Lacticaseibacillus casei strains, previously isolated from ripened Parmigiano Reggiano cheese, were characterized from a genetic and biochemical point of view. Two different prt genes existed in the genomes of both strains, but only one, named prtR1, was expressed. PrtR1 proteins extracted from both strains displayed the highest activity at 40 °C and pH 7. Interestingly, PrtR1 extracted from Lacticaseibacillus casei PRA205 retained some residual activity at 5 °C and at pH 4. These important biotechnological features can be exploited in the production of fermented dairy products. Peptidomic analysis revealed that both proteinases were able to release β- and αS1-casein-derived bioactive peptides, suggesting that Lacticaseibacillus casei can be a source of new proteinases that can be exploited for the formulation of dairy beverages or hydrolysates enriched in bioactive peptides.Lactic acid bacteria are nutritionally fastidious microorganisms typically used in the production of fermented dairy foods. The lactic acid bacteria’s proteolytic system is crucial for their growth in milk, and plays a paramount role in developing the organoleptic and healthy properties of fermented dairy foods. Cell-envelope proteinases are the first component of this system, responsible for the degradation of caseins into short peptides. In the present work, the cell-envelope proteinases of two highly proteolytic Lacticaseibacillus casei strains PRA205 and 2006 were characterized at both the biochemical and genetic levels. The genomes of both L. casei strains included two putative CEPs genes prtP2 and prtR1, but only prtR1 was transcribed. The extracted PrtR1 proteinases were serine proteinases with optimal activity at 40 °C and pH 7.5, and were activated by Ca2+ ions. Interestingly, PrtR1 from L. casei PRA205 exhibited high residual activity at pH 4 and at 5 °C, suggesting its possible exploitation for fermented food production. The caseinolytic activity against αS1- and β-casein indicated that both PrtR1s belonged to the PI/PIII type. These PrtR1s cleaved β-casein peptide bonds preferentially when amino acid M or N was present at the P1 subsite and amino acids A and D were at the P1′ subsite. Several bioactive peptides were found to be released from PrtR1 after αs1- and β-casein hydrolysis.In the present work, two cell-envelope proteinases (CEPs) from  Lactic acid bacteria (LAB) are a group of microorganisms generally found in nutrient-rich environments and are commonly used in the manufacturing of fermented dairy foods. LAB are nutritionally fastidious, and their growth is dependent on the presence of external sources of nitrogen , since they are auxotrophic for numerous amino acids . The lowThe LAB proteolytic system involves various components, such as (1) cell-envelope proteinases (CEPs), which are responsible for the first degradation of caseins into oligopeptides; (2) a specific transport system that internalizes peptides; and (3) a wide variety of internal peptidases, such as specific endopeptidases, aminopeptidases, tri- and dipeptidases, and proline-specific peptidases ,5.Lacticaseibacillus paracasei and Lactococcus lactis, PrtB typical of Lactobacillus delbrueckii subsp. bulgaricus, PrtH characteristic of Lactobacillus helveticus, PrtS from Streptococcus thermophilus, PrtR found in Lacticaseibacillus rhamnosus, and PrtL typical of Lactobacillus delbrueckii subsp. lactis , we, weL. caL. casei , while wg for 10 min, washed with 500 μL of TE buffer , and re-suspended in 200 μL of TE buffer with glass beads (diameter 0.106 mm). Subsequently, the cell suspension was vortexed with Vortex-Genie 2  for 4 min . Next, 15 μL of proteinase K (20 mg/mL) was added, and the mixture was incubated at 60 °C for 1 h. After incubation, 40 μL of 20% SDS was added, and the samples were incubated at 65 °C for 15 min. After cooling at room temperature, 90 µL of refrigerated 5 mol/L potassium acetate was added, and the mixture was centrifuged at 10,000× g for 10 min. After performing phenol–chloroform extraction and ethanol precipitation, DNA samples were suspended in 50 μL of TE buffer. Then, the suspension was mixed with 1.5 μL of RNAse (10 mg/mL) and incubated at 37 °C for 2 h. The concentration and purity of gDNA samples were determined using a NanoDrop ND-1000 spectrophotometer , while gDNA quality was evaluated by electrophoresis on 0.8% (w/v) agarose gel containing ethidium bromide (0.5 μg/mL) in 0.5X TBE buffer . gDNA samples were diluted to 50 ng/μL in ddH2O and stored at −20 °C for subsequent analysis.Genomic DNA (gDNA) was extracted from late exponential cultures grown in MRS, as described by Gala et al. . Brieflyprt genes in L. casei genomes was performed using the BLASTp algorithm against the NCBI database, using L. casei PrtP (AFJ15093.1) as the query sequence. A curated dataset containing 44 proteins from 23 strains was built, and sequences were aligned with the constraint-based alignment tool (COBALT) method [https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi; last access on 14 June 2021; E-Value threshold 0.01, max. hits 500) using the CDD database.The in silico search for putative ) method , using tprt genes, using Primer3 [w/v) agarose gel electrophoresis stained with ethidium bromide (5 μg/mL) and, when required, PCR amplicons were digested with the endonuclease EcoRV  in a 20 μL final volume, according to the manufacturer’s instructions. PCR products were purified with the DNA Clean & Concentrator™-5 Kit , and were sequenced on both strands through a DNA Sanger dideoxy sequencing process using both external and internal primers  model and the neighbor-joining (NJ) method. Bootstrap support values were calculated from 1000 replicates in MEGA6 [Primer pairs were designed on the conserved regions within or surrounding the catalytic domains of  Primer3   at a final OD600nm value of 1010 CFU/mL, and then spread on milk–citrate–agar (MCA) plates  in triplicate. After incubation for 48 h at 37 °C, cells were recovered from plates and re-suspended in physiological solution at a final concentration of 1010 CFU/mL. Samples were used for both RNA extraction and biochemical characterization.To induce the expression of CEPs, the strains were pre-cultured in 50 mL of MRS broth and incubated for 72 h at 37 °C under anaerobic conditions. After centrifugation at 10,000× w/v) agarose gel with formaldehyde (10 mL of 10× MOPS running buffer) and 18 mL of 37% formaldehyde (12 mol/L) on a pH 7.0 1× MOPS running buffer , after RNA treatment at 65 °C for 10 min.For RNA extraction, glassware was sterilized at 180 °C for at least 4 h to degrade RNases, and all the solutions were prepared with diethyl pyrocarbonate (DEPC)-treated water. MCA-induced cells were washed twice with DEPC-treated TE buffer , and cell pellets were maintained at −80 °C until thawed with 1 mL of Tri-reagent using the Direct-zol RNA MiniPrep Kit . Mechanical lysis was performed using a Vortex-Genie 2 for two rounds of 20 min at the highest speed, alternated with 3 min on ice. The quantity of total RNA was measured spectrophotometrically using a NanoDrop ND-1000 , while the integrity was checked via denaturing gel electrophoresis on a 0.9%  at 37 °C for 2 min in a preheated thermocycler with the lid temperature adjusted to 37 °C. After being chilled on ice and briefly centrifuged, 12.5 µL treated RNA samples were used as templates for first-strand cDNA synthesis with random hexamers , using RevertAid Reverse Transcriptase , according to the manufacturer’s instructions. CEP-specific RT-PCR assays were performed with the primers reported above . RT-PCR g for 10 min and re-suspended in 100 μL of Ca2+-free buffer  at a final concentration of 1010 CFU/mL. After 30 min of incubation at 37 °C under agitation, extracts were centrifuged , the supernatants collected, and the pellets re-suspended in the same volume of the Ca2+-free buffer for three additional cycles of incubation [Following induction of CEPs, cells were centrifuged at 12,000× cubation . The colcubation .p-nitroanilide. The assay mixture, containing 107 μL of 50 mmol/L sodium phosphate buffer (pH 7), 56 μL of 5 mol/L NaCl, 9.5 μL of 20 mmol/L of substrate and 15 μL of extract, was incubated at 40 °C for 120 min in a covered water bath. The reaction was stopped by adding 94 μL of 80% acetic acid. The released p-nitroanilide was measured at 410 nm using a microplate reader. Control reactions with the substrate but without CEPs were also prepared [p-nitroanilide per minute. Specific activity was expressed as units of proteinase activity per mg of protein.The proteinase activity of crude extracts was evaluated via a chromogenic assay. The activity was measured using the specific substrate succinyl-alanyl-alanyl-prolyl-phenylalanine-2, or MgCl2 was added to the reaction mixture at a final concentration of 2 mmol/L . Similarly, in order to evaluate the effects of inhibitors on proteinase activity, EDTA or PMSF was added to the mix, at a final concentration of 0.5 mmol/L .The effect of temperature on the CEPs’ activity was measured as described above, modifying the incubation temperature. The assay was carried out at four different temperatures , at a constant optimal pH value of 7. The effect of pH on enzyme activity was evaluated by modifying the pH of the reaction buffer, keeping the temperature constant at 40 °C. Sodium acetate buffer (50 mmol/L) was utilized for reactions carried out at pH 4–6 whereas sodium phosphate buffer (50 mmol/L) was used for the reaction performed at pH 8. To analyze the influence of metal ions on proteinase activity, KCl, CaClTo test the caseinolytic activity of CEPs, 5 mg/mL of αS1-or β-casein solution, dissolved in sodium phosphate buffer , was mixed at a 1:1 volume ratio with the crude extracts containing 1.5 U/mL of CEP activity. The mixtures were incubated at 40 °C and, after various intervals , aliquots of samples were withdrawn and stored at −20 °C for further analysis.2O) for 1 h under stirring. Subsequently, gels were de-stained with the de-staining solution  for 30 min under stirring. The de-staining step was repeated four times.The casein breakdown pattern was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Samples collected during the caseinolytic test described above were diluted in Laemmli buffer in order to load 2.5 μg of total protein per lane. Denaturation was completed by boiling the samples for 3 min. SDS–PAGE was carried out on 12% polyacrylamide gels on vertical electrophoresis cells for 1 h at 200 V. Gels were stained with the Coomassie blue staining method  equipped with a C18 column . Mass spectrometry (MS) and tandem MS experiments were performed on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer . The full description of the gradient, flow rate, and MS and MS/MS parameters was reported by Martini et al. .Peptide sequencing was carried out by using MASCOT  protein identification software, with the following search parameters: enzyme, none; peptide mass tolerance, ±5 ppm; fragment mass tolerance, ±0.12 Da; variable modification, oxidation (M) and phosphorylation (ST); maximal number of post-translational modifications permitted in a single peptide, 4. The assignment procedure was confirmed by the manual verification of MS/MS spectra.The peptides identified via mass spectrometry analysis were investigated in relation to previously identified bioactive peptides. The identification of bioactive peptides was carried out by using the Milk Bioactive Peptide Database (MBPDB)—an