Microsecond time-resolved X-ray scattering by utilizing MHz repetition rate at second-generation XFELs.

Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining Jɑ helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes.

Biodegradation of e-waste microplastics by Penicillium brevicompactum.

Electronic and electric waste (e-waste) management strategies often fall short in dealing with the plastic constituents of printed circuit boards (PCB). Some plastic materials from PCB, such as epoxy resins, may release contaminants, but neither potential environmental impact has been assessed nor mitigation strategies have been put forward. This study assessed the biodegradation of microplastics (1-2 mm in size) from PCB by the fungus Penicillium brevicompactum over 28 days, thus contributing to the discussion of mitigation strategies for decreasing the environmental impact of such plastics in the environment. The capacity of P. brevicompactum to induce microplastic fragmentation and degradation has been determined by the increased the number of smaller-sized particles and microplastic mass reduction (up to 75 % within 14 days), respectively. The occurrence of chain scission and oxidation of microplastics exposed to P. brevicompactum when compared with the control conditions (which occurred only after 28 days of exposure) can be observed. Furthermore, Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy performed in dried biomass put in evidence an increase in the absorption intensities in regions that could be attributed to functional groups associated with carbohydrates. The results underline the potential role of the genus Penicillium, particularly P. brevicompactum, in the biodegradation of microplastics from PCB, thus providing the basis for further exploration of its potential for e-waste bioremediation and research on the underlying mechanisms for sustainable approaches to mitigate e-waste pollution.

Microplastics from agricultural mulch films: Biodegradation and ecotoxicity in freshwater systems.

The application of bio-based biodegradable mulch films in agriculture has raised environmental concerns regarding their potential impacts on adjacent freshwater ecosystems. This study investigated the biodegradation of microplastics derived from a bio-based biodegradable mulch (bio-MPs) and its acute and chronic ecotoxicity considering relevant scenarios (up to 200 and 250 mg/kg of sediment, using pristine and/or UV-aged particles), using the fungus Penicillium brevicompactum and the dipteran Chironomus riparius as model organisms, respectively, due to their ecological relevance in freshwater environments. Fourier-transform infrared spectroscopy analysis suggested changes in the fungus's carbohydrate reserves and bio-MP degradation through the appearance of low molecular weight esters throughout a 28 day biodegradation test. In a short-term exposure (48 h), C. riparius larvae exposed to pristine or UV-aged bio-MPs had up to 2 particles in their gut. Exposure to pristine bio-MPs decreased larval aerobic metabolism (<20 %) and increased neurotransmission (>15 %), whereas exposure to UV-aged bio-MPs activated larval aerobic metabolism (>20 %) and increased antioxidant defences (catalase activity by >30 % and glutathione-s-transferase by >20 %) and neurotransmission (>30 %). Longer-term (28-d) exposure to UV-aged bio-MPs did not affect larval survival and growth nor the dipteran's emergence but increased male numbers (>30 %) at higher concentrations. This study suggests that the selected agricultural bio-based mulch film is prone to biodegradation by a naturally occurring fungus. However, there is a potential for endocrine disruption in the case of prolonged exposures to UV-aged microplastics. This study emphasises the importance of further research to elucidate the potential ecological effects of these plastic products, to ensure effective management practices, and to establish new regulations governing their use.

Solid-State Fermentation as Green Technology to Improve the Use of Plant Feedstuffs as Ingredients in Diets for European Sea Bass (Dicentrarchus labrax) Juveniles.

