ABSTRACT BOOK BIOLOGICAL TREATMENT OF SOLID WASTE. ETeCoS SUMMER SCHOOL 2014 JUNE 30 - JULY 4, CASSINO & GAETA, ITALY

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1 3 ETeCoS SUMMER SCHOOL 2014 BIOLOGICAL TREATMENT OF SOLID WASTE JUNE 30 - JULY 4, CASSINO & GAETA, ITALY ABSTRACT BOOK UNIVERSITY OF CASSINO AND SOUTHERN LAZIO Edited by: Francesco Di Capua, Bijit kumar Banik, Giovanni Esposito (UNICLAM, Cassino, Italy)

2 ETeCoS3 Summer School 2014 Biological Treatment of Solid Waste June 30 July 4, Cassino & Gaeta, Italy ABSTRACT BOOK Edited by: Francesco Di Capua, Bijit Kumar Banik, Giovanni Esposito Cassino, June 2014

3 Organizing Committee: Giovanni Esposito University of Cassino and Southern Lazio (Italy) Bijit Kumar Banik University of Cassino and Southern Lazio (Italy) Francesco Di Capua - University of Cassino and Southern Lazio (Italy) Giovanni de Marinis University of Cassino and Southern Lazio (Italy) Stefano Papirio University of Cassino and Southern Lazio (Italy) Giuseppe d'antonio University of Naples Federico II (Italy) Francesco Pirozzi - University of Naples Federico II (Italy) Massimiliano Fabbricino - University of Naples Federico II (Italy) Piet Lens UNESCO IHE (The Netherlands) Eric van Hullebusch University Paris Est (France) Antonio Panico PEGASO Telematic University (Italy) Luigi Frunzo - University of Naples Federico II (Italy) 2

4 Contents Organizing Committee...2 Contents...3 WASTEWATER TREATMENT...5 Anaerobic Oxidation of Methane and Sulphate Reduction by Sediments from Gulf of Cadiz and Grevelingen, North Sea (S. Bhattarai)... 6 Anaerobic Oxidation of Methane using Different Sulphur Compounds as Electron Acceptors (C. Cassarini)... 8 Bio-electro-Fenton: integrated electrochemical-biological process for removal of refractory organic pollutants from wastewater (A. Ganzenko) Biogenic Sulfide Production and Metal Recovery from Electronic Wastes Using Hydrometallurgical Methods (S. Janyasuthiwong) Calibrating Mixed-species Biofilm Models: Application to Wastewater Treatment (M.R. Mattei) 14 Characterization of EPS of Anammox biomass enriched from conventional activated sludge (Z. Ding) Coupling of Membrane Nanofiltration and Electrochemical Advanced Oxidation Processes for Removal of Pharmaceutical Residues from Wastewater (S.O. Ganiyu) Nitrogen Removal in Low ph and Heavy Metal Contaminated Wastewater (F. Di Capua) Optimization of sulphate reduction (J. Cassidy) Sewer Systems Management and Protection (B.K. Banik) Slow Release Electron Donors for Sulphate Removal from Wastewater in an Inverse Fluidized Bed Reactor (L.C. Reyes-Alvarado) Studies on the Fate of Pharmaceuticals in Aqueous Media: Electrochemical degradation and Biotransformation (H. Olvera-Vargas) GEOMICROBIOLOGY AND METALLURGY...28 Biogenic Selenium Nanoparticles: Production, Characterization and Applications (R. Jain) Evidence of Zn Isotopes Fractionation during Bioweathering of Metallurgical Slags (N.H. Yin). 30 Filamentous growth of neutrophilic bacterium Sphaerotilus natans and its role in the scavenging of inorganic pollutants (M. Seder-Colomina) Impact of electrocoagulation on biogenic colloidal selenium (L.C. Staicu) Leaching and Recovery of Molybdenum, Nickel and Cobalt from the Mineral Sludge Generated at a Metal Recycling Plant (M. Vemic) Metal Recovery from Electronic Waste (A. Işıldar) Metallurgical Sludges: from Mineralogical Characterization to Heavy Metals Bioleaching and Recovery (M. Sethurajan) Microbial synthesis of metal selenide nanoparticles using selenium oxyanion reducing bacteria... (J. Mal)

5 Mineralogy and environmental stability of copper pyrometallurgical slags (A. Potysz) The Role of Extracellular Polymeric Substances (EPS) from Biofilm in Heavy Metals Sorption (F. Cao) Use of Phanerochaete chrysosporium as a selenium-reducing organism for: the removal of selenite from water, the production of elemental selenium nanoparticles and its potential application as a new adsorbent for the removal of heavy metals (E.J. Espinosa-Ortiz) SOIL TREATMENT...48 Citric acid and TWEEN 80-assisted phytoremediation of multi-contaminated soils vegetated with alfalfa (A.C. Agnello) Emerging contaminants from water discharged in soil: effects and treatment strategies (L. Pontoni) HMs removal by soil washing: efficiency and economic optimization/engineering configuration (A. Ferraro) Landfarming of PAHs Contaminated Soils Combined with Composting of Organic Waste (B. Lukić) Treatment of soil washing / soil flushing solutions by electrochemical advanced oxidation processes (EAOPs) and integrated treatments (C. Trellu) SOLID WASTE TREATMENT...57 Biohydrogen production from dark and photo fermentation processes (A. Ghimire) Combination of Thermal Pretreatment and Biofilm Technology to Enhance Anaerobic digestion (M. Yeshanew) Dry Discontinuous Anaerobic Percolation Digestion DDAPD (S. Riggio) Fermentative bio-hydrogen production from complex organic waste by suspended and attached growth hyperthermophilic bacteria Thermotoga neapolitana (N. Pradhan) Pretreatment methods to enhance anaerobic digestion of food waste (J. Ariunbaatar) ETeCoS3 PhD STUDENT PROFILES...68 FIRST COHORT...69 SECOND COHORT...71 THIRD COHORT...83 FOURTH COHORT

6 WASTEWATER TREATMENT

7 Anaerobic Oxidation of Methane and Sulphate Reduction by Sediments from Gulf of Cadiz and Grevelingen, North Sea S. Bhattarai*, Z. Naangmenyele*, E.R. Rene*, G. Gonzalez-Gil* and P.N.L. Lens* * Pollution Prevention and Resource Recovery Chair Group, Department of Environmental Engineering and Water Technology, UNESCO-IHE Institute for Water Education, 2601 DA Delft, the Netherlands Keywords: methane; anaerobic methane oxidation; sulphate reduction; ANME; Membrane bioreactor Anaerobic methane oxidation (AOM) coupled to sulphate reduction (SR) is an important biochemical process performed by consortia of anaerobic methanotrophic archaea (ANME) and sulphate reducing bacteria (SRB). Previous research has identified that sulphate reducing AOM is a well-established phenomenon occurring in deep marine environments; nevertheless, till date the mechanism has not been fully understood, as the vast majority of deep sediments microbes and their roles are still unknown. In-depth knowledge of this process is still hampered by insufficient amounts of highly enriched cultures. Besides AOM relevance in marine environments, another aspect of AOM research is to use methane as an electron donor for sulphate removal from wastewater streams. High sulphate concentration in industrial wastewater, especially from paper mills, and refineries, is one of the challenges of waste water treatment industry. The treatment of these wastewaters demands for electron donors such as ethanol and hydrogen which eventually increase the cost of the treatment system. One of the biotechnological applications of this research is to use methane produced by anaerobic digestion in the system and utilize it for sulphate removal. This research aims to enrich the microorganisms mediating AOM and to investigate the performances of microbial community in batch and continuous systems. The biomass was obtained from (i) deep sea Gulf of Cadiz, Ginsburg mud volcano, (~900 m water depth, here referred to as GC) which is a known site for AOM, and (ii) estuarine Lake Grevelingen, North Sea (45 m water depth, here referred to as GV). In order to enrich and investigate the activity of ANME and SRB consortia, a series of experiments were performed as per the scheme illustrated in Figure1. A suspended biomass bioreactor (1.2 L) with external membrane (MBR) is presently in operation for more than 200 d (Figure1, Figure 2). The MBR is operated in sequencing batch mode for liquid and continuous supply of CH4. For the first 82 d, the MBR was operated in batch mode. During this period, the concentration of dissolved sulphide reached about 1 mm. This concentration appears to be toxic since sulphide production halted. Thus, the reactor operation was switched to continuous mode for 30 d while the sulphide production recovered and its concentration in the liquid reached about 0.8 mm in 180 d. The increment in sulphide concentration may indicate the activity of ANME, which are expected to be in enriched in the bioreactor. The microbial community of the different phases of reactor operation will be soon quantified by Q-PCR of functional genes related to SRB and AOM metabolism. Moreover, a 0.4 L packed bed trickling filter bioreactor (PBR) packed with polyurethane foam, and fed with CH4 and sulphate, is being continuously operated for 150 d (Figure 1). The reactor start-up phase took about 100 d of operation, and since then, sulphide production has been increasing reaching up to 0.9 mm in 160 d of operation. It is expected that AMNE will be also enriched in this PBR system. The results from the batch experiments with incubation of both GC and GV sediments with SO42- and CH4 showed slight increase of sulphide production and sulphate removal compared to biotic control ( incubation without any carbon source) and abiotic control ( incubation without any biomass and incubation with killed biomass), which may be attributed to the activity of ANME. After 120 d of incubation, the sulphide production was ~1mM and 0.6mM of carbon dioxide production in case of GC sediment; while with GV sediment ~1.8 mm of sulphide was produced. Furthermore, batch experiments for sulphate reduction using different e-donors (acetate, lactate and ethanol) than CH4 were performed using GC and GV sediments during 45 and 35 days, respectively. using. Sulphate reduction was observed in the presence of all the selected electron donors in both GC and GV sediment, whereas the control incubations had almost negligible amount of sulphide production. 6

8 Amongst the different electron donors, ethanol recorded the highest cumulative sulphide production, 3.92 mm, compared to acetate (1.6 mm) and lactate (1.21 mm), respectively in the case of GC sediment. In the case of GV sediment, the cumulative sulphide concentration reached up to about 5 mm with all three electron donors. It appeared that the microbial community of GV sediment is more sulphide tolerant than GC. In both sediments, ethanol showed higher sulphate reduction rate (volumetric rate: 0.35 mm SO42- L-1 d-1 in GC and 0.75 mm SO42- L-1 d-1 in GV). Ethanol appeared to be the preferable e-donor for the microbial community. To further characterize the GV biomass with respect to its tolerance to sulphide, the batch experiment with ethanol was spiked with 5 mm of ethanol and ~ 11 mm of sulphide production was observed which is almost equal to the amount of sulphate fed to the experiment. Microbial community analysis performed by fluorescence in situ hybridization (FISH) showed the presence of DBB cells, which are sulphate reducers commonly associated with ANME, and some archaeal cells which could be ANME cells. This suggests that this sediment has a potential for AOM enrichment. In summary, AOM activity was observed in both sediments. The membrane bioreactor showed interesting results in microbial activity and further microbial community quantification and characterization will provide detailed insight in AOM in continuous systems. Based on sulphate reduction with methane and enrichment of DBB, this research could provide the novel basis of AOM activity by GV sediment. Figure 1. Schematic of experimental design for AOM study Figure 2. Configuration of suspended biomass bioreactor with external membrane (MBR) 7

9 Anaerobic Oxidation of Methane using Different Sulphur Compounds as Electron Acceptors Chiara Cassarini*, Eldon R. Rene*, Graciela Gonzalez-Gil*, Piet N. L. Lens* *Pollution Prevention and Resource Recovery Chair Group, Department of Environmental Engineering and Water Technology, UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands; Keywords: Anaerobic methane oxidation; Sulphate reduction; Methanotrophic archea; Sulphate reducing bacteria; Waste water treatment Anaerobic methane oxidation (AOM) is a known process occurring in marine sediments, lake sediments and anaerobic reservoirs. Methane is a potent greenhouse gas and AOM is one of the major processes controlling its emission from the ocean (Conrad, 2009). AOM is mainly coupled to the reduction of sulphate as terminal electron acceptor and mediated by consortia of anaerobic methanotrophs (ANME) and sulphate reducing bacteria (SRB) (Knittel et al., 2009). Despite the known occurrence of AOM coupled to sulphate reduction (SR), the mechanism has not yet been fully understood. However, AOM investigation has another research direction; the desulphurization of industrial wastewater by using methane as the sole electron donor. Few researches have attempted to study AOM in bioreactors in order to achieve considerable higher AOM rates than those found in natural environments (maximum 10 µm cm-1 day-1 in Black Sea microbial reefs), but the slow growing nature of ANME remains as a major challenge for practical applications (Meulepas et al. 2009; Zhang et al. 2010). This research focuses on the development of a bioprocess for AOM using alternative sulphur compounds as electron acceptors. A biotrickling filter (BTF) will be used as bioreactor (Figure 1.1), where the biomass attaches onto a suitable packing material within the BTF. This facilitates the growth of slow growing microorganisms, by having less perturbation and more contact with the substrates (methane and sulphur compounds). The BTF column will be made of glass, with 12 cm of inner diameter and a packing height of 50 cm, and it will accommodate adequate sampling ports for biomass and liquid sample collection. The column is provided with enclosures made of glass, having ports for liquid and air supply and two metal perforated support material with 2 mm openings. The column will be packed with polyurethane foam cut in cubes of 2 cm3; the BTF is operated in counter-current mode. The gas is pumped through the column from bottom to top and the gas inflow will be controlled by a mass flow controller. The medium is trickled through a distributor to the bed from top to bottom. The trickled medium flows into a stirred tank, which is recirculated to the top using a peristaltic pump. In the recirculating liquid-tank, the ph and the temperature are controlled and adjusted. Different marine sediments, where natural AOM is occurring, will be used as inoculum for the BTF to facilitate microbial growth. Other oxidized forms of sulphur can be more energetically favorable than sulphate in the AOM process (Milucka et al., 2012). Therefore, the effect on AOM rate in the presence of different sulphur compounds, such as sulphate, elemental sulphur and thiosulphate, as electron acceptors, will be investigated in this research. 8

10 * Figure 1.1. Biotrickling filter configuration. Screening tests were performed in small sized fixed bed reactors (0.4 L) in order to select the electron acceptor for methane oxidation. The different electron acceptors that were screened are sulphate, elemental sulphur and thiosulphate, respectively. In the different reactors, sulphide, sulphate, thiosulphate, methane and carbon dioxide were monitored to select the preferred sulphur compound. As a preliminary conclusion, thiosulphate appears to be the best electron acceptor yielding, after ~20 days, ~7 times more sulphide than the test with sulphate, 4 mm and 0.6 mm, respectively. The selected electron acceptor will then be tested in the 5 L BTF reactor (Figure 1.1), by varying different process parameters, for the enrichment of the microbial community mediating AOM. The microbial consortia in the enriched biomass will be characterized, together with the investigation of the metabolic activity of the microorganisms involved using fluorescence in situ hybridization-nanometrescale secondary ion mass spectrometry (FISH-NanoSIMS) technique, stable isotope probing (SIP), lipid biomarkers analysis and the microbial communities will be further investigated using sequence analysis and bioinformatics. A mathematical model capable of simulating AOM-SR in the BTF will be developed and validated with the obtained experimental data. Moreover, sensitivity analysis and cost-benefit analysis will also be preformed to investigate the bioreactors performance and the possible application of this bioprocess. References Conrad, R. (2009). The Global Methane Cycle: Recent Advances in Understanding the Microbial Processes Involved. Environ. Microbiol. Rep., 1, Knittel, K.; Boetius A. (2009). Anaerobic Oxidation of Methane: Progress with an Unknown Process. Annu. Rev. Microbiol., 63, Meulepas, R. J. W.; Jagersma, C. G.; Gieteling, J.; Buisman, C. J. N.; Stams, A. J. M.; Lens, P. N. L. (2009). Enrichment of Anaerobic Methanotrophs in Sulfate-Reducing Membrane Bioreactors. Biotechnol. Bioeng., 104, Milucka, J.; Ferdelman, T. G.; Polerecky, L.; Franzke, D.; Wegener, G.; Schmid, M.; Lieberwirth, I.; Wagner, M.; Widdel, F.; Kuypers, M. M. M. (2012). Zero-Valent Sulphur is a Key Intermediate in Marine Methane Oxidation. Nature, 491, Zhang, Y.; Henriet, J.-P.; Bursens, J.; Boon, N. (2010). Stimulation of In Vitro Anaerobic Oxidation of Methane Rate in a Continuous High-Pressure Bioreactor. Bioresour. Technol., 101,

11 Bio-electro-Fenton: integrated electrochemical-biological process for removal of refractory organic pollutants from wastewater A. Ganzenko*, D. Huguenot*, N. Oturan*, E. D. van Hullebusch*, G. Esposito**, M.A. Oturan* * Université Paris-Est, Laboratoire Géomatériaux et Environnement, EA 4508, UPEMLV Marne-la-Vallée, France ** University of Cassino and Southern Lazio, Department of Mechanics, Structures and Environmental Engineering, Via Di Biasio, Cassino (FR), Italy Keywords: advanced oxidation, electro-fenton, biological, sequencing batch reactor, wastewater, pharmaceuticals Context Clean water is a basic need of humanity and as pollution becomes one of the biggest environmental challenges of the XXI century, water pollution becomes an alarming hardship. The most persistent and hazardous pollutants are coming from industrial activity and agriculture, therefore effective treatment of industrial wastewater is a well-recognized problem that requires an immediate action. Some of the newly emerging pollutants that have raised a great deal of public concern are pharmaceuticals. On one side, these chemicals are a basis of modern medicine, they sustain human life and health, but on the other side their uncontrolled release into environment has numerous negative consequences. One of the main ways of discharge is hospital effluent (Verlicchi et al. 2010). It has a really diverse and complex composition: different classes of pharmaceuticals, which are excreted by human body in almost unchanged form, and their metabolites, diagnostic agents, disinfectants, musks, corrosion inhibitors and other hardly degradable pollutants (Kovalova et al. 2012). Hospital effluents are normally discharged to the common sewage system and undergo conventional treatment at municipal wastewater treatment facilities. However, most of the pharmaceutically active compounds are not degraded at all, therefore they end up in the receiving natural waters (Heberer 2002). Despite tiny concentrations, these pollutants affect the ecosystem balance, as, for example, hormones from oral contraceptives shift gender balance in fish and cease their reproduction (McAvoy 2008). Presence of antibiotics in small concentrations, which are not high enough to destroy bacteria, cause habituation and trigger antibiotic resistance in microorganisms (Korzeniewska et al. 2013). In the light of the above, hospital effluent requires additional pretreatment before discharge to municipal sewage or a separate treatment system in order to provide sufficient and efficient degradation of pharmaceuticals. A group of physicochemical treatments, such as advanced o/xidation processes (AOP) have raised an elevated interest due to their ability to degrade hazardous substances on the contrary to other methods, which mainly only transfer pollution from wastewater to sludge, membrane filter or adsorbent. Moreover AOPs as pre-treatment can serve for elimination of pathogenic bacteria present in rather high amount in hospital effluent. The main drawback of AOP, which holds back its large-scale implementation, is their relatively high installation and operational costs. An interesting solution to high costs is a combination of AOP with biological treatment. Handling refractory organics with AOP often leads to formation of intermediate compounds, which are no longer toxic to microflora and are biodegradable. Therefore their further degradation with cost-intensive AOP is not economically viable. Nevertheless, a consecutive biological posttreatment represents a sound solution due to its low expenses and wellstudied technological process. Among a great variety of different AO processes, there is a perspective novel technology of electrochemical methods, in particular electro-fenton, which has shown very promising results for degradation and mineralization of refractory organic pollutants in relatively short treatment time. This method is simple in operation, require minimum of reagents and is environment-friendly. It also shows good removal results for pharmaceuticals (Ambuludi et al. 2013, Sires et al. 2008, Sirés et al. 2007) The type of biological process investigated in this study is aerobic treatment in sequencing batch reactor (SBR). All the stages of such aerobic treatment are taking place in one reactor; therefore using SBR reduces capital costs, necessary space and accordingly has a minimal footprint. Goal 10

12 The goal of this study is to get a deep insight into combined electro-fenton/biological system and to optimize the process for treatment of heavily polluted waters of hospitals. Besides an apparent value of this study for environment and human health, it is worth emphasizing novelty in general of studying such treatment method in detail and specifically for removal of pharmaceuticals. This work foresees promising results and is expected to trigger more research in this area. Methodology Research methodology comprises three main stages. First stage includes experimental work with electro-fenton on a synthetic solution of 15 most representative pharmaceuticals present in hospital wastewater. The degradation, mineralization and reaction kinetics are followed for better understanding of the treatment process. Secondly, electro-fenton is used as pre-treatment of real hospital effluent. The third phase is connected to operation of sequencing batch reactor feeded with already pre-treated real wastewater. Two operation modes batch and continuous are investigated. All data from experimental work will be summarized and analyzed during the final part of the project, which is based on process modeling. References Ambuludi SL, Panizza M, Oturan N, Ozcan A, Oturan MA Kinetic behavior of anti-inflammatory drug ibuprofen in aqueous medium during its degradation by electrochemical advanced oxidation. Environmental Science and Pollution Research 20: Heberer T Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data. Toxicology Letters 131: Korzeniewska E, Korzeniewska A, Harnisz M Antibiotic resistant Escherichia coli in hospital and municipal sewage and their emission to the environment. Ecotoxicol Environ Saf 91: Kovalova L, Siegrist H, Singer H, Wittmer A, McArdell CS Hospital wastewater treatment by membrane bioreactor: performance and efficiency for organic micropollutant elimination. Environ Sci Technol 46: McAvoy K Occurrence of Estrogen in Wastewater Treatment Plant and Waste Disposal Site Water Samples. Clear Waters 38. Sires I, Guivarch E, Oturan N, Oturan MA Efficient removal of triphenylmethane dyes from aqueous medium by in situ electrogenerated Fenton's reagent at carbon-felt cathode. Chemosphere 72: Sirés I, Oturan N, Oturan MA, Rodríguez RM, Garrido JA, Brillas E Electro-Fenton degradation of antimicrobials triclosan and triclocarban. Electrochimica Acta 52: Verlicchi P, Galletti A, Petrovic M, Barceló D Hospital effluents as a source of emerging pollutants. An overview of micropollutants and sustainable treatment options. Journal of Hydrology 389:

13 Biogenic Sulfide Production and Metal Recovery from Electronic Wastes Using Hydrometallurgical Methods S. Janyasuthiwonga, E. R. Renea, G. Espositob and P.N.L. Lensa a b Pollution Prevention and Resource Recovery Chair Group, Department of Environmental Engineering and Water Technology, UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands Department of Civil and Mechanical Engineering, University of Cassino and the Southern Lazio, Via Di Biasio, 43, Cassino (FR), Italy Keywords: sulfate reducing bacteria; sulfide precipitation; metal recovery; electronic wastes; The waste electrical and electronic equipment (WEEE) or commonly referred to as e-waste has begun to raise global concern and posed serious health problems. Not only have the bulky pieces caused solid waste management problems but the release of hazardous materials especially heavy metals in the environment accelerates the public awareness. On the other hand, metal resources depletion is another related issue due to improper resource management. The mining and electronics manufacturing industry have high demand of metals as raw material. The waste streams (wastewater, air emission and solid waste) generated from these industries usually contains high metal concentrations which can reflect the inefficiency of the technologies performance. As a sustainable resource management philosophy, the recovery of metals from waste stream is one of the chosen strategies. Sulfide precipitation is one of the famous methods which gained notice by many researchers and investors due to its characteristics of low solubility in a wide range of the ph, ease of use and fast reaction (Lewis, 2010). Hence, sulfidic technologies have been studied and modified for large applications especially with sulfate reducing bacteria (SRB) technology. Recently, there are many researchers moving toward the use of SRB bioreactors for metal contaminated wastewater treatment. In this study, the desktop computers PCBs were evaluated for their potential use as metal resource especially Cu with sulfide precipitation method. The batch tests were performed to identify the effects of WEEE particle size (Figure 1), temperature (5, 20, 30 and 60 C), liquid/solid ratio (10, 20 and 40) and contact time (16 h). The total concentration of Cu, Ni and Zn leached from microwave digestion with concentrated nitric acid, at a liquid/solid ratio of 20 were 1.88, 0.43 and 0.48 g/kg, respectively. The results from this study show that metal leaching and recovery from WEEEs could be used as a potentially clean and environmentally acceptable process to reduce the subsequent impacts of these heavy metals on the environment. Figure 1. Particle size used in the study: a) < 0.5 mm, b) mm, c) mm, and d) > 5.0 mm 12

14 References Lewis AE (2010) Review of Metal Sulphide Precipitation. Hydrometallurgy 104,

15 Calibrating Mixed-species Biofilm Models: Application to Wastewater Treatment MR Mattei*, B D Acunto**, G Esposito*, L Frunzo**, F Pirozzi*** *University of Cassino and Southern Lazio, Department of Civil and Mechanical Engineering, via Di Biasio 43, Cassino (FR) **University of Naples Federico II, Department of Mathematics and Applications, via Claudio 21, 80125, Napoli ***University of Naples Federico II, Department of Civil, Architectural and Environmental Engineering, via Claudio 21, 80125, Napoli Keywords: Biofilm; Multispecies; Bioreactors; Model; Calibration. Biofilm research had been neglected for a long time until microbiologists rediscovered these fascinating communities almost 40 years ago [1]. In the past decades, the number of studies performed on surface-associated microbes has increased considerably and today, we recognize that most, if not all, microbial species can form biofilms [2]. These sessile communities form anywhere there is a surface with a little moisture and some nutrients, and nearly always harbor a multitude of microbial species, which compete or coexist thanks to niche differentiation. Biofilm growth is a complex and dynamic process evolving in several steps and resulting from the balance of several physical, chemical and biological processes, all characterized by different time and space scales. Biofilms have been widely used to treat wastewater since the end of 19th century. The main advantage of using these aggregates derives from the interactions of several species (cometabolism) which are spatially close together but dominate different strata within the biofilm according to their metabolic activities and the local concentrations of dissolved substrates [3]. Indeed, the rate limitation of solute diffusion determines the formation of concentration gradients in and around the biofilm favoring the stratification of microbial species and reflecting on the design of biofilm reactors. In general, a fully developed biofilm can exhibit varying environmental and kinetic characteristics allowing the development of different microbial groups which contribute to the conversion of multiple organic and inorganic substrates. Biofilm structure and activities have been investigated using a broad variety of microscopic, physico-chemical and molecular biological techniques. Parallel to these investigations more and more complex mathematical models and simulations have been developed to describe the growth, structures and interactions of biofilms [4]. Calibration of a model to collected data at both macro and microscale, represents a key factor for the effective use of these prediction tools. The current work reflects this need and is aimed at highlighting the main critical components for biofilm reactor model calibration. References Battin, T.; Sloan, W.; Kjelleberg, S.; Daims, H.; Head, I.; Curtis, T.; Eberl, L. (2007). Microbial landscapes: new paths to biofilm research. Nat. Rev. Microbiol., 5, Kolter, R.; Greenberg, E. (2007). Microbial sciences: The superficial life of microbes. Nat., 441, Stewart, P.S.; Camper, A.K.; Handran, S.D.; Huang, C.-T.; Warnecke, M. (1997). Spatial distribution and coexistence of Klebsiella pneumonia and Pseudomonas aeruginosa in biofilms. Microb. Ecol., 33, Wimpenny, J.; Manz, W.; Szewzyk, U. (2000). Heterogeneity in biofilms. FEMS Microbiol. Rev., 24,

