Full-scale biological phosphorus removal: quantification of storage polymers, microbial performance and metabolic modelling

Dissertação para obtenção do Grau de Doutor em Engenharia Química e BioquímicaEnhanced biological phosphorus removal (EBPR) can be applied in wastewater treatment plants (WWTPs), as a sustainable and efficient way to remove phosphorus from wastewater and hence reduce its impact on eutrophication. This work characterises the performance, metabolism and identity of the microbial EBPR communities in full-scale WWTPs. The accurate quantification of the internal storage compounds, namely polyhydroxyalkanoate (PHA) and glycogen, is crucial to the characterisation of EBPR. The optimal glycogen and PHA quantification methods were sensitive to the heterogeneity of the sample, in terms of its microbial structure (floccular or granular) and, for PHA, in terms of the size and the number of substituents of the monomers forming the copolymer. Additionally, by characterising six full-scale EBPR WWTPs, in terms of their overall performance, microbial identity and metabolism, the composition of polyphosphate accumulating organisms (PAOs) was fairly similar in all plants. Also, a warmer climate was not sufficient to justify a higher presence of glycogen accumulating organisms (GAOs). Differing levels of denitrifying PAOs were obtained in different plants and the involvement of the tricarboxylic acid (TCA) cycle in the anaerobic metabolism of PAOs was observed. Furthermore, a metabolic model developed in this study, which incorporates the involvement of the anaerobic TCA cycle and a new description of the aerobic maintenance processes, was able to accurately describe the chemical cycling of soluble and intracellular compounds, while requiring a simple calibration procedure. A series of simulations demonstrated that lower acetate concentrations in the feed and higher aeration retention times would favour the TCA cycle metabolism over the glycolysis pathway, which would explain why the former has been more frequently encountered in WWTPs and the latter in lab-scale enriched cultures.Fundação para a Ciência e Tecnologia - PhD grant (SFRH/BD/29477/2006

Adjusting Organic Load as a Strategy to Direct Single-Stage Food Waste Fermentation from Anaerobic Digestion to Chain Elongation

Production of medium chain carboxylic acids (MCCA) as renewable feedstock bio-chemicals, from food waste (FW), requires complicated reactor configurations and supplementation of chemicals to achieve product selectivity. This study evaluated the manipulation of organic loading rate in an un-supplemented, single stage stirred tank reactor to steer an anaerobic digestion (AD) microbiome towards acidogenic fermentation (AF), and thence to chain elongation. Increasing substrate availability by switching to a FW feedstock with a higher COD stimulated chain elongation. The MCCA species n-caproic (10.1 ± 1.7 g L−1) and n-caprylic (2.9 ± 0.8 g L−1) acid were produced at concentrations comparable to more complex reactor set-ups. As a result, of the adjusted operating strategy, a more specialised microbiome developed containing several MCCA-producing bacteria, lactic acid-producing Olsenella spp. and hydrogenotrophic methanogens. By contrast, in an AD reactor that was operated in parallel to produce biogas, the retention times had to be doubled when fed with the high-COD FW to maintain biogas production. The AD microbiome comprised a diverse mixture of hydrolytic and acidogenic bacteria, and acetoclastic methanogens. The results suggest that manipulation of organic loading rate and food-to-microorganism ratio may be used as an operating strategy to direct an AD microbiome towards AF, and to stimulate chain elongation in FW fermentation, using a simple, un-supplemented stirred tank set-up. This outcome provides the opportunity to repurpose existing AD assets operating on food waste for biogas production, to produce potentially higher value MCCA products, via simple manipulation of the feeding strategy.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 66599

Selecting fermentation products for food waste valorisation with HRT and OLR as the key operational parameters.

