Analysis of micro- and nanoplastics in wastewater treatment plants: key steps and environmental risk considerations.

The analysis of micro- and nanoplastics (MNPs) in the environment is a critical objective due to their ubiquitous presence in natural habitats, as well as their occurrence in various food, beverage, and organism matrices. MNPs pose significant concerns due to their direct toxicological effects and their potential to serve as carriers for hazardous organic/inorganic contaminants and pathogens, thereby posing risks to both human health and ecosystem integrity. Understanding the fate of MNPs within wastewater treatment plants (WWTPs) holds paramount importance, as these facilities can be significant sources of MNP emissions. Additionally, during wastewater purification processes, MNPs can accumulate contaminants and pathogens, potentially transferring them into receiving water bodies. Hence, establishing a robust analytical framework encompassing sampling, extraction, and instrumental analysis is indispensable for monitoring MNP pollution and assessing associated risks. This comprehensive review critically evaluates the strengths and limitations of commonly employed methods for studying MNPs in wastewater, sludge, and analogous environmental samples. Furthermore, this paper proposes potential solutions to address identified methodological shortcomings. Lastly, a dedicated section investigates the association of plastic particles with chemicals and pathogens, alongside the analytical techniques employed to study such interactions. The insights generated from this work can be valuable reference material for both the scientific research community and environmental monitoring and management authorities.

Effects of inorganic ions on autotrophic denitrification by Thiobacillus denitrificans and on heterotrophic denitrification by an enrichment culture.

Salinity of nitrate-laden wastewaters, such as those produced by metal industries, tanneries, and wet flue gas cleaning systems may affect their treatment by denitrification. Salt inhibition of denitrification has been reported, while impacts of individual ions remain poorly understood whilst being relevant for wastewaters where often the concentration of a single ion rather than the salts varies. The aim of this study was to determine the inhibition by inorganic ions (Na+, Cl-, SO42- and K+) commonly present in saline wastewaters on denitrification and reveal its potential for the treatment of such waste streams, like those produced by NOx-SOx removal scrubbers. The inhibitory effects were investigated for both heterotrophic (enrichment culture) and autotrophic (T. denitrificans) denitrification in batch assays, by using NaCl, Na2SO4, KCl and K2SO4 salts at increasing concentrations. The half inhibition concentrations (IC50) of Na+ (as NaCl), Na+ (as Na2SO4) and Cl- (as KCl) were: 4.3 ± 0.3, 7.9 ± 0.5 and 5.2 ± 0.3 g/L for heterotrophic, and 1-2.5, 2.5-5 and 4.1 ± 0.3 g/L for autotrophic denitrification, respectively. Heterotrophic denitrification was completely inhibited at 20 g/L Na+ (as NaCl), 30 g/L Na+ (as Na2SO4) and 30 g/L Cl- (as KCl), while autotrophic at 8 g/L Na+ (as NaCl), 10 g/L Na+ (as Na2SO4) and 15 g/L Cl- (as KCl). In both cases, Cl- addition had the most important role in decreasing denitrification rate, while Na+ at 1 g/L stimulated autotrophic denitrification but rapidly inhibited the rate at higher concentrations. Nitrite reduction was less inhibited by the ions than nitrate reduction and both the osmotic pressure and the toxicity of the single ions played key roles in the overall inhibition of denitrification. Eventually, both autotrophic and heterotrophic denitrification showed potential for the treatment of a saline wastewater from a NOx-SO2 removal scrubber from a pulp mill.

Hydrochar and hydrochar co-compost from OFMSW digestate for soil application: 2. agro-environmental properties.

