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Environmental Processes

Ammonia monooxygenase-mediated cometabolic biotransformation and hydroxylamine-mediated abiotic transformation of micropollutants in an AOB/NOB co-culture Yaochun Yu, Ping Han, Li-Jun Zhou, Zhong Li, Michael Wagner, and Yujie Men Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02801 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018

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Environmental Science & Technology

Ammonia monooxygenase-mediated cometabolic biotransformation and hydroxylamine-mediated abiotic transformation of micropollutants in an AOB/NOB co-culture Yaochun Yu1, †, Ping Han2, †, Li-Jun Zhou2, 3, Zhong Li4, Michael Wagner2, Yujie Men1, 5, *

1

Department of Civil and Environmental Engineering, University of Illinois at

Urbana-Champaign, Urbana, IL 61801, USA 2

Department of Microbiology and Ecosystem Science, Division of Microbial Ecology,

Research Network “Chemistry meets Microbiology”, University of Vienna, 1090 Vienna, Austria 3

State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and

Limnology, Chinese Academy of Sciences, Nanjing 210008, China 4

Metabolomics Center, University of Illinois, Urbana, IL 61801, USA

5

Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL

61801, USA †

Equal contribution

* Corresponding Author Yujie Men Department of Civil and Environmental Engineering University of Illinois at Urbana-Champaign, Address: 205 North Mathews Ave Urbana, IL 61801-2352, USA Email: [email protected]; Phone: (217) 244-8259

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Abstract

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Biotransformation of various micropollutants (MPs) has been found to be positively

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correlated with nitrification in activated sludge communities. To further elucidate the roles

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played by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), we

5

investigated the biotransformation capabilities of an NOB pure culture (Nitrobacter sp.), and

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an AOB (Nitrosomonas europaea)/NOB (Nitrobacter sp.) co-culture for fifteen MPs, whose

7

biotransformation was reported previously to be associated with nitrification. The NOB pure

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culture did not biotransform any investigated MP, whereas the AOB/NOB co-culture was

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capable of biotransforming six MPs (i.e. asulam, bezafibrate, fenhexamid, furosemide,

10

indomethacin, and rufinamide). Transformation products (TPs) were identified and tentative

11

structures were proposed. Inhibition studies with octyne, an ammonia monooxygenase (AMO)

12

inhibitor, suggested that AMO was the responsible enzyme for MP transformation that

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occurred co-metabolically. For the first time, hydroxylamine, a key intermediate of all

14

aerobic ammonia oxidizers, was found to react with several MPs at concentrations typically

15

occurring in AOB batch cultures. All of these MPs were also biotransformed by the

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AOB/NOB co-culture. Moreover, the same asulam TPs were detected in both

17

biotransformation and hydroxylamine-treated abiotic transformation experiments, whereas

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rufinamide TPs formed from biological transformation were not detected during

19

hydroxylamine-mediated abiotic transformation, which was consistent with the inability of

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rufinamide abiotic transformation by hydroxylamine. Thus, in addition to cometabolism

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likely carried out by AMO, an abiotic transformation route indirectly mediated by AMO

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might also contribute to MP biotransformation by AOB.

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INTRODUCTION

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In recent years, with an increasing use of organic chemicals such as pesticides,

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pharmaceuticals and personal care products, there are emerging concerns about the

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potential risk of these contaminants to natural environments and public health.1-4 These

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compounds are typically detected at very low concentrations in aquatic systems (i.e., ng/L

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~ µg/L levels),5, 6 thus are also referred to as micropollutants (MPs). Conventional

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wastewater treatment plants (WWTPs) are one major sink of MPs.7 Biotransformation is an

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essential mitigation route of MPs in WWTPs, but most of these systems are not designed

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specifically for MP removal. Thus, MP removal in WWTPs is generally incomplete and its

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efficiency is highly compound specific.8-10 Remaining MPs and their transformation

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products (TPs) are discharged into aquatic systems, which may cause adverse effects.11-13

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Hence, further studies of MP biotransformation mechanisms and identification of their TPs

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are necessary and will in the long run help us to better understand the environmental fate of

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MPs, design removal or monitoring strategies, as well as establish precaution guidelines.2, 5,

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14

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Nitrification, the aerobic oxidation of ammonia via nitrite to nitrate, is an essential

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step for the removal of nitrogen compounds in WWTPs. In many WWTPs, this process is

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catalyzed by the concerted activity of ammonia-oxidizing bacteria (AOB) converting

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ammonia to nitrite, and nitrite-oxidizing bacteria (NOB) converting nitrite to nitrate,

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although ammonia-oxidizing archaea (AOA)15-18 the archaeal counterpart of AOB and the

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recently discovered complete ammonia oxidizers (comammox) converting ammonia to

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nitrate19-21 have also been detected in some WWTPs. Previous studies have shown 3 ACS Paragon Plus Environment


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correlations between nitrification and biotransformation of some MPs in nitrifying

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activated sludge communities.8, 11, 22-24 For example, MP biotransformation was enhanced

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when nitrification activity was stimulated.25 In addition, MP biotransformation in activated

48

sludge communities decreased when nitrification was completely inhibited by ammonia

49

monooxygenase (AMO) inhibitors.12, 25-27 In a recent study, the biotransformation of a

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number of MPs was inhibited by AMO inhibitors, and more than 50% inhibition was

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observed for four MPs (i.e., asulam, trimethoprim, monuron, and clomazone), providing

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strong indications that ammonia oxidizers (particularly AOB) were involved in the

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biotransformation of some MPs.8 However, the correlations observed in inhibition studies

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does not necessarily lead to causal relationships. In order to demonstrate the actual role

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played by AOB in MP biotransformation, and to identify the responsible enzymes and

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transformation pathways, complementary pure culture studies are required.