Acidogenic fermentation is attractive for food waste valorisation. A better understanding is required on how operation affects product selectivity. This study demonstrated that the hydraulic retention time (HRT) and organic loading rate (OLR) selected fermentation pathways in a single-stage, semicontinuous stirred tank reactor. Three combinations of HRT and OLR were tested to distinguish the effect of each parameter. Three fermentation profiles with distinct microbial communities were obtained. Predominantly n-butyric acid (13 ± 2 gCOD L-1, 55 ± 14% of carboxylates) was produced at an HRT of 8.5 days and OLR around 12 gCOD L-1 d-1. Operating at an HRT two days longer, yet with similar OLR, stimulated chain elongation (up to 13.6 gCOD L-1 of n-caproic acid). This was reflected by a microbial community twice as diverse at longer HRT as indicated by first and second order Hill number (1D = 24 ± 4, 2D = 12 ± 3) and by a higher relative abundance of genera related to secondary fermentation, such as the VFA-elongating Caproiciproducens spp., and secondary lactic acid fermenter Secundilactobacillus spp.. Operating at a higher OLR (20 gCOD L-1 d-1) but HRT of 8.5 days, resulted in typical lactic acid fermentation (34 ± 5 gCOD L-1) harbouring a less diverse community (1D = 8.0 ± 0.7, 2D = 5.7 ± 0.9) rich in acid-resistant homofermentative Lactobacillus spp. These findings demonstrate that a flexible product portfolio can be achieved by small adjustments in two key operating conditions. This improves the economic potential of acidogenic fermentation for food waste valorisation

Guidelines for addressing the interfaces between Technology Areas in the context of the EU-ETV Pilot Program

Environmental Technology Verification (ETV) is a new tool enabling the verification of the performance claims put forward by developers of innovative environmental technologies. As specified in the ETV General Verification Protocol (GVP), the EU-ETV programme, launched in 2011 by DG-ENV is covering three Technology Areas (TAs): Water treatment and monitoring, Materials, waste and resources, Energy Technologies. The verification bodies in charge of performing the verifications have to be accredited for one or more of these 3 TAs or part thereof. Three dedicated Technical Working Groups (TWGs) have been set up in order to provide technical guidance for the technologies falling under these TAs. Although the GVP is providing examples of technologies for each of the TA, the precise frontier between them is not always clear, in particular for technologies that relate to more than one distinct technology areas (e.g. energy efficiency in the domain of water treatment). This document provides guidance on this issue, in order to help decision making and ensure a harmonised approach thought the ETV scheme. This document, adopted on the on the 04/08/2015 by the TWGs, is a guidance document, with the meaning given in the General Verification Protocol of the EU ETV pilot programme (version 1.1), Section A.II.4.3. It has been produced by the EU ETV Technical Working Groups, chaired by the JRC, under the auspices of DG Environment. This document is also deliverable 2.1.6.4 under the Administrative Arrangement 070307/2011/630755/F4 between DG ENV and JRC (ref JRC No. 32937), “Scientific and technical support for the implementation of the EU Environmental Technology Verification (ETV) pilot programme” as modified by amendment 1 signed 16/06/2014.JRC.F.6-Energy Technology Policy Outloo

Use of the ETV logo and post-verification requirements when marketing a technology verified through the ETV scheme

Environmental Technology Verification (ETV) is a new tool enabling the verification of the performance claims put forward by developers of innovative environmental technologies. The Programme is set up foreseeing the existence of Technical Working Groups (TWGs), one for each technology area active under the Pilot Programme. These are chaired by the JRC and composed by Commission Invited Experts and by Experts representing the Verification Bodies with the overall aim to harmonise and exchange good practices among member states. ETV has a logo inherited from previous ETV FP7 projects, but it is not an official logo of the European Commission. ETV is not a certification nor a labialisation scheme. Therefore the logo should be used in well-defined conditions, specifying clearly what has been verified. This document defines and clarifies all elements relative to the logo and its use: design specifications, uses of the logo under different marketing circumstances and communication purposes. This document, adopted on the date by the TWGs on 08/04/2015, is a guidance document, with the meaning given in the General Verification Protocol of the EU ETV pilot programme (version 1.1), Section A.II.4.3. It has been produced by the EU ETV Technical Working Groups, chaired by the JRC, under the auspices of DG Environment. This document is also deliverable 2.1.6.4 under the Administrative Arrangement 070307/2011/630755/F4 between DG ENV and JRC (ref JRC No. 32937), “Scientific and technical support for the implementation of the EU Environmental Technology Verification (ETV) pilot programme” as modified by amendment 1 signed 16/06/2014.JRC.F.6-Energy Technology Policy Outloo