The work concerns the study of the hydrochar from digestate and hydrochar co-compost characterization as amendments. The processes for hydrochar and co-compost production were described in Part 1 of this work (Scrinzi et al., 2022). The amendment properties of hydrochar (produced at 180-200-220 °C for 3 h) and co-composts (25%, 50%, and 75% hydrochar percentage of digestate substitution) were assessed by phytotoxicity, plant growth bioassay, and soil effect. Different seeds species (Lepidium sativum, Cucumis sativus, and Sorghum bicolor sp.) were dosed at increased concentrations using both wet raw amendments and their water extracts. The chemical characterization showed phytotoxic compounds content depending on both the initial feedstock (digestate) and the HTC process; at the same time, the analysis highlighted the reduction of these compounds by composting (organic acid, polyphenols, salt concentration). The dose-response was analyzed by the Cedergreen-Streibig-Ritz model and the half-maximal effective concentration (EC50) was calculated based on this equation. The soil properties and GHG emissions measurements (CH4, CO2, N2O, and NH3) highlighted the effect on N dynamics and on soil respiration induced by substrates. The HC200 soil application determined a significant impact on CO2 and N2O emission and NH3 volatilization (10.82 mol CO2/m2; 51.45 mmol N2O/m2; 112 mol NH3/m2) and a significant reduction of total N and TOC (46% of TKN and 49% of TOC). The co-compost (75%) showed specific effects after soil application compared to other samples an increase of available P (48%), a greater content of nitrogen (1626 mg/kg dry basis), and a reduction of organic carbon (17%). Our results demonstrate the good quality of co-compost and at the same time the validity of this post-treatment for addressing many issues related to hydrochar use in the soil as an amendment, confirming the suitability of HTC process integration for digestate treatment in anaerobic digestion plants.

Hydrochar and hydrochar co-compost from OFMSW digestate for soil application: 1. production and chemical characterization.

The best available technique (BAT) for managing the organic fraction of municipal solid waste (OFMSW) is represented by anaerobic digestion (AD) and subsequent composting. This research explored a new industrial model in the framework of the C2Land international project, with the insertion of hydrothermal carbonization (HTC) as a post-treatment for OFMSW digestate. The reaction was set for 3 h at three different temperatures (180 ÷ 220 °C); the wet solid hydrochar obtained after filtration was then co-composted with greenery waste as a bulking agent and untreated OFMSW digestate in four different proportions in bench-scale bioreactors. The hydrochars and the hydrochar co-composts were suitable for agro-industrial applications, while the HTC liquors were tested in biochemical methane potential (BMP) for internal recirculation to AD. The scenarios proposed can be beneficial for plant enhancement and increased biogas production. This study reports results connected to the production phase. Mass balances confirmed that, during HTC, phosphorus precipitated into the solid products, organic nitrogen partially mineralized into ammonium, and oxidizable organic matter solubilized. The selected hydrochar obtained at 200 °C had mean (dry) solid, liquid, and gaseous yields equal to 77, 20, and 3 %db, respectively. The dynamic respirometric index (DRI) confirmed that the reproduced BAT for the composting process was effective in producing high-quality hydrochar co-composts in terms of biological stability. The BMP tests on HTC liquors showed some inhibitory effects, suggesting the need for future studies with inoculum adaptation and co-digestion, to dilute toxic compounds and enhance biogas production. Part 2 of this study describes the agro-environmental properties of hydrochars and hydrochar co-composts, including the beneficial effect of composting on hydrochars phytotoxicity.

Valorization of cow manure via hydrothermal carbonization for phosphorus recovery and adsorbents for water treatment.

The increased quantities of manure being generated by livestock and their extensive agronomic use have raised concerns around run-off impacting soil and groundwater quality. Manure contains valuable nutrients (especially phosphorus) that are critical to agriculture, but when directly land-applied the run-off of such nutrients contributes to eutrophication of waterways. This study investigates the hydrothermal carbonization of cow manure at two industrially feasible process extremes: 190 °C, 1 h and 230 °C, 3 h, to concentrate and then recover phosphorus from the solid hydrochar via acid leaching and precipitation. Up to 98 wt% of phosphorus initially present in the hydrochar (88% in the raw manure) can be recovered, with the dominant crystalline species being hydroxyapatite. Acid leached hydrochars were subsequently pyrolyzed at 600 °C for 30 min, and then evaluated as adsorbent materials for water remediation by using methylene blue as a model adsorbate. Although pyrolyzed hydrochars have surface areas an order of magnitude higher (160-236 m2/g) than the non-pyrolyzed acid leached hydrochars (11-23 m2/g), their adsorption capacity is three times lower. Furthermore, while the higher carbonization temperature leads to greater recovery of phosphorus, it likewise leads to higher heavy metal concentrations in the precipitate (ranging from 0.1 to 100 mgmetal/gppt). As such, lower temperature carbonization followed by acid-extraction - without further solid processing - is a potential pathway to recover phosphorus and adsorbent materials.

Anaerobic degradation of digestate based hydrothermal carbonization products in a continuous hybrid fixed bed anaerobic filter.

