Sulfamethoxazole Enhances Specific Enzymatic Activities under Aerobic Heterotrophic Conditions: A Metaproteomic Approach.

The growing concern about antibiotic-resistant microorganisms has focused on the sludge from wastewater treatment plants (WWTPs) as a potential hotspot for their development and spread. To this end, it seems relevant to analyze the changes on the microbiota as a consequence of the antibiotics that wastewater may contain. This study aims at determining whether the presence of sulfamethoxazole (SMX), even in relatively low concentrations, modifies the microbial activities and the enzymatic expression of an activated sludge under aerobic heterotrophic conditions. For that purpose, we applied a metaproteomic approach in combination with genomic and transformation product analyses. SMX was biotransformed, and the metabolite 2,4(1H,3H)-pteridinedione-SMX (PtO-SMX) from the pterin-conjugation pathway was detected at all concentrations tested. Metaproteomics showed that SMX at 50-2000 µg/L slightly affected the microbial community structure, which was confirmed by DNA metabarcoding. Interestingly, an enhanced activity of the genus Corynebacterium and specifically of five enzymes involved in its central carbon metabolism was found at increased SMX concentrations. Our results suggest a role of Corynebacterium genus on SMX risks mitigation in our bioreactors.

Principles, Advances, and Perspectives of Anaerobic Digestion of Lipids.

Several problems associated with the presence of lipids in wastewater treatment plants are usually overcome by removing them ahead of the biological treatment. However, because of their high energy content, waste lipids are interesting yet challenging pollutants in anaerobic wastewater treatment and codigestion processes. The maximal amount of waste lipids that can be sustainably accommodated, and effectively converted to methane in anaerobic reactors, is limited by several problems including adsorption, sludge flotation, washout, and inhibition. These difficulties can be circumvented by appropriate feeding, mixing, and solids separation strategies, provided by suitable reactor technology and operation. In recent years, membrane bioreactors and flotation-based bioreactors have been developed to treat lipid-rich wastewater. In parallel, the increasing knowledge on the diversity of complex microbial communities in anaerobic sludge, and on interspecies microbial interactions, contributed to extend the knowledge and to understand more precisely the limits and constraints influencing the anaerobic biodegradation of lipids in anaerobic reactors. This critical review discusses the most important principles underpinning the degradation process and recent key discoveries and outlines the current knowledge coupling fundamental and applied aspects. A critical assessment of knowledge gaps in the field is also presented by integrating sectorial perspectives of academic researchers and of prominent developers of anaerobic technology.

Enzymatic cometabolic biotransformation of organic micropollutants in wastewater treatment plants: A review.

Biotransformation of trace-level organic micropollutants (OMPs) by complex microbial communities in wastewater treatment facilities is a key process for their detoxification and environmental impact reduction. Therefore, understanding the metabolic activities and mechanisms that contribute to their biotransformation is essential when developing approaches aiming to minimize their discharge. This review addresses the relevance of cometabolic processes and discusses the main enzymatic activities currently known to take part in OMPs removal under different redox environments in the compartments of wastewater treatment plants. Furthermore, the most common methodologies to decipher such enzymes are discussed, including the use of in vitro enzyme assays, enzymatic inhibitors, the analysis of transformation products and the application of several -omic techniques. Finally, perspectives on major challenges and future research requirements to improve OMPs biotransformation are proposed.

Heterotrophic enzymatic biotransformations of organic micropollutants in activated sludge.

