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.

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.

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.

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.

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.

Role of methanogenesis on the biotransformation of organic micropollutants during anaerobic digestion.

Several studies showed that some organic micropollutants (OMPs) are biotransformed during anaerobic digestion (AD). Yet, most of them aim at reporting removal efficiencies instead of understanding the biotransformation process. Indeed, how each of the main AD stages (i.e., hydrolysis, acidogenesis, and methanogenesis) contribute to OMP biotransformation remains unknown. This study focuses on investigating the role of methanogenesis, the most characteristic step of AD, to OMP removal. More specifically, the sorption and the biotransformation of 20 OMPs by methanogenic biomass were analyzed determining their concentrations in both liquid and solid phases. Sorption onto methanogenic biomass displayed a similar behavior as reported for digested sludge. Most of the OMPs were biotransformed to a medium extent (35-70%) and only sulfamethoxazole was completely removed. Comparing these results with those reported for the complete AD process, methanogenesis was proven to play a key role, accounting for more than 50% of the OMP biotransformation (except for roxithromycin) during AD. An increase in the organic loading rate from 1 to 2gCOD/Ld, typical loads employed in sewage sludge anaerobic digesters, did not exert a clear cometabolic effect on the OMPs biotransformation. It is hypothesized that biotransformation occurs in both liquid and solid phases because no link between the partition coefficient (Kd) and the overall biotransformation efficiency was found. These findings allow a better understanding of the OMPs fate under anaerobic conditions, which is necessary to design efficient biological mitigation strategies.

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.