Biodegradation Products of a Sulfonated Azo Dye in Aerobic Granular

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Biodegradation products of a sulfonated azo dye in aerobic granular sludge sequencing batch reactors treating a simulated textile wastewater Rita Franca, M. Conceição Oliveira, Helena Maria Pinheiro, and Nídia Dana Lourenço ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.9b02635 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on August 5, 2019

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ACS Sustainable Chemistry & Engineering

Biodegradation products of a sulfonated azo dye in aerobic granular sludge sequencing batch reactors treating a simulated textile wastewater Rita D.G. Franca,*,† M. Conceição Oliveira,‡ Helena M. Pinheiro,† and Nídia D. Lourenço†,

†

iBB % Institute for Bioengineering and Biosciences, Department of Bioengineering,

Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa,

Portugal

‡

Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av.

Rovisco Pais, 1049-001 Lisboa, Portugal

UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e

Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica,

Portugal 1 ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

*

KEYWORDS:

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Corresponding author. E-mail: [email protected]

Acid

Red

14;

aerobic

granular

sludge;

biodegradation;

liquid

chromatography-tandem mass spectrometry; sequencing batch reactors; sulfonated

aromatic amines.

ABSTRACT: This study focused on the biodegradation of an azo dye (Acid Red 14, AR14)

in two anaerobic-aerobic sequencing batch reactors (SBRs) treating a synthetic textile

wastewater, operated with aerobic granular sludge under different hydrodynamic

regimens. The aim was to investigate the fate of the anaerobic AR14 breakdown products

(aromatic amines) during the SBRs’ aerobic reaction phase. Specifically, liquid

chromatography coupled with electrospray ionization tandem mass spectrometry (LC-

ESI-MS/MS) was used for structural characterization of AR14 biodegradation

metabolites, their molecular formulae being confirmed by accurate mass measurements.

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Nineteen molecules potentially related to AR14 were detected in the SBRs and their

relative abundances were followed along the aerobic stage of treatment cycles. The two

SBRs shared most of the identified compounds, but with differences in their metabolite

profiles. Biodecolorization through AR14 anaerobic azo bond reduction was confirmed by

the identification of the aromatic amine 4-amino-naphthalene-1-sulfonic acid, which was

further aerobically biodegraded, involving deamination and hydroxylation of the aromatic

ring. The other aromatic amine (1-naphthol-2-amino-4-sulfonic acid) was not detected,

being suggested to undergo autoxidation reactions forming dimeric, stable products. A

different AR14 biodegradation pathway was observed when nitrate was added to the

feed, a new intermediate product being detected (naphthalene-1-sulfonate).

INTRODUCTION

Textile wastewater (TWW) represents one of the main sources of pollution worldwide,

carrying high organic loads and recalcitrant dyes, azo dyes being the main type used (representing 60-70% of all the dye groups).1,2 Acid dyes (including azo, triarylmethane and

anthraquinone dyes), used to color protein fibers (wool, silk, angora, mohair, cashmere),



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nylon and certain modified synthetic fibers, are one of the largest classes of anionic dyes.3

In 1990, acid dyes were the type of dyes most commonly used, with more than 2000 entries in the colour index,1 the global import and export market for acid dyes being more recently estimated as 680,000 tonnes.2 Together with reactive dyes, acid dyes are

considered the most problematic anionic dyes for being recalcitrant to conventional treatment systems.4 This represents a critical environmental concern, owing to the high

degree of acid dyes’ loss to the textile wastewater after dyeing of typical fibers (namely, 5-20% when applied to polyamide).1 The need for efficient and environmentally friendly

dye removal processes led to the study of biological wastewater treatment systems.

Generally, these involve mixed bacterial cultures and include an anaerobic treatment

stage for the reductive cleavage of the dye’s azo bond, followed by an aerobic stage for degradation of potentially toxic, colorless aromatic amine products.5 The use of flocculent activated sludge in anaerobic-aerobic SBR systems has been studied in recent years,6

with notable success in the anaerobic decolorization stage. Yet, the scarce information

on the fate of the breakdown aromatic amines during the aerobic stage revealed that most are not degraded, thus constituting a health hazard.5,6 4 ACS Paragon Plus Environment