16 Characterization of EPS of Anammox biomass enriched from conventional activated sludge Z. Ding*; I. Bourven**; G. Guibeau**; A. Panico***; G, Esposito*; E, van Hullebusch**** * Department of Civil and Mechanical Engineering, University of Cassino and the Southern Lazio, via Di Biasio 43, Cassino (FR), Italy ** Groupement de Recherche Eau Sol Environnement, EA 4330, University of Limoges, 123 Avenue Albert THOMAS, Limoges, France *** Telematic University Pegaso, P.za Trieste e Trento, 48, Naples, Italy **** Université Paris-Est, Laboratoire Géomatériaux et Environnement, EA 4508, 5 bd Descartes, Marne la Vallée Cedex 2, France Keywords: ammonium; Anammox; AerAOB; salinity; EPS The use of nitrogen fertilizer and increased human induced nitrogen pollution has made the nitrogen removal an important element in the wastewater treatment process. The conventional nitrification/denitrification treatment has achieved the most widely full-scale application. However the discovery of Anaerobic Ammonium Oxidation (Anammox) provides a short-cut option of ammonia removal via the anaerobic oxidation of ammonium with nitrite as electron acceptor (Mulder et al., 1995). Ammonium is partially oxidized into nitrite by aerobic ammonium oxidation bacteria (aeraob) under oxygen limited condition. Then the present ammonium and nitrite compose the substrate for autotrophic Anammox bacteria and nitrogen gas is produced. The principle of partial nitritation (1) and Anammox (2) is described by the reactions below (Verstraete, 2007): NH O2 + 2HCO3- NO2- + 3H2O + 2CO2 (1) NH NO HCO3-1.02N H2O CH2O1.5N NO3- (2) Sequencing batch reactor (SBR) has been proven to be an effective approach to enrich Anammox biomass from wastewater activated sludge and anaerobic sludge (Strous et al., 1998). However, the enrichment period lasts from three months to one year according to literatures (Wang et al., 2011; Nutchanat et al. 2007). In the current research, two 4L SBRs are used to enrich the Anammox biomass while focus is on the investigation of the evolution of microbial community and change of extracellular polymer substance (EPS) composition. During the enrichment process, the influent nitrogen concentration, volumetric loading rate and sludge loading rate are strictly controlled according to the monitored reactor performance. The results obtained comply well with similar researches in literature (Wang et al., 2011; Nutchanat et al. 2007, Zhang et al., 2005). By the end of the 240 days enrichment period, about 55% total nitrogen removal was obtained with hydraulic retention time of 48 hours. Characterization of the enrichment was analysed through PCR-DGGE. EPS was extracted from sludge sampled on day 120 and day 240 by cation exchange resin (CER) method. Global protein, humic like substances, polysaccharide, uronic acid and DNA were quantified by photometric method. The amount of protein increased by six fold from day 120 to day 240. The amount of sugar and uronic acid has no significant change while amount of DNA increased by about 2/3. This proves that granulation did not happen during the enrichment. No humic like substance has been detected. The finger print of the protein is characterised by spectrofluorometer and size exclusion chromatography (HPLC-SEC). The results show that tyrosine like protein is the dominant species. The results will be better explained when PCR-DGGE results are available. References Mulder et al., Anaerobic ammonium oxidation discovered in a denitrifying fluidized-bed reactor. FEMS Microbiology Ecology, 1995, 16: Strous, M; Heijnen, J; Kuenen, J; Jetten, M.S.M. (1998) The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Applied Microbial Biotechnology, 50:

17 Verstraete, W. (2007) Course Notes for Biotechnology Process for Environmental Sanitation, University of Ghent, Belgium. Wang, T; Zhang, H; Gao, D; Yang, F; Yang, Shuai; Jiang, T; Zhang, G. (2011) Enrichment of Anammox bacteria in seed sludges from different wastewater treating process and start-up of Anammox process. Desalination, 271: Nutchanat Chamchoi, Suwanchai Nitisoravut. (2007) Anammox enrichment from different conventional sludges. Chemosphere, 66: Zhang, L.; Xiao, W. (2005) Cultivation of Anaerobic Ammonium Oxidation Bacteria and Their Performance in an Up-flow Anaerobic Sludge Bed Reactor. East China University of Science and Technology, 31,

18 Coupling of Membrane Nanofiltration and Electrochemical Advanced Oxidation Processes for Removal of Pharmaceutical Residues from Wastewater S.O. Ganiyu*, E. van Hullebusch*, N. Oturan*, M. Cretin**, G. Esposito***, M.A. Oturan* *Université Paris-Est Marne-la-Vallée, Laboratoire Géomatériaux et Environnement (LGE) 5 Boulevard Descartes, Marne-la-Vallée Cedex 2, France **IEM (Institut Européen des Membranes), UMR 5635 (CNRS-ENSCM-UM2), Université de Montpellier 2, Place E. Bataillon, F Montpellier, Cedex 5, France *** Department of Civil and Mechanical Engineering, University of Cassino and the Southern Lazio, Via Di Biasio 43, Cassino (FR), Italy Keywords: Coupling processes, membrane nanofiltration, electrochemical advanced oxidation processes, pharmaceutical residues, wastewater The PhD Thesis will focus on the development of a novel hybrid system that combines physical separation (ultrafiltration, UF; nanofiltration, NF) and electrochemical advanced oxidation processes (EAOPs) as a single unit for simultaneous separation and destruction of organic pollutants in aqueous medium. This hybrid system is unique because it combines the advantages of both UF/NF membrane technology and EAOPs (Zaky and Chaplin, 2013) as well as overcomes the challenges of individual treatment methods such as membrane fouling and treatment of huge concentrate generated in UF/NF (Van der Bruggen et al., 2008); formation of toxic intermediates and non-viability of EAOPs for treatment of low pollutants concentration wastewater (Synder et al, 2007). Nowadays pharmaceutical (PhAC) residues and hospital wastewaters are scientific object of researches due to growing concern on the persistence of PhACs, relatively large concentrations in aquatic environment as well as their impact on health and safety of both terrestrial and aquatic habitants. Effluents of wastewater treatment plants (WWTPs) remain the major source of these pollutants because of ineffectiveness of conventional treatment techniques employed to completely remove the PhACs accumulated in its feed stream via human urine and faeces and direct discharge of hospital wastewater (Daughton and Ternes, 1999; Heberer, 2002). The objective of this research is to develop an effective and efficient hybrid technique for treatment of PhACs residues and hospital wastewater. The first phase of the study focuses on anodic oxidation (AO) and electro-fenton (EF) degradation of synthetic solution of selected PhACs using wide range of anode materials. Similar studies will be carried out in the second phase but with tubular porous conductive membranes (TPCM) made, for example, from graphite as electrode, while the last part of the study deals with the development of an hybrid system in which TPCM act as both electrode and membrane for EAOPs and ultra/nanofiltration unit respectively and a pilot plant for treatment of real hospital wastewater. This research work is expected to give a deeper understanding of process engineering and environmental compatibility of a combined membrane filtration and EAOPs treatment of PhACs wastewater. Preliminary studies on degradation of one of the model PhACs Paracetamol (PCM) by AO and EF processes using boron doped diamond (BDD) and platinum (Pt) electrodes show faster degradation of the PCM in Pt cell than in BDD cell for EF process whereas mineralization (TOC removal) was much better with BDD anode compared to Pt anode under similar experimental conditions. The faster degradation of the PCM in EF with Pt anode is attributed to large amount of the strong oxidant ( OH) generated in the bulk solution through Fenton's reaction (Brillas et al., 2009; Oturan and Aaron, 2014) whereas in anodic AO PCM is oxidized by heterogeneous BDD( OH) generated on the anode surface by oxidation of water. In this later case the oxidation reaction is limited to the electrode surface (Panizza and Cerisola, 2009). Mineralization rate of the PCM increases with applied current, being higher than 96% after 8 h of electrolysis at 300 ma or higher current for both AO and EF with BDD anode and less than 73% for EF with Pt anode. With BDD anode, oxalic, oxamic, malonic, and malic acid were main carboxylic acids detected during 17

19 mineralization at optimum conditions using EF process whereas only oxalic and oxamic acids were found in analogous AO studies. References Brillas E., Sirés I., Oturan M.A. (2009). Electro-Fenton process and related electrochemical technologies based on Fenton s reaction chemistry. Chem. Rev., 109, Daughton, C.G.; Ternes, T.A. (1999). Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Perspect. 107, Heberer, T. (2002). Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: A review of recent research data. Toxicol. Lett. 131, Oturan, M.A.; Aaron, J.J. (2014). Advanced oxidation processes in water/wastewater treatment: Principles and applications. A review. Crit. Rev. Environ. Sci. Technol., in press. Panizza M., Cerisola G. (2009). Direct and mediated anodic oxidation of organic pollutants. Chem. Rev., 109, Snyder, S.A.; Adham, S.; Redding, A.M.; Cannon, F.S.; DeCarolis, J.; Oppenheimer, J.; Wert, E.C.; Yoon, Y. (2007). Role of membranes and activated carbon in the removal of endocrine disruptors and pharmaceuticals. Desalination 202, Van der Bruggen, B.; Manttari, M.; Nystrom, M. (2008). Drawbacks of applying nanofiltration and how to avoid them: A review, Sep. Purif. Technol. 63, Zaky, A. M.; Chaplin, B. P. (2013). Porous substoichiometric TiO2 anodes as reactive electrochemical membranes for water treatment. Environ. Sci. Technol. 47,

20 Nitrogen Removal in Low ph and Heavy Metal Contaminated Wastewater F. Di Capua*, S. Papirio*,**, G. Esposito*, P.N. L. Lens***, J.A. Puhakka** * Department of Civil and Mechanical Engineering, University of Cassino and Southern Lazio, Cassino, Italy. ** Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FIN Tampere, Finland. ***UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands. Keywords: mining waters; nitrogen; autotrophic denitrification; chemolithotrophic denitrifiers; metal toxicity. Introduction - This doctoral research will focus on nitrogen removal from acid mine drainage (AMD) through chemolithotrophic denitrification. AMD is a strongly acidic solution, high in sulfate and rich in metals (cadmium, nickel, copper, lead, zinc, arsenic, etc.). The extraction of groundwater to prevent flooding of mines, leachate discharge from barren rock piles and discharge of wastewater from the ore processing into tailing ponds result in large amounts of mine effluents in the order of several m3/min or million m3/year per mine. AMD is highly toxic to most life forms and if uncontrolled it may runoff into streams or rivers or leach into groundwater. AMD is further enriched by elevated levels of cyanide and nitrogen compounds (ammonium, nitrate and nitrite), mainly originated from heap leaching and blasting operations. The discharge of nitrogen loaded mine waters from working and abandoned mines and mine spoils is larger during spring and after heavy rainfall events. If overdischarged, NH4+ and NO3- favour the production of algal blooms and contribute to the eutrophication of receiving waters. Biological nitrate removal seems to be the most effective in terms of removal efficiencies and operating costs. Since AMD is typically organic deficient, autotrophic denitrification represents the most attractive and inexpensive solution for nitrate removal. Objectives - The main objective of this doctoral research is to investigate the ability of various inorganic electron donors to develop autotrophic denitrification in mining waters. Anaerobic batch bioassays and continuous reactors will be used for biomass enrichment and for experiments on electron donors, inhibitory ph and heavy metals toxicity. The best type of reactor, reactor configuration and electron donor suitable to carry out autotrophic denitrification will be investigated. Materials and Methods - Cultures of chemolithotrophic denitrifiers will be obtained by different sources and enriched in both anaerobic batch bioassays and continuous reactors. Cultures will be enriched on nitrate in a basal mineral medium with 2 ml/l of a trace elements solution as described by Cardoso et al. (2006). Five inorganic electron donors will be tested in batch bioassays. Preliminary tests will be carried out in order to choose the best values of ph, nitrate and biomass concentrations. Batch bioassays will be performed at room temperature (22 C) using 250 ml serum bottles placed on a gyratory shaker. Microcosms will be prepared in triplicate and electron donors will be supplied in stoichiometric excess. A different mineral medium will be prepared according to previous studies (Di Capua, 2013; Zou et al., 2013a; Zou et al., 2013b). Initially, the medium and the bottles will be sterilized. Electron donors, nitrate and bicarbonate ( mg/l of HCO3-), as inorganic carbon source and ph buffer will be added in each bottle. Nitrate concentration will be chosen according to the preliminary tests. 10 ml of enrichment cultures will be added. Anaerobic conditions will be established in each bottle by flushing with nitrogen gas (N2) for 15 minutes. After flushing, serum bottles will be aseptically sealed with butyl-lined rubber septa and aluminum crimps. In addition, further bottles will be prepared for electron-donor free and non-biological controls, respectively. In hydrogenotrophic batch experiments, two different methods will be used for H2 supply. Pyrite and sulfur will be tested at different particle sizes, after autoclaving. Ferrous iron and meta-arsenite stock solutions will be sterilized with 0.2 µm membrane filters. 19

21 Nickel and cobalt impact on iron- and arsenite-oxidizing cultures will be investigated. Fe2+ and As[III] will be also investigated as inhibitors, at higher concentrations than those used in the previous tests. Reactor experiments will be carried out based on the results obtained from the batch bioassays. Both up-flow fluidized bed reactors (FBRs) and membrane bioreactors (MBRs) will be operated. Feed ph will be gradually decreased in order to test denitrification performance under acidic conditions. A synthetic solution composed by nutrients, nitrate and electron donor will be fed to the reactors. The effect of hydraulic retention time (HRT) will be investigated in both FBRs and MBRs. Elemental sulfur and pyrite will be tested in FBRs both as biofilm carriers and electron donors. Zero-valent iron will be used as hydrogen source in a double-chamber FBR in which anaerobic iron corrosion and hydrogenotrophic denitrification will take place in two separate volumes. A MBR will be operated to test arsenite as electron donor. Both hollow fiber and flat sheet membrane will be tested. References Cardoso, R.B.; Sierra-Alvarez, R.; Rowlette, P.; Flores, E.R.; Gómez, J.; Field, J.A. (2006). Sulfide oxidation under chemolithoautotrophic denitrifying conditions. Biotechnol Bioeng., 95(6), Chung, J.; Amin, K.; Kim, S.; Yoon, S; Kwan, K.; Bae, W. (2014). Autotrophic denitrification of nitrate and nitrite using thiosulfate as an electron donor. Water Res., 58, Di Capua, F. (2013). Nitrogen removal in low ph and heavy metal contaminated mine wastewaters. M.Sc. thesis, Università degli Studi di Napoli "Federico II", Napoli, Italy. Zou, G.; Papirio, S.; Ylinen, A.; Di Capua, F.; Lakaniemi, A.M.; Puhakka, J.A. (2013). Fluidized-bed denitrification for mine waters. Part II: effects of Ni and Co. Biodegradation, 25(3): Zou, G.; Ylinen, A.; Di Capua, F.; Papirio, S.; Lakaniemi, A.M.; Puhakka, J.A (2013). Impact of heavy metals on denitrification of simulated mining wastewaters. Advanced Materials Research, 825,

22 Optimization of sulphate reduction J Cassidy*, HJ Lubberding*, G Esposito**, PNL Lens* *Pollution Prevention and Control Core, UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands **Department of Mechanics, Structures and Environmental Engineering, University of Cassino, Via Di Biasio, 34, Cassino (FR), Italy Keywords: Anaerobic oxidation of methane; sulphate reduction; biotechnology; bioreactor; bioprocess control The biological sulphate reduction process is mediated by a group of microorganisms known as sulphate reducing bacteria (SRB). Biological anaerobic reduction of sulphate has been successfully applied for the treatment of sulphate contaminated wastewater from industries on a larger scale for many years as it offers the possibility of an efficient treatment with low operation costs using various organic and easily utilizable carbon sources. The main limiting factor when building a large scale biological treatment process is the electron donor cost. Thus, it is desirable to look for the optimization of sulphate reduction process. In this work two approaches are studied: the applicability of a cheap carbon source, i.e., methane, and to optimize the input of the electron donor by using process control. Concentration (mg/l) For the design of a control strategy that uses the organic loading rate (OLR) as control input, feast and famine behaviour conditions need to be applied to the bioreactor to excite the dynamics of the process. These conditions have been shown to induce the accumulation of storage compounds in sulphate reducing bacteria (SRB) (Hai et al., 2004; Cypionka, 1989). Delays in the response time and a high control gain can be considered the most critical factors affecting the application of a sulphide control strategy in bioreactors. The delays may be caused by the induction of different metabolic pathways in the anaerobic sludge including the accumulation of storage products (VillaGomez et al., 2013). In the present study, sulphate and polyhydroxybutyrate (PHB) are considered as the main storage compounds accumulated in the biomass. To investigate the impact of substrate accumulation, feed shock loads were induced to an inverse fluidized bed reactor (IFB) performing biological sulphate reduction (Figure 1). A first feed shock load showed that even when no COD/sulphate was added to the influent, sulphide was still being produced after 15 days of operation. It is hypothesized that accumulation of polyhydroxybutyrate (PHB) and sulphate were the Dynamic COD loading sources for the continuous production of biological sulphide. The production of sulphide ceased when the 300 concentration of PHB was decreased to zero. A 200 second shock load was induced, by adding only COD. 100 This resulted in production of sulphide leading to the conclusion that there was more accumulation of 0 sulphate than of carbon sources. Activity tests showed the occurrence of sulphate accumulation in Time (days) the microbial cells and the usage of PHB as an electron donor for sulphate reduction. A mathematical Figure Sulphide, COD and sulphate model is currently being developed to obtain further profiles in the effluent during dynamic COD insight in these processes and their impact on the loading. Sulphate, CODout, Sulphide, bioprocess control system. The full understanding of CODin this phenomenon may lead to less usage of external electron donor, leading to a decrease in costs and chemicals. Anaerobic oxidation of methane coupled to sulphate reduction (AOM-SR) plays an important role in controlling the earth climate and marine ecosystem. Another important aspect of AOMSR is its application for desulfurization of wastewater where methane can be used as a sole electron donor. The use of methane for SR would close its cycle of utilization, decrease the emission of one of the most important greenhouse gases and reduce the risk of excess carbon source in the treatment effluent. Researchers have not been able to firmly establish the reaction mechanism or to 21

23 fully understand the factors that control oxidation/reduction rates. The main difficulty lies in the low growth yield (0.05 g dry weight/carbon oxidized) and long doubling time (up to 7 months) from in vitro incubations (Meulepas et al, 2009; Nauhaus et al, 2007; Thauer et al, 2008). So far, two groups of microorganisms have been identified to mediate in cooperation in the AOM-SR process: SRB and anaerobic methanotrophic archaea (ANME) with three distinct clusters (Zhang et al., 2011). In order to understand how environmental factors may impact the rates of oxidation and reduction, a high pressure continuous reactor was incubated with an inoculum known to perform AOM-SR. The variations on the operational conditions showed an effect on the bioconversion processes carried out by the consortium. By varying the latter, different processes are hypothesized to be occurring such as, sulphate reduction to sulphide, sulphate reduction to thiosulphate, anaerobic oxidation of methane and methanogenesis (trace methane oxidation). A mathematical model is being developed which will give further support to accept or reject these hypotheses. Sulphide (mmol) Sulphate + 13CH4 (control) Acetate +Sulphate + 13CH4 Sulphide (mmol) Another approach to understanding AOM-SR is to identify which intermediate chemical species might play a role in the process. To assess the potential of diverse compounds as intermediates in the AOM-SR process, in vitro incubations of the same inoculum at high pressure with several co-substrates were performed. Results showed that acetate stimulates the process which has not been reported for any other ANME/SRB communities (Figure 1.2). In addition, acetate was formed in the control group probably resulting from the reduction of CO2. These results are supported by a recent integrated analysis of the environmental metatranscriptome and single aggregate genome on this enrichment which suggest the presence of an acetate metabolism (Wang et al., 2013). These results support the hypothesis that acetate may serve as an intermediate in the AOM-SR process, at least in some groups of ANME/SRB Time (days) 100 Figure 1.2. Sulphide production in the absence (top) or presence (bottom) of acetate. References Cypionka, H. (1989). Characterization of sulfate transport in Desulfovibrio desulfuricans. Arch. Microbiol., 15: Hai, T.; Lange, D.; Rabus, R.; Steinbüchel, A. (2004). Polyhudroxualkanoate (PHA) accumulation in sulfate-reducing bacteria and identification of a class III PHA Synthase (PhaEC) in Desulfococcus multivorans. Appl Environ Microbiol., 70: Meulepas, R.J.; Jagersma, C.G.; Gieteling, J.; Buisman, C.J.N.; Stams, A.J.M.; Lens, P.N.L. (2009). Enrichment of anaerobic methanotrophs in sulfate-reducing membrane bioreactors. Biotechnol Bioeng., 104: Nauhaus, K.; Albrecht, M.; Elvert, M.; Boetius, A.; Widdel, F. (2007). In vitro cell growth of marine archaeal-bacterial consortia during anaerobic oxidation of methane with sulfate. Environ Microbiol., 9: Thauer, R.K.; Shima, S. (2008). Methane as fuel for anaerobic microorganisms. Ann NY Acad Sci., 1125: Villa-Gomez D.K.; Cassidy J.; Keesman K.; Sampaio R.; Lens P.N.L. (2013). Sulfide response analysis for sulphide control using a ps electrode in sulphate reducing bioreactors. Water Res., 50: Wang, F.P.; Zhang, Y.; Chen, Y.; He, Y.; Qi, J.; Hinrichs, K.U.; Zhang, X.X.; Xiao, X.; Boon, N. (2013). Methanotrophic archaea possessing diverging methane-oxidizing and electron-transporting pathways. The ISME journal, Zhang, Y., Maignien, L., Zhao, X., Wang, F., Boon, N. (2011) Enrichment of a microbial community performing anaerobic oxidation of methane in a continuous high-pressure bioreactor. BMC Microbiol., 11: 22

24 Sewer Systems Management and Protection B.K. Banik*, C. Di Cristo**, A. Leopardi** *ETeCoS3 PhD Student, Dipartimento di Ingegneria Civile e Meccanica, Università degli Studi di Cassino e del Lazio Meridionale, Cassino 03043, Italy ****Dipartimento di Ingegneria Civile e Meccanica, Università degli Studi di Cassino e del Lazio Meridionale, Cassino 03043, Italy Keywords: Toolkit; Sewer system; SWMM5; Optimization problems; Pollution source Wastewater systems in urban areas can be different in typology and size, but they all include a collection system, which can be combined or sanitary sewers. Recently, the waste-water and storm-water management is evolving from a simple flood and sanitary control to a whole environmental protection function. In many countries, such as US and many members of EU, operators are required to obtain a permit for discharges in sewer systems from the regulatory authority. So, a very important aspect of the sewer system management policy is represented by the detection and elimination of accidental or deliberate intrusions. Pollutant analyses, usually performed at the entrance of water treatment plant, can determinate if a contaminant intrusion is in act (Irvine et al., 2011). In particular, in an urban drainage system, a contamination event is represented by the intrusion of a pollutant substance, different from the usual composition of the waste water of that network. The identification of the contamination source in UDS is of particular interest because the pollutant can generate problems to waste water treatment plant and/or to the final recipient water body. For this reason, in UDSs the identification of the intrusion point and of the inflow characteristics, in both cases of accidental or deliberate discharge, is of particular interest. To date, most of the researches on pollution source identification problems are primarily focused on the water distribution systems (WDS) due to high concern about the public health in case a contaminant is introduced into the system either deliberately or accidentally. Very rare efforts have been spent for studying the effect of a pollutant injection in an Urban Distribution System (UDS) with particular reference to pollution source identification problems. The main goal of this research, which is basically a numerical modelling-based study, is to furnish a methodology for identifying the inflow characteristics and the source point provided that a contamination event occurs in a sewer network. The proposed procedure of pollution source identification involves not only the rapid recognition of the injection locations but also the characterization of start time, duration and magnitude of contaminants to effectively control the spread of contamination as well as remediating the contaminated area. However, insufficient data and countless possible contamination scenarios can pose challenge to the characterization process in terms of both accuracy and efficiency. To formulate the PSI problem, a time-dependent objective function, which minimize the difference between the estimated concentration values and the currently available concentration sensor data, was defined for the present case study. The objective function was computed by using the USEPA's SWMM (Rossman, 2010), which is a dynamic rainfall-runoff simulation model that computes runoff quantity and quality from, primarily, urban areas, while Genetic Algorithm (GA) of the GAlib (Wall, 1996) was used as an optimizer. A schematic description of the PSI methodology is shown in Figure 1. The primary goal of such a problem is to quickly estimate the source characteristics that best explain the observed contamination data at hand. It was assumed that the injected contaminants are conservative (i. e. have zero decay) and that the monitoring stations are ideal (i.e. provide prompt detection at very low contaminant concentrations). 23

25 Figure 1. Schematic representation of the PSI methodology In order to integrate the SWMM simulator with the proposed automated PSI methodology an ad-hoc SWMM-TOOLKIT is necessary to establish a communication from an outside environment. In doing that some 22 additional functions have been created for retrieving information about network nodes and time patterns, as well as for setting new values during the extended period simulation from a C++ platform. An application of the SWMM-Toolkit for running the proposed PSI methodology is presented performing an example test considering the literature network Example 8 of SWMM manual (Rossman, 2010) in dry weather condition. Applying the PSI methodology the obtained values of the four PSI variables are summarized in Table 1 as Case 1, showing that the exact solution is obtained. This result was expected, since it represents just a preliminary test for verifying the capability of the Toolkit in providing a useful instrument for performing the proposed PSI methodology or in general for running network analyses from other applications. Table 1. PSI results. Solution Node Concentration Starting Time Duration Exact Solution J1 (mg/l) 2.0 (hr) 8:00 (hr) 3 Case 1 J :00 3 Further investigations for testing the PSI methodology in more complex situations are necessary, such in wet weather conditions or considering measurement errors. Moreover, additional tests on a real case study as well as a robust uncertainty analysis on different parameters, like hydraulics and sensor errors, are also planned in future studies. References Irvine, K., Rossi, M. C., Vermette, S., Bakert, J., & Kleinfelder, K. (2011). Illicit discharge detection and elimination: Low cost options for source identification and track down in storm water systems. Urban Water Journal, 8(6), Rossman L.A., (2010). Storm water management model user's manual. Version 5.0. National Risk Management Research Laboratory, Office of Research and Development: US Environmental Protection Agency. Wall M., (1996). GAlib: A C++ library of genetic algorithm components. Mechanical Engineering Department: Massachusetts Institute of Technology. 24

