Operational strategies enhancing sewage sludge minimization in a combined integrated fixed-film activated sludge - oxic settling anaerobic system.

A lab-scale integrated fixed-film activated sludge (IFAS) reactor was mplemented with the oxic-settling anaerobic (OSA) cycle for reducing sewage sludge production through the addition of an anoxic/anaerobic sludge holding tank (SHT) along the sludge recycle line. The IFAS-OSA system was operated under the different hydraulic retention time (HRT) in the SHT (HRTSHT) of 12 h and 6 h, at an oxidation-reduction potential (ORP) < -91 mV and solid retention time (SRT) between 39 and 126 d. Furthermore, the effect of temperature increase in the SHT (TSHT) from ambient (19.8-25.6 °C) to mesophilic (35 °C) conditions was investigated. The system performances were monitored in terms of sludge minimization and dewaterability efficiencies as well as carbon and nutrients reduction. The observed sludge yield (Yobs) for the IFAS system was 0.37(±0.06) mg VSS/mg COD. After OSA implementation Yobs decreased by 32% and 46-65% at HRTSHT of 12 h and 6 h, respectively, indicating that prolonged exposure to anoxic/anaerobic conditions was not beneficial for sludge reduction. The lowest Yobs of 0.09(±0.05) mg VSS/mg COD (76% lower than that in the IFAS system) was obtained at an HRTSHT of 6 h and when TSHT was set at 35 °C. OSA implementation did not affect COD and NH4+ oxidation of the IFAS system (90-96% and 99%, respectively) and improved total nitrogen (TN) reduction (31-53%) due to improved denitrification in the SHT. On the contrary, sludge dewaterability worsened following OSA implementation, which was linked to the increased levels of exopolymeric substances in the suspended biomass.

A novel green approach for silver recovery from chloride leaching solutions through photodeposition on zinc oxide.

Silver is extensively used in electronics, industrial catalysis, and biomedical sector owing to its enhanced physicochemical properties. E-waste recycling may contribute significantly to enhance silver recovery in the view of a circular economy and limit the depletion of mineral sources. In this scenario, hydrometallurgical routes represent the most widely used techniques for silver extraction/recovery and require strong acidic solutions, high temperatures, and multiple operating units. An alternative sustainable route for silver recovery from leaching solutions used for silver extraction in industrial applications is herein proposed for the first time. The novel green process of silver recovery is based on the UV/vis light-driven photocatalytic deposition of pure metallic silver over low-cost and non-toxic ZnO photocatalyst. In the second step, ZnO is dissolved by slight acidification and pure metallic silver is easily recovered. Low environmental impact, mild operating conditions, and economic viability are among the major perks of the new silver recovery process developed. In the view of a full-scale implementation, several operating conditions of the recovery process (i.e., photocatalyst load, starting silver concentration, type of hole scavenger and irradiation) were thoroughly investigated. A mathematical model capable of describing the system behaviour under different operating conditions was also developed and allowed to estimate unknown kinetic parameters for the Ag-photodeposition process.

Phosphorus recovery as struvite and hydroxyapatite from the liquid fraction of municipal sewage sludge with limited magnesium addition.

Phosphorus (P) is an essential element to produce feed and fertilizers but also a nonrenewable resource. Both the predicted exhaustion of phosphatic rocks and the risk of eutrophication lead to an increasing necessity for P recovery methodologies to be applied in municipal wastewater treatment plants (WWTPs). One of the most promising solutions involves the precipitation of P-based minerals reusable as slow-release fertilizers. In this study, P recovery as struvite and hydroxyapatite from a municipal WWTP digestate liquid fraction (centrate) was investigated at varying pH (8-10), reagent typologies (MgCl2 , NaOH, Ca(OH)2 , and CaCl2 ), and concentrations under limiting magnesium doses through liquid- and solid-phase analyses and thermodynamical modeling. A maximum P recovery of 87.3% was achieved at pH 9 by adding NaOH and MgCl2 at a dose of 656 mg/L (the higher tested). According to these data, it was estimated that 92.0 tons/year of struvite and 33.2 tons/year of hydroxyapatite could be recovered from the WWTP centrate with a cost for reagent consumption being almost 50% of the mean P market value. An increase in P precipitation was observed while comparing experiments with the same pH values but with a higher Mg2+ dose. Ca2+ addition led to extensive P precipitation but mainly as amorphous phases that interfere with struvite formation.