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It has been hypothesized that AMO, a substrate-promiscuous key enzyme of all

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autotrophic ammonia oxidizers is responsible for nitrifier-mediated MP

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biotransformation.25, 28, 29 So far, whole-cell studies have been used to investigate AMO

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functionality due to the unavailability of purified AMO with retained activity.30 While it has

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been shown that AMO is able to oxidize some hydrocarbons and halogenated hydrocarbons

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co-metabolically,31, 32 direct evidence for MP transformation by this enzyme is largely

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lacking. Furthermore, the results of the inhibition studies could also suggest that the MP

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biotransformation was carried out by microorganisms whose growth is highly dependent on

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ammonia oxidizers. For example, the inhibition of ammonia oxidizers in nitrifying

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activated sludge will strongly reduce nitrite production, and thus also inhibit NOB. 4 ACS Paragon Plus Environment

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Therefore, there is an urgent need to investigate the MP biotransformation potential of

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nitrite oxidizers, too. Given that there is no nitrite accumulation in nitrifying activated

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sludge communities and the accumulated nitrite in AOB pure cultures may lead to abiotic

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nitration reactions of some MPs,33, 34 a co-culture system containing AOB and NOB species

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is more relevant to the actual nitrification condition, and will be used in this study to

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investigate the roles of nitrifiers.

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The goal of this study was (i) to investigate biotransformation capabilities of AOB

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and NOB using the model AOB species Nitrosomonas europaea and NOB species

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Nitrobacter sp.. Fifteen MPs including pharmaceuticals and pesticides were selected,

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whose biotransformation has been associated with nitrification in activated sludge by an

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inhibition study,8 (ii) to identify responsible enzymes for the transformation of selected

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MPs, and (iii) to further understand the respective transformation mechanisms.

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Biotransformation experiments were conducted using the NOB pure culture and a nitrifying

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co-culture containing the AOB and the NOB species. Metabolic and co-metabolic

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biotransformation mechanisms were tested by using the biotransformed MP as the sole

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energy source. AMO inhibition experiments were carried out to demonstrate the

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involvement of AMO in MP biotransformation. Abiotic transformation experiments using

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the key ammonia oxidation intermediates (e.g., hydroxylamine) were also conducted to

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further unravel mechanisms of MP biotransformation by ammonia oxidizers.

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MATERIALS AND METHODS

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MP Selection. Based on a previous inhibition study of MP biotransformation in activated

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sludge, 15 MPs (Table S1) were selected, whose biotransformation was inhibited by more 5 ACS Paragon Plus Environment


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than 20% when adding the bacterial AMO inhibitor octyne.8 These MPs are commonly

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used pesticides and pharmaceuticals, including acetamiprid, asulam, bezafibrate,

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carbendazim, clomazone, fenhexamid, furosemide, indomethacin, irgarol, levetiracetam,

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monuron, rufinamide, tebufenozide, thiacloprid, and trimethoprim. In addition, two

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compounds (mianserin and ranitidine) that were previously shown to be biotransformed by

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ammonia-oxidizing pure cultures,5 were included as positive controls for biomass activities

95

(Table S1). Reference compounds and methanol (HPLC grade) were purchased from

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Sigma-Aldrich (St. Louis, MO). Five internal standards including trimethoprim-d3,

97

metoprolol acid-d5, carbendazim-d4, and furosemide-d5 were obtained from C/D/N

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isotopes (Quebec, Canada) and Toronto Research Chemicals (Toronto, Canada). The

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bacterial AMO inhibitor 1-octyne35, 36 was purchased from Fisher Scientific. Stock

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solutions of MPs and internal standards were prepared individually in methanol at 1 g L-1,

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except for carbendazim and its corresponding internal standard carbendazim-d4, which

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were prepared in methanol at 0.1 g L-1. All stock solutions were stored at -20 °C. A mixture

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of standards (100 mg L-1 for each MP) was prepared via appropriate dilution of the stock

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solutions in methanol.

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Cultivation of Nitrifying Pure and Co-cultures. The AOB strain Nitrosomonas europaea

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Winogradsky (ATCC 19718) and the NOB stain Nitrobacter sp. (ATCC 25381) were

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obtained from the American Type Culture Collection (ATCC). They were maintained in 50

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mL suspension culture flasks with filter screw caps (VWR, Radnor, PA) containing 20 mL

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of a modified basal medium with 4.0 g L-1 CaCO3 for buffering the pH around 8.37 All

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cultures were incubated at 28 °C in a dark incubator and shaken at 120 rpm. Seven mM 6 ACS Paragon Plus Environment

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NH4Cl or NaNO2 was added as the growth substrate in the AOB or NOB cultures, which

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was re-amended upon depletion. The culture performance (ammonia or nitrite oxidation)

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was consistent and stable during each feeding cycle of the entire incubation period. The

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AOB/NOB co-culture was obtained by growing the two strains together, and maintained in

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the same basal medium with 2 mM NH4Cl as the substrate, which was re-added upon

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deletion of both NH4+ and NO2-. All cultures were sub-cultured (10%, v/v) into autoclaved

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fresh medium after three doses of the respective substrate (NH4Cl or NaNO2). Additional

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pure AOB and AOA species (i.e., Nitrosomonas nitrosa Nm90 and Nitrososphaera

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gargensis) were maintained as described previously.5

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Biotransformation Experiments. The capability of MP biotransformation by the NOB

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pure culture and the AOB/NOB co-culture were investigated in batch experiments.