While heterotrophic microorganisms constitute the major fraction of activated sludge biomass, the role of heterotrophs in the biotransformation of organic micropollutants (OMPs) has not been fully elucidated. Yet, such knowledge is essential, particularly when conceiving novel wastewater treatment plants based on a two-stage process including an A-stage under heterotrophic conditions and a B-stage based on anammox activity. Biotransformation of OMPs in activated sludge is thought to mostly occur cometabolically thanks to the action of low specificity enzymes involved in the metabolism of the primary substrates. For a better understanding of the process, it is important to determine such enzymatic activities and the underlying mechanisms involved in OMPs biotransformation. This task has proven to be difficult due to the lack of information about the enzymatic processes and the complexity of the biological systems present in activated sludge. In this paper, a continuous aerobic heterotrophic reactor following 20 OMPs at environmental concentrations was operated to (i) assess the potential of heterotrophs during the cometabolic biotransformation of OMPs, (ii) identify biotransformation reactions catalyzed by aerobic heterotrophs and (iii) predict possible heterotrophic enzymatic activities responsible for such biotransformations. Contradicting previous reports on the dominant role of nitrifiers in OMPs removal during activated sludge treatment, the heterotrophic population proved its capacity to biotransform the OMPs to extents equivalent to reported values in nitrifying activated sludge plants. Besides, 12 transformation products potentially formed through the activity of several enzymes present in heterotrophs, including monooxygenases, dioxygenases, hydrolases and transferases, were identified.

The organic loading rate affects organic micropollutants' cometabolic biotransformation kinetics under heterotrophic conditions in activated sludge.

Several studies have shown that organic micropollutants (OMPs) are biotransformed cometabolically in activated sludge systems. However, the individual role of heterotrophs in the microbial consortium is still not clear, i.e., there is still a gap regarding the influence of the heterotrophic activity on the cometabolic biotransformation kinetics and yield of the OMPs. Aiming to answer these questions, experiments with increasing primary substrate concentrations were performed under aerobic heterotrophic conditions in a continuous stirred tank reactor operated at several organic loading rates (OLR) with fixed hydraulic retention time. Moreover, the individual kinetic parameters were determined in batch assays with different initial substrate concentrations using the sludges from the continuous reactor. A set of 15 OMPs displaying a variety of physicochemical properties were spiked to the feeding in the ng L-1 - µg L-1 range. Results reveal that the biodegradation of the primary carbon source and the biotransformation of the OMPs occur simultaneously, in clear evidence of cometabolic behavior. Moreover, we conclude that the OMPs biotransformation kinetic constant (kbiol) shows a linear dependence with the OLR of the primary substrate for most of the compounds studied, suggesting that the heterotrophic activity seriously affects the OMPs biotransformation kinetics. However, under typical activated sludge systems operating conditions (hydraulic retention times above 8 h), their biotransformation yield would not be significantly affected.

Protein composition determines the preferential consumption of amino acids during anaerobic mixed-culture fermentation.

The valorisation of protein-rich residual streams by anaerobic mixed-culture fermentation (MCF) has been barely studied in contrast to carbohydrate-rich wastes. The aim of this work was, therefore, to investigate how protein composition, i.e. the amino acid (AA) profile, affects the individual consumption of amino acids and, consequently, the outcome of the process. Mixed-culture fermentations were performed with two model proteins (casein and gelatin) using continuous and batch reactors at neutral pH values and 25 °C. The acidification was incomplete for both proteins, with casein achieving a higher value than gelatin. Albeit dominated by acetic acid, product spectra were different as well, with n-butyric acid as the second major product for casein and propionic acid for gelatin. The preferential consumption of amino acids was demonstrated, which interestingly depends on protein composition. The previously accepted stoichiometry accurately describes iso and n-butyric acid production, but it fails for propionic, iso and n-valeric acid generation. Overall, this study offers a better understanding of protein fermentation mechanisms, which will help to improve degradation models and to design fermentation processes, based on optimal substrate selection.

Assessment of the fate of organic micropollutants in novel wastewater treatment plant configurations through an empirical mechanistic model.