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Sulfonated aromatic amines are resistant to biodegradation due to the hydrophilic nature of the sulfonate group.7 Biodegradation has mostly been demonstrated for relatively simple sulfonated amino-benzene/naphthalene compounds.6 This limitation has

generally been attributed to the lack of an adequate aerobic microbial population capable

of metabolizing such compounds. Yet, the operation of SBRs using the innovative aerobic granular sludge (AGS) technology8 was recently shown to be advantageous for azo dye degradation, as compared to conventional activated sludge (CAS).9 The excellent

biomass retention capacity of AGS systems can favor the establishment of a more diverse

microbial population, namely slow-growing bacteria, with the potential ability to

biodegrade recalcitrant aromatic amines, such as 4-amino-naphthalene-1-sulfonic acid (4A1NS) resulting from Acid Red 14 (AR14) bioreduction.9 Accordingly, 4A1NS

biodegradation was also accomplished in biofilm reactors, which allow high biomass retention.10 Moreover, the use of a plug-flow feeding regimen in AGS SBR systems was shown to play an important role in the selection of slow-growing bacteria.8,11

The aim of this study was to compare two anaerobic-aerobic AGS SBRs using different feeding regimens (static feed, anaerobic-aerobic reaction vs anaerobic plug-flow feed, 5 ACS Paragon Plus Environment


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anaerobic-aerobic reaction) regarding azo dye degradation performance, focusing on the

aerobic conversion of sulfonated aromatic amines resulting from azo bond anaerobic

reduction. The SBRs were fed with a synthetic TWW containing the azo dye AR14 with

changes in the feed solution along the experimental run, including an azo dye shock load

and nitrate supplementation. Based on preliminary HPLC analysis, cycles representative

of different 4A1NS profiles were further analyzed by liquid chromatography coupled with

electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the structural

characterization of AR14 reduction metabolites and subsequent degradation products

produced in the SBRs under different operational conditions. This allowed the study of

the fate of AR14 breakdown products regarding the effect of the hydrodynamic regimen

and also that of increasing the azo dye concentration and of the nitrate feed. A brief review

of the literature regarding mass spectrometry (MS) application to TWW sample analysis,

which served as a basis to select the analytical method and sample pre-treatment

procedures most suited to the objective of this work, is presented in Section 1 of the

Supporting Information.


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EXPERIMENTAL SECTION

SBR setup and operation

AGS, previously used in SBRs and subsequently stored at 4 ºC for 18 days, was used

to inoculate two 1.5-L SBRs (H/D=2.5). The SBRs were operated for 205 days using

different feeding regimens: statically fed (from top), anaerobic-aerobic SBR1 vs anaerobic plug-flow fed (from bottom), anaerobic-aerobic SBR2.12 They were operated in 6-h cycles:

30-min or 1-h feeding, followed by a 1.5-h or 1-h stirred anaerobic phase in SBR1 or

SBR2, respectively, 3.5-h aeration, 5-min settling, 1-min drain and idle. A synthetic textile wastewater12 containing a hydrolyzed starch-based sizing agent (Emsize E1), nutrients, pH buffering phosphates and 40 mg L-1 AR14 (Chromotrope FB, Sigma-Aldrich, 50% dye

content), was supplied to the SBRs at a 12-h hydraulic retention time and an organic loading rate, as chemical oxygen demand (COD), of 2.0 kg O2 m-3 d-1. Along the experimental run, changes were introduced in the feed composition of both SBRs: supplementation with 20 mg L-1 of silver nanoparticles (days 0-35); dye-shock load, with a three-fold increase in AR14 concentration to 120 mg L-1 (days 119-134); or



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supplementation with calcium nitrate to 60 and 120 mg NO3 L-1 (days 147-153 and 154205, respectively).

Sample selection

Samples were taken from the SBRs mixed liquor along selected treatment cycles,

centrifuged and the supernatant was stored at -20 ºC for subsequent examination. HPLC

analysis (as described in Section 3 of the Supporting Information) was performed to

assess the concentration-time profile of AR14 and of one of its reduction products

(4A1NS). Based on this preliminary analysis, three specific treatment cycles (I, II and III)

representative of different experimental periods and correspondent 4A1NS aerobic

profiles (Figure 1) were selected for further examination by MS. Specifically, cycle I (day

98) represents an operational period when the SBRs were under the base operational

conditions and 4A1NS was consistently converted in both reactors (Figure 1-I); cycle II

(day 134) represents the dye shock load period, when SBR1 consistently presented

incomplete 4A1NS conversion, while SBR2 sustained complete 4A1NS conversion

(Figure 1-II); cycle III (day 154) represents the period when the SBRs were supplemented

with nitrate and 4A1NS formation was not observed (Figure 1-III). For each selected cycle 8 ACS Paragon Plus Environment

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98), II (day 134) and III (day 154) from the sequencing batch reactors SBR1 (S) and SBR2

(T). Cycle I corresponds to the base operational conditions in feed composition (40 mg AR14 L-1), cycle II represents the dye shock load period (120 mg AR14 L-1), and cycle III represents the period of nitrate supplementation (120 mg NO3 L-1). The selected cycles cover the different 4A1NS aerobic fates observed: complete conversion (cycle I of both

SBRs and cycle II of SBR2), partial conversion (cycle II of SBR1) and not formed (cycle

III of both SBRs). The vertical lines in the charts represent the end of the anaerobic phase,

aeration onset. The arrows in the charts indicate the sampling times of the samples that

were further analyzed by mass spectrometry: A, AB and B correspond to the end of the

anaerobic phase, 1 hour of aeration and end of the aerobic phase, respectively. For a

detailed description of the different hydrodynamic regimens of SBR1 and SBR2 please

refer to section “SBR setup and operation”.