This study investigates the suitability of continuous hybrid fixed bed anaerobic filter reactor for treating sewage and agro-industrial digestate hydrothermal carbonization (HTC) products; hydrochar and HTC liquor (HTCL). The reactor was operated for 300 days under mesophilic conditions at different organic loading rates (OLR); maximum OLRs of 7.4 and 10 gCOD/L/d were reached while treating HTC liquor and slurry, respectively. 15 g/L hydrochar were added to the reactor as a supplement while treating HTCL solely thus increasing the biogas production up to 153%. The reactor was fed with HTCL and hydrochar with an increasing mixing ratio, and the co-digestion impact was dependent on hydrochar concentrations. The results of the study indicate that the hybrid fixed bed anaerobic filter reactor is a promising anaerobic digestion configuration for treating HTCL and overcoming the HTC upscaling challenges, and the suitability of digestate hydrochar utilization as supplement material for anaerobic digestion.

Coupling hydrothermal carbonization and anaerobic digestion for sewage digestate management: Influence of hydrothermal treatment time on dewaterability and bio-methane production.

Hydrothermal carbonization (HTC) technology is addressed in the framework of sewage digestate management. HTC converts digestate into a stabilized and sterilized solid (the hydrochar) and a liquor (HTCL) rich in organic carbon. This study aims to optimize the HTC operating parameters, namely the treatment time, in terms of hydrochar production, HTC slurry dewaterability, HTCL bio-methane yields in anaerobic digestion (AD), and process energy consumption. Digestate slurry was processed through HTC at different treatment times (0.5, 1, 2 and 3 h) at 190 °C, and the dewaterability of the treated slurries was addressed through capillary suction time and centrifuge lab-testing. In addition, biochemical methane potential (BMP) tests were conducted for HTCL under mesophilic conditions. Results show that by increasing the HTC treatment time the dewaterability was further improved, ammonium concentration in HTCL increased, and methane potential of HTCL decreased. 0.5 h HTCL had the highest bio-methane potential of 142 ± 3 mL CH4/g COD yet the treatment time was not sufficient for improving the slurry's dewaterability. HTC treatment time of 1 h at 190 °C was identified as the optimum trade-off for improved dewaterability and utilisation of HTCL for biogas production. 1 h HTCL bio-methane potential can cover around 25% of the HTC and AD thermal and electrical energy needs without considering the eventual use of the hydrochar as a biofuel.

Hydrothermal carbonization coupled with anaerobic digestion for the valorization of the organic fraction of municipal solid waste.

Hydrothermal carbonization (HTC) was evaluated as a promising treatment to enhance the biomethane potential during anaerobic digestion (AD) of the organic fraction of municipal solid waste (OFMSW). The OFMSW was carbonized at different conditions and HTC products were tested for biomethane potential into AD. Results proved that the use of HTC liquid and slurry into AD led to an increase in biomethane production up to 37% and 363%, respectively, compared to OFMSW. Methane production increased as the HTC process severity decreased, reaching its maximum at 180 °C, 1 h for both HTC products. Energy assessment demonstrated that the combustion of biogas produced by AD of HTC liquid and slurries covers up to 30% and 104% of the HTC thermal demand, respectively. When the energy from hydrochar and biogas combustion was recovered, the process efficiency reached 60%. Hence, HTC coupled with AD demonstrates to be an efficient way to valorize OFMSW.

A critical review of the current technologies in wastewater treatment plants by using hydrodynamic cavitation process: principles and applications.

In the last decade, hydrodynamic cavitation (HC) was increasingly used in the field of wastewater treatment. Due to its oxidative capability, HC was applied to treat aqueous effluents polluted by organic, toxic and bio-refractory contaminants, whereas its mechanical and chemical effects have allowed to disintegrate cells of microorganisms in biological applications. Due to their geometries, HC can be detected in some reactors, in which a variation of hydraulic parameters in the fluid such as flow pressure and flow velocity is induced. HC process involves the formation, growth, implosion and subsequent collapse of cavities, occurring in a very short period of time and releasing large magnitudes of power. In this paper, the vast literature on HC is critically reviewed, focusing on the basic principles behind it, in terms of process definition and analysis of governing mechanisms of both HC generation and pollutants degradation. The influence of various parameters on HC effectiveness was assessed, considering fluid properties, construction features of HC devices and technological aspects of processes. The synergetic effect of HC combined with chemicals or other techniques was discussed. An overview of the main devices used for HC generation and different existing methods to evaluate the cavitation effectiveness was provided. Knowledge buildup and optimization for such complex systems from mathematical modeling was highlighted.