26 Slow Release Electron Donors for Sulphate Removal from Wastewater in an Inverse Fluidized Bed Reactor L.C. Reyes-Alvarado*, E.R. Rene and P.N.L. Lens *Pollution Prevention and Resource Recovery Chair Group, Department of Environmental Engineering and Water Technology, UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands *Corresponding authors Keywords: Inverse fluidized bed reactor; sulphate reducing bacteria; carriers, polymers; slow release electron donors Acid mine drainage is an industrial wastewater and has very low ph, high oxidative potential, sulphate concentrations between 424 and 20,800 mg/l, very low COD content and in most cases has high concentrations of heavy metals. The recovery of sulphur under controlled condition is a necessity, but an electron donor is always needed. Now a day, metal recovery can also be done by simultaneous precipitation with sulphide. Nevertheless, process intensification and optimization techniques must be applied to find optimal electron donors, a suitable reactor configuration and better crystal formation which might be represent a cost benefit. In this research, the performance of an inverse fluidized bed reactor (IFB) containing polyethylene beads was evaluated for ~160 d under varying organic loading rates (OLRs) and at different COD/SO42- ratios. The IFB was acclimated at an hydraulic loading rate (HRT) of 24 h HRT (Table 1), with an initial volatile suspended solids (VSS) concentration of 2200 mg/l. As shown in Figure 1, COD was removed efficiently during the first period of operation. Sulphate removal did not take place in the first stage of operation due to the longer times required for biomass acclimation. It has been reported in the literature that carbon could be used for anabolism or cell maintenance and other dominant parallel process, as might be methane production. The last process, i.e., methane production, was possible due to the fact that the seeding microorganism was anaerobic sludge from a municipal wastewater treatment plant that was exposed to sulphidogenic conditions within the IFB at a stoichiometric COD/SO42- ratio of During the subsequent stages, when this ratio was increased to 1.3, sulphate reduction increased and high SO42- removal was maintained during the third period of operation. However, during period 4, when the HRT was reduced to 12 h, sulphate removal was affected initially, but their values were restored to high values yielding nearly steadystate conditions. Practically during all the experiments, COD removal efficiency was not clearly affected by the changes in COD/SO42- ratio, since it was in the range of 70-90%. Sulphate removal was better at a ratio >1, reaching values between 60 and 85% (Figure 1). On the other hand, sulphide production at the outlet of the bioreactor also showed similar fluctuating profiles depending on the amount of SO42- removed from the system. Separate batch activity tests were performed by collecting both attached and suspended biomass samples from the IFB. It was observed that suspended biomass was more active in reducing sulphate rather than the attached biomass. This can be explained by the residence time distribution analysis which had showed that the tracer, at HRT of 12 h, requires more than 40 h to completely leave the reactor. Furthermore, by using a HRT of 24 h, it can take longer than 4 d. The last findings can support the high content of planktonic or suspended biomass within the IFB, and its doubling time is shorter than the holding times of the reactor. During the entire test period of 160 d, no volatile fatty acid (VFA) content was found in the IFB. This reactor will be later used to study the effect of transient states on the sulphate reducing process. The use of electron donors such as lactate for SO42- can hamper the longevity of IFB operation in terms of increased operational costs. Thus, there is an urgent need to look for alternate, yet cheaper electron donors, which when combined with an appropriate reactor configuration can bring an optimal performance in terms of sulphate reduction, sulphide for other processes and therefore cost reduction. Potato, filter paper, and crab shells are sources of starch, cellulose and chitin, respectively. These electron donors were tested in batch experiments for SO42- reduction, COD release and sulfide production potential. For the case of potato, in the absence of sludge, the release 25

27 of soluble COD was found to be 405 and 492 mg/l, for particle size of 2 mm3 and 5 mm3, respectively. This available COD could be due to the presence of glucose in the potato, as well as proteins, which are soluble and can easily diffuse to the anaerobic mineral media. Filter paper released soluble COD of 214 and 231 mg/l for areas of 2 mm2 and 5 mm2, respectively. The influence of the anaerobic sludge could also bring more soluble COD available for other syntrophic bacteria. In the case of potato it was 600 and 811 m/l for both particle sizes, while from filter paper it was 1178 and 1348 mg/l for both areas used. Under similar experimental conditions, chitin gave 181 and 364 mg/l of COD for 2 mm and 4 mm diameter size, respectively. A third experiment in the presence of sulphate as the electron acceptor showed that hydrolysis-fermentation occurred within the test vials and the soluble COD released could be used by sulphate reducing bacteria. Thus, briefly summarizing, the results from this study showed different volumetric rates depending on the type of electron donor used. This can be attributed to the nature of carbohydrates, chemical composition, processing characteristics for the case of filter paper, the different areas exposed to the microorganism e.g. potato and crab shells differed from filter paper, etc. Their use in continuous biological reactors must be studied; nevertheless, this study proves that cheap lignocellulosic (bio)wastes could bring the necessary slow release and buoyancy properties to be used as carrier material in IFBs. Table 1. Operational conditions of the IFB Period I II III IV OLR (mg/l.d) Operational conditions SO42-LR HRT COD/ SO42(mg/L.d) (h) Remov al Effici I Recirculation rate (L/h) I 40 Time80(d) Operation Days I I Figure 1. Schematic of the IFB reactor and sulphate (------) and COD ( ) removal efficiency during 4 periods of operation. 26

28 Studies on the Fate of Pharmaceuticals in Aqueous Media: Electrochemical degradation and Biotransformation H. Olvera-Vargas*, N. Oturan*, D. Buisson**, E. van Hullebushc*, M.A. Oturan* * Université Paris-Est, Laboratoire Géomatériaux et Environnement (LGE), EA 4508, UPEM, Marne-la-Vallée, France. ** Muséum National d Histoire Naturelle, 63 rue Buffon, Paris Cedex 05, France. Keywords: Emerging pollutants, Anodic oxidation, BDD, Electro-Fenton, Biotransformation The electrochemical degradation of the Ranitidine and Fuosemide, in aqueous media was studied by Electrochemical Advanced Oxidation Processes (EAOPs) "electro-fenton" and "anodic oxidation" using different cells with H2O2 electrogeneration: Pt/carbon-felt (EF-Pt), BDD/carbon-felt without (AO-H2O2) or with ferrous iron (EF-BDD) and DSA/carbon-felt (EF-DSA). The higher oxidation power of the electro-fenton process using BDD anode was highlighted. The greater ability of BDD electrodes to produce active radicals, make this kind of anode materials able to completely degrade all the by-products, even those refractory to Pt/carbon-felt cell [1]. The oxidative degradation of the different drugs by the electrochemically generated OH radicals follows a pseudo-first order kinetics. The apparent rate constants of the oxidation of both pharmaceuticals by OH were determined according to pseudo-first order reaction kinetic model and the absolute rate constant of their oxidation reaction was determined by using competition kinetics method. It was found that the electrochemical degradation of the pharmaceuticals leads to the formation of aromatic by-products which are then oxidized to aliphatic carboxylic acids before their almost complete mineralization to CO2 and inorganic ions. The identification of these by-products allowed us to propose a general reaction mechanism for the drugs mineralisation. It was found that the applied EAOPs are viable environmentally friendly technology for the remediation of wastewaters containing pharmaceutical products. As microorganisms are a major source of enzymes, bioconversions, like enzymatic and microbial transformations, are considered as a competitive and ecologically effective approach in organic synthesis [2]. In this context, a combinatorial approach was conducted in order to produce different products from the biological transformation of Furosemide and Ranitidine. The microorganisms selection involved a series of 12 species that were used to make mixtures and only one of them was found to be potentially suitable to metabolize the drug Furosemide, while none of them resulted to be active for metabolising Ranitidine. Furosemide s bioconversion by Cunninghamella echunilata ATCC 9245 leads to the generation of three principal metabolites that were successfully identified by HPLC/MS and NMR analysis: aniline, a pyridinium derivate and a keto-enol derivate. The later haven t been identified before. References [1] Oturan, N.; Brillas, E.; Oturan, M.A. (2012). Unprecedented total mineralization of atrazine and cyanuric acid by anodic oxidation and electro-fenton with a boron-doped diamond anode. Environ. Chem. Lett., 10, [2] Wohlgemuth, R. (2010). Biocatalysis key to sustainable industrial chemistry. Curr. Opin. Biotech. 21,

29 GEOMICROBIOLOGY AND METALLURGY

30 Biogenic Selenium Nanoparticles: Production, Characterization and Applications R. Jain*, E.D. van Hullebusch**, F. Farges***, P.N.L. Lens* *UNESCO-IHE, Westvest 7, Delft, The Netherlands **Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM, Marne-la-Vallée, France ***Muséum National d'histoire Naturelle, USM 201, 61, rue Buffon, Paris, France Keywords: Biogenic, Aerobic, Anaerobic, Selenium, nanoparticles Selenium nanoparticles have applications in xerography, superconductivity, anti-fungal medication and heavy metal removal. However, chemical selenium nanoparticles production methods entail high production cost and are not environment friendly. On the other hand, biogenic selenium nanoparticles (BioSeNPs) can be produced by reduction of selenite and selenate containing wastewaters at ambient temperature and pressure, thus providing an environmental friendly and potential cost-effective process.1 In this PhD research, production, characterization and applications of BioSeNPs were studied. BioSeNPs were produced by reduction of selenite under aerobic conditions that led to entrapment of BioSeNPs in the sludge flocs.2 The effect of this entrapment led to better settling but poor dewaterability of the sludge. BioSeNPs were also produced by the reduction of selenite under anaerobic conditions at three different temperatures. The extracted and purified BioSeNPs had a coating of extra-polymeric substances on their surface leading to negative ζ-potential and colloidal stability.3 BioSeNPs were also found to be a very good adsorbent for zinc, copper and cadmium ions.4 X-ray absorption spectroscopy on Zn K-edge on BioSeNPs loaded with zinc suggested the important role of extra-polymeric substances on interaction of BioSeNPs and zinc ions. References: 1) Jain, R.; Gonzalez-Gil, G.; Singh, V.; van Hullebusch, E.D.; Farges, F.; Lens, P.N.L.; Biogenic Selenium Nanoparticles: Production, Characterization and Challenges. In Nanotechnology; Kumar, A., Govil, J.N.; Studium Press LLC, USA, 2014; pp 361:390 (in press). 2) Jain, R.; Matassa, S.; Esposito, G.; van Hullebusch, E.D.; Farges, F.; Lens, P.N.L; Reduction of selenite under aerobic conditions by activated sludge. To be submitted to Journal of Hazardous materials. 3) Jain, R.; Jordan, N.; Foerstendorf, F.; Weiss, S.; Heim, K.; Hübner, R; van Hullebusch, E.D.; Farges, F; Lens, P.N.L; Exo-polymeric substances govern the shape, size and surface properties of biogenic elemental selenium nanoparticles. To be submitted Applied Materials & Interfaces. (To be submitted). 4) Jain, R.; Jordan, N.; Schild, D.; van Hullebusch, E.D.; Weiss, S.; Carola, F.; Hübner, R.; Farges, F.; Lens, P.N.L; Adsorption of zinc by biogenic elemental selenium nanoparticles. Re-submitted to Environmental Science & Technology. 29

31 Evidence of Zn Isotopes Fractionation during Bioweathering of Metallurgical Slags N.H. Yina,b,c, Y. Sivryb, E.D. van Hullebuscha, and P.N.L. Lensc Laboratoire Géo matériaux et Environnement, Université Paris-Est, France Laboratoire de Géochimie des Eaux, IPGP et Université Paris Diderot, France c UNESCO-IHE Institute for Water Education, the Netherlands a b Keywords: Zn isotopes; fractionation; metallurgical slags; bioweathering. Industrial tailings of zinc and lead metallurgy were recently proved to release isotopically fractionated Zn, which allows tracing this anthropogenic source (Sivry et al. 2008, Sonke et al. 2008, and Ettler et al. 2006). Many biological and chemical processes are known to take place within these tailings and thus affect significantly the metal weathering (Barna et al. 2004). The present study focuses on the potential of zinc isotopes to trace processes involved in biological weathering of metallurgic slag. Pb and Zn slag originated from former pyrometallurgical activities of Imperial Smelting Furnace smelters (ISF, Noyelle-Godault, North of France). ISF contains 8.0 wt% of Zn which are embedded in a CaO-SiO-FeO matrix. Kinetic biological dissolution tests of metallurgical slags (ISF) were conducted in the presence of a pure heterotrophic bacterial strain (i.e. Pseudomonas aeruginosa) under three different experimental conditions: ultra-pure water (UPW), sterile growth media (GM) and growth media with bacteria (GM+B). The abiotic experiments were performed in both UPW and sterile GM to differentiate the changes in leaching chemistry caused either by growth medium or by the presence of the bacteria. Leachate compositions and Zn isotopes were measured by ICP-OES (icap 6200) and by Thermo-Scientific MC-ICP-MS (Neptune & Neptune +), respectively. Thermodynamic model Visual MinteQ v. 3.0 was used to predict the possible secondary precipitates. Primary and secondary solid phases were further studied by XRF, XRD and SEM-EDS. Zn dissolution from ISF slag can be analyzed in two different time-steps: far-from-equilibrium and near-equilibrium dissolution. Zn dissolution was clearly enhanced in the presence of Pseudomonas aeruginosa. The highest dissolution of Zn from ISF slags can be found under far-from equilibrium condition (24h); where the concentrations were 14 µm L-1 in GM+B medium, 5.5 µm L-1 in GM medium, and 2.4 µm L-1 in UPW medium, respectively. Later, the concentrations reduced to 9.5 µm L-1 in GM+B medium, 2.8 µm L-1 in GM medium, and 2.2 µm L-1 in UPW medium under nearequilibrium condition (192h). The percentage of dissolved Zn concentration decreased from 99% at 24h to 56.3% at 192h, indicating that more than 50% of Zn has been adsorbed onto bacteria surface during long term bio-weathering of slags. The bulk ISF slag displayed δ66zn signatures enriched in heavier isotopes i.e ± 0.04, meanwhile, lighter signatures can be found in the leachate in the presence of P. aeruginosa. The total Zn isotopic signatures of leachate (i.e. the acid digested leachate including bacteria bodies) displayed lighter Zn isotopes enrichment; ± 0.04 at 24h which became heavier over time, reaching ± 0.04 after 192h. On the other hand, the dissolved Zn isotopic signatures of leachate (i.e. the acid digested leachate excluding bacteria bodies via 0.2 µm filtration) showed 1.09 ± 0.04 at 24h and ± 0.04 after 192h. The offset values between total and dissolved δ66zn signatures was ± 0.04 when 14% of Zn in adsorbed form, and ± 0.04 when 45% of Zn in adsorbed form. Zn dissolution can be related to mineral phases present in ISF; Spinel Zn(Al0.8Fe0.2)O4 and Franklinite [(Zn,Mn2+,Fe2+)(Fe3+,Mn3+)2O4] where these phases are more resistant to weathering than ZnO phases embedded in slag matrix. The presence of the Pseudomonas aeruginosa not only elevated the concentration of Zn in the leachate but also served as the adsorbent for Zn during longer hours of weathering experiment (168 to 192 h). 30

32 Simultaneously, formation of white thin crust suggested that Zn in solution was also controlled by the precipitation of two main secondary phases: Zincite (ZnO) and Zinc carbonate (ZnCO3), and minor phases: alumino silicates (LDH layers of Al-Zn-Si) depositing back onto the weathered slags. Thus, the Zn isotopes fractionation during slag biological dissolution is expected to be related to its primary mineral phases present in initial slag, the presence of bacteria, and the formation of Zn secondary phases during longer weathering time. References Barna, R., et al. (2004). Leaching assessment of road materials containing primary lead and zinc slags. Waste Manage. 24, Ettler, V., et al. (2006). Geochemical and Pb isotopic evidence for sources and dispersal of metal contamination in stream sediments from the mining and smelting district of Príbram, Czech Republic. Environ. Pollut. 142, Sivry, Y., et al. (2008). Zn isotopes as tracers of anthropogenic pollution from Zn-ore smelters. The Riou Mort-Lot River system. Chem. Geol. 255, Sonke, J., et al. (2008). Historical variations in the isotopic composition of atmospheric zinc deposition from a zinc smelter. Chem. Geol. 252,

33 Filamentous growth of neutrophilic bacterium Sphaerotilus natans and its role in the scavenging of inorganic pollutants M. Seder-Colomina1, G. Morin2, G. Ona-Nguema2, J.J. Pernelle3, G. Esposito4, E. D. van Hullebusch1 1: Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM, Marne-la-Vallée, France. Fax: marina.sedercolomina@u-pem.fr, eric.vanhullebusch@u-pem.fr 2: Institut de Minéralogie, de Physique des Matériaux, et de Cosmochimie (IMPMC). Sorbonne Universités - UPMC Univ Paris 06, UMR CNRS 7590, Muséum National d Histoire Naturelle, IRD UMR 206, Paris, France. Fax: guillaume.morin@impmc.upmc.fr, georges.ona-nguema@impmc.upmc.fr 3: Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (Irstea), UR HBAN, CS Antony Cedex France. Tel: jean-jacques.pernelle@irstea.fr 4: University of Cassino and Southern Lazio, Department of Civil and Mechanical Engineering, Cassino (FR), Italy. Fax: giovanni.esposito@unicas.it Keywords: filamentous bacteria; oxygen; sorption; biogenic minerals; trace metals Sphaerotilus natans is a neutrophilic iron-relate.n d sheath-forming microorganism able to grow as planktonic or ensheathed cells, in the latter case forming filaments (1). Indeed, it can occasionally cause bulking problems in wastewater treatment plants due to the invasive proliferation of their filamentous growth form (2). Moreover, S. natans planktonic cells and filaments have also been reported as an excellent sorbent for inorganic pollutants (3). Hence, studying the factors affecting S. natans filamentation is crucial to understand not only the potential of planktonic cells to trigger the proliferation of filaments but also their influence on the scavenging of inorganic pollutants, either by direct biosorption or by sorption onto the biogenic minerals formed in its presence. The work presented here had three different objectives: 1) to evaluate the inducing effect of dissolved oxygen on S. natans filamentation by using a filtration system coupled to quantitative PCR (qpcr), 2) to study the role of this bacterium in the formation of biogenic minerals by using Scanning Electron Microscopy coupled to an Energy Dispersive X-ray detector (SEM-EDX) and X-ray absorption spectroscopy (XAS) and 3) to investigate the scavenging of trace metals by such biogenic iron minerals by using chemical analytical techniques and XAS. Results show that moderate oxygen depletion enhances the proliferation of S. natans filaments from planktonic cells. Moreover, the mineralogical studies lead to the identification of iron minerals presenting an amorphous nano-crystalline structure, potentially highly reactive when used for the scavenging of inorganic pollutants. Indeed, the removal of a trace metal was tested and results reveal high efficiency, which vary depending on the experimental conditions. The identification of moderate oxygen depletion as a factor affecting S. natans filamentation will help in better understanding the mechanisms of filamentation of this sheath-forming bacterium. Moreover, XAS results will shed light on the molecular environment of the trace metal when trapped by S. natans iron minerals and therefore will help to improve the design of strategies for the scavenging of radionuclides in near-neutral ph environments. References Seder-Colomina, M. et al. (2014) Sphaerotilus natans, a neutrophilic iron-related sheath-forming bacterium: perspectives for metal remediation strategies. Geomicrobiol J 31: Pellegrin, V. et al. (1999) Morphological and biochemical properties of a Sphaerotilus sp. isolated from paper mill slimes. Appl Environ Microbiol 65: Pagnanelli, F. et al. (2003) Metal speciation and ph effect on Pb, Cu, Zn and Cd biosorption onto Sphaerotilus natans: Langmuir-type empirical model. Water Research 37:

34 Impact of electrocoagulation on biogenic colloidal selenium L.C. Staicu*,**, P.N.L. Lens**, E.D. van Hullebusch*, M.A. Oturan* * Université Paris-Est, Laboratoire Géomatériaux et Environnement, France ** UNESCO-IHE Institute for Water Education, Delft, the Netherlands Keywords: Elemental selenium; Colloids; Electrocoagulation; Pseudomonas fluorescens; TCLP Colloidal elemental selenium, Se(0), adversely affects membrane separation processes and aquatic ecosystems. At the industrial level, biogenic Se(0) is generated by the biological treatment of soluble selenium-laden wastewaters (Sobolewski, 2013). In aquatic ecosystems, Se(0) is produced under anaerobic and reducing conditions (e.g. sediments) via microbial dissimilatory reduction of water-soluble Se oxyanions. Sediment disturbance can potentially release Se(0) in the water column (Zhang et al., 2004). For the current investigation, Se(0) was produced by a strain of Pseudomonas fluorescens and showed very limited gravitational settling. Our study investigated the sedimentation potential of colloidal Se(0) using electrocoagulation (EC). In EC, current is applied across an electrolytic cell leading to the dissolution of a sacrificial anode (Al or Fe) with the subsequent formation of trivalent cations that promote the in situ generation of coagulants. Concomitantly, electrolysis of water contributes to the evolution of hydrogen (at cathode) and oxygen (at anode) microbubbles. Iron and aluminum sacrificial electrodes were used under galvanostatic conditions (Fig. 1.1.). Because colloids are held in suspension by electrostatic repulsive forces, the presence of counterions brings about neutralization of their electric charge and diminishes their stability. Additionally, colloids are adsorbed onto metal oxides and (oxy)hydroxides forming large aggregates. As a consequence of both mechanisms, the pollutants agglomerate, increase their weight and settle (Mollah et al., 2001). Highly efficient sedimentation (97%) was observed at 200 ma for experiments with iron electrodes, whereas aluminum electrodes achieved the highest turbidity reduction (96%) at 300 ma. At the best turbidity removal, the aluminum electrodes consumed twice more energy than the iron electrodes, 3.42 kwh m-3 versus 1.56 kwh m-3. Owing to the less compact nature of the aluminum flocs, the sediment volume produced by aluminum treatment is three times higher than its iron counterpart. At 100 ma, leaching tests (Toxicity Characteristic Leaching Procedure, TCLP) show that Fe-Se sediment releases Se below the regulatory level (1 mg L-1), whereas Al-Se exceeds it by times (Fig. 2.2.). This result might be related to the mineralogical nature of the sediments. Environmental Electron Scanning micrographs (ESEM) show Fe-Se sediments having a reticular arrangement, whereas the Al-Se sediments appear not to possess an organized structure. Overall, our findings suggest that iron electrodes are a better option than aluminium electrodes for the sedimentation of colloidal Se(0). Iron passivation is an important factor that influences the performance of the process. 33

35 Figure 1.1. Schematic diagram of the electrocoagulation set-up. Note that: M = metal (e.g. Al, Fe), Mn+ = metal having the valence state n+, M(OH)n = metal (oxy)hydroxide. 60 Fe -1 metal concentration (mg L ) 50 Al 40 Al Fe Al Se Se Se Fe 10 Se Se 0 Al-Se (50 ma) Fe-Se (50 ma) Al-Se (100 ma) Fe-Se (100 ma) Current (ma) Figure 1.2. Leaching behavior (TCLP) of Al-Se and Fe-Se sediments. Sediments were produced at: 50 ma and 100 ma, 300 rpm, room temperature, [NaCl] = 42 mmol L -1, ph0 = 7.00, κ = 4.4 ms cm-1, initial turbidity = 500 NTU, followed by 24 h sedimentation in standard Imhoff cones. References Mollah, M.Y.A.; Schennach, R.; Parga, J.; Cocke, D.L. (2001). Electrocoagulation (EC) science and applications. J. Hazard. Mater., B84, Sobolewski, A. (2013). Evaluation of treatment options to reduce water-borne selenium at coal mines in West-Central Alberta. Microbial Technologies, Inc. Available from: Zhang, Y.; Zahir, Z.A.; Frankenberger Jr., W.T. (2004). Fate of colloidal-particulate elemental selenium in aquatic systems. J. Environ. Qual., 33,

36 Leaching and Recovery of Molybdenum, Nickel and Cobalt from the Mineral Sludge Generated at a Metal Recycling Plant M. Vemic*, F. Bordas***, G. Guibaud***, P.N.L. Lens**, E.D. van Hullebusch* *Université Paris-Est, Laboratoire Géomatériaux et Environnement (LGE), EA 4508, UPEM, Marne-la-Vallée, France **UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands ***Université de Limoges, Groupement de Recherche Eau-Sol-Environnement (GRESE), Faculté des Sciences & Techniques, 123 avenue Albert Thomas, Limoges, France Keywords: Mineral sludge; Metals; Chemical leaching; Sulfide precipitation; Leaching and recovery yields The leaching rate and yields of Mo, Ni and Co from mineral sludge sample collected from the collection basin of a catalyst, metallic oxide and battery recycling plant were quantified. The leaching experimental setup is presented in Figure 1.1. Mineral sludge sample Total metal concentrations (TMC) Mineral sludge leaching properties 1. Testing the effect of S/L ratio (1-50 g/l) under constant leaching time (2 h) and temperature (60 C): 2. Testing the effect of leaching time (1-5 h) under constant S/L ratio (1 g/l) and temperature (60 C): -HNO3 (A) -H2SO4 (B) -HCl (C) -Aqua regia (D) -HNO3+H2SO4 (1:1) (E) -H2SO4 (B) 4. Testing the leaching properties of different chemicals under the optimal leaching conditions (time 2 h, S/L ratio 0.25 g/l and temperature 60 C) -Acids: sulfuric, nitric, hydrochloric, citric, formic, acetic, ascorbic and oxalic. 3. Further testing the effect of S/L ratio ( g/l) under constant leaching time (2 h) and temperature (60 C): -H2SO4 (B) -HNO3+H2SO4+HCl (2:1:1) (F) 5. Further optimisation of H2SO4 leaching. Testing the effect of temperature ( C) under the optimal leaching conditions (time of 2 h, S/L ratio 0.25 g/l) Figure 1.1. Leaching experimental set up. Five sets of experiments were conducted: 1. Testing the effect of S/L ratio (150 g/l), 2. Testing the effect of time (1-5 h), 3. Testing the effect of S/L ratio ( g/l), 4. Testing the leaching properties of different chemicals under optimal leaching conditions, 5. Testing the effect of temperature ( C). 35

37 Different leaching reagents were tested at changing operational parameters (solid/liquid ratio (0.25, 0.5, 0.75, 1, 5, 10, 20, 30, 40 and 50 g/l), leaching time (1, 2, 3, 4 and 5 h) and temperature (20, 30, 40, 50, 60, 70, 80, 90 and 100 C)), in order to understand the leaching properties and select the suitable leaching reagent with the highest metal leaching yield. The tested leaching reagents were, respectively, stand-alone acids (sulfuric, nitric, hydrochloric, citric, formic, acetic, ascorbic and oxalic), acid mixtures (aqua regia (nitric + hydrochloric (1:3)), nitric + sulfuric (1:1), nitric + sulfuric + hydrochloric (2:1:1)), sodium carbonate, ammonium acetate, sodium hydroxide and hydroxylamine hydrochloride. Sulfuric acid (H2SO4) was found to be the best leachant with the highest metal leaching potential. The optimal leaching conditions were a successive leaching (three stage), temperature 80 C, leaching time 2 h and S/L ratio 0.25 g/l. Under these conditions, the leaching recovery yields from our mineral sludge sample reached 85.5, 40.5 and 93.8 % for Mo, Ni and Co, respectively. The recovery rate and yields of Mo, Ni and Co from mineral sludge sample collected from the collection basin of a catalyst, metallic oxide and battery recycling plant were also quantified. Chemical and biogenic sulfide precipitation were tested at changing operational parameters (metal/sulfide ratio (0.1, 0.2, 0.5, 1 and 2), rpm (0, 25, 50, 75 and 100), time (15, 30, 45, 60, 75, 90, 105 and 120 min) and ph (1, 2, 3, 4 and 5)) in order to understand the metal recovery properties. The 0.1 M Na2S was used for chemical sulfide precipitation with the sulfide concentration of 3200 mg/l. On the other hand the biologically produced sulfide had a concentration of 100 mg/l. From the results it was observed that the highest metal recovery potential was achieved at 100 rpm for the recovery time of 60 min and with the 0.1 m/s ratio for both the chemical and the biogenic sulfide precipitation. However, there was a big difference the recovery efficiencies between these two experiments. In case of a chemical sulfide precipitation, the recovery efficiencies achieved were 86.9, 61.7 and 55.4 % for Mo, Ni and Co, respectively (Figure 1.2 a). On the other hand, the biogenic sulfide precipitation was not efficient (the recovery potential was less than 1 %) with the recovery efficiencies of 0.8, 0.7 and 0.9 % for Mo, Ni and Co, respectively (Figure 1.2 b). a b Figure 1.2. Metal recovery yields (%) of the collection basin mineral sludge sample under the effect of chemical (a) and biogenic (b) sulfide (m/s ratio = 0.1-2, rpm = 100, time = 60 min, ph = 1). 36

