Polis_Msca-If-661429_Datasets

<strong>Plan name</strong> D1.2_POLIS_MSCA-IF-661429_DMP <strong>ID</strong> D1.2 <strong>Grant number</strong> 661429 - POLIS - H2020-MSCA-IF-2014 <strong>Project number</strong> 661429 <strong>Project acronym</strong> POLIS <strong>Project title</strong> Studying the bricks of microbial cities: characterization and structural properties of exopolysaccharides and their interaction with proteins and cations in anammox granular sludge <strong>Call (part) identifier</strong> H2020-MSCA-IF-2014 <strong>Topic</strong> MSCA-IF-2014-EF Marie Skłodowska-Curie Individual Fellowships (IF-EF) <strong>Fixed EC Keywords</strong> Environmental biotechnology, bioremediation, biodegradation <strong>Free keywords</strong> anammox, granular sludge, biofilm, exopolymeric substances, exopolysaccharides, EPS, rheology, exopolymeric proteins, mono-divalent cations <strong>Beneficiary</strong> Polytechnic University of Milan (PIC: 999879881) <strong>Department</strong> Department of Civil and Environmental Engineering (DICA) <strong>Supervisor</strong> Prof. Francesca Malpei <strong>Principal Investigator/Researcher</strong> Ing. Tommaso Lotti, PhD <strong>Principal Investigator/Researcher ID</strong> http://orcid.org/0000-0003-0379-4822 <strong>Plan data contact</strong> [e-mail]; [e-mail] <strong>Project description</strong> This research project is in the context of environmental engineering, in particular in the field of wastewater treatment, more specifically focused on biofilm-based innovative technologies for nitrogen removal. The anaerobic ammonium oxidation (anammox) bacteria are recently discovered players of the biogeochemical nitrogen cycle. The bioprocess based on anammox metabolism is an innovative technology for the removal of nitrogen from municipal and industrial wastewaters allowing important savings on operational costs due to the requirement of 60% less oxygen (aeration), no need for organic carbon and the production of 90% less excess sludge, compared with conventional nitrogen removal technologies. Its potential application to municipal wastewater (sewage) would allow a complete redesign of the present energy-consuming into an energy-yielding sewage treatment plant. Due to the slow growth rate of anammox bacteria, their retention in the system is one of the main concern for process stability. This is the reason why in most of the different anammox-based technologies currently applied, anammox bacteria are cultivated in the form of biofilm and in particular in the form of self-aggregating biofilm (i.e. granular sludge). Biofilm stability is closely related to the properties of the extracellular polymeric substances (EPS) constituting the matrix in which microorganisms live and grow. EPS are high-molecular weight compounds secreted by microorganisms establishing the functional and structural integrity of biofilms, and are considered the fundamental component that determines the physiochemical properties of a biofilm. EPS are mostly composed of polysaccharides and proteins, but also include other macro-molecules such as DNA, lipids and humic substances. The aim of the present research was to investigate the structural components of anammox EPS matrix unrevealing the mechanisms involved in anammox