Effects of conductive and non-conductive materials on the activity of a hydrogenotrophic methanogen

Several conductive materials (CM), such as carbon nanotubes (CNT), activated carbon (AC), and magnetite, have been reported to mediate interspecies electron transfer in methanogenic environments. However, CNT also accelerated methane production (MP) of pure cultures of methanogens. We hypothesize that other CM and also non-CM may affect the methanogenic activity of pure cultures. For that purpose, we incubated the hydrogenotrophic methanogen, Methanobacteriun formicicum strain DSM 1535T, with AC, zeolite (Zeo), sand and glass beads (at 0.5 g/L), and followed MP. All materials reduced lag phases preceding the MP, and the time for complete conversion of H2/CO2 to methane. The best results were obtained with Zeo, since total hydrogen conversion occurred in less than 5 days (instead of 8 days as in the control incubated without materials). Approximately 5 days with sand, and 6 days with glass beads and AC, were necessary to achieve the complete conversion. The lag phases with AC were quite short (1 day) when compared with the control assay without materials (5 days). The initial MP (determined during the first 3 days of incubation) was improved 16 times with Zeo and 11 times with AC, when compared with the cultures incubated without materials. The results show that there is not a direct relationship between conductivity and the improvement of methanogenic activity. Other physicochemical properties of the materials might be related with the beneficial effects towards methanogens.info:eu-repo/semantics/publishedVersio

Pure cultures of hydrogenotrophic methanogens are affected by modified activated carbons, zeolite, sand and glass beads

The metabolism of hydrogenotrophs has been showed to be improved in the presence of carbon nanotubes, which is relevant since they are crucial microorganisms in the conversion of waste to methane1. In this study, we investigated if other materials, with different physicochemical properties, also affect the hydrogenotrophic activity of Methanobacterium formicicum. M. formicicum was incubated separately with 0.5 g/L of sand, and commercial zeolite, glass beads and activated carbon (AC0) with and without modifications on the AC0 surface. Modifications were obtained by chemical oxidation with HNO3 (AC_HNO3), H2SO4 (AC_ H2SO4) or both (AC_HNO3_ H2SO4) and thermal treatments. All materials, with exception of AC_HNO3_ H2SO4, improved the methanogenic activity. Carbon-based materials significantly reduced the lag phases preceding methane production (MP) (from approximately 5 days in the control to circa 1 day). Zeolite, sand and glass beads also reduced the lag phases but less than carbon materials (i.e., from 5 days to 1.5, 2.7 and 3.5 days, respectively). Additionally, exponential MP rates were up to 1.5 times higher in the assays with non-carbon materials. All materials tested have different physical/chemical properties including conductivities, but all stimulated the methanogenic activity. Thus, further studies are necessary to identify the mechanisms behind the underlying observations.info:eu-repo/semantics/publishedVersio

Zeolite stimulates the activity of microbial enrichments converting butyrate to methane

Conductive materials have been tested as a strategy to improve methane production (MP) in anaerobic digestion (AD) processes1,2. The effect of zeolite (0.5 g/L) towards microbial enrichments converting butyrate (10 mmol/L) to methane was investigated and compared with a microbial enrichment in the absence of the material. The enrichments were initiated with granular anaerobic sludge from a brewery WWTP as inoculum. Incubations were carried out under strict anaerobic conditions (at 37 °C) and periodically transferred to fresh medium. The results showed that, after an initial adaptation period (3 transfers), the presence of zeolite significantly accelerated the total conversion of butyrate to methane, since it took approximately 30d with zeolite and around 45d without zeolite. However, both enrichment cultures after extended adaptation (more than 8 transfers) behaved similarly, degrading butyrate in approximately 15d. Nevertheless, zeolite addition to active butyrate enrichment cultures without previous contact with zeolite, slightly accelerated MP, while the highly adapted zeolite-enrichment decreased activity when incubated without zeolite. Thus, the presence of zeolite showed to stimulate the microbial activity enhancing MP from butyrate degradation. This material possess natural ion-exchange properties, absorptive capacity and could function as a support for biomass which makes its application very attractive to AD processes.info:eu-repo/semantics/publishedVersio

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples

The online version contains supplementary material available at: https://doi.org/10.1007/s00253-023-12627-9.The renewable, abundant, and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples' microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited $\beta$-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples.Open access funding provided by FCT|FCCN (b-on). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, the projects LIGNOZYMES—Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01–0145-FEDER-029773), and B3iS—Biodiversity and Bioprospecting of Biosurfactants in Saline Environments (PTDC/BII-BIO/5554/2020); and by RNCA Advanced Computing Project MetaLignoZymes, metagenomic analysis of lignocellulosic residues towards the discovery of novel enzymes (CPCA/A0/408464/2021).info:eu-repo/semantics/publishedVersio

Detoxification of ciprofloxacin in an anaerobic bioprocess supplemented with magnetic carbon nanotubes: contribution of adsorption and biodegradation mechanisms