Exploring the effect on the environment of encapsulated micro- and nano-plastics into asphalt mastics for road pavement.

A new environmental problem is represented by the huge transformation of plastic waste released into the environment into small fragments, the so called micro- and nano-plastics, due to atmospheric phenomena. The smaller the size of the plastic fragments, the more their spreading into environmental compartments. The aim of this study is to test encapsulation into asphalt mastics of waste plastic material (WPM) as sustainable strategy to obtain road flexible pavements and to evaluate the potential release in water of micro and nano plastics. A new mastic mixing method was developed to blend the WPM with the bitumen contained into a bitumen emulsion (BE60/40) by adopting low mixing temperatures. Three different WPM contents, equal to 5, 10 and 20% by the weight of the bitumen contained in the BE60/40, were adopted to produce the mastics; the mastics' rheological properties, obtained by frequency sweep and multiple stress creep and recovery tests, were compared to those of a traditional asphalt mastic containing limestone filler. The aging of asphalt mastics was analyzed by soaking them in water and gradually lowering and raising temperature between -10 and 60 °C at predefined intervals. The addition of WPM improved greatly the asphalt mastic performance; in particular, for a WPM content of 10%, the rheological response in terms of stiffness remained unchanged after the mastic underwent thermal excursions in water. Encapsulation of micro and nano plastics into mastics reduced of more than 99% their potential water release.

Microplastics in sewage sludge destined to anaerobic digestion: The potential role of thermal pretreatment.

Microplastics (MPs) are plastic particles with size smaller than 5 mm: their removal in municipal wastewater treatment plants mostly results in the accumulation of the coarser fraction into the sludge. The common application of the treated sludge as soil amendment raises the issue of the uncontrolled release of MPs into the environment, which depicts the need to identify suitable counteraction strategies. This work briefly reviews the most recent studies that focus on the fate of MPs during conventional sludge treatments, and, based on the results of this analysis, proposes the thermal pretreatment (120 °C, 30 min) of waste activated sludge (WAS) containing different kinds of MPs, in order to investigate its effect on the anaerobic biodegradability as well as on the abundance and physical features of MPs. Experimental results show that high temperatures did not alter polyethylene terephthalate (PET) MPs but the biodegradable-compostable ones (BIO-MPs), complying with the UNI EN 13432 standards. The profile of methane generation from thermally pretreated samples containing PET-MPs do not indicate any inhibition of the anaerobic process, which was positively influenced by the BIO-MPs in WAS: a 100% and 25% methane increase was observed over the control samples with and without the thermal pretreatment, respectively. Further studies are needed to better understand the mechanisms underlying biodegradable MPs behavior as well as to investigate the influence of high temperature treatments on smaller size MPs during anaerobic processes.

Sludge minimization in mainstream wastewater treatment: Mechanisms, strategies, technologies, and current development.

Excess sludge production in wastewater treatment plants has become an enormous environmental issue worldwide mainly due to the increased efforts towards wastewater purification. Researchers and plant operators are looking for technological solutions to reduce sludge production through the upgrading of existing technologies and configurations or by substituting them with alternative solutions. Several strategies have been identified to reduce sludge production, including the use of biological and physical-chemical methods (or a combination of them) and novel technologies, although many have not been sufficiently tested at full-scale. To select the most suitable system for sludge reduction, understanding the reduction mechanisms, advantages, disadvantages, and the economic and environmental impact of each technology is essential. This work offers a comprehensive and critical overview of mainstream sludge reduction technologies and underlying mechanisms from laboratory to full scale, and describes potential application, configuration, and integration with conventional systems. Research needs are highlighted, and a techno-economic-environmental comparison of the existing technologies is also proposed.

Valorisation of industrial hemp (Cannabis sativa L.) biomass residues through acidogenic fermentation and co-fermentation for volatile fatty acids production.

In line with the emerging circular bioeconomy paradigm, the present study investigated the valorisation of abundant hemp biomass residues (HBRs) such as hurds (HH) and a mix of leaves and inflorescences (Mix), and other organic wastes (i.e., cheese whey and grape pomace) through the volatile fatty acid (VFA) production in mono- and co-acidogenic fermentation. The highest VFA yields, measured as acetic acid (HAc) per unit of volatile solids (VS), were obtained with the untreated Mix in mono-fermentation (185 ± 57 mg HAc/g VS) and with the combination of Mix and CW in co-fermentation (651 ± 65 mg HAc/g VS), while the highest HAc percentage reached up to 94% of total VFAs. Finally, a preliminary techno-economic evaluation revealed that the mono-fermentation of alkali pretreated HH could lead to the highest revenues among HBRs, reaching up to 710-1810, 618-1577 and 766-3722 €/ha∙year for the production of HAc, single cell protein and polyhydroxybutyrates, respectively.

