Science of the Total Environment 792 (2021) 148361

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Evaluation of characteristics and microbial community of anaerobic

granular sludge under microplastics and aromatic carboxylic
acids exposure
Ronghua Qin a, Xumeng Lin a, Zhenpeng Chen a, Chengyuan Su a,b,⁎, Fenghua Zhu a, Wenjing Yang a,
Zhuxin Chen a, Pingping Lu a
a
Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Ministry of Education, 15 Yucai Road, Guilin 541004, PR China
b
University Key Laboratory of Karst Ecology and Environmental Change of Guangxi Province, Guangxi Normal University, 15 Yucai Road, Guilin 541004, PR China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Effects of aromatic carboxylic acids and

PES on AGS were investigated.
• Inhibitory effect of aromatic acids addi-
tion on the TTC activity was obvious.
• Methanobacterium and
Methanospirillum increased with aro-
matic acids and PES.
• Toxicity inhibition on the AGS was 1-NA
> HA > PA > BA.

a r t i c l e i n f o a b s t r a c t

Article history: The influences of polyether sulfone (PES) microplastics and different structures aromatic carboxylic acids such as
Received 3 March 2021 benzoic acid (BA), phthalic acid (PA), hemimellitic acid (HA), and 1-naphthoic acid (1-NA) on the performances
Received in revised form 5 June 2021 and characteristics of anaerobic granular sludge as well as the microbial community were investigated. The
Accepted 6 June 2021
chemical oxygen demand (COD) removal efficiency was the highest in the experimental group with 40 mg/L
Available online 8 June 2021
BA, reaching 90.1%. The inhibitory effect of aromatic carboxylic acids addition on the 2,3,5-triphenyltetrazolium
Editor: Frederic Coulon chloride (TTC) activity was more obvious than that on 2-para (iodo-phenyl)-3(nitrophenyl)-5(phenyl) tetrazo-
lium chloride (INT) activity. Compared with the control group (only 0.5 g/L PES microplastics, 60.6 mg TF·g
TSS·h−1), the inhibition effect of TTC activity was 32.5 mg TF·g TSS·h−1 and 44.3 mg TF·g TSS·h−1 in the 40
Keywords: mg/L HA and 40 mg/L 1-NA experimental groups, respectively. When aromatic carboxylic acids were added,
Polyether sulfone the activities of acetate kinase and coenzyme F420 in the anaerobic granular sludge decreased. The excitation-
Aromatic carboxylic acids emission matrix (EEM) fluorescence spectra indicated that loosely bound extracellular polymeric substances
Anaerobic granular sludge (LB-EPS) began to decay. After the addition of different aromatic carboxylic acids, the C\\C and C\\H functional
Extracellular polymeric substances
groups of the anaerobic granular sludge increased, suggesting that aromatic carboxylic acids migrated to the sur-
Microbial community
face of anaerobic granular sludge, such a transfer would lead to changes in anaerobic granular sludge perfor-
mance. High-throughput sequencing technology showed that the dominant microbial communities in the
anaerobic granular sludge were Proteobacteria, Methanothrix, and Methanomicrobia. After the addition of aro-
matic carboxylic acids, the relative abundances of Proteobacteria, Methanobacterium, and Methanospirillum in-
creased. In the presence of PES, 1-NA had the most serious toxicity to the anaerobic granular sludge.
© 2021 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: University Key Laboratory of Karst Ecology and Environmental Change of Guangxi Province, Guangxi Normal University, 15 Yucai Road, Guilin 541004, PR
China.
E-mail address: suchengyuan2008@126.com (C. Su).