biofilm (specifically granular sludge) formation and stability. Several extraction methods were tested to evaluate the extraction yield and the total carbohydrates/protein content. Mass spectrometry (e.g. MALDI MS) was used to investigate functional EPS and their fine structures, with special focus on hydrogel and film forming components. Rheometric analysis were used to evaluate the viscoelastic characteristics of anammox granular sludge in comparison with the hydrogel (potentially) formed by extracted EPS and their interaction with mono/divalent-cations. Microscopy techniques (mainly AFM, SEM) were used to image the morphology of the extracted biopolymer and the film structure. Considering that excess sludge is currently one of the main waste products of wastewater treatment facilities, the recovery of a bio-based polymer and its application in other industrial sectors would contribute to the transition towards a circular economy fostering sustainable economic growth. The results of the present research could identify the potential applications of a bio-based polymer recovered from excess biofilm sludge based on its properties. <strong>Open access to scientific publications and underlying data</strong> Scientific peer-reviewed publications such as journal articles will be deposited upon publication in Zenodo repository (https://zenodo.org/), an OpenAIRE/CERN repository. The deposition of the research data needed to validate the results presented in the deposited scientific publications ('underlying data') will be evaluated from time to time by the Researcher (Ing.Tommaso Lotti, PhD) and the Supervisor (Prof.Francesca Malpei) along the project development according to the approach of the European Commission “as open as possible, as closed as necessary”. The research data decided to be open up will be organized in separate data sets. For the description of each data set and the relative depositing procedure, the reader is referred to the “DATA SETS” section below. The list of openly shared data sets will be updated and/or modified whether necessary during the time course of the project. <strong>DATA SETS</strong> <strong>Data set name</strong> POLIS_MSCA-IF-661429_DATASET-1 <strong>Data set description</strong> Different protocols used to extract structural EPS from anammox sludge. EPSs were extracted from freeze-dried anammox granules (originating from the full-scale anammox reactor of the wastewater treatment plant of Rotterdam, Sluisjesdijk-Dokhaven) by the following methods: ultrasonication, heating with Na₂CO₃, cations exchange resin, ethylenediaminetetraacetic acid (EDTA), NaOH, formamide with NaOH, formaldehyde with NaOH, sulfuric acid. The extraction efficiency of each protocol was evaluated by the following parameters: - Extraction yield evaluated by gravimetric analysis - Carbohydrates and protein equivalent content of the EPS resulting from different extraction protocols Tests were conducted in triplicates. Dataset will be presented in the form of tables and saved in pdf format with the name “POLIS_MSCA-IF-661429_DATASET-1.pdf”. <strong>Standards and metadata</strong> Extraction protocols were derived from biofilm literature and in particular from biofilm systems applied in the field of wastewater treatment. Gravimetric