Sample pre-treatment

To achieve higher levels of separation and lower detection limits in LC-MS targeting

azo dye metabolites in complex matrices such as wastewater, several sample pre-


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treatment methods have been proposed (reviewed in Section 1 of the Supporting

Information). The detailed procedure and optimization of the sample pre-treatment,

including several liquid-liquid extraction (LLE) and solid-phase extraction (SPE) tests, are

provided in Section 2 of the Supporting Information. LLE, which was based on the method described by Chan et al. (2012),14 was discarded because no satisfactory results were

obtained, as opposed to SPE. After optimization, sample clean-up was performed by

SPE, using the generic Oasis HLB (Waters) method without the wash step. Briefly, the

SPE cartridge was initially conditioned with 1 mL methanol followed by 1 mL of MilliQ

water. Subsequently, 1 mL of the sample solution was loaded onto the cartridge at room

temperature, followed by elution with 1 mL of methanol, the eluted fraction being collected

for further analysis.

Liquid chromatography-mass spectrometry (LC-MS) analysis LC-MS2 and LC-high resolution MS/MS (LC-HRMS/MS) analyses were conducted on a

Dionex Ultimate 3000 HPLC system coupled inline to an LCQ Fleet ion trap mass

spectrometer equipped with an ESI source (Thermo Scientific), and on a UHPLC Elute

interfaced with a QqTOF Impact II mass spectrometer equipped with an ESI source 11 ACS Paragon Plus Environment


Page 12 of 37

(Bruker Daltoniks), respectively. The mass spectrometers were operated in the ESI

positive and negative modes. LC was performed on a CORTECS RP18 column (Waters)

using a gradient elution of 0.1% formic acid in water and acetonitrile. For details about LC-MS2 and LC-HRMS/MS settings see Sections 4 and 5 of the Supporting Information,

respectively.

RESULTS AND DISCUSSION

LC-MS analysis

Each SBR1 and SBR2 sample was analyzed by LC-MS in parallel with the

correspondent time sample from the dye-free control SBR in order to select the most

significant ions that were present in SBR1 or SBR2 samples but absent in the dye-free

SBR. The aim was to select uniquely the metabolites deriving from the azo dye AR14, as

opposed to other molecules resulting from the carbon source metabolism. In addition, an

AR14 solution was also analyzed by LC-MS in order to identify the impurities present in

the AR14 commercial product (section 6 of the Supporting Information). Analysis of the


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samples together with the controls resulted in the selection of 19 ions (Table 1) possibly

related to degradation products from AR14.

Table 1. Most significant ions detected in electrospray ionization (ESI) negative

(metabolites 1-16) and positive (metabolites 17-19) modes in each sequencing batch

reactor (SBR) sample from cycles I, II and III analyzed by LC-MS.

I

II

SBR1 A B

SBR2 A B

SBR1 A AB

B

SBR2 A AB

B

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Metabolite

RT (min)

m/z

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

15.9 10.0 3.7 12.7 6.7 12.3 11.9 12.8 13.7 15.6 10.1 10.7 17.3 12.7 14.7 19.5

189 207 222 228 239 252 268 377 379 391 393 394 419 457 592 689

X

17 18 19

7.3 12.6 13.7

173 174 189

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

III

X

X

X

SBR2 A B

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

SBR1 A B

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

RT: retention time; m/z: mass-to-charge ratio.



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Most of the selected ions were shared by different samples and SBRs. In addition, some

metabolites typically observed in cycles I and II were absent in cycle III (deprotonated molecules m/z 189, 222, 391, 393 and 394), and vice versa (deprotonated molecules m/z

228, 268, 379, 457, 592), suggesting that a change in the azo dye metabolism occurred upon nitrate supplementation of the SBRs. The deprotonated molecules m/z 222 and 457 (as well as 228, [M-2H]2-) detected in the ESI negative mode correspond to the

deprotonated aromatic amine 4A1NS and AR14, respectively, as confirmed by analysis

of the respective standards. The other aromatic amine resulting from AR14 azo bond

reduction, 1-naphthol-2-amino-4-sulfonic acid (1N2A4S) was not detected through LCMS (expected m/z 238, as deprotonated molecule), as expected from its unstable

character. The abundance of each selected ion (Table 1) in the analyzed samples is

graphically represented in Figures 2 and 3. Assuming that no significant variations

occurred during the analysis of all the samples in the low resolution MS (performed in a

single run to minimize variations), the results presented in Figures 2 and 3 can give an

indication of the changes in each ion abundance. Despite the relevant information that

can be extracted by analyzing each ion profile, it is important to note that relative 14 ACS Paragon Plus Environment

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abundance comparison between different ions is not appropriate owing to the fact that

ESI favors ionization of the most polar compounds.