38 Metal Recovery from Electronic Waste A. Işıldar*, E. D. van Hullebusch**, J. Puhakka***, J. van de Vossenberg*, E. R. Rene*, and P. N. L. Lens* * UNESCO-IHE Institute for Water Education, Pollution Prevention and Research Recovery, Westvest 7, 2611AX Delft, the Netherlands ** Université Paris-Est, Laboratoire Geomatériaux et Environnement (LGE), EA 4508, UPEMLV, Marne-la-Vallée, France *** Tampere University of Technology, Department of Chemistry and Bioengineering, Korkeakoulunkatu 8, Tampere, Finland Keywords: Electronic waste; metal recovery; secondary source; bioleaching; biorecovery Discarded electric and electronic devices comprise a growing segment of waste generated at households. Despite the growing awareness and deterring legislation, most of the waste electrical and electronic equipment (WEEE) is disposed in landfills. Inappropriate management of electronic waste is of global concern due to the nature of production and disposal of waste in a globalized world. In addition to toxic compounds present in WEEE, it also contains valuable metals. WEEE contains considerable quantities of valuable materials such as copper, gold, aluminum and nickel. Metal concentration of PCBs especially that of precious metals, is higher than those of the natural ores. Discarded electric and electronic devices and particularly PCBs have the potential to be a very promising secondary source of metals. The major economic driver for recycling of electronic waste is the recovery of valuable metals. Recovery of metals is traditionally carried out by pyrometallurgical and hydrometallurgical methods, which have their own drawbacks and limitations. A comparison of different metal recovery techniques are shown in Table 1. In this research, biological treatment of discarded boards, with the aim to recover metals of interest, will be investigated. Biohydrometallurgy, using microbes for metal recovery, enables environmentally sound and cost-effective processes to recover metals from waste material. In this context microbial leaching of metals from waste material (bioleaching) and biomass-based recovery of metals from leachate (biosorption, bioprecipitation) will be investigated. In addition, efficiency of a series of physico-chemical methods, e.g. acid leaching, thiourea/thiosulfate leaching, precipitation, and electrowinning will be assessed and compared to biomass based techniques. Copper (Cu) and gold (Au) are selected as metals of interest in this research. Table 1: Comparison of metal recovery techniques Parameters Pyrometallurgy Hydrometallurgy Biohydrometallurgy Environmental impact High, due to emissions Moderate, toxic reagents Low Economics Capital intensive Medium costs Low costs Social acceptance Low Medium High, cleaner processes Energy usage Very high Low, ambient conditions Low Recovery rate Low High recovery rates High recovery rates Final residue High Low Low-to-none Process conditions Harsh conditions Mild conditions Safe conditions 37

39 Due to distinct chemical behavior of base and precious metals, a multi-step bioleaching process shall be considered for this research. In the first step, base metals (e.g. copper) are leached out using chemolithotrophic acidophilic bacteria. In the following step, precious metals (e.g. gold) are leached out by cyanide producing bacteria. Recovery of metal ions from leachate solution will be subsequently carried out by precipitation using sulfide, biosorption using bacteria and fungi, as well as electro winning. Finally, a techno-economic assessment of the newlydeveloped technology as well as the environmental performance of the metal recovery process will also be evaluated. Moreover, the added-value of the novel technology for metal recovery from ewaste will be benchmarked and its suitability for the development of full-scale processes will be evaluated. References Cao, J.; Zhang, G.; Mao, Z.; Fang, Z.; Yang, C. (2009). Precipitation of valuable metals from bioleaching solution by biogenic sulfides, Minerals Engineering, 22, Ilyas, S.; Lee, J.C.; Chi, R. (2013). Bioleaching of metals from electronic scrap and its potential for commercial exploitation, Hydrometallurgy, , Park, Y., J.; Fray, D., J. (2009). Recovery of high purity precious metals from printed circuit boards, Journal of Hazardous Materials, 164, Ting, Y., P.; Pham, V., A. (2009). Gold bioleaching of electronic waste by cyanogenic bacteria and its enhancement with bio-oxidation, Advanced Materials Research, 73, Tuncuk, A.; Stazi, V.; Akcil, A., Yazici, E., Y.; Deveci, H. (2012). Aqueous metal recovery techniques from e-scrap: Hydrometallurgy in recycling, Minerals Engineering, 25,

40 Metallurgical Sludges: from Mineralogical Characterization to Heavy Metals Bioleaching and Recovery M. Sethurajan*, E.D. van Hullebusch*, H.A. Horn**, L.H.ArimuraFigueiredo***, P.N.L. Lens**** *Université Paris-Est, Laboratoire Géomatériaux et Environnement (LGE), EA 4508, UPEMLV, Marne-la-Vallée, France. ** Universidade Federal de Minas Gerais, NGqA-CPMTC, Instituto de Geociências, Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte - MG, , Brazil. *** Universidade Estadual de Montes Claros,Solos e nutrição de plantas, Avenida Reinaldo Viana, 2630, Bico da Pedra Janauba - MG, , Brazil. ****UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands. Keywords: Metallurgical wastes, Metals recovery, Secondary resources, Biohydrometallurgy, Metal sulfide precipitation Introduction - In this electronic era, absolutely everything humans depend on is either made of minerals or metals and so their demand in the day-to-day life is increasing. Considering the fact that the gradual depletion of worldwide reserves for high grade ores has resulted in the urge to look for alternative resources to recover metals. The wastes generated in the metallurgical industries can be used as a secondary resource as it contains high concentration of metals. From the economic point of view, conventional pyro-metallurgical approach will not be a better tool for the extraction of metals from the wastes as it requires high capital investment. Recently, the use of micro-organisms (biohydrometallurgy) to facilitate the extraction and recovery of base and precious metals from fly ash, wastes, dusts, has developed into a successful and expanding area of biotechnology. This technology holds the promise of reducing the fixed capital costs dramatically, and also offers the opportunity to reduce environmental pollution. Objective(s) -The ultimate aim of this research project is to recover the valuable metals from the metallurgical wastes. In order to achieve this objective, the research plan has several subobjectives (i) To understand about the various physico-chemical, and mineralogical characteristics, (ii) To investigate in detail about the leachability (chemical and biological) of the heavy metals from the muds and (iii) To study about the selective recovery of the metals from the leachates. Methodology -Samples were dried and ground to ensure that the particle size is below 1mm in diameter. Mineralogy of the samples were studied by X-Ray diffraction. Presence and determination of Total solids, volatile and fixed solids (US EPA 1684) and carbonate content in the metallurgical wastes were calculated. Hotplate aqua regia acid digestion was done to know about the total metal concentrations of the samples. Toxicity characteristics leaching procedure (TCLP) of the metal wastes were also investigated by US EPA 1311 procedure. Ultrasound acceleratedsequential extraction procedure (Perez-Cid et al. 1998) wasdone to understand about the amounts of acid extractable, oxidisable, reducible and residual fractions of the metals. Effect of ph on the leaching behavior was studied by US EPA 1313 procedure. Metals concentration in the leachates were determined by ICP-OES. Results Our initial results reveal that the sludges contain significant concentration of Zinc (2.5% to 5%), Lead (1.7% to 2.3%) and metals such as Manganese, Copper, and Aluminium in detectable fractions. The residues contain high concentration of Cu (47%), Zn (28%), Cd (9%) and Pb (5%). Both the sludges and residues are Hazardous wastes, releasing higher concentration of Pb and Cd into the environment, than the permissible concentration suggested by EPA. Sequential extraction analytes contain Cd (99%), Cu and Zn morethan 50% in acid exchangeable fractions, Mn> Zn Cu > Fe > Al >Pb in the reducible fraction and Pb> Cu > Zn > Al > Cd >Mn in the oxidisable fraction. Metals such as Fe, Pb and Al were mostly observed in the residual fractions. Leaching of metals is high at low ph and the release of metals was decreased with increase in ph Conclusions and Future directions The results suggested that these are hazardous wastes 39

41 and significant concentration of Zn can be leached from these wastes. Currently, a study to recover the heavy metals by biologically produced hydrogen sulfides from sulfuric acid based heap leachates is on-going. Future directions will include optimizing the parameters for the maximum leaching of metals and finding suitable conditions to achieve the selective recovery of metals from the leachates. References: Lee, J.C.; Pandey, B.D.; (2012). Bio-processing of solid wastes and secondary resources for metal extraction a review. Waste Manage., 32, Lewis, A.E. (2010). Review of metal sulfide precipitation, Hydrometallurgy, 104, Lottermoser, B.G. (2003). Mine Wastes: Characterization, Treatment, and Environmental Impacts, Springer, Verlag Berlin Heidelberg New York. Pérez-Cid, B.; Lavilla, I.; Bendicho, C. (1998). Speeding up of a three-stage sequential extraction method for metal speciation using focused ultrasound, Anal. Chim. Acta., 360, United States Environmental Protection Agency (1992).Toxicity Characteristic Leaching Procedure (TCLP), Test Method 1311-TCLP, Washington, DC. United states environmental protection agency (2012). Liquid-Solid Partitioning As A Function Of Extract ph Using A Parallel Batch Extraction Procedure, United states environmental protection agency (2001).Total, Fixed, and Volatile Solids in Water, Solids, and Biosolids

42 Microbial synthesis of metal selenide nanoparticles using selenium oxyanion reducing bacteria J. Mal*, V.N. Yarlagadda*, E.D. van Hullesbusch**, P.N.L. Lens* * Pollution Prevention and Resource Recovery Chair Group, Department of Environmental Engineering and Water Technology, UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands ** Laboratoire Géomatériaux et Environnement, Université Paris-Est Marne-la-Vallée, Institut Francilien des Sciences Appliquées, Bât. IFI 5, Boulevard Descartes Champs sur Marne Marne La Vallée, Cedex 2 France Keywords: Microbial selenium reduction; metal selenide; nanoparticles; quantum dots; anaerobic granular sludge. Selenium oxyanion containing wastewaters are generated in various industries such as acid mine drainage, acid seeps, exhaust flue gas of SO2, agriculture drainage. Treatment of such wastewaters is desired to meet the environmental discharge guidelines. Among the available treatment options, microbial reduction appears to be promising for removal of selenate and selenite from wastewaters. In this PhD research, it is proposed to investigate microbial metabolism for the removal and recovery of selenium oxyanions as economically valuable elemental selenium and metal selenide nanoparticles. Development of semiconductor nanocrystals known as quantum dots (QDs) has been of great interest for fundamental research and industrial development. Due to unique semiconducting properties induced by the onset of quantum confinement in the size range 1 20 nm, the QDs of different sizes, shapes and composition have been used in solar cells and optoelectronic sensors as well as fluorescent biolabelling, including their use in cancer diagnosis. The chemical synthesis of metal selenide nanoparticles involves the use of highly toxic solvents and high temperatures. Also the present chemical synthetic procedures are energy intensive. Besides, it is not easy to obtain nanoparticles of the desired size because of their high growth rate in chemical synthesis. Recent reports have shown that microbial synthesis provides increased controllability of the formation and growth of the QDs (Fellowes et al., 2013, Ayano et al., 2013). Moreover, the use of microorganisms for production nanoparticles provides a low cost, environmental friendly method because the synthesis occurs at ambient condition, without the use of toxic reducing agents. In this PhD research, we propose a new methodology and technology for selenium oxyanion reduction and metal selenide nanoparticles production. Anaerobic granular sludge will be used as the source of microbial metabolism for the production of metal selenide nanoparticles. The effect of different temperature and different electron donor on metal selenide nanoparticles production and physical, chemical and biological properties of nanoparticles will be explored. UV vis, photoluminescence, X-ray diffraction and transmission electron microscopy analysis will be done to confirm metal selenide synthesis. The production of metal selenide nanoparticles will be studied on both batch and lab-scale bioreactor. Previously, upflow anaerobic sludge blanket reactor (UASB) was used for biological removal of selenate and selenite. To the best of our knowledge, no study has been carried out on the use of anaerobic granular sludge for production of metal selenide nanoparticles. So, the reduction of selenium oxyanions for the production of metal selenide nanoparticles will be investigated in a UASB bioreactor. Optimization and development of product recovery methods will also be investigated. References Fellowes, J.W.; Pattrick, R.A.D.; Lloyd, J.R.; Charnock, J.M.; Coker, V.S.; Mosselmans, W.; Weng, T.C.; Pearce, C.I. (2013). Ex situ formation of metal selenide quantum dots using bacterially derived selenide precursors. Nanotechnology, 24(14), Ayano, H.; Miyake, M.; Terasawa, K.; Kuroda, M.; Soda, S.; Sakaguchi, T.; Ike, M. (2013). Isolation of a selenite reducing and cadmium-resistant bacterium Pseudomonas sp. strain RB for microbial synthesis of CdSe nanoparticles. J. Biosci.Bioeng 117(5),

43 Mineralogy and environmental stability of copper pyrometallurgical slags A. Potysz*, J. Kierczak**, Y. Fuchs*, M. Gryboś****, G. Guibaud****, P. Lens***, E.D. van Hullebusch* * Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEMLV, Marne-la-Vallée, France. ** University of Wroclaw, Institute of Geological Science, Cybulskiego 30, Wroclaw, Poland *** Chair group Pollution Prevention and Resource Recovery, UNESCO-IHE Institute for Water Education, P.O. Box 3015, 2601 DA Delft, The Netherlands **** Université de Limoges, Groupement de Recherche Eau Sol Environnement (EA 4330), Faculté des Sciences et Techniques, 123 Avenue A. Thomas, Limoges Cedex, France. Keywords: Cu-metallurgical slags; mineral phases; metallic elements; ph-dependent leaching Copper pyrometallurgical slags constitute important by-products with respect to their volume of production and high residual content of metallic elements inefficiently recovered during industrial process. Due to lack of sustainable practices in the past, slags disposal has been a common manner. As the result, there are many industrial areas where slags have been proved to be a source of metallic pollution for ambient environment. For this reason, nowadays main prerequisite in undertaking any waste management strategies is determination of environmental stability of slags. Geochemical approach combining chemical and mineralogical studies together with leaching experiments representing ph-specific case scenario was applied for environmental risk assessment of Cu-metallurgical slags. Two groups of slags: historical (HS) and modern one including: shaft furnace slag (SFS), granulated slag (GS) and lead slag (LS) were evaluated with relevant attention given to distribution of metallic elements in mineral phases as well as slags stability with respect to release of selected contaminants (Cu, Zn, Pb). Results demonstrated that slags display considerable concentrations of metallic elements varying from one slag type to another (Figure 1.1). Mineralogical study revealed that slags are composed of different mineral phases possessing impurities of metallic elements in various quantities. HS and SFS slags are mainly composed of synthetic analogues of silicates such as fayalite (HS) and diopside (SFS) as well as minor sulfides (e.g. bornite, chalcopyrite). GS is an amorphous material composed of glass containing copper droplets, whereas LS is an exceptional type characterized by volumetrically major sulfides and minor fayalite. Due to important diversity of slags, they behave variously under exposure to different ph conditions. Strong acidic conditions (ph 2) were found to be the least stable for analysed materials (Figure 1.2), whereas leachability at alkaline conditions showed lower importance (e.g. Cu: up to 114 mg/kg for HS; Zn: up to 243 mg/kg for LS; Pb: up to 5539 mg/kg for LS). Slightly alkaline ph of 10.5 was found to be optimal conditions for any slag management actions due to their low susceptibility to release contained metals. According to results of this study it may be concluded that environmental stability of slags depends on both, their bulk chemistry and mineralogy. However, factor playing superior role in metal leachability is mineral phase being its carrier. For, this reason separate consideration of individual slags behaviour appears to be important for preventing environmental contamination and should be regarded as priority. 42

44 Figure 1.1. Comparison of metallic elements content in Cu-pyrometallurgical slags: HS-Historical slag, SFS- Shaft furnace slag, GS- Granulated slag, LS- Lead slag. Figure 1.2. Leaching of metallic elements under exposure to ph 2 HS-Historical slag, SFS- Shaft furnace slag, GS- Granulated slag, LS- Lead slag. 43

45 The Role of Extracellular Polymeric Substances (EPS) from Biofilm in Heavy Metals Sorption F. Cao*, Y. Pechaud*, G. Guibaud**, P.N.L Lens***, E.D. van Hullebusch* *Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM, Marne-la-Vallée, France ** Université de Limoges, Groupement de Recherche Eau Sol Environnement (EA 4330), Faculté des Sciences et Techniques, 123 Avenue A. Thomas, Limoges Cedex, France. ***Pollution Prevention and Control Core, UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, the Netherlands Keywords: extracellular polymeric substances; biosorption; heavy metals; characterization The importance of heavy metals in the environment and biological processes both as potential toxic contaminant as well as essential nutrients is of current concern. In biological aggregates, metals interact significantly with the extracellular polymeric substances (EPS) which are mainly secreted by micro-organisms (Flemming and Wingender, 2010; Sheng et al., 2010). To propose innovative solutions to remove and recover heavy metals (Li et al., 2014) or to improve the biogas production yields of anaerobic digestion (van Hullebusch et al., 2006; Zandvoort et al., 2004), a better understanding of the mechanisms involved in these interactions is required. Extracellular polymeric substances (EPS) are metaphorically called house of biofilm cells (Flemming et al., 2007). Within this house, the immediate conditions of life of biofilm cells are determined. EPS influence porosity, density, water content, charge, sorption properties, hydrophobicity, and mechanical stability of biofilm (Flemming and Wingender, 2010). EPS are biopolymers which are not only comprised of polysaccharides and proteins. According to the origin, a certain amount of nucleic acid, lipids, humic-like substances can be found in EPS (Frølund et al., 1996). EPS are known to play a key role in biosorption of metals in biological aggregates. The negatively charged functional groups harbored in EPS, such as carboxyl, phosphate, amine and hydroxyl, provide abundant binding sites for metal ions (Guibaud et al., 2008). Even if during the last decades, studies have improved our understanding on the interactions between EPS and metals, the influence of several factors such as EPS composition, and environmental conditions (ph, temperature, nutrients, etc.) which govern metal speciation is not well known. In this study, Cupriavidus metallidurans CH34 strain was chosen as the model bacterium, which can resist high concentrations of several metals (including Ni, Co, Zn, Cd) (Mergeay et al., 1985). Nickel and cobalt are the targeted metals. The main aim of our study is to improve the knowledge of the relationship between nutrients, electron donors and EPS composition, and, in turn, the role of EPS composition on metal sorption by the model strain and the EPS secreted. EPS extracted from this strain will be characterized by potentiometric titration, HPSEC and 3DEEM techniques. References Flemming, H.C.; Wingender, J. (2010) The biofilm matrix. Nat. Rev. Microbiol. 8, Flemming, H.C.; Neu, R.T.; Wozniak, D.J. (2007) The EPS matrix: The House of biofilm cells. J. Bacteriol., 189, Frølund, B.; Palmgren, R.; Keiding, K.; Nielsen, P.H. (1996) Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res., 30, Guibaud, G.; Bordas, F.; Saaid, A.; D abzac, P.; van Hullebusch, E. (2008) Effect of ph on cadmium and lead binding by extracellular polymeric substances (EPS) extracted from environmental bacterial strains. Colloids Surf. B Biointerfaces, 63, Li, W.W.; Yu, H.Q. (2014) Insight into the roles of microbial extracellular polymer substances in metal biosorption. Bioresour. Technol., Special Issue on Biosorption, 160,

46 Mergeay, M.; Nies, D.; Schlegel, H.G.; Gerits, J.; Charles, P.; van Gijsegem, F. (1985) Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol., 4, Sheng, G.P.; Yu, H.Q.; Li, X.Y. (2010) Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review. Biotechnol. Adv., 28, van Hullebusch, E.; Gieteling, J.; Zhang, M.; Zandvoort, M. H.; Daele, W. V.; Defrancq, J.; Lens, P.N.L. (2006) Cobalt sorption onto anaerobic granular sludge: Isotherm and spatial localization analysis. J. Biotechnol., 121, Zandvoort, M.H.; Gieteling, J.; Lettinga, G.; Lens P.N.L. (2004) Stimulation of methanol degradation in UASB reactors: In situ versus pre-loading cobalt on anaerobic granular sludge. Biotechnol. Bioeng., 87,

47 Use of Phanerochaete chrysosporium as a selenium-reducing organism for: the removal of selenite from water, the production of elemental selenium nanoparticles and its potential application as a new adsorbent for the removal of heavy metals E.J. Espinosa-Ortiz*, E.R. Rene*, G. González-Gil*, E.D. van Hullebusch **, and P.N.L. Lens* *Pollution Prevention and Resource Recovery Chair Group, Department of Environmental Engineering and Water Technology, UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands e.espinosaortiz@unesco-ihe.org, e.raj@unesco-ihe.org, g.gonzalez@unesco-ihe.org, p.lens@unesco-ihe.org **Université Paris-Est, Laboratoire Géomatériaux et Environnement, EA 4508, UPEMLV, Marne-la-Vallée, France Eric.vanHullebusch@univ-paris-est-fr Keywords: selenium, nanoparticles, fungi, wastewater, pelleted bioreactor Concentration levels of selenium (Se) oxyanions, selenite (SeO32-) and selenate (SeO42-) are highly variable in industrial wastewaters, leading to the necessity of developing feasible and successful removal and recovery strategies. Traditionally, physical and chemical techniques are used for treating Se polluted streams; however, very little attention has been given to its recovery from wastewater. Some microorganisms possess the ability to reduce selenium oxyanions to elemental Se (Se0) [1], making the biological treatment an effective alternative method for Se removal. Different biological agents have been use for Se removal; however, the use of fungi is still in its infancy. In this study batch and continuous experiments were performed to investigate and exploit the use of Phanerochaete chrysosporium as a selenium-reducing organism for: the removal of selenite from water, the production of elemental selenium nanoparticles and its potential application as a new adsorbent for the removal of heavy metals. The white-rot fungus P. chrysosporium was selected because of its well known ability to degrade organic compounds [2,3], to synthesize metal nanoparticles [4,5] and to self-immobilize as pellets, providing a series of advantages in technical applications in comparison to dispersed mycelial growth [6]. P. chrysosporium was found to be highly sensitive to selenite, but not to selenate. Under batch conditions (ph 4.5, 10 mg glucose L-1, 10 mg Se L-1, 30 C) Se removal efficiency by P. chrysosporium was about 40-50% for SeO32-, and only about 10% for selenate. Biomass production and substrate consumption were inhibited for SeO32- incubations, which also showed different fungal pellet morphology compared to incubations without Se. Moreover, SeO32- incubations resulted in the intracellular synthesis of elemental selenium nanoparticles in the nano-size range between nm (Figure 1). Use of fungal pellets of P. chrysosporium in an up-flow bioreactor for the continuous removal of selenite was investigated. The system was operated over 40 days (1 g COD L-1 d-1, 10 mg Se L-1 d-1, flow rate L h-1, HRT 24 h, ph 4.5, 30 C) achieving steady-state removal profiles (8 d, 70% removal). The response of the fungal-pelleted bioreactor to shock-loads and intermittent spikes of selenite was tested, showing good resilience to overstressed selenite concentrations, as well as good adaptability and flexibility by recovering to every intermittent spike of SeO32-, achieving 70% total Se removal. As a parallel line of research, the potential of using selenium nanoparticles immobilized fungal pellets (SNIFP) of P. chrysosporium as biosorbent to remove Zn2+ from wastewater was assessed. In batch experiments (50 mg Zn2+ L-1, 150 rpm, 24 h, ph 4.5, 30 C), the metal uptake capacity of SNIFP (11.3 mg g-1) was found to be higher than fungal pellets grown without any source of selenium (8.5 mg g-1). The effect of different parameters (i.e. ph, ionic strength, biomass dose) on the adsorption capacity of SNIFP was determined, as well as the isotherms and the adsorption kinetics. 46

48 P. chrysosporium pellets grown with selenite Intracellular nse0 Figure 1. Fungal pellets of P.chrysosporium incubated with selenite containing elemental selenium nanoparticles. References Espinosa-Ortiz E, Lens PNL (2013). Selenium stress in fungi: potential applications. In: Proceeding of the 3 rd International Conference on Research Frontiers in Chalcogen Cycle Science and Technology, Delft, The Netherlands, 27th-28th May 2013, Rene ER, Kijjanapinich P, Lens PNL, (Eds): Moldes D, Rodríguez S, Cameselle C, Sanromán MA (2003) Study of the degradation of dyes by MnP of Phanerochaete chrysosporium produced in a fixed-bed bioreactor. Chemosphere 51: Wang C, Sun H, Li J, Li Y, Zhang Q (2009) Enzyme activities during degradation of polycyclic aromatic hydrocarbons by white rot fungus Phanerochaete chrysosporium in soils. Chemosphere 77: Sanghi R, Verma P, Puri S (2011) Enzymatic formation of gold nanoparticles using Phanerochaete chrysosporium. Adv Chem Eng Sci 1(3): Vigneshwaran N, Kathe AA, Varadarajan PV, Nachane RP, Balasubramanya RH (2006) Biomimetics of silver nanoparticles by white rot fungus, Phanerochaete chrysosporium. Colloids Surf B Biointerfaces 53: Pazouki M, Panda T (2000) Understanding the morphology of fungi. Bioprocess Eng 22: 47

