drastic decrease in richness and diversity of microbial communities after the enrichment with crude oil. Enriched microbial communities were mainly dominated by genera known to degrade hydrocarbons, namely Alcanivorax, Pseudomonas, Acinetobacter, Rhodococcus, Flavobacterium, Oleibacter, Marinobacter, and Thalassospira, without significant differences between geographic areas and locations. Predictive functional profiling of the enriched microbial consortia showed a high potential to degrade the aromatic compounds aminobenzoate, benzoate, chlorocyclohexane, chlorobenzene, ethylbenzene, naphthalene, polycyclic aromatic compounds, styrene, toluene, and xylene. Only a few genera contributed for more than 50% of this genetic potential for aromatic compounds degradation in the enriched communities, namely Alcanivorax, Thalassospira, and Pseudomonas spp. This work is a starting point for the future development of prototype consortia of hydrocarbon-degrading bacteria to mitigate oil spills in the Iberian NW coast.

Diversity and Bioactive Potential of Actinobacteria Isolated from a Coastal Marine Sediment in Northern Portugal.

Natural compounds have had increasing applications in the biotechnological sector, with a large fraction of these substances being channeled to the pharmaceutical industry due to their important pharmacological properties. The discovery of new bioactive molecules with novel mechanisms of action constitutes a promising solution for the design of alternative therapeutic solutions. Actinobacteria are a large group of morphologically and physiologically diverse bacteria well known for their production of biotechnologically relevant compounds. The Portuguese coast is scantly explored in terms of Actinobacteria diversity and respective bioactive potential, offering a good opportunity to find new Actinobacteria taxa and bioactive natural products. In this study, we investigated the Actinobacteria diversity associated with a sediment sample collected from the intertidal zone of a beach in northern Portugal, through a cultivation-dependent approach, and screened its antimicrobial and cytotoxic potential. A total of 52 Actinobacteria strains were recovered from the marine sediment, with the largest fraction of the isolates belonging to the genus Micromonospora. Bioactivity screening assays identified crude extracts of six Streptomyces strains active against C. albicans, exhibiting minimum inhibition concentration (MIC) values in the range of 3.90-125 µg mL-1. Twenty-five Actinobacteria crude extracts (obtained from strains of the genera Micromonospora, Streptomyces and Actinomadura) exhibited significant effects on the viability of at least one tested cancer cell line (breast ductal carcinoma T-47D and liver hepatocellular carcinoma HepG2). The Actinobacteria extracts demonstrating activity in the antimicrobial and/or cytotoxic assays were subjected to metabolomic analysis (Mass spectrometry (MS)-based dereplication and molecular networking analyses), indicating the presence of four clusters that may represent new natural products. The results obtained demonstrate the importance of bioprospecting underexplored environments, like the Portuguese coast, for enhancing the discovery of new natural products, and call attention to the relevance of preserving the natural genetic diversity of coastal environments.

Microbial degradation of two highly persistent fluorinated fungicides - epoxiconazole and fludioxonil.

Biodegradation of two highly persistent fluorinated fungicides, epoxiconazole (EPO) and fludioxonil (FLU), by microbial consortia enriched from estuarine sediment and agricultural soil is reported. After an enrichment period of 6 months, four microbial consortia were able to completely remove and defluorinate the fungicides in co-metabolic conditions. Defluorination was biologically mediated and results suggest it is not a primary catabolic step, as fungicide removal was always faster than its defluorination. Three of the four enriched consortia had similar biodegradation performances in the absence of a co-substrate. Biodegradation kinetics revealed that microbial degradation followed a first-order kinetics, with cultures being capable of biodegrading concentrations up to 10 mg L-1 of EPO or FLU, in a maximum of 21 days. Estimated half-life values for these compounds were significantly lower than those reported in literature, highlighting the unique metabolic performance of the obtained consortia. Analysis of their microbial composition revealed that they integrate several bacterial species belonging to the Proteobacteria phylum, with the most common genera being Pseudomonas, Ochrobactrum and Comamonas. This is the first study providing clear evidence on the biodegradation of EPO and FLU, opening doors for the design of bioremediation technologies for the recovery of ecosystems polluted with such recalcitrant compounds.

Biodegradation of enrofloxacin by microbial consortia obtained from rhizosediments of two estuarine plants.

This study aimed to investigate the potential of microbial communities from the rhizosediment of two plants - Phragmites australis and Juncus maritimus - occurring in an estuarine area subjected to a high anthropogenic impact, to biodegrade ENR, a commonly used veterinary antibiotic. An enrichment process with 1 mgL-1 of ENR was conducted during ca. 9 months, using acetate as a co-substrate. After this, the enriched microbial consortia were challenged with higher ENR concentrations of 2 and 3 mgL-1. Microbial cultures enriched with 1 mgL-1 of ENR were capable of biodegrading this antibiotic, though not completely. By the end of the enrichment phase, microbial cultures were defluorinating an average of 50% of the ENR supplemented. Higher ENR concentrations led to lower biodegradation performances, suggesting a possible toxic/inhibitory effect in the microbial cultures. Phylogenetic identification of the microorganisms isolated from microbial cultures enriched with ENR revealed a high taxonomical diversity, with microorganisms belonging mainly to Proteobacteria and Bacteroidetes phyla. Assemblage of the obtained isolated strains (according to the enriched cultures from which they were isolated) revealed that the resulting consortia were also capable of degrading ENR, indicating that the main microbial players in the biodegradation of this antibiotic were isolated. These consortia also showed to be more robust to degrade higher concentrations of ENR than the corresponding enriched cultures. This study shows that microorganisms derived from rhizosediments of the selected plants, exhibit capacity to biodegrade ENR, though not completely for the concentrations tested, and may be further explored for the development of bioremediation strategies for the treatment of this antibiotic.