According to Figures 2 and 3, the deprotonated molecules whose abundance was typically observed to decrease along the aerobic phase in both SBRs include m/z 207, 222, 377, 393 and 592. In contrast, deprotonated molecules corresponding to m/z 239,

252, 268, 379, 394 and 689 generally increased in abundance along the aerobic phase

in both SBRs, representing good candidates for transformation products of the former

ions. Specifically, taking into account their relatively symmetrical abundance variation along the aerobic phase (Figure 2), a correlation might exist between the ions m/z 207

and 252. In contrast, none of the ions whose abundance increased in the aerobic phase could be directly correlated with the disappearance of m/z 222, supporting the hypothesis

of complete mineralization of 4A1NS in cycle I in both SBRs and cycle II in SBR2.



m/z 189

m/z 207 800 800

m/z 189

700 700

250 250

450 450

m/z 207

400 400

150 150

300 300

500 500

Intensity

Intensity

250 250

400 400

200 200

300 300

100 100 50 50

100 100

100 100

50 50

00

00

SBR1 SBR2 SBR1 SBR2 SBR1 SBR2

I

II

150 150

200 200

I

III

m/z 228

70

II

300 300

m/z 228

m/z 239

50

30 30

Intensity

200 200 Intensity Intensity

Intensity

Intensity

50

40 40

150 150

100 100

20 20

50 50

10 10 00

00

SBR1 SBR2 SBR1 SBR2 SBR2 SBR1 SBR1 SBR2 SBR1 SBR2 SBR1 SBR2

II

I

II

m/z 377

m/z 268

1400 1400

30 30

1

800 800

200 200

II

60 60 40 40 20 20 00

I

II


I

III

m/z 393

m/z 391

1200

m/z 391

300

450

m/z 393

400 400

1000

350 350

800

300 300

250

250 Intensity

800

200

200 150

150

m/z 394

250 250

600 600

200 200

150 150

400 400

100

III

450

1000

300

II m/z 394

1200

Intensity

350 350

80 80


III

III

m/z 379

00

2

SBR1 SBR2 SBR1 SBR1 SBR2 SBR1 SBR2 SBR2

II

m/z 379

100 100

600 600

10 10

I

1 1

400 400

0

m/z 377

1000 1000

20 20

m/z 252

I

1200 1200 Intensity

Intensity

40 40

SBR1 SBR1 SBR2 SBR2

III

1 120 120

Intensity Intensity

m/z 268

50 50

0

II


III

1600 1600

60 60

100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 00


III

70

70

SBR1 SBR2 SBR2 SBR1 SBR1 SBR2 SBR1 SBR2

100

250 250

60

I

SBR1 SBR1 SBR2 SBR2

I

III

m/z 239

70 60

00


Intensity

Intensity Intensity

600 600 200 200

m/z 222

350 350

Intensity

300 300

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100 100

50 50

200 200

00

00


I

II

III

50 50 00 2 SBR1 SBR2 SBR1 SBR2 SBR1 SBR2

I

II


I

III

II

III

Figure 2. Abundance (intensity) of ions m/z 174 to 394 (ESI negative mode) in samples

harvested in cycles I, II and III from the sequencing batch reactors SBR1 and SBR2 at 2

2

the end of the anaerobic phase (darkest blue bars; sample B), after 1 hour of aeration

16 ACS Paragon Plus Environment 2

2

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Page 18 of 37

blue or orange bars; sample AB; only analyzed on cycle II), and at the end of the aerobic

phase (lightest blue or orange bars; sample B).

Although both SBRs shared most of the identified compounds in each experimental

period, specific differences were observed in their metabolites’ aerobic profiles. For

instance, some deprotonated molecules presented opposite variation trends in the two SBRs (Figures 2 and 3). Specifically, the abundance of ions with m/z 189, 391 and 419

increased along the aerobic phase in SBR1, while decreasing in SBR2. Regarding

protonated molecules, while the abundance of the majority was maintained along the aerobic phase in SBR1, the number of ions with m/z 174 and 189 decreased in SBR2, as opposed to m/z 173 and 346, the abundance of which increased in the latter. Overall, a

decrease in metabolite abundance along the aerobic phase was more often observed in

SBR2 than in SBR1, indicating a potential advantage in using a plug-flow feeding

regimen.