This study aimed to evaluate the utilization by juvenile European sea bass of a SSFed PF mixture with Aspergillus niger CECT 2088. A 22-day digestibility and a 50-day growth trial were performed testing four diets, including 20 or 40% of an unfermented or SSFed PF mixture (rapeseed, soybean, rice bran, and sunflower seed meals, 25% each). SSF of the PF added cellulase and ß-glucosidase activity to the diets. Mycotoxin contamination was not detected in any of the experimental diets except for residual levels of zearalenone and deoxynivalenol (100 and 600 times lower than that established by the European Commission Recommendation-2006/576/EC). In diets including 20% PF, SSF did not affect growth but increased apparent digestibility coefficients of protein and energy, feed efficiency, and protein efficiency ratio. On the contrary, in diets including 40% PF, SSF decreased growth performance, feed intake, feed and protein efficiency, and diet digestibility. SSF decreased the intestinal amylase activity in the 40% SSFed diet, while total alkaline proteases decreased in the 20% and 40% SSFed diets. Hepatic amino acid catabolic enzyme activity was not modulated by SSF, and plasma total protein, cholesterol, and triglyceride levels were similar among dietary treatments. In conclusion, dietary inclusion of moderate levels of the SSFed PF, up to 20%, improves the overall feed utilization efficiency without negatively impacting European sea bass growth performance. The replacement of PF with the SSFed PF mixture may contribute to reducing the environmental footprint of aquaculture production.

Enrichment of a Plant Feedstuff Mixture's Nutritional Value through Solid-State Fermentation.

Plant feedstuffs are the main ingredients of animal feed. Owing to food-feed competition, increasing the utilization efficiency of these feedstuffs is important for animal nutrition. This can be achieved via solid-state fermentation (SSF). SSF of a plant feedstuff mixture (PFM) (25% rapeseed meal, soybean meal, rice bran, and sunflower meal) by three fungi (Aspergillus ibericus MUM 03.29, Aspergillus niger CECT 2088, and Aspergillus niger CECT 2915) resulted in an increase in protein content by 5%, irrespective of fungi, a reduction in cellulose content by 9 to 11%, and of hemicellulose content by 21 to 34%, relative to unfermented PFM. Enzyme production was measured: the highest cellulase (123.7 U/g), xylanase (431.8 U/g), and beta-glucosidase (117.9 U/g) activity were achieved with A. niger CECT 2088. Principal component analysis showed a positive correlation between all fermented PFMs and enzyme production, protein content, digestibility, and fiber reduction. Bioprocessing of the PFM by SSF increased its nutritional value and digestibility, making it more appealing for animal feeds.

Facemasks: An insight into their abundance in wetlands, degradation, and potential ecotoxicity.

Disposable facemasks represent a new form of environmental contamination worldwide. This study aimed at addressing the abundance of facemasks in an overlooked natural environment with high ecological and economic value - the wetlands (Ria de Aveiro, Portugal, as study case), evaluating their potential biodegradation using naturally occurring fungi and assessing the potential ecotoxicity of released microfibres on local bivalves. All masks collected within 6500 m2 area of Aveiro wetland were 100 % disposable ones (PP-based, confirmed by Fourier transform infrared spectroscopy - FTIR) with an initial abundance of 0.0023 items/m2 in Sept. 2021, which was reduced by ∼40 % in Apr. 2022 and ∼87 % in Sept. 2022, as a reflection of the government policies. Analysis of the carbonyl index (0.03 to 1.79) underlined their state of degradation, primarily due to sun exposure during low tides. In laboratory conditions, 1 mm2 microplastics obtained from new disposable facemasks were prone to biodegradation by Penicillium brevicompactum and Zalerion maritimum inferred from microplastics mass loss (∼22 to -26 % and ∼40 to 50 %, respectively) and FTIR spectra (particularly in the hydroxyl and carbonyl groups). In addition, microfibres released from facemasks induced sublethal effects on the clam, Venerupis corrugata, mostly in their UV-aged form when compared to pristine ones, characterised by a decrease in cellular energy allocation (CEA) and an increase in aerobic energy metabolism (ETS). Concomitantly, clams exposed to 1250 items/L of UV-aged microplastics (similar to field-reported concentrations) expressed greater clearance capacity, indicating a need to compensate for the potential energy unbalance. This study provides the first baseline monitoring of facemasks in wetlands while bringing new evidence on their biodegradation and ecotoxicity, considering environmentally relevant conditions and keystone organisms in such environments. Such studies require scientific attention for rapid regulatory action against this emerging and persistent pollutant, also targeting remediation and mitigation strategies considering these items under pandemic scenarios.