In anaerobic bioreactors, the electrons produced during the oxidation of organic matter can potentially be used for the biological reduction of pharmaceuticals in wastewaters. Common electron transfer limitations benefit from the acceleration of reactions through utilization of redox mediators (RM). This work explores the potential of carbon nanomaterials (CNM) as RM on the anaerobic removal of ciprofloxacin (CIP). Pristine and tailored carbon nanotubes (CNT) were first tested for chemical reduction of CIP, and pristine CNT was found as the best material, so it was further utilized in biological anaerobic assays with anaerobic granular sludge (GS). In addition, magnetic CNT were prepared and also tested in biological assays, as they are easier to be recovered and reused. In biological tests with CNM, approximately 99% CIP removal was achieved, and the reaction rates increased ?1.5-fold relatively to the control without CNM. In these experiments, CIP adsorption onto GS and CNM was above 90%. Despite, after applying three successive cycles of CIP addition, the catalytic properties of magnetic CNT were maintained while adsorption decreased to 29 ± 3.2%, as the result of CNM overload by CIP. The results suggest the combined occurrence of different mechanisms for CIP removal: adsorption on GS and/or CNM, and biological reduction or oxidation, which can be accelerated by the presence of CNM. After biological treatment with CNM, toxicity towards Vibrio fischeri was evaluated, resulting in ? 46% detoxification of CIP solution, showing the advantages of combining biological treatment with CNM for CIP removal.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte. Ana Rita Silva holds an FCT grant SFRH/BD/131905/2017. Cátia S.N. Braga holds an FCT grant SFRH/BD/132003/2017. This work was also financially supported by: Base Funding-UIDB/50020/2020 of the Associate Laboratory LSRE LCM-funded by national funds through FCT/MCTES (PIDDAC). OSGPS acknowledges FCT fund ing under the Scientific Employment Stimulus-Institutional Call CEECINST/00049/2info:eu-repo/semantics/publishedVersio

Modulation of butyrate-degrading methanogenic communities by conductive materials

Butyrate is a volatile fatty acid commonly present in anaerobic bioreactors. Previous research showed that methane production (MP) rates from butyrate, by lake sediment microbiomes, doubled by addition of carbon nanotubes, which was accompanied by changes in the microbial community composition, with enrichment of typical fatty-acid degrading bacteria (Syntrophomonas spp.), well known to exchange electrons with methanogens via hydrogen or formate formation1. But the authors suggested that electrons exchange via conductive materials (CM) may take place instead. In our study, anaerobic butyrate-degrading enrichment cultures were developed with other CM, namely activated carbon (AC) and magnetite (Mag) at 0.5 g/L. MP started earlier in AC enrichment and complete degradation was achieved faster in Mag enrichment. Syntrophomonas spp. were enriched in all cultures (representing 60 to 80 % of the total bacterial community), but hydrogenotrophic methanogens were highly stimulated by AC (78 % of Methanomicrobiales), while the methanogenic community of Mag culture was more diverse in acetoclastic methanogens (43% of Methanosarcina and Methanosaeta). It is still unclear if the improvement on butyrate degradation is associated to the role of CM in interspecies electron transfer, but it is undoubtful that they differentially modulate the methanogenic communities towards faster MP.info:eu-repo/semantics/publishedVersio

Differential effects of carbon-based and iron-based conductive materials in anaerobic butyrate-degrading enrichments

Introduction Conductive materials (CM) accelerate methane production (MP), probably by promoting more efficient interactions between bacteria and methanogens. This work investigates the effects of activated carbon (AC) and magnetite (Mag) in microbial enrichments degrading butyrate. Three different butyrate-degrading enrichments were developed: 1) without CM, 2) with AC, or 3) with Mag. It was also investigated if the effect of CM persisted when CM-adapted enrichments were transferred to new medium without CM, and if CM affected the activity of stable enrichments without previous contact with CM. Methodology Enrichment series were initiated with granular anaerobic sludge as inoculum, butyrate (10 mmol/L) as substrate, and CM (0.5 g/L AC or 0.5 g/L Mag), or without CM, and incubated at 37 °C, under strict anaerobic conditions. The following parameters were monitored: methane by gas chromatography; butyrate and acetate by high performance liquid chromatography; oxidation-reduction potential; pH and conductivity. RNA was extracted and taxonomic composition of the microbial communities was obtained by 16S rRNA gene sequencing. Results During the first incubations, AC-enrichment consumed hydrogen derived from butyrate degradation within 4 days, which was much faster than the enrichments with Mag and without CM, which presented lag phases (LP), preceding MP, longer than 11 and 7 days, respectively. Thus, Mag probably inhibited butyrate-degrading bacteria and/or hydrogenotrophic methanogens. Conversely, after the lag phase, Mag-enrichment was the fastest converting acetate to methane (3 times faster than in AC-enrichment), suggesting a stimulatory effect of Mag towards acetoclastic methanogens. Nevertheless, once the enrichments were adapted to the growth conditions, more efficient butyrate conversion was observed by all enrichments, with lag phases lower than 4 days, even in the control-enrichment. No significant changes on butyrate degradation were observed when highly adapted CM-enrichments were transferred to fresh medium without CM. On the other hand, when active enrichments (without previous contact with CM), were incubated with AC, it became slightly faster (0.7 times shorter LP), and with Mag were greatly inhibited (12 times longer LP). Syntrophomonas spp. represented 60 to 80 % of the total bacterial communities in all enrichments. Hydrogenotrophs were more abundant in AC-enrichment (78 % of Methanomicrobiales) and Mag-enrichment was highly enriched in acetoclastic methanogens (43 % of microorganisms assigned to Methanosaeta and Methanosarcina). Conclusions The presence of CM affects the performance of butyrate-degrading communities, with AC accelerating particularly butyrate conversion to methane (via H2/CO2) and acetate, and Mag inhibiting that first step but stimulating acetate conversion to methane.info:eu-repo/semantics/publishedVersio