Comparing the abundance of the same ion between samples harvested in cycles I and II, it was possible to observe that the number of deprotonated molecules with m/z 189,


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239 and 391 (as well as 4A1NS, m/z 222) significantly increased in both SBRs upon the

three-fold increase in AR14 concentration (Figure 2). This result further supports a

possible direct (or indirect) relation of the respective compounds to AR14 or its reduction

products, 4A1NS and 1N2A4S. Furthermore, a comparison between cycles III and I

(same AR14 concentration in the SBRs feed), allowed the identification of compounds

whose presence possibly resulted from the supplementation of the SBRs with nitrate. Specifically, deprotonated molecules with m/z 207, 252, 268, 379 and 592 (besides AR14,

m/z 228 and 457) were only significantly observed in samples from cycle III. In contrast, deprotonated molecules with m/z 189, 391, 393 and 394 (besides 4A1NS, m/z 222), as well as protonated molecules with m/z 189 were absent in cycle III (Figures 2 and 3).

LC-HRMS/MS analysis

For each SBR1 and SBR2 sample, precursor ions were selected (Table 1) and collision-

induced dissociation was performed to obtain the respective product ions spectra. After analyzing all LC-MS2 data, the fragmentation pattern found for each of the 19 selected

ions was summarized in Table S5 of the Supplementary Information. In addition, LC-

HRMS/MS was conducted in ESI negative and positive modes for some of the samples, 19 ACS Paragon Plus Environment


Page 20 of 37

covering the 19 precursor ions previously selected, the results being summarized in Table

2. Tandem MS experiments performed on a QqTOF mass spectrometer allowed to

propose fragmentation pathways for selected precursor ions with higher accuracy than in an ion trap, which was particularly relevant to distinguish ions with very similar m/z values,

as well as to confirm some of the neutral losses through their accurate mass values (Table

2).

Fragmentation patterns characteristic of compounds containing the sulfonate group

were repeatedly observed in the ESI negative mode, as neutral losses of 64 u and 80 u

typically correspond to the loss of SO2 and SO3, respectively (Table 2). This specific fragmentation pattern further indicates that most of the selected ions originated from the

azo dye AR14, since this was the only compound in the SBR feed that contained sulfonate

groups. In addition, other fragments possibly involved the loss of NH3 (17 u), CH4 (16 u), CHN (27 u), and CO (28 u), among others. Based on the RT, m/z and fragmentation

pattern of each precursor ion, it was possible to suggest some correlations between the metabolites (Table 2). Specifically, metabolites 6 (m/z 252) and 7 (m/z 268) probably

correspond to a similar molecule, metabolite 7 containing one extra oxygen atom. Owing 20 ACS Paragon Plus Environment

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to the similarities between their fragmentation patterns, metabolite 8 (m/z 377) probably corresponds to the AR14 molecule (metabolite 14, m/z 457) without one sulfonic acid group. In addition, metabolite 13 (m/z 419) is possibly an adduct of metabolite 8 (m/z 377) with C2H2O. Also, metabolites 8 (m/z 377) and 9 (m/z 379) likely have a similar structure, with the difference of the latter containing two extra protons. The same can be hypothesized for metabolites 10 (m/z 391) and 11 (m/z 393). Furthermore, metabolite 12 (m/z 394) possibly corresponds to metabolite 11 (m/z 393) but containing a nitrogen atom instead of an oxygen atom. The same can be inferred regarding metabolites 17 (m/z 173) and 18 (m/z 174). Based on the fragmentation patterns detailed in Table 2 and on these

assumptions, the tentative attribution of some of the ions previously selected as relevant

in the AR14-fed SBRs was performed, the proposed chemical structures being summarized in Figure 4 (cf. sections 8 and 9 of the Supporting Information for the

respective fragmentation pattern, MS and MS/MS spectra). Although it is not possible to

precisely determine the molecular structure of the metabolites with the available

information, Figure 4 gives an indication of the general structure. Confirmation of the

proposed structures would require further study, namely comparison with commercial 21 ACS Paragon Plus Environment

Page 22 of 37


standards, when available (confirmed for metabolites 3, 4 and 14), Nuclear Magnetic

Resonance (NMR) and further MS/MS analysis.


Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. LC-HRMS/MS analysis results. Measured accurate mass of precursor ions,

respective product ions (the most relevant are highlighted in bold) and neutral loss is

indicated, along with attributable species.