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Pre-grown biomass was harvested by centrifugation at 10,000 × g (4 °C for 15 min). The

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biomass was re-suspended in autoclaved fresh medium and concentrated to reach a similar

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ammonia or nitrite oxidation rate (~ 2 mM per 8 hours). Individual MP or mixed MP stock

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solution was added to empty culture flasks. After the organic solvent methanol was

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completely evaporated, 25 mL of thoroughly mixed concentrated NOB pure culture or

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AOB/NOB co-culture was inoculated. As the mixture of tested MPs inhibited the

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physiological activity of N. europaea at concentrations over 30 µg/L (each), and did not

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show inhibition on Nitrobacter sp. at a concentration up to 100 µg/L (each), an initial MP

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concentration of 100 µg/L (each) was used for the NOB experiments, while 10 µg/L (each)

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was applied for the AOB/NOB co-culture. The culture flasks were shaken at 150 rpm for 5

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min to re-dissolve the MPs. Weight loss due to evaporation in each flask was measured and 7 ACS Paragon Plus Environment


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compensated by adding sterile nanopure water every 24 hours. Two mM NH4Cl and 2 mM

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NaNO2 were added to the AOB/NOB co-culture and the NOB culture at the beginning,

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respectively, and were re-added afterwards to keep the ammonia/nitrite oxidation activity.

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For the AOB/NOB co-culture, 2 mM NH4Cl was re-added every 12 hours during the first

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two days, and every 24 hours afterwards. For the NOB pure culture, 2 mM NaNO2 was

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re-added every 24 hours. Samples (1 mL) were taken immediately after the first addition of

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NH4Cl or NaNO2, and centrifuged at 16,000 × g (4 °C for 15 min). Supernatant was

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collected and stored at 4 °C for LC-MS/MS and nitrogen species (i.e., NH4+, NO2-, and

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NO3-) measurements. Cell pellets were stored at -20 °C for quantitative PCR (qPCR)

142

analysis. Samples were taken using the same procedure at subsequent time points during an

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incubation period of 168 hours. At the end of incubation, cells in 10 mL culture were

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collected by centrifugation, washed with sterile medium for three times, and stored at

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-20 °C for intracellular MP and TP analysis. Biomass-free sterile fresh medium control and

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heat-inactivated biomass (autoclavation at 121 °C for 20 min) control were set up in

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triplicates in the same way as the biotransformation experiments described above. Briefly,

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in both medium and heat-inactivated controls, the same MPs were added, and ammonium

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(2 mM), nitrite (2 mM) and nitrate (14 mM) were added to mimic the levels of N-species

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present in the biotransformation experiments.

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Asulam Inhibitory Effect Tests. Biotransformation experiments were performed as

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described above using the same pre-grown co-culture with different initial concentrations

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of asulam (0, 10, 20, 30, 40, 50, and 80 µg/L). Two mM ammonia was supplied every 24

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hours. Supernatant samples in a time series were taken over a 72-h incubation period, and 8 ACS Paragon Plus Environment

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ammonia degradation and asulam biotransformation were determined as described above.

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AMO Inhibition Experiments. To investigate the role of AMO in asulam

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biotransformation, the bacterial AMO inhibitor octyne (~10 mg/L)8, 38 was added to the

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AOB/NOB co-culture with 10 µg/L asulam after a 72-h incubation. We chose octyne as the

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AMO inhibitor instead of the commonly used allylthiourea (ATU) and acetylene because (i)

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ATU has been found not specific to AMO;8 (ii) acetylene is in gaseous phase, thus it is

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difficult to keep its inhibitory effect in aerobic reactors with fast air exchange; (iii) octyne

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is added as liquid, and was found to have more rapid inhibition than acetylene.38 All

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inhibition experiments were performed in quadruplicates, three of which were sampled in a

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time series over a 168-h incubation period for LC-MS/MS analysis, cell growth, and

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N-species measurements. The remaining replicate was sampled every 24 hours to monitor

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the ammonia oxidation activity using Hach test tubes for NH4- and NO3-N according to the

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manufacturer’s instructions. Octyne was re-added as soon as recovery of ammonia

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oxidation activity was detected.

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Role of Ammonia Oxidation Intermediates in MP Transformation. To investigate the

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effects of the ammonia oxidation intermediates, hydroxylamine (NH2OH) and nitric oxide

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(NO) on MP biotransformation, MP transformation experiments after addition of either

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NH2OH or NO were carried out in sterile fresh medium. Based on previously measured

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concentrations of extracellular NH2OH produced by ammonia oxidizers in batch reactors

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and the observation that 5 µM NH2OH was decomposed within 10 hours in the medium

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used,39

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comparison to AMO-mediated intermediate NH2OH, MP transformation by NO, a

5 µM NH2OH was added every 12 hours over a 168-h incubation period. As a

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non-AMO-mediated intermediate was also carried out. Excess NO was added in the form

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of 114.3 µM NONOate (Fisher Scientific, Hampton, NH), which releases ~ 200 µM NO

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during the 168-h incubation (first-order process with half-life of 56 hours at 25 °C). A

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mixture of all 17 MPs were added at a concentration of 20 µg/L for each compound.

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Samples at 0, 12, and 168 h were taken for LC-HRMS analysis.

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Analytical Method. MPs were analyzed by liquid chromatography coupled to a

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high-resolution quadrupole orbitrap mass spectrometer (LC- HRMS/MS) (Q Exactive,

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Thermo Fisher Scientific). For LC analysis, 50 µL sample was loaded onto a C18 Atlantis-

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T3 column (particle size 3 µm, 3.0 × 150 mm, Waters), and eluted at a flow rate of 350

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µL/min with nanopure water (A) and acetonitrile (B) (both amended with 0.1% formic

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acid), at a gradient as follows: 5% B: 0 − 1 min, 5% − 100% B: 1 − 8min, 100% B: 8 − 20

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min, and 5% B:20 − 26 min. The compounds were measured in full scan mode on HRMS

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at a resolution of 70,000 at m/z 200 and a scan range of m/z 50 -750 in a positive/negative

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switching mode. The limit of quantification (LOQ) for each MP is determined as the lowest

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concentration with a detection variation < 20%, which was listed in Table S1.