online database of human milk- and dairy-derived bioactive peptides .The cleavage probability and the positive or negative influence on the cleavage of a specific amino acid in the P1 and P1′ subsites were calculated as described by Solieri et al. . The amiA is in the position n (P1 or P1′ subsite), the cleavage probability of the P1-P1′ bond will be as follows:The amino acid residue in position P1 or P1′ influences the CEP cleavage probability. If the amino acid residue The mean cleavage probability was defined by the following formula:A in the P1 and P1′ subsites was quantified by the coefficient Kn:The positive or negative influence of an amino acid residue Kn values > 0 indicate a favorable influence of the amino acid A in the specific subsite on the cleavage of the P1–P1′ bond, whereas Kn values < 0 suggest a negative effect on the cleavage.p < 0.05.Data are shown as the mean ± standard deviation (SD) for three replicates. Univariate analysis of variance (ANOVA) with Tukey’s post hoc test was applied using GraphPad Prism 6.0 . The differences were considered significant at prt gene exists in the genomes of several LAB species, and that the pattern of prt genes is highly variable at the inter-strain level. For instance, L. helveticus possesses four unique types of CEP—namely prtH1, prtH2, prtH3, and prtH4—while L. rhamnosus CGMCC11055 possesses both prtP and prtR genes, although prtP was initially detected only in L. lactis and L. paracasei/L. casei species [Most LAB are thought to possess only one type of CEP , but it  species ,24,25,26L. casei PrtP (AFJ15093.1) amino acid sequence as a query in BLASTp search, we built a dataset consisting of 44 putative Prt-encoding genes in L. casei. Protein alignment with COBALT showed that 22 proteins exhibited the insert domain, and were categorized as PrtP (data not shown). The remaining 22 proteins lacked the insert domain, and were categorized as PrtR, with three different lengths of approximately 1500, 1800, and 2200 amino acids (data not shown). This result suggests that both prtP and prtR paralogs exist in several L. casei genomes, including in the type strain ATCC 393T. Clustering analysis showed two distinct clades for L. casei PrtP proteins, referred to as PrtP1 and PrtP2, while PrtR proteins were grouped into three different clusters, referred to as PrtR1, PrtR2, and PrtR3 (Using the nd PrtR3 .“CD-search” in the Conserved Domain Database (CDD) predicted that PrtP1 and PrtP2 would share the same functional domains, except for the FIVAR domain (pfam07554)—which is present in PrtP2, but not in PrtP1—and the CHB_HEX_C domain, which is present in PrtP1, but not in PrtP2 . PrtR prprt genes in 44 L. casei genomes, CEP-specific primer pairs were designed across the putative active domains of five different Prt-encoding genes, and were used to assess the type and distribution of prtP and prtR paralogs in L. casei PRA205 and 2006. Both strains were positive for PrtP2- and PrtR1-specific PCR assays, as they exhibited two bands with the expected lengths of approximately 2630 bp and 1723 bp, respectively, while no PCR products were obtained with the primer pairs targeting the prtP1, prtR2, and prtR3 genes  to the S8-peptidase-encoding genes in the recently released genomes of L. zeae strains FBL8 and CECT 9104. L. zeae is a recently restored species closely related to L. casei, and is indistinguishable from L. casei based on 16S rRNA gene sequencing [L. zeae strain FBL8; 99.81%). Furthermore, PRA205 and 2006 PrtP2 partial proteins diverged from L. casei LC5 PrtP2 due to 13 substitutions, namely, E120K, N122S, V143A, D229A, T237A, S454R, T551A, D652A, A771T, R776G, S837G, K860R, N874S  were also proven to be plasmid-encoded in Lactococcus lactis [S. thermophilus, the prtS gene is part of a genomic island flanked by conserved insertion sequence (IS) elements [prt gene patterns observed in lactobacilli. Remarkably, PRA205 and 2006 PrtR1 partial proteins diverged from those of the L. casei group, and clustered separately , proving that the proteolytic activity reported was due to the action of CEPs rather than intracellular peptidases. However, we cannot exclude the possible occurrence of cell-wall-associated peptidases. The presence of cell-wall aminopeptidases has been reported for L. lactis, L. helveticus, and Streptococcus cremoris [L. casei.Peptide-rich media generally repress proteinase activities in several lactobacilli, including L. casei ,35. Thercremoris ,37,38. Hcremoris  did not prt gene is actively transcribed in MCA-grown cells, we carried out an RT-PCR assay. prtR1, but not prtP2, suggesting that the CEP activity detected above mainly came from PrtR1 proteinase when the strains were grown on MCA medium. This result differed from that found in L. rhamnosus strain CGMCC11055, which has both prtP and prtR genes, but only prtP gene transcription was detected when grown in a synthetic medium [L. rhamnosus EN1 was not released following incubation with Ca2+-free buffer.To qualitative establish which c medium . Moreovec medium , which sL. casei 2006, whereas the proteinase extracted from L. casei PRA205 retained 48% of its activity. The high proteolytic activity at 5 °C seems to be a peculiar feature of L. casei PRA205 PrtR1, since the majority of previously characterized CEPs almost completely lost their activity at temperatures around 20 °C [L. casei PRA205 showed a significantly higher residual proteolytic activity compared to that extracted from L. casei 2006.The proteolytic activity of PrtR1 was analyzed by modifying the incubation temperature and the pH of the reaction buffer in order to determine the best conditions of substrate hydrolysis. The PrtR1 extracted from each strain displayed the highest activity at 40 °C, and the enzymatic activity at 40 °C was taken as the reference (100%) for the calculation of residual (%) activity A. A slignd 20 °C ,8,19,41.L. casei PRA205 in the production of bioactive peptides in fermented dairy foods such as cheese, yogurt, and fermented milk, which are characterized by low pH values. This observation could also explain the fact that L. casei PRA205 whole cells presented higher proteolytic activity in fermented milk than L. casei 2006 whole cells, despite the latter strain showing the highest CEP specific activity [L. casei PRA205 PrtR1 retained almost 50% of its activity at low temperatures suggests that the enzyme might also be active during the cold storage of fermented dairy foods, promoting the continuous release of bioactive peptides from caseins during cold storage.This property could be of paramount importance because it suggests a possible exploitation of activity . Moreove+  and Mg2+  ions when added at 2 mmol/L, and vice versa—PrtR1 activity was enhanced by 2 mmol/L Ca2+ ions , as reported for numerous other extracted CEPs [L. casei PRA205 and L. casei 2006 PrtR1, respectively), suggesting that both PrtR1s were members of the serine proteinase family. The chelator EDTA at 1 mmol/L concentration also inhibited the PrtR1 activity , indicating that cations  are required for their activity.The proteolytic activity of both PrtR1s was inhibited by KThe proteolytic activity of PrtR1 was assessed against αS1- and β-caseins as substrates. Aliquots of samples were taken after 0, 8, 24, 30, 48, and 56 h, and analyzed via SDS–PAGE and mass spectrometry experiments. As shown in L. casei PRA205 and L. casei 2006 belonged to the PI/PIII type. These results are inconsistent with the findings of Kojic et al. [L. casei HN14, while they are consistent with the mixed PI/PIII-type CEP isolated from L. casei IFLP 731 [Based on the specific hydrolysis patterns of the αS1-, β-, and κ-caseins, CEPs were classified in three different types: CEPs belonging to the PI type primarily hydrolyze β-casein, which is cleaved into more than 100 different oligopeptides, ranging from 4 to 30 amino acid residues . PIII-tyc et al. , who chaIFLP 731 .Samples collected during the casein hydrolysis assay were submitted to UHPLC/HRMS analysis for peptide identification in order to study the casein breakdown patterns produced by the PrtR1 extracted from the selected strains. The full list of identified peptides at the different time points for the both β- and αS1-caseins, along with the mass spectrometry data, are reported in L. casei PRA205 and L. casei 2006, respectively  of the cleavage sites were still in the C-terminal region. At the end of the hydrolysis, the cleavage sites were distributed throughout the entire β-casein sequence for both of the PrtR1s; however, no cleavage sites were identified in the polyphosphorylated region between residues 8 and 28. It is worth noting that the majority of the previously characterized lactobacilli proteinases have a proven preference for hydrolyzing the C-terminal region of β-casein [The time-course analysis indicated that the hydrolysis of β-casein began at the hydrophobic C-terminal region. After 8 h of reaction, 80% and 76% of the cleavage sites produced by β-casein .Kn coefficients, which measure the positive or negative effects of amino acids on the P1–P1′ cleavage probability, were calculated , and for positively charged amino acids (R and K) in the P1 position. In particular, the amino acids M and N showed the highest Kn values for both PrtR1s, therefore exerting the strongest positive effect on cleavage probability at the P1 subsite. The only difference between the two PrtR1s was the strong positive effect of amino acid A on PrtR1 extracted from L. casei 2006. In position P1, the amino acids I, T, P, V, and H, as well as the negatively charged (D and E) and aromatic (F and Y) amino acids, exhibited a strong negative effect on cleavage probability for both of the extracted PrtR1s.PrtR1 extracted from both Kn values. The strongest negative effect at position P1′ was exhibited by the polar uncharged amino acids  and by the amino acid E. Some of the identified preferred amino acids have already been reported in previous studies; for example, the positive effect of amino acid Q in position P1 has been already described for CEPs characterized from L. delbrueckii CRL581, L. helveticus BGRA43, L. paracasei BGHN14, L. rhamnosus BGT10 and PRA331, and Lc. lactis NCDO763 [L. lactis NCDO763 and L. rhamnosus PRA331, respectively [L. casei PRA205 [The strongest positive effect on cleavage probability at subsite P1′ was found for the small aliphatic amino acids A and G, as well as the amino acids H and D, for both of the extracted PrtR1s. In both cases, the amino acids D and A showed the highest  NCDO763 ,23,43,44L. casei PRA205 and L. casei 2006, respectively . The hydrolysis of β-casein by PrtR1 of ectively .L. casei strains commonly released a total of 13 bioactive peptides, whereas 4 were uniquely produced by L. casei 2006 PrtR1 and 1 by L. casei PRA205 PrtR1. In contrast, the hydrolysis of αS1-casein by PrtR1 of L. casei PRA205 and L. casei 2006 generated 9 and 11 bioactive peptides, respectively, among which 8 were commonly released by the PrtR1 of both strains.The PrtR1 of both The identified bioactive peptides showed several functional properties, as 13 peptides were ACE inhibitors, 13 were antimicrobial, 6 were antioxidants, 5 had immunomodulatory activity, 1 showed dipeptidyl peptidase IV (DPPIV)-inhibitory activity, and 1 showed anxiolytic activity. Most of the identified bioactive peptides were tested in vitro for their bioactivity, whereas some of them also exhibited their activities in vivo. For example, the tripeptide