Pseudomonas abieticivorans sp. nov.), that can grow on resin acids as a sole carbon source. Our results illuminate key stages in degradation of an abundant renewable resource, and how defensive extractive compounds have major roles in shaping microbiomes.The bark is the outermost defense of trees against microbial attack, largely thanks to toxicity and prevalence of extractive compounds. Nevertheless, bark decomposes in nature, though by which species and mechanisms remains unknown. Here, we have followed the development of microbial enrichments growing on spruce bark over six months, by monitoring both chemical changes in the material and performing community and metagenomic analyses. Carbohydrate metabolism was unexpectedly limited, and instead a key activity was metabolism of extractives. Resin acid degradation was principally linked to community diversification with specific bacteria revealed to dominate the process. Metagenome-guided isolation facilitated the recovery of the dominant enrichment strain in pure culture, which represents a new species ( The bark is the outermost defense of trees against microbial attack, largely due to toxicity of extractive compounds. Here, Ristinmaa et al. study microbial community dynamics and chemical changes during degradation of spruce bark over six months, showing that the microbial degradation of extractive compounds, such as resin acids, has a major role in shaping the microbial community. Extractives are secondary metabolites with diverse properties that can be extracted using hydrophilic or hydrophobic solvents. Compared to wood, the chemical composition of bark remains less well understood and it also differs widely among tree species, especially regarding the extractives, and may also differ depending on season and sampling point4. Spruce is one of the dominant tree species in northern hemisphere forests and of high economic value, and its bark comprises around 10–15% of the volume at harvest. In the Nordic countries alone, bark is produced in close to 400 million m3 per year5, but it is largely seen as an industrial side stream and is either left to rot in the forest or burnt for energy at the mill. In addition to wood polymers, spruce bark contains a high amount of lipophilic extractives, mainly resin acids (>10 mg/g dry bark), triglycerides (~8 mg/g), steryl esters (~5 mg/g), fatty acids (~2.5 mg/g), and sterols (~1.3 mg/g)  and so-called extractive compounds , in addition to the regular wood polymers cellulose, hemicelluloses, and lignin7. Resin acids in particular are known to be toxic to microorganisms8 as well as water-living animals with LC50 values  of sub-mg/L9. Despite the antimicrobial properties of bark, it is degraded in nature over time, but compared to the extensive literature on the biological degradation of wood, data on the molecular details of microbial bark degradation are sparse. Snapshot studies of which microbial communities can colonize bark of different trees have been conducted, for instance, analyzing bark remaining on logs or samples buried in soil after years of incubation12. However, how these communities correlate to degradation stage is currently unclear, as simultaneous detailed chemical analyses of the bark have not been conducted over time.Extractives generally inhibit microbial attack through cellular toxicity or by precipitating proteins, and this is also true for spruce extractivesPhlebiopsis gigantea, Trametes versicolor, and Bjerkandera spp14. Additionally, sterols and unsaturated fatty acids were degraded by Phanerochaete velutina and Stropharia rugosoannulata15, and free and esterified sterols by Phlebia radiata and Poria subvermispora16. Bacteria have also been shown to degrade triglycerides, sterols, and resin acids17. More detailed investigations of resin acid degradation by Pseudomonas and Paraburkholderia suggested a degradation pathway of abietane-type resin acids19. However, it is presently unclear whether certain extractive groups have a larger effect on microbial communities and their development during bark degradation, and whether individual microorganisms tolerate, metabolize, and/or detoxify bark extractives. The composition and development of microbial communities during active decomposition of bark cannot be directly compared to similar studies of wood degradation, due to the large proportion of toxic extractive compounds in the bark material21.Regarding the degradation of individual spruce extractives, there are reports of non-toxic triglycerides and fatty acids, as well as toxic resin acids. Triglycerides have been shown to be degraded by the fungi Pseudomonas abieticivorans. We confirmed that P. abieticivorans can grow on resin acids as the sole carbon source, which is attributed to a large gene cluster encoding this dedicated function. Our holistic study paves the way for detailed understanding of microbial degradation of bark, which is important information concerning both the overall carbon cycle in forests and future development of valorization strategies for complex renewable bark biomass.In order to understand how bark can be degraded, the process needs to be monitored over time and both microbial and chemical analyses need to be combined, to answer the questions of what type of species may be involved or dominate and what bark compounds are mainly affected. Here, we have investigated spruce bark degradation over 6 months using an enriched microbial community sourced from an industrial bark pile, and combined several methods to obtain a broad picture of the entire process. The enrichment development was studied by marker gene and metagenomics sequencing, and the simultaneous chemical changes of the carbohydrates, lignin, and extractives were monitored. We identified resin acids as key molecules to be degraded to allow the expansion of microbial diversity, while carbohydrates were metabolized to a lesser extent. Bacteria rather than fungi were linked to resin acid degradation, and we successfully isolated and genome-sequenced a strain of the main resin acid-degrading bacterium, representing the new species Bark was sourced from a pulp and paper mill after having been stripped from the logs. To sterilize the bark without excessively disrupting its structure, it was gamma-irradiated instead of autoclaved, as elevated temperatures and pressure would release or modify the extractives, and doses over 20 kGy were found to be enough to stop any growth. An active inoculum was prepared from unsterilized bark, and separate cultures were inoculated and collected over 24 weeks, with one fraction per sample analyzed by DNA extraction, and one fraction analyzed chemically.2 or other volatiles. As a portion of each sample was sent for DNA extraction and sequencing, and the ash cannot disappear from the material, the proportional ash content was used to normalize the data in the biotic samples. The overall mass loss represents 3.6 g per 10 g sample, but the total bark consumption should be considered higher as microbial cells also represent a fraction of the total carbon. The bark carbohydrates were expected to be a major nutrient source for the consortium, and the monosaccharide composition was determined. Surprisingly, there were only small decreases in hemicellulose-derived monosaccharides   bark samples, very little differences could be observed overall throughout the process. The ash content, however, appeared to increase by ~53% in the biotic samples over the entire cultivation Fig. , which we) Figs. , S2. Howe) Figs. , S2, whees Figs. , S2. The4. As hypothesized, the amount of extractives decreased significantly over time for the biotic sample with 46% decrease in extractive dry weight after 2 weeks and 76% decrease after 24 weeks. In the abiotic sample no significant differences were observed , Penicillium (14%), Trichoderma (6.4%), and Mucor (6%), which are common cosmopolitan fungal genera.To compare the chemical changes in the bark to the development of the microbial consortium enriched on it, we analyzed its composition by long-read sequencing of marker gene regions of both bacteria (16S rRNA) and fungi ITS2), followed by taxonomical assignment, and a principal component analysis showed good agreement between replicates Fig. . After q, followe25. Overall, the bacterial microbial community was dominated by the genera Pseudomonas, Burkholderia-Caballeonia-Paraburkholderia, and Nitrospirillum where the former two have previously been linked to resin acid degradation19. To gain further insight into the microbiome at 2 weeks, this sample was further shotgun metagenome sequenced.The alpha diversity of the bacterial community was higher than that of the fungi throughout the experiment. In contrast to the fungal community however, it sharply decreased in the 2-week sample and then gradually increased to almost the initial value Figs. . This suPseudomonas and Burkholderia22.The combined observation of resin acid degradation and lowered bacterial diversity during initial growth suggests enrichment of highly specialized microorganisms that can tolerate, detoxify, or fully metabolize resin acids. To gain deeper biological insight on the process, short-read metagenome sequencing was first performed on the 2-week sample, and later followed by long-read sequencing to improve quality and enable reconstruction of metagenome-assembled genomes (MAGs) Table  and S5. Pseudomonas abietaniphila BKME-9T, Pseudomonas diterpeniphila A19-6a, and Paraburkholderia xenovorans LB400T (formerly: Burkholderia xenovorans) a so-called dit gene cluster has been attributed to this function from gene knockout studies  in the metagenome . Many of the MAGs classified as Pseudomonadota  have a ditA1 homolog which is a gene shown to be essential for resin acid-degradation through gene knockout analyses in both P. abietaniphila and P. xenovorans26. Comparing all ditA1 homologs among the MAGs showed sequence identities around 30% for the vast majority, which corroborates previous studies suggesting that ditA1 is not predominantly shared via horizontal gene transfer28. In the metagenome, MAG7 was the second most abundant (12.6%) and classified as Paraburkholderia. It encodes a putative partial dit cluster comprising ditA1, ditB, ditD, ditL, and ditQ  score of 99.06%, which despite only being 4.3% relative abundant has a considerable amount of putative dit genes, including two ditA1 genes found in two different clusters, together with ditF and ditJ homologs. Curiously, MAG4 encodes a seven-gene cluster comprising ditA1, ditA2, ditC (24.5% seq. id.), ditB, ditI, ditL, and ditQ, while only representing 2% of the metagenome reads.Resin acid-degradation is sparsely described in literature but within the genomes of es Table 19,26,27.ed Figs. , S9, andome Fig. . VaryingitQ Fig. . Despiteion Fig. . MAG6 wa29. In total, 1783 putative CAZymes were identified, including 777 glycoside hydrolases (GHs), 752 glycosyltransferases (GTs), 135 carbohydrate esterases, 81 auxiliary activities, and 38 polysaccharide lyases , in ten different PULs  present in the MAGs using dbCAN2ses Fig. . Interesses Fig. 1–S15. Ovses Fig. , S17. MAULs Fig. . Two of ULs Fig. , which aPseudomonas genus, while taxonomic classification analyses using