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.

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.

Cometabolic Enzymatic Transformation of Organic Micropollutants under Methanogenic Conditions.

Anaerobic digestion (AD) has been shown to have the biological potential to decrease concentrations of several organic micropollutants (OMPs) in sewage sludge. However, the mechanisms and factors behind these biotransformations, which are essential for elucidating the possible transformation products and to foster the complete removal of OMPs via operational strategies, remain unclear. Therefore, this study investigated the transformation mechanisms of 20 OMPs during the methanogenic step of AD with a focus on the role of acetate kinase (AK), which is a key enzyme in methane production. The results from lab-scale methanogenic reactors showed that this step accounts for much of the reported OMP biotransformation in AD. Furthermore, enzymatic assays confirmed that AK transforms galaxolide, naproxen, nonylphenol, octylphenol, ibuprofen, diclofenac, bisphenol A, and triclosan. Except for galaxolide, for which further studies are required to refine conclusions, the OMP's chemical structure was a determinant for AK action because only compounds that contain a carboxyl or hydroxyl group and have moderate steric hindrance were enzymatically transformed, likely by phosphorylation. For these seven compounds, this enzymatic mechanism accounts for 10-90% of the measured methanogenic biotransformation, suggesting that other active enzymes of the AD process are also involved in OMP biotransformation.

The ManureEcoMine pilot installation: advanced integration of technologies for the management of organics and nutrients in livestock waste.

Manure represents an exquisite mining opportunity for nutrient recovery (nitrogen and phosphorus), and for their reuse as renewable fertilisers. The ManureEcoMine proposes an integrated approach of technologies, operated in a pilot-scale installation treating swine manure (83.7%) and Ecofrit® (16.3%), a mix of vegetable residues. Thermophilic anaerobic digestion was performed for 150 days, the final organic loading rate was 4.6 kgCOD m-3 d-1, with a biogas production rate of 1.4 Nm3 m-3 d-1. The digester was coupled to an ammonia side-stream stripping column and a scrubbing unit for free ammonia inhibition reduction in the digester, and nitrogen recovery as ammonium sulphate. The stripped digestate was recirculated daily in the digester for 15 days (68% of the digester volume), increasing the gas production rate by 27%. Following a decanter centrifuge, the digestate liquid fraction was treated with an ultrafiltration membrane. The filtrate was fed into a struvite reactor, with a phosphorus recovery efficiency of 83% (as orthophosphate). Acidification of digestate could increment the soluble orthophosphate concentration up to four times, enhancing phosphorus enrichment in the liquid fraction and its recovery via struvite. A synergistic combination of manure processing steps was demonstrated to be technologically feasible to upgrade livestock waste into refined, concentrated fertilisers.

Influence of hydraulic retention time on the psychrophilic hydrolysis/acidogenesis of proteins.

The influence of the hydraulic retention time (HRT) on the anaerobic hydrolysis of complex substrates has been studied under psychrophilic conditions. For this purpose, a continuous stirred tank reactor was operated at 15 °C and neutral pH and gelatin was considered as a model protein. Three HRTs have been tested: 12, 21 and 36 h. Gelatin hydrolysis was greatly dependent on HRT, increasing from 40% at 12 h-HRT to a maximum of 65% at 36 h-HRT. Molecular size distribution analyses of the effluent showed that hydrolysation of compounds larger than 10 kDa was poor at 12 h-HRT, whereas the fraction of 1-10 kDa was completely transformed into compounds smaller than 1 kDa. Higher HRT (36 h) improved the degradation of the recalcitrant fraction (>10 kDa), obtaining an effluent with around 95% of soluble molecules (<1 kDa). In that way, the use of membrane bioreactors for the treatment of this type of macromolecules could improve the degradation efficiencies by enabling to increase the residence time of the non-hydrolyzed molecules, with what would be possible to achieve higher organic loading rate operation.

Towards a standardization of biomethane potential tests.

Production of biogas from different organic materials is a most interesting source of renewable energy. The biomethane potential (BMP) of these materials has to be determined to get insight in design parameters for anaerobic digesters. Although several norms and guidelines for BMP tests exist, inter-laboratory tests regularly show high variability of BMPs for the same substrate. A workshop was held in June 2015, in Leysin, Switzerland, with over 40 attendees from 30 laboratories around the world, to agree on common solutions to the conundrum of inconsistent BMP test results. This paper presents the consensus of the intense roundtable discussions and cross-comparison of methodologies used in respective laboratories. Compulsory elements for the validation of BMP results were defined. They include the minimal number of replicates, the request to carry out blank and positive control assays, a criterion for the test duration, details on BMP calculation, and last but not least criteria for rejection of the BMP tests. Finally, recommendations on items that strongly influence the outcome of BMP tests such as inoculum characteristics, substrate preparation, test setup, and data analysis are presented to increase the probability of obtaining validated and reproducible results.

Influence of transitional states on the microbial ecology of anaerobic digesters treating solid wastes.

A better understanding of the microbial ecology of anaerobic processes during transitional states is important to achieve a long-term efficient reactor operation. Five wastes (pig manure, biodiesel residues, ethanol stillage, molasses residues, and fish canning waste) were treated in five anaerobic reactors under the same operational conditions. The influence of the type of substrate and the effect of modifying feeding composition on the microbial community structure was evaluated. The highest biomethanation efficiency was observed in reactors fed with fish canning waste, which also presented the highest active archaeal population and the most diverse microbial communities. Only two Bacteria populations could be directly related to a particular substrate: Ilyobacter with biodiesel residues and Trichococcus with molasses residues. Results showed that the time to achieve steady-state performance after these transitional states was not dependent on the substrate treated. But reactors needed more time to handle the stress conditions derived from the start-up compared to the adaptation to a new feeding. Cluster analyses showed that the type of substrate had a clear influence on the microbiology of the reactors, and that segregation was related to the reactors performance. Finally, we conclude that the previous inoculum history treating solid waste and higher values of active Archaea population are important factors to face a successful change in substrate not entailing stability failure.