1

m/z 189.0585 *

MS Precursor ion [M-H]-

RT (min) 12.3

2

207.0143

[M-H]-

8.1

3

222.0280

[M-H]-

1.7

4

228.0112

[M-2H]2-

10.8

5

239.0072

[M-H]-

4.7

Metabolite

6

252.0030

[M-H]-

10.3

7

267.9923

[M-H]-

9.9

8

377.0599

[M-H]-

10.9

9

379.0362

[M-H]-

12.0

10

391.0483

[M-H]-

13.5

11

393.0636

[M-H]-

10.0

12

394.0470

[M-H]-

8.9

13

419.0705

[M-H]-

15

14

457.0251

[M-H]-

10.7

m/z 173.0280 145.0329 143.0529 115.0606 158.0644 128.0555 221.0203 170.0291 220.9974 174.0352 159.0468 145.0323 129.0378 222.0039 206.0095 188.0397 250.9898 220.9915 192.9973 188.0364 170.0253 361.0407 313.0982 297.1035 281.0850 270.0927 313.1053 299.0787 271.0834 327.0847 310.0823 299.0894 329.0918 313.0989 314.0890 296.0779 286.0939 377.0604 355.1088 339.1143 324.0905 313.0986 297.1029 377.0649 297.1066

MS/MS Product ions [(M-H)-CH4][(M-H)-CH4-CO][(M-H)-SO2][(M-H)-C2H4][(M-H)-SO2][(M-H)-SO2-CH2O][(M-H)-C10H6NO4S•]•[(M-H)-C10H7O3S•-SO3]•[(M-H)-H2O][(M-H)-SO2H•]-• [(M-H)-SO3][(M-H)-SO2-CH2O][(M-H)-SO3-CH2O][(M-H)-CH2O][(M-H)-CH2O2][(M-H)-SO2][(M-H)-NH3][(M-H)-NH3-CH2O][(M-H)-NH3-CH2O-CO][(M-H)-SO3][(M-H)-SO3-H2O][(M-H)-CH4][(M-H)-SO2][(M-H)-SO3][(M-H)-SO3-CH4][(M-H)-SO3-CHN][(M-H)-SO2H2][(M-H)-SO3][(M-H)-SO3-CO][(M-H)-SO2][(M-H)-SO3H•]-• [(M-H)-SO2-CO][(M-H)-SO2][(M-H)-SO3][(M-H)-SO3][(M-H)-SO3-H2O][(M-H)-SO3-CO][(M-H)-C2H2O][(M-H)-SO2][(M-H)-SO3][(M-H)-SO2H•-CH2O][(M-H)-C2H2O-SO2][(M-H)-C2H2O-SO3][(M-H)-SO3][(M-H)-SO3-SO3]-

Neutral loss (u) 16.0305 44.0256 63.9614 91.9537 63.9636 93.9725 236.0099 287.0011 18.0098 64.9720 79.9605 93.9749 109.9694 29.9991 45.9935 63.9633 17.0025 47.0008 74.9950 79.9559 97.967 16.0192 63.9617 79.9564 95.9749 106.9672 65.9309 79.9575 107.9528 63.9636 80.9660 91.9589 63.9633 79.9562 79.9580 97.9691 107.9531 42.0101 63.9617 79.9562 94.9800 105.9719 121.9676 79.9602 159.9185



15

592.0349

[M-H]-

12.7

16

689.1617

[M-H]-

15.8

17

173.0739

[M+H]+

5

18

174.0571

[M+H]+

11.1

19

189.0572

[M+H]+

12.3

Page 24 of 37

[(M-H)-C10H7NO3S••]••[(M-H)-C10H6NO4S•]•[(M-H)-SO3-SO3][(M-H)-SO2][(M-H)-SO2-SO2][(M-H)-NH3][(M-H)-SO3][(M+H)-CHN]+ [(M+H)-CHN-H2O]+ [(M+H)-CO]+ [(M+H)-CHN-H2O]+ [(M+H)-CO]+

236.0060 221.0180 170.0280 528.0725 464.1096 672.1355 609.2059 146.0621 128.0547 146.0629 129.0457 161.0619 146.0391 143.0521

221.0191 236.0071 286.9971 63.9624 127.9253 17.0262 79.9558 27.0118 45.0192 27.9942 45.0114 27.9953 43.0181 46.0051

[(M+H)-CO-CH3•]+• [(M+H)-CO-H2O]+

* Precursor ion absent in respective MS/MS spectra indicating total fragmentation; RT: retention time.