both the Genome Taxonomy32 and SILVA33 databases supported our strain being a new species, which we named Pseudomonas abieticivorans sp. nov. A phylogenetic analysis based on 16S rRNA similarity between P. abieticivorans and the most closely related Pseudomonas species, which do not include previously identified resin acid-metabolizing species, showed P. abieticivorans being located on a separate phylogenetic branch , P. baetica a390T (81.4%), P. putida NBRC 14164T (81.6%), and P. koreensis Ps 9-14T (81.6%) , and it is interesting to speculate what limits growth on these substrates, but as the solubility of resin acids is very low (1.7–5 mg/L), solubilization is likely a major factor limiting utilization9.With resin acid degradation being a major activity in early spruce bark degradation and MAG15 being numerically dominant, we pursued to study this specific population in more detail. Based on the previous chemical and genomic information, we designed culture conditions with abietic acid as a carbon source which enabled us to successfully isolate a bacterial strain (designated PIA16) from the 2-week sample which was further characterized via genome-sequencing and phenotypic analysis. Long sequencing reads were assembled into a reference-grade, closed 6,715,763 bp genome, with a 62% G + C content and 5987 predicted protein-coding genes Tables , S8. A snch Fig. , again s%) Table , well beP. abieticivorans revealed few degradative CAZymes, and only suggests the ability to grow on starch, with enzymes from GH13 (7 homologs), GH31 (1), GH77 (1), in addition to several putative enzymes involved in bacterial cell wall lysis/remodeling , GH73 (2), GH103 (2))  which is composed of around 80 protein-coding genes ) Fig. . The API2)) Fig. , which frce Fig. , but intait Fig. 22. To gais Table , identifis Table , definednes Fig. . When anids Fig. .Fig. 4ThP. abieticivorans encodes a complete dit cluster (24/24 expected genes), which was compared to those found in known resin acid-degrading strains using BLAST and collinearity analyses  was also identified in P. resinovorans, but in P. multiresinovorans, homologs to dit genes were not found in a cluster but rather spread out in the genome.As expected from the metagenomics, ses Fig. . Strikinans Fig. . A lower36. Despite its fundamental protective role, how bark can be degraded by microorganisms is not known. Previous studies have shown that microbial communities differ between bark and wood, as well as the specific sampling point on the tree, and also that the presence of bark slows down the degradation of the underlying wood37. The importance of the bacterial communities that exist on bark of living trees was recently highlighted, where it was shown that bark-dwelling methanotrophic bacteria decrease methane emissions from trees, thus mitigating global warming38. Fungal bark communities have been less investigated but shown to change over time as wood degrades through measurements of wood decay stage, density, dry matter content, and C:N ratio12. Despite the importance of extractives in the antimicrobial properties of bark, they have, however, previously been overlooked.Bark is vital for the protection of trees and disruption of this outer barrier by e.g., wood-boring insects can lead to enhanced microbial access, decimated coniferous forests worldwide, and disruption of global ecosystems8.In this study, we monitored the development and dynamics of a bark-sourced microbial community on fresh spruce bark over 6 months, as well as the chemical changes to the material to identify important degradation stages, compounds, and microorganisms for this process. The minor degradation of polysaccharides was surprising, as especially non-cellulosic and non-crystalline carbohydrates are regarded as readily degradable by plant biomass-metabolizing enzymes, while slow lignin degradation was observed as expected. It is worth pointing out that some of the measured monosaccharides likely originate from the microorganisms, as these could not be separated from the bark itself, and possibly the acid insoluble residue could partially comprise microorganism-derived molecules in addition to lignin. It appears that for the microbial enrichment used in this study, the bark extractives are the main nutrients in the initial degradation stages, or possibly inhibit the growth of specialized carbohydrate-degrading species. The initial sharp decrease in alpha-diversity in the bacterial community, and that it only recovered after the resin acids had begun being metabolized, suggests that resin acids in particular may act as “gate-keeper” molecules and that their detoxification is a key step in bark degradation. This agrees well with resin acids being regarded as major defensive compounds with broad antimicrobial properties39, and the apparent increase in the concentration of especially dehydroabietic acid at the initial degradation stage is interesting. It could indicate that the resin acids are in fact inaccessible in fresh bark and possibly weakly bound to other molecules, polymerized, or otherwise tightly encapsulated within the bark before being released. Currently, there is no information on resin acids being for instance ester-bonded to any structural component within the bark, but it could be speculated that their release is instead linked to the degradation of triglycerides, as these can help solubilize the hydrophobic resin acids in the resin itself. Recently, extractive compounds have been shown to either inhibit or enhance the activity of carbohydrate-active enzymes, such as lytic polysaccharide monooxygenases40, so it is possible that the extractives may exert general enzyme-inhibitory rather than toxic effects that block wider carbohydrate utilization.Resin acids are thought to mainly be encapsulated within resin canals in wood25, though a clear shift in actual carbohydrate-degrading capacity in the enrichment did not become apparent during our study. Direct comparison with previous studies is not trivial as they typically have analyzed single time points, combined bark and wood, and/or omitted detailed chemical analyses12. However, in some studies, we see similar profiles of microbial abundance as reported here, with a dominance of Pseudomonadota for bacteria11 and mainly basidiomycetes and ascomycetes for fungi12, though without chemical analyses of the bark itself knowing what stage of degradation these previous studies correspond to is not possible. Without complementary techniques, such as quantitative metaproteomics, the biological roles of the fungi remain more elusive as the number of reads from sequencing does not necessarily reflect biological importance, and fungi are often regarded as main drivers of boreal forest biomass conversion. Follow-up studies of bark-degrading microbiomes will be crucial to determine how diverse such communities can be, and whether our results reflect general trends for microbial spruce bark metabolism. While the studied cultures were fully exposed to air, we cannot exclude the possibility that (micro)anaerobic pockets were also formed in the material during the experiment, though as full conversion of resin acids has not been observed anaerobically22, such micro-environments would likely not be highly abundant. The dry matter content used in the experiment (30%) was close to that of fresh spruce bark 42, which is not known as an anaerobic tissue.Over a longer time frame, the amplicon data suggest that the bacterial populations begin shifting from an extractive focus to a carbohydrate conversion, given the increase of Bacteroidotadit cluster-related genes, assumed to encode enzymes and proteins involved in resin acid (diterpene) degradation26, which further indicates that resin acid metabolism is crucial to thrive on fresh spruce bark. Interestingly, the number of encoded degradative CAZymes in our reconstructed MAGs was relatively low in the MAGs of highest abundance , from a bark pile resulting from stripping of spruce logs at the mill after harvest, with the average age of trees at harvest being ~70 years. The bark was left to dry at room temperature for seven days. Thereafter, the bark was milled in a Wiley-type mill, mesh size <0.1 mm, and was afterwards sterilized with 25 kGy by gamma-irradiation (Mediscan GmbH & Co KG). To validate sterilization, bark samples were subjected to 0, 10, 20, and 30 kGy, respectively and then added to either liquid lysogeny broth (LB) or potato dextrose agar plates. No growth was observed from bark samples that had received 20 or 30 kGy after several weeks of incubation.42). The solid-state cultures were incubated aerobically in the same conditions as the inoculum, above, and individual samples were collected after 0, 2, 4, 8, 12, 16, 20, and 24 weeks of growth in biological triplicate experiments. The moisture was adjusted if needed using water based on regular weighing of the samples. At sampling, ~4 g bark samples (dry weight) were weighed and used for chemical analysis of the carbohydrates, lignin, and extractives present in the bark, while the remaining culture was frozen at −20 °C and used for 16S rRNA and ITS profiling. Non-inoculated blank samples sterilized by gamma-irradiation in the same manner as the inoculum treated sample were used as controls.To partially mimic growth conditions on spruce bark in nature, solid-state cultivation was used. First, a bark inoculum was generated by growing unsterilized, milled bark in 2.3 mL M9 minimal medium  per g bark for 6 months , to obtain an actively growing culture . 1 g (wet weight) of the ~30 g growing culture was added to 10 g sterile bark samples with 2.3 mL M9 medium per g bark, yielding a dry matter content of ~30%, somewhat below bark sampled on trees  had been subjected to dry oxidation at 575 °C was measured.For evaluation of the effect of microbial growth on the ash content present in the bark, the ash concentration was measured according to National Renewable Energy Laboratory standard method45. The insoluble residue was determined gravimetrically. The filtrate from the hydrolysis was used to determine monosaccharide and acid-soluble lignin content. The acid-soluble lignin was determined by absorbance measurements at 205 nm on a Specord 205 , using an extinction coefficient of 110 dm3 g−1 cm−146. The monosaccharide composition of the bark was monitored using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on an IC5000 system (Thermo Scientific). A 2 × 250 mm Dionex Carbopac PA1 column (Thermo Scientific) with a 2 × 50 mm guard column (Thermo Scientific) was used at a column temperature of 30 °C. The eluents were A – water, B – 200 mM NaOH, and C – 200 mM NaOH and 170 mM sodium acetate47. The samples were eluted using eluent A (0.26 mL/min) and detected using a post-column addition of eluent B (0.13 mL/min). Thereafter, the column was washed with eluent C and equilibrated with eluent A. The monosaccharide concentrations were determined using an internal fucose standard and pure external standards of arabinose, mannose, galactose, glucose, and xylose. Peak analysis was performed using Chromeleon software 7.2.10 (Thermo Scientific).To elucidate if the microorganisms were degrading carbohydrates or lignin in the spruce