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 μmol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .−) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ρ0 strain, lacking mitochondrial DNA, was used. The ρ0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 .− precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013

Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling

IF/00780/2015 PTDC/BIA-MIB/31028/2017 UIDP/04378/2020 UIDB/04378/2020 LA/P/0140/2020 SFRH/BD/140394/2018 PD/BD/142847/2018 PD00065/2013 DL 57/2016 ROTEIRO/0031/2013-PINFRA/22161/2016 BFU2016-75633-P PID2019-105451GB-I00 E34_R17 LMP58_18 to R.H-G RTI2018-099592-B-C21 ITN, GA-642157 COST Action GLYCONanoProbes (CA18132) ERC-2017-AdG, project number 788143-RECGLYCANMR RTI218-094751-B-C21) DNRF107The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addition of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our experiments suggest that during this process, neighboring tandem repeats are critical for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addition, in-cell analysis shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodology established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to determine the specificity toward complex mucin acceptor substrates.publishersversionepub_ahead_of_prin

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b

Funding Information: This work was supported by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B; ERC-2018-StG 804236-NEXTGEN-IO to A.P.) and the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement No 956758 to K.S.; ITN BactiVax under grant agreement no. 860325 to U.A. and ITN DIRNANO grant agreement No 956544 to F.C.). X-ray diffraction experiments described in this paper were performed using beamlines XALOC synchrotron at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland). F.M., C.S. and H.C. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding projects: PTDC/BIA-MIB/31028/2017 and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy—i4HB project (LA/P/0140/2020), to the CEEC contracts 2020.00233.CEECIND and 2020.03261.CEECIND for F.M. and H.C., respectively, and to PhD grant 2022.11723.BD of C.S. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 22161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). F.M. and J.J.-B. acknowledge to the European funding for the GLYCOTwinning project (No. 101079417) and -COST Action GLYCONANOPROBES. A.P.’s research is funded by “La Caixa” Foundation (HR21-00925), AECC (LABAE211744PALA), Fundación FERO, Ikerbasque, and BIOEF EITB MARATOIA BIO19/CP/002. We thank Agencia Estatal de Investigación of Spain for grants PID2019-107956RA-I00 (A.P.), PID2019-107770RA-I00 (J.E.-O.), RTI2018-099592-B-C21 (F.C.), ID2020-114178GB (R.B. and J.D.S.), RYC2018-024183-I (A.P.), and the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain) A.A.-V. receives funding from “La Caixa” Foundation (ID 100010434, LCF/BQ/DR20/11790022). A. B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC). F.C. acknowledges the Mizutani Foundation for Glycoscience (Grant 220115). Funding Information: This work was supported by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B; ERC-2018-StG 804236-NEXTGEN-IO to A.P.) and the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement No 956758 to K.S.; ITN BactiVax under grant agreement no. 860325 to U.A. and ITN DIRNANO grant agreement No 956544 to F.C.). X-ray diffraction experiments described in this paper were performed using beamlines XALOC synchrotron at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland). F.M., C.S. and H.C. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding projects: PTDC/BIA-MIB/31028/2017 and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy—i4HB project (LA/P/0140/2020), to the CEEC contracts 2020.00233.CEECIND and 2020.03261.CEECIND for F.M. and H.C., respectively, and to PhD grant 2022.11723.BD of C.S. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 22161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). F.M. and J.J.-B. acknowledge to the European funding for the GLYCOTwinning project (No. 101079417) and -COST Action GLYCONANOPROBES. A.P.’s research is funded by “La Caixa” Foundation (HR21-00925), AECC (LABAE211744PALA), Fundación FERO, Ikerbasque, and BIOEF EITB MARATOIA BIO19/CP/002. We thank Agencia Estatal de Investigación of Spain for grants PID2019-107956RA-I00 (A.P.), PID2019-107770RA-I00 (J.E.-O.), RTI2018-099592-B-C21 (F.C.), ID2020-114178GB (R.B. and J.D.S.), RYC2018-024183-I (A.P.), and the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain) A.A.-V. receives funding from “La Caixa” Foundation (ID 100010434, LCF/BQ/DR20/11790022). A. B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC). F.C. acknowledges the Mizutani Foundation for Glycoscience (Grant 220115). Publisher Copyright: © 2023, The Author(s).Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.publishersversionpublishe