Biodegradation of oxytetracycline and enrofloxacin by autochthonous microbial communities from estuarine sediments.

This work investigated the potential of microbial communities native to an estuarine environment to biodegrade enrofloxacin (ENR) and oxytetracycline (OXY). Sediments collected from two sites in the Douro river estuary (Porto, Portugal) were used as inocula for the biodegradation experiments. Experiments were carried out for one month, during which ENR and OXY (1 mg L-1) were supplemented individually or in mixture to the cultures at 10-day intervals. Acetate (400 mg L-1) was added to the cultures every 3 days to support microbial growth. A series of experimental controls were established in parallel to determine the influence of abiotic breakdown and adsorption in the removal of the antibiotics. Removal of antibiotics was followed by measuring their concentration in the culture medium. Additionally, next-generation sequencing of the 16S rRNA gene amplicon was employed to understand how microbial communities responded to the presence of the antibiotics. At the end of the biodegradation experiments, microbial cultures derived from the two estuarine sediments were able to remove up to 98% of ENR and over 95% of OXY. The mixture of antibiotics did not affect their removal. ENR was removed mainly by biodegradation, while abiotic mechanisms were found to have a higher influence in the removal of OXY. Both antibiotics adsorbed at different extents to the estuarine sediments used as inocula but exhibited a higher affinity to the sediment with finer texture and higher organic matter content. The presence of ENR and OXY in the culture media influenced the dynamics of the microbial communities, resulting in a lower microbial diversity and richness and in the predominance of bacterial species belonging to the phylum Proteobacteria. Therefore, microbial communities native from estuarine environments have potential to respond to the contamination caused by antibiotics and may be considered for the recovering of impacted environments through bioremediation.

Biodegradation of mono-, di- and trifluoroacetate by microbial cultures with different origins.

This work focused on the biodegradation of three structurally related fluoroacetates (FAs), mono- (MFA), di- (DFA) and trifluoroacetate (TFA), using as microbial inocula samples collected from a site with a long history of industrial contamination and activated sludge obtained from a municipal wastewater treatment plant. Biodegradation experiments were carried out under different modes of substrate supplementation, which included (i) FAs fed as sole carbon sources; (ii) FAs (only for DFA and TFA) fed in co-metabolism with sodium acetate; and (iii) mixtures of MFA with DFA or TFA. Biodegradation of the target compounds was assessed through fluoride ion release. Defluorination was obtained in the cultures fed with MFA, while DFA and TFA were recalcitrant in all tested conditions. When present in mixture, DFA was shown to inhibit biodegradation of MFA, while TFA had no effect. A total of 13 bacterial isolates obtained from MFA degrading cultures were found to degrade 20mgL-1 of this compound, as single strains, when supplemented as a sole carbon source. Sequencing of the 16S rRNA gene indicated that among these degrading bacteria only Delftia acidovorans had been previously reported to be able to degrade MFA. This work shows that, despite their similar chemical structures, biodegradation of the three tested FAs is very distinct and draws attention to the unknown impacts that the accumulation of DFA and TFA may have in the environment as a result of their high recalcitrance.

Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics.

Fluoroquinolones and cephalosporins are two classes of veterinary antibiotics arising as pollutants of emerging concern. In this work, the microbial degradation of two representative antibiotics of both these classes, enrofloxacin (ENR) and ceftiofur (CEF), is reported. Biodegradation of the target antibiotics was investigated by supplementing the culture medium with ENR and CEF, individually and in mixture. Microbial inocula were obtained from rhizosphere sediments of plants derived from experimental constructed wetlands designed for the treatment of livestock wastewaters contaminated with trace amounts of these antibiotics. Selected microbial inocula were acclimated during a period of 5months, where the antibiotics were supplemented every three weeks at the concentration of 1mgL-1, using acetate as a co-substrate. After this period, the acclimated consortia were investigated for their capacity to biodegrade 2 and 3mgL-1 of ENR and CEF. Complete removal of CEF from the inoculated culture medium was always observed within 21days, independently of its concentration or the concomitant presence of ENR. Biodegradation of ENR decreased with the increase in its concentration in the culture medium, with defluorination percentages decreasing from ca. 65 to 4%. Ciprofloxacin and norfloxacin were detected as biodegradation intermediates of ENR in the microbial cultures supplemented with this antibiotic, indicating that defluorination of at least part of ENR in these cultures is not an immediate catabolic step. Abiotic mechanisms showed high influence in the removal of CEF, affecting less ENR degradation. The acclimation process with the target antibiotics led to significant shifts in the structure and diversity of the microbial communities, predominantly selecting microorganisms belonging to the phyla Proteobacteria (e.g. Achromobacter, Variovorax and Stenotrophomonas genera) and Bacteroidetes (e.g. Dysgonomonas, Flavobacterium and Chryseobacterium genera). The results presented in this study indicate that biodegradation can be an important mechanism for the environmental removal of the tested compounds.