49 SOIL TREATMENT

50 Citric acid and TWEEN 80-assisted phytoremediation of multicontaminated soils vegetated with alfalfa Ana Carolina Agnello1,2,*, David Huguenot2, Eric van Hullebusch2, Giovanni Esposito1 1 Dipartimento di Ingegneria Civile e Meccanica, Università degli Studi di Cassino e del Lazio Meridionale, Italia. Géomatériaux et Environnement, Université Paris-Est Marne-la-Vallée, France. *Corresponding author ac.agnello@unicas.it 2 Laboratoire Keywords: heavy metals; hydrocarbons; organic acids; phytoremediation; surfactants. Introduction: The combination of phytoextraction and rhizodegradation phytotechnologies can be employed together with the aim to remediate soils multi-contaminated by inorganic and organic pollutants [1]. However, low bioavailability of contaminants represents a significant limitation in phytoremediation [2]. To overcome this constraint, biodegradable amendments such as low molecular weight organic acids with metal chelating ability and surfactants, which enhance the solubility of organics, can be added to soils [3]. Objectives: To assess the phytoremediation potential of alfalfa (Medicago sativa L.) in a multicontaminated soil as well as the effects of citric acid and Tween 80 (polyethylene glycol sorbitan monooleate), applied individually and in combination, on the phytoremediation process. Methodology: The experimental design included a pot experiment in the growth chamber. One month old alfalfa seedlings were transplanted to a soil contaminated by heavy metals (Cu, Pb, Ni and Zn) and petroleum hydrocarbons. Pots were fortnightly treated with citric acid (15 mmol kg-1 dry soil), Tween 80 (0.036 mmol kg-1 dry soil), or the combination of both compounds. Vegetated and non-vegetated controls received distilled water instead of amendments. Each condition was repeated in triplicates. Plants were harvested after 30, 60 and 90 days, every time three days after amendment application. Soil samples (from the rhizosphere in the case of vegetated pots) were taken at the same times. Plant parts were dried, weighed, mineralized and analyzed for their content in Cu, Ni, Pb and Zn by ICP-OES. The number of aliphatic hydrocarbon degraders and the activity of lipase enzyme were determined in soil samples by the most-probable-number method and a colorimetric assay, respectively [4, 5]. Results: The results showed that alfalfa plants could tolerate and grow in the multi-contaminated soils. Over the 90-day experimental time, shoot and root biomass increased and negligible plant mortality arose. Heavy metals were uptaken by alfalfa to a limited extent, and mostly by plant roots. Heavy metal concentration in plant tissues were in the following order: Zn > Cu > Pb > Ni. Alfalfa rhizosphere effect was manifest, enhancing both microbial population (alkane degraders) and activity (lipase enzyme), with rhizosphere effects of and 2.04, respectively, after 90 days. Soil amendments did not significantly enhance plant metal concentration or total uptake. By contrast, the combination of citric acid and Tween 80 significantly improved alkane degraders (5.3-fold increase) and lipase activity (1.0-fold increase) in the rhizosphere of amended plants, after 30 days of experiment. Conclusions: This evidence supports the phytoremediation potential of alfalfa species to facilitate the remediation of multi-contaminated soils and the possibility to enhance the phytoremediation process through the joint application of citric acid and Tween 80. References: [1] Salt, D.E., Smith, R.D., Raskin, I., Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology 49, [2] National Research Council, Bioavailability of Contaminants in Soils and Sediments: Processes, Tools, and Applications. The National Academies Press, Washington, DC. [3] Agnello, A.C., Huguenot, D., van Hullebusch, E.D., Esposito, G., Enhanced phytoremediation: a review of low molecular weight organic acids and surfactants used as amendments. Critical Reviews in Environmental Science and Technology. DOI: / [4] Wrenn, B.A., Venosa, A.D., Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure. Canadian Journal of Microbiology 42, [5] Margesin, R., Feller, G., Hämmerle, M., Stegner, U., Schinner, F., A colorimetric method for the determination of lipase activity in soil. Biotechnology Letters 24,

51 Emerging contaminants from water discharged in soil: effects and treatment strategies L. Pontoni*, G. Esposito*, P. N. Lens**, F. Pirozzi*** *University of Cassino and Southern Lazio, Cassino, Italy **UNESCO IHE Institute for water education, Delft, The Netherlands ***Univesity of Naples Federico II, Naples, Italy Keywords: emerging contaminants; tertiary treatment; wastewater reuse; soil/water equilibria; bioavailability; Since the world population is steadily increasing, water demand is in rapid growth. Projections until 2025 are stating that many countries are entering a period of severe water shortage. On the other hand, most of water consumption is destined to irrigation purposes (Seckler 1998). In most places, the water used for agriculture is inefficient and consumptive. For these reason strategies for water recovery and reuse are being developed since the last decades in many countries around the world, primarily in arid and semi-arid regions (Avnimelech 1993). One possible mechanism is the recycling of wastewaters and drainage waters that can be used instead of fresh water sources for irrigation. Municipal wastewater reuse is the most promising reuse prospect, since it improves the security of supply; it reduces the resource constraints due to variable rainfalls, and reduces the need for expensive water storage construction and freshwater supply infrastructure. This kind of strategy while showing undeniable advantages gives place to concerns and unknowns about the impact of the quality of the recycled water, both on the receiving soil and on the crop itself. Water quality issues that can create real or perceived problems in agriculture include nutrients and sodium concentration, heavy metals and the presence of contaminants such as infective pathogens for human and animal, pharmaceutical and endocrine disruptors (Toze 2006). This wide range of pollutants can be adsorbed on both inorganic and organic fraction of irrigated soils (Afifi et al. 2011) giving often rise to bioaccumulation and biomagnification effects (Sinha et al. 2006), with related health concerns (Chen et al. 2004). Hence, as stated by the Hyderabad declaration on Wastewater use in Agriculture: without proper management, wastewater use poses serious risks to human health and the environment. In the last decades several researches are breaking out, identifying a class of both organic and inorganic contaminants described as Contaminants of Emerging Concerns (CECs), that are often not ruled by precise water re-use guidelines and same time of not completely known behaviour in the environment as well as against the human health (Muñoz et al. 2009). The main problem is thus related to their very low concentration in treated waters, requiring, these compounds, advanced analytical techniques for detection and quantitation. The approach is anyway to reduce, or to avoid when possible, the release of micro-pollutants in the soil. The fate of micro-pollutants in the environment is in fact really hard to predict, since mechanisms of mobility through the soil are very heterogeneous and strongly related to its physical-chemical nature, to the soil-water retention and its fluid transmission characteristics (Mingorance et al. 2007). Soil water sorption-release equilibria are hence really complex and uneasy to model or generalize (McGechan and Lewis 2002). It follows that at actual knowledge a soil-by-soil as well as water by water experimental approach is required. The present research project aims to find new strategies to remove soil-accumulable micropollutants and disinfection by-products from waters intended for reuse. It also aims to investigate the effect on soil of treated wastewater and mobility and bio-availability of pollutants through soils: after the initial literature study some of the most common CECs found present in treatment facilities outputs are identified and selected to be studied. The treatment technologies that will be object of study will include photolysis (Jo et al. 2011) and ozonation (Huber at al. 2005) at high dosage and adsorption using Carbon Nanotubes (CNTs) (Chen et al. 2007) as sorbent material for emerging contaminants. A second part will concern the evaluation of the effect of reclaimed wastewater on irrigation of soils. Thus the interactions with all the components of soil will be evaluated, in order to model the possible accumulation of the studied pollutants even at trace concentration. In order to have generalizable data and repeatable experiments an artificial model soil (Wilhelm and Maibach 50

52 2008) will be built in order to make easier separable, and thus compatible with mathematical modelling, the effects of all the variables that are involved in the complex interaction between soil and pollutants. The results obtained from the model soil will be compared with a selection of real soils in order to verify the reliability of the obtained data and their transferability to real systems. The ability of retaining trace pollutants will be evaluated at changing the soil properties by operating on the model soil composition in order to find correlations between the mobility of pollutants and the chemical composition and structure of both organic and mineral matters composing the soil. References Afifi, A.A., El-Rheem, K.M.A., Youssef, R.A. (2011). Influence of Sewage Water Reuse Application on Soil and the Distribution of Heavy Metals. Nat. and Sci., 9, Avnimelech, Y Irrigation with sewage effluents: The Israeli experience. Environ. Sci. Technol., 27, Chen, Y., Wang, C., Wang, Z., Huang, S. (2004). Assessment of the contamination and genotoxicity of soil irrigated with wastewater. Plant Soil, 261, Chen, W., Duan, L., Zhu, D. (2007). Adsorption of polar and nonpolar organic chemicals to carbon nanotubes. Environ. Sci. Technol., 41, Huber, M.M., GÖbel, A., Joss, A., Hermann, N., LÖffler, D., McArdell, C.S., Ried, A., Siegrist, H., Ternes, T.A., von Gunten, U. (2005). Oxidation of pharmaceuticals during ozonation of municipal wastewater effluents: a pilot study. Environ. Sci. Technol., 39, Jo, C.H., Dietrich, A.M., Tanko, J.M. (2011). Simultaneous degradation of disinfection byproducts and earthy-musty odorants by the UV/H< sub> 2</sub> O< sub> 2</sub> advanced oxidation process. Water Res., 45, McGechan, M., Lewis, D. (2002). SW Soil and water: Transport of particulate and colloid-sorbed contaminants through soil, Part 1: General Principles. Biosys. Eng., 83, Mingorance, M., Gálvez, J.F., Peña, A., Barahona, E. (2007). Laboratory methodology to approach soil water transport in the presence of surfactants. Coll. Surf. A: Physicochem. Eng.Asp., 306, Muñoz, I., Gómez-Ramos, M.J., Agüera, A., Fernández-Alba, A.R., García-Reyes, J.F., Molina-Díaz, A. (2009). Chemical evaluation of contaminants in wastewater effluents and the environmental risk of reusing effluents in agriculture. TrAC, Trends Anal. Chem., 28, Seckler, D.W. (1998) World water demand and supply, 1990 to 2025: Scenarios and issues, Iwmi Sinha, S., Gupta, A., Bhatt, K., Pandey, K., Rai, U., Singh, K. (2006). Distribution of metals in the edible plants grown at Jajmau, Kanpur (India) receiving treated tannery wastewater: relation with physico-chemical properties of the soil. Environ. Mon. Assess., 115, Toze, S. (2006). Reuse of effluent water benefits and risks. Agric. Water Manage., 80, Wilhelm, K.-P., Maibach, H.I OECD guidelines for testing chemicals. Dermatoxicology, 7th edn. CRC Press, Boca Raton,

53 HMs removal by soil washing: efficiency and economic optimization/engineering configuration A. Ferraroa,b,c, G. Espositoa, M. Fabbricinob, E. van Hullebuschc c University of Cassino and Southern Lazio, Department of Civil and Mechanical Engineering, Cassino, Italy University of Naples Federico II, Department of Civil, Construction and Environmental Engineering, Naples, Italy c Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM, Marne-la-Vallée, France Mail: alberto.ferraro3@gmail.com b Keywords: soil washing; heavy metals; heavy metal:chelant complexes; chelants recovery; biodegradability tests; The release of heavy metals and other pollutants made soil contaminations one of the most important environmental problem [1] and biogeochemical mobilization of these elements can contaminate drinking water supplies or results in uptake by vegetation and potential food chain impacts [2], then, the treatment of contaminated soil is necessary. Chemical-enhanced soil washing has been proved to be a promising technology for the remediation of contaminated soils with potential applicability and economic feasibility [3]. In this research, a soil washing process, involving Ethylenediamine-N,N -disuccinic (EDDS) acid as extracting agent, has been employed for the remediation of an heavy metals (HMs) contaminated agricultural soil. Samples of the naturally contaminated soil has been collected from Castel San Giorgio (Italy). Tests has been carried out for the determination of the soil properties (ph, particle size distribution, cation exchange capacity, field moisture, electrical conductivity, volatile solids) and contaminants characteristics in terms of total concentration and fractionation in soil. This last has been determined through a modified BCR three-step sequential extraction procedure [4]. The process has been focused on the removal of copper (Cu) and chromium (Cr) which show a concentration above the limit values of Italian normative (D.lgs 152/06). In a first step, the soil washing has been carried out in CSTR conditions through batch tests with a lifetime of 96 hours to investigate the kinetics of HMs release. These tests have been done changing the values of the process parameters (liquid to soil ratio, EDDS:HMs molar ratio) to find the optimal washing conditions and study the relation between removal efficiency and process parameters; the optimal conditions (liquid to soil ratio, EDDS volume, retention time) has been employed in a second washing configuration. New tests simulated a plug-flow washing through a multi-step washing with different fresh solutions on the same soil. The sum of EDDS volume and retention time of all the steps was equal to the total EDDS volume and retention time considered as optimum from the CSTR tests to make a comparison in terms of efficiency/cheapness between the two configuration. In a second step of the research, electrochemical treatment has been involved for the remediation of the washing solution after the soil treatment. The aim is to remove the HMs from to solution for its reuse in further soil treatments. In order to optimize the process electrodeposition tests have been made on synthetic solutions of EDDS and metals (Cu, Ca, Fe, Mn, Mg) in the same concentrations resulting from the maximum removal achieved in the optimal conditions selected from the CSTR tests. The experiments has been carried out using iron electrodes and changing process parameters such as ph of the solution, current density, conductivity in order to investigate the relation between the removal efficiency and the parameter values, evaluate the kinetics of the process and to find the optimal treatment conditions. Later, the optimal values will be employed for a treatment of a real washing solution after the remediation of a contaminated soil; the effectiveness of the process will be evaluated reusing the treated washing solution and determining its removal efficiency in further soil washing steps. 52

54 References [1] Z. Arwiddson, K. Elgh-Dalgren, T. von Kronhelm, R. Sjoberg, B. Allard, P. van Hees, Remediation of heavy metal contaminated soil washing residues with amino polycarboxylic acids, Journal of Hazardous Material 173 (2010) [2] H.A. Elliot, N. L. Shastri, Extractive decontamination of metal-polluted soils using oxalate, Water, Air and Soil Pollution 110 (1999) [3] R. J. Tobia, Final report: Pilot-scale soil washing study, Rep. No. EPA Contract , U.S. Environmental Protection Agency, Washington, D.C., [4] M. Pueyo, J. Mateu, A. Rigol, M. Vidal, J.F. Lopez-Sanchez, G. Rauret, Use of the modified BCR three-step sequential extraction procedure for the study if trace element dynamics in contaminated soils, Environmental Pollution 152, 2008,

55 Landfarming of PAHs Contaminated Soils Combined with Composting of Organic Waste Lukić B.1, Panico A.2, Huguenot D.3, van Hullebusch E.D.3, Esposito G.1, Pirozzi F.4 1Department of Civil and Mechanical Engineering, University of Cassino and the Southern Lazio, Via Di Biasio, 43, Cassino, FR, Italy; 2Telematic University Pegaso, Piazza Trieste e Trento 48, Napoli, Italy; 3Université Paris-Est, Laboratoire Géomatériaux et Environnement (LGE), EA 4508, UPEM, Marne-la-Vallée, France; 4Department of Civil, Architectural and Environmental Engineering, University of Naples Federico II, via Claudio 21, 80125, Napoli, Italy. Keywords: polycyclic aromatic hydrocarbons; bioremediation; landfarming technology; composting of organic waste; synthetic soil. Polycyclic Aromatic Hydrocarbons (PAHs) are included among the most common organic pollutants in the environment as they are frequently detected in soils and sediments as well as in groundwater and atmosphere. Such compounds are highly hydrophobic and tend to be easily adsorbed onto the organic matter of solid particles forming persistent organic pollutants that are toxic, and some of them even cancer-causing. For this reason a large number of studies have been recently conducted in order to find the most suitable techniques for PAHs removal from contaminated soils. Bioremediation processes are innovative methods used to remove organic pollutants from contaminated soils: they need a low technology to be performed even if they need long times for a successful biodegradation. Among biological treatments landfarming is very promising since it is based on naturally occurring bio-processes that consume or immobilize contaminants rather than transfer them to other environmental medium contributing thus to cost reduction. Although landfarming has been already used successfully to reduce the concentration of PAHs in contaminated soil, concerns remain about the long process time, its effectiveness in reducing the level of recalcitrant PAHs and the potential toxicity of metabolites generated during the degradation process. The use of combined processes, i.e. landfarming combined with composting of organic waste, might contribute to achieve a higher PAHs removal rate, a shorter removal time and, at the same time, reduce the quantity of waste landfilled. The present research is actually focused on investigating the effects of such combined process on the remediation of PAHs contaminated soils. For this reason four different types of organic wastes, i.e. fruit and vegetable waste, food and kitchen waste, sewage sludge from wastewater treatment plant and buffalo manure, have been selected and separately added in the ratio 1 to 5 as dried mass to a synthetic soil artificially contaminated with 4 different PAHs, i.e. anthracene, chrysene, benzo[k]fluoranthene and benzo[a]pyrene, in high concentration. The experiments have been conducted in bench scale reactors under controlled conditions. Through this research work it might be expected to achieve new insights and knowledge in the implementation of environmental technologies such as landfarming and composting process using an integrated approach. The limiting factors such as process time, nutrients, ph, moisture level, presence of microorganisms and toxic metabolites during composting processes should be investigated, considered and adequately managed in order to optimize the biodegradation of both low and high molecular weight PAHs. 54

56 Treatment of soil washing / soil flushing solutions by electrochemical advanced oxidation processes (EAOPs) and integrated treatments Clément TRELLU*, Emmanuel MOUSSET*, David HUGUENOT*, Yoan PECHAUD*, Nihal OTURAN*, Eric D. VAN HULLEBUSCH*; Giovanni ESPOSITO**, Mehmet A. OTURAN* * Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM, Marne-la-Vallée, France, clement.trellu@u-pem.fr ** University of Cassino and the Southern Lazio, Department of Civil and Mechanical Engineering, Via Di Biasio, 43, Cassino, FR, Italy, giovanni.esposito@unicas.it Keywords: soil washing / soil flushing; electrochemical advanced oxidation processes; integrated treatments; polycyclic aromatic hydrocarbons; humic substances. The persistence of hydrophobic organic compounds (HOCs) in soils is an issue of significant public, scientific and regulatory concern. Indeed, some HOCs have high persistence in the environment, potential toxicity, mutagenicity, carcinogenicity and are able to be bioaccumulated and bioconcentrated (EU - Stockholm Convention, 2004). Therefore, this pollution should be treated to avoid its adverse impacts. Regarding the removal of HOCs from soil, it is delicate to deal with economic, environmental and health issues. The achievement of sufficient efficiency involves the use of expensive treatments (Colombano et al., 2010). This is the reason for which industrial stakeholders are still looking for effective, less expensive and more environmentally friendly processes. During the two last decades, promising results have been obtained concerning the use of extracting agents (surfactants, biosurfactants, cyclodextrins, cosolvents, etc.) in order to improve the removal of HOCs from soil by using soil washing (SW) (ex-situ) or soil flushing (SF) (in-situ) processes (Mulligan et al., 2001; Paria, 2008; Mousset et al., 2014). However, SW/SF solutions must be treated as they are highly contaminated (Villa et al., 2010). Moreover, it is necessary to reuse extracting agents for further SW/SF steps in order to perform the treatment in a cost-effective way (Gómez et al., 2010a; Gómez et al., 2010b). The main goal of this work is the study of an integrated process for the treatment of SW/SF solutions. Particularly, it is aimed at assessing the efficiency of the combination of the following processes: selective adsorption step of target pollutants in order to recover extracting agents; electro-fenton and/or anodic oxidation (EAOP) processes for the degradation of organic compounds refractory to conventional oxidation techniques; post-biological treatment for the mineralization of by-products formed during EAOPs in a costeffective way. This research project also focuses on a better understanding of hydroxyl radical oxidation mechanisms during SW/SF solution treatment by EAOPs (Brillas et al., 2009; Oturan and Aaron, 2014). Particularly, the influence of nature and concentration of extracting agents and soil organic matter on target HOCs removal will be investigated. The degradation of each compound will be studied individually and compared to results obtained in synthetic SW/SF solutions. Thus, competitive and/or synergistic effect will be highlighted. References Brillas; E.; Sirés, I.; Oturan, M.A. (2009). Electro-Fenton process and related electrochemical technologies based on Fenton s reaction chemistry. Chem. Rev., 109, Colombano, S.; Saada, A.; Guerin, V.; Bataillard, P.; Bellenfant, G.; Beranger, S.; Hube, D.; Blanc, C.; Zornig, C.; Girardeau, I. (2010). Quelles techniques pour quels traitements Analyse coûts-bénéfices. BRGM/RP, 58609, FR. 55

57 EU - Stockholm Convention (2004). The global Stockholm Convention on Persistent Organic Pollutants, opened for signatures in May 2001 and entered into force on 17 May Gómez, J.; Alcántara, M.T.; Pazos, M.; Sanromán, M.A. (2010a). Remediation of polluted soil by a two-stage treatment system: desorption of phenanthrene in soil and electrochemical treatment to recover the extraction agent. J. Hazard. Mater., 173, Gómez, J.; Alcántara, M.T.; Pazos, M.; Sanromán, M.A. (2010b). Soil washing using cyclodextrins and their recovery by application of electrochemical technology. Chem. Eng. J., 159, Mousset, E.; Oturan, M.A.; van Hullebusch, E.D.; Guibaud, G.; Esposito, G. (2014). Soil washing/flushing treatments of organic pollutants enhanced by cyclodextrins and integrated treatments: state of the art. Crit. Rev. Environ. Sci. Technol., 44, Mulligan, C.N.; Yong, R.; Gibbs, B. (2001). Surfactant-enhanced remediation of contaminated soil: a review. Eng. Geol., 60, Oturan, M.A.; Aaron, J.J. (2014). Advanced oxidation processes in water/wastewater treatment: Principles and applications. A review. Crit. Rev. Environ. Sci. Technol., in press. Paria, S. (2008). Surfactant-enhanced remediation of organic contaminated soil and water. Adv. Colloid Interface Sci., 138, Villa, R.D.; Trovó, A.G.; Nogueira, R.F.P. (2010). Soil remediation using a coupled process: soil washing with surfactant followed by photo-fenton oxidation. J. Hazard. Mater., 174,

58 SOLID WASTE TREATMENT

59 Biohydrogen production from dark and photo fermentation processes Anish Ghimire*, Giovanni Esposito*, Francesco Pirozzi**, Luigi Frunzo*** *Department of Civil and Mechanical Engineering, University of Cassino and Southern Lazio, via Di Biasio 43, Cassino (FR), Italy **Department of Civil, Architectural and Environmental Engineering, University of Naples Federico II, via Claudio 21, Naples, Italy ***Department of Mathematics and Applications Renato Caccioppoli, University of Naples Federico II, via Claudio 21, Naples, Italy Keywords: biohydrogen yield; dark fermentation; photo fermentation, biomass valorization Biohydrogen (H2) from renewable sources such as biomass has been envisioned as the future fuel. Among biological H2 production systems, combined dark and photo fermentation process is a promising technology which can deliver higher conversion efficiencies and treat organic waste with valuable H2 as a product. However, lower yield and high cost of production are two existing major barriers for its development. A major challenge for scaled-up biohydrogen process is to achieve higher yield and utilization of dark fermentation (DF) residues (Das & Veziroglu, 2008; Ren et al., 2011). However, the limitations of DF development can be overcome by using low cost substrates such as waste biomasses, pre-treatment of substrates, inoculum enrichment methods and by optimizing bioreactor design and operation (De Gioannis et al.i, 2013; Li & Fang, 2007; Ntaikou et al., 2010; Show et al., 2012; Wong et al., 2014). Moreover, the volatile organic acids obtained as byproducts of dark fermentation can be converted to biohydrogen (H2) in photo fermentation process improving the overall H2 yield (Redwood et al., 2012). The present study is aimed at investigating different operating conditions for dark fermentative and photofermentative biohydrogen production and assess the biohydrogen potential (BHP) of range of complex real substrates such as food waste, olive mill wastewater (OMWW), cheese whey and rice straw. Different operating conditions of temperature and ph were tested to determine the optimum conditions for dark fermentative H2 production and the potential of the DF residues to be utilized in photofermentation process was evaluated to enhance the total yield of H2. The study of effect of initial ph on BHP showed acidic ph of favored dark fermentative H2 production. Similarly, the effect of food and microorganisms (F/M) ratios evaluated at two acidic and neutral ph of 5 and 6.5 respectively influenced the BHP and the fermentation pathways. The results of the studies with different inoculum sources and culture temperatures using OMWW revealed that the activated sludge as a hydrogen producing seed inocula under thermophilic conditions can offer higher BHP in comparison to anaerobic sludge and mesophilic conditions of dark fermentation. From the results of the effect of Organic Loading Rates (OLR), Hydraulic Retention Time (HRT) on continuous dark fermentative H2 production studied with semi-continuous bioreactor have shown the influence on the quantity and the quality of H2 production and fermentation pathways. Moreover, the effect of addition of buffalo manure as co-substrate with cheese whey affected the quantity and the quality of daily biohydrogen production in another study with thermophilic semi-continuous reactor. Furthermore, combining the photofermentative biohydrogen production with DF by utilizing its byproducts offered higher H2 yield. References Das, D., & Veziroglu, T. (2008). Advances in biological hydrogen production processes. International Journal of Hydrogen Energy, 33(21), doi: /j.ijhydene De Gioannis, G., Muntoni, a, Polettini, a, & Pomi, R. (2013). A review of dark fermentative hydrogen production from biodegradable municipal waste fractions. Waste Management (New York, N.Y.), 33(6), doi: /j.wasman Li, C., & Fang, H. H. P. (2007). Fermentative Hydrogen Production From Wastewater and Solid Wastes by Mixed Cultures. Critical Reviews in Environmental Science and Technology, 37(1), doi: / Ntaikou, I., Antonopoulou, G., & Lyberatos, G. (2010). Biohydrogen Production from Biomass and Wastes via Dark Fermentation: A Review. Waste and Biomass Valorization, 1(1), doi: /s

60 Redwood, M. D., Orozco, R. L., Majewski, A. J., & Macaskie, L. E. (2012). An integrated biohydrogen refinery : Synergy of photofermentation, extractive fermentation and hydrothermal hydrolysis of food wastes. Integrated Biohydrogen Refinery Projected net power kw / ha Sustainable power On-shore wind 20 kw / ha Crop-deri. Bioresource Technology, 119, doi: /j.biortech Ren, N., Guo, W., Liu, B., Cao, G., & Ding, J. (2011). Biological hydrogen production by dark fermentation: challenges and prospects towards scaled-up production. Current Opinion in Biotechnology, 22(3), doi: /j.copbio Show, K. Y., Lee, D. J., Tay, J. H., Lin, C. Y., & Chang, J. S. (2012). Biohydrogen production: Current perspectives and the way forward. International Journal of Hydrogen Energy, 37(20), doi: /j.ijhydene Wong, Y. M., Wu, T. Y., & Juan, J. C. (2014). A review of sustainable hydrogen production using seed sludge via dark fermentation. Renewable and Sustainable Energy Reviews, 34, doi: /j.rser

61 Combination of Thermal Pretreatment and Biofilm Technology to Enhance Anaerobic digestion Martha Yeshanew1, Giovanni Esposito1, Luigi Frunzo2, Piet Lens3,Francesco Pirozzi4 1Department of Mechanics, Structures and Environmental Engineering, University of Cassino and southern Lazio, via Di Biasio 43, Cassino (FR), Italy 2Department of Mathematics and Applications Renato Caccioppoli, University of Naples Federico II, Via Claudio, 21, 80125, Naples, Italy 3UNESCO-IHE Institute for Water Education, P.O. Box 3015, 2601 DA Delft, The Netherlands 4Department of Civil, Construction and Environmental Engineering (DICEA) University of Naples Federico II, Via Claudio, 21, 80125, Naples, Italy Keywords: Anaerobic Digestion; Thermal Pretreatment; Biofilm reactors; Trace Metals; Mathematical Model; Anaerobic digestion processes have been used for over a century for the effective treatment of wastewater and organic wastes and are currently recognized as a major treatment technology for solid wastes. The main product of anaerobic digestion is a mixture of gas (i.e. biogas) characterized by a high potential energy. With the depletion of fossil fuel reserve and the increasing doubts about the biofuels produced from energy crops, biogas more and more appears as a real valuable energy source to be developed (Tilche & Galatola 2008). On the other hand the slow growing bacteria, methanogens, require large reactors and longer retention time to ensure complete degradation of organic matter in conventional anaerobic digesters (Poh & Chong, 2009). Recently considerable attention has been paid towards the development of high rate reactors in order to decrease reactor volume and maximizing methane production rate (Rajeshwari et al., 2000). Even though there were different kinds of high rate digesters, one of the most interesting studied technologies is anaerobic biofilm based processes, reported as a good technology for the optimization of AD process. In these systems, microorganism are attached to a solid surface and each other forming a micro colonies or biofilm. The adhesion of microorganisms over solid carriers with large specific surface areas, leads to high biomass concentration and reaction rates, thus less reactor volume is needed (Wang et al., 2010; Gong et al., 2011). Many researchers identified hydrolysis as a rate limiting step in most anaerobic digestion processes especially for particulate substrate. In order to improve hydrolysis rate, one possibility is to pretreat substrate. Thermal pretreatment represents one of the most promising technologies able to increase hydrolysis rate by splitting up the organic part of substrate into short chain fragments (Myint et al., 2007). For a biofilm reactor the pretreated substrate can easily diffuse into the active biofilms and methanization can takes place relatively at a faster rate. Trace metal requirement had also become a major concern for the stable operation and better performance of anaerobic digestion (Zhang et al., 2011). Trace metals play a crucial role in the growth and metabolism of anaerobic microorganisms where they are essential for many physiological and biochemical processes (Zandvoort et al.,2006). The degradation of volatile fatty acids (VFAs), particularly propionate had become faster by supplementing a single or combination of trace element solution. This research focuses on developing a two stage system constituted by a thermal pretreatment/hydrolysis phase (first stage) combined with a biofilm methanation technology (second stage) in order to optimize methane production and process stability. The optimal pretreatment temperature and time will be assessed by using appropriate BMP tests. Biofilm anaerobic digesters continuously fed performance will be investigated at different OLRs and HRTs. Also the effect of addition of trace metals on the pretreated substrate will be studied using batch tests. Moreover mathematical model will be developed for the integrated system by extending ADM1 model. 60