analysis according to American Public Health Association (APHA), Standard Methods for the Examination of water and Wastewater (2005). Data are expressed as milligrams of extracted-EPS dry weight per gram of volatile suspended solids (VSS) of the original biofilm (mg-EPS/g-VSS). Total carbohydrate content was determined by a phenol-sulphuric acid assay with D-glucose used as standard (Dubois et al., 1956). Data are expressed as milligram of total carbohydrates as D-glucose equivalent per gram of extracted-EPS dry weight (mg/g-EPS). Protein content was measured by the biconchoninic acid (BCA) protein assay with bovine serum albumin (BSA) used as standard (Interchim Uptima BC assay quantitation kit). Data are expressed as milligram of total proteins as BSA equivalent per gram of extracted-EPS dry weight (mg/g-EPS). To the best of author knowledge Metadata standards are missing in this particular field. Therefore, our results were reported using acronym and units of measurements according to the most renowned journals in the field (e.g. Water Research, Environmental Science and Technology, Bioresource Technology, etc.). In order to facilitate data sharing and use, metadata describing the nomenclature used for the storage of research data is given in a separate text file named "DATASET-1_metadata" in .pdf format. The file " DATASET-1_metadata" contains the list of acronyms and symbols used for data presentation as well as the description of the materials and methods used during the experimental phase. <strong>Data sharing</strong> A selection of research data will be deposited, upon publication of the scientific publication reporting the elaborated research data, in Zenodo repository (https://zenodo.org/), an OpenAIRE/CERN repository. Zenodo provides to each data set a persistent identifier (DOI). Peer-reviewed scientific publications will be deposited in self archiving/green "OA" repositories such as Tommaso Lotti's page on ResearchGate (https://www.researchgate.net/profile/Tommaso_Lotti) and Polytechnic University of Milan institutional self-archiving system (https://re.public.polimi.it/). Research data as well as scientific publications and conference papers are available upon request via e-mail. <strong>Data set name</strong> POLIS_MSCA-IF-661429_DATASET-2 <strong>Data set description</strong> Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules such as biopolymers. MALDI methodology is a three-step process. First, the sample is solubilized in a solvent, mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into the mass spectrometer. The present dataset comprises the mass spectra of the extracellular polymeric substances (EPS) samples extracted from anammox granular sludge. EPS were extracted from freeze-dried anammox granules originating from the full-scale anammox reactor of the wastewater treatment plant of Rotterdam, Sluisjesdijk-Dokhaven (van der Star et al., 2007). EPS was dissolved in ammonium bicarbonate (5 mM) and run in a conventional SDS-PAGE gel and stained with Coomassie Blue. Stained gel bands were cut and trypsin digestion was performed following the Protease-Max digestion protocol (Promega). α-Cyano-4-hydroxycinnamic acid (HCCA) was used as matrix and dissolved to a final concentration of 10 mg/mL in a solution