LLY demonstrated a positive influence in vivo in Swiss albino mice against ethanol-induced oxidative stress . The β-cWith the sole exception of the αS1-casein-derived peptides LGY and RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEK, all the other identified bioactive peptides were found at the end of the hydrolysis, suggesting their resistance to further hydrolysis by PrtR1 . In addiTo exert their physiological effect, bioactive peptides should be stable under gastrointestinal conditions and resistant to the hydrolysis by gastrointestinal proteases. In a recent study, it was demonstrated that most of the peptides identified after in vitro gastro-pancreatic digestion contained 1–4 proline residues near the carboxylic ends of their sequences. In addition, the simultaneous presence of DPP-IV-inhibitory peptides causes a strong decrease in the intestinal prolyl peptidases’ activity . Among tL. casei PRA205 and L. casei 2006, previously isolated from ripened Parmigiano Reggiano cheese, were characterized. Both strains possessed two different prt genes in their genome—prtP2 and prtR1—but the evidence collected in this study suggests that only one gene, prtR1, is actively transcribed in MCS-induced cells after 48 h of incubation. In both strains, the predicted protein catalytic domain sequences of PrtR1 showed six amino acid substitutions compared with the reference sequence, suggesting that these proteases were peculiar to the selected strains. In addition, PrtR1 identified in L. casei PRA205 and 2006 showed high similarity with PrtR1-like sequences which are plasmid-encoded in other LAB species. The presence of PrtR1 on plasmids could confer an important evolutionary advantage to these strains, but further analysis is required to establish the true gene position.In this work, two unique CEPs from the highly proteolytic strains L. casei PRA205 retained 48% of its activity at 5 °C and showed higher activity at pH 4 than strain L. casei 2006. These important biotechnological features can be exploited to produce fermented dairy products with low pH and low storage temperatures, such as fermented milk and yoghurt.PrtR1 extracted from both strains displayed the highest activity at 40 °C, pH 7; interestingly, PrtR1 extracted from Remarkably, peptidomic analysis assisted us in demonstrating that these CEPs are able to release β- and αS1-casein-derived bioactive peptides. Most of these peptides matched the sequences of previously reported bioactive peptides, and some of them were resistant to gastro-intestinal hydrolysis.L. casei species, which is poorly explored in comparison with thermophilic lactobacilli or lactococci. Furthermore, PrtR1 from both strains were able to release some bioactive peptides, suggesting that L. casei can be a source of new proteases exploitable as enzymes for the formulation of dairy beverages or hydrolysates enriched in bioactive peptides, and with improved digestibility of milk proteins. Thereby, NS-LAB can be considered as a source of new enzymes, and the knowledge of CEPs developed in this work can be used as a tool for the selection of new proteinase-proficient strains, as well as for the improvement of functional lactobacilli performance—particularly in the L. casei species.Overall, the results presented in this study provide new knowledge on the proteolytic systems of two strains belonging to the


The maximum carboxylation rate (VCmax), which is limited by Rubisco activity, decreased earlier than the light-saturated potential electron transport rate. This decrease in VCmax was caused by decreased Rubisco activation state under HTS. Mg application reduced yield loss by stabilizing Pn. Rubisco activation was enhanced by increasing Rubisco activase activity following Mg application, thereby stabilizing Pn. We conclude that Mg maintains Rubisco activation, thereby helping to stabilize Pn under HTS.Inhibited photosynthesis caused by post-anthesis high-temperature stress (HTS) leads to decreased wheat grain yield. Magnesium (Mg) plays critical roles in photosynthesis; however, its function under HTS during wheat grain filling remains poorly understood. Therefore, in this study, we investigated the effects of Mg on the impact of HTS on photosynthesis during wheat grain filling by conducting pot experiments in controlled-climate chambers. Plants were subjected to a day/night temperature cycle of 32°C/22°C for 5 days during post-anthesis; the control temperature was set at 26°C/16°C. Mg was applied at the booting stage, with untreated plants used as a control. HTS reduced the yield and net photosynthetic rate (P Wheat is a C3 crop that grows during the winter and spring seasons, with an optimum grain filling temperature of 20–24°C . The temUnder favorable conditions, approximately 70–90% of the final grain yield is obtained from photosynthates produced during grain filling . Longer 2 during photosynthesis, and its catalytic limitations compromise photosynthesis efficiency  is involved in several physiological and biochemical processes of plant growth and development . Up to 1fixation . Mg mainfixation . Mg defiis rates , whereasis rates . Thus, MHigh-temperature stress frequently occurs during the grain filling stage; however, few studies have examined Mg functions involved in photosynthesis under HTS during the grain filling stage. We hypothesized that under HTS, Mg application would enhance Rubisco activation, contributing to HTS alleviation. Therefore, we examined variation in Rubisco content and Rubisco activation state, light energy utilization efficiency under HTS to evaluate the effects of Mg application on Rubisco carboxylation activity, and electron transport capacity under HTS.Triticum aestivum L.) was grown in plastic pots with a volume of 0.015 m3 , each with three holes at the bottom. Every pot was filled with 9 kg of air-dried and uniformly mixed clayey loam soil sieved through a 0.5-mm mesh. The soil contained 11.78 g kg–1 organic matter, 0.87 g kg–1 total nitrogen (N), 81.37 mg kg–1 available potassium (K), 19.25 mg kg–1 available phosphate (P), and 110.9 mg kg–1 available Mg. After soil filling, 5 g of compound fertilizer  was applied to each treatment. At the jointing (Feekes 6.0) and booting stages (Feekes 10.0) , during the 2016–2018 growing season. The local widely grown cultivar of Yangmai-16 (es 10.0) , 0.45 g 4⋅7H2O was applied at 0.85 g per pot (0.11 g⋅kg–1) at the booting stage (Feekes 10.0), and pots without additional Mg application were used as controls (CK). The pots were moved to climate-controlled chambers set at a different temperature for 16–20 days after anthesis (DAA). HTS of the air temperature was simulated at 32°C/22°C, and 26°C/16°C was applied as the optimal temperature (OT). The temperature setting during treatment days and the leaf temperature are shown in –2 s–1 with a photoperiod of 16 h. Each pot was watered about 2–4 l each morning and evening during post-anthesis. Following treatment, pots were relocated in the OT growth chamber until maturity. Thus, the four treatments were CK-OT , CK-HT (control fertilizer with HTS), Mg-OT , and Mg-HT .Magnesium fertilizer in the form of MgSOPlant sampling were conducted at 15DAA (prior to HTS treatment), 17DAA (2 days after treatment), 20DAA (5 days after treatment), and 25DAA. The flag leaves were detached from five randomly selected pots for each treatment and immediately submerged in liquid nitrogen for fresh sample measurements. Dry samples were collected from whole plants in five randomly selected pots of each treatment. We manually cut the plants at ground level using pruning scissors and then dried the samples at 105°C for 15 min in the oven, followed by 70°C until constant weight. The dried samples were ground for N and Mg content determination. Yield measurements at the maturity stage were tracked using the same five pots in each treatment.–2 s–1. The vapor pressure deficit was between 0.5 and 1.0 kPa. The reference CO2 conference level was set at 400 μmol mol–1, and the relative humidity of the leaf chamber was set to 55–65%. The temperature of the leaf chamber was set according to real-time temperature in the growth chamber. The equipment was preheated for 0.5 h prior to measurement, and data were recorded three times after cuvette acclimatization for at least 5 min.The gas exchange parameters were measured in flag leaves using the red and blue light source chamber (LI6400-02B) of a gas exchange machine  in this study. The gas exchange parameter measurements were according to the methods by 2 concentration (A–Ci) curve was determined according to 2 injection system was used to control the CO2 concentration. Leaves were placed in the chamber for 10 min for adaptation. The CO2 concentration in the leaf chamber was then adjusted to values of 400, 200, 100, 50, 100, 150, 200, 400, 600, 800, 1000, 1200, and 1600 μmol mol–1, and data were recorded for about 3 min per setting. Plotting Pn as the vertical coordinate and Ci as horizontal coordinate, the initial slope of the curve (Ci < 200 μmol mol–1) represented carboxylation efficiency (CE). The Rubisco maximum carboxylation rate (VCmax) and maximum electron transport rate (Jmax) were calculated according to modified equations (The net photosynthesis rate/intercellular COquations . The A–Cs) and then applied a flash (∼8000 μmol photons m–2 s–1) to record the maximum fluorescence under light (Fm’). We maintained the leaves in the dark for 3 s, turned on the far-red light, then measured the initial fluorescence Fo’ under the light. Next, the leaves were shaded and dark-adapted for more than 30 min, and the minimum (Fo) and maximum (Fm) chlorophyll fluorescence were recorded. Then, we calculated PSII efficiency (ΦPSII) as ΦPSII = (Fm’ – Fs)/Fm’, maximum fluorescence as Fm = Fm – F0/Fm (0’/Fs × (Fm’ – Fs)/(Fm’ – F0’) (m’ – Fs)/Fm’ × PPFD × 0.85 × 0.5  , and the85 × 0.5 .3 and HCl solution (1:1 v/v) for 4 h at 50°C, and then the volume was adjusted to 20 mL using ddH2O. Five biological repeats were conducted separately. Mg content was analyzed using the ICP-OES, Optima 8000.We used 100 mg ground dried samples to determine Mg content. The samples were extracted using 5 mL of HNOTotal N analyses were conducted with the method from micro-Kjeldahl . Dried sChlorophyll concentrations were measured spectrophoto metrically according to 2 frozen leaf samples and extracted each sample in 5 mL of buffer solution prepared according to the methods. Then, we centrifuge the mixture at 15,000 × g for 15 min at 4°C and mixed the crude enzymatic extract with 5 × loading buffer. We boiled 1 mL of the mixture for 1 min prior to electrophoresis. The amount of each sample was 10 μL used in the electrophoresis which was at 100 V for about 8 h. After that, the gels were stained with 0.25% (w/v) Coomassie Blue R-250. The gels were destained with 25% ethanol and 8% glacial acetic acid solution. Subunits of 55 and 15 kDa were put into a container with 2 mL of formamide. Then, the samples were kept in a 50°C water bath overnight. The absorbance at 595 nm (OD595) was measured. The calibration curve is shown in the Rubisco content was measured using SDS-PAGE following the methods of 450 within 15 min after adding the termination solution.Rubisco activase content was determined using an RCA Kit  according to the manufacturer’s instructions. The double-antibody sandwich method was used to determine the RCA content of the plant according to 14CO2 fixation assay, but the NADH-linked assays were