Altering operational conditions during protein fermentation to volatile fatty acids modifies the associated bacterial community.

In recent years, the production of volatile fatty acids (VFA) through mixed culture fermentation (MCF) has been gaining attention. Most authors have focused on the fermentation of carbohydrates, while other possible substrates, such as proteins, have not been considered. Moreover, there is little information about how operational parameters affect the microbial communities involved in these processes, even though they are strongly related to reactor performance and VFA selectivity. Hence, this study aims to evaluate how microbial composition changes according to three different parameters (pH, type of protein and micronutrient addition) during anaerobic fermentation of protein-rich side streams. For this, two continuous stirred tank reactors (CSTR) were fed with two different proteins (casein and gelatine) and operated at different conditions: three pH values (5.0, 7.0 and 9.0) with only macronutrients supplementation and two pH values (5.0 and 7.0) with micronutrients' supplementation as well. Firmicutes, Proteobacteria and Bacteroidetes were the dominant phyla in the two reactors at all operational conditions, but their relative abundance varied with the parameters studied. At pH 7.0 and 9.0, the microbial composition was mainly affected by protein type, while at acidic conditions the driving force was the pH. The influence of micronutrients was dependent on the pH and the protein type, with a special effect on Clostridiales and Bacteroidales populations. Overall, this study shows that the acidogenic microbial community is affected by the three parameters studied and the changes in the microbial community can partially explain the macroscopic results, especially the process selectivity.

Biodegradation kinetic constants and sorption coefficients of micropollutants in membrane bioreactors.

In order to elucidate the capability of biomass developed in membrane bioreactors (MBR) to degrade and sorb emerging micropollutants, biodegradation (kbiol) and sorption (ksor) kinetic constants as well as solid-liquid partition coefficients (Kd) of 13 selected pharmaceutical and personal care products (PPCPs) were determined with MBR heterotrophic biomass adding a pulse (100 ppb of each compound) and following the liquid and solid phase concentrations over time. The results obtained were compared to literature data referring to conventional activated sludge (CAS) systems. Two experiments were performed: one in the MBR itself and the second one in a batch reactor with the same type and concentration of biomass as in the MBR. Overall, both biodegradation and sorption coefficients were in the same range as previously reported by other studies in CAS systems, indicating that MBR biomass does not show better capabilities for the biological degradation and/or sorption of PPCPs compared to the biomass developed in CAS reactors. Therefore, the higher PPCPs removal efficiencies found in MBRs are explained by the high biomass concentrations obtained at the long sludge retention times at which this type of reactors are usually operated.

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.

Should we pretreat solid waste prior to anaerobic digestion? An assessment of its environmental cost.

Many studies have shown the effectiveness of pretreatments prior to anaerobic digestion of solid wastes, but to our knowledge, none analyzes their environmental consequences/costs. In this work, seven different pretreatments applied to two types of waste (kitchen waste and sewage sludge) have been environmentally evaluated by using life cycle assessment (LCA) methodology. The results show that the environmental burdens associated to the application of pretreatments prior to anaerobic digestion cannot be excluded. Among the options tested, the pressurize-depressurize and chemical (acid or alkaline) pretreatments could be recommended on the basis of their beneficial net environmental performance, while thermal and ozonation alternatives require energy efficiency optimization to reduce their environmental burdens. Reconciling operational, economic and environmental aspects in a holistic approach for the selection of the most sustainable option, mechanical (e.g., pressurize-depressurize) and chemical methods appear to be the most appropriate alternatives at this stage.

Correlations between molecular and operational parameters in continuous lab-scale anaerobic reactors.

In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably, the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and high biogas production.

Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm.

The oxygen-limited autotrophic nitrification/denitrification (OLAND) process comprises one-stage partial nitritation and anammox, catalyzed by aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), respectively. The goal of this study was to investigate whether quorum sensing influences anoxic ammonium oxidation in an OLAND biofilm, with AnAOB colonizing 13% of the biofilm, as determined with fluorescent in situ hybridization (FISH). At high biomass concentrations, the specific anoxic ammonium oxidation rate of the OLAND biofilm significantly increased with a factor of 1.5 ± 0.2 compared to low biomass concentrations. Supernatant obtained from the biofilm showed no ammonium-oxidizing activity on itself, but its addition to low OLAND biomass concentrations resulted in a significant activity increase of the biomass. In the biofilm supernatant, the presence of long-chain acylhomoserine lactones (AHLs) was shown using the reporter strain Chromobacterium violaceum CV026, and one specific AHL, N-dodecanoyl homoserine lactone (C(12)-HSL), was identified via LC-MS/MS. Furthermore, C(12)-HSL was detected in an AnAOB-enriched community, but not in an AerAOB-enriched community. Addition of C(12)-HSL to low OLAND biomass concentrations resulted in a significantly higher ammonium oxidation rate (p < 0.05). To our knowledge, this is the first report demonstrating that AHLs enhance the anoxic ammonium oxidation process. Future work should confirm which species are responsible for the in situ production of C(12)-HSL in AnAOB-based applications.

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.

Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox.

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.

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.

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.

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.