To the Editor:Eosinophilic airway inflammation is present in approximately half of patients with asthma, and it is particularly associated with asthma attacks. IL-5 is a major cytokine involved in eosinophil differentiation, proliferation, activation, and eosinophil-mediated inflammatory responses . Severe Pharmacodynamic studies of mepolizumab and benralizumab have only been undertaken over longer time periods, and their findings are difficult to compare, as baseline and change in blood eosinophils have been expressed differently –9. Theren = 6) for the treatment of poorly controlled eosinophilic asthma or commencing maintenance treatment with mepolizumab 100 mg subcutaneously (n = 6) or benralizumab 30 mg subcutaneously (n = 6) for poorly controlled severe eosinophilic asthma . We assessed blood eosinophil counts at baseline , at 2 and 4 hours after the first dose for all patients, and at 6, 8, 24, and 96 hours if the blood eosinophil count remained higher than 150 cells/μl. A 30-day blood eosinophil count was obtained for those who received the mepolizumab or benralizumab injection. There is no previous information on the onset of action of mepolizumab and benralizumab; however, the effect of prednisolone starts in about 2 hours, so we included the time points 2 and 4 hours, and later time points to show the predicted slower onset of mepolizumab. Hematological analysis was done by laboratory staff using an Abbott Architect ci8200 analyzer.This was a substudy of the Oxford Airways Study . The Oxford Airways Study is an observational study of patients with asthma and chronic obstructive pulmonary disease, which allows blood, sputum, and bronchoscopy sampling at stable state and during exacerbations, before and after clinical treatment. The main outcome of this substudy was time to reach a 50% reduction in blood eosinophil count after administration of mepolizumab, benralizumab, and prednisolone. Patients were recruited from the Oxford Special Airways Clinic. Inclusion criteria were having a known diagnosis of severe asthma and baseline blood eosinophil count above 300 cells/μl. Subjects who had taken prednisolone in the preceding 2 weeks or who were already on a biologic treatment were excluded. We recruited 18 patients who were either commencing prednisolone 30 mg daily for 5 days   and higher than in the prednisolone arm at 4 hours . The geometric mean (geometric SD) blood eosinophil counts 30 days after benralizumab and mepolizumab were 8 (2.8) and 92 (1.7) cells/μl, respectively (P = 0.002).Demographics, inhaled corticosteroid dose, lung function, baseline blood eosinophil count, and exhaled nitric oxide levels were not significantly different between groups . The meaoc test) . A bloodTo the best of our knowledge, this is the first study comparing the rate of depletion of blood eosinophils in the first 24 hours after treatment with mepolizumab and benralizumab. We found that, in contrast to mepolizumab, benralizumab caused rapid and near complete depletion of eosinophils with a speed of onset of effect very similar to that seen with oral prednisolone. The blood eosinophil count 30 days after the first injection was also significantly lower after benralizumab compared with mepolizumab. These findings are consistent with greater efficacy and a different mechanism of eosinophil depletion.Mepolizumab and benralizumab have proven and similar efficacy and safety in the longer-term treatment of severe eosinophilic asthma , 3, sugg


Within this study, a set of multiple structure-based pharmacophore models for HPPD inhibitors were developed. The ZINC and natural product database were virtually screened, and 29 compounds were obtained. The binding mode of HPPD and its inhibitors obtained through molecular docking study showed that the residues of Phe424, Phe381, His308, His226, Gln307 and Glu394 were crucial for activity. Molecular-mechanics-generalized born surface area (MM/GBSA) results showed that the coulomb force, lipophilic and van der Waals (vdW) interactions made major contributions to the binding affinity. These efforts will greatly contribute to design novel and effective HPPD inhibitory herbicides. In recent years, crops severely suffer from infestations of various weeds, and the weeds resistance are increasing seriously . The dev4-Hydroxyphenylpyruvate dioxygenase (HPPD) is one of the most important herbicide target enzymes . It is aArabidopsis thaliana HPPD (AtHPPD) were identified, uploaded and stored in the protein database (RCSB PDB), including 11 protein–ligand complexes and 10 resolutions less than three angstroms  and ligand (fitting protein and ligand itself) during the 100 ns simulations is shown in Gbind, ΔGbind coulomb, ΔGbind Hbond, ΔGbind lipo and ΔGbind vdW parameters involved in the calculation are shown in Gbind value was 55.129 kcal/mol, the ΔGbind coulomb value was −48.956 kcal/ mol, the ΔGbind Hbond was −0.530 kcal/mol, nonpolar solvation (ΔGbind lipo) value was −26.162 kcal/mol and van der Waals (ΔGbind vdW) value was −41.324 kcal/mol. From the parameters calculated for each compound, it could be easily concluded that ΔG bind coulomb and ΔG bind vdW were the main contributors to the binding free energy of compounds. Noticeably, it was also detected that ΔG covalent, providing unfavorable energy, was negative to the binding of protein.MM/GBSA is a molecular mechanics of binding energy, solvation model, nonpolar solvation term, nonpolar solvent accessible surface area and van der Waals interaction method to define the free energy of binding . MM/GBSASix crystallographic structures of HPPD protein were collected from the RCSB PDB database . The interaction between the HPPD protein and potential ligands was calculated using LS. Although these models have similar chemical characteristics, they all describe slightly different interaction patterns that may occur within the HPPD binding site. For example, both the 1TG5 and 1TFZ models contain a hydrogen bond (green) effect with HIS287 and a hydrophobic (yellow) effect with Phe398 and Phe360, but the 1TFZ model has one more aromatic ring effect (blue) than the 1TG5 model, the same as 5YY6. Among the amino acids in the 5YWK and 5YWH models, Phe424, Phe381, Phe392 and Met335 all produced hydrophobic groups (yellow), but the amino acid His308 in 5YWH produced hydrogen bonds (green) as shown in The co-crystallized ligand benquitrione was redocked into the corresponding 5YY6 protein binding pocket . The RMSFlexible docking was performed with the 29 selected candidates against the multiple conformers of the receptor protein 5YY6. Six small molecules with optimal comprehensive conditions were obtained according to the conditions of docking score and the action mode of amino acid. Docking score for the 6 compounds is shown in Analysis of the cocrystal structures shows some interactions predicted by our ensemble docking model A. SimulaProtein interactions with the ligand were monitored and normalized by a timeline representation over the course of the trajectory  energy term was the major contributor to molecular binding. Overall, the results provide a set of guidelines for design novel and more potent HPPD inhibitors.In conclusion, a reasonable and hierarchical virtual screening process was successfully constructed to identify potential HPPD inhibitors. Molecular docking revealed that compounds ZINC00004910836, ZINC000040310216 and ZINC000003830381 all produced metal coordination with amino acids His226, His308 and Glu394 and formed π–π interactions with amino acids Phe424 and Phe381, and all of them produced more important hydrogen bond interaction with the protein. MD simulation proved the existence of metal coordination bonds and the influence of π–π interaction, which was consistent with the previous molecular docking results. The MD simulation also confirmed that three of the six compounds had high binding efficiency with HPPD. The binding free energy calculated by MM/GBSA demonstrated that the van der Waals  . Then optimize these models to optimize their prediction performance. In the first optimization stage, the size of the features were deleted or adjusted to allow as many active ingredients as possible to draw pharmacophores and exclude inactive compounds according to the contribution map (ex.html)  and the The 6 pharmacophores obtained above are subjected to multiple virtual screening of 98,133 compounds from the ZINC and Natural Product database. If the number of molecules obtained by the primary screening is too large, the first 200 matching high scores are taken and finally, the selected groups of compounds are summarized to obtain a common intersection compound.A total of 29 compounds out of 98,133 were found to be–in high fitness with the pharmacophore model. Using LigPrep, the ionization states, tautomeric forms and 3D conformation of 29 intersection compounds were generated . The default conditions were maintained, except for the ionization states which were generated at the target pH of 7.0 ± 0.2 using Epik including the original state . The resThe protein preparation workflow in Maestro (Schrödinger) was used to preprocess the HPPD protein structure to assign bond sequences and optimize the structure, including hydrogen bond optimization and constrained minimization. Use Schrödinger Prime to add missing side chains when necessary. For each structure, a protein chain with the co-crystal ligand was retained, and more than 5 Å of water molecules were removed from the heteroatom group . The intFor the obtained six HPPD protein structures representations, Schrodinger Glide were used to generate docking-grids around the cocrystal binding sites under the default settings. Each cocrystal ligand was re-docked into its corresponding structure validated model; in each case, the cocrystal posture was reproduced with root mean square deviation (RMSD) value <1.5 Å.The docking simulation was performed in Glide. The ligand representation of all compounds obtained through LigPrep was docked with the extra precision (XP) of the default settings of all 6 HPPD models prepared using Glide (Schrodinger), except writing out at most 5 poses per ligand representation and including 32 poses per ligand for post-docking minimization . The obtThe complex was introduced into the MD simulations in the Desmond (v3.8) module of Schrödinger software. The simulation system was built in an auto calculated trapezoidal box, which was dissolved with simple point charge (SPC) water and neutralized with appropriate number of counter ions. In addition, the OPLS_2005 force field was used to minimize the energy of complex systems, where the maximum interaction was set to 2000 and convergence threshold was set to 1.0 kcal/mol/Å . The steGbind was calculated with the MM/GBSA methodology was applied based on stable MD trajectory.The initial MM/GBSA calculation uses initial and MD to optimize the receptor ligand complex. Use all default parameters, including the VSGB 2.0 solvation model. Calculation of Schrodinger software suite ,44. The Gcomplex is the free energy of the complex; Greceptor is the free energy of the receptor; Gligand is the free energy of the ligand.