62 References Gong W.; Liang H.; Li W., Wang Z.(2011). Selection and evaluation of biofilm carrier in anaerobic digestion treatment of cattle manure. Energy, 36, Myint M.; Nirmalakhandan N.; Speece R. E.(2007). Anaerobic fermentation of cattle manure: Modeling of hydrolysis and acidogenesis. Water Research, 41, Poh P.E. and Chong M. F.(2009). Development of anaerobic digestion methods for palm oil mill effluent (POME) treatmenta Review. Bioresource Technology, 100,1 9. Rajeshwari K. V.; Balakrishnan M.;Kansal A.;Kusum L.; Kishore V.V.V. (2000). State-of-the-art of anaerobic digestion technology for industrial wastewater treatment. Renewable and Sustainable Energy Reviews, 4, Tilche A. and Galatola M. (2008). The potential of bio-methane as bio-fuel/bio-energy for reducing greenhouse gas emissions: a qualitative assessment for Europe in a life cycle perspective. Water Science & Technology, Wang W.; Hou H.; Hu S.; Gao Z. (2010). Performance and stability improvements in anaerobic digestion of thermally hydrolyzed municipal biowaste by a biofilm system. Bioresource Technology, 101:, Zandvoort M.H.; Hullebusch E.D.V.; Gieteling J.; Lens, P.N.L. (2006). Granular sludge in full-scale anaerobic bioreactors: Trace element content and deficiencies. Enzyme and Microbial Technology, 39, Zhang L.; Lee Y. W.; Deokjin J. (2011). Anaerobic co-digestion of food waste and piggery wastewater: Focusing on the role of trace elements. Bioresource Technology, 102,

63 Dry Discontinuous Anaerobic Percolation Digestion DDAPD S. Riggio*, R. Escudié*, M. Torrijos*, G. Esposito**, E. van Hullebusch*** *INRA-LBE Avenue des Etangs Narbonne, France **Università degli studi di Cassino e del Lazio Meridionale Viale dell'università, Loc. Folcara Cassino (FR), Italy *** Université Paris-Est, 5 Bd Descartes Marne-la-Vallée cedex 02, France Keywords: dry anaerobic digestion, percolation system, multi-stage, manure. Anaerobic digestion is a natural process based on the synergy of several microbial species taking place in anaerobic and reducing environmental conditions. During that process, in which organic matter (OM) is slowly decomposed to simpler compounds, a big part of the energy contained in it is transferred in very energetic molecules like methane CH4 [1], [2]. Broadly studied over the past 50 years, Anaerobic Digestion (AD) is a very known and appreciated process because of its unique common response to several actual problems: waste management, human pollution and energy source differentiation. Technologies based on this process have never stopped to change and improve their performances since the start. If, originally, AD was created to treat liquid effluent mainly, nowadays many AD plants are designed to treat waste in their natural dry form (MSW, rural waste, green waste, etc). Dry AD is overtaking Wet Anaerobic Digestion (WAD) in last years because of some important advantages: smaller reactor size, less mechanical parts and lower investment [3]. Dry Discontinuous Anaerobic Digestion Percolation digestor (DDAPd), a reactor where the solid phase is static and the liquid one is percolating through the bulk, seems to answer better than other to the need of farmers in treating animal wastes which are often under dry form. Moreover, manure, a mixture of feces, urine and straw, is a very common resource in French farms and anaerobic digestion responds to the manure environmental disposal obligations on one side and the need in energy and good quality fertilizer on the other side. DDAPd, and in particular the adaptation of this technology to manure, is not something well studied and a lot of scientific parameters, often set empirically by industries, need nowadays to find a scientific support in order to improve their performances. Two parameters play a major role in DDAPD : first the recirculation which controls the mass transfer, and second the microbial seedling. Since the mass transfer is the major problem in dry reactor, parameters like the recirculation frequencies and the flows as well as variations of these during the digestion or the technical ways to perform it are crucial [4] [6]. The seedling on the other side can influence the kinetics of the process; so the inoculum state (liquid or solid), the way of mixing it to fresh substrate and its origin are important to be defined [6] [8]. A third parameter, whose role is on one side to overcome the limitation of batch reactors, a discontinuous biogas production, and on the other side the poorer performances due to nonoptimal conditions, is the coupling strategies of several reactors in what is called a multi-stage process [9], [10]. Through the study of the evoked parameters this work aims at giving a scientific base at the setting and at the process control of AD plants utilizing DDAPD technology for manure digestion. References [1] R. Moletta, La Méthanisation, 2nd ed. Lavoisier, 2008, p [2] Y. Li, S. Y. Park, and J. Zhu, Solid-state anaerobic digestion for methane production from organic waste, Renew. ans Sustain. Energy Rev., vol. 15, pp , [3] L. De Baere, B. Mattheeuws, and F. Velghe, State of the art of anaerobic digestion in Europe, vol. 32, no. 0,

64 [4] S. Kusch, H. Oechsner, and T. Jungbluth, Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates Sigrid Kusch * Hans Oechsner Thomas Jungbluth, Int. J. Environ. Waste Manag., vol. 9, pp , [5] H. Benbelkacem, R. Bayard, A. Abdelhay, Y. Zhang, and R. Gourdon, Bioresource Technology Effect of leachate injection modes on municipal solid waste degradation in anaerobic bioreactor, Bioresour. Technol., vol. 101, no. 14, pp , [6] E. ten Brummeler and H. C. J. M. Horbach, Dry Anaerobic Batch Digestion of the Organic Fraction of Municipal Solid Waste, J. Chem. Tech. Biotechnol, vol. 50, pp , [7] E. ten Brummeler and I. W. Koster, Enancement of Dry Anaerobic Batch Digestion of the Organic Fraction of Municipal Solis Waste by Aaerobic Pretreatment Step, Biol. Wastes, vol. 31, pp , [8] Z. Cui, J. Shi, and Y. Li, Bioresource Technology Solid-state anaerobic digestion of spent wheat straw from horse stall, Bioresour. Technol., vol. 102, no. 20, pp , [9] D. P. Chynoweth, J. Owens, D. O. Keefe, J. F. K. Earle, G. Bosch, and R. Legrand, SEQUENTIAL BATCH ANAEROBIC COMPOSTING OF THE ORGANIC FRACTION OF MUNICIPAL SOLID WASTE, Wat. Sci. Tech., vol. 25, no. 7, pp , [10] H. S. Shin and S. K. Han, Multi-step sequential batch two-phase anaerobic composting of food waste, Environ. Technol., vol. 22, pp ,

65 Fermentative bio-hydrogen production from complex organic waste by suspended and attached growth hyperthermophilic bacteria Thermotoga neapolitana Pradhan N.1, Panico A.2, Dipasquale L.3, d'ippolito G.3, Esposito G.1, Lens P.4, Pirozzi F5. 1Department of Civil and Mechanical Engineering, University of Cassino and the Southern Lazio, Via Di Biasio, 43, Cassino, FR, Italy; 2Telematic University Pegaso, Piazza Trieste e Trento 48, Napoli, Italy; 3Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Pozzuoli, Napoli, Italy; 4UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, the Netherlands; 5Department of Civil, Architectural and Environmental Engineering, University of Naples Federico II, via Claudio 21, 80125, Napoli, Italy. Keywords: bio-hydrogen; dark fermentation; fermentation by-products; suspended growth; attached growth; mathematical modelling. Bio-hydrogen is globally considered by researchers as one of the clean energy vector that can face the current environmental issues: i) reduce the green house gas emission responsible for the global warming; ii) find an environmentally friendly substitute to the depleting non-renewable resources to provide the future the necessary energy supply. Bio-hydrogen production by dark and photo fermentation has enormous potential to replace current hydrogen production technologies such as pyrolysis and gasification, which mostly depend on fossil fuels. Batch and continuous tests have been conducted in laboratories using pure and mixed cultures of inoculum under various operating conditions and have shown promising results. The use of pure culture for bio-hydrogen production is interesting since the potential bio-hydrogen yield is close to its theoretical maximum value, and at the same time, it is challenging as the inoculum needs more specific environmental conditions unlike mixed culture inoculum: actually, any deviation from the standard environmental conditions can easily inhibit the process pathways thus resulting in decrease of net bio-hydrogen production. The current research has planned to use a hyperthermophilic bacteria namely Thermotoga neapolitana for fermentative bio-hydrogen production. The research will investigate the engineering aspects to produce bio-hydrogen and other valuable biological fermentation byproducts, i.e. lactic acid, using suspended as well as attached growth biological system in continuous stirred tank reactors. The effect of operating parameters such as hydraulic retention time, organic loading rate, ph, pressure of gas in the headspace and type of gas sparged to create anaerobic condition will be studied in detail. In addition to the previous aim, mathematical modelling will be performed to optimize the process conditions for hyperthermophilic fermentative bio-hydrogen production and design the full scale biological reactors. 64

66 Pretreatment methods to enhance anaerobic digestion of food waste Javkhlan Ariunbaatara,f, Antonio Panicob, Luigi Frunzoc, Franceso Pirozzie, Daniel Yehd, Giovanni Espositoa, Piet Lensf a Department of Civil and Mechanical Engineering, University of Cassino and the Southern Lazio, Via Di Biasio, Cassino (FR) - ITALY b Telematic University Pegaso, P.za Trieste e Trento, 48, Naples (Italy) c Department of Mathematics and Applications Renato Caccioppoli, University of Naples Federico II, Via Claudio, 21, 80125, Naples, Italy d Department of Civil and Environmental Engineering, University of South Florida, USA e Department of Civil, Architectural and Environmental Engineering, University of Naples Federico II, Via Claudio, 21, 80125, Naples, Italy f UNESCO-IHE Institute for Water Education (UNESCO-IHE), Westvest 7, 2611 AX Delft, The Netherlands Keywords: Food waste; pretreatment methods; trace metals; one-stage and two-stage continuously stirred tank reactor; anaerobic membrane bioreactor The growing global concerns on the increasing amount of waste, energy demand, and climate change have stimulated the research on the acceleration and enhancement of the anaerobic digestion (AD) process of organic solid waste. Food waste (FW) has a high moisture content and is readily biodegradable, it serves as a perfect substrate for AD, thus it was chosen as a substrate. Pretreatment methods to increase the biodegradability of FW, addition of trace metals to enhance the biochemical process of AD and various bioreactor configurations have been studied in the scope of this research. The effect of thermal and ozonation pretreatments of FW, addition of trace metals as well as the possibility to enhance the mesophilic batch reactor performance by applying thermophilic/hyperthermophilic pretreatments are studied through batch experiments. Physically separating the hydrolytic and methanogenic microbes is considered as a biological pretreatment, hence the comparison of one-stage and two-stage continuously stirred tank reactors (CSTR) is also tested in semi-continuous mode. In line with trace metals addition and biological pretreatment, it is expected that a co-digestion of FW with a manure of carnivores and herbivores could yield an interesting results. Hence, manures from big cat carnivores (tiger), omnivore (sloth bear), as well as herbivores (giraffe and llama) are also studied and compared with the results of a commonly reported cow manure. Furthermore, since the anaerobic membrane bioreactors (AnMBRs) can be operated at low hydraulic retention time (HRT) the biogas from FW could be recovered in a shorter time with a better quality digestate (permeate) for nutrient recovery, which could be a promising technology to enable the sustainable water-energy-food nexus. During the series of batch experiments the biomethane potential of untreated FW was in the range of ± 8.5 and ± 4.3 mlch4/gvs. All tested conditions of both thermal and ozonation pretreatments resulted in an enhanced biomethane production. The best result of ± 10.6 mlch4/gvs was achieved with 80 C, 90 min. On the basis of net energy calculations, the enhanced biomethane production could cover the energy requirement of the thermal pretreatment. In contrast the enhanced biomethane production with ozonation pretreatment is insufficient to supply the required energy for the ozonator. It was also found that the advantages of both mesophilic and thermophilic AD processes could be accomplished in a single batch digester. Based on batch experiments on the effect of thermophilic/hyperthermophilic hydrolysis prior to mesophilic biomethanation the best result of 44.1 ± 0.6% higher cumulative biomethane was achieved with the pretreatment at 50 C for 12h. Moreover, as biological pretreatment, the two-stage CSTR was compared with one-stage CSTR as control. Two-stage CSTR was more stable with better ph control, resistant to organic loading shocks, and has a higher biomethane production. Also a small amount of hydrogen was detected from the first stage of two-stage system makes it an attractive system for biohythane production. Re-circulation of digestate supernatant provided the necessary alkalinity, thus preventing an eventual failure by VFA accumulation. However, re-circulation also resulted in ammonium accumulation, yielding a lower biomethane production. Supplementation of trace metals 65

67 (Ni, Cu, Mn, Co, Zn, Fe, Al, B, W, Se and Mo) was found out to be a very effective way to enhance the AD of FW. Metals (II) could result in 15 40%, whereas metals (VI) could result in 7-31 % higher biomethane production, and metals (III) are toxic for anaerobes. Supplementing ug/l of Fe (II) and Se (VI) resulted in the best results of ± 2.87% and ± 5.69%, higher biomethane as compared to the control. 66

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69 ETeCoS3 PhD STUDENT PROFILES

70 FIRST COHORT

71 Treatment of high strength selenium-laden wastewaters Social relevance Selenium (Se) is a chemical element that belongs to the chalcogen family (together with sulfur, S, and tellurium, Te). As a consequence of its complex biogeochemistry, Se is cycled through different compartments of the environment in liquid, solid and gaseous forms. When rocks and minerals are mined and processed, or when coal is burned to generate energy, selenium is released in the environment. The margin between Se dietary deficiency and Se toxicity stretches only one order of magnitude Due to its bioaccumulative capacity, even low levels of selenium build up along the trophic levels (i.e. biomagnification) producing ecological disasters (e.g. Lake Belews; Kesterson Reservoire). All these incidents triggered intensive research and a set of actions to enforce strict regulatory Se discharge limits. The wastewaters generated mainly by mining, oil and metal refining, as well as by the coal-fired power generation sector, contain levels of selenium that are high above the permissible discharge limits. Treating selenium-laden wastewaters poses a great challenge because of their complexity. Our study focuses not only on treating soluble selenium oxyanions, selenate, (Se[VI], SeO42-), and selenite, (Se[IV], SeO32-), but also investigates the solid-liquid separation potential of biogenic elemental selenium, Se(0). Biogenic Se(0) exhibits colloidal properties that make its treatment challenging. We perform the treatment of selenium species by a mixed approach. Soluble selenium oxyanions are reduced to Se(0) by microbial metabolism (mixed and pure cultures). Colloidal Se(0) is separated from the bulk liquid by chemical dosing and by electrocoagulation. Technological challenges The physical-chemical treatment of toxic selenate and selenite is costly. Some physical-chemical treatment options generate high amounts of sediments needing further processing and disposal. Large amounts of wastewaters generated by the mining sector are difficult to treat using the conventional treatment approach. Complex wastewaters (e.g. flue gas desulfurization) contain also other toxic metals and have high total dissolved solid content. Colloidal Se(0) must be removed from the treated wastewater prior discharge because of its deleterious effects on filter feeders. CV researcher (PhD student) PhD Student: Lucian Staicu Contact: l.staicu@unesco-ihe.org or staiculucian@gmail.com Promoter: Professor Piet Lens (UNESCO-IHE, The Netherlands) Co-supervisors: Dr. Eric van Hullebusch (Université Paris-Est, France) Professor Mehmet Oturan (Université Paris-Est, France) 70

72 SECOND COHORT

73 PHD PROJECT: ASSISTED PHYTOREMEDIATION OF MULTI-CONTAMINATED SOILS (NOVEMBER 2011 OCTOBER 2014) SOCIAL IMPACT: As a result of different anthropogenic activities, soil resources remain contaminated with heavy metals and petroleum hydrocarbons, which represents a global environmental issue of great concern, particularly when such contaminants are present simultaneously in soils. Phytoremediation can be defined as the use of plants to remove pollutants from the environment or to make them harmless. The use of plants to treat environmental concerns is an emerging and eco-friendly remediation technology, whose main advantages include relatively low costs, few secondary wastes, low environmental impact and high public acceptance; while its main drawback is the longer restoration time that may be required to achieve clean-up goals. Phytoremediation strategies such as phytoextraction and rhizodegradation can be used to deal with heavy metal and hydrocarbon contaminated soils, respectively. However, a key factor limiting phytoremediation is the low bioavailability of these contaminants. To increase the ability of pollutants to be transferred from a soil compartment to plants and soil microorganisms, amendments like low molecular weight organic acids (LMWOAs) and surfactants can be added to soils and, as a consequence, it is possible to improve the effectiveness of the phytoremediation process. In addition, through bioaugmentation techniques it is also possible to enhance the performance of the remediation process. This approach consists in the introduction of bacteria able to metabolize the pollutants of concern. TECHNOLOGICAL CHALLENGE: This research plan proposes the use of LMWOAs and surfactants added as soil amendments as well as bioaugmentation techniques to introduce organic acid and/or surfactant-producing bacteria to contaminated soils. The general aim is to assist the phytoremediation process to attain an improved remediation of multi-contaminated soils. METHODOLOGIES: Phytoremediation experiments are performed at growth chamber. Effect of soil amendments on plant growth and development, are evaluated by a seed germination test, observation of plant visual toxicity symptoms, measurement of plant biomass and chlorophyll quantification. Bioavailability of heavy metals is studied by quantifying the bioavailable fraction of metals in soil and soil solution. Phytoextraction of metals is assessed by the determination of plant biomass, quantification of heavy metals in plant parts and calculation of phytoextraction parameters. Hydrocarbon rhizodegradation is assessed by quantification of hydrocarbons in soil, calculation of contaminant removal rate, quantification of soil alkane degraders and soil lipase activity. ANA CAROLINA AGNELLO ac.agnello@unicas.it Promoter: Prof. Giovanni ESPOSITO Co-Promoters: Prof. Eric VAN HULLEBUSCH & Prof. David HUGUENOT 72

74 Optimization of sulphate reduction PhD research November October 2014 Social Impact Sulphate pollution is a concern sin ce it can be the origin of several effects in the environment. Excessive quantities may pose health threats. In the absence of oxygen or nitrate it acts as an electron acceptor and is converted to hydrogen sulphide by anaerobic microorganisms. Hydrogen sulphide causes an unpleasant smell, corrosion problems, may lead to the mobilization of toxic metals and is fatally toxic to humans. Thus, it is important to desulphurize the wastewater before its discharge to the surface water. Biological anaerobic reduction of sulphate has been successfully applied for the treatment of sulphate contaminated wastewater from industries on a larger scale for many years as it offers the possibility of an efficient treatment with low operation costs using various organic and easily utilizable carbon sources. The end product is hydrogen sulphide; hence this technique is suitable for treatment of water containing dissolved metals which can be precipitated simultaneously and removed as stable precipitates of sulphide. The main limiting factor when building a large scale biological treatment process is the electron donor cost. Technological Challenge It is desirable to look for the optimization of the sulphate reduction process. In this study two approaches are proposed: the applicability of a cheap carbon source, i.e., methane and the optimization of the electron donor input by using process control. Additionally, the use of methane would close its cycle of utilization, decrease the emission of one of the most important greenhouse gases and reduce the risk of excess carbon source in the treated effluent. For biotechnological application some challenges need to be overcome. The current research will do so by using different strategies: 1) the use of microbial mats with active anaerobic oxidation of methane obtained from marine sediments as enrichment starting material; 2) the enrichment of the microorganisms in a membrane bioreactor; 3) the use of alternative substrates for sulphate reducing bacteria; 4) the modification of environmental conditions. In addition, bioprocess control will be applied to optimize the input of substrate in the biosystem. Controlling the production of sulphide in a bioreactor will be the first step for an automated heavy metal precipitation/recovery. The main objective is to develop a feasible control strategy for the control of biogenic sulphide production in sulphate reducing bioreactors. Two different strategies will be tested: a PID controller with gain scheduling and a model-based adaptive controller. CV-researcher (PhD student) Name: Nationality: Promoter: Co-promoters: Graduated: Joana Cassidy Portugal Prof Piet Lens (UNESCO-IHE, The Netherlands) Dr Henk Lubberding (UNESCO-IHE, The Netherlands) Prof Giovanni Esposito (University of Cassino, Italy) Dr Yu Zhang (Shanghai Jiao Tong University, Republic of China) MSc Environmental Engineering, University of Aveiro, Portugal j.cassidy@unesco-ihe.org 73

75 Studies on the fate of pharmaceuticals in aqueous media: synthesis, characterization and detection of abiotic transformation products using advanced oxidation processes PhD research November 2011-october 2014 Social and ecological impact Pharmaceuticals are classified as emerging pollutants of increasing concern due to possible negative impacts on human health and the environment. They are constantly introduced in sewage treatment plants either through excretion or disposal by flushing of unused or expired medication, or directly within the sewage effluents of wastewater treatment plants or hospitals. They have complex chemical structures, showing a wide range of persistence and being capable of reacting in an aqueous medium under the action of chemical, biological or physical agents. They end up in surface and ground water and can even be found in drinking water, representing adverse effects on terrestrial and aquatic organisms. Moreover, the transformation products from degradation of parent drug replace gradually the initial molecule in the environment which could cause a significant effect by the appearance of new pollutants that may be even more toxic that the parent one. Wastewaters treatments are mostly performed by oxidation reactions using chemical oxidants, such as chlorine or ozone, and also by biologic degradation, which are not efficient enough for the destruction of these compounds, provoking their release to the environment and the generation of some intermediates from their partial oxidation. Technological challenge Faced with this problem, we believe it is necessary to synthesize the transformation products (TPs) to both enable the development of methods for their determination, and the assessment of their potential toxicity. My thesis project aims to develop a multidisciplinary methodology to be drawn from the study of four selected pharmaceuticals on risk criteria and representative chemical structures, frequently encountered in water resources. We propose firstly, to prepare the largest number or TPs of a particular drug using three complementary approaches: bioconversion, electrochemical and chemical oxidation, and advanced oxidation processes. In a second step, analysis of all the obtained compounds is intended to provide most likely environmental transformation products which will be synthesized in order to develop analytical methods for their detection in environmental matrices and to study their potential toxicity. Expected results are the development of a predictive methodology applicable to the study of any environmentally questioned molecule. The benefits will be related to the pharmaceutical, cosmetic, agrochemical, chemical industries, as well as organizations and industries specializing in the management of water resources. 74

76 Leaching of Heavy Metals from Mining/Metallurgical Wastes and Metals Recovery PhD Research November October 2014 Social impact The proposed PhD research is done in collaboration with a recycling plant (France), which is mainly focused on catalyst and metallic oxide recycling and in the latest period it includes also the crushing step of battery recycling. In 23rd October 2001 European directive No. 2001/81/CE restricted the emissions of pollutants such as sulphur dioxide, nitrogen oxides, etc. into the atmosphere. Consequently, levels of sulphur in petrol's and diesels should not exceed 10 ppm as from The result of this directive is a significant increase in the amount of used catalysts, which are used in greater numbers by oil companies to achieve the threshold fixed by the European Commission. The used catalysts contain the components (alumina, sometimes silica, molybdenum, nickel, tungsten, phosphorus, etc.) and the harmful elements captured during the refining process (sulphur, carbon, hydrocarbons, nickel, vanadium, arsenic, etc.). Thus, disposal of spent catalysts represent an increasing environmental problem due to their metallic content, which is being considered as hazardous waste. In order to achieve recycling of this large amount of used catalysts, the recycling plant designed a process allowing the recovery of metals which is based on intelligent combinations of pyrometallurgy and hydrometallurgy. However, rainfall is causing leaching of the metals from the recycling plant site and storage area. Rainwater is collected in the collection reservoir and then taken by the wastewater treatment plant, treated and discharged into the nearby river. Rainwater in the reservoir is passing through the settling stage thus forming the sludge which is containing increased concentrations of different metals. Since some of the metals (Mo, Ni, Co, Cr, Zn and W) present in the sludge are especially valuable to the recycling plant (economically and ecologically), recovery of those metals is of primary importance in this PhD research. Technological challenge Goals of research are: Theoretical and experimental description of the various speciation, leaching and recovery processes Comparative review of their efficiency Critical analysis in terms of their advantages, disadvantages and limitations Focus on Mo recovery and additionally on recovery of other metals CV-researcher (PhD student) Name: Promotor: Supervisors: Graduated: Miss Mirjana Vemic Prof. Stéphanie ROSSANO (Université Paris-Est, UPE) Dr. Eric van HULLEBUSCH (Université Paris-Est, UPE) Dr. François BORDAS (Université de Limoges, UNILIM) Prof. Gilles GUIBAUD (Université de Limoges, UNILIM) Prof. Piet LENS (UNESCO-IHE) MSc. Environmental Science, Water Quality UNESCO-IHE, Delft, The Netherlands mirjana.vemic@gmail.com Management, 75

77 Biogenic Sulfide Production and Selective Metal Precipitation at low ph for E-waste Leachate Treatment PhD Research November October 2014 Social relevance Metal contamination in water is a wide spread issue around the world and these phenomena become a main concern in many areas due to the adverse effects on health especially human health. Anthropogenic sources have more effects than the natural processes, especially with industrial activities. The waste streams generated from these sources is usually low in ph and contains high metal concentrations. On the other hand, metal resource depletion also became a serious issue recently. The growth of these industries is increasing every year because of demand and advancement in new technologies. The proper wastewater treatment and the resource recovery from waste streams should thus be considered. Biological sulfide precipitation by sulfate reducing bacteria (SRB) in bioreactors has been developed to cope with these two objectives. Technological challenge Though there is many research on using SRB bioreactor to treat metal contaminated wastewaters using SRB bioreactor exists as two compartments. The first is to enrich the SRB to generate sulfide and pass the sulfide into another reactor which is set up to precipitate the metal and let it settle down. As a consequence, operating two reactors requires more space and operational costs. The Inversed Fluidized Bed (IFB) reactor is a solution which is developed and can satisfy both needs on metal treatment and recovery within one reactor. The requirement for this set up is less than the existing technology. Moreover, the biomass which is attached on the carrier material also benefits in easiness for separation of the metal precipitates from the biomass. The main objective of this research is to evaluate the potential use of this technology for wastewater treatment, especially high level metal contaminated wastewater like e-waste leachate. Name: Suthee Janyasuthiwong Host institute: UNESCO-IHE, the Netherlands Promotor: Prof. Piet Lens (UNESCO-IHE, the Netherlands) Co-promotor: Prof. Giovanni Esposito (UNICAS, Italy) Contact: s.janyasuthiwong@unesco-ihe.org 76