Direct nitrogen stripping and upcycling from anaerobic digestate during conversion of cheese whey into single cell protein.

The environmental impact of the dairy industry is heavily influenced by the overproduction of nitrogen- and carbon-rich effluents. The present study proposes an innovative process to recover waste contaminated nitrogen from anaerobic digestate while treating excess cheese whey (CW) and producing high-quality, clean single cell protein (SCP). By relying on direct aeration stripping techniques, employing an airflow subsequently used in the aerobic cheese whey fermentation step, the investigated process was able to strip 41-80% of the total ammonium nitrogen (N-NH4+) from liquid digestate. The stripped ammonia gas (NH3) was completely recovered as N-NH4+ in the acidic CW, and further upcycled into SCP having a total protein content of 74.7% and a balanced amino acids profile. A preliminary techno-economic analysis revealed the potential to directly recover and upcycle nitrogen into SCP at costs (4.3-6.3 €·kgN-1) and energetic inputs (90-132 MJ·kgN-1) matching those of conventional feed and nitrogen management processes.

Phosphorous removal and recovery from urban wastewater: Current practices and new directions.

Phosphate rocks are an irreplaceable resource to produce fertilizers, but their availability will not be enough to meet the increasing demands of agriculture for food production. At the same time, the accumulation of phosphorous discharged by municipal wastewater treatment plants (WWTPs) is one of the main causes of eutrophication. In a perspective of circular economy, WWTPs play a key role in phosphorous management. Indeed, phosphorus removal and recovery from WWTPs can both reduce the occurrence of eutrophication and contribute to meeting the demand for phosphorus-based fertilizers. Phosphorous removal and recovery are interconnected phases in WWTP with the former generally involved in the mainstream treatment, while the latter on the side streams. Indeed, by reducing phosphorus concentration in the WWTP side streams, a further improvement of the overall phosphorus removal from the WWTP influent can be obtained. Many studies and patents have been recently focused on treatments and processes aimed at the removal and recovery of phosphorous from wastewater and sewage sludge. Notably, new advances on biological and material sciences are constantly put at the service of conventional or unconventional wastewater treatments to increase the phosphorous removal efficiency and/or reduce the treatment costs. Similarly, many studies have been devoted to the development of processes aimed at the recovery of phosphorus from wastewaters and sludge to produce fertilizers, and a wide range of recovery percentages is reported as a function of the different technologies applied (from 10-25% up to 70-90% of the phosphorous in the WWTP influent). In view of forthcoming and inevitable regulations on phosphorous removal and recovery from WWTP streams, this review summarizes the main recent advances in this field to provide the scientific and technical community with an updated and useful tool for choosing the best strategy to adopt during the design or upgrading of WWTPs.

Biohythane production from food waste in a two-stage process: assessing the energy recovery potential.

ABSTRACTBiohythane (hydrogen + methane) production in a two stage dark fermentation (DF) and anaerobic digestion (AD) process from food waste (FW) has been studied. This paper investigated the effect of operation temperature, i.e. mesophilic (34 °C) and thermophilic (55 °C) , on biohythane yield and total energy recovery carried out at the initial culture pH 5.5 and pH 7, respectively for DF and AD batch tests. The mesophilic DF tests gave a higher hydrogen yield of 53.5 (±4) mL H2/g VS added compared to thermophilic DF tests, i.e. 37.6 (±1) mL H2/g VS added. However, higher methane yields, i.e. 307.5 (± 10) mL CH4/g VS, were obtained at thermophilic AD tests compared to mesophilic AD, i.e. 276.5 (±4.3) mL CH4/g VS. The total energy recovery from thermophilic DF + AD was higher (11.4 MJ/kg VS) than the mesophilic (10.4 MJ/kg VS) combined process. Interestingly, the analysis of kinetic parameters of mesophilic tests, determined from the Modified Gompertz equation, showed that mesophilic DF had faster H2 production kinetics, which can be attributed to a faster adaptation of the heat-shocked inoculum used in the tests to the incubation temperature. However, thermophilic AD tests exhibited faster kinetics for methane production.

Sustainable asphalt mastics made up recycling waste as filler.