Fiber–matrix interfacial adhesion is one of the key factors governing the final properties of natural fiber-based polymer composites. In this work, four extrusion reactive agents were tested as potential compatibilizers in polyhydroxylbutyrate (PHB)/cellulose composites: dicumyl peroxide (DCP), hexamethylene diisocyanate (HMDI), resorcinol diglycidyl ether (RDGE), and triglycidyl isocyanurate (TGIC). The influence of the fibers and the different reactive agents on the mechanical properties, physical aging, and crystallization behavior were assessed. To evaluate the compatibilization effectiveness of each reactive agent, highly purified commercial cellulose fibers (TC90) were used as reference filler. Then, the influence of fiber purity on the compatibilization effect of the reactive agent HMDI was evaluated using untreated (U_RH) and chemically purified (T_RH) rice husk fibers, comparing the results with the ones using TC90 fibers. The results show that reactive agents interact with the polymer matrix at different levels, but all compositions showed a drastic embrittlement due to the aging of PHB. No clear compatibilization effect was found using DCP, RDGE, or TGIC reactive agents. On the other hand, the fiber–polymer interfacial adhesion was enhanced with HMDI. The purity of the fiber played an important role in the effectiveness of HMDI as a compatibilizer, since composites with highly purified fibers showed the greatest improvements in tensile strength and the most favorable morphology. None of the reactive agents negatively affected the compostability of PHB. Finally, thermoformed trays with good mold reproducibility were successfully obtained for PHB/T_RH/HMDI composition. The development of biobased biodegradable thermoplastic materials is a topic research of special interest because it can represent a cost-effective and environmental-friendly alternative to commodities . Among tHowever, PHB presents some shortcomings that limit its industrial applicability. PHB is a semicrystalline polymer that is capable of a high degree of crystallinity but has a relatively low crystallization rate. Hence, PHB suffers an appreciable embrittlement with time due to secondary crystallization and physical aging ,15,16,17Lignocellulosic fibers are hydrophilic materials composed by bundles of cellulose fibers embedded in a matrix of other non-cellulosic materials such as lignin, hemicelluloses, pectin, waxes, and other minor components . The advIt is well known that the resultant properties of fiber-based composites depend not only on the properties of the constituents but are also determined by the fiber–matrix adhesion. The hydrophilic nature of the lignocellulosic fibers lowers the compatibility with the hydrophobic polymer. Nevertheless, according to Bhardwaj et al. , the relSome attempts to improve interfacial adhesion are physical treatments (plasma or corona discharge), chemical purification treatments (dewaxing and delignifying treatments) of the fibers, grafting, or the use of additives such as compatibilizers or coupling agents ,28,29. RAnother strategy could be by using radical generators that could arouse random linkages between the matrix and the reinforcement via radical intermediate species, such as peroxides. Dycumil peroxide (DCP) has been used to compatibilize PHBV/Miscanthus fiber composites  or PHB aIn this work, the efficiency as compatiblizers of four different reactive agents in fiber-based PHB composites was tested. The reactive agents used were dicumyl peroxide (DCP), hexamethylene diisocyanate (HMDI), resorcinol diglycidyl ether (RDGE), and tryglicidyl isocyanurate (TGIC). The chemical structures of them are shown in The effect of the different reactive agents on the PHB/cellulose interfacial interactions was studied by scanning electron microscopy (SEM), tensile tests, and dynamic mechanical analysis (DMA). Indeed, the effect of aging was assessed for all compositions. As maintained biodegradability is an important requirement for the applicability of these systems, the effect of the different reactive agents on the biodisintegration under standard composting conditions (ISO 20200) was also evaluated. With the aim of analyzing the influence of fiber purity on the compatibilization efficiency, untreated rice fibers (U_RH) and chemically purified rice husk fibers (T_RH) according to a previous work  were useFinally, since packaging is one of the potential application fields for these composites, the suitability of PHB/T_RH/HMDI composites to be processed by thermoforming was tested. This process has been chosen for both its difficulty and for being one of the most popular forming techniques used in the packaging industry.®  in pellet form (P309). Purified alpha-cellulose fiber grade with an alpha-cellulose content >99.5% (TC90) was purchased from CreaFill Fibers Corp. . Rice husk (RH) by-product from the rice production process was kindly provided by Herba Ingredients . The four reactive agents used (dicumyl peroxide (DCP), hexamethylene diisocyanate (HMDI), resorcinol diglycidyl ether (RDGE), and triglycidyl isocyanurate (TGIC)) were purchased from Sigma Aldrich . Sodium hydroxide , hydrogen peroxide , glacial acetic acid , and sulfuric acid  were purchased from Sigma Aldrich .Poly(3-hydroxybutyrate) was supplied by Biomerwt/v, fiber/liquid ratio of 1:20, 80 °C, 2 h). This treatment was applied twice. The second stage consisted of an oxidative attack with peracetic acid (PAA) . The peracetic acid was prepared by the mixing of 30%(v/v) hydrogen peroxide and acetic acid in the reaction medium with a volume ratio of 3:1 at room temperature and 1% (w/w) of sulfuric acid as catalyzer. This procedure was adapted from the literature [RH fibers were ground in a mechanical knife mill and then sieved in 140 µm mesh. These untreated RH fibers were named U_RH. A fraction of the ground and sieved RH fibers were subjected to a two-stage purification treatment in order to remove the major parts of impurities and non-cellulosic components such as waxes, lignin, and hemicelluloses. The first stage consisted of an alkaline attack with NaOH  were prepared with all reactive agents. The effect of the cellulose purity was studied on rice husk fibers, with and without chemical treatment, using HMDI as the reactive agent. For the sake of comparison and to evaluate the effects of the compatibilizers on the matrix, blank compounds (without cellulose) were prepared as controls. All the compositions studied are summarized in The compounds were prepared by melt extrusion in a twin-screw co-rotating extruder  with an L/D ratio of 24 and a diameter of 2.5 cm. All the components were dried before extrusion; PHB pellets were dried in a dehumidifier Piovan DPA50  at 60 °C following the producer’s drying recommendations and the fibers  were dried in an oven at 100 °C for at least 2 h. The formulations were manually premixed in zip-bags. The temperature profile of the extruder was set as follows: 165/170/175/180 °C (from the hopper to the extruder die), and the screw speed was kept constant at 40 rpm. The extrudate material was pelletized and dried following the same considerations as pure PHB.From the extruded pellets, different samples were obtained by compression molding in a parallel plate hot-press : bars of 50 × 12.5 × 3.5 mm for dynamic mechanical analysis tests, films of 0.4 mm nominal thickness for uniaxial mechanical tests, films of 0.2 mm nominal thickness for composting tests, and films of 0.8 mm nominal thickness for thermoforming essays. Samples with neat PHB were processed and tested at the same conditions as the compounds.The morphology of PHB/TC90, PHB/U_RH, and PHB/T_RH composites with and without reactive agents was examined by scanning electron microscopy (SEM), using a high-resolution field-emission microscope . The samples were prepared by cryofracturing after immersion in liquid nitrogen and then coated by sputtering with a thin layer of Pt. c), melting temperatures (Tm), and melting enthalpies (ΔHm) were calculated from all respective heating/cooling scans. The crystallinity (Xc) of the PHB–reactive agent compositions was determined by applying the expression (1) [m (J/g) is the melting enthalpy of the polymer matrix, ΔH°m is the melting enthalpy of 100% crystalline PHB  (146 J/g) [Differential scanning calorimetry (DSC) experiments were conducted on a DSC2  with an intracooler Julabo FT900  calibrated with Indium standard before use. Samples were analyzed at 0 days (after hot-pressed films obtention) and after 100 days, to account for physical aging at room temperature. The samples weighing typically 6 mg were first heated from −20 °C to 200 °C at 10 °C/min, kept for 5 min to erase thermal history, and cooled down to −20 °C at 10 °C/min. Then, a second heating scan to 200 °C at 10 °C/min was performed. Crystallization temperatures (Tsion (1) :(1)Xc(%)146 J/g) , and w iTensile tests were conducted in a universal testing machine Shimatzu AGS-X 500N  at room temperature with a crosshead speed of 10 mm/min. Dumbbell 400 µm-thick samples were die-cut from the hot-pressed films and tested according to ASTM D638 (Type IV) standard. The samples were tested immediately after processing (0 days) and after 15 days of aging at room temperature. All the samples were stored in a vacuum desiccator at ambient temperature until tested.Dynamic mechanical analysis (DMA) experiments were conducted on hot-pressed sample bars (55 × 12.5 × 3.5 mm) in an AR G2 oscillatory rheometer  equipped with a clamp system for solid samples (torsion mode). Samples were heated from −20 °C to melting temperature with a heating rate of 2 °C/min at a constant frequency of 1 Hz. The maximum deformation (γ) was set to 0.1%. 