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Cell Extraction for Intracellular Concentration Measurement. A cell extraction

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procedure from a previous study40 was used with slight modification. Briefly, internal

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standards were spiked in cell pellets collected from 10 mL culture (at a final concentration

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of 4 µg/L for each) followed by an addition of 2 mL lysis solvent containing methanol (0.5%

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formic acid): nanopure water (0.1% w/w EDTA), 50: 50 (v/v). The cells were disrupted by

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ultrasonication at 50 °C for 15 min, centrifuged at 10,000 × g for 10 min. The supernatant

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was collected in a glass vial. This procedure was repeated twice for a better recovery. 10 ACS Paragon Plus Environment

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Finally, ~ 6 mL supernatant was evaporated to dryness under a gentle steam of dinitrogen

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gas at 40 °C. The analytes were re-dissolved in 0.5 mL filter-sterilized fresh medium

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without CaCO3, which were then centrifuged at 10,000 × g at 4 °C for 10 min. The

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supernatant was collected for LC-HRMS measurement.

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Transformation Product Identification. Both suspect screening and non-target screening

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were carried out to identify TPs. Suspect screening was done by TraceFinder 4.1 EFS

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software (Thermo Fisher Scientific). TP suspect lists were compiled using an automated

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metabolite mass prediction script, which considered a number of known redox and

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hydrolysis reactions, as well as several conjugation reactions at primary and secondary

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levels. Plausible TPs were identified according to the following criteria: i) mass accuracy

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tolerance < 5ppm; ii) isotopic pattern score > 70%; iii) peak area > 5 × 106; iv) increasing

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trend over time, or first increase then followed by a decrease; v) absent in biological

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samples without MP addition or heat-inactivated controls. Sieve 2.2 software (Thermos

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Fisher Scientific) was used for non-target screening and the TP candidates were selected

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based on the same criteria. MS2 fragment profiles of TP candidates were obtained using

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data-dependent MS/MS scan, to help elucidate TP structures. MarvinSketch (NET6.2.0,

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2014) was used for drawing, displaying, and characterizing chemical structures,

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ChemAxon (http://www.chemaxon.com).

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Ammonium, Nitrite, and Nitrate Measurements. Ammonia/ammonium was measured

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by a colorimetric method.41 Nitrite was measured by photometry with the sulfanilamide

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N-(1-naphthyl) ethylenediamine dihydrochloride (NED) reagent method.42 Nitrate was first

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reduced to nitrite by vanadium chloride, and then detected by the acidic Griess assay.43 The 11 ACS Paragon Plus Environment


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detection limit for ammonium, nitrite, and nitrate was 10 µM.

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Quantitative PCR. Genomic DNA was extracted by DNeasy Blood & Tissue Kit

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(QIAGEN, Germantown, MD) according to manufacturer’s instructions. Cell growth was

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measured by qPCR. Specific primers targeting the 16S rRNA genes of N. europaea,

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(forward: 5’-GCAGCAGAGGGGAGTGGAAT-3’, reverse:

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5’-CGTGCATGAGCGTCAGTGTC-3’), and Nitrobacter sp. (forward:

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5’-CCATGACCGGTCGCAGAGAT-3’, reverse: 5’-AACTAAGGACGGGGGTTGCG-3’)

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were designed and validated using the web-based tool, Primer-BLAST

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(https://www.ncbi.nlm.nih.gov/tools/primer-blast/). FAST SYBR Green reagents (Thermo

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Fisher Scientific) were used for qPCR according to the manufacturer’s instructions. In

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general, each 20-µL reaction mixture contained 2.5 µL of gDNA sample or serially diluted

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standard, 10 µL 2× Fast SYBR Green master mix, and 0.625 µM of the forward and reverse

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primers. The PCR procedure included an initial deactivation at 95 °C for 20s, followed by

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40 thermal cycles at 95 °C for 20s, then at 60 °C for 30s. Genomic DNA of N. europaea

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and Nitrobacter sp. were quantified by NanoDrop One (Thermo Fisher Scientific) and

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served as qPCR standards.

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RESULTS AND DISCUSSION

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MP Biotransformation by the AOB/NOB Co-culture and the NOB Pure Culture.

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According to a previous study,8 the biotransformation of four selected compounds (i.e.,

240

asulam, monuron, clomazone, and trimethoprim) in a nitrifying activated sludge

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community was highly associated with nitrification (50 − 90% inhibition when nitrification

242

was inhibited). To further demonstrate the causal relationship between nitrifiers and MP 12 ACS Paragon Plus Environment

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biotransformation, we used an AOB/NOB co-culture that mimics the complete nitrification

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system in WWTPs to investigate the biotransformation of the four selected MPs. This

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co-culture setup also reduces the potential for biologically mediated abiotic nitritation that

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has been observed for some MPs previously.33, 34 As AOB generally represent the dominant

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ammonia-oxidizers in municipal WWTPs,15 the AOB model organism N. europaea was

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selected to construct the co-culture with the NOB model organism Nitrobacter sp..

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Nitrobacter frequently occurs in nitrifying activated sludge.44, 45 However, it should be

250

noticed that NOB of the genera Nitrotoga 44, 46, 47 and Nitrospira 48-50 often outnumber

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Nitrobacter in nitrifying activated sludge systems. As biomass production for

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biotransformation experiments is relatively slower with members of these genera, a

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Nitrobacter strain was selected for the experiments, but future studies should also include

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other NOB species. As expected, the co-culture converted all added ammonium to nitrate at

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a conversion rate of 2 mM per 8 hours with no nitrite accumulation. A relative removal of

256

20% of a MP was arbitrarily selected as a threshold for a positive biotransformation result,

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in order to make sure that other factors like measurement variations did not cause false

258

positive results. Among the four MPs, only asulam (as well as mianserin and ranitidine that

259

were included as positive controls for MP biotransformation) were biotransformed (> 20%

260

removal) by the co-culture during a 168-h incubation (Fig. 1A). Inconsistent with the

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activated sludge inhibition study,8 no significant transformation of monuron, clomazone,

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and trimethoprim was observed by the co-culture (Fig. S1). Intracellular asulam

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concentrations of the co-culture in the biotransformation experiments were below the