78 PreTreatment Methods To Enhance Anaerobic Digestion of Organic Solid Waste PhD Research November 2011 October 2014 Social impact Anaerobic digestion (AD) is one of the oldest and well-studied technologies for stabilizing organic wastes. AD offers advantages over other available treatment technologies, due to its limited environmental impacts and high potential for energy recovery. Such positive aspects coupled with the recent concerns on rapid population growth, increasing energy demand, and global warming have promoted further research on the AD process development and improvement in order to enhance biogas production, achieve faster degradation rates and reduce the amount of final residue to be disposed. Food waste (FW) is the single largest fraction of municipal solid waste, and FAO suggests that 1.3 billion ton of FW is generated every year. Effective treatment of such huge amount of FW with AD has a potential to produce 894 TWh/year, which is approximately 5% of the total global electrical energy utilization. Technological challenges The main concern of treating OSW with AD is the long retention time, as the anaerobes must be given a sufficient time to reproduce. To reduce the retention time while maximizing the biogas production, pretreatment methods of OSW can be applied. Moreover, addition of trace metals enables the anaerobic microbes to produce the appropriate enzymes for the degradation of OSW, which leads to an enhanced AD process. Therefore, the main objective of this research is to investigate the various methods to enhance AD of FW by applying pretreatment methods as well as adding trace metals. Nevertheless, microbial wash out due to excessive loading of OSW could still be a drawback. Hence, to prevent from such wash out a novel anaerobic membrane technology is also studied. Considering the huge potential of energy recovery, this research focuses solely on the AD of FW. CV - Researcher: Name: Nationality: Graduated: Javkhlan Ariunbaatar Mongolia MSc Environmental Engineering and Management, Asian Institute of Technology, Thailand Promoter: Giovanni Esposito (University of Cassino and Southern Lazio, Italy) Co-promoters: Francesco Pirozzi (University of Naples, Federico II, Italy) Piet N.L. Lens (UNESCO-IHE, Netherlands) Daniel H. Yeh (University of South Florida, USA) jaka@unicas.it, j.ariunbaatar@unesco-ihe.org 77

79 Filamentous growth of neutrophilic bacterium Sphaerotilus natans : role in the scavenging of inorganic pollutants Erasmus Mundus Joint Doctorate Programme (ETeCoS3) : November 2011-October 2014 Social impact Heavy metals and radionuclides are present in ecosystems worldwide due to the release from natural sources or to anthropogenic activities, such as mining, ore processing and disposal. The use of microorganisms to restore those polluted ecosystems (process known as bioremediation) is nowadays of increasing interest. Bioremediation has been specially studied in acidic mining environments, were bacteria participate in the immobilization of inorganic pollutants such as As, Sr, Cd, Cs, Pb, U, etc. However, in nearneutral ph environments, which are more frequent in nature, little is known about such scavenging processes, especially those related to the bacteriogenic iron oxides (BIOS) formed by iron-related bacteria due to the challenge presented by Fe reactivity under those conditions. Technological challenge The aim of this project is to study the formation of BIOS by neutrophilic ironrelated sheath-forming filamentous bacteria, and to elucidate factors affecting such filamentation. It is also of interest to investigate biosorption and BIOS scavenging of heavy metals and radionuclides on water systems. In addition, comparison between BIOS and their abiotic hydrous ferric oxides (HFO) analogues will reveal the most efficient way to scavenge these inorganic pollutants, as biofilm and filamentous growth of the bacteria might simplify the pollutants recovery. In this context, special interest is focused on the neutrophilic iron-related sheath-forming bacterium Sphaerotilus natans, as unlike other iron-related bacteria such as Gallionella or Leptothrix, it is easily grown under laboratory conditions. Mechanisms of S. natans filamentation, biomineralization and biosorption its potential in the scavenging of inorganic pollutants are thus of major interest. and The microbiology approach suggested, based on molecular biology, will shed light on the physiology and factors affecting sheath formation of S. natans. In parallel, mineralogical investigations together with chemical analyses will be used to improve the present knowledge on iron biomineralization, the nature of the forming BIOS and the efficiency of the (bio)sorption of heavy metals and radionuclides at circumneutral ph. Microbiology Lectin binding observed under Confocal Laser Scanning Microscopy (CLSM) Quantitative Polymerase Chain Reaction (qpcr). Mineralogy Synchrotron-based spectroscopies: o XANES o EXAFS Scanning Electron Microscopy - Energydispersive X-ray detection (SEM-EDX) Analytical techniques Infrared spectroscopy (IR) Inductively coupled plasma mass spectrometry/optical emission spectrometry (ICPMS/OES) Flame atomic absorption spectrometry (F-AAS) 78

80 Contributions of Isotopic Techniques to the Study of Metallurgical Tailings Bio/weathering PhD Research November 2011 October 2014 Research background Metallurgical tailings or slags are the vitreous by-products or wastes resulting from the metal smelting industries. This research focuses on the study of Pb and Zn in slags resulting from the pyrometallurgical activities of Lead Blast Furnace and Imperial Smelting Furnace (LBF and ISF, resp., Noyelle-Godault, N. of France). Slags are often enriched in potentially toxic elements (Pb, Zn) which can be leached out through weathering or alteration processes and poses the treat to the environment. Stable isotopes & analytical techniques Isotopic techniques will be used in this study to identify the slags bioweathering mechanisms in natural environment, due to Pseudomonas aeruginosa. The main issue will be to decipher between the biological and chemical processes (dissolution, precipitation, complexation and adsorption) affecting the elements behavior. Hence, the analytical techniques to be applied are HR-ICP-MS (Neptune and Element II), ICP-OES, XRF, XRD, and SEM/EDS. In parallel to the chemical batch experiment, the metal speciation in the leachates will be determined using the Visual Minteq software. Interesting outcomes Zn isotopes fractionation during slag chemical dissolution is expected to be related to its mineral phases present in both slags. Both ISF and LBF displayed δ66zn signatures enriched in heavier isotopes (i.e ±0.04 and ±0.03, respectively). Zn dissolution is related to mineral phases present in each slag: in ISF as Spinel Zn(Al0.8Fe0.2)O4 and in LBF as Franklinite [(Zn,Mn2+,Fe2+)(Fe3+,Mn3+)2O4]. Zn from Spinel phases were more resistant to weathering comparing to Zn from sulfide phases and Zn from glassy silicates matrix of the slags. Formation of white thin crust suggested that Zn in solution is widely controlled by the precipitation of two main secondary phases: Zincite (ZnO) and Zinc carbonate (ZnCO3) where the source of CO2 mostly comes from the atmosphere. CV-researcher (PhD student) Name Miss Nang Htay YIN Promotor Dr. Eric van Hullebusch (Université Paris-Est) Co-promotors Dr. Yann Sivry (Université Paris-Diderot) Prof. Piet Lens (UNESCO-IHE, The Netherlands) Graduated MSc. in Environmental Engineering and management, Asian Institute of Technology (AIT), Thailand 79 79

81 MATHEMATICAL MODELLING OF MULTISPECIES BIOFILMS: APPLICATION TO WASTEWATER TREATMENT PhD research November2011-October2014 INTRODUCTION Biofilms, commonly defined layer like aggregations of microorganisms, have been widely used to treat wastewater in attached growth systems since the end of the 19th century. Attached and suspended growth systems are based on the same biological metabolic processes to remove carbon and nutrients in wastewater treatment plants, but there are some inherent differences that provide several advantages and some challenges for the application of attached growth processes. Indeed, bacteria growing in biofilms show some advantages: i) they cannot be washed away with the water flow but they grow in locations where their food supply remains abundant; ii) they show an increased resistance to antimicrobial agents and allow to achieve a higher biomass concentration value in bioreactors; iii) their synergistic interactions characterizing natural biofilm communities have shown to facilitate the simultaneous removal of various pollutants in wastewater treatment reactors. The biofilm growth is a complex and dynamic process characterized by several steps and governed by complex systems of nonlinear partial differential equations. Biofilm formation results from the balance of several physical (substrate transport), chemical and biological factors (growth yields and substrate conversion rates). The main biofilm expansion is due to bacterial growth and to extracellular polymer production; the nutrients necessary for bacterial growth are dissolved in the liquid flow and to reach the cells they are transported by diffusion. Biofilms represent the basis of all attached growth systems since the efficiency and robustness of a wastewater treatment plant mainly depends on the composition and activity of its microbial community. In this contest, mathematical models represent the perfect means for analysing and predicting microbial interactions and optimizing the control criteria of biofilm processes. RESEARCH OBJECTIVES This research project is aimed at developing a mathematical model and numerical simulations to predict the growth of multi-species and multi-substrate biofilms. The model will deal with different microbial kinetics and biomass detachment mechanisms; it will predict the short-term responses of biofilm performance to substrate variations in the bulk liquid as well as the long-term development of film thickness and of microbial species. The model will link microscale phenomena occurring within the biofilm with macroscale indicators of full-scale process performance. The mathematical modelling will be combined with experimental research in order to acquire the knowledge and ability to engineer the biofilm structure and its function. A lab-scale biofilm reactor is planned to be set-up and run. Techniques for measuring biofilm will be based on two types of analyses: chemical analysis of bulk water and local chemical and physical measurements inside the biofilm. The data collected through experimental observations will be used to calibrate and validate the mathematical model. The validated model could be used to evaluate novel process designs without the cost, time, and risk of building a physical prototype of the process and to test a wide range of operating strategies for a process. It could be used to individuate the best operational conditions to optimize the management of biofilm based wastewater treatment plants. CV-researcher Name: Maria Rosaria Mattei Promotor: Prof. Giovanni Esposito Co-Promotors: Prof. Berardino D Acunto, Prof. Francesco Pirozzi Graduated: Environmental Engineering, University Federico II of Naples (Italy) Contact: mariarosariamattei@gmail.com tel:

82 Biogenic production of selenium nanoparticles and their applications PhD research November October 2014 Social Impact Nanoparticles demonstrate novel physical, chemical, biological, mechanical, optical and electrical properties that are different from bulk material owning to their large surface to volume ratio. Due to these novel properties, nanoparticles are finding application in energy generation, electronic industry for ever smaller chips, improving storage capacity of device, waste remediation, medicine - to name a few. Selenium nanoparticles are useful in xerography, semiconductor industry, medicine as an antifungal agent and dietary supplements. From an environmental perspective, selenium nanoparticles can be used to remove Hg, Cu and Zn from the environment. Technological Challenge The biggest challenge in the biogenic production of selenium nanoparticles (BioSeNPs) is to achieve a desired product quality - size less than 100 nm, desired surface charge and shape. The stabilizing agents can be the solution to this challenge. This research focused on understanding the role of extra-polymeric substances in stabilizing BioSeNPs at macro as well micro-level. With this understanding, BioSeNPs were used for adsorption of Zn, Cu and Cd. The interaction was studied with batch adsorption experiments and spectroscopic methods. The success of these experiments led to scale up of the process to produce selenium nanoparticles in different reactor configuration and integration of adsorption of heavy metals in continuous reactor process. CV-researcher (PhD student) Name: Rohan Jain Supervisors: Prof Piet Lens (UNESCO-IHE, The Netherlands) Prof. Eric Van D Hullebusch (UPE, Marne La Vallée, France) Prof. François Farges (MHNH, Paris, France, Italy) Graduated: MTech, Biochemical Engineering and Biotechnology, IIT Delhi, India r.jain@unesco-ihe.org 81 81

83 Engineering and Biochemical Aspects of Anaerobic Ammonium Oxidation (Anammox) process in wastewater treatment PhD research April 2012 March 2015 Denitrification has been believed to be the only pathway for the return of reactive nitrogen to its non-reactive form N2. This view was challenged when Anammox was firstly observed by Mulder et al (1995) in a denitrifying wastewater treatment plant in the Netherland in Anammox is a process which ammonium is anaerobically oxidized into nitrogen gas with nitrite as electron acceptor mediated by autotrophic bacteria which belongs to the phylum Planctomycetes. Coupled to partial nitrification, the Anammox process provides a short-cut option of ammonia removal in comparison to the conventional nitrification/denitrification process. With the increase of nitrogen rich wastewater from municipal and agriculture use, the Anammox based treatment provides an opportunity of process optimization in terms of low energy consumption, low space occupation and low cost due to the absence of organic carbon source addition. Technological challenge Although Anammox technology has attracted worldwide research interest in both wastewater treatment application and global nitrogen balance, the technology is still subject to long start-up due to the low growth rate of the bacteria and process sensitivity. As a consequence, large scale application becomes difficult and is limited. In the current stage of the PhD research, the chemical and microbial aspects of the enrichment of Anammox bacteria is investigated in search of opportunities of intervention strategy to accelerate the process. Future research also includes the study of environmental parameters especially the effect of salinity in the performance of Anammox reactor. Marine Anammox will also be studied with an attempt to its application under low temperature and high salinity. Name: Ding Zhiji Supervisor: Prof. Giovanni Esposito (UNICAS, Italy) Co-supervisor: Prof. Eric van Husbusch (UPE, France) Mentor: Antonio Panico (UNINA) 82 82

84 THIRD COHORT

85 Landfarming of PAHs Contaminated Soils Combined with Composting of Organic Waste EMJD Research October September 2015 Introduction Polycyclic Aromatic Hydrocarbons (PAHs) are one of the most common pollutants in the environment as they are frequently found in soils and sediments as well as in groundwater and atmosphere. Incomplete combustion of fossil fuels as anthropic factor, accidental forest fires and volcanic eruptions as natural factors, are the main causes of their presence in soils. PAHs are highly hydrophobic and tend to be easily adsorbed onto the organic matter of solid particles forming persistent pollutants. As they are toxic, some of them even cancer-causing, a large number of studies on remediation of PAHs contaminated soils have been conducted in the recent years with the aim of studying the most suitable techniques for their removing from contaminated soils. Proposed Landfarming Technique Bioremediation is a well known process, commonly used to remove organic pollutants from contaminated soils. This process is attractive because needs low capital costs and low technology to be performed, but the extremely long time to be naturally finalized makes it not suitable for most of practical uses. Therefore, the aim of this research is to optimize the bioremediation process occurring in landfarming technique by adding organic waste to PAHs-contaminated soils in order to enrich the soil with organic matter, nutrients as well as microorganisms. Thus, landfarming of PAHs-contaminated soils combined with composting of organic waste could represent an interesting solution to improve the biodegradation process of PAHs, and for eco-friendly disposal of organic waste as well. The focus of this research is actually to study the effectiveness of organic wastes added to soil with the aim of optimizing the PAHs removal by landfarming. PhD Researcher Name: Borislava Lukić Graduated: Master of Engineering in Exploration of Mineral Resources, Faculty of Mining and Geology, University of Belgrade (2008); M.Sc. Degree second level in Engineering and Environmental Health, Faculty of Engineering, University of Naples Federico II (2010) ETECOS3 Promotor: Prof. Giovanni Esposito (UNICAS, Italy) Co-Promotors: Prof. Eric van Hullebusch (UPE, France), Prof. Francesco Pirozzi (UNINA, Italy), Eng. Antonio Panico (University Pegaso, Italy), Associate Prof. David Huguenot (UPE, France) Contact: b.lukic@unicas.it; borislaval@yahoo.com 84 84

86 IMPACT OF MICROBIAL ACTIVITY ON GEOCHEMICAL STABILITY OF Cu-RICH INDUSTRIAL WASTES IN SOILS ENVIRONMENTAL RELEVANCE Industrial activity related to mining and processing of copper is a reason of growing environmental concern. Apart from advantages arising from effective production, this industrial sector produces huge amounts of metallurgical wastes (slags). Pyrometallurgical process is intended to extract metals from ores, however depending on the process efficiency some metal concentrations still remain in the waste materials. Usually, slags are dumped close to the centre of activity, but in the case of inappropriate isolation of disposal site, it may entail serious environmental consequences. Biogeochemical weathering and physical erosion may mobilize metallic elements contained in wastes and consequently lead to pollution of local environment (soils, surface and ground waters, sediments). Further migration of pollutants and transfer to large distances, make remediation of polluted areas difficult or in some cases even impossible to perform. Therefore, appropriate isolation of wastes as well as frequent environmental monitoring of disposal sites are necessary to prevent deterioration of environmental quality. Furthermore, recent attention given to sustainable waste management has focused on the methods allowing reuse metallurgical wastes instead of their disposal. Bio/recovery of metals remaining in the waste materials seems to be a promising technology for the future. Besides advantages derived from the recovery of valuable compounds, it is also important that extraction of metals decreases harmfulness of metallurgical materials and consequently allows further reuse or safe disposal. RESEARCH OBJECTIVES Comparison of chemical and phase composition of Cu-slags originating from different time periods of industrial activity and different smelting technologies Assessment of the leaching behavior of slags Examination of the impact of microbial activity on mobilization/immobilization of metals in the weathering conditions Determination of the most important physico-chemical and biological factors affecting the waste alteration Analysis of slags weathering products Study of the process efficiency of metals bio/recovery PhD Student: Anna Potysz Graduated: MSc Environmental Protection (University of Wroclaw, Poland) Contact: Anna.Potysz@univ-mlv.fr or apotysz@gmail.com Promoter: Professor Eric van Hullebusch (Université Paris-Est, France) Co-supervisors: Professor Gilles Guibaud (Université de Limoges, France) Professor Piet Lens (UNESCO-IHE, The Netherlands) Dr. Malgorzata Grybos (Université de Limoges, France) Dr. Jakub Kierczak (University of Wroclaw, Poland) 85 85

87 Bio-electro-Fenton: integrated electrochemical-biological process for removal of refractory organic pollutants from wastewater PhD research October 2013 October 2016 Biological processes are by far the most wide-spread and conventional methods for any wastewater treatment. They possess important advantages which could not be overcome by any other treatment so far: cost-effective, well-studied and therefore easily modified according to the local needs. However they have some serious limitations for degradation of refractory pollutants and as wastewater discharge regulations get stiffer, a need for proper treatment becomes an urge. Until now the only available option for degradation of non-biodegradable organic pollutants is advanced oxidation processes (AOP). Among a great variety of AOP, electro-fenton appears to be a novel technology, which has shown high removal and mineralization of refractory organics. This environment-friendly technology is easy to operate, requires only two reagents (Fe2+ and H2O2), one of which is produced in situ (H2O2). However, electro-fenton is an electrochemical process and it consumes energy, which leads to relatively high operational costs. A practical solution between high removal efficiency and low costs is an integrated electrochemical-biological process: electro-fenton/sequencing batch reactor (SBR). SBR is a type of aerobic reactor, which have previously demonstrated good removal results and has one of lowest environmental footprints among other biological processes. Such an integrated system has a synergetic effect as two methods enhance each other s advantages. The scientific object of research is hospital wastewater, which recently has attracted great deal of interest and research. It is heavily charged with pharmaceuticals, disinfectants, corrosion inhibitors, musks etc. Those contaminants pose as serious threat to ecosystem balance and particularly to human health. Effluents from hospitals are normally discharged untreated or passes through the municipal treatment facility, where the pharmaceutically active compounds are poorly degraded and/or removed. Therefore most of the pharmaceuticals and their metabolites end up practically unchanged in natural water system, from where they can be finally found in drinking water. Results of this work can be possibly used for designing an appropriate treatment of other types of wastewater simply by adjusting running parameters. Name: Alexandra Ganzenko Supervisor: Prof. Mehmet A. Oturan Co-promotor: Dr. David Huguenot Prof. Giovanni Esposito Contact: alexandra.ganzenko@gmail.com 86 86

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89 Metallurgical Wastes Bio/Weathering And Heavy Metals Bioleaching And Recovery PhD Research November October 2015 Social impact Metallurgical industries are producing metals from various types of ores, which also end up in generating huge loads of metal bearing wastes. According to a survey by EU statistics, the wastes generated from mining and quarrying industries play a big contribution in the total wastes generated in the EU. These metallurgical wastes are usually deposited in a storage deposit or dams by the metal production industries. The storage dams can act as a potential reservoir of toxic heavy metals. Accidental collapse and leakage in the storage dam is a serious environmental threat. For example, dam collapses of Aznalcóllar (Spain, 1998), Baia Mare and Baia Borsa (Romania, 2000) caused harmful adverse effects on the environment. Also due to natural weathering, the release of the toxic heavy metals causes adverse effects in the environment, contaminating the surrounding soil, the surface water and also the ground waters. Secondly, demand and usage of metals nowadays increasing constantly, thereby resulting in the gradual depletion of the worldwide of the metal resources. Since these wastes are rich in metals, there is a possibility in using them as secondary resources for metal production and so the environmental impact and consequences due to the storage of the wastes can also be reduced. Technological challenge In the recent past decades, there were many research reports investigated the use of flue dusts, furnace sludges and slags for the extraction and recovery of metals, but there are only very few research articles examined with the metal producing industrial muds and sludges for the metals recovery. There is a lack of fundamental studies in the literature in the context of using metallurgical muds as the secondary resource of metal recovery and most of the metallurgical residues were found in oxidized state, so metals cannot be extracted by oxidative dissolution. Also no sulfidic forms of metals (like pyrite) are present, which bares the usage of metal solubilisation by chemolithotrophic bacteria. Acidic leaching and acid producing fungal leaching will be applied for the extraction of the heavy metals from the wastes and biologically produced metal sulfide precipitation will be performed for the recovery of the metals Name: Promotor: Co-promotors: Netherlands) Contact: Manivannan Sethurajan Prof. E. van Hullebusch (UPE,France) Prof. Piet Lens (UNESCO-IHE, the Prof. Heinrich A.Horn (UFMG, Brazil) biotek_mani@yahoo.co.in Manivannan.Sethurajan@univ-mlv.fr 88 88

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91 Anaerobic Methane Oxidation in the Presence of Sulfate as an Electron Acceptor PhD Research March 2013-March 2016 Societal Relevance Methane (CH4) is the most abundant hydrocarbon in the atmosphere, and it is an important greenhouse gas, which has so far contributed an estimated 20% of post-industrial global warming. Sources of methane are mixed from human related activities and natural process. The potential of global warming due to CH4 is 25 times more than that of carbon dioxide (CO2). The effects of global warming are the ecological and social changes caused by the rise in global temperatures. So the research on anaerobic oxidation of methane as a global methane sink can provide relevant information on methane cycle and recommend for the interventions, if required. Another important applicable aspect of the research is to use methane as an electron donor for sulphate removal from waste water system. High sulphate concentration in industrial wastewater, especially from paper mills, and refineries, is one of the challenges of waste water treatment industry. It demands for the electron donors like ethanol, hydrogen which eventually increase the cost of treatment system. This research provides an opportunity to utilize methane produced by anaerobic digestion to perform sulphate reduction which can save cost involved with expensive electron donors. Technological Challenges CH4 and CO2 are major end products of organic matter decomposition in deep waters and sediment. The biogeochemical cycle of methane is receiving most attention these days as it can be part of natural process with biotechnological application. There is still a knowledge gap in understanding the methane source and sink because majority of methane production and consumption involves microbial community in the deep marine environment. Vast majority of these microbial communities are less known. In recent past scientific community achieved success to enrich and cultivate highly active groups of methane producers known as methanogens, however the major part of methane consumers (ANME) are not fully understood till the date as any of this group has not been cultivated yet. Thus the study of ANME and anaerobic methane oxidation has gained most attention these days as it is an important phenomenon to understand the gap in methane biogeochemical cycle. The overall objective of this research work is to perform anaerobic methane oxidation and sulphate reduction at higher rates (compared to the existing in situ and ex situ studies) in laboratory scale bioreactor(s) and elucidate the biotechnological mechanism of the process. Name: Susma Bhattarai (UNESCO-IHE, the Netherlands) Promoter: Prof. Piet N. L. Lens (UNESCO-IHE, the Netherlands) Co-promoters: Prof. Giovanni Esposito (UNICAS, Italy) Dr. Eldon Raj Rene (UNESCO-IHE, the Netherlands) Dr. Graciela Gonzalez-Gill (UNESCO-IHE, the Netherlands) Contact: s.bhattarai@unesco-ihe.org 90 90

92 Mycogenic production of elemental selenium nanoparticles PhD Research October October 2015 Social impact The use of materials at the nano-size scale has increased in the last decades due to their enhanced properties. In particular, the use of elemental selenium nanoparticles (nse0) has attracted the attention of researchers due to their unique photoelectric, semiconducting and X ray sensing properties, being used in rectifiers, solar cells, glass industry, etc. The most common physicochemical methods to produce nanoparticles are complex, expensive and non eco-friendly. Along with the necessity of investigating more eco-friendly, simple and suitable methods for the production of nse 0, there is the requirement of new cost-effective and environmentally friendly technologies to reduce toxic forms of selenium (selenite and selenate) from water streams. Technological challenge The biological synthesis of nanoparticles has become very popular in the recent years. Bacteria are the preferred microorganisms for biogenic production of nanoparticles, however, the use of fungi is also very promising. Mycogenic production of nanoparticles has proven to be feasible and to provide even higher productivity rates compared to bacteria. Nevertheless, the use of fungi to synthesize nanoparticles is still in its infancy. The effect of different operational parameters on the removal of selenium oxyanions and the synthesis of nse0 by fungi has not yet been addressed. Moreover, all of the fungal studies have used batch tests, and there is no a single one investigation that shows the continuous removal of selenium and the production of nanoparticles in a fungal bioreactor. This PhD research aims to provide a solution for both the removal of selenium pollutants and the production of selenium nanoparticles by developing a novel biotechnological system based on a fungal pelleted bioreactor to remove selenium oxyanions in effluents, while producing and recovering Se0 nanoparticles. Moreover, this research also aims to explore the use of the selenium nanoparticles immobilized fungal as a new biosorbent material for the removal of heavy metals from wastewater. CV-researcher (PhD student) Name: Nationality: Promoter: Co-promoters: Project: Erika J. Espinosa Ortiz Mexico Prof. Piet Lens (UNESCO-IHE, The Netherlands) Prof. Eric van Hullebusch (Universite Paris-Est, France) Mycogenic production of selenium nanoparticles e.espinosaortiz@unesco-ihe.org 91 91

93 BIOHYDROGEN PRODUCTION FROM SEQUENTIAL DARK AND PHOTO FERMENTATION Social Impact In light of depleting global reserves of fossil fuels and pollution problems from the byproducts of use of fossil fuels, environmental friendly alternative energy carriers and sources became highlighted. The current research interests have been directed towards renewable energy technologies like biogas and biohydrogen production utilizing the renewable resources like biomass residues and organic waste. For its high energy content and environmental friendliness of production, biohydrogen could hold a future promise of dream fuel. Technological Challenge The biological processes, i.e. dark fermentation processes are limited by its lower yield compared to its theoretical maximum ideal yield of 4 moles of H2 per moles of glucose. The by-products of dark fermentation which mostly include volatile fatty acids need to be further treated for complete conversion. In addition, there are some setbacks in the optimum operation of the process for higher H2 production rates. The use of low cost materials such as lignocellulosic materials from agricultural and forest residues, agroindustrial waste, and organic wastewater in the dark fermentation process can give cost competitiveness its scaled-up development. Moreover, the process could be optimized with suitable ph and temperature control. The dark fermentation effluents can be further converted to biohydrogen via photo fermentation to increase the total yield of biohydrogen. The main objective of this study is to use low cost biomass waste in sequential two stages dark and photofermentation process in order to overcome the high cost and low yield limitations of the process. Name: Promoter: Co-promoters: Contact: Anish Ghimire Prof. Giovanni Esposito (UNICAS, Italy) Prof. Francesco Pirozzi (UNINA, Italy) Dr. Eric Trably (INRA, Narbonne, France) anishghimire@gmail.com 92 92