correlated strongly with the radiometric assay  polyvinylpyrrolidone)], assay buffer solution , and activating solution  were according to 2 frozen leaf samples and extracted each sample using 5 mL of pH 8.0 buffer solution. The mixture was centrifuged at 15,000 × g for 1 min at 4°C. The initial Rubisco activity was measured using a cuvette containing 100 μL of the crude enzymatic extract, 700 μL of assay buffer, and 200 μL of 10 mmol RuBP. The change in absorbance at 340 nm was monitored for 60 s (3 s per measurement) at room temperature. To determine the total Rubisco activity, 100 μL of crude enzymatic extract was activated for 10 min at 25°C in RuBP assay buffer by adding 100 μL of activating solution. Next, 100 μL of this mixture was added with 700 μL of assay buffer and 200 μL of 10 mmol RuBP sequentially, and the OD value at 340 nm was monitored for 1 min (3 s per measurement). The ratio of the initial activity over the total activity was calculated as the Rubisco activation state.Rubisco activity was determined following the method of ic assay . NADH-liic assay , so we u3350 and 4 mM RuBP and then incubated for 5 min at 4°C. Next, the extract was added to two parallel (A and B) reactions at 25°C. The recipes of the A and B solutions were according to Rubisco activase activity measurements were processed following the process from g for 2 min at 4°C. The supernatant was collected. The ATP detection working solution was prepared according to the kit protocol. To each well, we added 100 μL of extract, followed by 100 μL of working solution. The luciferase signals were detected for 30 s using a multifunctional microplate reader (SpectraMax M2). A standard ATP concentration curve ranging from 1 pM to 1 M was prepared by gradient dilution. ADP concentration measurements were conducted by conversion to ATP.We used ATP and ADP Bioluminescence Assay Kit  to determine ATP and ADP contents, respectively, following the manufacturer’s instructions. Twenty milligrams of wheat leaf samples were collected in 200 μL pre-chilled lysis buffer, and a glass homogenizer was used to fully lyse the leaf tissue. The samples were centrifuged at 10,000 × P < 0.05. We used a minimum of three biological replicates. All graphs presented were produced using the GraphPad Prism 9 , and tables were produced using Microsoft Excel 2016 .All data were analyzed using the SPSS ver. 10.0 software . Two-way analysis of variance was performed for all data, and the means were compared using Duncan’s multiple-comparison tests at a significance level of High-temperature stress treatment significantly affected the 1000-grain weight of wheat plants, resulting in decreased yield . The 100After anthesis, CK-HT and Mg-HT treatment durations were shortened by about 3 days due to HTS . HoweverThe effects of Mg application on Mg and N content in flag leaves under HTS are shown in n of wheat flag leaves under all treatments generally decreased during growth (n (9.93%), transpiration rate (Tr) (5.64%), leaf stomatal conductance (Gs) (2.64%), and chlorophyll content (3.33%) and increased Ci (8.84%) at 17DAA, although the changes in Gs, Tr, and chlorophyll contents were not significant. At 20DAA, Pn, Gs, Tr, and chlorophyll contents decreased under HTS by 17.89, 9.14, 13.11, and 9.57%, respectively, compared with CK-OT, whereas Ci increased significantly, by 11.10%. Similar trends were observed at both 25DAA and 30DAA. At 17DAA in the Mg-HT treatment, Pn (4.30%), Gs (2.08%), Tr (4.25%), and chlorophyll contents (2.63%) were decreased and Ci (2.34%) was increased, but not significantly. At 20DAA, Mg-HT treatment stabilized Ci , and Pn, Gs, Tr, and chlorophyll contents decreased by 9.92, 7.33, 8.55, and 6.10%, respectively, compared to Mg-OT. Plants were also less affected by HTS in the Mg-HT treatment than in the CK-HT treatment at 25DAA and 30DAA. The above results indicated that Mg could enhance the photosynthetic capability and stabilize that under HTS.The Pg growth . HTS decCmax (21.71%) at 17DAA in CK-HT and also decreased Jmax (5.02%), but not significantly  and Vficantly . CE, VCmv/Fm) at 17DAA, but ΦPSII and ETR decreased due to a reduction in qL in the CK-HT treatment  stress  stress . Mg applg with K . In thisCmax, and Jmax decreased under HTS  consume ATP and NADPH to regenerate NADP+ and ADP, reducing the light-dependent reaction and accepting transferred electrons during stable forward photosynthesis (+ and ADP reduction. Next, electron transfer was inhibited, which led to photoinhibition (The COynthesis . Under Hynthesis . The posynthesis . HTS dirhibition . TherefoThe results of the present study indicate that HTS caused a decrease in Rubisco carboxylation activity which inhibited photosynthesis during the wheat senescence stage, whereas Mg application maintained Rubisco carboxylation by enhancing its activation state and stabilizing the electron transfer rate. Thus, photosynthesis was sustained by Mg application under HTS. These results suggest that Mg plays an indispensable role in sustaining photosynthesis during grain filling by improving Rubisco activation state under HTS conditions.The original contributions presented in the study are included in the article/YS, JH, CS, ZT, and TD conceived and designed the experiments. YS, JH, CS, SXL, and SYL developed methodologies. LG, SYL, JH, and JG analyzed the experimental data. SYL, LG, AU, YS, and XL wrote the manuscript. SXL, LG, AU, DJ, WC, ZT, and TD revised the manuscript. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


UFGT, F3H and LDOX) coupled with High-resolution melting (HRM) analysis aiming the varietal identification. The Alvarinho wines revealed to have the same haplotype using this marker set, demonstrating its applicability for genetic identification. In addition, to assess their geographical provenance, a multi-elemental approach using Sr and Pb isotopic ratios of wine, soil and bedrock samples was used. The isotopic data suggest a relation between Sr and Pb uptake by vine roots and soil’s texture and clay content, rather than with the whole rock’s isotopic ratios, but also highlights the potential of a discriminating method based on the combination of selected isotopic signatures.The interest in developing reliable wine authenticity schemes is a hot-topic, especially for wines with recognized added-value. In order to accomplish this goal, two dimensions need to be considered: the grapevine variety determination and the geographical provenance. The aim of this study was to develop a multidisciplinary approach applicable to wines from the sub region Melgaço and Monção of the demarcated Vinho Verde region and from the demarcated Douro region. The proposed scheme consists on the use of DNA-based assays to detect Single Nucleotide Polymorphisms (SNPs) on three genes of the anthocyanin pathway ( Global food products and beverages markets have increased the need to establish reliable and quick authenticity systems so they can be tracked, throughout the production and/or commercial chain, to its origin. The development of such a system has the advantage of guaranteeing consumers that the purchased products are in accordance to their expectations and avoid the enormous economic damages caused by forgeries and even health risks, due to the presence of dangerous compounds and contaminants in the counterfeited food products .Botrytis cinerea tolerance and, in 1756, the first region with denomination of origin (DO), Porto, was created in Portugal. From then on, several appellation and wine control systems have been implemented worldwide. These denominations are defined based on both the terroir features  and in the grapevine varieties used to produce such wines, giving them unique characteristics that can differentiate those wines in relation to other, having important economic returns [To assure the consumers needs’ and requirements in such differentiated food products, several certification labels  have been regulated by the European Union with different denominations  ,4. The wThe need to control the denomination of origin requires the implementation of a robust authenticity system, aiming to control both the grapevine variety and the geographical origin used in a given wine ,8,9. SinIn Portugal, there are contrasting features between the geological and soil environments of the Melgaço and Monção sub region ,27 of th3 medium-grained two-mica granite, yielding a strong penetrative foliation developed under ductile deformation conditions; (b) at Monção, a post-D3 coarse- to medium-grained porphyritic biotite granite, without major evidences of ductile deformation, but affected by some fracturing. The Douro demarcated region is essentially characterized by metasediments of neoproterozoic-cambrian age, which, in the area of interest of this study, are subdivided in the following formations: (a) Rio Pinhão Formation, formed by a succession of thick metagreywackes interbedded with phyllites and some levels of microconglomerates; (b) Pinhão Formation, composed by chloritic phyllites, quartzo-chloritic phyllites and metagreywackes; (c) Desejosa Formation, typically consisting of millimetre- to centimetre-scale alternations of parallel-laminated metapelites and fine-grained psammites, resulting in a striped appearance, that makes it very distinct from the two previous formations [2 and lower Al2O3 average contents than the latter [3 granites also occur. An example is given by the medium-grained slightly porphyritic two-mica granite that appears in Seixo de Ansiães area, which yields an internal northwest—southeast magmatic foliation, concordant with the structure of the host metasediments [In northern Portugal, two-mica granites and biotite-rich granites intruded neoproterozoic to devonian metasedimentary rocks during the Variscan orogeny ,29. In trmations ,31. Geoce latter . Althougediments ,34.In this study, different samples of wine (11), soil (11) and bedrock (11) were collected from seven vineyards of the two demarcated wine-producing regions: (1) Monção and Melgaço sub region and (2) Douro region . The soiThe grapevine varieties Touriga Franca and Touriga Nacional at Quinta do Portal and Quinta do Confradeiro vineyards (both implanted in the Desejosa Formation) and Alvarinho and Touriga Nacional at Quinta da Abelheira vineyards (both implanted in the Rio Pinhão Formation) were used as a control in order to assess a possible influence of metabolic processes from plants on Sr and Pb isotopic signatures’ fractionation of wines produced in a particular geological/soil environment.2; 60 mg/L) was added to both red and white musts. White grape-must was clarified by cold settling at 4 °C for 36 h. Red grape-musts  and white grape-must were transferred to 2 L containers filled to 2/3 of their volume and fitted with a rubber cork fixed with an air locker. All grape-musts were supplemented with 1 g/L of di-ammonium phosphate (DAP). Alcoholic fermentations were conducted at 17 °C (white grape-musts) and at 25 °C (red grape-musts) by the commercial yeast strain S. cerevisiae QA23 inoculated at an initial cell count of 106 CFU/mL. The vinifications were daily monitored by weight loss, as an estimate of CO2 production, and were allowed to proceed until no further weight loss was observed. After the end of alcoholic fermentation, the wines were racked off the lees and cold settled at 4 °C. Before bottling, SO2was added to wines to adjust free sulfur dioxide content to 35 mg/L and to 50 mg/L, for red and white