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detection limit. This excludes active biosorption/uptake by living cells, a phenomenon 13 ACS Paragon Plus Environment


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observed for heavy metals.51 To elucidate whether the NOB species Nitrobacter sp. contributed to the

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biotransformation in the co-culture, we tested the biotransformation of asulam, as well as

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all the other 16 investigated MPs by the Nitrobacter sp. pure culture. Although the nitrite

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oxidation of Nitrobacter sp. was not inhibited by the added MPs, there was no obvious

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biotransformation (> 20% removal) for any of the tested MPs over a 168-h incubation

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period (Fig. S2). Therefore, although NOB was co-inhibited with AOB in previous

272

inhibition studies due to the inhibited nitrite production,8, 10, 25, 52 they seem less likely

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contribute to the biotransformation of the selected MPs, which showed a correlation with

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nitrification activity. These results also indicate that the MP biotransformation in the

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co-culture was carried out by the AOB species N. europaea.

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Although biotransformation of monuron, clomazone, and trimethoprim was

277

strongly associated with nitrification in a previous study,8 these MPs were not

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biotransformed by the ammonia oxidizers tested in this study. The inability of trimethoprim

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biotransformation by N. europaea is consistent with findings by Khunjar et al.28 This

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discrepancy between pure culture studies and community inhibition studies is possibly

281

because the nitrifying activated sludge in the inhibition study contained ammonia-oxidizers

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with different biotransformation capabilities. This seems less likely, given the same

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biotransformation compound specificity (i.e., of the four compounds, only asulam was

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biotransformed at a 144-h removal of 25-40%) observed for the two AOB species (N.

285

europaea and Nitrosomonas nitrosa Nm90 isolated from activate sludge53) and the very

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distantly related AOA species (Nitrososphaera gargensis)37 tested in this study (Fig. S3). 14 ACS Paragon Plus Environment

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Nevertheless, another recently discovered ammonia oxidizer, comammox was also detected

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in some WWTPs, which carries out complete ammonia oxidation to nitrate, and is

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physiologically distinct from the other ammonia oxidizers (i.e., AOB and AOA).54

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Although beyond the scope of this study, it is worthwhile to investigate their roles in MP

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biotransformation in future studies. Another possible reason for the discrepancy between

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pure culture studies and community inhibition studies is that NOB species other than

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Nitrobacter, or heterotrophic microorganisms growing dependently on nitrifiers55 in the

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nitrifying activated sludge were responsible for the biotransformation of the MPs that were

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not transformed by the tested ammonia oxidizers. For example, studies have indicated that

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trimethoprim can undergo various biotransformation pathways under both aerobic

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(nitrifying) and anoxic (denitrifying) conditions.56 Therefore, it is highly likely that

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heterotrophic microorganisms physiologically interacting with nitrifiers carried out the

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biotransformation of trimethoprim in nitrifying activated sludge communities. Although

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lack of previous bacterial biotransformation studies, the biotransformation of monuron and

301

clomazone, similar with trimethoprim biotransformation was likely carried out by

302

heterotrophs.

303

Transformation Product Identification. To identify TPs of asulam, suspect screening was

304

first carried out. The self-compiled suspect list includes ~ 1000 compounds derived from

305

asulam via various reactions, including conjugation at primary and secondary levels. By

306

suspect screening, two asulam TPs were identified in the nitrifying co-culture, with exact

307

masses of [M-H] at 230.0129 (denoted “TP230”), and at 274.9979 (denoted “TP274”) (Fig.

308

1B). The formula of TP230 is C8H9O5NS (−NH+O from asulam), and the formula of 15 ACS Paragon Plus Environment


309

TP274 is C8H8O7N2S (−2H+3O from asulam). Both TPs were likely formed from oxidation

310

reactions, and their hypothetical structures are proposed (Fig. S4). TP230 was probably

311

formed by the oxidation of the amine group (−NH2) of asulam to a hydroxyl group (−OH),

312

and TP274 likely contains a nitro group from oxidation of the amine group, as well as a

313

hydroxyl group added on the aromatic ring (Fig. S4). It is worth noting that the removal of

314

asulam did not lead to a complete breakdown of the main structure into simpler molecules,

315

as both identified TPs have similar or even larger masses than asulam. MS2 analysis were

316

further carried out to validate the proposed structures of the two TPs (Fig. S4). However,

317

the MS2 fragments of the two TPs did not provide clear evidence for the position of the

318

adding −OH or nitro group. As the corresponding standard compounds with the proposed

319

TP structures were unavailable, there were no reference MS2 fragmentation profiles to

320

compare with, thus the exact TP structures remain unclear.

321

According to the peak areas, the two TPs account for ~ 40% of the removed asulam,

322

resulting in an incomplete mass balance. Therefore, we further carried out non-target

323

screening to identify TP candidates that might be missing from the suspect screening.

324

However, except the above two TPs, no additional plausible TP was detected. We also

325

analyzed possible TPs in the intracellular samples using suspect and non-target screening.

326

According to the same screening criteria, no TP candidate was detected in the intracellular

327

samples, which made it unlikely of active uptake of TPs into living cells. As asulam is a

328

relatively small molecule (m/z 231.0434, [M+H]), possible reasons for the incomplete mass

329

balance include: (1) formation of even smaller molecules that are not detectable by LC-MS;

330

(2) asulam and the two TPs were detected in two ESI modes (positive for asulam, and 16 ACS Paragon Plus Environment

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negative for the TPs). The ionization efficiency under negative mode is usually lower than

332

positive mode, resulting in lower response intensities for the same concentration of

333

compounds.

334

The incomplete biotransformation of asulam and poor mass balance nicely illustrate

335

that it is crucial to elucidate TP profiles during transformation processes to obtain a

336

comprehensive understanding of the environmental fate of parent MPs. Future research is

337

needed to better understand the ecological impact and putative health risks associated with

338

TPs from incomplete MP transformation.