3) obtained from hot-pressed plates. Solid synthetic waste was prepared by mixing 10% of activated mature compost , 40% sawdust, 30% rabbit feed, 10% corn starch, 5% sugar, 4% corn seed oil, and 1% urea. The water content of the mixture was adjusted to 55%. The samples were placed inside mesh bags to simplify their extraction and allow the contact of the compost with the specimens; then, they were buried in compost bioreactors at 4–6 cm depth. Bioreactors were incubated at 58 °C. The aerobic conditions were guaranteed by mixing the synthetic waste periodically and adding water according to the standard requirements. Two replicates of each sample were removed from the boxes at different composting times for analysis. Samples were washed with water and dried under vacuum at 40 °C until reaching a constant mass. The disintegration degree was calculated by normalizing the sample weight to the initial weight with Equation (2):i is the initial dry mass of the test material and mf is the dry mass of the test material recovered at different incubation stages. The disintegration study was completed taking photographs for visual evaluation.Disintegration tests under standard composting conditions (ISO 20200 ) were caThe thermoformability of PHB/T_RH/HMDI was tested by a vacuum-assisted thermoforming technique in a pilot plant  equipped with an infrared emitter heating device. The mold used was a female circular tray that was 55 mm in diameter and 15 mm in depth with an edge radium of 5 mm. Rectangular hot-pressed sheets of a typical thickness of 800 μm were used for this study. The sheets were stamped with a square grid pattern (0.5 × 0.5 cm) in order to track the deformation that occurred during their mold conformation. The infrared heater was set to 600 °C, whereas the heating and vacuum times  were optimized in each case to obtain the best results. In order to assess the role of the reactive agents, blends with TC90 were prepared as detailed in the experimental section. The morphology of the PHB/TC90 composites with and without the reactive agents has been analyzed by SEM. Low magnification images were used to study the distribution of the fibers within the polymer matrix, and high magnification ones were used to examine the fiber/matrix interface. The micrographs of the different composites are shown in As it can be observed in Regarding the reactive agents, no remarkable differences in morphology are detected in PHB/TC90 composites with reactive agents compared with the composite without them. Although the major part of the fibers seems to be well embedded into the polymer matrix, some pull-out and detached fibers are detected, as well as a small gap between the fibers and the matrix. With regard to the use of DCP, contrary to other works reported in literature ,36,42, iDSC experiments were run in all samples. The thermograms were obtained from the films recently processed (0 days) and after 100 days of storage at room temperature, when it is supposed that all secondary crystallization and physical aging phenomena has taken place ,43. It cThe melting behavior and crystallinity index during first heating scans of the composites are affected in different extents by the different components. However, it must be considered that the crystal morphologies developed during cooling correspond to processing conditions, which implies higher cooling rates with respect to DSC-controlled cooling at 10 °C/min.c) of 49% at 0 days. After aging, Xc increases to 59%, and the melting peak temperature changes to 170 °C, due to secondary crystallization [c of 91 J/g, which corresponds to an Xc value of 63% (for either aged or unaged samples).Neat processed PHB presents a melting peak temperature of 175 °C and a crystallinity index  seems to partially hinder the secondary crystallization , as the c values than the other compositions, finding a reduction of Tc from 117 °C to 106 °C in PHB/HMDI and 111ºC in PHB/TC90/HMDI samples. These findings can be related with some hindered motion of the polymer chains, thus suggesting the interaction of HMDI with the polymer matrix and the fibers [After erasing the thermal history, no remarkable differences in crystallization temperatures or enthalpies were observed among the compounds, except in the case of HMDI addition. Compounds with HMDI showed lower Te fibers . m and Xc was detected, being in agreement with some crosslinking of the PHB matrix with the peroxide initiator DCP [With respect to melting in second heating scans, after low cooling rates where polymer chains had enough time and mobility to develop high crystallinity (10 °C/min during DSC test conditions), the crystallinity indexes and melting temperatures of the different compositions were similar to those corresponding to neat PHB, except for the compositions containing DCP. For DCP-containing compounds, significant reductions of Tator DCP . PHB is known by its physical aging and secondary crystallization ,16,43. SNeat PHB shows a high variation in its tensile behavior due to aging. The elastic modulus increased more than 100% after 15 days, and the deformation at break decreased five times. In both cases, the failure mode is brittle, with unstable fracture and without showing necking nor evidence of shear yielding. The cause for such behavior is attributed to secondary crystallization and the physical aging of the amorphous region ,43.When reactive agents are added to PHB, at 0 days, there is a minimum increase in elastic modulus in the case of addition of DCP, RDGE, and TGIC and a more pronounced one with HMDI . HoweverWhen cellulose (TC90) is added to neat PHB (at 0 days), an increase in elastic modulus and a reduction of tensile strength and ductility is observed . The incWhen reactive agents are added, the elastic modulus rises in all cases with respect to either cellulose without reactive agents or PHB with reactive agents. Similarly, the tensile strength also rises; both suggest an increase of affinity between the matrix and reinforcement. However, deformation at break does not increase, and it remains at a similar value as uncompatibilized TC90. To better understand the role of TC90 and the different reactive agents on the mechanical behavior of PHB after aging (15 days), the elastic modulus, tensile strength, and elongation at break values of the composites are depicted in After aging, the incorporation of TC90 fibers to PHB results in a slight improvement of elastic modulus and tensile strength, while showing a comparable elongation at break. In The mechanical characterization of the composites was completed by DMA testing. Their storage modulus (G’) and the damping factor (tan δ) evolution with temperature are represented in g) in semicrystalline polymers [g values around 10 °C. Nevertheless, the tan δ versus temperature plot suggest certain restricted mobility of the polymer chains in composites, since the tan δ peaks are broader than neat PHB [No appreciable differences in storage modulus among the different samples are observed. These results are in agreement with the elastic modulus observed in mechanical tests. The tan δ represents the energy dissipated during the dynamic tests, and the tan δ peak is usually used to determine glass transition . In this section, the compatibilization ability of HMDI is tested using an unpurified rice husk fiber (U_RH) and a chemically treated rice husk fiber (T_RH) to study the combined effect of purification treatment and compatibilizer on the interfacial PHB/fiber interactions. SEM micrographs shown in  allow viAs it can be observed in Uniaxial tensile tests of PHB/U_RH and PHB/T_RH with and without HMDI were performed on 15-day aged samples at room temperature. Elastic modulus, tensile strength, elongation at break, and the static toughness obtained from the area below the stress–strain representative curves for each composite are depicted in The addition of either U_RH or T_RH fibers produces a reinforcement effect with respect to neat PHB, since an increment of the modulus of elasticity is detected in all cases. On the other hand, the tensile strength seems to increase only when the treated fibers are the ones added, where the use of HMDI on such treated fibers produced an additional improvement of this parameter. For composites with treated fibers, the elongation at break remains simmilar to that of neat PHB, whereas for the untreated ones, this parameter is reduced .PHB-based composites reinforced with fibers are considered an attractive alternative to commodities for short-term life applications such as packaging. For this reason, it is of particular interest to test their processability by thermoforming, which is a conventional technique that is usually applied in this industrial field. In this regard, PHB/T_RH/HMDI films have been thermoformed into trays following the procedure described in the experimental section, and the best results obtained are presented in As it can be observed in One of the strengths of the PHB/fiber composites that make them especially attractive for packaging applications is their biodegradability and specifically their compostability. For this reason, it is of special interest to evaluate the effect of the reactive agents on the behavior under normalized composting conditions of the composites. Biodisintegration tests were conducted according to the ISO 20200 standard. The weight loss over time of the tested materials is represented in As shown in As shown in According to these results, it can be concluded that neither the fibers nor the reactive agents have a negative effect on the biodisintegrability of PHB in composting conditions. Analyzing together the results obtained for the composites prepared with TC90, U_RH, and T_RH fibers with and without HMDI, it seems clear that the compatibility efficiency of the HMDI depends to a large extent on the purity of the fibers. Probably, this dependence is due to a greater presence of OH groups on the surface of the purified fibers, since it is assumed that compatibilization occurs through the formation of urethane bonds between the isocyanate groups of the HMDI and the hydroxyl groups of the fibers . Tran etAs reported in previous works , U_RH fic by about 17% in neat PHB samples, but also to the physical aging of the amorphous fraction. Both fibers and reactive agents partly hinder secondary crystallization, leading to lower crystallinity in aged composites with respect to neat PHB. The melting temperature and crystallinity index for the compositions containing DCP were reduced at high cooling rates (processing conditions) and low cooling rates (DSC conditions), suggesting crosslinking of the PHB matrix. In this work, PHB/TC90 composites were obtained by reactive extrusion using four different reactive agents: dicumyl peroxide (DCP), hexamethylene diisocyanate (HMDI), resorcinol diglycidyl ether (RDGE), and triglycidyl isocyanurate (TGIC). The effect of aging attending on the influence of the reactive agents and the fibers on the mechanical and crystallization behavior of the composites was assessed. Aging produced a drastic embrittlement of the PHB characterized by increased elastic modulus and decreased tensile strength, elongation at break, and toughness. This embrittlement was attributed to secondary crystallization, which increased the value of XNo clear compatibilization effect was found for the reactive agents DCP, RDGE, and TGIC. Elastic modulus, tensile strength, and elongation at break remained practically unchanged for PHB/TC90/DCP and PHB/TC90/TGIC compared with PHB/TC90. In the case of PHB/TC90/RDGE, those values were even lower than PHB/TC90. On the contrary, the tensile strength in PHB/TC90/HMDI was improved by about 40% with respect to neat PHB and around 18% with respect to the PHB/TC90 composite. The improved mechanical performance together with the SEM observations indicate enhanced interfacial adhesion between the TC90 fibers and PHB. A clear influence of the purity of the fibers on the effectiveness of HMDI at improving interfacial adhesion was observed. HMDI only demonstrated effectiveness in purified cellulose fiber composites, the greatest improvements in the mechanical performance being the ones obtained with highly purified commercial cellulose fibers (TC90). The different reactive agents did not negatively affect the compostability of PHB. Finally, thermoformed trays with good mold reproducibility were successfully obtained for PHB/T_RH/HMDI composite.