bark, the acetone-extracted bark was dried overnight, followed by hydrolysis using 72% sulfuric acid according to established protocolsN,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA), 50 µL trimethylchlorosilane (TMCS), 20 µL pyridine, and heated for 20 min at 70 °C.To determine the effect of microbial growth on the lipophilic extractive groups present in the spruce bark acetone extracts, the bark extractives were analyzed by GC coupled to a flame ionization detector (FID) and GC-mass spectrometry (MS) by MoRe Research Örnsköldsvik AB. The bark extracts were derivatized and analyzed as trimethylsilyl derivatives. The internal standards , at a concentration of 200 µg/mL, were added to 50 µL samples containing 30 mg/mL of bark extractives dissolved in acetone. Thereafter, the vials were evaporated under nitrogen and derivatized using 100 µL The concentrations of extractive groups  were evaluated on a Thermo Trace 1300 chromatography system coupled to an FID using an on-column Programmable Temperature Vaporization (PTV) injection method. The temperature program of the oven started at 100 °C, which was held for 1.5 min, followed by increasing the temperature to 340 °C using a gradient of 10 °C/min, and holding this temperature for 3 min. The PTV temperature increased from 80 °C to 340 °C at 10 °C/min, which was held for 10 min. The fatty acids, resin acid, and sterol ester concentrations were evaluated on a HP-5ms  column relative to the peak area of the internal standard heptadecanoic acid. High boiling compounds, such as steryl ester and triglyceride concentrations were evaluated using a HP-1 SIM/Dist  column based on the relative peak area towards the internal standards cholesteryl heptadecanoate and 1,3-dipalmitoyl-2-oleylglycerol, respectively. Peak evaluation was performed using Chromeleon software (Thermo Fisher Scientific).GC-MS was used for the identification of individual compounds in the fatty acids, resin acids, and sterols present in the bark extracts. Quantification of the individual compounds was based on the FID total ion peak, relative to the peak area of the internal standard heptadecanoic acid. GC-MS analysis was performed on an Agilent 8890 chromatography system with a CTC Analytics CombiPAL (Autosampler) coupled to an Agilent 5975 C MSD mass spectrometer with split-less injection using the same column and as the long column FID experiment, a HP-5ms  column and helium as a carrier gas at a rate of 1.2 mL/min. The temperature program of the oven started at 100 °C, was held for 1 min, thereafter, the temperature was increased to 340 °C at 10 °C/min, which at the end was held for 3 min. The injector was kept at 230 °C. Compounds were identified in the MS by comparing their mass spectra with those of the Wiley and NIST libraries on MassHunter (Agilent).1H-measurements were conducted using an 8 µs 1H-detection pulse, 2 s acquisition time, 5 s recycle delay, and 32 scans. 13C-measurements were conducted using a 14 µs 13C-detection pulse, 1 s acquisition time, 5 s recycle delay, and 8096 scans. Correlation spectroscopy was recorded using 2048 scans with 256 increments. Heteronuclear single quantum coherence was recorded using 64 scans with 384 increments and a transfer delay of 3.425 ms corresponding to a 145 Hz J-coupling. Heteronuclear multiple bond coherence was recorded using 64 scans with 512 increments and a transfer delay of 62.5 ms, corresponding to an 8 Hz J-coupling. For all samples, the solvent was used as chemical shift reference with the acetone-d6 methyl signal referenced to 2.05 ppm and 29.84 ppm for 1H and 13C, respectively.All NMR measurements were conducted on a Varian Inova 500 MHz operating at 11.7 T with a 5 mm HFX-probe. Measurements were performed at 298 K with acetone-d6 as solvent. The 48. Gel electrophoresis using Tapestation 2200 and Genomic DNA screentapes (Agilent) was used to validate product size and purity of a subset of DNA extracts, with the majority of the DNA being >5000 bp and of good quality for amplicon sequencing. DNA concentration and purity were measured using Qubit dsDNA HS/BR Assay kit (Thermo Fisher Scientific).For each timepoint, duplicate spruce bark samples were subjected to DNA extraction and community analysis performed by DNASense ApS (Denmark) using the standard protocol for FastDNA Spin kit for Soil  with the following exceptions: 500 µL of sample, 480 µL sodium phosphate buffer, and 120 µL MT buffer were added to a Lysing Matrix E tube, and bead beating was performed at 6 m/s for 4 × 40s52. The forward and reverse primers used include custom 24 nt barcode sequences followed by the sequences targeting the fungi ITS2: [ITS29] TACACACCGCCCGTCG and [ITS4] TCCTSCGCTTATTGATATGC53.Amplicon libraries for the bacteria 16S rRNA gene variable regions 18 (bV18A) and fungi ITS2 were prepared using a custom protocol, and performed by DNASense ApS. Up to 25 ng of extracted DNA was used as template for PCR amplification, and each PCR reaction (50 μL) contained 0.2 mM dNTP mix, 0.01 units of Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific), and 500 nM of each forward and reverse primer in the supplied SuperFI Buffer. PCR was done with the following program: Initial denaturation at 98 °C for 3 min, 25 cycles of amplification  and final elongation at 72 °C for 5 min. The forward and reverse primers used include custom 24 nt barcode sequences followed by the sequences targeting bV18A: [8F] AGRGTTYGATYMTGGCTCAG and [1391R] GACGGGCGGTGWGTRCAThe resulting amplicon libraries were purified using the standard protocol for CleanNGS SPRI beads  with a bead-to-sample ratio of 3:5. DNA was eluted in 25 μL of nuclease-free water . Sequencing libraries were prepared from the purified amplicon libraries using the SQKLSK114 kit  according to the manufacturer’s protocol with the following modifications: 500 ng total DNA was used as input, and CleanNGS SPRI beads for library cleanup steps. DNA concentration was measured using Qubit dsDNA HS Assay kit . Gel electrophoresis using Tapestation 2200 and D1000/High sensitivity D1000 screentapes  was used to validate product size and purity of a subset of amplicon libraries.The resulting sequencing library was loaded onto a MinION R10.4.1 flowcell and sequenced using the MinKNOW 23.04.6 software . Reads were basecalled and demultiplexed with MinKNOW guppy g6.5.7 using the super accurate basecalling algorithm (config r10.4.1_400bps_sup.cfg) and custom barcodes.55. Potential generic placeholders and dead-end taxonomic entries were cleared from the taxonomy flat file, i.e., entries containing uncultured, metagenome or unassigned, were replaced with a blank entry. The filtered reads were mapped to the SILVA 138.1 99% NR database with Minimap2 v2.24r1122 using the ax Mapont command56 and downstream processing using samtools v1.1457. Mapping results were filtered such that query sequence length relative to alignment length deviated <5%. Noteworthy, low-abundant operational taxonomic units (OTUs) making up <0.01% of the total mapped reads within each sample were disregarded as a data denoising step. Further bioinformatic processing was done via RStudio IDE (2023.3.0.386) running R version 4.3.1 (20230616) and using the R packages: ampvis2 (2.8)48, tidyverse (2.0.0), seqinr (4.2.30), ShortRead (1.58.0), and iNEXT (3.0.0)59.The sequencing reads in the demultiplexed and basecalled fastq files were filtered for length (320–2000 bp) and quality (phred score >15) using a local implementation of filtlong v0.2.1 with the settings –min_length 320 –max_length 2000 –min_mean_q 97. The SILVA 16S/18S rRNA 138 SSURef NR99 full-length database in RESCRIPt format was downloaded from the QIIME on 29 September 202260 with Minimap2 v2.24r1122 using the ax mapont command56 and downstream processing using samtools v1.1457. Potential generic place holders and dead-end taxonomic entries were cleared from the taxonomy flat file, i.e., entries containing uncultured, metagenome or unassigned, were replaced with a blank entry. Mapping results were filtered such that alignment length was >125 bp and mapping quality >0.75. Noteworthy, low abundant OTUs making up <0.01% of the total mapped reads within each sample were disregarded as a data denoising step. Further bioinformatic processing was done as for the bacterial gene regions, above.The sequencing reads in the demultiplexed and basecalled fastq files were filtered for length (320–2000 bp) and quality (phred score >15) using a local implementation of filtlong v0.2.1 with the settings –min_length 320 – max_length 2000 –min_mean_q 97. The filtered reads were mapped to the QIIME formatted and 99% identity clustered UNITE database release 9.0DNA from the week 2 sample was extracted using the DNeasy PowerSoil Pro Kit following the manufacturer’s recommendations , by DNASense ApS. For Oxford Nanopore sequencing, a custom SPRi-bead size-selection protocol was further used to clean up and size-select (approx. cut-off 1500–2000 bp) DNA prior to sequencing. Gel electrophoresis on Genomic DNA ScreenTapes and using the Tapestation 2200  was used to validate DNA size distribution of a subset of DNA extracts. DNA concentration and purity were measured with the Qubit dsDNA HS Assay kit  and on the NanoDrop One .61 and trimmed for adapters using Cutadapt v. 3.762. Rasusa v. 0.6.1 was used to subset the Illumina data to 100 gb (gigabases)63. Raw Oxford Nanopore Fast5 files were basecalled in Guppy v. 6.1.5 using the Super-accurate basecalling algorithm. Basecalled fastq were was then adapter-trimmed in Porechop v. 0.2.4 Porechop using default settings. NanoStat v.1.6.064 was used to assess the quality parameters of the basecalled data. The trimmed data were then filtered in Filtlong v. 0.2.0 with min_length set to 1000 bp and min_mean_q set to 95.For Illumina sequencing, the DNA was quantified using Qubit  and fragmented to ~550 bp using a Covaris M220 with microTUBE AFA Fiber screw tubes and the settings: duty factor 10%, peak/displayed power 75 W, cycles/burst 200, duration 40 s and temperature 20 °C. The fragmented DNA was used for metagenome preparation using the NEB Next Ultra II DNA library preparation kit. The DNA library was paired-end sequenced (2 ×151 bp) on a NovaSeq S4 system . For Oxford Nanopore sequencing, a long-read sequencing library was prepared according to the SQK-LSK110 protocol . Approximately 50–100 fmole was loaded onto primed FLO-MIN106D (R9.4.1) flow cells and sequenced on the GridION platform using MinKNOW Release 22.05.7 (MinKNOW Core 5.1.0). Raw Illumina reads were filtered for PhiX using Usearch1165. 