339

Co-metabolic Biotransformation of Asulam. To investigate the biotransformation

340

mechanism of asulam by N. europaea in the nitrifying co-culture, limited ammonium

341

(one-time addition of 0.05 mM NH4Cl at the beginning of the experiment) was added to the

342

co-culture only as the nitrogen source, and asulam was added as a putative energy source.

343

Under these conditions, no asulam biotransformation was observed (Fig. 2A), with no

344

ammonia oxidation (Fig. 2B) and little cell growth (Fig. 2C). In comparison, asulam was

345

continuously biotransformed with active ammonium oxidation and cell growth in the

346

control with ammonium as the energy and nitrogen source (Fig. 2). These results suggest

347

that asulam biotransformation by N. europaea in the co-culture occurred via co-metabolism.

348

Similarly, previous studies have shown that AOB can co-metabolically biotransform a

349

number of other MPs, which has often been hypothesized to be carried out by AMO given

350

its broad substrate spectrum.5, 11, 12, 32, 57-59 However, experimental support for this

351

hypothesis is scarce.

352

Experimental Support for AMO as the Responsible Enzyme for Asulam 17 ACS Paragon Plus Environment


353

Biotransformation. To investigate whether AMO is the responsible enzyme for asulam

354

biotransformation, inhibitory effects of asulam on ammonia oxidation was first examined.

355

Compared to the no-asulam control, no inhibition of ammonia oxidation was observed for

356

asulam at 10, 20, and 30 µg/L (Fig. S5). The ammonia oxidation rate started to decrease

357

with 40 µg/L asulam by 4.7%, and the inhibition increased up to 15.5% at a concentration

358

of 80 µg/L (Fig. S5). Interestingly, the inhibitory effect of asulam on ammonia oxidation

359

decreased gradually after re-feeding the co-culture with ammonium (Fig. S5). This is

360

possibly because, the ratio of ammonia to asulam increased after re-feeding with

361

ammonium as asulam was removed over time which led to a decrease in potential substrate

362

competition for AMO. Moreover, newly synthesized AMO during cell growth could also

363

lead to the recovery of ammonia oxidation with a longer incubation. The inhibitory effect

364

of asulam on ammonia oxidation suggested that asulam might be a competitive substrate

365

for AMO, which was likely the responsible enzyme for asulam biotransformation. A

366

substantial inhibitory effect of asulam was observed at 80 µg/L (c.a., 348 nM) (Fig. S5),

367

indicating that asulam possibly has an even higher affinity to AMO than ammonia (the

368

ammonia affinity of the AMO of N. europaea is reported between 20 − 70 µM NH3, c.a., ~

369

330 − 1200 µM of total ammonium at pH 8).54

370

In addition, a specific mechanistic inhibitor of bacterial AMO, octyne,35, 36 was used

371

to further examine whether AMO was the responsible enzyme for the cometabolic asulam

372

biotransformation. Octyne was added after 72 hours during asulam biotransformation in the

373

nitrifying co-culture. As the addition of octyne resulted in instantaneous inhibition of AMO

374

activity, we hypothesized that, if AMO was the responsible enzyme, immediate inhibition 18 ACS Paragon Plus Environment

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of asulam biotransformation would occur after octyne addition, while if other enzymes

376

carried out the biotransformation, a lagged inhibition would be expected as their activities

377

likely persist for a while after AMO inhibition.30 Ammonia oxidation and asulam

378

transformation was determined 2 hours after the octyne addition. Compared to the

379

no-octyne-treated control, in the octyne-treated co-culture, complete inhibition of ammonia

380

oxidation was observed (Fig. 3A), and asulam biotransformation also completely ceased

381

(Fig. 3B). Consistently, no growth of AOB and NOB occurred after octyne exposure (Fig.

382

3C&D). These results provide strong support for the assumption that AMO is the enzyme

383

involved in asulam biotransformation by N. europaea in the co-culture.

384

Abiotic MP Transformation by NH2OH, the Intermediate Formed by AMO. In

385

addition to being converted by AMO as non-specific substrates, it is also possible that MPs

386

abiotically react with hydroxylamine, the product of ammonia oxidation by AMO.39 In

387

order to test this, we conducted abiotic MP transformation experiments with the addition of

388

NH2OH at concentrations occurring in AOB batch cultures.39 We also tested MP

389

transformation by NO, a non-AMO-mediated ammonia oxidation intermediate of AOB.

390

NO can be formed by nitrite reductase (NirK) in AOB.60-62 Moreover, it is produced as an

391

obligate intermediate by hydroxylamine dehydrogenase (HAO) of AOB. 63, 64 All 15 MPs

392

and the two control MPs mianserin and ranitidine were exposed to hydroxylamine and NO,

393

respectively. The abiotic transformation was compared to the biological transformation by

394

the AOB/NOB co-culture (Fig. 4). Eight MPs were biotransformed by the AOB/NOB

395

co-culture (Fig. 4, Fig. S6), whereas no biotransformation was observed in the tested NOB

396

pure culture for all MPs (Fig. S2). Six out of the eight MPs were also transformed 19 ACS Paragon Plus Environment


397

abiotically in NH2OH-added fresh medium (with even higher removals), and only

398

rufinamide and bezafibrate did not exhibit transformation by NH2OH (Fig. 4). In contrast,

399

only three of the eight MPs, i.e., indomethacin, ranitidine and fenhexamid, were

400

transformed by NO, taken into consideration the abiotic removal of mianserin and

401

indomethacin (61% and 22%, respectively) in fresh medium with no NH2OH or NO

402

addition (Fig. 4). Transformation of those three compounds by NO were less pronounced

403

than by NH2OH (Fig. 4). Furthermore, one cannot exclude the possibility that the

404

transformation of the three MPs was attributed to reactions with the NONOate residual

405

rather than the released NO.