Novel wastewater treatment plants (WWTPs) are expected to be less energetically demanding than conventional ones. However, scarce information is available about the fate of organic micropollutants (OMPs) in these novel configurations. Therefore, the objective of this work is to assess the fate of OMPs in three novel WWTP configurations by using a plant-wide simulation that integrates multiple units. The difference among the three configurations is the organic carbon preconcentration technology: chemically enhanced primary treatment (CEPT), high-rate activated sludge (HRAS) combined or not with a rotating belt filter (RBF); followed by a partial-nitritation (PN-AMX) unit. The simulation results show that the three selected novel configurations lead mainly to comparable OMPs removal efficiencies from wastewater, which were similar or lower, depending on the OMP, than those obtained in conventional WWTPs. However, the presence of hydrophobic OMPs in the digested sludge noticeably differs among the three configurations. Whereas the configuration based on sole HRAS to recover organic carbon leads to a lower presence of OMPs in digested sludge than the conventional WWTP, in the other two novel configurations this presence is noticeable higher. In conclusion, novel WWTP configurations do not improve the OMPs elimination from wastewater achieved in conventional ones, but the HRAS-based WWTP configuration leads to the lowest presence in digested sludge so it becomes the most efficient alternative.

Metabolic modeling for predicting VFA production from protein-rich substrates by mixed-culture fermentation.

Proteinaceous organic wastes are suitable substrates to produce high added-value products in anaerobic mixed-culture fermentations. In these processes, the stoichiometry of the biotransformation depends highly on operational conditions such as pH or feeding characteristics and there are still no tools that allow the process to be directed toward those products of interest. Indeed, the lack of product selectivity strongly limits the potential industrial development of these bioprocesses. In this work, we developed a mathematical metabolic model for the production of volatile fatty acids from protein-rich wastes. In particular, the effect of pH on the product yields is analyzed and, for the first time, the observed changes are mechanistically explained. The model reproduces experimental results at both neutral and acidic pH and it is also capable of predicting the tendencies in product yields observed with a pH drop. It also offers mechanistic insights into the interaction among the different amino acids (AAs) of a particular protein and how an AA might yield different products depending on the relative abundance of other AAs. Particular emphasis is placed on the utility of this mathematical model as a process design tool and different examples are given on how to use the model for this purpose.

A metabolic model for targeted volatile fatty acids production by cofermentation of carbohydrates and proteins.

Anaerobic mixed-culture fermentations are interesting processes to valorise organic wastes by converting them to volatile fatty acids. One of the main issues is that certain operational conditions (e.g. pH or different substrate concentrations) can vary significantly the product spectrum. So far, there are no tools that take into the account the characteristic features of cofermentation processes, which hinders the possibility of designing processes that use real wastes as substrates. In this work a mathematical model was developed for the production of volatile fatty acids from organic wastes with a high concentration of carbohydrates and proteins. The model reproduces satisfactorily experimental results and is also able of giving mechanistic insight into the interactions between carbohydrates and proteins that explain the observed changes in the product spectrum. We envision this model as the core of an early-stage design tool for anaerobic cofermentation processes, as shown in this work with different examples.

Acidogenesis is a key step in the anaerobic biotransformation of organic micropollutants.

Understanding the role of the different anaerobic digestion stages on the removal of organic micropollutants (OMPs) is essential to mitigate their release from wastewater treatment plants. This study assessed the fate of 21 OMPs during hydrolysis and acidogenesis to elucidate the contribution of these stages to the overall anaerobic removal. Moreover, the removal mechanisms and factors influencing them were investigated. To this purpose, a fermentation reactor was operated and fed with two different substrates: starch (to jointly evaluate hydrolysis and acidogenesis) and glucose (to isolate acidogenesis). Results indicate that sulfamethoxazole was highly biotransformed (>80 %), while galaxolide, celestolide, tonalide, erythromycin, roxithromycin, trimethoprim, octylphenol and nonylphenol achieved a 50-80 % biotransformation. Since no significant differences in the biotransformation efficiencies were found between starch and glucose fermentation, it is stated that the enzymatic activities involved in starch hydrolysis do not significantly contribute to the cometabolic biotransformation of OMPs, while acidogenesis appears as the major player. Moreover, a higher biotransformation (≥15 percentage points and p ≤ 0.05) was found for galaxolide, celestolide, tonalide, erythromycin and roxithromycin during acidogenesis in comparison with the efficiencies reported for the acetogenic/methanogenic step. The biotransformation of some OMPs was explained considering their chemical structure and the enzymatic activities.