The continuous growth of waste is generating worldwide more and more increasing related environmental concerns. Anything that is not recycled or recuperated from waste represents a loss of raw materials and other production factors used in the manufacture, transport and consumer phases of the product. This research explored the potential of three waste namely Construction and Demolition (CD) waste, Fly Ash (FA), and Jet Grouting (JG) waste as fillers in comparison to the traditional limestone one for making hot asphalt mastics for road pavement, through a rheological analysis and environmental compatibility tests towards the release of potentially toxic elements. A total of eight asphalt mastics were prepared by using two filler-to-binder weight ratios (f/b) of 0.5 and 1 for blending each filler with a neat bitumen 50/70 penetration grade. The Frequency Sweep test and the Multiple Stress Creep and Recovery (MSCR) test were carried out to investigate the rheological properties of the asphalt mastics. Asphalt mastics containing FA and JG fillers were found to be more mechanically and environmentally efficient than traditional limestone mastic in particular by adopting an f/b equal to 1 where it was observed higher complex shear modulus values, G*, (on average 50% compared to the traditional asphalt mastic) and lower non-recoverable creep compliance values, Jnr, (on average 35% compared to the traditional asphalt mastic) at all test temperatures investigated. Based on the suggested ranking methodology, CD emerged as the filler performing in the same way of the traditional one. All the waste containing mastics, showed up noticeable environmental compatibility, being the potentially toxic elements completely immobilized into the mastics' structure e practically not releasable into acidic water, highlighting the waste recycling for road pavements as primary strategy to immobilize hazardous wastes.

Air side-stream ammonia stripping in a thin film evaporator coupled to high-solid anaerobic digestion of sewage sludge: Process performance and interactions.

Air side-stream ammonia stripping in a thin-film evaporator (TFE) is proposed for the first time to control total ammonia nitrogen (TAN) concentration in a centralized full-scale plant performing high-solid anaerobic digestion (HSAD) of sewage sludge (SS). In this process, anaerobically digesting sludge (ADS) is continuously recirculated from the digester to the TFE unit where ammonia is stripped by an air stream. The stripped ammonia reacts with sulfuric acid in an absorption unit to produce ammonium sulfate. Overall, HSAD coupled to air side-stream ammonia stripping results in a twofold production of fertilizers (i.e. ammonium sulfate and SS digestate) recycling nutrients from organic wastes in agreement with the principles of circular economy. This study evaluates the influence of different operational airflow rates, temperatures and CO2 concentrations on air side-stream ammonia stripping in the TFE without alkali addition and the impact of air-based ammonia stripping on HSAD performance at full-scale. The study also investigates the chemistry and the interaction between the ammonia and carbonate subsystems of ADS and clarifies the stoichiometric relationship between ammonia and CO2 stripping.

Bioaugmentation strategy to enhance polycyclic aromatic hydrocarbons anaerobic biodegradation in contaminated soils.

This paper proposes an innovative bioaugmentation approach for the remediation of polycyclic aromatic hydrocarbon (PAH) contaminated soils, based on a novel habitat-based strategy. This approach was tested using two inocula (i-24 and i-96) previously enriched through an anaerobic digestion process on wheat straw. It relies on the application of allochthonous microorganisms characterized by specific functional roles obtained by mimicking a natural hydrolytic environment such as the rumen. The inocula efficiency was tested in presence of naphthalene alone, benzo[a]pyrene alone, and a mix of both of them. In single-contamination tests, i-24 inoculum showed the highest biodegradation rates (84.7% for naphthalene and 51.7% for benzo[a]pyrene). These values were almost 1.2 times higher than those obtained for both contaminants with i-96 inoculum and in the control test in presence of naphthalene alone, while they were 3 times higher compared to the control test in presence of benzo[a]pyrene alone. In mixed-contamination tests, i-96 inoculum showed final biodegradation efficiencies for naphthalene and benzo[a]pyrene between 1.1 and 1.5 higher than i-24 inoculum or autochthonous biomass. Total microbial abundances increased in the bioaugmented tests in line with the PAH degradation. The microbial community structure showed the highest diversity at the end of the experiment in almost all cases. Values of the Firmicutes active fraction up to 7 times lower were observed in the i-24 bioaugmented tests compared to i-96 and control tests. This study highlights a successful bioaugmentation strategy with biological components that can be reused in multiple processes supporting an integrated and environmentally sustainable bioremediation system.

Insights into bioflocculation of filamentous cyanobacteria, microalgae and their mixture for a low-cost biomass harvesting system.