16S rRNA gene sequences were extracted using Barrnap v. 0.9 and classified against the SILVA SSU 138.1 database33. Forward reads were classified with Kaiju v. 1.8.2 against the nr_euk 2021-02-24 database66 and visualized with Krona v. 2.8.167.A short-read metagenome was assembled with Megahit v. 1.2.9 using a predefined kmer-list 68 by setting the metagenome parameter (– meta). The assembled metagenome was subsequently polished with quality-filtered Oxford Nanopore data using one round of polishing with Minimap2 v. 2.2456 and Racon v.1.4.2069 and two rounds of polishing with Medaka (v.1.6.1). The metagenome was finally polished with Minimap2 v. 2.2456 and Racon v.1.4.2069 using Illumina data. The metagenome was visualized in RStudio IDE (4.2.0 (2022-04-22)) running R version 4.0.4 (2021-02-15) and using the mmgenome2 R package v. 2.1.3. Metagenome-assembled genomes (MAGs) were manually extracted in mmgenome2 v. 2.2.1. Completeness and contamination values were assessed using CheckM v. 1.1.370. MAG abundance was calculated using CoverM v. 0.6.1. MAGs were classified using the Genome Taxonomy Database toolkit v. 2.1.0 against the r207_v2 Genome Taxonomy Database72. Ribosomal RNA genes were identified and extracted using Barrnap v. 0.9. MAGs were annotated using Prokka v. 1.14.673.Draft metagenomes were assembled with Flye v.2.9dit cluster genes from Pseudomonas abietaniphila BKME-9 and Paraburkholderia xenovorans  was performed in eggNOG-mapper using usegalaxy.eu75.Putative resin acid-degrading proteins present in the MAGs were identified by using Basic Local Alignment Search Tool (BLAST) with protein sequences from ns Table  against Pseudomonas Isolation Agar (PIA) . 5 g of the partially degraded bark was diluted in 45 mL of water, left to soak for 30 min at room temperature, and 100 mL inoculum from the resulting liquid was spread onto the plates with tenfold dilutions up to 10−5. Plates were incubated at room temperature for 5 days. Obtained colonies were re-streaked on M9 minimal medium agar plates (15 g/L agar) containing 10 g/L of abietic acid. Single colonies were obtained by sequential streaking on fresh abietic acid plates, and for initial identification these were picked, boiled for 10 min, and used for PCR amplification of 16S rRNA and the signature Pseudomonas gene rpoD. Each PCR reaction (50 mL) contained Phusion polymerase (Thermo Scientific), 5X HF buffer (Thermo Scientific), water, 1 mL of boiled colonies, and 10 nM of each forward and reverse-tailed primer T primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1470 R (5′-TACGGYTACCTTGTTACGACT T-3′), and the partial rpoD sequence using primers PsEG30F (5’-ATYGAAATCGCCAARCG-3’) and PsEG790R (5′-CGGTTGATKTCCTTGA-3′), as previously described76. PCR was conducted with the following program: initial denaturation at 94 °C for 20 s, 35 cycles of amplification  and a final elongation at 72 °C for 7 min. The PCR products were analyzed by gel electrophoreses, purified, and sequenced using Sanger sequencing (Macrogen). Sequence results were identified using BLAST at NCBI77. An isolate, designated PIA16, was selected for genome sequencing and physiological tests, based on its ability to grow on abietic acid and sequence dissimilarity (16S rRNA and rpoD) to known species. The species was later named Pseudomonas abieticivorans, which is used henceforth. P. abieticivorans sp. nov. is deposited at the Culture Collection University of Gothenburg (CCUG 76343T) and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM 114633).Resin acid degrading bacteria were isolated by serial dilution from one sample after 2 weeks of bark degradation, that had been stored at −20 °C, using plates containing 64. Raw Illumina reads were filtered for PhiX using Usearch1161 and trimmed for adapters using cutadapt (v. 2.862). A draft de novo assembly was produced with Flye v. 2.9-b176868. The draft assembly was subsequently polished one time with Racon v. 1.4.2069 and Minimap2 v. 2.22-r110156 and twice with Medaka v. 1.4.3 (Oxford Nanopore Technologies). The genome was finally polished with Minimap2 v. 2.22-r110156 and Racon v.1.4.1369 using Illumina data. Assembly graph and draft assembly coverage were visualized and inspected with Bandage v. 0.8.178. Genome annotation was conducted using Prokka (https://github.com/tseemann/prokka) v. 1.14.673. Sample genomes were classified with the GTDB (Genome Taxonomy Database) tool Kit v. 1.7.071 against the GTDB r20272. The genome was functionally annotated (COG) using WebMGA79.DNA extraction, sequencing, and analysis were performed by DNASense ApS. DNA was extracted using the DNeasy PowerSoil Pro kit following the manufacturer’s recommendations (Qiagen) with minor modifications. Gel electrophoresis using Tapestation 2200  was used to validate product size and purity of a subset of DNA extracts. DNA concentration was measured using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). A rapid (SQK-RBK 110.96) library was prepared according to the manufacturer’s protocol, except for minor modifications (Oxford Nanopore Technologies). The library was loaded on a primed R9.4.1 MinION flow cell and sequenced on the GridION platform using MinKNOW Release 21.10.4. Sample DNA concentrations were measured using the Qubit dsDNA HS kit and the DNA quality was evaluated using TapeStation with the Genomic ScreenTape (Agilent Technologies). Sequencing libraries were prepared using the NEB Next Ultra II DNA library prep kit for Illumina (New England Biolabs) following the manufacturer’s protocol. Library concentrations were measured in triplicate using the Qubit dsDNA HS kit and library size estimated using TapeStation with D1000 HS Screen-Tape. The sequencing libraries were pooled in equimolar concentrations and diluted to 4 nM. The samples were paired end sequenced (2 × 301 bp) on a MiSeq (Illumina) using a MiSeq Reagent kit v3, 600 cycles (Illumina) following the standard guidelines for preparing and loading samples on the MiSeq. Fast5 raw data were basecalled and demultiplexed in Guppy (Oxford Nanopore Technologies) v. 6.0.1 using the sup algorithm. Basic read statistics including amount of data produced, median read quality and read N50 were assessed with NanoPlot v. 1.36.2P. abieticivorans and the most closely related known species, a phylogenetic analysis of all the Pseudomonas species reported in NCBI: GenBank80 was performed. BLASTn on NCBI web server80 was used to acquire 16S ribosomal RNA sequences, with Pseudomonas (taxid:286) sequences database selected, and limited to a maximum of 1000 target sequences. Multiple sequence alignment was executed using CLUSTALW with msa package81 in R v4.2.282. Alignments were processed by using bios2msd83 and adegenet84 R packages. Phylogenetic trees were constructed using ggtree85 and ggplot286 R packages. Tree estimation was calculated by the neighbor-joining method supported by ape87 package. Collinear analysis of P. abieticivorans compared to five species reported to be able to utilize resin acids  were rendered hydrophilic by glow discharge before applying 5 µL of cell suspension  for 3 min. The suspension was removed by blotting onto Whatman filter paper and the remaining bacteria were immediately fixed by applying 5 µL of 2% glutaraldehyde for 2 min. After blotting away the fixative and washing the grids twice with distilled water, the samples were stained for 1 min with 1% uranyl acetate. Excess stain was removed by blotting and the grids were air-dried before imaging. The samples were examined with a Talos L120C transmission electron microscope (Thermo Fisher Scientific) operating at 120 kV, and images were captured with a 4k × 4k BM-Ceta CMOS camera.Negative staining and transmission electron microscopy (TEM) were used to examine the morphology of 600 after 4 days of growth, with pH adjusted using NaOH or HCl. Fluorescence was studied by plating on King A or King B media (Millipore). Additional physiological tests were conducted using the API 20 NE and API ZYM systems according to the manufacturer’s instructions (bioMérieux). All tests were performed in duplicate.Growth at various temperatures  was investigated on LB plates over 4 days and growth was assessed based on the presence of visible colonies. Growth based on salt concentration (NaCl 0-12% w/v) and the pH-growth range was assessed in LB medium by monitoring OD92. The fatty acid methyl esters mixtures were analyzed by GC-FID using Sherlock Microbial Identification System (MIS) . Peaks were automatically integrated, and fatty acid names and percentages were calculated by the MIS Standard Software .Analysis of cellular fatty acids was carried out by DSMZ Services, Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH . The fatty acid methyl esters of the PIA16 strain were obtained from cells grown in tryptic soy broth (Millipore) at 28 °C for 24 h. Cellular fatty acids were analyzed after conversion into fatty acid methyl esters by saponification, methylation, and extraction using minor modifications of established methods600) of 0.01. The concentration of the resin acids (100 mg/L) was deliberately kept low to minimize background as resin acids are present as insoluble particles in the medium. Prior to inoculation, P. abieticivorans was grown in 10 mL LB medium at room temperature overnight. To follow growth and resin acid degradation over time, 1 mL of culture was sampled for evaluation of growth using protein concentration and degradation of resin acids using GC-MS. Intracellular protein concentration was used as a pseudo-value for growth due to the resin acids’ low solubility in water, making OD measurements unreliable. Briefly, cells were spun down, the pellet was resuspended in 1 M NaOH, boiled, and neutralized using HCl. Protein concentration was measured using the Bio-Rad BCA kit following the manufacturer’s instruction. Resin acid extracts were analyzed using a modified method according to ref. 93. Briefly, the resin acids were acidified using 40 µL HCl and spiked with an internal standard of 0.1 mg isopimaric acid (Carbosynth) and extracted three times using 1 mL ethyl acetate. Resin acids were dried over nitrogen and resuspended in 500 µL acetone and silylated using 25 µL BSTFA and TMCS (Sigma). Resin acid identification and quantification were performed by capillary GC (Agilent 7890A). Helium, the carrier gas, had a flow rate of 1 mL/min and the MS source was operated at 230 °C with the quadrupole at 150 °C. Analytes were separated using a HP-5 column with a temperature program starting at 70 °C, which was held for 2.25 min, was increased to 200 °C at 20 °C/min, thereafter 5 °C/min until the temperature reached 230 °C. The final ramp was at 35 °C min to 300 °C which was held for 10 min. Injector temperatures were at 300 °C. Helium, the carrier gas, had a flow rate of 1 mL/min and the MS source was operated at 230 °C with the quadrupole at 150 °C. The NIST MS Search Programme (Vers. 2.2) was used for identification using the library NIST/EPA/NIH Mass Spectral Library (NIST 11). Semi-quantification of the resin acids was done as using the internal standard and the following relationship: WS = AS × WI/AI, where W stands for the mass fraction, A for the area in the chromatogram, I the internal standard (isopimaric acid) and S the species, respectively.Growth on dehydroabietic acid and abietic acid was done in cultures of M9 medium containing 100 mg/L of dehydroabietic acid (Megazyme ~90%) or abietic acid (Sigma ~75%) and inoculated with a starting optical density at 600 nm , using continuous imaging and pixel-based growth determination, in a 24-well plate (6 × 4) using 100 mg/L of each resin acid: dehydroabietic acid (Megazyme ~90%), abietic acid (Sigma ~75%), and isopimaric acid (Carbosynth). Growth was screened at 30 °C, and the cultures inoculated from an overnight culture of P. abieticivorans grown on LB broth to a starting OD600 of 0.01 in a total volume of 1.2 mL.The growth of Pseudomonas abieticivorans . Cells are Gram-negative, rod-shaped , with a singular flagellum, and catalase and oxidase negative. Growth occurs at 4-30 °C. Optimal growth temperature is between 25 and 28 °C. The species grows at pH 5-9 and in the presence of 6 w/v NaCl. The species is negative for reduction of nitrates to nitrate and reduction of nitrates to nitrogen, for indole production, fermentation of glucose, urease, and proteases for gelative hydrolysis. The species is positive for assimilation of d-glucose, l-arabinose, d-mannitol, potassium gluconate, capric acid, malate, and trisodium citrate, and negative for assimilation of d-mannose, N-acetyl-d-glucosamine, d-maltose, adipic acid, and phenylacetic acid. The enzymatic activities which were positive were alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, acid-phosphatase, arginine dihydrolase, and catalase. The ones which displayed a negative test were lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, α-mannosidase, α-fucosidase. Growth is detected in R2A, PIA, and LB. The major fatty acids are summed feature 3 , summed feature 8 , C16:0 (31.57%), C17:0 cyclo (12.79%), C12:0 (4.11%), C12:0 2-OH (3.05%), C12:0 3-OH (3.49%), C10:0 3-OH (3.00%), C14:0 (0.47%). The type strain, PIA16  was isolated from spruce bark on abietic acid. The G + C content of the type strain is 61.1 mol% and the genome size is 6,715,763 bp.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data File