Comprehensive comparison of chemically enhanced primary treatment and high-rate activated sludge in novel wastewater treatment plant configurations.

Novel wastewater treatment plants (WWTPs) are designed to be more energy efficient than conventional plants. One approach to becoming more energy efficient is the pre-concentration of organic carbon through chemically enhanced primary treatment (CEPT) or high-rate activated sludge (HRAS). This study compares these approaches in terms of energy demand, operational costs, organic micropollutants (OMP), and virus removal efficiency. A CEPT pilot-scale plant was operated at a hydraulic retention time (HRT) of 30 min, and a lab-scale HRAS reactor was operated at an HRT of 2 h and a solid retention time (SRT) of 1 d in continuous mode. A minimum dose of 150 mg/L ferric chloride (FeCl3) was required to achieve a threshold chemical oxygen demand (COD)-to-ammonium ratio below 2 g COD to 1 g of NH4+ -N (fulfilling the requirement for a partial nitritation-anammox reactor), reaching high phosphate (PO43-)-removal efficiency (>99%). A slightly lower COD recovery was attained in the HRAS reactor, due to the partial oxidation of the influent COD (15%). The lower PO43- removal efficiency achieved in the HRAS configuration (13%) was enhanced to a comparable value of that achieved in CEPT by the addition of 30 mg/L FeCl3 at the clarifier. The CEPT configuration was less energy-intensive (0.07 vs 0.13 kWh/m3 of wastewater) but had significantly higher operational costs than the HRAS-based configuration (6.0 vs 3.8 c€/m3 of wastewater). For OMPs with kbiol > 10 L/gVSS·d, considerably higher removal efficiencies were achieved in HRAS (80-90%) than in CEPT (4-55%). For the remaining OMPs, the biotransformation efficiencies were generally higher in HRAS than in CEPT but were below 55% in both configurations. Finally, CEPT was less efficient than HRAS for virus removal. HRAS followed by FeCl3 post-treatment appeared to be a more effective alternative than CEPT for COD pre-concentration in novel WWTPs.

Air-side ammonia stripping coupled to anaerobic digestion indirectly impacts anaerobic microbiome.

Air-side stripping without a prior solid-liquid phase separation step is a feasible and promising process to control ammonia concentration in thermophilic digesters. During the process, part of the anaerobic biomass is exposed to high temperature, high pH and aerobic conditions. However, there are no studies assessing the effects of those harsh conditions on the microbial communities of thermophilic digesters. To fill this knowledge gap, the microbiomes of two thermophilic digesters (55°C), fed with a mixture of pig manure and nitrogen-rich co-substrates, were investigated under different organic loading rates (OLR: 1.1-5.2 g COD l-1  day-1 ), ammonia concentrations (0.2-1.5 g free ammonia nitrogen l-1 ) and stripping frequencies (3-5 times per week). The bacterial communities were dominated by Firmicutes and Bacteroidetes phyla, while the predominant methanogens were Methanosarcina sp archaea. Increasing co-substrate fraction, OLR and free ammonia nitrogen (FAN) favoured the presence of genera Ruminiclostridium, Clostridium and Tepidimicrobium and of hydrogenotrophic methanogens, mainly Methanoculleus archaea. The data indicated that the use of air-side stripping did not adversely affect thermophilic microbial communities, but indirectly modulated them by controlling FAN concentrations in the digester. These results demonstrate the viability at microbial community level of air side-stream stripping process as an adequate technology for the ammonia control during anaerobic co-digestion of nitrogen-rich substrates.