Solid-State Fermentation of Plant Feedstuff Mixture Affected the Physiological Responses of European Seabass (Dicentrarchus labrax) Reared at Different Temperatures and Subjected to Salinity Oscillation.

This study aimed to evaluate the effects of dietary inclusion of plant feedstuff mixture (PFM) pre-treated by solid-state fermentation (SSF) on the physiological responses of European seabass. For that purpose, two diets were formulated to contain: 20% inclusion level of non-fermented plant ingredients mixture (20Mix) and 20Mix fermented by A. niger in SSF conditions (20Mix-SSF). Seabass juveniles (initial body weight: 20.9 ± 3.3 g) were fed the experimental diets, reared at two different temperatures (21 and 26 °C) and subjected to weekly salinity oscillations for six weeks. Growth performance, digestive enzyme activities, humoral immune parameters, and oxidative stress indicators were evaluated. A reduction in weight gain, feed intake, and thermal growth coefficient was observed in fish fed the fermented diet (20Mix-SSF). Salinity oscillation led to an increase in weight gain, feed efficiency, daily growth index, and thermal growth coefficient, regardless of dietary treatment. Higher rearing temperatures also increased daily growth index. No dietary effect was observed on digestive enzymes activities, whereas rearing temperature and salinity oscillation modulated digestive enzyme activities. Oxidative stress responses were significantly affected by experimental diets, temperature, and salinity conditions. Catalase and glutathione peroxidase activities showed an interactive effect. Fish reared at 21 °C showed higher enzymatic activity when fed the 20Mix-SSF. Conversely, fish reared at 26 °C showed higher GPx activity when fed the 20Mix diet. Fish reared at 26 °C showed reduced peroxidase and lysozyme activities, while salinity fluctuation led to increased lysozyme activity and decreased ACH50 activity. ACH50 activity increased in fish fed the 20Mix-SSF. Overall, the dietary inclusion of PFM fermented by A. niger was unable to mitigate the impact of environmental stress on physiological performance in European seabass. In fact, fermented feed caused an inhibition of growth performances and an alteration of some physiological stress indicators.

Shelf-Life Performance of Fish Feed Supplemented with Bioactive Extracts from Fermented Olive Mill and Winery By-Products.

Fortifying fish feeds with bioactive compounds, such as enzymes and antioxidants, has been an adopted strategy to improve feed nutritional quality and sustainability. However, feed additives can lose activity/effectiveness during pelleting and storage processes. This work aimed to monitor functional activity stability in feeds supplemented with a bioactive extract, including cellulases, xylanases, and antioxidants. This bioactive extract (FBE) was produced by Aspergillus ibericus under solid-state fermentation of olive mill and winery by-products. Two isoproteic and isolipidic diets were formulated and unsupplemented or supplemented with lyophilized FBE (0.26% w/w). Both diets were stored at room temperature (RT) or 4 °C for 4 months. Results showed that feed storage at 4 °C enhanced the stability of the enzymes and cellulase was more stable than xylanase. Compared to RT, storage at 4 °C increased cellulase and xylanase half-life by circa 60 and 14%. Dietary FBE supplementation increased antioxidant activity and storage at 4 °C reduced antioxidant activity loss, while in the unsupplemented diet, antioxidant activity decreased to the same level in both storage temperatures. Dietary supplementation with FBE reduced lipid peroxidation by 17 and 19.5% when stored at 4 °C or RT, respectively. The present study is a step toward improving the storage conditions of diets formulated with bioactive compounds.

A full-length infectious cDNA clone of a dsRNA totivirus-like virus.