Marine biofouling remains an unsolved problem with a serious economic impact on several marine associated industries and constitutes a major vector for the spread of non-indigenous species (NIS). The implementation of biofouling monitoring programs allows for better fouling management and also for the early identification of NIS. However, few monitoring studies have used recent methods, such as metabarcoding, that can significantly enhance the detection of those species. Here, we employed monthly monitoring of biofouling growth on stainless steel plates in the Atlantic Port of Leixões , over one year to test the effect of commercial anti-corrosion paint in the communities. Fouling organisms were identified by combining morpho-taxonomy identification with community DNA metabarcoding using multiple markers . The dominant colonizers found at this location were hard foulers, namely barnacles and mussels, while other groups of organisms such as cnidarians, bryozoans, and ascidians were also abundant. Regarding the temporal dynamics of the fouling communities, there was a progressive increase in the colonization of cyanobacteria, green algae, and red algae during the sampled period with the replacement of less abundant groups. The tested anticorrosion paint demonstrated to have a significant prevention effect against the biofouling community resulting in a biomass reduction. Our study also reports, for the first time, 29 NIS in this port, substantiating the need for the implementation of recurring biofouling monitoring programs in ports and harbours. This colonization process is most intense in coastal or shallow waters, where species diversity, temperatures, nutrient levels, and availability of submerged substrata are usually higher compared to offshore areas3. Biofouling represents a major economic issue for maritime industries and it raises important environmental concerns due to increased drag, exhaust emissions, and operational costs5 while creating also the potential for invasion by non-indigenous species (NIS)8. Stainless steel surfaces are frequently used as a material in vessels, port installation surfaces, cooling water circuits, ships or related equipment, and are known to be extensively colonized by fouling organisms when submerged in water10. Biofouling monitoring programs are therefore essential to help in the understanding and mitigation of such negative impacts. Until recently, the diversity of biofouling organisms has been mainly assessed through morphological identification11. However, the implementation of methods that allow for earlier and more comprehensive detection of the micro- and macro-fouler colonizers is desirable13. Metabarcoding approaches are useful to validate species identification based on morphological traits, with the possibility of “sight-unseen” detection of target species15, and detecting of species with a high resolution while processing all the specimens from a sample simultaneously16. Moreover, metabarcoding approaches can simultaneously employ different gene-markers to minimize taxonomic bias18, which is very useful to accurately characterize global routes of indigenous and non-indigenous species19. Such strategies have recently been successfully applied to unravel the composition and structure of eukaryotic macrofouling assemblages22, to detect introduced species26 and to perform port biodiversity surveys28. Regarding the latter, only a few studies looked into the effect of antifouling or anticorrosion paints30.Marine biofouling organisms colonize immersed surfaces and form communities on diverse biogenic habitats31 requires solid knowledge about the presence and distribution of NIS to achieve accurate environmental status32. Furthermore, the International Convention for the Control and Management of Ships' Ballast Water and Sediments , adopted in 2004 by the International Maritime Organization33, which aims to reduce the spread and the new introduction of harmful organisms, requires the monitoring of invasive species transported by ships. The BWM Convention has developed a sampling protocol for fouling organisms that has been used to perform a Port Biological Baseline Survey (PBBS) and is capable of supporting NIS management strategies35. Notwithstanding, the Portuguese list of NIS reflects a clear lack of information about northern Portuguese ports, encompassing only species (133 marine and brackish) monitored in central or southern estuaries  and the archipelagos (Madeira and Azores)36.Biofouling monitoring programs are still seldom used in Portuguese ports. This is particularly relevant considering that the “European Marine Strategy Framework Directive”37. The settlement of fouling communities and the presence of NIS in the port water were assessed and the colonization of surfaces covered with and without anti-corrosion paint was compared. The temporal succession of the fouling taxa colonizing steel plates was evaluated with a complementary metabarcoding and morphological assessment. Our results show that the employed four gene marker combination, together with the morphological identification is a powerful approach to accurately assess the biofouling diversity since several phyla/taxa were only retrieved with a particular marker. Additionally, this study revealed for the first time a list of 29 possible non-indigenous species present in Northern Portuguese waters.Taking this into consideration, this study aimed to assess the colonization of biofouling species (indigenous and NIS) in the cruise-ship terminal marina of the Port of Leixões, Northern Portugal, on a monthly basis and over one year. The Port of Leixões is the second most important Portuguese port in terms of volume of shipping and marine traffic, and its traffic is steadily increasing every year, representing around 25% of the Portuguese international tradeThe analysis of the biofouling colonization on the immersed steel panels was conducted in the cruise terminal of the Port of Leixões  . At the Port of Leixões, a pier that docks both large cruise passenger ships and freighter ships was selected as the study site  and to a previous empirical study40. The PBBS survey suggests that while working with PVC plates, these should be sanded before the deployment to provide a more hospitable substrate for organisms. As we were working with steel plates, we did not sand the plates beforehand. Besides this consideration, we followed most of the recommendations advised by the guide for PVC plates as they are similar to previous studies that employed steel surfaces42.The experimental design for the detection of biofoulers was selected according to a previous guidelineThe employed artificial structure was constituted by 10 stainless-steel (316 2B) square panels (20 cm × 20 cm × 2 mm), attached by carabineers and supported by a grey PVC tube  the two painted panels (n = 3 for each panel), and (ii) four random bare steel panels (n = 3 for each panel). The selected grids were scraped using a sterile stainless-steel surgical blade and the same grid was not scraped in the subsequent months. The sampling was conducted monthly from April 2016 until March 2017. All panels were permanently immersed throughout the sampling campaign, only re-surfaced for the sampling of the selected grids as detailed above and were then vertically submerged with the same orientation. A total of 216 grids were scraped throughout the one-year sampling period (i.e. 6 painted panel grids and 12 bare panel grids pre month). The scraped biomass was placed into sterile five-millilitre microcentrifuge tubes, placed on ice coolers, and transported into the laboratory. In the last sampling month, the bare panel labelled as P6, was fully scraped on both Southwest- and Northeast-facing sides (n = 2) making a total of 218 scraped samples. Aliquots of the scraped samples were used for morpho-taxonomic analysis and the remaining biomass was lyophilized and stored at − 80 °C until DNA extraction procedures 45 and AlgaeBase46 databases.To visually identify biofouling species, a qualitative analysis of the samples was performed under a light microscope at × 10, × 40, and × 100 magnifications. Photographs were obtained using a digital camera unit Olympus U-TV1X-2 DP72 attached to an Olympus SZX10 microscope or an Olympus BX41 phase-contrast microscope. Identification was performed to the lowest possible taxonomic level, by consulting taxonomic guides48. The obtained three-dimensional images were converted to integrate single two-dimensional (2D) images through the process of “Maximum Intensity Projection (MIP)”. The biofouling coverage was calculated using the MIP 2D by converting the images to 8-bit grey images, which were then processed into binary images through the “process-make binary” command. Noisy signals were removed using the “Filters” option and biofouling colonization percentage was calculated using the “Analyze particles” option.The biofouling colonization was evaluated by photographing all panels in both facing sides using a digital camera (Canon PowerShot G11). To quantitatively determine the colonization percentage of the panels, the image processing package Fiji which includes the ImageJ software v.1.46 was used2) were not considered, resulting in a total area of 336 cm2.Biofouling biomass was calculated by weighing individually wet-panels every month in a stainless-steel digital scale  at the study site before each sampling, by always taking into account the preservation of the biofouling cover. The weight of the panels was corrected by subtracting the corresponding non-fouled panel weight and divided by the panel area. The area where the carabineers attached to the panel  and confirmed by visualization on a 1% agarose (w/v) gel. Table Lyophilized material from each scraped grid samples (n = 216) was homogenized using a bleached pestle and a mortar. The dry weight (20 to 250 mg) was then transferred to PowerSoil DNA Isolation Kit PowerBead tubes . Additionally, six additional samples (three from each side) of the fully scraped bare panel from the last sampling month were subjected to the same DNA extraction protocol. All the material used in the weighing was previously sterilized by submerging it on a 10% bleach solution for 10 min and thoroughly rinsing it with ultrapure water before being used. To achieve physical lysis of cell material, samples were submitted to two cycles of 5,600 g for 15 s in a PreCellys®24 homogenizer  and, subsequently, DNA was extracted, according to the kit manufacturer’s instructions, making a total of 222 DNA extracts. Then, DNA extracts from each panel were pooled (n = 3) to yield one painted and one bare panel sample per month. Lastly, DNA extracts were