Thermal hydrolysis of sewage sludge partially removes organic micropollutants but does not enhance their anaerobic biotransformation.

Pretreatment technologies prior to anaerobic digestion (AD) have been developed with the aim of enhancing biogas productivity and reducing the presence of pathogens in digested sludge. Among them, thermal hydrolysis (TH) appears as the most promising one. In wastewater treatment plants (WWTPs) sludge is the end point of many organic micropollutants (OMPs), which was proved to lead to important environmental and human risks since sludge is commonly used in agriculture. The objective of this work is to determine the fate OMPs in TH and subsequent AD. Sewage sludge was pretreated in a TH pilot plant at 170 °C for 20 min. Afterwards, two anaerobic digesters with a working volume of 14 L fed with fresh and pretreated sludge were operated in parallel in mesophilic conditions. TH proved to be an effective technology to partially or totally remove the dissolved fraction of OMPs as well as the fraction sorbed into those suspended solids that are solubilised after this pretreatment. However, it did not affect the OMPs sorbed concentration into solids that are not solubilised. Globally, the OMPs removal efficiency during TH appears to be linked to the solids solubilisation during this process. Afterwards, the OMPs biotransformation efficiency in AD of fresh and pretreated sludge was determined. Noticeable differences between the microbiome of both reactors was determined, but the anaerobic biotransformation was not substantially different for most of the OMPs. However, it affected musk fragrances, which presented considerably lower biotransformation efficiency in the reactor fed with pretreated sludge. Therefore, TH was proved effective in partially removing OMPs but not in enhancing their bioavailability and subsequent anaerobic biotransformation.

Energetic and economic assessment of sludge thermal hydrolysis in novel wastewater treatment plant configurations.

Novel wastewater treatment plants (WWTPs) are aimed to be more energetically efficient than conventional ones. Their first step is a chemical oxygen demand (COD) preconcentration stage with different alternatives, such as rotating belt filters (RBF), chemically enhanced primary treatment (CEPT), high-rate activated sludge (HRAS), or combinations thereof, in which energy requirements are substantially reduced. The COD recovered as sludge allows a noticeable increase of biogas production in anaerobic digestion (AD). In conventional WWTPs, sludge anaerobic biodegradability can be significantly enhanced by applying sludge pretreatment methods, such as thermal hydrolysis (TH), before AD. However, considering that novel-sludges are more anaerobically biodegradable than conventional ones, the impact of TH on their methane production is expected to result significantly lower. In this study, an energetic and economic assessment of applying TH in novel WWTPs was performed. We found that TH is only justified to reduce operational costs as long as sludge TS concentration in the feeding to the TH unit is higher than 1-2%. The HRAS-based WWTP is the scenario that leads to the lowest treatment costs (below 1c €/ m3 wastewater if sludge is thickened over 10% of TS). However, the WWTP based on CEPT for COD preconcentration leads to the lowest electricity consumption (below 0.01 kWh/m3 of wastewater), but even in the most favourable conditions the energy autarky was not achievable. Results show that the main impact of TH is mainly due to sludge disposal savings (270,000-430,000 €/year for a 500,000 inhabitants WWTP) rather than the increase of energy production (achieves maximum savings of 35,000-60,000 €/year). Payback time is very dependent on the WWTP size, ranging from 15 to 30 years for a 100,000 inhabitants WWTP and from 2 to 4 years for a 1,000,000 inhabitants WWTP.

Organic overloading affects the microbial interactions during anaerobic digestion in sewage sludge reactors.