Cardiac structure and function were investigated by echocardiogram. Myocardial glycolysis, the aerobic and anaerobic pathways and glycogen were analyzed by enzymatic assay, Western blot, and enzyme-linked immunosorbent assay (ELISA). The following were observed: increased left ventricular hypertrophy; early diastolic function change and severe systolic and diastolic dysfunction in HF; increased HIF-1α in the 2nd week and in HF; precocious alteration and intensification of glycolysis with a shift to anaerobic metabolism from the 6th week onwards; association between HIF-1α, glycolysis, and the anaerobic pathway. Our hypothesis was confirmed as there was an early change and intensification in glucose metabolism, alteration in HIF-1α, and an association between data during the progression from hypertrophy to heart failure.In pathological cardiac hypertrophy, the heart is more dependent on glucose than fatty acids. This shift in energy metabolism occurs due to several factors, including the oxygen deficit, which activates hypoxia-inducible factor-1α (HIF-1α), a critical molecule related to glucose metabolism. However, there are gaps regarding the behavior of key proteins in the glycolytic pathway and HIF-1α during the transition from hypertrophy to heart failure (HF). This study assesses the hypothesis that there is an early change and enhancement of HIF-1α and the glycolytic pathway, as well as an association between them during cardiac remodeling. Sham and aortic stenosis Wistar rats were analyzed at 2, 6, and 18 weeks and in HF ( The heart can meet energy demands using various substrates, such as glucose, lactate, fatty acids, amino acids, and ketone bodies ,2. Aroun2) deficit  of the following signs: RV hypertrophy, left atrial thrombus, pleural effusion, hemorrhagic liver, and ascites [HF group presented all the signs that characterized severe HF. At the end of each experimental period , animals were fasted for 8 h and were anesthetized with a mixture of ketamine  and xylazine  and euthanized by decapitation.Supravalvular aortic stenosis (AoS) in young rats has been used as an experimental model to study chronic HF, initially with preserved ejection fraction which may progress to cardiac systolic insufficiency ,58,59,600th week ,64. Mort ascites . AnimalsThe final body weight (BW), lung weight, and lung/tibia ratio were measured at the end of each experimental period. Cardiac hypertrophy was determined by macroscopic analysis of the following parameters: atrium (AT), LV, and RV weights, as well as their ratio with tibia length.Echocardiogram was performed before each euthanasia using a commercially available echocardiograph  equipped with a 5–11.5 MHz multifrequency transducer as previously described ,58,59,61g for 10 min at 4 °C using an enzyme-linked immunosorbent assay (ELISA) kit , according to manufacturer instructions.Blood samples were collected from the tail tip before euthanasia for glucose analysis and were assessed using a handheld glucometer . Fasting serum insulin concentrations were measured in blood samples collected after euthanasia and centrifuged at 1620× g for 20 min at 4 °C and supernatant collected. For the extraction of GLUT1 and GLUT4, protein membranes were homogenized in extraction buffer containing 1M Tris-HCl, 25 mM pentetic acid (DTPA), β-Mercaptoethanol, 0.1 M Phenylmethylsulfonyl Fluoride (PMSF), and protease inhibitor. The homogenate was centrifuged at 1000× g for 3 min at 4 °C to remove insoluble material. The supernatant was then centrifuged at 18,000× g for 10 min at 4 °C and transferred to an ultracentrifuge  at 100,000× g for 45 min at 4 °C. After ultracentrifugation, the pellet was resuspended in 100 μL of cold RIPA lysis buffer. Protein concentrations were determined using a Pierce BCA Protein Assay kit . Samples (50 μg) were subjected to SDS-PAGE in polyacrylamide gels (6% or 10% depending on protein molecular weight). After electrophoresis, proteins were electrotransferred to nitrocellulose membrane . The blotted membranes were blocked with 5% nonfat dry milk in Tris-buffered saline/Tween-20  for 2 h at room temperature. Membranes were then incubated overnight at 4 °C–8 °C with primary antibody against: HIF-1α ; IR ; pIR ; PI3K ; AKT ; pAKT ; HKII ; PFK2 ; PK ; LDH ; PDH ; GLUT1 ; and GLUT4 . Primary antibody binding was detected using peroxidase-conjugated secondary antibodies  incubated for 1.5 h at room temperature. A control rat was present on all gels at the standard internal sample control (ISC). Protein bands were visualized via chemiluminescent detection  in a Western blot detection system , and quantified by densitometry using Image J analysis software version 1.52. Targeted bands were normalized to the expression of cardiac β-actin .Cardiac expression of HIF-1α and the proteins involved in glucose metabolism were evaluated by Western blot. LV tissue samples were homogenized in cold RIPA lysis buffer  containing protease  and phosphatase  inhibitors. The homogenate was centrifuged at 12,000× 2, 0.8 mM EDTA, 1.5 mM KCl, 4 mM 2 mercaptoethanol, 0.4 mM NADP+, 2.5 mM ATP, 1 mM glucose, 1.4 units glucose-6-phosphate dehydrogenase, and 0.05% Triton X-100. Total assay volume was 165 µL, and 10 µL of homogenate was added. The assay was initiated by adding glucose [−1 × cm−1).Activities of key glucose metabolism enzymes were analyzed, including HKII, LDH, and CS. Heart LV samples were homogenized 1:20 (wt/vol) in 50mM Tris-HCl, 1mM EDTA, and protease inhibitor cocktail, pH 7.4 using a Polytron instrument . The lysate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected. All enzyme activities were determined at 25 °C using a Spectra Max 250 microplate spectrophotometer , and assay buffer without sample was used as blank. HKII assay medium consisted of 75 mM Tris-HCl, 7.5mM MgCl glucose . The ratCardiac glycogen content was determined as described by Trinder (1969) . The tesData are expressed as mean ± SD (standard deviation) or median . Echocardiographic, cardiac metabolic, and structural variables, considering both sources of variation, i.e., AoS (absence or presence) and euthanasia moment  were analyzed by ANOVA using a 2 × 4 factorial scheme with a completely randomized design; this was complemented with Tukey’s multiple comparisons test  and non-parametric analysis of variance technique (ANOVANP) followed by Dunn’s post hoc test . The choice of parametric or non-parametric procedure was based on the Kolmogorov–Smirnov normality test . RegardiIn conclusion, our hypothesis was confirmed as there was an early change and intensification of the glucose metabolism, observed by increased activity and/or expression of glucose transporters, glycolysis enzymes, and proteins involved in lactate production and the intracellular insulin signaling cascade, concomitant with high levels of HIF-1α during the progression from hypertrophy to heart failure.


Behavioural measurement for evoked pain, morphological analyses of nerve fibre loss in the epidermis, measurement of reactive oxygen species (ROS) levels, and antioxidant molecules were analysed over several months in SUMOylation-deficient and control mice. Our longitudinal analysis at 30 weeks post-high-fat diet revealed that SNS-Ubc9−/− mice exhibited earlier and more pronounced thermal and mechanical sensation loss and accelerated intraepidermal nerve fibre loss compared to control mice. Mechanistically, these changes are associated with increased levels of ROS both in sensory neuronal soma and in peripheral axonal nerve endings in SNS-Ubc9−/− mice. In addition, we observed compromised detoxifying potential, impaired respiratory chain complexes, and reduced levels of protective lipids in sensory neurons upon deletion of SUMOylation in diabetic mice. Importantly, we also identified mitochondrial malate dehydrogenase (MDH2) as a SUMOylation target, the activity of which is negatively regulated by SUMOylation. Our results indicate that SUMOylation is an essential neuroprotective mechanism in sensory neurons in type 2 diabetes, the deletion of which causes oxidative stress and an impaired respiratory chain, resulting in energy depletion and subsequent damage to sensory neurons.Diabetic peripheral neuropathy (DPN) is the prevalent type of peripheral neuropathy; it primarily impacts extremity nerves. Its multifaceted nature makes the molecular mechanisms of diabetic neuropathy intricate and incompletely elucidated. Several types of post-translational modifications (PTMs) have been implicated in the development and progression of DPN, including phosphorylation, glycation, acetylation and SUMOylation. SUMOylation involves the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins, and it plays a role in various cellular processes, including protein localization, stability, and function. While the specific relationship between high blood glucose and SUMOylation is not extensively studied, recent evidence implies its involvement in the development of DPN in type 1 diabetes. In this study, we investigated the impact of SUMOylation on the onset and progression of DPN in a type 2 diabetes model using genetically modified mutant mice lacking SUMOylation, specifically in peripheral sensory neurons (SNS-Ubc9 Diabetes is a chronic medical condition that requires ongoing management. It is associated with significant healthcare costs, which are expected to rise further as the prevalence of diabetes increases . In the Type 2 diabetes is the most prevalent form; it usually occurs in adulthood, and it is related to obesity and a sedentary lifestyle. It results from insulin resistance causing a burden on pancreatic beta cells, which gradually lose their ability to produce insulin. If left untreated, both types 1 and 2 diabetes can