Cyanobacteria and microalgae are considered as interesting feedstocks for either the production of high value bio-based compounds and biofuels or wastewater treatment. Nevertheless, the high costs of production, mainly due to the harvesting process, hamper a wide commercialization of industrial cyanobacteria and microalgae based products. Recent studies have found in autoflocculation and bioflocculation promising spontaneous processes for a low-cost and environmentally sustainable cyanobacteria and microalgae biomass harvesting process. In the present work, bioflocculation process has been studied for three different inocula: filamentous cyanobacteria, microalgae and their mixture. Their cultivation has been conducted in batch mode using two different cultivation media: synthetic aqueous solution and urban wastewater. The removal of nutrients and flocculation process performance were monitored during the entire cultivation time. Results have proved that bioflocculation and sedimentation processes occur efficiently for filamentous cyanobacteria cultivated in synthetic aqueous solution, whereas such processes are less efficient in urban wastewater due to the specific characteristics of this medium that prevent bioflocculation to occur. Besides different efficiencies associated to cultivation media, this work highlighted that bioflocculation of sole microalgae is not as effective as when they are cultivated together with filamentous cyanobacteria.

Supramolecular aggregation of colloidal natural organic matter masks priority pollutants released in water from peat soil.

Natural organic matter (NOM) from Sphagnum peat soil is extracted in water and subjected to several investigations to obtain structural and conformational information. Data show that the extracted NOM is self-organized in colloidal aggregates of variable sizes (from nano to micro scales, depending on the solvent composition, i.e., ultrapure water, solutions with denaturing agents, acetone, ethanol). Aggregates are formed by highly heterogeneous classes of organic compounds. According to the results of nuclear magnetic resonance and fluorescence measurements, the three-dimensional structure of aggregates, revealed by scanning electron microscope imaging, is supposed to be stabilized by the exposition of polar functional groups to the solvent, with consequent formation of hydrogen bonds, dipole-interactions and cation bridging. In contrast, the inner part of the aggregates displays hydrophobic features and is hypothesized to be further reinforced by the establishment of π-stacking interactions. The structure is assumed to be a supramolecular aggregation of small-medium oligomeric fragments (Max 750 Da) in which priority pollutants are entrapped by dispersive forces. The structures are shown to be nanosized spheroidal particles further aggregated to form higher dimension supra-structures. Carbohydrates play primary role, stabilizing the structure and giving marked hydrophilic properties to the aggregates.

Dynamics of bacterial communities and substrate conversion during olive-mill waste dark fermentation: Prediction of the metabolic routes for hydrogen production.

The aim of this work was to study the biological catalysts and possible substrate conversion routes in mesophilic dark fermentation reactors aimed at producing H2 from olive mill wastewater. Bacillus and Clostridium were the most abundant phylotypes during the rapid stage of H2 production. Chemical analyses combined with predictive functional profiling of the bacterial communities indicated that the lactate fermentation was the main H2-producing route. In fact, during the fermentation process, lactate and acetate were consumed, while H2 and butyrate were being produced. The fermentation process was rich in genes that encode enzymes for lactate generation from pyruvate. Lactate conversion to butyrate through the generation of pyruvate produced H2 through the recycling of electron carriers via the pyruvate ferredoxin oxydoreductase pathway. Overall, these findings showed the synergy among lactate-, acetate- and H2-producing bacteria, which complex interactions determine the H2 production routes in the bioreactors.

Effect of nickel on the comparative performance of inverse fluidized bed and continuously stirred tank reactors for biogenic sulphur-driven autotrophic denitrification.

The comparative performance of an inverse fluidized bed reactor (IFBR) having high density polyethylene beads as carrier materials for biofilm formation and a continuous stirred tank reactor (CSTR), both maintaining autotrophic denitrification using biogenic sulphur (ADBIOS) in the absence and presence of nickel (Ni2+), was studied. The reactors were compared in terms of NO3--N and NO2--N removal and SO42--S production throughout the study. A simulated wastewater with an inlet NO3--N concentration of 225 mg/L and a decreasing concentration of biogenic sulphur (bio-S) from 1.5 to 0.375 g/L was used. Both reactors were operated at a hydraulic retention time (HRT) of 48 h for 140 days and at an HRT of 42 h for the following 68 days. A more efficient ADBIOS was observed in the CSTR than IFBR throughout the study due to a better mixing of the feed wastewater in the bulk liquid and a higher availability of bio-S to the suspended cells. The NO3--N removal efficiency in the IFBR decreased by approximately 41% when the feed bio-S was reduced to 0.375 g/L, while it remained unaffected in the CSTR. Conversely, the presence of Ni2+ did not significantly affect NO3--N removal in both reactors even at a feed Ni2+ concentration of 120 mg/L. The highest NO3--N removal rates achieved were 86 and 108 mg NO3--N/(L·day) in the IFBR and CSTR, respectively, in the presence of 120 mg/L of feed Ni2+ at an HRT of 42 h. Batch studies conducted with acclimatized biomass showed that the continuous-flow operation mode in both reactors played a major role in helping the autotrophic denitrifiers to tolerate Ni2+ toxicity.