of pure acetonitrile and 0.1% trifluoroacetic acid (70/30% v/v). The digested EPS solution was mixed with the matrix (50/50% v/v), spotted on the target plate and then analyzed by means of a MALDI TOF/TOF Ultraflex III (Bruker). After complete drying, the samples were submitted to <em>mass</em> spectrometry analysis on a MALDI TOF TOF Ultraflex III (Bruker Daltonics, Germany), which was operated in <em>positive reflectron</em> for peptide analyses. The peptidic fraction of the venom was acquired in the range m/z 900-4500. <strong>Standards and metadata</strong> Metadata describing the nomenclature used for the storage of research data are given in a separate text file named "DATASET-2_metadata". The file "DATASET-2_metadata" contains the list of acronyms and symbols used for data presentation as well as the matrix and solvent used for sample preparation. The mass spectrometer output files is presented in the standard format “mzML” (Mass Spectrometry Markup Language) in order to facilitate data sharing and reuse and saved in a compressed folder named "POLIS_MSCA-IF-661429_DATASET-2". The format “mzML” is a unified open file format containing the peaks list and information about the precursor MS signals and about the associated metadata (i.e., instrument settings and description, acquisition mode, etc). <strong>Data sharing</strong> A selection of research data are deposited, upon publication of the scientific publication reporting the elaborated research data, in Zenodo repository (https://zenodo.org/), an OpenAIRE/CERN repository. Peer-reviewed scientific publications will be deposited in self archiving/green "OA" repositories such as Tommaso Lotti's page on ResearchGate (https://www.researchgate.net/profile/Tommaso_Lotti) and Polytechnic University of Milan institutional self-archiving system (https://re.public.polimi.it/). Research data as well as scientific publications and conference papers are available upon request via e-mail.计划名称： D1.2_POLIS_MSCA-IF-661429_DMP 编号： D1.2 资助编号： 661429 - POLIS - H2020-MSCA-IF-2014 项目编号： 661429 项目简称： POLIS 项目标题： 研究微生物城市的构成单元：厌氧氨氧化（anaerobic ammonium oxidation, anammox）颗粒污泥中胞外多糖的表征与结构特性及其与蛋白质和阳离子的相互作用 征集（部分）标识： H2020-MSCA-IF-2014 研究主题： MSCA-IF-2014-EF 玛丽·斯克洛多夫斯卡-居里个人奖学金（IF-EF） 欧盟委员会固定关键词： 环境生物技术、生物修复、生物降解 自由关键词： anammox、颗粒污泥、生物膜、胞外聚合物（exopolymeric substances, EPS）、胞外多糖、流变学、胞外蛋白质、一价/二价阳离子 受资助方： 米兰理工大学（PIC: 999879881） 院系： 土木与环境工程系（DICA） 指导教师： 弗朗西斯卡·马尔佩教授（Prof. Francesca Malpei） 首席研究员/研究人员： 工程师汤姆马索·洛蒂，博士（Ing. Tommaso Lotti, PhD） 首席研究员/研究人员标识： http://orcid.org/0000-0003-0379-4822 计划数据联系人： [e-mail]; [e-mail] 项目描述： 本研究项目隶属于环境工程领域，尤其聚焦于污水处理方向，更具体地说，是针对脱氮的生物膜基创新技术。厌氧氨氧化（anaerobic ammonium oxidation, anammox）细菌是近年发现的生物地球化学氮循环参与者。基于anammox代谢的生物工艺是一种创新的市政与工业废水脱氮技术，与传统脱氮技术相比，其仅需60%的曝气氧量、无需有机碳源且剩余污泥产量降低90%，可显著节约运行成本。若将其应用于市政污水（生活污水）处理，可将现有高能耗污水处理厂重构为产能型污水处理设施。 由于anammox细菌生长速率缓慢，其在系统中的留存是保障工艺稳定性的核心关切之一。这也是当前多数应用的anammox基工艺中，anammox细菌以生物膜形式，尤其是自聚集生物膜（即颗粒污泥）形式培养的原因。生物膜的稳定性与构成微生物生存与生长基质的胞外聚合物（extracellular polymeric substances, EPS）的特性密切相关。EPS是微生物分泌的高分子量化合物，构成生物膜的功能与结构完整性基础，被视为决定生物膜物理化学特性的核心组分。EPS主要由多糖与蛋白质组成，同时包含DNA、脂质与腐殖质等其他大分子。 本研究旨在解析anammox EPS基质的结构组分，揭示anammox生物膜（特指颗粒污泥）形成与稳定的相关机制。本研究测试了多种提取方法，以评估提取产率与总碳水化合物/蛋白质含量。采用质谱技术（如基质辅助激光解吸电离质谱（matrix-assisted laser desorption/ionization mass spectrometry, MALDI MS））研究功能性EPS及其精细结构，重点关注水凝胶与成膜组分。