2

5

3

7

6

NH2

OH

NH

N OH

O

O

OH O

–

S

O O

–

S

O

O

–

S

O

O

–

OH

S

O O

O

O O

8

O

O

C10H8NO3S-

C10H7O3S-

C10H7O5S-

O O

S

O

–

O S

O

H OH

OH N

O

N

O

O

NH

S

O

S

11 O

H OH

C10H6NO6S-

C10H6NO5S-

10

–

O

–

N

N N –

O

C20H13N2O5S-

C20H11N2O5S-

13

12 O

O

O

C20H13N2O4S-

4 & 14

–

O

NH

–

O

OH H

O

O

S

N

N

O

O S

S

H OH

HO

O

N

N

O –

O

OH

C20H12NO6S-

C22H15N2O5S

O

18

17

+

NH + NH2

18’

+ NH2 NH

NH2 18’’ NH

O

C20H13N2O7S2

19

++

OH

S O

O H+ +

OH

O

CH3

C10H9N2O+

OH

OH

C10H8NO2+

OH

C11H9O3+

+ OH2 OH


Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Summary of some of the plausible molecular formulas and chemical structures

proposed for metabolites detected in the electrospray ionization (ESI) negative and

positive modes (blue and orange squares, respectively).

The molecular formula of each proposed chemical structure (Figure 4) was assessed

on accurate mass measurements of the corresponding precursor taking into account the

accuracy and precision of the measurement parameters, such as error (ppm) and

mSigma (Table 3).The molecular formulas were validated through the fragmentation

patterns, supporting the respective proposed chemical structure (Figure 4). Although

future work is needed to confirm their chemical structure, correlations between the

identified metabolites are suggested (Figure 5), in light of the predicted degradation

pathway

for

AR14

(described

in

EAWAGBBD

Pathway

Prediction

System;

http://umbbd.ethz.ch/predict/) and the associations previously established based on the

LC-MS analysis. Table 3. Classification of the correspondence between the measured accurate m/z value

in the LC-HRMS analysis, and the proposed ion chemical formula (Score = 100%).



Metabolite

Page 26 of 37

Measured m/z

Attribution

Ion Formula

Calculated m/z

> (ppm)

mSigma

2 3 4 5 6 7 8 10 11 12 13 14 17 18

207.0139 222.0280 228.0112 239.0072 251.9979 267.9956 377.0602 391.0483 393.0625 394.0449 419.0785 457.0251 173.0739 174.0566

[M-H][M-H][M-2H]2[M-H][M-H][M-H][M-H][M-H][M-H][M-H][M-H][M-H][M+H]+ [M+H]+

C10H7O3S C10H8NO3S C20H12N2O7S2 C10H7O5S C10H6NO5S C10H6NO6S C20H13N2O4S C20H11N2O5S C20H13N2O5S C20H12NO6S C22H15N2O5S C20H13N2O7S2 C10H9N2O C10H8NO2

207.0121 222.0230 228.0048 239.0020 251.9972 267.9921 377.0602 391.0394 393.0551 394.0391 419.0707 457.017 173.0709 174.0550

-8.5 -22.6 -28.1 -22 -2.6 -12.8 -0.2 -22.7 -18.9 -14.7 -18.7 -17.8 -16.9 -9.4

10.1 32.5 17.9 16.6 31.3 n.a. 19.1 9.9 8.2 48.3 36.8 24.0 n.a. 5.8

19

189.0561

[M+H]+

C11H9O3

189.0546

-7.8

n.a.

n.a.: not attributed. Identification of the sulfonated aromatic amine 4A1NS in both SBRs confirmed the

occurrence of AR14 reduction in the anaerobic stage. Accordingly, the first step of AR14

biodegradation (Figure 5) apparently corresponded to the anaerobic AR14 azo bond

reduction, at least in cycles I and II, when 4A1NS was detected. However, it is possible

that AR14 (corresponding to compounds 4 and 14) suffered other transformations before

the cleavage of its azo bond. Specifically, desulfonation of AR14 may have occurred

resulting in metabolite 11 and, subsequently, metabolite 8, in addition to the incomplete

cleavage of the AR14 azo bond (Figure 5). Since these metabolites were observed at the

end of the anaerobic phase (Figure 2), the hypothetical desulfonation of the aromatic ring

occurred under anaerobic conditions. 26 ACS Paragon Plus Environment

Page 27 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


On the other hand, when the original AR14 molecule was completely reduced, two

aromatic amines were formed, as confirmed by the presence of 4A1NS (metabolite 3).

The complete disappearance of the recalcitrant 4A1NS from the SBR samples indicated

that it was further transformed during the aerobic stage (Figures 1 and 2), representing a progress relative to previous work.13 In accordance with the predicted AR14 degradation

pathway (EAWAGBBD Pathway Prediction System), 4A1NS probably underwent

deamination and aromatic ring hydroxylation forming metabolite 5, as previously suggested.14 Subsequently, metabolite 5 is expected to suffer aromatic ring cleavage

(Figure 5), with further oxidation eventually leading to the production of aliphatic carboxylic acids and ultimately mineralization.14,15 Based on the putative structures

proposed for metabolites 18’’ and 6, another aromatic ring opening pathway was

suggested (Figure 5), although the probability of these transformations must be further

addressed.

