Toward Characterizing the Genetic Basis of Trace Organic Contaminant Biotransformation in Activated Sludge: The Role of Multicopper Oxidases as a Case Study

Abstract

Activated sludge treatment leverages the ability of microbes to uptake and (co)-metabolize chemicals and has shown promise in eliminating trace organic contaminants (TrOCs) during wastewater treatment. However, targeted interventions to optimize the process are limited as the fundamental drivers of the observed reactions remain elusive. In this work, we present a comprehensive workflow for the identification and characterization of key enzymes involved in TrOCs biotransformation pathways in complex microbial communities. To demonstrate the applicability of the workflow, we investigated the role of the enzymatic group of multicopper oxidases (MCOs) as one putatively relevant driver of TrOCs biotransformation. To this end, we analyzed activated sludge metatranscriptomic data and selected, synthesized, and heterologously expressed three phylogenetically distinct MCO-encoding genes expressed in communities with different TrOCs oxidation potentials. Following the purification of the encoded enzymes, we screened their activities against different substrates. We saw that MCOs exhibit significant activities against selected TrOCs in the presence of the mediator compound 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and, in some cases, also in the presence of the wastewater contaminant 4′-hydroxy-benzotriazole. In the first case, we identified oxidation products previously reported from activated sludge communities and concluded that in the presence of appropriate mediators, bacterial MCOs could contribute to the biological removal of TrOCs. Similar investigations of other key enzyme systems may significantly advance our understanding of TrOCs biodegradation and assist the rational design of biology-based water treatment strategies in the future.

Can AI Help Improve Water Quality? Towards the Prediction of Degradation of Micropollutants in Wastewater.

“…When the respective laccase sequences extracted from wastewater were cloned and expressed in E. coli, the respective purified enzymes indeed showed increased activity in combination with a typical laccase mediator compound, i.e., 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS). [41] It has been suggested that, in such laccase-mediator systems, the laccase itself oxidizes ABTS to the doubly charged cation ABTS 2+ , which then in turn oxidizes other chemicals with lower oxidation potential than the mediator itself. [40] Since previous data suggest that, most likely, the latter reaction of the activated mediator with the chemical is the rate-limiting step in this process, the system reduces again to a purely chemical reaction system, which, in principle, should be predictable from suitable QC descriptors.…”

“…We then compared the experimental outcomes of this study with a number of poten-tially relevant QC descriptors to see whether they can predict reactivity in the experimental system. In the experimental study, [41] degradation reactions were examined for 20 structurally diverse micropollutants (1−20 of Scheme 1) with MCO and ABTS as a mediator (21, shown in a solid box of Scheme 1) in ammonium acetate buffer at pH 4-6 (Fig. 3).…”

“…One possibility is measurement informatics (MI), where AI technology is applied to various measurement techniques, e.g., spectroscopy. [48][49][50] The aims of MI include extracting more in- [41] Compounds 13−20 were degraded, while 1−12 were not. Those in the dotted box were degraded.…”

“…Scheme 1. Compounds examined experimentally [41]. Compounds 13−20 were degraded, while 1−12 were not.…”

Investigation of Water Splitting Reaction by a Multicopper Oxidase through X‐ray Absorption Nanospectroelectrochemistry

“…These sites facilitate the dynamic reactions according to the microenvironment of amino acid residues close to the copper sites. [9][10][11] In particular, bilirubin oxidase (BOD) can reduce molecular oxygen (native reaction) to water while maintaining its ability to oxidize water molecules. The WOR, a non-native reaction, depends on the electrode potential and the H + availability on the enzyme cofactor.…”