grape-must, respectively. The standard physico-chemical analyses were performed prior to and at the end of alcoholic fermentation, according to the Organisation International de la Vigne et du Vin (OIV) official methods [All wines resulted from monovarietal micro-vinifications to avoid possible multi-isotopic variability . Grapes http://www.stabvida.com, accessed on 1 April 2021) using three previously studied genes: UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) [flavanone 3-hydroxylase (F3H) and leucoanthocyanidin dioxygenase (LDOX) [Young leaves of Alvarinho, Touriga Franca and Touriga Nacional grapevine varieties were collected from different vineyards and immediately frozen in liquid nitrogen, in order to be used as a reference material to develop the HRM assays. Total genomic DNA was extracted from the frozen young leaf samples using the CTAB method . DNA same (UFGT) ,33, flave (LDOX) .http://www.stabvida.com, accessed on 1 April 2021). The CodonCode Aligner 4.0.4  was used to generate consensus sequences, and sequence alignment was performed using Geneiousv5.6.4. The HRM data was analysed using the High-Resolution Melt Software v3.0.1 . After normalization and determining the temperature shift, the different melt curves of the several plots were generated.PCR reactions and HRM analysis were performed in a StepOne™ Real-Time PCR System  according to Pereira et al. ,37 and G3) mixture in pressurized teflon capsules (Savillex®) on a heating plate for three days, then dried and taken up in hydrochloric acid (HCl) for chemical separation. The wines were also digested on a heating plate, but through a reflux system with HNO3 and hydrogen peroxide (H2O2) in successive stages. After the total digestion of the different samples, the resulting solutions were evaporated and the residues were dissolved with HCl (6.2 N) and dried again. Cation exchange resin (BioradAG50W) in quartz glass columns was then used to separate the Sr fraction by ion chromatography. All reagents used in the preparation of the samples were purified by bi-distillation. The ultrapure water (18.2 MΩ.cm) was obtained by a Milli-Q Element system® . 87Sr/86Sr isotopic ratios were measured on a VG Sector 54™ mass spectrometer  operating in dynamic mode. The different Sr samples were deposited in a Ta filament with phosphoric acid (H3PO4) (0.5 N) and by maintaining a 1–2 V 88Sr beam for 50–100 cycles, an internal precision of 20 ppm (at 2σ level) on 87Sr/86Sr was consistently achieved. Fractionation was corrected with an exponential law relative to 86Sr/88Sr = 0.1194. During the measuring campaign, the value determined for the Standard Reference Material® (SRM) 987 was 87Sr/86Sr = 0.710256 ± 0.000005  and 0.710271 ± 0.000013 . Analytical blanks for Sr are lower than 250 pg.Strontium isotopic analyses of bedrock, soil and wine samples were obtained at the Laboratory of Isotope Geology of the University of Aveiro (LGI-UA), Portugal, following the methodology described by Costa et al. , with so3. For geological samples, the solutions were conveniently diluted to obtain a concentration of about 200 ng of about Pb/g solution. The samples were introduced as wet aerosols into a Neptune™ MC-ICP-MS  using an ESI 100 µL/min PFA nebulizer  and a dual cyclon-ic-Scott double pass spray chamber . Lead amounts from wine samples were lower than 100 ng/g solution. Therefore, wine samples were diluted to a 20 ng/g solution and introduced as dry aerosols with an ESI 50 µL/min PFA nebulizer  and an ESI Apex-IR desolvating unit . The spectrometric measurement comprised a static multi-collection routine of 105 cycles with an integration time of 8 s per cycle. Spectrometric  blanks were subtracted with the On-Peak-Zeroes routine, measuring a blank 0.32 N HNO3 solution before each sample for 60 s. Instrumental mass bias was corrected online after the addition of a proportional amount of a solution of the National Bureau of Standards® (NBS) 997 thallium certified reference material and using 205Tl/203Tl = 2.3889 according to Thirlwall et al. [206Pb/204Pb = 19.9442 ± 0.0022; 207Pb/204Pb = 15.5016 ± 0.0011; 208Pb/204Pb = 36.7317 ± 0.0056; 208Pb/206Pb = 2.16781 ± 0.00010; 207Pb/206Pb = 0.91486 ± 0.00006.The Pb isotopic analyses of bedrock and wine samples were performed at the Geochronology and Isotope Geochemistry-SGIker Facility of the Universidad del País Vasco UPV/EHU (Spain). Samples were prepared in PP Class-A (ISO-5) laminar flow bench within an ISO-7 clean lab. Geological and wine samples were digested following procedures adapted from Marchionni et al. . Lead wal et al. . The accThe particle size analysis of selected metasedimentary and granitic soils was carried out on full robotized equipment from Skalar Analytical  at the Laboratory of Soils and Plants—Joaquim Quelhas dos Santos of University of Trás-os-Montes e Alto Douro, Portugal, following the International Organization for Standardization (ISO) 11277 method by the sedimentation/pipetting option.To confirm the varietal composition of Alvarinho, Touriga Franca and Touriga Nacional wine samples, a DNA–based method was tested, using high–resolution DNA melting analysis to detect variants/haplotypes, based on specific SNPs detected within the nucleotide sequence of three genes previously studied, UFGT, F3H and LDOX ,38. To eThe grapevine varietal identification in wine samples is extremely relevant ,38,42. S87Sr/86Sr isotopic ratios in whole rocks, bulk soils and wines from vineyards of eleven different locations are reported in 87Sr/86Sr values from the studied areas range from 0.727938 ± 0.000020  to 0.783222 ± 0.000020 (two-mica granite–Melgaço), whereas those from soils are within the range of 0.728944 ± 0.000020 (Rio Pinhão Formation–Quinta da Abelheira) and 0.779035 ± 0.000019 (two-mica granite–Melgaço)  . The varion line A, fact t to soil A. Furthe87Sr/86Sr values are within the range of 0.713981 ± 0.000024 (Alvarinho–Melgaço) and 0.721478 ± 0.000017 (Touriga Franca–Pinhão Formation) and, generally, there are no significant differences between grape varieties  values, which are more resistant to chemical weathering [87Sr/86Sr values than the wine  ,18,24,47According to Mihaljevič et al.  the totaSome recent studies stated that lead uptake in plants occurs mainly through the foliage and in a lesser extend through the root system ,60. Neve206Pb/204Pb, 207Pb/204Pb and 208Pb/204Pb ratios because 204Pb is the only stable non-radiogenic isotope of Pb [206Pb/204Pb and 207Pb/204Pb values are always higher  than those of metasedimentary rocks (18.3067 ± 0.0009 to 18.5879 ± 0.0009 and 15.6475 ± 0.0007 to 15.6797 ± 0.0008), but no distinction can be made with respect to their 208Pb/204Pb values  to 18.7847 ± 0.0008 (Alvarinho–Rio Pinhão Formation); 15.6244 ± 0.0009 (Touriga Franca–Pinhão Formation) to 15.6699 ± 0.0008 (Alvarinho–Rio Pinhão Formation); and 38.3963 ± 0.0021 (Alvarinho–Monção) to 38.8573 ± 0.0020 (Alvarinho–Rio Pinhão Formation), respectively. Furthermore, there are no significant isotopic differences within the two monovarietal wines  produced from the vineyards of Quinta do Portal and Quinta do Confradeiro (Desejosa Formation)  for the results obtained by Almeida and Vasconcelos in wine Port, table and red fortified wines from Douro region [206Pb/204Pb = 17.84 ± 0.32, 207Pb/204Pb =15.24 ± 0.31 and 208Pb/204Pb = 37.31 ± 0.52,54 and also from those determined by Epova et al. [206Pb/204Pb = 18.191 ± 0.146, 207Pb/204Pb = 15.621 ± 0.014 and 208Pb/204Pb = 38.207 ± 0.156.In the studied areas, the wines’ rmation) C, but atnal wine D,E, whico region  in winesa et al.  in authe207Pb/204Pb and 208Pb/204Pb values from whole rock samples to the respective wines stands out, that is extended to the 206Pb/204Pb values of granites and respective wines. However, this latter isotopic ratio increases from the metasediments to the respective wines. These isotopic discrepancies observed between geological substrata and their respective wines can be due to mineralogical and textural specificities of each bedrock and respective derived soils. In fact, Marchionni et al. [206Pb and 207Pb in metasediments and granites, lower 206Pb/204Pb and 207Pb/204Pb values would be expected for their respective wines [206Pb/204Pb values than the respective whole rock samples, would be the higher rate of adsorption of lead in their enriched clay fraction, enabling a concomitant isotopic uptake by vine roots [The Pb isotopic ratios of both bedrock and wine samples were determined at the same uncertainty level, so their relation can be depicted in the diagrams of i et al. ,19 statei et al. . In thisve wines . Therefone roots ,51.206Pb/204Pb and 208Pb/204Pb values  than those from granitic areas , but they are indistinct with the respect to their 207Pb/204Pb values (206Pb/204Pb and 208Pb/204Pb values of those wines produced in vineyards emplaced in the less permeable soils, derived from metasediments, suggest, as for Sr isotopic results, a correlation between the Pb uptake by vine roots and texture and clay content of soils, rather than with the whole rock’s Pb isotopic ratios. However, within granitic areas, the Alvarinho wine from Melgaço yields higher 206Pb/204Pb, 207Pb/204Pb and 208Pb/204Pb values than that from Monção (In the variation diagrams of b values D,E. As pm Monção D,E. ConsIn general, the achieved results for Pb and Sr isotopic systems show an identical behavior within the two studied geological environments and respective wines. However, the Desejosa Formation’s metasediments/soils and wines yield Sr isotopic ratios similar to those from both granites of Monção and Melgaço B, but di207Pb/206Pb and 208Pb/206Pb signatures it is possible to obtain a more perceptible and reliable differentiation scheme to distinguish wines according to their geographical origin. In fact, two wine/bedrock series, with distinctive linear regressions, can be identified among the studied samples (By combining wine and bedrock’s  samples F, which The present study represents the basis for the development of an authenticity and geographical origin assessment system applied to wines from the sub region Melgaço and Monção of the demarcated Vinho Verde region and also to those from the demarcated Douro region. This system requires that both varietal composition and geographical provenance are correctly determined.UFGT, F3H and LDOX genes [The use of DNA-based markers has proven to be efficient in the discrimination of the varietal composition of wines, as long as a panel of molecular markers is previously established ,38. MicrOX genes ,38. The In terms of geographical provenance, the potential of a discriminating method based on the use of selected Sr and Pb isotopic signatures has been demonstrated, combined with textural and clay content analysis, which enable to distinguish wines produced from vineyards implanted in soils derived from metasedimentary and granitic environments. Concomitantly, these isotopic studies have also emphasized the importance of soil’s texture and clay content role in Sr and Pb uptake by vine roots.However, further detailed studies, involving a higher number of bedrock, soil and wine samples of vineyards from different locations and distinct winemaking process, are still required to improve the reliability of authenticity criteria for these wines. In the future, these studies could benefit the provenance and authenticity assessments for Alvarinho, Porto and other Portuguese wines, and therefore help to avoid fraud and adulteration.