lead to serious complications, such as nerve damage (diabetic neuropathy), kidney damage (diabetic nephropathy), and cardiovascular disease ,4,5. DPNSUMOylation is a process in which a small ubiquitin-like modifier (SUMO) is covalently attached to the lysine residue of a target protein. This modification regulates cellular processes, such as transcription, DNA repair, and protein localization ,21. A se2 in a humidified incubator. The cells were split 24 h prior to transfection to achieve 70% confluency at the time of transfection. HEK293 cells were transfected with 20 µg of plasmid DNA (pSENP1 or control plasmid) using the calcium chloride transfection method described in [2, 510 µL of 1× BBS (pH 6.9), and 441 µL of H2O. The mixture was incubated at 37 °C for 10 min and subsequently added dropwise to the cells in 6 mL of DMEM without supplements. After 8 h, the medium was changed to full DMEM. Twenty-four hours post-transfection, the cells were harvested for Western blot analysis, or assays for activity of malate dehydrogenase (MDH) were performed. Human embryonic kidney cells  were cultured in Dulbecco’s modified Eagle medium , 4 mM L-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin. The cells were grown at 37 °C and 5% COribed in . Briefly−/− and Ubc9fl/fl mice. DRG were isolated from 4–5-week-old mice and incubated in a mixture of digestive enzymes containing trypsin, collagenase, and DNase at 37 °C for 30 min. The neurons were enriched from non-neuronal cells using the Percol purification procedure described in Simonetti et al. [Dorsal root ganglion (DRG) cultures were made from SNS-Ubc9i et al. . Brieflyg and the supernatant was collected. The collected lysate was further diluted to 0.1% SDS concentration and subjected to immunoprecipitation using either 5–7 µg of anti-SUMO1  or IgG control antibody  and 25 µL of either Myc-Trap  or control-Trap beads  and incubated for 24 h at 4 °C or for 2 h for the Trap beads. In the case of OxPhos and PKG1, DRG tissue was lysed in 0.1% SDS RIPA buffer without DTT [Immunoprecipitation was performed either from HEK cells or mouse DRG tissues, as described previously . Brieflyhout DTT . The lyshout DTT . The des−/− and Ubc9fl/fl mice using a commercial kit as per manufacturer’s protocol . Briefly, cells and DRG tissue were lysed in 1× reaction buffer from the kit using mild sonication. The lysate was centrifuged at 13,000× g for 10 min at 4 °C and the supernatant was transferred into a new tube. MDH activity was determined in supernatants by generating a product with absorbance at 450 nm proportional to the enzymatic activity present following the manufacturer’s instructions. Furthermore, the same lysate samples were used in Western blot analysis to determine the expression levels of MDH2 and actin. The MDH2 protein levels in each sample were normalised over control protein actin prior to the final quantification of MDH activity. The analyses were derived using 4–7 biological samples with 2–3 technical replicates in each experiment.The assay for determining MDH activity was performed either with transfected HEK cells or from the DRG tissue isolated from SNS-Ubc9−/− and Ubc9fl/fl mice were used for all experiments. Two to four littermates were housed in individually ventilated enriched cage-rack systems in a temperature- and humidity-controlled room with an inverse light/dark cycle (12/12). All animals had free access to water and food. Mice of both sexes were used in all of the experiments in equal numbers. All animal experimental procedures were approved by the Regierungspräsidium Karlsruhe: Abteilung 3, Landwirtschaft, Ländlicher Raum, Veterinär und Lebensmittelwesen’, approval numbers 35-9185.81/G-133/19, All animal experiments followed institutional guidelines and were conducted in accordance with German Law, which regulates animal welfare and the protection of animals used for scientific purposes .Age-matched 8–10-week-old SNS-Ubc9−/− mice), the only known E2-conjugating enzyme in the SUMOylation [−/− was previously described by Agarwal et al. [fl/fl mice) were mated with mice expressing the Cre recombinase under the control of the Nav 1.8 promoter (SNS-Cre mice) [−/− and control Ubc9fl/fl mice were used for all experiments.To investigate the role of SUMOylation, we employed mice lacking Ubc9 protein  . HomozygDiabetes was induced in mice via a high-fat diet (HFD) comprising 45% kcal as saturated fat from Lard  . Five weAll behavioural experiments were performed with awake, unrestrained, and age-matched mice. Both male and female mice were included in behavioural recordings. The experimenter was blinded for both the gender and treatment in the experimental groups. Mice were acclimatised to the recording setup twice a day for four days before the recording. Mechanical sensitivity was measured using Von Frey filaments at 10, 17, 23, and 30 weeks post-HFD. Mice were placed on the elevated metal grid, and Von Frey monofilaments exerting a specific force of 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, 2.0, and 4.0 g were tested on the plantar hind paw. Each filament was applied five times at a time interval of 3–4 min on the hind paw. Sixty percent response frequency was calculated at 10, 17, 23, and 30 weeks post-HFD, as described previously ,39,40. T−/− and Ubc9fl/fl mice as basal or 30 weeks post-HFD, as previously described [Immunohistochemistry was performed on skin biopsies of the plantar surface of the hind paws from the diabetic and non-diabetic SNS-Ubc9escribed ,41. Brieescribed ,42.−/− and Ubc9fl/fl mice were isolated for metabolomics analysis, as previously described in [2. Frozen sample material was extracted with 190 µL 100% methanol containing 10 µL 0.02 mg/mL Ribitol for 15 min at 70 °C with vigorous shaking. After the addition of 100 µL 100% chloroform, samples were shaken at 37 °C for 5 min. To separate polar from organic phases, 200 µL of HPLC-grade water was added, and samples were centrifuged for 10 min at 11,000× g while avoiding the interphase containing cellular debris. In total, 350 µL of the polar (upper) phase was transferred to a fresh glass vial and dried using a vacuum concentrator  without heating. Sciatic nerves from diabetic and non-diabetic SNS-Ubc9ribed in . Mice weSequential online methoximation and silylation reactions were performed using an MPS autosampler . Methoximation was achieved by adding 20 µL of 20 mg/mL methoxyamine hydrochloride (Sigma 226904) in pyridine (Sigma 270970) and incubating at 37 °C for 90 min in a Gerstel MPS Agitator Unit (250 rpm). For sialylation reactions, 45 µL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide  was added, and samples were incubated at 37 °C for 30 min with gentle shaking. Before injection, samples were incubated at room temperature for 45 min. m/z) of 50–600 with an acquisition rate of 17 spectra/second. ChromaTof v5.50 software  was used for data processing. For GC/MS analysis, a GC-ToF system was used consisting of an Agilent 7890 Gas Chromatograph  fitted with an Rxi-5Sil MS column  coupled to a Pegasus BT Mass Spectrometer . The GC was operated with an injection temperature of 250 °C, and a 1 µL sample was injected in split less mode. The GC temperature program started with a 1 min hold at 40 °C followed by a 6 °C/min ramp up to 210 °C, a 20 °C/min ramp up to 330 °C, and a bake-out at 330 °C for 5 min using Helium as carrier gas with constant linear velocity. The ToF mass spectrometer was operated with ion source and interface temperatures of 250 °C, a solvent cut time of 9 min, and a scan range  and nociceptive peptidergic marker  on the hind paw skin sections from SNS-Ubc9uishable a–d, and uishable b,d.−/− and Ubc9fl/fl mice under basal and diabetic conditions by assessing cGMP-dependent PKG1 dimer/monomer ratios, which serve as an ROS sensor in neurons [−/− and Ubc9fl/fl mice at basal or 30 weeks post-HFD, whereby detection of fluorescence indicates ROS generation and mitochondrial dysfunction [−/− and Ubc9fl/fl mice. Both genotypes had increased ROS with diabetes progression at 30 weeks post-HFD  in DRG of SNS-Ubc9/fl mice h. These −/− mice, deletion of SUMOylation led to increased MDH2 activity compared to control mice  and mitochondrial malate dehydrogenase 2 (MDH2) regulate this ratio . In a prrol mice d. Howeverol mice f. This i−/− and Ubc9fl/fl mice in basal and diabetic states. Linoleic acid and eicosatrienoic acid are crucial omega-6 and omega-3 fatty acids that play vital roles in maintaining neuronal membrane integrity and fluidity, which are essential for proper neuron function [−/− mice compared to controls  remains poorly understood. Notably, SUMOylation has been linked to neurodegenerative diseases, like Alzheimer’s and Huntington’s disease ,49,50. Cfl/fl diabetic mice when compared to non-diabetic control mice, but this increase was reduced in SUMOylation-deficient mice. Consequently, our findings suggest that SUMOylation serves as a protective mechanism against the loss of redox balance, the absence of which contributes to neuronal dysfunction. A number of interesting mechanistic processes may contribute to this outcome. SUMOylation influences glucose metabolism, including insulin signalling, gluconeogenesis, glycolysis, and glucose uptake. SUMOylation of insulin receptor substrate (IRS-1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase M2 (PKM2), and glucose transporter 4 (GLUT4) promote glucose homeostasis ,51. DeSUHyperglycaemia, mitochondrial membrane potential disruption, or dysfunction of ETS complexes contribute to excessive ROS production . We expl−/− as compared to control Ubc9fl/fl mice demonstrate that SUMOylation is a crucial biological process required to maintain the physiological functions of the respiratory chain under pathophysiological conditions. An inefficient ETS chain could also result from an imbalanced NADH/NAD+ ratio [Indeed, the reduced levels of the ETS complex we observed in diabetic SNS-Ubc9D+ ratio . NADH isD+ ratio . In our D+ ratio . HoweverD+ ratio , the incOur results show that diabetic neuropathy is associated with mitochondrial dysfunction, and it is regulated by SUMOylation. SUMOylation-deficient mice exhibit substantial impairment in the mitochondrial oxidative phosphorylation complexes III, IV, and V within the respiratory chain under diabetic conditions. However, establishing causal links between mitochondrial dysfunction, oxidative stress, and sensory loss in diabetes remains a crucial area of investigation. Exploring the potential therapeutic role of mitochondria-targeted antioxidants, like MitoTEMPOL  or ROS sFrom a therapeutic viewpoint, enhancing SUMOylation pathway enzymes in peripheral neurons could potentially prevent or reverse diabetic neuropathy. Presently, there are no highly specific drugs activating these enzymes. However, certain compounds, like Gingko extract, N106 (specific for SUMO-activating enzyme E1 ligase) ,69, or SBased on our results, we conclude that SUMOylation is an essential neuroprotective mechanism in sensory neurons in type 2 diabetes, the loss of which results in oxidative stress at nerve terminals and suppresses the efficiency of the respiratory chain. Consequently, this impairment contributes significantly to nerve damage in DPN.