There is still a lack of information about microbial interactions of anaerobic digestion microbiome during process disturbance which limits our ability to predict the mechanisms that drive community dynamics on these events. This paper aims to determine how an organic overloading affects these interactions and to characterize in detail the microbiome structure and diversity in sewage sludge anaerobic reactors during an acidosis event. Two identical sewage sludge anaerobic reactors were subjected to an organic loading shock by adding glycerol waste. As consequence, volatile fatty acids accumulated after only 24 h (up to 2.5 g/L) while Bacteroidales and Methanomicrobiales became displaced by Firmicutes and Methanosaeta sp, showing that reactor acidosis can occur without an immediate decline of this methanogen. Network analysis revealed 9 clusters of co-occurring microorganisms with different behaviors during overloading. At first, Veillonellaceae family, the main glycerol degrading, associated with Candidatus Cloacimonetes, volatile fatty acids fermenters, increased their relative abundance in detriment of the syntrophic bacteria; although as conditions become more acidic, these groups were displaced by other fermenters like Porphyromonadaceae and Chitinophagaceae. Eventually, the methanogenesis failed 72 h after organic overloading, when pH reached values lower than 6. Overall, our results showed a succession of functionally redundant microorganisms, most likely because of niche specialization during organic overloading. The detailed temporal analysis elucidated the processes governing the dynamics anaerobic digestion microbiome, a knowledge required to develop anaerobic digestion management strategies based on its microbiome during process disturbances.

Reversibility of enzymatic reactions might limit biotransformation of organic micropollutants.

Biotransformation of many organic micropollutants (OMPs) in sewage treatment plants is incomplete leading to their release into the environment. Recent findings suggest that thermodynamic aspects of the reaction as chemical equilibrium limit biotransformation, while kinetic parameters have a lower influence. Reversibility of enzymatic reactions might result in a chemical equilibrium between the OMP and the transformation product, thus impeding a total removal of the compound. To the best of our knowledge, no study has focused on proving the reversible action of enzymes towards OMPs so far. Therefore, we aimed at demonstrating this hypothesis through in vitro assays with bisphenol A (BPA) in the presence of kinase enzymes, namely acetate kinase and hexokinase, which are key enzymes in anaerobic processes. Results suggest that BPA is phosphorylated by acetate kinase and hexokinase in the presence of ATP (adenosine 5-triphosphate), but when the concentration of this co-substrate decreases and the enzymes loss their activity, the backward reaction occurs, revealing a reversible biotransformation mechanism. This information is particularly relevant to address new removal strategies, which up to now were mainly focused on modifying the kinetic parameters of the reaction.

Biotransformation of organic micropollutants by anaerobic sludge enzymes.

Biotransformation of organic micropollutants (OMPs) in wastewater treatment plants ultimately depends on the enzymatic activities developed in each biological process. However, few research efforts have been made to clarify and identify the role of enzymes on the removal of OMPs, which is an essential knowledge to determine the biotransformation potential of treatment technologies. Therefore, the purpose of the present study was to investigate the enzymatic transformation of 35 OMPs under anaerobic conditions, which have been even less studied than aerobic systems. Initially, 13 OMPs were identified to be significantly biotransformed (>20%) by anaerobic sludge obtained from a full-scale anaerobic digester, predestining them as potential targets of anaerobic enzymes. Native enzymes were extracted from this anaerobic sludge to perform transformation assays with the OMPs. In addition, the effect of detergents to recover membrane enzymes, as well as the effects of cofactors and inhibitors to promote and suppress specific enzymatic activities were evaluated. In total, it was possible to recover enzymatic activities towards 10 out of these 13 target OMPs (acetyl-sulfamethoxazole and its transformation product sulfamethoxazole, acetaminophen, atenolol, clarithromycin, citalopram, climbazole, erythromycin, and terbutryn, venlafaxine) as well as towards 8 non-target OMPs (diclofenac, iopamidol, acyclovir, acesulfame, and 4 different hydroxylated metabolites of carbamazepine). Some enzymatic activities likely involved in the anaerobic biotransformation of these OMPs were identified. Thereby, this study is a starting point to unravel the still enigmatic biotransformation of OMPs in wastewater treatment systems.