Escherichia coli that are in line with German regulations for fertilizers. However, one finding of Salmonella spp. exceeded the threshold value. Cultivation of bacteria from the mature compost resulted in 200 isolates with 36.5% of biosafety level 2 (BSL-2) species. The majority is known as opportunistic pathogens that likewise occur in different environments. A quarter of the isolated BSL-2 strains exhibited multiresistance to different classes of antibiotics. Molecular analysis of total DNA before and after composting revealed changes in bacterial community composition and ARGs. 16S rRNA gene amplicon sequencing showed a decline of the two most abundant phyla Proteobacteria  and Firmicutes , whereas the abundance of Chloroflexi, Gemmatimonadetes, and Planctomycetes rose. Groups containing many human pathogens decreased during composting, like Pseudomonadales, Bacilli with Bacillus spp., or Staphylococcaceae and Enterococcaceae. Gene-specific PCR showed a decline in the number of detectable ARGs from 15 before to 8 after composting. The results reveal the importance of sufficiently high temperatures lasting for a sufficiently long period during the thermophilic phase of composting for reducing Salmonella to levels matching the criteria for fertilizers. However, most severe human pathogens that were targeted by isolation conditions were not detected. Cultivation-independent analyses also indicated a decline in bacterial orders comprising many pathogenic bacteria, as well as a decrease in ARGs. In summary, thermophilic composting could be a promising approach for producing hygienically safe organic fertilizer from ecological sanitation.In times of climate change, practicing sustainable, climate-resilient, and productive agriculture is of primordial importance. Compost from different resources, now treated as wastes, could be one form of sustainable fertilizer creating a resilience of agriculture to the adverse effects of climate change. However, the safety of the produced compost regarding human pathogens, pharmaceuticals, and related resistance genes must be considered. We have assessed the effect of thermophilic composting of dry toilet contents, green cuttings, and straw, with and without biochar, on fecal indicators, the bacterial community, and antibiotic resistance genes (ARGs). Mature compost samples were analyzed regarding fecal indicator organisms, revealing low levels of  Declining natural resources, high energy demand of mineral fertilizer production and wastewater treatment, as well as increasing soil degradation raise the need for alternative ideas regarding sanitation and agriculture . EcologiBeneficial effects on soils have also been described for biochar, which is charcoal derived from organic matter through pyrolysis. In contrast to charcoal, however, biochar is produced not as a fuel but as an amendment for agriculture  . AdditioHowever, there are major concerns regarding the safety of compost from ecological sanitation when used for crop production, concerning especially the hygiene, as well as the fate of pharmaceutical residues and antibiotic resistances .via contaminated vegetables, e.g., the EAHEC outbreak via sprouts between May and July 2011 with almost 4,000 infected persons in Germany only . Although composting parameters are diverse and results differ, mposting .Acquired antibiotic resistance and the accumulation of multiple resistances in pathogenic bacteria are a growing health problem causing an estimated minimum of 700,000 deaths every year . There aTo assess the hygienization potential of thermophilic composting for ecological sanitation, especially regarding human pathogenic bacteria and ARGs, we have conducted composting trials with human excrements together with other organic wastes. In addition, the effect of biochar as a co-substrate during the composting process was evaluated.C/N-ratio of the substrates are shown in n = 3): 459 ± 10 kg/m3 density; 37.1 ± 1.1% dry matter; 79.1 ± 10.6% volatile solids; pH 9.4 ± 0.1; 0.5 ± 0.1 mS/cm conductivity; 72.0 ± 5.6% dry matter carbon; 0.57 ± 0.07% dry matter nitrogen; 1.84 ± 0.24% dry matter hydrogen; 0.07 ± 0.01% dry matter sulfur. The high carbon content of around 70% of dry matter and high amount of volatile solids of around 80% dry matter match the properties of biochar  was set up twice at an interval of 2 weeks, with E1 and E1-B referring to the replicates, which were set up first, and E2 and E2-B referring to the replicates set up 2 weeks later. “-B” indicates the biochar treatment. E1 and E1-B were set up on August 15, 2018 and E2 and E2-B set up on August 29, 2018. The dimensions of the piles as measured from the ground (length × width × height) were 6 m × 3 m × 0.9 m for E1, 5 m × 3 m × 0.9 m for E1-B, 8 m × 3 m × 0.8 m for E2, and 5 m × 2 m × 0.8 m for E2-B. Moisture of the compost material on the day of experimental set-up was 61, 64.3, 66.5, and 67.1% for E1, E1-B, E2, and E2-B, respectively. Temperature curves of the averaged measurements are shown in Sampling was conducted at the day of the experimental set-up (“start” samples), after 2 weeks (only repetition 1), and at the end of the experimental trials after 5.5 (repetition 1) and 5 months (repetition 2), respectively. Samples were taken from the center of each compost pile at a depth of approximately 0.6 m at three different positions , respectively. Therefore, a hole was dug into the pile and approximately 20 L of substrate/compost were excavated and homogeneously mixed on a tarp. From this homogenous mixture circa 300 g was sampled for analysis. All samples were stored at 4°C for the isolation studies and at −20°C for molecular analyses.Salmonella and Escherichia coli were detected by means of the most probable number technique (MPN) according to the German Federal Ministry for the Environment, Nature Conservation, and Nuclear Safety (–1 dilution) and shaken at 150 rpm at 4°C overnight. A 10-fold dilution series up to 10–9 was prepared. Triplicates of each dilution were applied to a series of cultivation steps as follows.For the assessment of compost hygiene, r Safety . StatistSalmonella, 1 mL of each dilution was added to a test tube with 9 mL buffered peptone water (Carl Roth GmbH), vortexed for 10 s, and incubated at 37°C for 24 h ± 1 h. A total of 100 μL of this enrichment was transferred into 10 mL Rappaport-Vassiliadis broth , vortexed for 10 s, and incubated at 42°C for 24 h ± 1 h. One loopful of the sample was spread on two specific agars for identification: brilliant green-lactose-sucrose agar  and xylose-lysine-deoxycholate agar . Plates were incubated at 37°C for 24 h ± 1 h. Putative positive colonies were transferred to standard-I-agar , and incubated at 37°C overnight for verification via slide agglutination test with the monoclonal test serum “Anti-Salmonella A-67 + Vi, omnivalent”  according to the manufacturer’s instructions.For E. coli was performed in parallel from the same dilution series. One milliliter of each dilution transferred to 9 mL MacConkey broth , vortexed for 10 s, and incubated at 37°C for 24 h ± 1 h. If a color change to yellow, indicating lactose fermentation, was observed, samples were streaked on MacConkey agar  for verification.Detection of Salmonella and E. coli results were documented indicating the confirmed positive (+) or negative (−) results for each of the triplicates in each dilution step. From the number of positive replicates in the highest three dilutions showing positive results, an index number was generated to be used with the respective tables of Both –2 – 10–7) were plated in triplicates on R2A agar and CHROMagar™ ESBL , respectively. Plates were incubated at 37°C overnight. Colonies were classified according to their morphology and subsets of each group transferred to selective agar for specific isolation of severe human pathogenic bacteria  in 0.9% sodium chloride solution and shaken overnight at 4°C and 150 rpm. Afterward, a 10-fold dilution series was prepared. One hundred μL of each dilution  using the protein extraction procedure according to the manufacturer’s instructions . Mass spectra were compared with the MALDI-BDAL Database (Version 3.1). Scores between 2.0 and 3.0 were interpreted as high-confidence identification as recommended by the manufacturer. Lower scores were considered not sufficient for identification.Isolates were identified All by mass spectrometry not sufficiently identified and all BSL-2 isolates with a MALDI score below 2.3 were subjected to 16S rRNA gene Sanger sequencing . For this, single colonies of the isolates were suspended in 300 μL sterile distilled water and incubated at 95°C for 15 min to disrupt the cells. There was 0.5 μL template used in a 10 μL PCR reaction containing 0.2 U Taq polymerase , 1 x PCR reaction buffer S , 0.2 μM of each primer (16S rRNA gene standard primers 27f and 1492r), 250 μM of each dNTP, and 1 μg bovine serum albumin (BSA). PCR was initiated by heating to 95°C for 5 min, followed by 30 cycles of 30 s at 95°C, 30 s at 50°C, and 10 s at 72°C. The final elongation step was carried out for 7 min at 72°C. The PCR products were sequenced by Eurofins Genomics Germany GmbH, Ebersberg, Germany. Sequences were aligned against the nucleotide database (nt) of BLAST sequence analysis tool .1). Isolates were tested against the antibiotics recommended by EUCAST for the respective genus  were performed to analyze the antibiotic resistance of the isolates. Sixteen frequently used antibiotics, ampicillin, meropenem, imipenem, ciprofloxacin, erythromycin, vancomycin, clindamycin, doxycycline, tetracycline, tigecycline, kanamycin, gentamicin, sulfamethoxazole, norfloxacin, ofloxacin, and nitrofurantoin were selected for analysis according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing  as equivalent to a cell number of 1 × 108 CFU/mL, which was applied in the assay accordingly. CASO agar with 10% newborn calf serum as suggested by the authors was tested in comparison with CASO agar without the serum. Since CFUs did not vary for the isolates tested, CASO agar without additives was used for the assay. The plates were incubated at 37°C for 16–20 h before measuring the halo diameters.Compost was stored at −20°C after sampling. Due to the very inhomogeneous structure, especially of the material prior to composting, pretreatment was necessary before DNA extraction. Therefore, samples were dried at 40°C until weight loss was no longer detectable (between 14 and 22 days) and grinded using a vibratory disk mill (Fritsch) with an agate p-9 grinding set for 5 min at 750 rpm. Grinding jars were cleaned after each sample through a 2-min grinding step with quartz sand and subsequent washing.