At the same time, the durability test verified the sustainability of the AC electrochemical method. For Cr (VI)-polluted wastewater with an initial concentration of 50 mg·L−1, the effluent concentration could still reach drinking water grade (<0.05 mg·L−1) after 10 cycling experiments. This study provides an innovative approach for the rapid, green and efficient removal of Cr (VI) containing wastewater at low and medium concentrations.A large amount of Cr (VI)-polluted wastewater produced in electroplating, dyeing and tanning industries seriously threatens water ecological security and human health. Due to the lack of high-performance electrodes and the coulomb repulsion between hexavalent chromium anion and cathode, the traditional DC-mediated electrochemical remediation technology possesses low Cr (VI) removal efficiency. Herein, by modifying commercial carbon felt (O-CF) with amidoxime groups, amidoxime-functionalized carbon felt electrodes (Ami-CF) with high adsorption affinity for Cr (VI) were prepared. Based on Ami-CF, an electrochemical flow-through system powered by asymmetric AC was constructed. The mechanism and influencing factors of efficient removal of Cr (VI) contaminated wastewater by an asymmetric AC electrochemical method coupling Ami-CF were studied. Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR), and X-ray photoelectron spectroscopy (XPS) characterization results showed that Ami-CF was successfully and uniformly loaded with amidoxime functional groups, and the adsorption capacity of Cr (VI) was more than 100 times higher than that of O-CF. In particular, the Coulomb repulsion effect and the side reaction of electrolytic water splitting were inhibited by the high-frequency anode and cathode switching (asymmetric AC), the mass transfer rate of Cr (VI) from electrode solution was increased, the reduction efficiency of Cr (VI) to Cr (III) was significantly promoted and a highly efficient removal of Cr (VI) was achieved. Under optimal operating conditions , the asymmetric AC electrochemistry based on Ami-CF can achieve fast (30 s) and efficient removal (>99.11%) for 0.5–100 mg·L Among them, is a large amount of chromium-polluted wastewater produced from metal plating, leather manufacturing, textile dyeing and other industries, which seriously threatens human and environmental health . In the  HCrO4−) , it can  HCrO4−) ,7. The WO4−) [−1 , and the [−1 [−1 . Therefo4, Na2S2O5 and HS2, etc.) and hydroxides 2, etc.) will produce a large amount of Cr-containing sludge and high alkaline solution, resulting in potential secondary pollution [2O72− and HCrO4−) so it cannot be effectively reduced on the electrode surface, which decreases the reduction and removal efficiency of Cr (VI). At the same time, water cracking on the electrode surface leads to a large amount of energy loss, which is a common problem in electrochemical methods [2+, Zn2+, Pb2+, Cd2+). However, there is no research on the application of AC electrochemical technology to Cr (VI) removal from wastewater.In general, Cr (VI) is highly mobile and soluble in a wide pH range, increasing its migration and potential harm, whereas Cr (III) has about 500–1000-times lower toxicity and mobility than Cr (VI) and is very easy to be removed by precipitation and adsorption . Recentlollution . In contollution ,17,18. Tollution , electroollution  and elecollution /reductioollution  for sacr methods . In addi methods . The dev methods , lithium methods  and reme methods . For exa methods  designed−1) of Cr (VI) with a high flux of 300 L h−1 m−2, which is superior to other methods reported. Furthermore, the removal mechanism of Cr (VI) in AC electrochemical system was thoroughly discussed combined with advanced characterizations. This study provides a new idea for the treatment of Cr (VI) containing wastewater by AC electrochemical technology in the future.Herein, amidoxime modified carbon felt electrode (Ami-CF) was prepared, and a novel penetrating asymmetric AC electrochemical system was constructed as a research platform to reveal an AC electrochemistry-mediated Cr (VI) removal mechanism. Due to the existence of amidoxime groups, Ami-CF is very hydrophilic and could make full use of the high surface area of the electrode. Meanwhile, amidoxime groups on Ami-CF provide strong chelating sites that can bind Cr (VI), resulting Ami-CF a 2.1–5.5 times saturated adsorption capacity than other adsorbents reported in the literature. The influences of applied voltage, duty ratio, initial Cr (VI) concentration, pH, flow rate and other coexisting ions on Cr (VI) removal by asymmetric AC electrochemical system were investigated and discussed. By employing Ami-CF as working electrode and coupled with high frequent cathode–anode conversion, AC electrochemistry not only enhances the mass transfer process while reducing side reactions, but also periodically attracts Cr (VI) at the active site under positive bias and reduces Cr (VI) to Cr (III) and repels Cr (III) under negative bias, thereby releasing the active site and constantly regenerating the Ami-CF. Under optimal operating conditions, the asymmetric AC electrochemical system based on Ami-CF can achieve fast (30 s) and efficient removal (>99.11%) for wastewater with a wide concentration , purchased from Carbon Energy Technology Co., LTD. , Super P Carbon Black purchased from Alfa Aesar , polyacrylonitrile, N, N-dimethylformamide, hydroxylamine hydrochloride, sodium carbonate, dibenzoyl hydrazine, potassium dichromate, anhydrous copper sulfate, anhydrous zinc sulfate, anhydrous calcium sulfate, copper nitrate, phosphoric acid and sulfuric acid were all purchased from Alding Chemical Reagent Co., LTD . DC and AC power supply were purchased from UNI-T Co., LTD . The peristaltic pump was purchased from River Fluid Technology Co., LTD . The experimental water was deionized.The original carbon felt (O-CF) was cut into discs with a diameter of 1.0 cm, and then polyacrylonitrile (PAN), Super P carbon black and N, n-dimethylformamide (DMF) were mixed and stirred at a mass ratio of 1∶1∶30 for 12 h to form a uniform slurry. The PAN-CF electrode was prepared by dipping the round carbon felt sheet with slurry and drying it in the oven (70 ℃). Then, the dried PAN-CF was put into a water bath at 70 ℃ (100 mL), and 8 g hydroxylamine hydrochloride and 6 g sodium carbonate were added to the water bath successively for hydroxylamine reaction (90 min). After the reaction, the carbon felt sheet was washed with deionized water and dried in a vacuum furnace (80 ℃) to obtain an amidoxime functionalized electrode (Ami-CF).−1 and a scanning accuracy of 2 cm−1. The surface chemical properties of the electrodes were analyzed by X-ray photoelectron spectroscopy .The electrode surface morphology was characterized by scanning electron microscopy . Surface functional groups were determined by Fourier to transform infrared spectroscopy  with a scanning range of 400 to 4000 cm−1 (pH = 6 ± 0.05). A total of 0.6 mL equal samples were taken at the time points 0 min, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h, respectively. Samples were filtered and diluted properly before analysis of final Cr (VI) concentration. Each data point, including blanks , was run in triplicate. The quasi-first-order kinetic equation (Equation (1)) and the quasi-second-order kinetic equation (Equation (2)) were used to fit the adsorption kinetic data, respectively. The formula is as follows [Qe and Qt are the adsorption capacity (mg·g−1) of Cr (VI) on the electrode at adsorption equilibrium and at adsorption time t, respectively. Reaction time is t (h); The adsorption rate constants of K1 and K2 for the quasi-first order and quasi-second order kinetics, respectively.Adsorption experiments were conducted using a batch approach as described in our previous studies ,29. The  follows ,30:Qt = −1, 0.5 mg·L−1, 1 mg·L−1, 2.5 mg·L−1, 5 mg·L−1, 10 mg·L−1, 25 mg·L−1, 50 mg·L−1, 100 mg·L−1, 250 mg·L−1, 500 mg·L−1 and 1000 mg·L−1 Cr (VI) solution (pH = 6 ± 0.05) were added afterwards. Then the centrifuge tubes were placed in the water bath thermostatic oscillator and agitated under the same condition as described above. The adsorption time was 36 h (equilibrium time) obtained in the kinetic experiment. After the adsorption is completed, 0.6 mL equal samples are respectively taken for the determination of Cr (VI) content, calculation of adsorption capacity and removal rate. The Langmuir model (Equation (3)) and the Freundlich model (Equation (4)) are used to fit the adsorption isotherm data [Ce is the equilibrium concentration (mg·L−1), Qe is the equilibrium adsorption capacity (mg·g−1), Qm is the maximum adsorption capacity (mg·g−1), KL is the Langmuir constant (L·mg−1), KF is the Freundlich adsorption coefficient (mg·g−1·L1/n·mg−1/n). N is a heterogeneous factor.For adsorption isotherm, 20~25 mg O-CF, PAN-CF, and Ami-CF were weighed and placed in the centrifuge tubes, 50 mL of 0 mg·Lerm data ,31,32. T−1) is adjusted through the peristaltic pump. The solution slowly flows to the electrochemical processing device through the silicone tube. Appropriate AC power parameters  are set. Cr (VI) is finally reduced to Cr (III) and flows into the discharge bottle. As shown in Qe of O-CF, PAN-CF and Ami-CF on Cr (VI) is calculated according to Equation (5). The removal rate (R) of Cr (VI) is calculated according to Equation (6).C0 refers to the initial concentration of Cr (VI) (mg·L−1), Ct refers to the concentration of Cr (VI) at moment t (mg·L−1), V refers to the volume of solution (L) and M refers to the mass of carbon felt electrode (g).Cr (VI) was determined and analyzed by dibenzoyl dihydrazine spectrophotometry (GB 7467-87). Total chromium was determined and analyzed by ICP-OES . The adsAll experimental data were expressed as an average of three replicates with standard deviation. To compare the pollutant removal efficiencies of different treatment strategies; statistical analyses were performed through the statistical program SPSS Statistics GradPack , including analysis of variance, Bartlett’s and Levine’s tests for homogeneity of variance and normality. Differences between individual means were identified using Tukey HSD-procedure at the 5% significance level. Tamhane’s T2 was selected for that equal variance between groups was not assumed.−1 and 2854 cm−1 are due to the symmetric and asymmetric stretching of -CH2− on the O-CF surface [−1 indicates that PAN is coated in CF before the hydroxylamine reaction, and -C≡N disappears after the hydroxylamine reaction. Meanwhile, peaks at 3100–3500 cm−1, 1641 cm−1 and 903 cm−1, respectively, represent -NH/-OH, -C=N-, and N-O in amidoxime [SEM characterization showed that both O-CF and Ami-CF were composed of fibers with a diameter of about 10 μm a,b. In c surface . The appmidoxime , verifyimidoxime .−1, the adsorption capacities of O-CF, PAN-CF and Ami-CF on Cr (VI) show obvious differences with the change of adsorption time. The adsorption of Cr (VI) on Ami-CF is a typical kinetic process. The adsorption capacity increases rapidly within the first 12 h, and the adsorption equilibrium is reached in about 36 h , and the highest adsorption capacity is 33.86 mg·g−1. It was much higher than the equilibrium adsorption capacity of O-CF and PAN-CF (~1.1 mg·g−1). 2 are 0.976 and 0.993, respectively. The pseudo-first-order kinetics are based on the assumption that the adsorption rate is controlled by the diffusion step and the pseudo-second-order kinetics are based on the assumption that the adsorption rate is controlled by the chemisorption mechanism [The adsorption kinetics and isothermal adsorption process of Cr (VI) on carbon felt electrode material are shown in echanism ,36,37. Techanism .