How inoculation affects the development and the performances of microalgal-bacterial consortia treating real municipal wastewater.

To date, little is known about the start-up of photobioreactors and the progressive development of stable microalgal-bacterial consortia with a view to the full-scale treatment of real wastewater. Two photo-sequencing bioreactors, one inoculated with Chlorella vulgaris (RC) and one with the absence of inoculum (RW), were fed with real municipal wastewater and run in parallel for 101 days. The influence of the inoculation was evaluated in terms of pollutant removal efficiency, excess sludge production, solids settleability and microbial community characteristics. No significant differences were observed in the removal of COD (89 ± 4%; 88 ± 3%) and ammonium (99 ± 1%; 99 ± 1%), mainly associated with bacteria activity. During the first weeks of acclimation, Chlorella vulgaris in RC promoted better P removal and very high variations of DO and pH. Conversely, under steady-state conditions, no significant differences were observed between the performances of RC and RW, showing good settleability and low effluent solids, 7 ± 8 and 13 ± 10 mg TSS/L respectively. Microbiome analysis via 16S rRNA gene sequencing showed that, despite a different evolution, the microbial community was quite similar in both reactors under steady state conditions. Overall, the results suggested that the inoculation of microalgae is not essential to engender a photobioreactor aimed at treating real municipal wastewater.

How suspended solids concentration affects nitrification rate in microalgal-bacterial photobioreactors without external aeration.

The use of microalgae for the treatment of municipal wastewater makes possible to supply oxygen and save energy, but must be coupled with bacterial nitrification to obtain nitrogen removal efficiency above 90%. This paper explores how the concentration of Total Suspended Solids (TSS, from 0.2 to 3.9 g TSS/L) affects the nitrification kinetic in three microalgal-bacterial consortia treating real municipal wastewater. Two different behaviors were observed: (1) solid-limited kinetic at low TSS concentrations, (2) light-limited kinetic at higher concentrations. For each consortium, an optimal TSS concentration that produced the maximum volumetric ammonium removal rate (around 1.8-2.0 mg N L-1 h-1), was found. The relationship between ammonium removal rate and TSS concentration was then modelled considering bacteria growth, microalgae growth and limitation by dissolved oxygen and light intensity. Assessment of the optimal TSS concentrations makes possible to concentrate the microbial biomass in a photobioreactor while ensuring high kinetics and a low footprint.

Optimisation of Fe2+/H2O2 ratio in Fenton process to increase dewaterability and solubilisation of sludge.

Fenton is one of the advanced oxidation processes that can oxide organic compounds efficiently increasing the dewaterability of sludge. This study reports the optimisation of two reagents, Fe2+ and H2O2, involved in the Fenton process in order to increase sludge dewaterability and solubilisation, which were evaluated in terms of SRF, CST and DS. The study was divided into two sets of tests. First the optimal ratio Fe2+/H2O2 was set varying from 0.5-2.0. Results showed that the best Fe2+/H2O2 was equal to 0.8 corresponding to 2.1 s of CST, 2.1·1013 m kg-1 of SRF and 3.1% of DS. In the second set of tests, the Fe2+/H2O2 ratio was maintained fixed to 0.8 while the concentration of reagents was decreased up to 98% in order to verify the efficiency of the process. Results showed that performing Fenton process with a concentration of H2O2 and Fe2+ of, respectively, 6000 and 5000 mg L-1 the SRF and CST could be reduced up to 88% and 76%, respectively, and a DS equal to 3.1% could be obtained. A reduction in the Fenton reagents down to 300 and 250 mg L-1, respectively, for H2O2 and Fe2+ showed a little decrease in efficiency of the process. However, the Fenton process could be still performed thus obtaining an economic saving.

Remediation of Soil Polluted by Organic Compounds Through Chemical Oxidation and Phytoremediation Combined with DCT.