® Soil gDNA extraction kit (Macherey-Nagel) according to the manufacturer’s protocol. Lysis buffer SL2 was applied without enhancer. For the mechanical disruption of the samples a FastPrep-24™ 5G homogenizer  was used at 5 m/s for 30 s. Extracts were eluted in 50 μL nuclease-free water, purified using a magnetic separation device NucleoMag SEP (Macherey-Nagel) and CleanNGS kit (CleanNA) according to the manufacturer’s instructions and stored at −20°C.Total DNA was extracted from 250 mg compost samples using the NucleoSpinThirty-two different ARGs were searched for using PCR with gene-specific primers . Every P® High-Fidelity PCR Kit  was used with 0.02 U/μL polymerase, 1× buffer GC, 0.2 μM of each primer, 200 μM of each dNTP, 0.2 mM MgCl2, 5% DMSO, and 0.6 μg BSA. For the first round, 0.5 μL of the 10-fold diluted purified DNA extracts (1–5 ng) were used in 10 μL PCR reactions consisting of an initial denaturation of 5 min at 95°C, 20 cycles of 30 s at 95°C, 30 s at 50°C, and 10 s at 72°C and a final elongation of 7 min at 72°C. For the second round, 1 μL of PCR product from the first round was used as a template in a 50-μL reaction. Thermal cycling was carried out at 98°C for 1 min, followed by 8 cycles consisting of 45 s at 98°C, 45 s at 60°C, and 10 s at 72°C, and a final elongation at 72°C for 5 min. PCR products were quantified by means of agarose gel electrophoresis and purified using the NucleoMag® 96 clean-up kit (Macherey-Nagel) according to manufacturer’s instructions and eluted in 50 μL of sterile, nuclease-free water. Triplicates were pooled in equal volumes for sequencing.For Illumina sequencing, 16S rRNA gene amplicons were generated in a two-step PCR approach using primers 341f (5′-CCTACGGGNGGCWGCAG-3′) and 805r (5′- GACTACHVGGGTATCTAATCC -3′) without adapters in a first round to generate considerable amounts of PCR product. The PCR product was used as a template in a second round with primers S-D-Bact-0341-b-S-17 (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACG GGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTA CHVGGGTATCTAATCC -3′) . PCR wasPREP Plus Magnetic Beads  were used for purification of the indexed products as recommended by the manufacturer. Normalization of all libraries to the same concentration was performed using the Janus Automated Workstation from Perkin Elmer . Sequencing was conducted using Illumina MiSeq platform using dual indexing and MiSeq reagent kit v3 (600 cycles) as recommended by the manufacturer.Indices and Illumina sequencing adapters were attached to the PCR products using the Nextera XT Index kit . Index PCR was performed using 5 μl of template PCR product, 2.5 μl of each index primer, 12.5 μl of 2× KAPA HiFi HotStart ReadyMix, and 2.5 μl PCR grade water. The thermal cycling scheme was as follows: 95°C for 3 min, 8 cycles of 30 s at 95°C, 30 s at 55°C and 30 s at 72°C and a final extension at 72°C for 5 min. Quantification of the products was performed using the Quant-iT dsDNA HS assay kit and a Qubit fluorometer  following the manufacturer’s instructions. MagSi-NGSde novo chimera and reference-based chimera removal against the SILVA SSU 138 NR database (® (® (Demultiplexing and clipping of adapter sequences from the raw amplicon sequences were performed with the CASAVA software (Illumina). The program fastp (v0.20.0)  with a mdatabase . Sequencdatabase  and RStuabase  under the NCBI BioProject study number Salmonella spp. and E. coli as indicator organisms for compost hygiene. Salmonella was detected in one triplicate of pile E1, resulting in a mean of <120 CFU/g for E1. E. coli was present in all piles in small numbers , Achromobacter (2.5%), Stenotrophomonas (2.5%), Staphylococcus (1%), and Streptomyces (1%). In addition, Castellaniella, Cellulosimicrobium, Dermacoccus, Lysinibacillus, and Rhodococcus are represented by one isolate each. The number of isolates per compost pile varied between 33 and 63, but numbers derived from biochar and non-biochar treatments were similar . No clear patterns were observed regarding distribution of genera and abundances in biochar-containing piles and biochar-free ones. Serratia was an exception, with 18 isolates from the biochar-containing piles and 6 from the non-biochar ones. Although less abundant, Bordetella  and Stenotrophomonas  also showed notable differences in their distribution between biochar and non-biochar treatments. In the non-biochar treatments, a higher diversity of genera was found (13 vs. 9). Besides, most genera were slightly more abundant in the non-biochar treatments, except Serratia, Achromobacter, and Lysinibacillus spp. that were more abundant in the biochar treatments. Eleven genera were found in the first repetition and nine in the second one. Nineteen Serratia isolates were derived from the first composting repetition vs. five from the second repetition. Bacillus (55 vs. 19) also mostly occurred in the first repetition. Pseudomonas isolates peaked in the second repetition (14 vs. 6), as well as Achromobacter (5 vs. 0) and Stenotrophomonas (4 vs. 1). Forty different species were identified, 10 of which are assigned to BSL-2 and 3 that are not assigned to a biosafety level yet . Seventy-three isolates in total belong to BSL-2 (36.5% of identified isolates), slightly more BSL-2 isolates were found in the non-biochar treatments (n = 39). The largest group within BSL-2 isolates consists of B. intermedia , followed by Serratia marcescens (n = 11) and Pseudomonas mendocina (n = 6). Bacteria were isolated from the mature compost (two replicates per pile) through targeted cultivation on bacterial groups containing human pathogens. In total, 200 isolates were obtained and phylogenetically affiliated Acinetobacter baumannii, Bacillus anthracis, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, and Staphylococcus aureus) only B. cereus and P. aeruginosa could be isolated. In terms of the genera of interest, Bacillus, Pseudomonas, and Staphylococcus spp. were identified. Potential serious human pathogens such as Bacillus anthracis, Staphylococcus aureus, Acinetobacter spp., and Enterococcus spp. were not isolated.Of the species of interest for which the culture conditions were selected , 14 days samples  and end samples (triplicates from each pile). We aimed to compare the bacterial community before and after composting, as well as the treatments with and without biochar as a co-substrate of the composting process.x-axis, which explains 74.1% of the differences observed. Fourteen-day samples are closer to the start samples, indicating higher similarity with the bacterial community from the beginning of composting than with the mature compost community.The composition of the bacterial community changed from the start to the end as depicted by principal coordinates analysis of all samples analyzed . Start, Proteobacteria , Firmicutes (13–33% → 12–16%), Actinobacteria (13–18% → 16–18%), Chloroflexi (1–5% → 13–15%), Bacteroidetes (7–14% → 8–10%), Gemmatimonadetes (0.5–1.1% → 3.7–5.1%), and Planctomycetes (0.1–0.7% → 3.7–4.7%)  . This coGammaproteobacteria represented the most abundant group within the phylum Proteobacteria, followed by Alpha- and Deltaproteobacteria. The first accounted for 23–35% of the sequences in the samples from the start and declined by half toward the end of composting (11–12%). This trend was due to strong reductions in the most abundant orders Pseudomonadales (5–16% → 1%) and Xanthomonadales (3–5% → 1–2%), whereas other orders within the Gammaproteobacteria increased in relative abundance toward the end of composting. Among them, Steroidobacterales (0–0.3% → 3.1–4.2%) was the most prominent one with slightly higher numbers in the non-biochar treatments  as the most abundant genus, and Rhodobacteraceae with Pseudofulvimonas as the most abundant genus (0.11–0.56% → 0–0.02%).eatments . The ordPseudomonas to 0.6–1.1% in the end samples, Pseudomonas spp. made up 10% of all isolates. Serratia, representing 12% of the isolates, were not found in the 16S rRNA gene amplicons. However, the family they belong to, the Enterobacteriaceae, were present .Despite the decline in abundance of Alphaproteobacteria increased in relative abundance from 6 to 7% in the start samples to 10–11% after composting. This trend was mainly visible in the most abundant order Rhizobiales (2.7–4.8% → 6.5–7.2%). However, the relative abundance of the most prominent familiy in both start and end samples, Rhizobiaceae, declined (2.3–4.1% → 1.9–2.1%), whereas the following families showed rising numbers .Sphingomonadales with only one assigned family (Sphingomonadaceae) and a declining trend (1.2–2.4% → 0.5–0.9%) and Rhodovibrionales, which showed a small increase in most compost piles (0.3–0.7% → 0.5–0.6%). Interestingly, abundance of members of one of the two families declined (Fodinicurvataceae), whereas numbers of the other rose (Kiloniellaceae).Next in abundance were the orders Deltaproteobacteria rose over the course of composting from 4–6 to 6–8%. This tendency could be found in 10 of the 11 orders detected, Bradymonadales being the exception. However, abundance trends within the trials differed for the dominant order Myxococcales  and PB19 (max. 0.03%). Myxococcales were not only the most abundant, but also the most diverse order with 11 families, mostly consisting of only one affiliated genus.The abundance of Firmicutes, Bacilli were the dominant group, showing a decline in relative abundance from start to end of composting . The most abundant order was Bacillales with highest abundances in the 14-day samples, but an overall decline toward the end of composting . Three orders were affiliated with Clostridia: Clostridiales being dominant with 2.5–4.8% of sequences in the start samples and 3.0–3.6% in the mature compost.Actinobacteria did not change, but the occurrence of its dominant class Actinobacteria declined (12–17% → 6–7%), whereas other groups rose in numbers (Acidimicrobiia (0–1% → 7–9%), Thermoleophilia, Nitriliruptoria). The three most abundant orders within the class Actinobacteria were Micrococcales, Streptosporangiales, and Corynebacteriales, all of which declined during composting. Corynebacteriaceae with their only genus Corynebacterium showed increased numbers in the 14-day samples but disappeared at the end of composting.Relative abundance of 16S rRNA gene sequences of the phylum Bacteroidetes with the number of Bacteroidia declining (7–14% → 7–9%) and the other two classes Rhodothermia (0–0.1% → 0.6–0.9%) and Ignavibacteria (0 → 0.1–0.5%) rising. The three most abundant orders within the Bacteroidia were Cytophagales, Flavobacteriales, and Sphingobacteriales of which the first one rose and the latter two declined over the course of composting. Interestingly, all three orders showed relative abundances in the 14-day samples opposing the general trend from start to end, meaning lowest levels in the Cytophagales 14-day samples and highest levels in the respective samples for Flavobacteriales (Flavobacteriaceae) and Sphingobacteriales (Sphingobacteriaceae). Besides, 14-day samples jumping out of the general trends were revealed for the order Bacteroidetes, as well as for orders Corynebacteriales and Propionibacteriales within the Actinobacteria, for Clostridiales within the Firmicutes, and within the Proteobacteria for Rhizobiales and Sphingomonadales (Alphaproteobacteria), Myxococcales (Deltaproteobacteria), Cellvibrionales, Oceanospirillales, and Pseudomonadales (Gammaproteobacteria). This list is based on individual observation of E1 and E1-B with averaged end samples (without E2 and E2-B), since 14-day samples were only available for repetition E1/E1B. When samples of each treatment were further averaged with start and end samples of E2 and E2-B, this pattern applies to more groups, including Bacillales and Bacilli within Bacillales . Numbers rose strongly from start to end of composting (1–5% → 13–15%). This trend was seen in all classes, especially in Anaerolineae (0–1% → 9–11%) and Chloroflexia (1–4% → 3–4%).Among the most abundant phyla, Gemmatimonadetes and Planctomycetes showed a rising tendency from start to end of composting. Both groups held many uncultured representatives and a relatively low diversity at higher taxonomic resolution.All classes and orders within Differences in terms of relative