采用流变分析，对比anammox颗粒污泥与提取EPS（潜在）形成的水凝胶的粘弹性特性，以及EPS与一价/二价阳离子的相互作用。采用显微技术（主要为原子力显微镜（AFM）、扫描电子显微镜（SEM））对提取的生物聚合物形貌与膜结构进行成像。 考虑到剩余污泥是污水处理厂的主要副产物之一，回收生物基聚合物并将其应用于其他工业领域，将有助于推动循环经济转型，促进可持续经济增长。本研究成果可基于其特性，确定从剩余生物膜污泥中回收的生物基聚合物的潜在应用场景。 科学出版物及原始数据开放获取： 经同行评审的科学出版物（如期刊论文）将在发表后上传至Zenodo知识库（https://zenodo.org/），该库隶属于OpenAIRE/欧洲核子研究中心（CERN）。研究人员（工程师汤姆马索·洛蒂，博士）与指导教师（弗朗西斯卡·马尔佩教授）将在项目推进过程中，依据欧盟委员会“尽可能开放，必要时封闭”的原则，适时评估用于验证已发表科学出版物中成果的研究数据（即原始数据）的开放方案。确定开放的研究数据将整理为独立数据集。关于各数据集的描述与相关上传流程，请参阅下文的“数据集”部分。公开共享的数据集列表将在项目周期内根据需要进行更新与调整。 数据集： 数据集名称： POLIS_MSCA-IF-661429_DATASET-1 数据集描述： 本数据集包含从anammox污泥中提取结构性EPS的多种方案。采用以下方法从冻干anammox颗粒污泥（取自鹿特丹Sluisjesdijk-Dokhaven污水处理厂的全规模anammox反应器）中提取EPS：超声法、碳酸钠（Na₂CO₃）加热法、阳离子交换树脂法、乙二胺四乙酸（ethylenediaminetetraacetic acid, EDTA）法、氢氧化钠（NaOH）法、甲酰胺-氢氧化钠法、甲醛-氢氧化钠法、硫酸法。采用以下参数评估各方案的提取效率： - 通过重量分析法评估提取产率 - 不同提取方案所得EPS的碳水化合物与蛋白质当量含量 实验设置三次重复。数据集将以表格形式呈现，并保存为名为"POLIS_MSCA-IF-661429_DATASET-1.pdf"的PDF文件。 标准与元数据： 提取方案源自生物膜相关文献，尤其来自污水处理领域的生物膜系统研究。 重量分析法依据美国公共卫生协会（American Public Health Association, APHA）《水与废水检验标准方法》（2005版）执行。数据以每克原生物膜挥发性悬浮固体（volatile suspended solids, VSS）对应的提取EPS干重毫克数表示（mg-EPS/g-VSS）。 总碳水化合物含量采用苯酚-硫酸法测定，以D-葡萄糖作为标准品（Dubois等，1956）。数据以每克提取EPS干重对应的D-葡萄糖当量总碳水化合物毫克数表示（mg/g-EPS）。 蛋白质含量采用二喹啉甲酸（bicinchoninic acid, BCA）蛋白质测定法，以牛血清白蛋白（bovine serum albumin, BSA）作为标准品（Interchim Uptima BC检测定量试剂盒）。数据以每克提取EPS干重对应的BSA当量总蛋白质毫克数表示（mg/g-EPS）。 据研究者所知，该领域目前缺乏统一的元数据标准。因此，本研究结果采用该领域顶尖期刊（如《Water Research》《Environmental Science and Technology》《Bioresource Technology》等）所使用的缩写与计量单位进行报告。为便于数据共享与复用，本研究将数据存储所用的命名规范相关元数据整理至单独的PDF文本文件"DATASET-1_metadata"中，该文件包含数据展示所用的缩写与符号列表，以及实验阶段所用材料与方法的说明。 数据共享： 在发表阐述本数据集研究成果的科学出版物后，部分研究数据将上传至Zenodo知识库（https://zenodo.org/），该库隶属于OpenAIRE/欧洲核子研究中心（CERN）。Zenodo将为每个数据集提供永久标识符（DOI）。 经同行评审的科学出版物将上传至自存档/绿色开放获取（OA）知识库，例如汤姆马索·洛蒂在ResearchGate上的个人页面（https://www.researchgate.net/profile/Tommaso_Lotti）以及米兰理工大学机构自存档系统（https://re.public.polimi.it/）。 研究数据、科学出版物与会议论文可通过邮件申请获取。 数据集名称： POLIS_MSCA-IF-661429_DATASET-2 数据集描述： 基质辅助激光解吸电离（matrix-assisted laser desorption/ionization, MALDI）是一种用于质谱分析的软电离技术，可实现生物分子（如生物聚合物）的分析。MALDI方法分为三个步骤：首先，将样品溶解于溶剂中，与合适的基质材料混合后点样于金属靶板；其次，脉冲激光辐照样品，引发样品与基质材料的烧蚀与解吸；最后，分析物分子在烧蚀气体的热羽流中发生质子化或去质子化而电离，随后被加速进入质谱仪。 本数据集包含从anammox颗粒污泥中提取的EPS样品的质谱图谱。EPS取自鹿特丹Sluisjesdijk-Dokhaven污水处理厂全规模anammox反应器中的冻干anammox颗粒污泥（van der Star等，2007）。 将EPS溶解于碳酸氢铵（5 mM）溶液中，经常规十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后，采用考马斯亮蓝染色。切取染色后的凝胶条带，按照Protease-Max消化方案（Promega公司）进行胰蛋白酶消化。采用α-氰基-4-羟基肉桂酸（α-Cyano-4-hydroxycinnamic acid, HCCA）作为基质，将其溶解于乙腈与0.1%三氟乙酸的混合溶液（体积比70/30）中，终浓度为10 mg/mL。将消化后的EPS溶液与基质溶液按体积比50/50混合，点样于靶板后，采用MALDI TOF/TOF Ultraflex III（布鲁克公司）进行分析。样品完全干燥后，在MALDI TOF TOF Ultraflex III（德国布鲁克道尔顿公司）上进行质谱分析，该仪器采用正离子反射模式进行肽段分析。肽段信号采集范围为m/z 900-4500。 标准与元数据： 本数据集将数据存储所用的命名规范相关元数据整理至单独的文本文件"DATASET-2_metadata"中，该文件包含数据展示所用的缩写与符号列表，以及样品制备所用的基质与溶剂信息。质谱仪输出文件采用标准格式“mzML”（质谱标记语言，Mass Spectrometry Markup Language），以方便数据共享与复用，保存于名为"POLIS_MSCA-IF-661429_DATASET-2"的压缩文件夹中。mzML格式是一种统一的开放文件格式，包含峰列表、前体质谱信号信息及相关元数据（如仪器设置与说明、采集模式等）。 数据共享： 在发表阐述本数据集研究成果的科学出版物后，部分研究数据将上传至Zenodo知识库（https://zenodo.org/），该库隶属于OpenAIRE/欧洲核子研究中心（CERN）。 经同行评审的科学出版物将上传至自存档/绿色开放获取（OA）知识库，例如汤姆马索·洛蒂在ResearchGate上的个人页面（https://www.researchgate.net/profile/Tommaso_Lotti）以及米兰理工大学机构自存档系统（https://re.public.polimi.it/）。 研究数据、科学出版物与会议论文可通过邮件申请获取。