406

Further analyses on TPs focused on MPs transformed by NH2OH. For asulam,

407

TP230 and TP274 identified during biotransformation in the co-culture were also detected

408

in the NH2OH-treated fresh medium. The similar compound specificities and TP profiles

409

between biotransformation and NH2OH-mediated abiotic transformation suggest that

410

abiotic MP transformation by NH2OH might be an important transformation route

411

indirectly mediated by AMO, which was not recognized previously. The abundance of

412

TP230 in the NH2OH-treated fresh medium after 168 hours was higher than that in the

413

co-culture when adding the same concentration of asulam (20 µg/L) (Fig. 5). One should

414

notice that more asulam was converted in the NH2OH-treated fresh medium (> 80%) than

415

in the co-culture (~ 60%) after 168 hours (Fig. 4), which might result in the higher

416

formation of TP230 in the NH2OH-treated fresh medium. In other words, if asulam

417

removal in the co-culture reached the same level as that in the NH2OH-treated fresh

418

medium, the abundance of TP230 under both conditions would be similar. Under the 20 ACS Paragon Plus Environment

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419

assumption that the abundance of TPs formed from non-enzymatic transformation by

420

NH2OH in the co-culture was similar as that observed in fresh medium with NH2OH

421

addition, this result suggests that the formation of TP230 was more likely via the

422

NH2OH-mediated abiotic transformation route. Nevertheless, the abundance of TP274 in

423

the AOB/NOB co-culture was substantially higher than that in the NH2OH-treated fresh

424

medium (Fig. 5), which indicates the co-occurrence of non-enzymatic and enzymatic MP

425

transformation pathways for the formation of TP274.

426

Among the other five MPs (i.e. mianserin, indomethacin, ranitidine, furosemide,

427

and fenhexamid) transformed abiotically in NH2OH-added fresh medium, no plausible TP

428

could be detected for indomethacin, furosemide, and fenhexamid in both the AOB/NOB

429

co-culture and the NH2OH-added fresh medium. For mianserin and ranitidine, consistent

430

with the previous study,5 the same TPs: TP279 for mianserin, and TP273, TP289, TP330

431

for ranitidine were detected in the AOB/NOB co-culture, as well as in the NH2OH-added

432

fresh medium, but at lower concentrations compared to those in the AOB/NOB co-culture

433

(Fig. S7). One possible reason is that in the AOB/NOB co-culture the same TPs were

434

formed via both AMO-mediated biotransformation and NH2OH-mediated abiotic

435

transformation, leading to a higher TP formation.

436

Moreover, by suspect screening, two major TPs of rufinamide with exact masses of

437

[M + H] at 240.0597 (TP240), and 312.0790 (TP312) (Fig. S8) detected in the AOB/NOB

438

co-culture were not detected in the NH2OH-treated fresh medium, which was in agreement

439

with no transformation of rufinamide by NH2OH abiotically (Fig. 4).

440

With NH2OH treatment, it is surprising that the identified TPs were in oxidized 21 ACS Paragon Plus Environment


441

forms of the respective MPs, given that NH2OH is nucleophilic and typically serves as a

442

reducing agent. Moreover, no reduced forms of asulam TPs could be detected when

443

targeting the corresponding TP suspects based on reported reductive reactions of NH2OH

444

with compounds that also have carboxylic ester functional groups as asulam.65 The

445

oxidized forms of TPs were probably formed from reactions of the MPs with some

446

oxidizing intermediates from the reaction of NH2OH with dissolved O2. In this context, it is

447

interesting to note that although the initial dose of NH2OH (5 µM) was in excess compared

448

to the MP concentrations (~ 0.06 - 0.12 µM), the abiotic transformation of MPs by NH2OH

449

did not reach the highest level after 12 hours with the addition of a single dose of NH2OH,

450

but further increased upon reamendment of NH2OH during a longer exposure (Fig. 4). This

451

could be due to faster degradation of NH2OH when reacting with dissolved O2 than reacting

452

with the MPs, or due to the possibility that the MPs reacted with intermediates from

453

NH2OH decomposition in water, which could be formed at lower concentrations. However,

454

the exact pathways and mechanisms of NH2OH-mediated abiotic MP transformation need

455

to be further elucidated.

456

Environmental Relevance and Implications. Nitrifiers are ubiquitously distributed in

457

natural environments, as well as engineered systems such as WWTPs. Although positive

458

correlations have been previously observed between MP biotransformation and nitrification

459

activities,8 the responsible enzymes and involved pathways are not well understood. Here,

460

we demonstrate that the NOB Nitrobacter sp. did not contribute to the biotransformation of

461

all tested MPs whose biotransformation was associated with nitrification activity according

462

to nitrifying activated sludge inhibition studies.8 We also show that only 6 of 15 MPs, 22 ACS Paragon Plus Environment

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463

whose biotransformation positively correlated with the activity of nitrifiers in a nitrifying

464

activated sludge community were transformed by the AOB species in an AOB/NOB (N.

465

europaea/Nitrobacter sp.) co-culture. Similar results were observed in other studies,11, 28, 52

466

where more MPs were biotransformed by complex communities such as nitrifying

467

activated sludge than by various pure ammonia-oxidizing cultures, including AOB and

468

AOA. Besides potential contributions of other physiologically diverse ammonia oxidizers

469

and NOB, including the recently discovered complete ammonia oxidizers20, 21 to MP

470

biotransformation, it is highly likely that heterotrophic bacteria interactively co-existing

471

with nitrifiers also make important contributions to MP transformation in nitrifying

472

activated sludge. Thus, keeping the diversity and complexity in biological systems could be

473

important to obtain a higher overall removal of MPs. Further studies are needed to

474

investigate effects of microbial diversity on MP biotransformation, and to identify the key

475

heterotrophs that co-exist with nitrifiers and contribute to MP biotransformation.

476

Importantly, we present evidence favoring that AMO of N. europaea is the

477

responsible enzyme for asulam biotransformation, which likely occurred via co-metabolism.