scraped from two whole bare plates, one from plates facing Northeast and other from plates facing Southwest 50, COI (mlCOIintF51- dgHCO219852) and 23S rRNA (p23SrVF- p23SrVR)53 genes were selected to amplify the main fouling groups ; annealing at 70 °C (2 min) for 16S rRNA, 70 °C (1 min) for 18S rRNA, 62 °C (1 min) for 23S rRNA, and 62 °C (30 s) for COI. The extension step was conducted at 72 °C (1 min) repeated for 40 cycles and a final extension at 72 °C for 10 min. DNA amplification for each of the gene markers was confirmed by 1% agarose (w/v) gel electrophoresis. All PCR reactions included negative and positive controls; the positive controls were samples from mock communities and composed of fouling target taxa chosen for each of the gene markers  runs were performed using one protocol (see below) and adjusting annealing temperatures as needed, for each gene marker region. Amplicons derived from the 16S rRNA, 18S rRNA, 23S rRNA, and the mitochondrial cytochrome oxidase subunit I (COI) genes were selected for the metabarcoding approach Table . IndividThe bioinformatics analyses were subdivided in three main stages, pre-processing and clean-up of raw datasets, OTU clustering, and OTU identification. The pre-processing was conducted at LGC genomics, and initially, all raw reads (in FASTQ format) were subjected to base calling and demultiplexing pre-processing (Illumina bcl2fastq v.1.8.4). After that, the forward and reverse sequences were merged using BBMerge (v.34.48).54 (using default settings). All reads containing less than 100 bp of length and a phred quality score of < Q2055 were discarded. Chimeric sequences were identified and removed using UCHIME v.4.2.40. At the end of this clean-up stage, taxonomic unit (OTU) picking was performed by using UCLUST v1.2.22q56 within QIIME using the closed-reference method. The canonical clustering threshold of 97% for 16S rRNA was adopted57, and the same threshold was made for other markers following recent metabarcoding studies that tried to identify non-indigenous species in marine fouling communities18. To classify the 16S and 18S rRNA reads, Greengenes (release gg_13_5)58 and SILVA (release 108)59 were used, respectively, as implemented in the QIIME pipeline and using the default QIIME-based wrapper classifier, the RDP naive Bayesian classifier60.The clean-up of 16S and 18S rRNA gene sequences was done with the Quantitative Insights Into Microbial Ecology  software61 ; 3) all blast hits with low confidence scores , OTUs with more than one taxonomic genus blast hit assigned, and/or classified as “uncultured, unidentified, unclassified or environmental”, were removed from the analyses; 4) lastly, in dubious cases (e.g. two different species hits to the same OTU) the blast parameters were re-evaluated  and at the same time OTUs were manually scrutinized/selected via alignment  for the remaining OTUs the taxonomy classification was based on the top hit.The clean-up and clustering of 23S rRNA and COI gene marker sequences were carried out using the USEARCH sequence analysis tool (v.8.1.1831), namely the read quality filtering and fastq_filter scripts to remove reads containing less than 100 bp of length and phred quality score of < Q20. Next, the UCHIME v.4.2.40 software was used to remove chimeric sequences. The classification of OTUs and taxonomy assignment of both the 23S rRNA and COI gene marker was carried out as follows: 1) all OTU sequences were searched against the NT-NCBI database (downloaded on 25/11/2018)) using the BLASTn algorithm (v.2.5.0) ; 2) OTUs without a hit in the NT-NCBI database were blasted against the BOLD System databaseshttps://www.iucngisd.org/gisd/), the Invasive Species Compendium (CABI: www.cabi.org/isc), the Information system on aquatic non-indigenous and cryptogenic species (AquaNIS: www.corpi.ku.lt/databases/index.php/aquanis/) and the National Exotic Marine and Estuarine Species Information System (NEMESIS: //invasions.si.edu/nemesis/ ), all accessed on March 2020. All positive hits were validated using two approaches. First, COI sequences were processed in Translator X server63 using variable genetic codes and following NCBI tables. All sequences without translation and/or with stop codons were removed from the analyses. Second, all sequences with positive matches and encoding an interrupted protein were subject to manual curation, through alignment at the protein and nucleotide levels, against the reference.NIS detection was only based on the COI sequences. The COI OTUs, previously classified, were compared against the following reference databases: the Global Invasive Species Database , both in RStudio, R statistical software (version 2.15.2)66. A one-way ANOVA analysis was used to compare the diversity indices values, plates biomass, and the abundance of fouling organisms for the treatments (presence vs. absence of paint). If the data did not follow a normal distribution , differences among treatments were analyzed using the non-parametric Kruskal–Wallis test, followed by a two-tailed Mann–Whitney-Wilcoxon test for pairwise comparisons. All statistical data analysis was performed using R statistical software66 (version 2.15.2). A multivariate version of Levene’s analysis of multivariate homogeneity of group dispersions (function ‘betadisper’ in R vegan package65) was used to test for homogeneity of variances of the groups (plates with paint vs plates without paint). A permutational multivariate analysis of variance (PERMANOVA)67 was subsequently used to test the taxonomic dissimilarity between the plates with anti-corrosion paint and the plates without anti-corrosion paint. For the PERMANOVA analysis, Bray–Curtis resemblance matrices were produced using the untransformed raw data, and p-values were calculated from 9,999 random permutations and dummy variables (0.0001) were used to account for zero. This test was performed using the ‘adonis’ function provided by the vegan package65 and ran in the R statistical software66.The Alpha-diversity indices  for the four-gene markers were calculated using the Phyloseq package (version 1.5.3)Chthamalus sp., followed by the bivalve Mytilus spp. , two hydrozoan genera, i.e. Ectopleura and Obelia, and the red alga C. cilliatum were also observed. During the summer period (June–August 2016) those plates were colonized by the green algae Ulva sp. and by the bell-shaped ciliate Pseudovorticella spp.. In the last three months (January to March 2017), macroalgae and crustacean larvae were observed again   were successfully amplified for almost all samples. The only exception was the COI gene, which did not amplify for one painted panel sample . The amount of DNA per sample ranged from 12.9 to 114 (ng/μL). The quality control steps of the bioinformatic approach showed some quality differences among markers. While the 16S rRNA and 18S rRNA quality filtering saw a relatively small fraction of reads removed, the 23S rRNA and COI genes datasets were drastically reduced, from 1,167,426 to 332,609 (23S rRNA) and from 1,464,221 to 250,245 (COI gene). The main factor for this reduction was the Q > 20 parameter . The average length of the merged reads (as counted using the fastx-info command of usearch) was of 310 bp for 16S rRNA, 300 bp for 18S rRNA, 368 bp for 23S rRNA and 313 bp for COI. As expected, the number of OTUs assigned were also highly uneven: 7,329 OTUs for the 16S rRNA, 40,092 OTUs for the 18S rRNA gene, 925 OTUs for the 23S rRNA gene, and 1,614 OTUs for the COI gene all based on 97% similarity . In this study, a total of twenty-six phyla were exclusively detected using a single marker: nine phyla with the 16S rRNA gene; eight phyla for the 18S rRNA gene; three phyla for 23S rRNA gene and six phyla for the COI gene, but no exclusive phyla identified using the morphological identification Fig. . Non-excRegarding alpha-diversity, no significant differences were found for any of the tested indices  for each of the four markers when comparing the diversity between the plates with and without the employed anti-corrosion paint. The dispersion between groups using the betadisper test was not significant for any of the gene marker dataset, meaning that group dispersions are homogenous (p > 0.05) for all the gene marker datasets. The subsequent PERMANOVA analysis demonstrated a significant dissimilarity between the taxa growing in surfaces with and without anti-corrosion paint, both for the 16S rRNA (p = 0.014) Table  and the Regarding prokaryotes, the 16S rRNA gene dataset retrieved, as expected, a higher number of OTUs for all the phyla when compared to all other markers (p < 0.01) except for the Firmicutes and Verrumicrobia phyla, for which no significant differences were found when compared to the 23S rRNA gene data Fig. a. The reIn terms of eukaryotic OTUs Fig. a, no maj68, namely the red algae Polysiphonia brodiei, the incrusting bryozoan Watersipora subtorquata and the barnacle Austrominius modestus. Two NIS were also morphologically identified, i.e., the hydroid Ectopleura crocea and the bryozoan Watersipora subtorquata. All the NIS found are tertiary colonizers and macrofoulers, except for five species, namely the Bristly crab Pilumnus hirtellus, the copepod Oithona similis, the mudshrimp Monocorophium sextonae, and the annelids Ctenodrilus serratus and Chaetogaster diaphanus. The Australian tubeworm Ficopomatus enigmaticus69  that resulted in NIS identification, as well as the blast-hits results used for the taxonomic classification of NIS, are provided in Supplementary Table Potential NIS were only detected among the COI-derived OTUs. During the taxonomic classification of COI gene-derived OTUs, two databases (NT-NCBI and BOLD) were used. Despite our effort to classify the OTUs sequences without classification in NT-NCBI database, against BOLD database, no new classifications were found. In summary, 27 species, representing 1.7% of the total retrieved COI gene OTUs, were found to be potential NIS Table . Three o69 Table  and the 72 and is less likely to under-detect small organisms73. By the same token, the taxonomic classification pipelines continue to be computationally intensive and highly dependent on huge computational resources. Due to the computational limitations we have applied a homology-based approach to perform the taxonomic classification and used conservative parameters to mitigate their limitations in terms of accuracy. Overall, our multi-marker metabarcoding data confirms that this is a powerful tool to assess biofouling community biodiversity, enhancing the resolution of taxa by providing richer taxonomic information about this Portuguese port. Nevertheless, the detection of nonindigenous species was only