Simultaneous denitrification, phosphorus recovery and low sulfate production in a recirculated pyrite-packed biofilter (RPPB).

The simultaneous removal of nitrate (15 mg N-NO3- L-1) and phosphate (12 mg P-PO43- L-1) from nutrient-polluted synthetic water was investigated in a recirculated pyrite-packed biofilter (RPPB) under hydraulic retention time (HRT) ranging from 2 to 11 h. HRT values ≥ 8 h resulted in nitrate and phosphate average removal efficiency (RE) higher than 90% and 70%, respectively. Decrease of HRT to 2 h significantly reduced the RE of both nitrogen and phosphorus. The RPPB showed high resiliency as reactor performance recovered immediately after HRT increase to 5 h. Solid-phase characterization of pyrite granules and backwashing material collected from the RPPB at the end of the study revealed that iron-phosphate, -hydroxide and -sulfate precipitated in the bioreactor. Thermodynamic modeling predicted the formation of S0 during the study. Residence time distribution tests showed semi-complete mixing hydrodynamic flow conditions in the RPPB. The RPPB can be considered an elegant and low-cost technology coupling biological nitrogen removal to the recovery of phosphorus, iron and sulfur via chemical precipitation.

Bioflocculation and settling studies of native wastewater filamentous cyanobacteria using different cultivation systems for a low-cost and easy to control harvesting process.

Bioflocculation phenomena for filamentous cyanobacteria were studied and analysed in two different cultivation systems (i.e. based on air-bubbling and on shaking) and for different initial biomass concentrations. Floc formation and biomass settling were monitored during batch cultivation tests according to an innovative protocol. Results showed that the two cultivation systems enhanced two different flocculation behaviours: air bubbling led to the formation of small and dense flocs, while the shaking table resulted in larger (14 mm2 vs 4 mm2) but mechanically weaker flocs. Floc analysis evidenced that the different mixing systems also affected the speciation of biomass. A mathematical model was developed to simulate and predict the settling performance during the bioflocculation process of filamentous cyanobacteria. Natural settling was examined at different phases of biomass growth. Optimal conditions were obtained at the end of the exponential growth phase, when 70% of the total cultivated biomass could be recovered.

Effect of carbon-to-nitrogen ratio on simultaneous nitrification denitrification and phosphorus removal in a microaerobic moving bed biofilm reactor.

In this study, long-term simultaneous nitrification denitrification (SND) and phosphorous removal were investigated in a continuous-flow microaerobic MBBR (mMBBR) operated at a dissolved oxygen (DO) concentration of 1.0 (±0.2) mg L-1. The mMBBR performance was evaluated at different feed carbon-to-nitrogen (C/N) ratios (2.7, 4.2 and 5.6) and HRTs (2 days and 1 day). Stable long-term mMBBR operation and chemical oxygen demand (COD), total inorganic nitrogen (TIN) and phosphorous (P-PO43-) removal efficiencies up to 100%, 68% and 72%, respectively, were observed at a feed C/N ratio of 4.2. Lower TIN removal efficiency and unstable performance were observed at feed C/N ratios of 2.7 and 5.6, respectively. HRT decrease from 2 days to 1 day resulted in transient NH4+ accumulation and higher NO2-/NO3- ratio in the effluent. Batch activity tests showed that biofilm cultivation at a feed C/N ratio of 4.2 resulted in the highest denitrifying activity (189 mg N gVSS-1 d-1), whereas the highest nitrifying activity (316 mg N gVSS-1 d-1) was observed after cultivation at a feed C/N ratio of 2.7. Thermodynamic modeling with Visual MINTEQ and stoichiometric evaluations revealed that P removal was mainly biological and can be attributed to the P-accumulating capacity of denitrifying bacteria.