EU Environmental Technology Verification pilot programme - Guidance documents: Guidelines for the eligibility assessment of technologies proposed to the EU-ETV scheme

Environmental Technology Verification (ETV) is a new tool to enable the verification of the claims provided by environmental technologies. The Programme is set up foreseeing the existence of Technical Working Groups (TWGs), one for each technology area active under the Pilot programme. These are chaired by the JRC and composed by Commission Invited Experts and by Experts representing the Verification Bodies with the overall aim to harmonise and exchange good practices among member states. This document summarises the outcome of the discussion of the Technical Working Groups concerning the assessment of eligibility criteria by Verification Bodies. It clarifies and provides guidance to help Verification Bodies evaluate whether a technology proposed for ETV meets the minimum requirements imposed by the programme such as fitting the scope of the programme, presenting innovative features and an environmental added value, being “ready-to-market” and meeting user’s needs and legal requirements. This document, adopted on the on the 23rd April 2014 by the TWGs, is a specific guidance document, with the meaning given in the General Verification Protocol of the EU ETV pilot programme (version 1.0), Section A.II.4.3. It has been produced by the EU ETV Technical Working Groups, chaired by the JRC, under the auspices of DG Environment.JRC.F.6-Energy Technology Policy Outloo

EU Environmental Technology Verification pilot programme - Guidance documents: Guidelines for the workflow of documents and information between Verification Bodies, Technical Working Groups and Commission Services

Environmental Technology Verification (ETV) is a new tool to enable the verification of the claims provided by environmental technologies. The Programme is set up foreseeing the existence of Technical Working Groups (TWGs), one for each technology area active under the Pilot programme. These are chaired by the JRC and composed by Commission Invited Experts and by Experts representing the Verification Bodies with the overall aim to harmonise and exchange good practices among member states. This document summarises the outcome of the discussion of the Technical Working Groups concerning the the streamline of information and agrees on a workflow for the communication of documents between Verification Bodies, Technical Working Groups and Commission Services. This document, adopted on the on the 15th October 2013 by the TWGs, is a specific guidance document, with the meaning given in the General Verification Protocol of the EU ETV pilot programme (version 1.0), Section A.II.4.3. It has been produced by the EU ETV Technical Working Groups, chaired by the JRC, under the auspices of DG Environment.JRC.F.6-Energy Technology Policy Outloo

Denitrification Activity of Polyphosphate Accumulating Organisms (PAOs) in Full-Scale Wastewater Treatment Plants

A comprehensive assessment of full-scale enhanced biological phosphorus removal (EBPR) plants (five plants, 19 independent tests) was undertaken to determine their effectiveness in terms of aerobic and anoxic P removal. By comparing parallel P uptake tests under only aerobic or under anoxic-aerobic conditions, results revealed that introducing an anoxic stage led to an overall P removal of on average 90% of the P removed under only aerobic conditions. This was achieved with negligible higher PHA and glycogen requirements, 30% lower overall oxygen consumption and with the simultaneous removal of nitrate, reducing up to an estimate of 70% of carbon requirements for simultaneous N and P removal. Varying fractions of denitrifying polyphosphate accumulating organisms (DPAOs), from an average of 25% to 84%, were found in different plants. No correlation was found between the DPAO fractions and EBPR configuration, season, or the concentration of any of the microbial groups measured via quantitative fluorescence in situ hybridisation. These included Type I and Type II Ca. Accumulibacter and glycogen accumulating organisms, suggesting that chemical batch tests are the best methodology for quantifying the potential of anoxic P removal in full-scale wastewater treatment plants

Early life vitamin D depletion alters the postnatal response to skeletal loading in growing and mature bone