Upon SBRs supplementation with nitrate (cycle III), 4A1NS was no longer detected in

SBR1 and SBR2 (Figures 1 and 2). Instead, new metabolites emerged (Table 1),

suggesting that a different AR14 biodegradation pathway took place in the presence of 27 ACS Paragon Plus Environment


Page 28 of 37

nitrate. In fact, in light of the proposed structure for metabolite 2, direct deamination of the

4A1NS was suggested to occur upon AR14 reduction under anaerobic conditions. This

observation is in accordance with a previous study reporting the degradation of some aromatic amines under denitrifying conditions.16 Spefically, this finding in supported by Seymour et al. (2002) who showed that nitrite, which is produced during the first step of

denitrification, reacts with aromatic amines, resulting in deamination, thus improving their biodegradation potential.17

In contrast to 4A1NS, the other aromatic amine derived from AR14 azo bond reduction

(1N2A4S) was not detected. In this sense, some transformations of 1N2A4S are

proposed, involving metabolite 18’ (Figure 5). In addition, similarly to 4A1NS, deamination

and aromatic ring hydroxylation can be proposed to generate metabolite 5. However, the presence of a hydroxyl group in the ortho-position relatively to the amino group in 1N2A4S provides an unstable character to this molecule,18 which explains why it was not detected

by LC-MS. Consequently, 1N2A4S is likely to undergo spontaneous autoxidation

reactions forming dimeric, stable products, which might be at the origin of complex

aromatic structures containing sulfonate groups, such as metabolites 10 and 12. 28 ACS Paragon Plus Environment

Page 29 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Overall, these results represent a relevant contribution towards the elucidation of AR14

biodegradation pathways, involving mechanisms whose understanding may be crucial for

efficient TWW treatment.



Page 30 of 37

–

O

–

O S

H OH

13

H OH

8

O S

O

O N

O

N

N N

O

AR14

H HO

H HO O

O

H OH

H OH

4 & 14

OH

O

H NH

N

H NH

N

N

–

–

–

O

O

S

O

O

OH

n.d.

–

S

NH2

3

NH2

O

S

O

O

O

O

O

–

S

2

O

O

–

+

OH

OH

5

++ NH NH2

S

O

O

O

O

18’

H OH

n.d.

O

N

+ NH+2 NH

18’’

+

OH

O

OH

O S

S

S

11

O

–

S

OH

O

OH

O

O

12

–

O

NH

O

O S

10

–

O

NH

O

n.d.

S

O

6

O O

N

–

O OH

O

OH

N O

OH

O

S O O

–

O

O

–

S

O O

O

Figure 5. Proposed conversion relations between the putative structures proposed for the

different metabolites detected through LC-MS/MS, with a focus on the biodegradation of

Acid Red 14 (AR14) and the aromatic amines resulting from its reduction, namely 4-


Page 31 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


amino-naphthalene-1-sulfonic acid (4A1NS; metabolite 3), according to the EAWAGBBD

Pathway Prediction System. n.d.: not detected in LC-MS analysis.

ASSOCIATED CONTENT

Supporting Information:

The following files are available free of charge. The Supporting Information is

available free of charge on the ACS Publications website at DOI: XXX.

Review of liquid chromatography-mass spectrometry application to textile wastewater;

methodology and results regarding sample pre-treatment optimization (LLE and SPE); detailed experimental information (HPLC, LC-MS2 and LC-HRMS/MS); Summary of results obtained in LC-MS2; total ion chromatograms, MS spectra, MS/MS spectra, and

fragmentation patterns according to LC-HRMS/MS analysis. (PDF)

ACKNOWLEDGMENT

This work was financed by Fundação para a Ciência e a Tecnologia (FCT, Portugal),

projects PTDC/AAG-TEC/4501/2014 and UID/BIO/04565/2013. Funding from Programa



Page 32 of 37

Operacional Regional de Lisboa 2020 (Projects N. 007317 and RNEM-Lisboa-01-0145-

Feder-402-022125) is also acknowledged. R.D.G. Franca and N.D. Lourenço

acknowledge

FCT

for

doctoral

(SFRH/BD/95415/2013)

and

post-doctoral

(SFRH/BPD/88095/2012) research grants, respectively. The authors thank Ms. Ana Dias

for running the low resolution MS data. This article was presented at the 13th International

Chemical and Biological Engineering Conference (CHEMPOR 2018). The authors

acknowledge the Scientific and Organizing Committees of the Conference for the

opportunity to present this work.

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Biodegradation Products of a Sulfonated Azo Dye in Aerobic Granular

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