abundances in the starting material from repetition 1 (E1 and E1-B) and repetition 2 (E2 and E2-B) were often observed. The repetitions were set up independently 2 weeks apart from each other. The averaged end samples, however, are usually very similar regardless of the starting relative abundance. Most of the outliers could be seen in only one of the four compost piles (E2-B) and in most of the cases in one of the biochar duplicates .Biochar treatment did neither show different composition or abundance of species compared with the non-biochar treatment, nor different tendencies of decrease or increase over composting. However, the alpha diversity was slightly higher as compared to the non-biochar samples. Nevertheless, the strongest change in diversity was observed between start and end samples of the composting process .To get an insight into the number of different ARGs, their presence or absence in the compost, differences between non-biochar and biochar treatments, as well as the development and/or dissemination of resistance genes in the composting process, gene-specific PCRs were conducted.aac6-aph2a, aph(2)-Ic], erythromycin , sulfamethoxazole , and tetracycline (tetL) were detected in all samples. Both kanamycin resistance genes tested, as well as ermB and tetracycline resistance genes tetB, tetK, tetM, and tetS, were present in the start and partly the 14-day composting samples, but not in the end samples. tetA was present in starting and 14-days samples and in 1 out of 12 samples from the mature compost. ampC, aph(2)-Ib, blaZ, ermC, ermD, mecA, vanB, qnrA, qnrB, and vanC1/C2 genes, chloramphenicol resistance genes catpIP501, and catLM, as well as carbapenemase-genes blaIMP, blaKPC, blaNDM-1, blaOXA-48, and blaCTX-M-15 were not present in any of the samples. No clear differences between biochar and non-biochar treatments were observed. The number of different resistance genes detected decreased from 15 in the starting samples to 8 in the mature compost  due to one positive result in one of the triplicates analyzed. Therefore, the requirement of most German regulations of 0 CFU/50 g of fertilizer was not met for pile E1  that are not easily degradable. Therefore, temperatures remained rather low and did not match the German regulations for compost [minimum temperatures of 55°C for 2 weeks, 60°C for 6 days, or 65°C for 3 days, Salmonella enterica ssp. enterica serotype Senftenberg during composting. Besides, moisture content and microbial competition were found to influence survival of Salmonella. The authors identified a combination of 60°C and 60–65% moisture during composting as sufficient to inactivate Salmonella within 10 h . In general, moisture contents between 50 and 60% are considered to be optimal  shows (intermediate) resistance to ampicillin, sulfamethoxazole, and vancomycin. Clinical strains are often resistant to beta-lactam antibiotics, which is in line with the ampicillin resistance in this isolate . However, one notable difference is the occurrence of multiple resistant isolates, of which two-thirds originate from the biochar treatments. Interestingly, no differences between the treatments have been detected in terms of the molecular analysis of antibiotic resistance genes in compost DNA extracts. Different biochar types, as well as the combination of biochar type with different manures, were shown to influence the behavior of ARGs over composting . BiocharB. cereus and P. aeruginosa), and other BSL-2 isolates were present only in small numbers. The identified species also occur naturally in soil environments and are known as opportunistic rather than strict pathogens. Of BSL-2 isolates, 79% exhibited no or only few resistances. All these findings point to a hygienization of the substrates over the course of composting.Although more than one third of the isolated bacteria belong to BSL-2, only a few serious human pathogens targeted by the isolation strategy, were identified , although relative abundances of the respective phyla vary across experiments and treatments. In many studies, relative abundance of Proteobacteria in the mature compost varies between around 10 and 30%  can be found in the article/EG and KW designed and supervised the laboratory experiments. KW, AP, DS, KE-S, MW, and IL performed the laboratory experiments and analyzed the data. KP, NB, and TH designed the composting trial. TH conducted process control. KW, DS, AP, TH, and EG wrote the manuscript and designed all figures and tables. RD contributed with insightful discussions on analysis and interpretation of the data. All authors interpreted the results, read, and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The acidity of all the isomers was evaluated by both spectrophotometric and potentiometric titrations. The introduction of the -OCF3 group influences the acidity, depending, however, on the position of a substituent, with the ortho isomer being the least acidic. Molecular and crystal structures of ortho and para isomers were determined by the single crystal XRD method. Hydrogen bonded dimers are the basic structural motives of the investigated molecules in the solid state. In the case of the ortho isomer, intramolecular hydrogen bond with the -OCF3 group is additionally formed, weaker, however, than that in the analogous -OCH3 derivative, which has been determined by both X-Ray measurements as well as theoretical DFT calculations. Docking studies showed possible interactions of the investigated compounds with LeuRS of Escherichia coli. Finally, the antibacterial potency of studied boronic acids in vitro were evaluated against Escherichia coli and Bacillus cereus.Three isomers of (trifluoromethoxy)phenylboronic acids were studied in the context of their physicochemical, structural, antimicrobial and spectroscopic properties. They were characterized by  It is not, however, the case of the ortho isomer, primarily due to the steric hindrance. Very little is known, however, about the properties of boronic acids containing the -OCF3 group. The syntheses of all isomers of (trifluoromethoxy)phenylboronic acids have been previously reported ..ortho- aErot) for the twist of boronic group is 16.2 kJ mol−1 and 16.6 kJ mol−1 for para (3) and meta (2) O-CF3 substituted phenylboronic acids, respectively (ortho isomer (Erot = 33.0 kJ mol−1). Concordantly, molecule 1 is more stable than 2 and 3. Taking into account the boronic group resonance stabilization effect, the intramolecular hydrogen bond energy was estimated to 17 kJ mol−1. This is in general agreement with the value calculated from topological features of electron density at the bond critical point (BCP) using the Espinosa–Lecomte estimation   direction leads to the formation of a characteristic molecular ladder …B interactions. Finally, an interesting observation can be made while analyzing the mechanical features of single crystals. Compound 3 forms typical cuboidal crystals, which are easily crushed under applied force. In contrast, 1 crystallizes in the form of very tiny needles, which easily undergo mechanical plastic deformation under force . The E. coli LeuRS (4ARI) complex with a boronic ligand (AN2679) was taken from the protein databank   take into account a correction to the dilution of the analyte during the measurement. All measurements were performed at 298 K. The dissociation constants values obtained with the potentiometric method were calculated using a STOICHIO version CV EQUID computer program which uses the non–linear least-squares Gauss–Newton–Marquardt method for the fitting procedure /wR[F] (I ≥ 3σ(I)) = 5.97%/12.65%; Δρmax/min = +0.34/−0.26 eÅ3.3: C7H7BF3O3, Mr = 205.93 a.u.; T = 100 K; triclinic, P-1, a = 4.9858(7) Å, b = 7.2976(9) Å, c = 11.5769(13) Å, α = 86.779(9)°; β = 87.374(10)°; γ = 85.973(10)°; V = 419.14(9) Å3; dcalc = 1.632 gcm−3; μ = 1.470 mm−1; F(000) = 208; θ full: 67.7 o; number of collected/independent/unique reflection (Rint = 1.40%) = 2592/1689/1467; no. of parameters/restrains = 133/2; GOF = 1.044; R[F]/wR[F] (I ≥ 3σ(I)) = 3.73%/9.92%; Δρmax/min = +0.31/−0.21 eÅ3.To calculate the dimer interaction energies, a single-point calculations on M062X  6–311 + Bacillus cereus (CCM 2010) and Gram-negative bacteria—Escherichia coli (CCM 5172), obtained from the Czech Collection of Microorganisms (Masaryk University). Bacteria were routinely maintained at 34 °C on nutrient broth which consists of peptone (5 g) and meat extract (3 g) in 1000 mL of distilled water.The antibacterial activity of compounds was tested against Gram-positive bacteria—6–107 bacterial cells was uniformly spread out on the surface of the solidified nutrient agar medium and allowed to dry. In such prepared cultures, 3-mm wells were cut and filled with DMSO containing 100, 50, 25 or 10μg of the tested compound. As the controls, DMSO and water solutions of 50 μg of streptomycin were used. Cultures of bacteria were incubated for 24 h at 34 °C. The antimicrobial activity was estimated by the measurement of the inhibition zone of bacterial growth around the place of application of the tested substances. The experiments were performed in triplicate.The activity of tested compounds was established using both the agar well diffusion method and by determining the minimal inhibitory concentration (MIC) values. In the case of the diffusion tests, 0.5mL of a 24-h culture containing 106 CFUs (colony forming units) per 1 mL of nutrient broth were prepared. The number of bacterial cells in medium was estimated using optical density measurements (OD) at an absorbance wavelength of 600 nm. To obtain the set of experimental concentrations of investigated compounds, the stock solution was serially diluted and placed to cultures, ensuring that the final concentration ranged from 1 to 500.0 µg  mL−1. Appropriate cultures were incubated for 24 h at 34°C. The experiments were performed in triplicate. The MIC was assessed based on the lowest concentration of tested compounds required to inhibit bacterial growth (detected as the lack of visible turbidity) and by spectrophotometry by measuring the OD600.The minimal inhibitory concentration (MIC) was determined by a serial dilution method. For this purpose, 2 mL of bacterial cultures containing 10 1–3 were characterized by 1H, 13C, 11B and 19F-NMR spectroscopy. The commercial sample of 2 contained considerable amounts of the corresponding boroxine, whereas the acetone solution of 1 and 3 exclusively contained signals corresponding to boronic acids. The addition of a drop of D2O to its acetone solution resulted in shifting the equilibrium towards boronic acid as well as shifting all the signals in the NMR spectra. The introduction of the -OCF3 group influences the acidity, depending, however, on the position of a substituent with the ortho isomer being the least acidic. Typical hydrogen bonded dimers are the basic primary structural motives of compounds 1 and 3 in the solid state. Amongst the three isomeric (trifluoromethoxy)phenylboronic acids studied, compound 1, containing an ortho substituent, displays an intramolecular hydrogen bond present in the solid state. However, due to a strong electron-withdrawing effect of the OCF3 substituent, that interaction is weaker than analogous one found in 1a. Despite the promising results of docking studies, none of the studied compounds reveal considerable activity against Escherichia coli. At the same time, compounds 2 and 3 display limited activity against Bacillus cereus with MIC values of 125 µg/mL.Compounds