achieved with one of the gene markers (COI) employed in the study. Furthermore, and despite the clear advantages of metabarcoding approaches for the monitoring of micro- and macro-foulers, there are problems inherent to such approaches, ranging from primer bias74 to differences in resolution among markers, to the lack of reference databases and universal bioinformatics pipelines to deal with next-generation sequencing (NGS)-generated datasets75. Nevertheless, our findings demonstrate that all employed markers added important data to the biofouling survey by detecting unique taxonomic groups. A high level of marine diversity detection was achieved by the metabarcoding approach: we retrieved 7,329 OTUs for 16S rRNA, 40,092 OTUs for 18S rRNA, 925 OTUs for 23S rRNA and 1,614 OTUs for COI. Overall, the choice of data filtering, clustering algorithms, and specific parameters can have a significant impact on the obtained OTUs for the different genes employed in the metabarcoding approach. Results based on length variable regions, such as the 18S rRNA, are strongly influenced by the data processing workflow such as the clustering methods which can result in the overestimation of OTU numbers76. As for the detected species, COI retrieved more species (637 species) than the 23S rRNA marker (28 species) and COI was the only gene contributing to the detection of NIS in the Port of Leixões. The deficiency of taxonomic coverage in reference databases is one of the major obstacles to large-scale application of metabarcoding in biodiversity research and biofouling monitoring29. In this study, for each of the gene markers we used one, or a combination of two databases  taking in consideration the respective gene and the target taxa to be amplified  and the polychaeta Ficopomatus enigmatus were only visually detected. The absence of these prevalent groups in the metabarcoding data could probably be explained by the natural complexity of marine biofouling matrices, which may not have allowed complete homogeneity of the samples before DNA extraction29. Another explanation could be the randomly chosen panel grids used in our sampling. Hence, our findings indicate that morphological identification is still a valuable strategy to be implemented in monitoring programs to identify fouling and NIS species. Almost all rarefaction curves for the alpha diversity reached the saturation plateau  compared to the 28 genera and 11 species which were morphologically identified. It should be stated that taxonomy assignment at the lowest levels, particularly at a species level has some degree of uncertainty due to the restricted specificity/resolution of markers and incompleteness of currently available reference sequence databases. The morphological approach employed here did not provide any new information to assess biofouling community biodiversity, at the phylum level, in the sampled port. Nevertheless, important biofouling organisms, such as the macroalgae eau Fig. , suggest80, with a more significant effect in the prokaryotic communities than in the eukaryotic communities30 Proteobacteria, Cyanobacteria, and Bacteroidetes are known to be the dominant bacterial phyla colonizing different surfaces at diverse sampling locations, particularly in the early stages of colonization82. In our study, we found a significantly higher presence of the phylum Bacteroidetes in the plates with anti-corrosion paint when compared to the plates without paint. Bacteroidetes are efficient surface colonizers of steel surfaces in the marine environment and are known to be associated with biological corrosion83. Moreover, Firmicutes are other well-known colonizers of immersed artificial surfaces84, and here we report a significantly higher abundance of this phylum in the plates without anti-corrosion paint. Usually, studies characterizing prokaryotic communities colonizing artificial surfaces involve sampling attached communities growing for weeks or a few months1, while our work involved a longer sampling period which could explain why for the bare panels such a high abundance of sequences was classified as cyanobacteria/plastids in the Greengenes database. The particular dominance of photoautotrophic organisms in the panels facing Northeast is also in line with the effect of sunlight in their colonization. At the same time, the colonization of these groups was limited in the plates with anti-corrosion paint and delayed for approximately six months, which is in line with previous studies79.Regarding the effect of the anti-corrosion paint, there is the absence of significant differences for any of the alpha-diversity indices  for all the markers. On the other hand, the PERMANOVA analysis results demonstrated that for the 16S rRNA and 23S rRNA gene markers, there was a strong dissimilarity between the communities growing in the presence and the absence of paint, but this was not observed for the other markers. Some previous studies employing commercial anti-fouling coatings reported a lower diversity of the fouling communities as a result of the presence of those commercial products in the surfaces22. The data from the 18S rRNA and 23S rRNA gene markers suggests a community stabilization that initiated in the fourth month of the deployment of the panels, whereby the main taxonomic groups demonstrate little variation in their abundance until the last sampling month. In terms of seasonal patterns, and as revealed by 18S rRNA gene data, green algae (Chlorophyta) are dominant in Spring and Summer in the bare panels when compared to other algae (Rhodophyta or Ochrophyta) in agreement with previous studies85. Among the marine fouling communities, red algae (Rhodophyta) are believed to settle after the hard-foulers and filamentous algae3, and in our study, the settlement of these organisms initiated after a few months of the immersion of the plates.Regarding the eukaryotic communities, Rhodophyta, Chlorophyta, Ochrophyta, Cnidaria, Arthropoda, and Mollusca were the most abundant taxa along the year, which is consistent with previous macrofouling studies3. The COI gene marker data revealed more extensive colonization by the phototrophic taxonomic group Bacillariophyta in the Northeast panels, with the Southwest panels showing the dominance of Arthropoda. Visual inspection indicated that barnacles , and mussels (Mytilus spp.) were dominant all year in all plates. Other hard-fouler organisms like bryozoans are more reported in temperate climates during the warmer seasons of Spring or in Summer, and our findings are in line with this, particularly for the 18S rRNA gene dataset86. The results from the 18S rRNA and COI gene markers reveal stronger colonization of Cnidaria in the Summer, with increased abundance for the Spring season, similar to previous studies87. No significant presence of sponges was detected in our results, a group that is often reported in biofouling assemblages89. In general, no consistent seasonal patterns of abundance were identified among most of the analyzed taxa, suggesting stabilization of communities after the early months of immersion92. Nevertheless, our data clearly shows that, as expected, bare panels harbour higher biomass than the painted panels, which means that the employed commercial anti-corrosion paint has a significant impact in the colonization of the communities in employed steel plates. A significant effect of the paint in the early colonization of the communities was also observed. This difference decreased during the sampling period and converged with the levels of bare panels around the tenth month of colonization. Regarding the rate of colonization, panels facing northwest showed a total coverage of around 100% in the fifth month of sampling, with a decline of surface coverage in the subsequent months. On the other hand, panels facing Northeast were never fully colonized, reaching close to 90% coverage in the ninth month of sampling. Previous studies from fouling communities sampled from temperate areas suggest a rapid increase in colonization reaching 75–100% after one year, whereas at higher latitudes this colonization process is much slower93. Regarding the colonization of biomass per area, there were significantly higher biomass colonization values for the bare panels, with higher access to light, also likely due to the importance of phototrophic organisms in the structuring of the fouling community.Regarding hard-fouler colonization, a higher presence of barnacles in the Southwest-facing plates (with no direct exposure to the sunlight) was observed when compared to the Northeast facing plates (with direct exposure to the sunlight). On the other hand, there was a more abundant and earlier colonization of algae in the surfaces facing Northeast  we selected the sampling location in a high priority area in Northern Portugal. For correct monitoring of fouling communities, PBBS suggests taking in consideration the seasonality and life cycle patterns of those species and conduct sampling in certain seasons of the year, when species are mature and can be identified by morphology. However, metabarcoding approaches have allowed the detection of conspicuous organisms as well of unseen organisms95 which was also the case in our study. Furthermore, the year-long approach to the monitoring of the fouling species revealed to be important for future monitoring studies as some taxonomic shifts were detected. As expected, our results underline that morpho-taxonomy identification is particularly well suited for the detection of conspicuous organisms, such as macrofauna or macroalgae which can be readily identified but are occasionally not detected by the metabarcoding approach. Lastly, and taking in consideration our results, we suggest that the development of a regional reference database of non-indigenous species would enhance the metabarcoding approach accuracy for future monitoring studies.Finally, a list of 29 NIS present in the Port of Leixões was for the first time compiled in this work , after comparing our outputs with the ICES, ISSG, GISD, CABI, AquaNIS and NEMESIS databases. Currently, baseline studies of biofouling monitoring programs are being globally conducted and implemented at portsBiofouling monitoring of ports gathers essential information for the planning and the maintenance of marine structures and allows for early NIS identification. Here, we report the biofouling diversity and their succession in the Port of Leixões for one year using a combined morphological and multi-marker metabarcoding approach. These approaches proved to be complementary since a higher number of retrieved OTUs were obtained with the metabarcoding approach while some of the NIS were only visually detected. Key to our analysis was the use of four different gene markers and different reference databases. This allowed for a high reported diversity and has the additional benefit of enabling cross-verification of species detections, such as for rare or non-indigenous species of interest. This study also includes the first published NIS list of the Port of Leixões which can be a starting point for future biofouling monitoring programs. Hence, we conclude that a combination of morphological and metabarcoding multi-marker methodologies is an advantageous strategy for biofouling monitoring programs and the detection of potential NIS in future studies.Supplementary information