Soils contaminated with organic substances is an important issue across Europe: In some areas, these are the main causes of pollution, or the second after contamination from waste disposal. This paper included an experimental application that compared three methods of remediation of contaminated sites, based on electric fields: A single treatment (electroremediation); and two combined treatments, phyto-electrochemical and electrooxidation (a combination of chemical treatment and a DCT-direct current technology). The contaminated soil was taken from a former industrial area devoted to oil refining, located between two roads: The one national and the other one for industrial use. Nine soil samples were collected at two depths (0.2 and 0.4 m). The initial characterization of the soil showed a density of 1.5 g/cm³ and a moisture of about 20%; regarding grain size, 50% of the soil had particles with a diameter less than 0.08 mm. The electrochemical treatment and electrooxidation had an efficiency of 20% while the two combined methods had efficiencies of 42.5% for electrooxidation (with H2O2) and 20% for phyto-electroremediation (phyto-ER) with poinsettias.

Effects of hydrodynamic cavitation, low-level thermal and low-level alkaline pre-treatments on sludge solubilisation.

WAS is a polluting and hazardous waste generated in WWTPs that must be treated to prevent pollution and human health risks. Anaerobic digestion is the most used process for sludge stabilization. However, it must be improved in terms of both speed and extend of degradation. With the purpose of reducing the energy and chemical consumption linked to sludge treatment, in this study, different anaerobic digestion pre-treatments such as low-level mechanical (hydrodynamic cavitation, 2 bar), low-level thermal (50 °C) and low-level alkaline (NaOH, KOH and Ca(OH)2, pH 10) methods, and a combination thereof, were tested as strategies to improve sludge solubilisation. When the pre-treatments were used alone, the alkaline pre-treatment showed the highest sludge solubilisation. Among the alkaline reagents tested, NaOH and KOH led to higher DDPCOD (41.6 and 39.4%), while only 8.4% was achieved by using Ca(OH)2. However, the low-level hydrodynamic cavitation assisted thermo-alkaline pre-treatment was the most efficient in terms of both sludge solubilisation (DDPCOD = 53.0%) and energy efficiency (EE = 64.5 mgΔSCOD kJ-1). The synergetic effects of the combined pre-treatment were also confirmed by the highest release of EPS. Furthermore, cytometric analyses showed that the main mechanism involved in sludge solubilisation for the investigated pre-treatments was flocs disintegration rather than cell lysis.

One stage olive mill waste streams valorisation via hydrothermal carbonisation.

An olive waste stream mixture, coming from a three phase-continuous centrifugation olive oil mill industry, with a typical wet basis mass composition of olive pulp 39 wt%, kernels 5 wt% and olive mill waste water 56 wt%, was subjected to hydrothermal carbonisation (HTC) at 180, 220 and 250 °C for a 3-hour residence time in a 2-litre stainless steel electrically heated batch reactor. The raw feedstock and corresponding hydrochars were characterised in terms of proximate and ultimate analyses, higher heating values and energy properties. Results showed an increase in carbonisation of samples with increasing HTC severity and an energy densification ratio up to 142% (at 250 °C). Hydrochar obtained at 250 °C was successfully pelletised using a lab scale pelletiser without binders or expensive drying procedures. Energy characterisation (HHV, TGA), ATR-FTIR analysis, fouling index evaluation and pelletisation results suggested that olive mill waste hydrochars could be used as energy dense and mechanical stable bio-fuels. Characterisation of HTC residues in terms of mineral content via induced coupled plasma optical emission spectroscopy (ICP-OES) as well as Total and Dissolved Organic Carbon enabled to evaluate their potential use as soil improvers. Nutrients and polyphenolic compounds in HTC liquid fractions were evaluated for the estimation of their potential use as liquid fertilisers. Results showed that HTC could represent a viable route for the valorisation of olive mill industry waste streams.

Correction to: Strict anaerobic side-stream reactor: effect of the sludge interchange ratio on sludge reduction in a biological nutrient removal process.

The correct Fig. 1 is shown in this paper.

Anaerobic Digestion of Cattle Manure Influenced by Swirling Jet Induced Hydrodynamic Cavitation.

In this work, a modified swirling jet-induced cavitation has been employed for increasing anaerobic digestion efficiency of cattle manure. The hydrodynamic cavitation (HC) treatment improved the organic matter solubilization and the anaerobic biodegradability of cattle manure. The degree of disintegration increased by 5.8, 8.9, and 15.8% after the HC treatment at 6.0, 7.0, and 8.0 bars, respectively. However, the HC treatment at 7.0 bars had better results in terms of methane production. This result may be attributed to the possible formation of toxic and refractory compounds at higher inlet pressures, which could inhibit the methanization process. Further, total Kjeldahl nitrogen content was found to decrease with increasing inlet pressures, as the pH and the turbulent mixing favored the ammonia stripping processes. HC treatment decreased the viscosity of the treated cattle manure, favoring the manure pumping and mixing. Considerations on the energy input due to the HC pre-treatment and the energy output due to the enhanced methane yield have been presented. A positive energy balance can be obtained looking at the improved operational practices in the anaerobic digesters after the implementation of the HC pre-treatment.