Resource recovery from pig manure via an integrated approach: A technical and economic assessment for full-scale applications.

Intensive livestock farming cannot be uncoupled from the massive production of manure, requiring adequate management to avoid environmental damage. The high carbon, nitrogen and phosphorus content of pig manure enables targeted resource recovery. Here, fifteen integrated scenarios for recovery of water, nutrients and energy are compared in terms of technical feasibility and economic viability. The recovery of refined nutrients with a higher market value and quality, i.e., (NH4)2SO4 for N and struvite for P, coincided with higher net costs, compared to basic composting. The inclusion of anaerobic digestion promoted nutrient recovery efficiency, and enabled energy recovery through electricity production. Co-digestion of the manure with carbon-rich waste streams increased electricity production, but did not result in lower process costs. Overall, key drivers for the selection of the optimal manure treatment scenario will include the market demand for more refined (vs. separated or concentrated) products, and the need for renewable electricity production.

Why are organic micropollutants not fully biotransformed? A mechanistic modelling approach to anaerobic systems.

Biotransformation of most organic micropollutants (OMPs) during wastewater treatment is not complete and an unexplained steady decrease of the biotransformation rate with time is reported for many OMPs in different biological processes. To minimize and accurately predict the emission of OMPs into the environment, the mechanisms and limitations behind their biotransformations should be clarified. Aiming to achieve this objective, the present study follows a mechanistic modelling approach, based on the formulation of four models according to different biotransformation hypotheses: Michaelis-Menten kinetics, chemical equilibrium between the parent compound and the transformation product (TP), enzymatic inhibition by the TP, and a limited compound bioavailability due to its sequestration in the solid phase. These models were calibrated and validated with kinetic experiments performed in two different anaerobic systems: continuous reactors enriched with methanogenic biomass and batch assays with anaerobic sludge. Model selection was conducted according to model suitability criteria (goodness of fitting the experimental data, confidence of the estimated parameters, and model parsimony) but also considering mechanistic evidences. The findings suggest that reversibility of the biological reactions and/or sequestration of compounds are likely the causes preventing the complete biotransformation of OMPs, and biotransformation is probably limited by thermodynamics rather than by kinetics. Taking into account its simplicity and broader applicability spectrum, the reversible biotransformation is the proposed model to explain the incomplete biotransformation of OMPs.

A combination of ammonia stripping and low temperature thermal pre-treatment improves anaerobic post-digestion of the supernatant from organic fraction of municipal solid waste treatment.

Municipal Solid Waste is mostly composed of organic material which is often treated in anaerobic reactors in waste treatment plants. In most cases, the obtained digestate undergoes a solid/liquid separation step, producing a liquid fraction (known as anaerobic supernatant) rich in ammonium nitrogen that has to be further treated in order to meet discharge standards. The objective of the present work was to evaluate the feasibility of anaerobic post-digestion (37 °C) of a supernatant rich in carbon and nitrogen from a municipal waste treatment plant. In order to increase the efficiency of the process, a combined pre-treatment consisting of a low-temperature thermal process (75 °C) and ammonia stripping (1.3 Lair Lsupernatant-1 min-1) was applied. The effects of pre-treatment contact time (4 and 8 h) and the hydraulic retention time (HRT) in the anaerobic reactor (20-40 d) were studied. Supernatant pre-treatment with 8-h contact time caused 13% organic matter solubilisation, thus improving methanisation by 18% when the HRT was 40 d. At the same time, ammonia stripping allowed to maintain ammonia concentration in the digester below inhibitory values (less than 100 mg N-NH3 L-1) enabling therefore high methanogenic activity (>0.23 g COD g-1 VS d-1). The final effluent characteristics (low total ammonia nitrogen and aerobically biodegradable organic matter levels) would permit implementing subsequent less energy intensive and more environmental-friendly technologies (such as partial nitritation/anammox) to comply with discharge limits.