Synechocystis salina was used along with the weaker biofilm-forming Cyanobium sp. Biofilms were developed in defined hydrodynamic conditions using glass (a model hydrophilic surface) and a polymeric epoxy coating (a hydrophobic surface) as substrates. Biofilms developed in both surfaces at lower shear conditions contained a higher number of cells and presented higher values for wet weight, thickness, and chlorophyll a content. The impact of hydrodynamics on biofilm development was generally stronger than the impact of surface hydrophobicity, but a combined effect of these two parameters strongly affected biofilm formation for the weaker biofilm-producing organism. The antibiofilm performance of the polymeric coating was confirmed at the hydrodynamic conditions prevailing in ports. Shear forces were shown to have a profound impact on biofilm development in marine settings regardless of the fouling capacity of the existing flora and the hydrophobicity of the surface.Understanding the conditions affecting cyanobacterial biofilm development is crucial to develop new antibiofouling strategies and decrease the economic and environmental impact of biofilms in marine settings. In this study, we investigated the relative importance of shear forces and surface hydrophobicity on biofilm development by two coccoid cyanobacteria with different biofilm formation capacities. The strong biofilm-forming  Marine biofouling is an area of intense research particularly due to the considerable economic impacts on marine transport. Biofouling on ship hulls increases frictional drag and may result in a fuel consumption increase ranging from 6% to 45%, depending on the size of the vessel ,2,3,4. TBiofouling by macrofouling organisms such as bryozoans, mollusks, polychaeta, tunicates, coelenterates, or fungi occurs after biofilm formation by microfouling organisms such as cyanobacteria and diatoms which are early colonizers ,8,9,10. Port authorities in different countries are moving towards a “clean hull” policy where vessels must provide evidence of biofouling management before they arrive ,15,16. TSeveral parameters have been indicated as modulators of biofilm development, including surface hydrophobicity and hydrodynamic conditions ,19. ReceIn this work, we have tested a polymeric epoxy resin commonly used to coat the hulls of small recreational vessels  ,21. PartThe main goal of this study was to evaluate the relative importance of shear forces and surface hydrophobicity on cyanobacterial biofilm development in marine settings. For that purpose, we have followed biofilm development in defined hydrodynamic conditions (including those that can be found in harbors), using two cyanobacterial coccoid strains with different biofilm-forming capacities and two model surfaces with different hydrophobicity (glass and an epoxy polymeric coating). These surfaces can be found in ship hulls and also in the windows of underwater sensors and measuring devices.®  solution, an amphoteric disinfectant used for cleaning and disinfecting surfaces [In order to assess the cyanobacterial biofilm development, two different surfaces, glass and a polymeric epoxy resin, were used. Glass coupons  were immersed in a 2% (v/v) TEGO 2000surfaces , for 20 surfaces  under agsurfaces . For theThe water contact angle of both surfaces was determined in three independent measurements performed at 25 ± 2 °C, by the sessile drop method using a contact angle meter , as described in Gomes et al. 2015 . In eachSynechocystis salina LEGE 00041  was originally obtained from a seawater sample, collected on June 2000, at Espinho beach (41.00847 N 8.646958 W) located in the north coast of Portugal [Cyanobium sp. LEGE 06097  was isolated from the intertidal zone, on green macroalga, collected on July 2006, at Martinhal beach (37.01869 N 8.926714 W) located in Vila do Bispo, Portugal [−1 of synthetic sea salts (Tropic Marin) and B12 vitamin . Cultures were grown under 14 h light /10 h dark cycles at 25 °C. Cyanobacterial strains were obtained from the Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CCB) deposited at the Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Porto, Portugal . SynechoPortugal . CyanobiPortugal . CyanobaPortugal  suppleme8 cell/mL. Microtiter plates were incubated at 25 °C in an orbital shaker with a 25 mm orbital radius  at 40 and 185 rpm and under alternate light cycles of 14 h light (10–30 mol photons m−2 s−1)/10 h dark. The selection of the hydrodynamic conditions was based on a previous study describing that a shaking frequency of 185 rpm in this incubator corresponds to an average shear rate of 40 s−1 and a maximum of 120 s−1, while 40 rpm corresponds to an average shear rate of 4 s−1 and a maximum of 11 s−1 as determined by computational fluid dynamics [−1 was estimated for a ship in a harbor [Biofilm assays were performed on 12-well plates  under previously optimized conditions . Brieflya harbor , and lowa harbor ,34, bothBiofilm formation was followed for six weeks (42 days), every seven days. During the incubation period, the culture medium was replaced twice a week. Biofilm formation experiments were performed with two technical replicates and in two independent assays .a content. The biofilm structure was analyzed at day 42 by optical coherence tomography (OCT). Before sampling the culture medium was carefully removed and, then, the coupons were gently rinsed with a sterile sodium chloride solution  in order to remove loosely attached cyanobacteria. At each sampling point, two coupons of each experimental condition were analyzed concerning (i) the number of biofilm cells, (ii) biofilm wet weight, (iii) biofilm thickness, and iv) chlorophyll −1 sodium chloride solution and vortexing for 3 min at maximum power. Then, 10 µL of cellular suspension was placed on each side of a Neubauer chamber and observed under the microscope . After vortexing, the coupons were observed by microscopy in order to confirm complete cell detachment.Cyanobacterial cells were detached from the coupons by dipping each coupon in 2 mL of 8.5 g·LTo determine the biofilm wet weight, coupons were detached from the wells with a sterile tweezer and weighted. Biofilm wet weight was obtained by the difference between initial coupon weight, determined prior to inoculation, and the weight after sampling.Biofilm thickness was assessed using a Nikon Eclipse LV100 microscope coupled to a joystick , connected to a camera , and analyzed using the NIS-Elements AR (Advanced Research) 4.13.05 software package. This tool features fully automated acquisition and device control through multi-dimensional image acquisition and analysis. For each coupon, a minimum of five representative independent fields were analyzed to obtain accurate and reproducible results.a quantification is a common method to estimate the biomass on marine environments because this pigment is unique and predominant in all groups of cyanobacteria [g, for 5 min at room temperature) and the supernatant discarded. Since chlorophyll pigments are light-sensitive, the following chlorophyll extraction procedures were performed in the dark, as previously reported [a extraction. The absorbance at 750 nm (turbidity), 665 nm (chlorophyll a) and 652 nm (chlorophyll b) were measured on a V-1200 spectrophotometer . The chlorophyll a concentration (μg·cm−2) was calculated using the following Equation (1) [Chlorophyll bacteria . Detachereported . Brieflytion (1) .Chl a (−1) and imaged. The captured volume was 3.66 × 1.52 × 2.98 mm3 (509 × 313 × 1024 pixels). The refractive index was set to 1.40, since this value produced optimal results in a previous study [On day 42, the biofilms were imaged by OCT using a Thorlabs Ganymede instrument  with a central wavelength of 930 nm. After the gentle rinsing, the wells were filled with 3 mL of a sterile sodium chloride solution (8.5 g·Lus study . For eaca content). Results were presented as the percentage increase between shear forces (obtained at 40 and 185 rpm). Descriptive statistics were used to compute mean and standard deviation for sample parameters . Since the distribution of some variables was not normal, both parametric and nonparametric tests were used. Student’s t-test was used to compare biofilm formation under lower and higher shear forces, either for glass or epoxy-coated glass surfaces. For the determination of the impact of the hydrodynamic conditions and surface hydrophobicity on biofilm formation, the Mann–Whitney test was used  and represented in radar charts. Radar charts were divided into four quadrants, where each one depicts the average values obtained in each sampling point (days) under the following experimental conditions: Q1) glass at 40 rpm (Gla/40), Q2) epoxy-coated glass at 40 rpm (Epx/40), Q3) epoxy-coated glass at 185 rpm (Epx/185), and Q4) glass at 40 rpm (Gla/185). The impact of the hydrodynamic conditions was calculated by subtracting the values obtained at different shear forces for both glass (Q1 vs. Q4) and epoxy-coated glass (Q2 vs. Q3); whereas the impact of the surface hydrophobicity was determined by subtracting the values obtained for two different surfaces at lower shear (Q1 vs. Q2) and higher shear (Q4 vs. Q3). All positive differences were considered as increments resulting from hydrodynamic condition or surface hydrophobicity and represented by a colored area . The combined effect (green area) has been plotted whenever the surface effect overlapped the hydrodynamic effect.The impact of the hydrodynamic condition and surface hydrophobicity on biofilm development was estimated for each analyzed parameter  in an orbital incubator generating average shear rates of 4 and 40 sdynamics . Surfacedynamics ,38.S. salina (high biofilm former) and Cyanobium sp. (low biofilm former), the number of biofilm cells was higher at lower shear for all the time points tested. Biofilms developed on glass at lower shear displayed on average a higher number of cells of S. salina   B–D. HoweCyanobium sp. a combined effect resulting