94 HMs removal by soil washing: efficiency and economic optimization/engineering configuration HMs contamination problem. The environmental concern about heavy metals (HMs) presence is high, especially in the soil, because soil contamination is largely caused by them. The increasing contamination of soils by HMs is mainly due to industrial and anthropogenic activities. The contamination of soil can be widely extended representing an important risk for water and earthly ecosystems since it can be source of acute effects and also chronic effects for different kind of organisms and for entire ecosystems; an alteration of the soil can affect not only soil productivity, but also the quality of water and agricultural products that are used every day. Soil remediation represents an important industrial activity in Europe and the United States and the research is aimed at analyzing several ways to make it increasingly promising and competitive among other soil remediation technologies. Contaminated soil remediation by soil washing procedure. Soil washing is a technology aimed to remove contaminants from the soil involving different operational options, such as physical separation, chemical extraction and a combination of both of them. Chemical extraction allows a transfer of the contaminants to a liquid phase by desorption and solubilizationa and high removal efficiency results have been obtained involving aminopolycarboxylate (APCs) chelants as chemicals. EDDS, among various APCs, showed good contaminant removal capacity and biodegradability characteristics. The aim of this research is to investigate about the best process conditions for the HMs removal from soil through EDDS involvement, identifying a suitable reactor configuration and carrying out studies about the recovery of the washing solution and the biodegradability of the HM-chelant complex after treatment, in order to consider also the economic and environmental aspect of the process. PhD student: Alberto Ferraro (alberto.ferraro3@gmail.com) Master degree in Environmental Engineering at University of Naples " Promoter: Prof. Giovanni Esposito (University of Cassino and Southern Lazio) Supervisors: Prof. Giovanni Esposito (University of Cassino and Southern Lazio) Prof. Eic vanhullebusch (University of Paris-Est) Prof. Massimiliano Fabbricino (University of Naples Federico II ) 93 93

95 FOURTH COHORT

96 Emerging contaminants from water discharged in soil: effects and treatment strategies Social impact: Since the world population is steadily increasing, water demand is in rapid growth. Projections until 2025 are stating that many countries are entering a period of severe water shortage. On the other hand, most of water consumption is destined to irrigation purposes. In most places, the water used for agriculture is inefficient and consumptive. For these reason strategies for water recovery and reuse are being developed since the last decades in many countries around the world, primarily in arid and semi-arid regions. One possible mechanism is the recycling of wastewaters and drainage waters that can be used instead of fresh water sources for irrigation. Municipal wastewater reuse is the most promising reuse prospect, since it improves the security of supply; it reduces the resource constraints due to variable rainfalls, and reduces the need for expensive water storage construction and freshwater supply infrastructure. This kind of strategy while showing undeniable advantages gives place to concerns and unknowns about the impact of the quality of the recycled water, both on the receiving soil and on the crop itself. Water quality issues that can create real or perceived problems in agriculture include nutrients and sodium concentration, heavy metals and the presence of contaminants such as infective pathogens for human and animal, pharmaceutical and endocrine disruptors. In the last decades several researches are breaking out, identifying a class of both organic and inorganic contaminants described as Contaminants of Emerging Concerns (CECs), that are often not ruled by precise water re-use guidelines and same time of not completely known behaviour in the environment as well as against the human health. This wide range of pollutants can be adsorbed on both inorganic and organic fraction of irrigated soils giving often rise to bioaccumulation and biomagnification effects, with related health concerns. Hence, as stated by the Hyderabad declaration on Wastewater use in Agriculture: without proper management, wastewater use poses serious risks to human health and the environment. Aims and scopes of the research project: New strategies and technologies are needed to be studied to remove soil-accumulable micro-pollutants and disinfection by-products from waters intended for reuse. Is also needed a deeper investigation of the the effects on soil of treated wastewater and mobility and bio-availability of pollutants through soils need advanced clarifications: Among the treatment technologies possible to be applied, photolysis, ozonation at high dosage and adsorption using Carbon Nanotubes (CNTs) are selected for emerging contaminants removal. The evaluation of the effect of reclaimed wastewater on irrigation of soils needs the molecular level study of the interactions with all the components of soil, in order to model the possible accumulation of the pollutants, even at trace concentration. The ability of retaining trace pollutants at changing the soil properties should be sensibly helpful to find correlations between the mobility of pollutants and the chemical composition and structure of both organic and mineral matters composing the soil. PhD student: Contact: Website: Ludovico Pontoni ludovico.pontoni@unina.it Promoter: Prof. Francesco Pirozzi, Supervisors: Prof. Francesco Pirozzi, Prof. Giovanni Esposito Prof. Piet N. Lens University of Naples Federico II (Naples, Italy) University of Naples Federico II (Naples, Italy) University of Cassino and Southern Lazio (Cassino, Italy) UNESCO-IHE Institute fo Water Education (Delft, The Netherlands) 95

97 Microbial synthesis of metal selenide nanoparticles using selenium oxyanion reducing microorganisms PhD research October 2013-November 2016 Social impact Selenium pollution is a recurrent problem in effluents from mining, coal combustion, petrochemical, electronic and agricultural industries. But, conventional treatments for removal of Se from wastewater have often failed as they are not effective to meet the regulatory requirement. Moreover, metal selenide quantum dots (QDs) have unique semiconducting properties that differ from those of the bulk materials. The photo-optical and photo-voltaic properties these QDs are particularly suited for their application in solar cells and nanodot-based gas sensors as well as novel bioassays, fluorescent biolabelling, including their use in cancer detection. Technological challenge Microbial synthesis of metal selenide nanoparticles is relatively a new area of research. Anaerobic granular sludge will be used as the source of microbial metabolism for the production of metal selenide nanoparticles. However, till date, no study has been carried out on the use of anaerobic granular sludge for production metal selenide nanoparticles. The biggest challenge is to obtain nanoparticles of the desired size. The effect of different temperature and different electron donor on metal selenide nanoparticles production and physical, chemical and biological properties of nanoparticles will be explored. Furthermore, scaling up of the process to produce metal selenide in UASB will also be investigated. Optimization and development of product recovery methods will also be investigate. This microbiological method of production of metal selenide nanoparticles are regarded as safe, cost-effective and environment-friendly processes. Anaerobic Sludge Metal Selenide nanoparticles Metal (Zn/Pb/Cd) Spectral properties of nanoparticles will be studied by UV-visible spectroscopy; photoluminescence, whereas elemental analysis and molecular arrangement will be carried out by XRD, XPS and EDX. SEM and TEM will be used to investigate the shape and size of nanoparticles. CV-researcher Name: Promoter: Co-promoter: Graduated: Joyabrata Mal Prof Piet Lens (UNESCO-IHE, The Netherlands) Prof Eric D. van Hullebusch (UPE, France) M.Tech Biotechnology, IIT Guwahati, Guwahati, India j.mal@unesco-ihe.org 96

98 Treatment of soil washing / soil flushing solutions by electrochemical advanced oxidation processes (EAOPs) and integrated treatments PhD research October 2013 September 2016 Social impact The release of hydrophobic organoxenobiotics such as polycyclic aromatic hydrocarbons, petroleum hydrocarbons or polychlorobiphenyls results in long-term contamination of soils and groundwaters. This becomes a common concern since they have high potential toxicological impact. Therefore, the development of a cost effective process with high pollutant removal efficiency is a major challenge for remediation companies. Soil washing (SW) and soil flushing (SF) processes enhanced by the use of extracting agents (surfactants, biosurfactants, cyclodextrins etc.) are conceivable and efficient approaches. However, this generates highly contaminated effluents containing high extracting agent concentrations. For the treatment of these SW/SF solutions, it is aimed at removing target pollutants and recovering extracting agents for further SW/SF steps. Technological challenge The main general objective of the research plan is to improve the cost effectiveness of SW/SF solutions treatment. Particularly, it will be assessed the efficiency of the combination of the following processes: selective adsorption step of target pollutants in order to recover extracting agents; electro-fenton and/or anodic oxidation (EAOPs) processes for the degradation of organic compounds refractory to conventional oxidation techniques; post-biological treatment for the mineralization of by-products formed during EAOPs in a cost-effective way. Mechanisms occuring during the treatment of SW/SF solutions by EAOPs will be also studied in order to highlight competition/synergistic effects. PhD student Clément Trellu clement.trellu@u-pem.fr Graduated in Environmental School of Engineering, speciality water treatment and civil engineering, Poitiers (France), in Promotor Prof. Mehmet A. Oturan (Université Paris-Est, France) Co-promotor Dr. David Huguenot (Université Paris-Est, France) Dr. Hab. Giovanni Esposito (Uniclam, Italy) Dr. Hab. Eric D. Van Hullebusch (Université Paris-Est, France) 97

99 Anaerobic oxidation of methane with different sulphur compounds as electron acceptors PhD research October 2013-September 2016 Social impact Methane is a well known greenhouse gas and it has 25 times the global warming potential of carbon dioxide over 100 year time frame. Therefore, small changes in its atmospheric concentration have large implications for future climate change. Its concentration is increasing at an alarming rate of about 1% per year. Large quantity of methane are biologically produced by marine sediments (85300TgCH4/year); however, the emissions of methane are controlled by anaerobic methane oxidation mainly coupled to sulphate reduction (AOM-SR), which consumes more than 90% of methane. AOMSR is a known process mediated by consortia of microorganisms, but the mechanism has not yet been fully understood and further research is needed to define and understand the natural and biochemical cycle of this green house gas. AOM investigation have another research direction; the desulphurization of industrial wastewater. Sulphur compounds such as sulphate, thiosulphate, sulphide, sulphite and dithionite are common contaminants discharged in fresh water due to industrial activities. Biological anaerobic sulphate reduction has been successfully applied for the treatment of these sulfur compounds; the main limiting factor is the cost of the electron donor, which can be reduced with the use of a cheaper and more available carbon source, such as methane. Technological challenges Most of the previous literature reports focused on the investigation of the microbial community involved in AOM-SR process to further understand the mechanism and to isolate the archaea involved. AOM-SR can also be potentially applied in industrial wastewater treatment. Few researches have attempted to study AOM in bioreactors achieving considerable higher AOM rates than those found in natural environments, but showing the slow growing nature of ANME as major challenge for practical application. This research focuses on the development of a bioreactor for AOM coupled to different sulphur compounds, such as sulphate, elemental sulphur and thiosulphate, as electron acceptors. The microbial consortia in the enriched biomass will be characterized, together with the investigation of the metabolic activity of the microorganisms involved using FISH-NanoSIMS technique, stable isotope probing (SIP) and lipid biomarkers analysis. More insights will be gained on the mechanisms of AOM and the bioreactor design will be adapted to obtain faster biomass growth and AOM higher rates for environmental applications. CV researcher (PhD candidate) Name: Chiara Cassarini Promotor: Prof. Piet N. L. Lens (UNESCO-IHE, the Netherlands) Co-Promotors: Prof. Giovanni Esposito (University of Cassino, Italy) c.cassarini@unesco-ihe.org 98

100 Metal Recovery from Electronic Waste PhD Research from October 2013 October 2016 Social Impact Discarded electric and electronic devices comprise a growing segment of waste generated at households. According to the new Waste Electrical and Electronic Equipment Directive (WEEE Directive, 2012/19/EU) by the European Community (EC), waste electrical and electronic equipment (WEEE) should be separately collected from municipal waste. Despite the growing awareness and deterring legislation, most WEEE is disposed in landfills. Significant proportions of WEEE still go unreported, most notably in Southeast Asia and West Africa. Inappropriate management of e-waste is of global concern due to the nature of production and disposal of waste in a globalized world. When e-waste is disposed of without adequate treatment, there are predictable negative impacts on the environment and human health. WEEE contains more than 1000 different substances, many of which are toxic, for instance heavy metals, along with CFCs (chlorofluorocarbons) and BFRs (bromated flame retardants). In addition to being a high toxic material, e-waste also contains high amount of metals. Technological Challenge The main objective of this research is to develop an appropriate biomass-based method to recover metals from e-waste. Biological treatment promises more cost-effective and environmentally sound processes as compared to other technologies. A group of microorganisms are known to leach metals from their ores, and are utilized on full-scale in biomining processes. In this context microbial leaching of metals from waste material (bioleaching) and biomass-based recovery of metals from leachate (biosorption, bioprecipitation) will be investigated. Acidophilic chemolithotrophic bacteria and cyanogenic bacteria will be selected to leach metals from electronic waste in a multi-step process. Moreover, leached metals in solution will be recovered by biological methods such as bioprecipitation, biosorption. In addition, efficiency of a series of physico-chemical methods, e.g. acid leaching, thiourea/thiosulfate leaching, precipitation, and electrowinning will be assessed and compared to biomass-based techniques with regard to metal leaching efficiency. Researcher: Name: Arda Işıldar Contact: a.isildar@unesco-ihe.org, ardaisildar@gmail.com Country: Turkey Education: M.Sc. Sustaianble Resource Management, B.Eng. Environmental Engineering Promoter: Prof Piet N. L. Lens (UNESCO-IHE) Copromoters: Dr. Eric van Hullebusch (University of Paris) Prof. Jaakko Puhakka (Technical University of Tampere) 99

101 Combination of Thermal Pretreatment and Biofilm Technology to Enhance Anaerobic Digestion PhD Research October 2013 September 2016 Social Impact Anaerobic digestion is one of the known biological treatment for organic solid waste and wastewater since decades. It offers significant advantages in decreasing amount of waste while keeping small sludge production and recovering energy. Anaerobic biofilm reactors are among the technologies designed to retain higher biomass concentration inside the reactors. These technologies had the benefits of reducing the volume of the digester and increasing economic performance in terms of methane production and operational stability. Immobilization of cells on the surface of the media also improves their resistance to toxic shock load. With the increasing generation of solid waste by different activities, the use of thermal pretreatment largely alleviated the problem of hydrolysis of solid organics causing rate limitations. Technological Challenges Due to the low growth rate of anaerobic microorganism, most conventional anaerobic digester requires longer hydraulic retention time and larger reactor volume to achieve complete degradation of substrates. The poor operational stability also decreased the attractiveness of the processes from being widely commercialized. It is possible to produce higher amount of methane as well as operate at short hydraulic retention time and high loading rate conditions using biofilm reactors. The microbe immobilization in biofilm reactors largely decreased the risk of microbe losses particularly the slow growing methanogens. Maximizing biomass concentration in the reactor with biofilm had not the only way to obtain optimum methane yield since hydrolysis stage had been also found as the rate limiting step especially with particulate substrate. The thermal pretreatment improve the solubility by increasing the solid liquid separation of the substrate and thus promote easier diffusion to the biofilms. The general objectives of the research is to improve the performance and stability of anaerobic digestion through integration of thermal pretreatment as a first stage and biofilm reactor as a second stage. PhD Student Name : Martha Minale Yeshanew Promoter : Prof. Giovanni Esposito (UNICAS, Italy) Co-Promoter : Prof. Piet Lens (UNESCO-IHE, Netherlands); Prof. Francesco Pirozzi (UNINA, Italy) Contact: martaminale@gmail.com 100

102 Fermentative bio-hydrogen production from complex organic waste by suspended and attached growth hyperthermophilic bacteria Thermotoga neapolitana Erasmus Mundus Joint Doctorate Programme (ETeCoS3): October 2013-September 2016 Social Impact Biological processes to produce hydrogen have shown a great potential to be an alternative source of green energy for the future. Over the last few decades, actually, hydrogen production by dark and photo-fermentation has shown promising results as sustainable energy suitable to meet the escalating gap in the demand-supply of energy. The dark and photo-fermentation can effectively replace the conventional technologies to produce hydrogen such as gasification, and pyrolysis which indeed depend on non-renewable energy resources. Furthermore, the dark-fermentation produces bio-hydrogen from almost all types of organic waste by using either mixed or pure cultures of hydrogen producing bacteria and, moreover, forms as biological process by-products, various organic acids that can have further scientific applications or can be used to feed cascading processes such as a photo-fermentation to produce hydrogen or, as an alternative, an anaerobic digestion to produce methane. The use of pure culture for bio-hydrogen production is more interesting than mixed culture, since the potential bio-hydrogen yield is close to it s theoretical maximum value. Technological Challenge The key challenge of biological fermentative hydrogen production remains the economic aspect at full scale that mostly depends on various operating parameters and engineering design aspects. A further problem concerns post production of hydrogen such as purification, storage and transport. The current research has planned to use a hyperthermophilic bacteria, namely Thermotoga neapolitana for fermentative bio-hydrogen production from various organic waste. The research mainly focuses on further optimization of the operational parameters and engineering design aspects to produce bio-hydrogen and other valuable biological fermentation by-products, i.e. lactic acid, using suspended as well as attached growth biological systems in continuous stirred tank reactors. The effect of operating parameters such as hydraulic retention time, organic loading rate, ph, pressure of gas in the headspace and the type of gas sparged to create anaerobic condition will be studied in detail. In addition to the previous aim, mathematical modeling will be performed to optimize the process conditions for hyperthermophilic fermentative bio-hydrogen production and design the full scale biological reactors. Name: Promoter: Co-promoter: Co-supervisors: Contact: Nirakar PRADHAN Giovanni ESPOSITO (UNICAS, Italy) Piet LENS (UNESCO-IHE, the Netherlands) Antonio PANICO (UNINA, Italy) Giuliana D'IPPOLITO (ICB-CNR, Italy) Laura DIPASQUALE (ICB-CNR, Italy) n.pradhan@unicas.it 101

103 Coupling of membrane nanofiltration and electrochemical advanced oxidation processes for the removal of pharmaceutical residues from wastewater EMJD research October November 2016 Social impacts The occurrence of pharmaceuticals (PhACs) in wastewater, surface and underground water has been proven for many years. The most significant entry route for PhACs into the aquatic environment is the effluents of wastewater treatment plants (WWTPs) because a large proportion of medication taken by patients passes through their body partially or unmodified and travels via urine and faeces to wastewater. Other source include direct disposal of expired drugs, production sites and hospital effluents. The potential threat posed by PhACs and its active metabolites include abnormal physiological processes and reproductive impairment in aquatic organisms, increased incidences of cancer and the development of antibiotic resistant bacteria. Conventional wastewater treatment techniques applied in WWTPs are not adequate to remove these pollutants, even with the combination of post treatment disinfection of the effluents. Additionally, single water treatment techniques such as membrane separation and advanced oxidation processes (AOPs) has been proven to either not technically adequate due to incomplete removal of the PhACs and formation of toxic intermediates or economically not viable because huge operation cost. Technical Challenge Application of sequence of treatments or combined treatments is a promising technique in wastewater reclamation and drinking water treatment for removal of refractory pollutants such as PhACs and endocrine disruptor hormones. This approach not only combined the advantages of the constituent treatment techniques but also eliminates the drawbacks of one another. Integrated membrane filtration EAOPs is one of such combined or sequential treatments with huge potential for efficient and effective removal of PhACs from wastewater. Membrane filtration is one of most technically flexible wastewater treatment technique and has provided an affordable alternative for sustainable water reclamation, but the problem of membrane fouling and need for treatment of rejected concentrate prior to disposal is a serious issue in this technique. EAOPs on the other hand, are highly efficient and eco-friendly electrochemical methods that utilize in situ generated hydroxyl radicals as main oxidant for total mineralization of organics to CO2. However, these techniques are less viable for treatment of large volumes of low concentration pollutants. Therefore combined membrane filtration EAOPs is an exciting transformative technology that is capable of concurrent separation and degradation of pollutants in a continuous process with EAOPs assist in eliminating membrane fouling and remediation of rejected organics via oxidation while membrane filtration not only concentrate the pollutants to the level that can be efficiently treated by EAOPs but also confine the toxic intermediates within the membrane to ensure prolong contact with the oxidants. Name: Soliu Oladejo Ganiyu (UPE, France) Supervisor: Prof. Mehmet A. Oturan (UPE, France) Co-promoter: Dr. Eric van Hullebusch (UPE, France) Prof. Govanni Esposito (Uniclam, Italy) Prof. Marc Cretin (IEM, France) Contact: Soliu.Ganiyu@u-pem.fr 102

104 The Role of Extracellular Polymeric Substances (EPS) in Heavy Metals Sorption PhD Research Oct Oct Social Impact High frequencies of anthropogenic activities and industrialization have generated a large amount of waste effluents containing heavy metals. These discharged heavy metals have caused severe pollution and posed a threat to the ecosystem. Many remediation techniques have been applied to remove or recover heavy metals. However, conventional wastewater treatment like physical and chemical techniques may be time-consuming and not eco-friendly. Although these traditional techniques are efficient, they only partially remove heavy metals and it is difficult to recover these treated metals. Thus, innovative solutions such as biosorption have been proposed to remove and recover heavy metals. Among different types of biosorbents, extracellular polymeric substances (EPS) are of special concern in the last few decades, as they are ubiquitous and eco-friendly. Negatively charged functional groups harbored in EPS provide abundant binding sites for metal ions. The functional groups are ascribed to the different components of EPS like proteins and polysaccharides. Nevertheless, the composition of EPS is affected by several factors (source, metal speciation, etc.), and the mechanisms of which are not well-characterized. On the other hand, trace amounts of metals, like Fe, Zn, Ni, Cu, Co, have been found to stimulate microbial growth and increase methane productivity during anaerobic digestion. These metals are sorbed and/or precipitate within biological aggregates, the process of which is closely associated with the functions of EPS. It is therefore very demanding to study the interactions between EPS and metals, not only aiming at the removal and recovery of heavy metals, but also for the manipulation of engineering processes. Research objectives Investigate the influences of nutrient conditions and bacterial growth phase on EPS composition and metal sorption performance. Identify the components and functional groups of different EPS samples. Compare metal binding abilities of biomass with/without EPS, as well as extracted EPS samples; characterize the speciation of metals sorbed by EPS samples. PhD Student: Feishu CAO Contact: feishu.cao@u-pem.fr Promoter: Prof. Eric van Hullebusch (Université Paris-Est) Co-promoters: Dr. Yoan Pechaud (Université Paris-Est) Prof. Gilles Guibaud (Université de Limoges) Prof. Piet Lens (UNESCO IHE) 103

105 Dry Discontinuous Anaerobic Percolation Digestion (DDAPD) Erasmus Mundus Joint Doctorate Programme (ETeCoS3) : October 2013 September 2016 Social context Broadly studied over the past 50 years, Anaerobic Digestion (AD), a natural process through which organic matter in slowly decomposed and CH4 is produced, is a very known and appreciated process in the environment field because of its unique common response to several actual problems: waste management, human pollution and energy source differentiation. In France, the high amount of solid animal dejections, manure, push researcher and engineers to adapt AD, firstly implemented in a liquid reactor, to solid substrates by trying to keep their solid properties. Dry Discontinuous Anaerobic Digestion Percolation digestor (DDAPd), a reactor where the solid phase is static and the liquid one is percolating through the bulk, seems to answer better than other possible configurations to these actual needs. DDAPd, and in particular the adaptation of this technology to manure, is not something well studied and a lot of scientific parameters, often set empirically by industries, need nowadays to find a scientific support Fig. 1 : one-stage DDAPd in order to improve their performances. Objectives Two parameters play a major role in DDAPD: first the recirculation which controls the mass transfer inside the reactor, and second the microbial seedling, which controls the kinetics. However, in order to overcome the discontinuous biogas production and the poorer performances due to non-optimal conditions, the coupling strategies of several reactors in what is called a multistage process need to be studied for real industrial sites. Thus, the PhD thesis objectives are summarizes as follows: - Acquire knowledge and management of DDAPD process one-stage in particular with manure, - Improve one-stage process performances through the parameters like seeding and recirculation, - Increase efficiency and overcome the one-stage limited possibilities through several coupling - reactors strategies, - Implement an IT tool based on recognized AD wet and dry models able to forecast manure degradation in coupled reactors (wet and/or dry). Name Silvio Riggio Nationality Italian Promotor Giovanni Esposito ( Università di Cassino, Italy) Co-promotors Renaud Escudié (INRA-LBE, France), Eric van Hullebusch (Université ParisEst, France) Michel Torrijos (INRA-LBE, France), Romain Debord (Naskeo Env., France), Co-supervisors Graduated Joaquim Comas ( LEQUIA, Spain) MSc Energy and Environment, National Institut of Applied Sciences (INSA), France silvio.riggio88@gmail.com 104

106 Nitrogen Removal from Low ph and Heavy Metal Contaminated Wastewater PhD Research from October 2013 December 2016 Research context The extraction of groundwater to prevent flooding of mines, leachate discharge from barren rock piles and discharge of wastewater from the ore processing into tailing ponds result in large amounts of mine effluents in the order of several m3/min or million m3/year per mine. The oxidation of sulfur-reduced minerals such as pyrite forms an extremely acidic leaching solution known as acidic mine drainage (AMD). AMD is a strongly acidic solution, high in sulfate and rich in metals (cadmium, nickel, copper, lead, zinc, arsenic, etc.) and highly toxic to most life forms. If uncontrolled, it may runoff into streams or rivers or leach into groundwater. AMD is further enriched by elevated levels of cyanide and nitrogen compounds (ammonium, nitrate and nitrite), mainly originated from heap leaching and blasting operations. The discharge of nitrogen-loaded mine waters from working and abandoned mines and mine spoils is larger during spring and after heavy rainfall events. If over-discharged, NH4+ and NO3favor the production of algal blooms and contribute to the eutrophication of receiving waters. Biological nitrate removal seems to be the most effective in terms of removal efficiencies and operating costs. Since AMD is typically organic deficient, autotrophic denitrification represents the most attractive and inexpensive solution for nitrate removal. Technological Challenge This doctoral research aims to investigate the ability of various inorganic electron donors (ferrous iron, Fe(0), sulfur-reduced compounds, pyrite, hydrogen and arsenite) to develop autotrophic denitrification in mining waters. Anaerobic batch bioassays and different types of continuous reactors (FBR, MBR, etc.) will be used for biomass enrichment and for experiments on electron donors, inhibitory ph and heavy metals toxicity. The best type of reactor, reactor configuration and electron donor suitable to carry out autotrophic denitrification will be investigated. Researcher profile Solfatara volcano, Pozzuoli, Napoli, Italy. Name: Francesco Di Capua Contact: f.dicapua@unicas.it Country: Italy Education: M.Sc. & B.Sc. in Environmental Engineering, University of Naples Federico II Promoter: Prof Giovanni Esposito (University of Cassino and Southern Lazio, UNICLAM) Copromoters: Prof. Piet Lens (UNESCO-IHE, The Netherlands) Prof. Jaakko Puhakka (Tampere University of Technology, TUT) 105

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108 ABSTRACT BOOK of the ETeCoS3 Summer School 2014 University of Cassino and Southern Lazio (UNICLAM) June 30 July 4, Cassino & Gaeta, Italy Edited by: Francesco Di Capua, Bijit Kumar Banik, Giovanni Esposito. Printed by: Copisteria di Ateneo G.F. Services