478

Furthermore, we report for the first time that abiotic MP transformation by hydroxylamine,

479

the product of AMO-mediated ammonia oxidation, likely represents another transformation

480

mechanism of MPs by ammonia oxidizers. It expands our knowledge on MP

481

transformation pathways and highlights that reactive intermediates of different

482

environmental microbial guilds might play an important yet understudied role in MP

483

transformation. As an NH4-N concentration (2 mM, c.a. 28 mg/L as N) relevant to typical

484

influent of municipal WWTPs was used in the batch reactors of this study, similar MP 23 ACS Paragon Plus Environment


485

transformation pathways might be occurring in nitrifying activated sludge in continuous

486

flow reactors in WWTPs. However, it needs to be further investigated whether the actual

487

free NH2OH available for abiotic MP transformation in WWTPs was similar with that in

488

lab-scale batch reactors. Altogether, these findings give a better understanding of roles

489

played by nitrifiers (i.e., AOB and NOB) in MP biotransformation, the responsible enzyme,

490

and the underlying mechanisms, which will provide important insights into the fate of MPs

491

in various nitrifying environments.

492

SUPPORTING INFORMATION

493

The Supporting Information is available on the ACS Publication Website, including Table

494

S1, and Fig. S1-S8.

495

ACKNOWLEDGEMENTS

496

We would like to thank Mengwei Han from the Department of Civil and Environmental

497

Engineering at University of Illinois at Urbana-Champaign and Dr. Zhenyu Tian from the

498

Center for Urban Waters at University of Washington Tacoma for their thoughtful

499

discussion on LC-MS/MS data analysis. P.H. and M.W. were supported by the European

500

Research Council Advanced Grant project NITRICARE 294343. L.J.Z. was supported by

501

the National Natural Science Foundation of China (31770551).

502

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A

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B

5x107

Asulam TP230 TP274

1.0

Peak Area

Asulam C/C0

4x107

0.8 0.6 0.4 Active biomass Medium ctrl Heat-inactivated ctrl

0.2

3x107 2x107 1x107 0

0 0

24

48

72 96 Time (h)

120

144

0

168

24

48

72

96

120

144

168

Time (h)

Figure 1. Biotransformation (A) and TP formation (B) of asulam (initial concentration: 10 µg/L) in the AOB/NOB co-culture (n=3; TP274 and TP230 were not detected in the medium or the heat-inactivated control).


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1.2

A

Asulam C/C0

1.0 0.8 0.6 0.4 10 µg/L asulam + 2 mM NH4-N

0.2

10 µg/L asulam + 0.05 mM NH4-N

0

B

Nitrate (mM)

20 15 10

0 µg/L asulam + 2 mM NH4-N

5

10 µg/L asulam + 2 mM NH4-N 10 µg/L asulam + 0.05 mM NH4-N

16S rRNA gene (copies/mL)

0

C

8x108

N. europaea 0 µg/L asulam + 2 mM NH4-N

6x108

10 µg/L asulam + 2 mM NH4-N 10 µg/L asulam + 0.05 mM NH4-N

4x108

2x108

108

0

24

48

72 96 Time (h)

120

144

168

Figure 2. Asulam biotransformation by the AOB/NOB co-culture grown with reamended and limited NH4-N (A: asulam removal; B: nitrate formation; C: cell growth of the AOB N. europaea; 2 mM ammonium was added repeatedly as indicated by arrows; 0.05 mM ammonium was only added at 0 h; n=3).



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20 A 0 µg/L asulam 10 µg/L asulam 10 µg/L asulam + octyne

0.8 Asulam C/C0

Nitrate (mM)

15

B

1.0

10

0.6 octyne added 0.4

5 octyne added

10 µg/L asulam 10 µg/L asulam + octyne

0.2

0

0

0 30 60 70

72

74

96 120144168

0 30 60 70

Time (h)

5x108

N. europaea

C

108 octyne added 0 µg/L asulam 10 µg/L asulam 10 µg/L asulam + octyne

2x107 0

24

48

72

96

74

96 120144168

Time (h)



5x108

72

120

Nitrobacter sp.

D

octyne added 108

0 µg/L asulam 10 µg/L asulam 10 µg/L asulam + octyne 107

144

0

168

24

48

72

96

120

144

168

Time (h)

Time (h)

Figure 3. Nitrate formation from a total of 14 mM ammonium addition (nitrite was below detection limit under all conditions) (A), asulam removal (B), growth of N. europaea (C) and Nitrobacter sp. (D) in the AOB/NOB co-culture with and without the addition of octyne (72h results were measured before octyne addition, n=3).


Page 37 of 39


100

** *

Removal (%)

80

*

* *

*

*

* *

60

*

*

NH2OH + fresh medium_12h

*

*

*

40

*

20

AOB/NOB co-culture_168h NH2OH + fresh medium_168h

*

*

* * * *

NO + fresh medium_168h Fresh medium_168h

* *

*

* 0 -20

ne As Fu ula m ro se m Fe nh i de ex a R uf m i d in a Be mid e za fi b C ar r b e a te nd Tr i m az i m et ho pr M im on u C lo ron m a Ac zo ne e L e ta m ip ve ri ti r ac d et am Te Irg bu aro fe no l Th zid e ia cl op rid

n ci

i ti di

ha

an R

et m

do In

M

ia

ns

er in

-40

Figure 4. Comparison of MP transformation in NH2OH- or NO-added sterile fresh medium and in the AOB/NOB co-culture (* indicates significant difference from the 168h removal in the fresh medium control, p < 0.05, n = 3).



1.2x107 1.0x107

AOB/NOB co-culture_168h NH2OH + fresh medium_168h

Peak area

8.0x106 6.0x106 4.0x106 2.0x106 0

TP274

TP230

Figure 5. Formation of asulam TPs in the AOB/NOB co-culture and in the NH2OH-treated fresh medium after a 168-h incubation with the same addition of asulam (neither of the two TPs was detected in the medium or heat-inactivated biomass control, n=3).


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