Coupling pathway prediction and fluorescence spectroscopy to assess the impact of auxiliary substrates on micropollutant biodegradation.

Some bacteria can degrade organic micropollutants (OMPs) as primary carbon sources. Due to typically low OMP concentrations, these bacteria may benefit from supplemental assimilation of natural substrates present in the pool of dissolved organic matter (DOM). The biodegradability of such auxiliary substrates and the impacts on OMP removal are tightly linked to biotransformation pathways. Here, we aimed to elucidate the biodegradability and effect of different DOM constituents for the carbofuran degrader Novosphingobium sp. KN65.2, using a novel approach that combines pathway prediction, laboratory experiments, and fluorescence spectroscopy. Pathway prediction suggested that ring hydroxylation reactions catalysed by Rieske-type dioxygenases and flavin-dependent monooxygenases determine the transformability of the 11 aromatic compounds used as model DOM constituents. Our approach further identified two groups with distinct transformation mechanisms amongst the four growth-supporting compounds selected for mixed substrate biodegradation experiments with the pesticide carbofuran (Group 1: 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde; Group 2: p-coumaric acid, ferulic acid). Carbofuran biodegradation kinetics were stable in the presence of both Group 1 and Group 2 auxiliary substrates. However, Group 2 substrates would be preferable for bioremediation processes, as they showed constant biodegradation kinetics under different experimental conditions (pre-growing KN65.2 on carbofuran vs. DOM constituent). Furthermore, Group 2 substrates were utilisable by KN65.2 in the presence of a competitor (Pseudomonas fluorescens sp. P17). Our study thus presents a simple and cost-efficient approach that reveals mechanistic insights into OMP-DOM biodegradation.

Assessing the substrate specificity of a micropollutant degrading strain: generalist or specialist?

Natural dissolved organic matter (DOM) can serve as an additional substrate for organic micropollutant (OMP) degrading bacteria, thus influencing OMP biodegradation in aquatic systems. DOM biodegradation depends on the OMP degrader's ability to grow on different DOM constituents, and on its capability to compete for DOM constituents against the rest of the resident aquatic microbial community. This study aimed to investigate the growth of a model OMP degrader strain, Novosphingobium sp. KN65.2 (assumed specialist), isolated for its ability to mineralize carbofuran, on thirteen DOM constituents; compare its metabolic capabilities to those of a common freshwater strain (Pseudomonas fluorescens sp. P17) (generalist); and to evaluate competition for specific compounds. Growth experiments were carried out in pure- and mixed culture batch experiments. The DOM constituents tested included aromatic amino acids and a range of phenolic acids (lignin derivatives). The OMP degrader could biodegrade approximately half of the tested compounds. It showed a high specialization for substrates containing a hydroxyl-group in the para-position of the primary aromatic ring substituent. However, its broad substrate range enabled the strain to grow on the same number of auxiliary substrates as the generalist. Moreover, the OMP degrader was able to successfully compete against the generalist for the biodegradation of one (4-hydroxybenzaldehyde) out of three substrates (4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, L-tyrosine), which were biodegraded by both strains. The study results provide insight on the substrate specificity of a model OMP degrader, which can inform development of modeling frameworks investigating the influence of DOM on OMP biodegradation.

Role of Ammonia Oxidation in Organic Micropollutant Transformation during Wastewater Treatment: Insights from Molecular, Cellular, and Community Level Observations.

Organic micropollutants (OMPs) are a threat to aquatic environments, and wastewater treatment plants may act as a source or a barrier of OMPs entering the environment. Understanding the fate of OMPs in wastewater treatment processes is needed to establish efficient OMP removal strategies. Enhanced OMP biotransformation has been documented during biological nitrogen removal and has been attributed to the cometabolic activity of ammonia-oxidizing bacteria (AOB) and, specifically, to the ammonia monooxygenase (AMO) enzyme. Yet, the exact mechanisms of OMP biotransformation are often unknown. This critical review aims to fundamentally and quantitatively evaluate the role of ammonia oxidation in OMP biotransformation during wastewater treatment processes. OMPs can be transformed by AOB via direct and indirect enzymatic reactions: AMO directly transforms OMPs primarily via hydroxylation, while biologically produced reactive nitrogen species (hydroxylamine (NH2OH), nitrite (NO2-), and nitric oxide (NO)) can chemically transform OMPs through nitration, hydroxylation, and deamination and can contribute significantly to the observed OMP transformations. OMPs containing alkyl, aliphatic hydroxyl, ether, and sulfide functional groups as well as substituted aromatic rings and aromatic primary amines can be biotransformed by AMO, while OMPs containing alkyl groups, phenols, secondary amines, and aromatic primary amines can undergo abiotic transformations mediated by reactive nitrogen species. Higher OMP biotransformation efficiencies and rates are obtained in AOB-dominant microbial communities, especially in autotrophic reactors performing nitrification or nitritation, than in non-AOB-dominant microbial communities. The biotransformations of OMPs in wastewater treatment systems can often be linked to ammonium (NH4+) removal following two central lines of evidence: (i) Similar transformation products (i.e., hydroxylated, nitrated, and desaminated TPs) are detected in wastewater treatment systems as in AOB pure cultures. (ii) Consistency in OMP biotransformation (rbio, µmol/g VSS/d) to NH4+ removal (rNH4+, mol/g VSS/d) rate ratios (rbio/rNH4+) is observed for individual OMPs across different systems with similar rNH4+ and AOB abundances. In this review, we conclude that AOB are the main drivers of OMP biotransformation during wastewater treatment processes. The importance of biologically driven abiotic OMP transformation is quantitatively assessed, and functional groups susceptible to transformations by AMO and reactive nitrogen species are systematically classified. This critical review will improve the prediction of OMP transformation and facilitate the design of efficient OMP removal strategies during wastewater treatment.

Investigating Fluorescent Organic-Matter Composition as a Key Predictor for Arsenic Mobility in Groundwater Aquifers.

Dissolved organic matter (DOM) is linked to the heterogeneous distribution of elevated arsenic (As) in groundwater used for drinking and irrigation purposes, but the relationship between DOM characteristics and arsenic mobility has yet to be fully understood. Here, DOM from groundwater sampled in the Bengal Basin region was characterized using both conventional bulk emission-excitation (EEM) spectroscopy and high-performance size-exclusion chromatography coupled to spectroscopy (HPSEC-EEM). Notably, application of the novel HPSEC-EEM approach permitted the total fluorescence of individual samples to be independently resolved into its underlying components. This allowed the external validation of the bulk-sample fluorescence decomposition and offered insight into the molecular size distribution of fluorescent DOM. Molecular size distributions were similar for the UVA fluorescent (C310 and C340) as well as the three visible fluorescent (C390, C440, and C500) components. There was a greater visible fluorescence in shallow aquifer samples (10-33 m) with high As (SH, up to 418 µg/L) than in samples from the same depth with lower As (up to 40 µg/L). This indicated a link between DOM quality and As mobility within the shallow aquifer. The deep aquifer samples (170-200 m) revealed DOM characteristics similar to SH samples but had low As concentrations (<4 µg/L), signifying that the deep aquifer is potentially vulnerable to As contamination. These findings pave the way for a more comprehensive assessment of the susceptibility of drinking water aquifers, thereby supporting the management of groundwater resources.