There is increasing evidence of persistent effects of early life vitamin D exposure on later skeletal health; linking low levels in early life to smaller bone size in childhood as well as increased fracture risk later in adulthood, independently of later vitamin D status. A major determinant of bone mass acquisition across all ages is mechanical loading. We tested the hypothesis in an animal model system that early life vitamin D depletion results in abrogation of the response to mechanical loading, with consequent reduction in bone size, mass and strength during both childhood and adulthood. A murine model was created in which pregnant dams were either vitamin D deficient or replete, and their offspring moved to a vitamin D replete diet at weaning. Tibias of the offspring were mechanically loaded and bone structure, extrinsic strength and growth measured both during growth and after skeletal maturity. Offspring of vitamin D deplete mice demonstrated lower bone mass in the non loaded limb and reduced bone mass accrual in response to loading in both the growing skeleton and after skeletal maturity. Early life vitamin D depletion led to reduced bone strength and altered bone biomechanical properties. These findings suggest early life vitamin D status may, in part, determine the propensity to osteoporosis and fracture that blights later life in many individuals

MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the ‘MiDAS 4’ database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus.Fil: Dueholm, Morten Kam Dahl. Aalborg University; DinamarcaFil: Nierychlo, Marta. Aalborg University; DinamarcaFil: Andersen, Kasper Skytte. Aalborg University; DinamarcaFil: Rudkjøbing, Vibeke. Aalborg University; DinamarcaFil: Knutsson, Simon. Aalborg University; DinamarcaFil: Arriaga, Sonia. Instituto Potosino de Investigación Científica y Tecnológica; MéxicoFil: Bakke, Rune. University College of Southeast Norway; NoruegaFil: Boon, Nico. University of Ghent; BélgicaFil: Bux, Faizal. Durban University of Technology; SudáfricaFil: Christensson, Magnus. Veolia Water Technologies Ab; SueciaFil: Chua, Adeline Seak May. University Malaya; MalasiaFil: Curtis, Thomas P.. University of Newcastle; Reino UnidoFil: Cytryn, Eddie. Agricultural Research Organization Of Israel; IsraelFil: Erijman, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; ArgentinaFil: Etchebehere, Claudia. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Fatta Kassinos, Despo. University of Cyprus; ChipreFil: Frigon, Dominic. McGill University; CanadáFil: Garcia Chaves, Maria Carolina. Universidad de Antioquia; ColombiaFil: Gu, April Z.. Cornell University; Estados UnidosFil: Horn, Harald. Karlsruher Institut Für Technologie; AlemaniaFil: Jenkins, David. David Jenkins & Associates Inc; Estados UnidosFil: Kreuzinger, Norbert. Tu Wien; AustriaFil: Kumari, Sheena. Durban University of Technology; SudáfricaFil: Lanham, Ana. University of Bath; Reino UnidoFil: Law, Yingyu. Singapore Centre For Environmental Life Sciences Engineering; SingapurFil: Leiknes, TorOve. King Abdullah University of Science and Technology; Arabia SauditaFil: Morgenroth, Eberhard. Eth Zürich; SuizaFil: Muszyński, Adam. Politechnika Warszawska; PoloniaFil: Petrovski, Steve. La Trobe University; AustraliaFil: Pijuan, Maite. Catalan Institute For Water Research; EspañaFil: Pillai, Suraj Babu. Va Tech Wabag Ltd; IndiaFil: Reis, Maria A. M.. Universidade Nova de Lisboa; PortugalFil: Rong, Qi. Chinese Academy of Sciences; ChinaFil: Rossetti, Simona. Istituto Di Ricerca Sulle Acque (irsa) ; Consiglio Nazionale Delle Ricerche;Fil: Seviour, Robert. La Trobe University; AustraliaFil: Tooker, Nick. University of Massachussets; Estados UnidosFil: Vainio, Pirjo. Espoo R&D Center; FinlandiaFil: van Loosdrecht, Mark. Delft University of Technology; Países BajosFil: Vikraman, R.. VA Tech Wabag, Philippines Inc; FilipinasFil: Wanner, Jiří. University of Chemistry And Technology; República ChecaFil: Weissbrodt, David. Delft University of Technology; Países BajosFil: Wen, Xianghua. Tsinghua University; ChinaFil: Zhang, Tong. The University of Hong Kong; Hong KongFil: Nielsen, Per H.. Aalborg University; DinamarcaFil: Albertsen, Mads. Aalborg University; DinamarcaFil: Nielsen, Per Halkjær. Aalborg University; Dinamarc