Strict anaerobic side-stream reactor: effect of the sludge interchange ratio on sludge reduction in a biological nutrient removal process.

In order to investigate the impact of the sludge interchange ratio (IR) on both the sludge reduction process and the carbon and nutrient removal efficiencies, an anaerobic side-stream reactor (ASSR) at 20 °C and - 400 mV was operated for 300 days coupled to a sequencing batch reactor (SBR) for urban wastewater treatment. It was found that a 100% interchange rate, corresponding to an anaerobic solid retention time (SRTASSR) of 2.5 days, was the most suitable case in terms of sludge reduction and wastewater treatment process, achieving a 66% sludge reduction compared to a control system simulated as an SBR. Chemical oxygen demand (COD), ammonium nitrogen, total nitrogen, and phosphate removal efficiencies of 86.1 ± 7.2, 82.5 ± 11.2, 81.7 ± 12.0, and 62.6 ± 15.0%, respectively, were achieved. When the interchange rate was increased, more ammonium nitrogen and soluble extracellular polymeric substance concentrations were released in the ASSR. This implies that cell lysis and hydrolysis of particulate organic matter in the ASSR were processes of fundamental importance with the increasing mass of sludge cycled to the ASSR. Compared to the release of ammonia, soluble COD release was detected to a lesser extent, due to its consumption by microorganisms in the ASSR. There was also a simultaneous increase in slow-growing microorganisms which use organic carbon for metabolic activities, above all sulfate-reducing bacteria and denitrifying phosphate-accumulating organisms. This increase contributed significantly to sludge reduction in the SBR-ASSR system.

A swirling jet-induced cavitation to increase activated sludge solubilisation and aerobic sludge biodegradability.

In this work, a modified swirling jet induced hydrodynamic cavitation (HC) has been used for the pre-treatment of excess sludge. In order to both improve the HC treatment efficiencies and reduce the energy consumption, the effectiveness of the HC reactor on sludge disintegration and on aerobic biodegradability has been investigated at different operating conditions and parameters, such as temperature, inlet pressure, sludge total solid (TS) content and reactor geometry. The inlet pressure was related to the flow velocity and pressure drop. The best results in terms of sludge solubilisation were achieved after 2h of HC treatment, treating a 50.0gTSL-1 and using the three heads Ecowirl system, at 35.0°C and 4.0bar. Chemical and respirometric tests proved that sludge solubilisation and aerobic biodegradability can be efficiently enhanced through HC pre-treatment technique. At the optimum operating conditions, the specific supplied energy has been varied from 3276 to 12,780kJkgTS-1 in the HC treatment, by increasing the treatment time from 2 to 8 h, respectively. Low endogenous decay rates (bH) were measured on the excess sludge at low specific supplied energy, revealing that only an alteration in floc structure was responsible for the sludge solubilisation. On the contrary, higher bH values were measured at higher specific supplied energy, indicating that the sludge solubilisation was related to a decreasing biomass viability, as consequence of dead cells and/or disrupted cells (cell lysis).

Shift in microbial community structure of anaerobic side-stream reactor in response to changes to anaerobic solid retention time and sludge interchange ratio.

A laboratory scale nutrient removal activated sludge system coupled with an anaerobic side-stream reactor was operated for 300 days treating real urban wastewater. A significant decrease in sludge production was obtained increasing the anaerobic solid retention time (SRTASSR) and decreasing the sludge interchange ratio (IR). In this study, the microbial community structure was analyzed and compared with the sludge reduction performance. Quantitative polymerase chain reaction analyses encoding 16 ribosomal RNA and functional genes revealed a wide diversity of phylogenetic groups in each experimental period, resulting from long solids retention time and recirculation of sludge under aerobic, anoxic and anaerobic conditions. However, decreasing SRTASSR from 10 to 2.5d and increasing IR from 27 to 100%, an increasing selection of both fermenting bacteria able to release extracellular polymeric substances and hydrolyze organic matter and slow growing bacteria involved in nutrient removal were detected and linked to the sludge reduction mechanisms.