from hydrodynamics and surface hydrophobicity was responsible for a higher number of biofilm cells, biofilm wet weight and thickness, and chlorophyll a content  presented a higher number of biofilm cells when compared to those developed at higher shear (185 rpm), both on glass and epoxy-coated glass surfaces. Similar results were obtained for wet weight and thickness. This later parameter has a strong impact on the performance of underwater devices , and thea content as a good indicator of cyanobacterial biofilm growth [a, which is consistent with the higher number of biofilm cells obtained in this condition. These results are corroborated by a previous study demonstrating that biofilm development by filamentous cyanobacteria is also promoted at low shear forces [Likewise, several studies have proposed the determination of chlorophyll m growth ,40. Our r forces .a content were higher on glass than on epoxy-coated glass. This result suggests that, in this assay, the hydrophilic surface promoted biofilm formation. Other studies referred that adhesion and consequent biofilm formation may occur to a greater extent on hydrophobic surfaces rather than on hydrophilic surfaces [Concerning the surface hydrophobicity, results demonstrated that biofilms formed on glass displayed a higher number of cells than on epoxy-coated glass. Similarly, the biofilm wet weight and thickness, and chlorophyll surfaces . Howeversurfaces . S. salina and Cyanobium sp. independently of their capacity to form a biofilm. However, our data analysis demonstrated that, for S. salina, the increase in biofilm parameters is mainly due to shear forces (yellow shadowed area is greater than blue shadowed area, Cyanobium sp., a combined effect resulting from the shear force and surface hydrophobicity is responsible for the biofilm development behaviour (green shadowed area prevails over yellow and blue shadowed areas, The tendencies verified for the analyzed parameters were validated for both For strains with low biofilm formation capability, although hydrodynamics also play an important role in biofilm development, surface hydrophobicity becomes more important than in high biofilm producers. In this case, a combined effect between hydrodynamics and hydrophobicity becomes relevant. Surface properties such as hydrophobicity may promote cell/surface interactions that, even in a low magnitude, may facilitate bacterial retention and contribute to biofilm development when combined with lower shear . FurtherOCT analysis highlighted the impact of shear forces on cyanobacterial biofilm development, demonstrating that higher biofilm amounts were obtained at lower shear. It was also possible to observe the presence of three-dimensional structures of streamers in biofilms formed under these conditions on both surfaces. According to Drescher et al., the presence of these structures may contribute to biofilm growth by facilitating the capture of new cells and other components to the biofilm . In ship hulls, epoxy composites are used due to their strong adhesion to the construction material, high strength, and great chemical resistance . Our res


Zea mays L.) with zinc (Zn), iron (Fe) and selenium (Se) grown on a micronutrient deficient soil under greenhouse conditions. Factorial design experiment was set under greenhouse conditions. The experiment consisted of two levels of each N, Zn, Fe and Se. The levels for N were 125 and 250 mg N kg−1 soil; Zn were 1 and 5 mg Zn kg−1 soil; levels of Fe were 0 and 10 mg Fe kg−1 soil; levels of Se were 0 and 0.02 mg Se kg−1 soil. An additional experiment was also conducted to study the effect of the Zn form applied as a ZnO or ZnSO4 on shoot growth, shoot Zn concentration and total shoot Zn uptake per plant. Shoot Zn concentrations increased by increasing soil Zn application both with ZnSO4 and ZnO treatments, but the shoot Zn concentration and total Zn uptake were much greater with ZnSO4 than the ZnO application. Under given experimental conditions, increasing soil N supply improved shoot N concentration; but had little effect on shoot dry matter production. The concentrations of Zn and Fe in shoots were significantly increased by increasing N application. In case of total uptake of Zn and Fe, the positive effect of N nutrition was more pronounced. Although Se soil treatment had significant effect, N application showed no effect on Se concentration and accumulation in maize shoots. The obtained results show that N fertilization is an effective tool in improving the Zn and Fe status of silage maize and contribute to the better-quality feed.Agronomic biofortification is one of the main strategies for alleviation of micronutrient deficiencies in human populations and promoting sustainable production of food and feed. The aim of this study was to investigate the effect of nitrogen (N)fertilization on biofortification of maize crop ( Agronomic biofortification of field crops with micronutrients is one of the main strategies in sustainable production of healthy and nutrient rich food and feed . Human hThanks to successful impacts of green revolution, farmers have managed to grow more high-yielding cereal crops, leading to the increased feed and food production and decline in cereal prices. However, this trend had a negative side effect, and resulted in dilution in the concentrations of micronutrients in the food and feed and unintentionally enhanced hidden hunger problem ,12,13.Zinc, Fe and Se deficiencies in soils are common in both developed and developing countries ,8,14. SoDairy cattle nutrition is highly affected by Zn, Fe and Se presence in the feed, because these micronutrients have crucial role in different metabolic processes ,16. ManySelenium, which acts as an antioxidant, makes an integral part of several enzymes. Selenium as selenocysteine (Se-Cys) is incorporated in the active center of at least 25 selenoproteins ,20. AnalZea mays L.), the most grown field crops worldwide, provide dietary staple food for > 200 million people with about 20% of their calories needs  in a closed-vessel microwave system . The digested solution was diluted with DI water. For the determination of Zn, Fe and Se in the digested solution, inductively coupled plasma optical emission spectrometry (ICP-OES)  was used. A dry combustion method (950 °C) using a LECO Tru-Spec C/N Analyzer  was used for the determination of N concentration in the samples. The certified standard reference materials, obtained from the National Institute of Standards and Technology , were used for checking the precision in mineral nutrient analysis.Plant shoot materials, after washing with deionized water, were dried at 70 °C for the dry mater determination of shoot weight and analysis of micronutrients. Dried and ground plant samples (0.2 g) were digested with acid [a mixture containing 2 mL of 30% (v/v) HAs standard sample, the SRM 1547 peach leaves, were used and the deviation was below 2%. The total uptake per plant was calculated by multiplying the concentration with dry mater yield. The R commander program was used for statistical analysis of the results obtained. Analysis of variance (ANOVA) was used to assess the significance level of the effects of treatments and their interactions on the reported traits. Tukey test at 5% level (P ≤ 0.05) was used for significant difference among means, whenever ANOVA  indicated significant effect of treatments. The relationship between the treatments was assessed by linear regression model.Zinc deficiency symptoms  appeared in plants grown under low supply of Zn . The symBy contrast, soil N and Zn applications did not significantly affect shoot Se concentration and total Se uptake .Variation in N supply did not show a significant impact on the shoot dry matter yield of the plants at both low and adequate Zn treatment , indicatOn the other hand, variation in Zn soil application both under low and adequate N supply significantly affected shoot dry mater production . Table 2Both Zn concentration and total Zn uptake by shoots were increased by increasing soil Zn application . At the Selenium soil treatment had significant effect on Se concentration and shoot Se uptake in maize shoot , while bShoot Fe concentration and uptake were greatly affected by N and Zn supply . Both ZnThe relationship between N-Zn and N-Fe concentrations in shoot is shown in The relation between N-Zn in shoot is highly dependent on availability of N in the soil irrespective of Zn soil availability. Although Zn application decreased shoot Fe concentration , still i4 application when compared to the soil ZnO application  of plants were significantly increased through the increase in N fertilization of plants . These rIn a short-term experiment, Erenoglu et al. (2011)  showed t4 and FeEDTA had no clear effect on shoot and grain Fe concentrations of wheat plants; but at a given Fe dose, increasing N fertilization significantly improved shoot and grain Fe [Like Zn, increasing soil N supply also positively affected shoot Fe concentrations, even at much higher level than Zn . Losak egrain Fe . The N-dgrain Fe . It is kgrain Fe ,56,57.In contrast to Zn and Fe, increasing N fertilization did not affect plant Se concentration , which sPlants are very responsive to soil Se fertilization and show substantial increases in shoot and grain concentrations of Se as shown in field-grown maize plants by Chilimba et al. (2012)  in Malaw−1 (Considering the average concentrations of observed micronutrients in experimental maize plants and dietary requirement of the dairy cattle, we can suggest that biofortified maize grown on the deficient soils can meet the dairy cattle requirements to certain extent. Average Se maize shoot DM levels ranged between 0.04–0.88 mg kg−1 , while t−1 DM, for heifers 31 mg kg−1 DM and dry cows 22.8 mg kg−1 DM [−1. According to this comparison, Zn biofortified silage could meet the requirements of dry cows and heifers but not those of milking cows. Required dietary concentration of Fe for milking cow is 24 mg kg−1 DM [−1 DM could easily meet the dietary requirements of all cattle categories.This indicates that maize silage could be very successfully Se biofortified after optimization of the application rates. In case of Zn, required dietary concentration for milking cows is 63 mg kgThe present study showed that N nutritional status represents a key factor in biofortification of silage maize with Zn and Fe. It is suggested that an optimum N fertilization of feed and food crops are required to contribute to better human and animal dietary intake of Zn and Fe. The results also highlighted importance of use of water-soluble Zn fertilizers in Zn biofortification of plants grown on high pH soils.