−1, whereas that of the Ami-CF-containing amidoxime group on Cr (VI) can reach 101.73 mg·g−1. It is 2.1–5.5 times the saturated adsorption capacity of biochar, oxidized composite materials and other adsorbents reported in the literature (2 of the Langmuir model for Ami-CF adsorption of Cr (VI) is 0.992, whereas that of the Freundlich model is 0.968 .Langmuir and Freundlich isotherm models are often used to reveal the interaction mechanism between heavy metal ions and adsorbents. Among them, the Langmuir model assumes that metal ions occur at a uniform interface through monolayer deposition. In contrast, the Freundlich isotherm model describes adsorption at a non-uniform interface . The adsterature  [30,38,3is 0.968 . TherefoRL is 0–1, it indicates favorable adsorption, RL > 1 indicates unfavorable adsorption, RL = 1 indicates linear adsorption, and RL = 0 indicates irreversible adsorption [RL value ranges from 0.08 to 0.9, which indicates that Ami-CF is favorable for the adsorption of Cr (VI). In addition, with the increase in initial Cr (VI) concentration, RL value decreases continuously, indicating that the higher the concentration of pollutants, the more favorable the adsorption.When sorption . The resIn the traditional DC-mediated treatment of wastewater containing heavy metals, parameter optimization is limited to the difference of control voltage, whereas in the AC mediated treatment, the regulated parameters generally include positive and negative bias, frequency and duty ratio. The preliminary experimental results show that when the AC frequency is controlled at 400 Hz, the side reaction of electrolytic water is minimal. Therefore, the square-wave AC frequency was controlled to be 400 Hz, and the influences of positive and negative bias, duty cycle, solution flow rate, initial solution pH, initial concentration of Cr (VI) and other coexisting ions on Cr (VI) removal by AC electrochemical system were investigated. Two parallel treatments were set for each group of experiments.−1) and duty ratio (20%) in the liquid inlet bottle were kept unchanged, and the positive bias was fixed at 1V. The reduction and removal efficiency of Cr (VI) by AC electrochemical system under a negative bias voltage of −1.5 V, −2 V, −2.5 V, −3 V and −4 V were studied successively. The results are shown in −1, which meets the limit value of Cr (VI) of the national sanitary standard for drinking water (<0.05 mg·L−1). When a negative bias is further increased, the residual concentration of Cr (VI) remains below 4 μg·L−1. Therefore, −2.5 V is the best voltage for negative bias in the subsequent experiments. Similarly, by fixing negative bias as −2.5 V, the electrochemical removal effects of positive bias 0.5 V, 1 V, 1.5 V, 2 V and 2.5 V on Cr (VI) are studied successively. As shown in An AC electrochemical system removes Cr (VI) in solution by applying asymmetric square wave bias. The volume of Cr (VI) solution (50 mL), solution pH (2 ± 0.05), a flow rate of a peristaltic pump (0.5 mL·minThe duty cycle refers to the ratio of the high-level (positive bias) duration in a cycle. Other conditions were controlled in accordance with the above studies. The duty ratio was controlled as 10%, 20%, 30%, 40% and 50% separately to study its influence on the electrochemical reduction of Cr (VI). As shown in −1, 0.3 mL·min−1), Cr (VI) in the solution could be removed entirely due to the long residence time between electrodes and sufficient contact between Cr (VI) and Ami-CF surface. When the flow rates were 0.5 mL·min−1 and 0.7 mL·min−1, the removal efficiency of Cr (VI) was about 99.98% and 95.76%, respectively. When the flow rate is further increased, the higher flow rate will reduce the adsorption-reduction process of Cr (VI) on the electrode surface, decreasing removal efficiency. Therefore, the flow rate of the peristaltic pump is set as 0.5 mL·min−1.The effect of flow rate on Cr (VI) removal by penetrating electrochemical treatment devices is shown in −1). When the pH of the feed solution was adjusted to 2 ± 0.05, the Cr (VI) removal rate decreased slightly. Still, it remained at a high Cr (VI) removal rate (more than 85.04%) in the range of experimental concentration (0.5–250 mg·L−1) and Cr (VI) in the range of 0.5–100 mg·L−1 could almost be completely removed. The residual Cr (VI) is lower than 0.05 mg·L−1, which meets the hygienic limit of drinking water. When the pH of the feeding solution is adjusted to 3 ± 0.05, the removal rate of Cr (VI) at 0.5–50 mg·L−1 concentration can reach more than 93.74%. As shown in 2O72− and HCrO4− and participates in the reduction reaction from Cr (VI) to Cr (III) [3 precipitate until pH > 4 [3 films or colloids may be generated on the electrode surface (Equation (11)) [−1 m−2, and the contact time between Cr (VI) and Ami-CF in solution is only 30 s. Therefore, this method can achieve rapid and efficient removal effect on medium and high concentration Cr (VI) wastewater at low pH, which is superior to adsorption, precipitation, photocatalytic reduction and other methods [However, when the concentration of Cr (VI) in the feed solution is too high, the removal rate obviously decreases. The main reasons for the reduction of Cr (VI) removal rate with the increase in pH are as follows: (1) According to E -pH diagram for chromium speciation predominance , the redCr (III) . The reaCr (III) ; and (3)l pH > 4 , the hydl pH > 4 . Thus, ion 11)) , thus hion (11)) . Althougon (11)) ,44. Theron (11)) ,41. It s) , thus  methods .Cr2O72−2+, Zn2+ and Ca2+ were taken as representative coexisting heavy metals and alkali metal cations, and SO42−, CO32− and NO3− were taken as representative anions. The influence of coexisting ions on the removal of Cr (VI) by this method was discussed. The optimized electrochemical and solution parameters were selected as the experimental conditions; namely, the AC frequency was 400 Hz, the bias voltage was  V, the duty ratio was 20%, the pH of the solution was 2 ± 0.05 and the flow rate of the peristaltic pump was 0.5 mL·min−1. An amount of 50 mL Cr (VI) containing 100 mg·L−1 and different concentrations of Cu2+/Zn2+/Ca2+/SO42−/CO32−/NO3− solutions were treated, and two parallel treatments were set up in each group.Considering the complexity of actual wastewater components , Cu2+, Z−1. The removal rate of Cr (VI) was further improved when the solution contained 50 mg·L−1 co-existing Cu2+/Zn2+/Ca2+. This may be because the introduction of appropriate Cu2+/Zn2+/Ca2+ can improve the ionic strength and conductivity of the solution. The reduction potentials of Zn2+ and Ca2+ are relatively high, so they only act as electrolytes in the whole reaction system and do not participate in chemical reactions. Relatively speaking, Cu2+ is quickly reduced to Cu+/Cu0 and acts as a reducing agent to enhance the reduction removal of Cr (VI) [2+, Zn2+ and Ca2+ was further increased, the removal rate of Cr (VI) began to decrease. This may be because a large number of positively charged Cu2+, Zn2+ and Ca2+ will gather on the cathode surface through electrostatic attraction and compete with Cr (VI) for reaction sites on the electrode surface [As shown in  Cr (VI) . However surface , resulti−1 coexisting SO42−/CO32−/NO3−, the removal rate of Cr (VI) does not decrease, although it has no promoting effect. There are two possible reasons: (1) Compared with the reduction of S6+/C4+ and N5+ in SO42−/CO32−/NO3−, the reduction of Cr6+ to Cr3+ in HCrO4− is more likely [2+/Zn2+/Ca2+) and anions (SO42−/CO32−/NO3−) with the same concentration involved in this experiment, the ionic strength obtained from the conversion of anions is smaller than that of cations, and the conductivity of the solution containing the coexisting anions is relatively small. When the concentrations of SO42−, CO32− and NO3− were further increased, the removal rate of Cr (VI) decreased slightly. This may be because a large number of negatively charged SO42−, CO32− and NO3− will accumulate on the anode surface through electrostatic attraction, which inhibits the reduction of Cr6+ when the electrode is switched to the cathode later. In addition, Visual MINTEQ 3.1 was used to simulate the distribution of chromium species in 100 mg·L−1 Cr (VI) solution at pH 2. The results showed that Cr2O72− and HCrO4− were the dominant chemical species, accounting for 6.4% and 93%, respectively. When different concentrations of Cu2+/Zn2+/Ca2+/SO42−/CO32−/NO3− were present in the solution, the species distribution of Cr (VI) did not change significantly  and negative bias voltage  were consistent. The removal effects of Cr (VI) from 0.5–250 mg·L−1 were compared between DC and AC electrochemical methods. The results are shown in −1 Cr (VI), 100% removal was achieved by both DC and AC methods. However, when the initial concentration of Cr (VI) increased further, the removal efficiency of the DC method decreased significantly. For example, when the infusion concentration was 100 mg·L−1 and 250 mg·L−1, the removal efficiency of the DC method was 77.22% and 64.65%, respectively. In contrast, the AC method can still maintain the removal rate of 99.97% for Cr (VI) of 100 mg·L−1, and even the Cr (VI) of 250 mg·L−1 can still reach 85.37%, which is more than 20% higher than the DC method, which further highlights the superiority of AC method.The solution conditions  solution was continuously injected into the electrochemical filtration device at the rate of 0.5 mL·min−1 by a peristaltic pump and 50 mL was taken as a single dose. The removal efficiency in the long-term operation process shown in −1, which met the safety standard of Cr (VI) for drinking water stipulated by the World Health Organization. This excellent performance indicates that the functional electrode Ami-CF has excellent recycling performance, and the electrochemical treatment device can maintain excellent removal efficiency for a long time. Meanwhile, Cr (VI) was converted into Cr (III), which could be easily separated and recovered through precipitating with alkali, consequently reduced the overall cost. Thus the long time and stable performance, together with the concept of turning waste into resource, demonstrated the proposed method has broad application prospects.In order to test the stability of the functional electrode Ami-CF and the electrochemical treatment device, 50 mg·LIn order to explore the removal mechanism of Cr (VI) by this method, the Ami-CF after Cr (VI) adsorption, Cr (VI) treatment by direct current electrochemistry, and AC electrochemistry were characterized by XPS. After carbon correction, there was no characteristic peak of Cr on the surface of unused Ami-CF as a control group a. In Fig−1. Coupled with the Ami-CF electrode, the Coulomb repulsion effect and the side reaction of electrolytic water were suppressed under the high frequency anode and cathode conversion (asymmetric AC), and the diffusion mass transfer rate of Cr (VI) in the solution was promoted. Consequently, the asymmetric AC electrochemistry based on Ami-CF can rapidly (in 30 s) turning 0.5–100 mg·L−1 Cr (VI) into the safety standard of WHO drinking water with a high flux of 300 L h−1 m−2. Long-term operation of a total of 10 cycles demonstrated a high, stable and efficient removal performance. These results indicate that the asymmetric AC electrochemistry coupled with Ami-CF exhibit great application potential for the treatment of medium-to-high concentration Cr (VI)-containing wastewater. Further study is needed to scale-up the asymmetric AC electrochemical system and treat real wastewater with a much more complex composition.In this study, we provide a stable and efficient treatment method for wastewater containing Cr (VI). To improve adsorption and subsequent eletroreduction of Cr (VI) to Cr (III), facile fabrication of Ami-CF from commercial carbon felt by functionalization with amine oxime was developed and has excellent hydrophilicity and a strong adsorption capacity for Cr (VI) with saturated adsorption capacity of 101.73 mg·g