Totivirus-like viruses are a group of non-segmented double-stranded (ds)RNA viruses with two open reading frames, which were recently discovered and provisionally assigned to the Totiviridae family. Unlike yeast and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first infectious full-length cDNA clone of a totivirus-like virus, the Omono River virus (OmRV), and produced infectious particles using an RNA-transcript-based method. Compared with the parent wild-type particles from nature, the infectious-cloning OmRV particles have presented strong cytopathic effects, infectivity and similar morphology. Thus far, the established system is one of the few reported systems for generating a non-segmented dsRNA virus cDNA clone.

Are mulch biofilms used in agriculture an environmentally friendly solution? - An insight into their biodegradability and ecotoxicity using key organisms in soil ecosystems.

Biobased and biodegradable plastic mulch films (aka, mulch biofilm) have emerged as a sustainable alternative to conventional plastic mulch films in agriculture, promising to reduce soil contamination with plastic residues through in situ biodegradation. However, current standards certifying biodegradable plastics cannot predict biodegradability in natural settings. The scarce studies considering the possible biodegradation and ecotoxicity of mulch biofilms in soil systems question the environmental friendliness of these alternative options. This study assessed the biodegradation of a commercially available mulch biofilm by the soil-dwelling fungus Penicillium brevicompactum (in solid culture media and soil for 15 and 28 days, respectively), and the ecotoxicological effects of mulch biofilm microplastics on the earthworm Eisenia andrei (pristine or UV-weathered, at 0.125-0.250-0.500 g/kg). Results (from microplastics' mass loss, microscopy, and FTIR spectroscopy) suggest that the presence of P. brevicompactum promotes mulch biofilm's biodegradation. Exposure to environmental concentrations of pristine biofilm microplastics (and its ingestion) increased earthworms' sensitivity to touch, induced physiological alterations, decreased energy reserves, and decreased their reproduction (>30%). Conversely, exposure to weathered biofilm microplastics slightly increased earthworms' sensitivity, as well as carbohydrate reserves,without affecting their reproduction. The tested mulch biofilm seems to be, at first sight, an environmentally friendly alternative as it presented susceptibility for biodegradation by a widespread fungus, and the absence of ecotoxicological chronic effects on a key macroinvertebrate species in soil ecosystems when considering environmental relevant concentrations and plastics weathered conditions. Notwithstanding, the obtained results highlight the need to revise current standards, as they often neglect the role of, and their chronic effects on, naturally occurring organisms.

Are Biobased Plastics Green Alternatives?-A Critical Review.

Environmental sustainability is driving an intense search for "green materials". Biobased plastics have emerged as a promising alternative. Their building blocks can now be obtained from diverse biomass, by-products, and organic residues due to the advances in biorefineries and bioprocessing technologies, decreasing the demand for fossil fuel resources and carbon footprint. Novel biobased polymers with high added value and improved properties and functionalities have been developed to apply diverse economic sectors. However, the real opportunities and risks of such novel biobased plastic solutions have raised scientific and public awareness. This paper provides a critical review on the recent advances in biobased polymers chemistry and emerging (bio)technologies that underpin their production and discusses the potential for biodegradation, recycling, environmental safety, and toxicity of these biobased solutions.

Preliminary comparative analysis of the resolving power of COX1 and 16S-rDNA as molecular markers for the identification of ticks from Portugal.

The utility of mitochondrial cytochrome oxidase subunit I (COX1) and 16S ribosomal DNA (16S-rDNA) sequence analyses as a complementary/alternative tool to classical taxonomy, for the identification of some of the most prevalent hard tick species from Portugal was evaluated using BOLD-ID (COX1 only), BLASTn and phylogenetic tree reconstruction based on multiple nucleotide sequence alignments. Both molecular markers proved suitable for identifying ticks to a species level, but specific aspects that limit their resolving power must be considered. Their accuracy of tick identification in all life stages and of the other tick species described in the South of Europe is required.