https://doi.org/10.1016/j.scitotenv.2021.148361
0048-9697/© 2021 Elsevier B.V. All rights reserved.
R. Qin, X. Lin, Z. Chen et al.
1. Introduction
Plastics are widely used in various fields, such as food packaging,
medical devices, personal care products, children's toys, industrial pro-
duction, etc. While plastic products bring convenience to people's
lives, they also cause considerable plastic pollution (Anderson et al.,
2016; Yu et al., 2020). Microplastics are defined as synthetic organic
polymers of plastics with a particle size less than 5 mm (Zhang and
Chen, 2020; Dodson et al., 2020; Zhang et al., 2020a; Frias and Nash,
2019). Compared with ordinary plastics, microplastics are more danger-
ous plastic pollutants, which are more harmful to the ecosystem
(Andrady, 2011; Lin et al., 2018; Kurniawan et al., 2021). Due to the
chemical stability and long residence time, plastic particles are fre-
quently deposited in sludge and other sediments, while a variety of ma-
rine organisms absorb microplastic particles and toxins through the
food chain, which results in impaired growth and development
(Claessens et al., 2011; Wang et al., 2018a; Oliviero et al., 2019; Pena
et al., 2016). Microplastics can occur in freshwater systems and are dis-
tributed in the water and sediments of rivers and lakes (Dafne et al.,
2015; McCormick et al., 2014; Triebskorn et al., 2019). In order to
strengthen the control of plastic pollution and establish a sound long-
term management mechanism for plastic products, the Ministry of
Ecology and Environment of China issued the “Opinions on Further
Strengthening the Control of Plastic Pollution”, the production of daily
chemical products containing plastic microbeads was prohibited.
Currently, wastewater treatment plants (WWTPs) are potential
sources of microplastics in the environment and are receiving increas-
ing attention (Mason et al., 2016; Carr et al., 2016). Wastewater contain-
ing microplastics is eventually transferred to WWTPs, and although
more than 90% of microplastics entering WWTPs are eventually re-
moved, most remain in the sludge (Lares et al., 2018). As a result, sludge
becomes source of microplastics in the environment, acting as a
microplastics carrier (Li et al., 2018a; Rolsky et al., 2020). Plastic parti-
cles are frequently deposited in sludge and other sediments (About
75.7% MPs, 36.3–46.3 particles MPs/g) accumulated in sludge phase
from a WWTP in China (Jiang et al., 2020). In Denmark, the estimated
annual microplastic load for all treatment plants was 4000 tons per
year, but determined at 3100 tons per year, incorporated into the sew-
age sludge (Vollertsen and Hansen, 2017). Therefore, the effects of
microplastics, aging or refractory organic additives on sludge remain
to be explored. In most plastic products, basic polymers are mixed
with chemical additives with different effects to improve their perfor-
mance (Hahladakis et al., 2018). These additives can leach from the
plastics into water, potentially harming aquatic ecosystems (Oliviero
et al., 2019; Jang et al., 2016; Liu et al., 2021). However, plasticizer, as
an additive, is not particularly stable in these plastic products and will
leach from the final product. The degradation of aging plastics and the re-
lease of toxic chemicals make the distribution of these substances in the
environment and the human body likely to lead to long-term environ-
mental and health problems (Hahladakis et al., 2018). As an important
source of plasticizers, phthalic acid esters do not covalently bond with
polymer chains, they are easy to release aromatic substances of phthalates
when aged and decomposed (Hu et al., 2020). In addition, polyethylene
terephthalate will be decomposed into non-biodegradable solid complex
aromatic substances such as benzoic acid and terephthalate during ther-
mal degradation (Sarker et al., 2011).
Anaerobic granular sludge is commonly used in wastewater treat-
ment. Anaerobic granular sludge-based anaerobic reactors, such as the
up-flow sludge bed reactor, anaerobic baffled reactor and anaerobic ex-
panded granular sludge bed reactor, have been widely used for high
concentration organic wastewater treatment (Zhang et al., 2020b). Nev-
ertheless, when treating wastewater containing microplastics, anaero-
bic granular sludge is negatively affected (such as reduced COD
removal efficiency, anaerobic digestion and microbial community).
Zhang et al. (2020b) found that 75–300 MPs/L of polyethylene tere-
phthalate reduced the chemical oxygen demand (COD) removal
2
Science of the Total Environment 792 (2021) 148361
efficiency of anaerobic granular sludge by 17.4–30.4%, and methane
yield of anaerobic granular sludge by 17.2–28.4%. At present, the focus
of attention is the research on the influence mechanism of different
types of microplastics on anaerobic granular sludge, polyvinyl chloride
(PVC) microplastics caused negative effects on anaerobic digestion
through leaching the toxic bisphenol A, and polyethylene terephthalate
(PET) microplastics carried on their negative influence mainly through
leaching the toxic di-n-butyl phthalate (DBP) (Wei et al., 2019a; Wei
et al., 2019b). Especially, the combined pollution of microplastics and
organic pollutants on anaerobic granular sludge has attracted people's
attention. In real WWTPs, the temperature of the wastewater, the sub-
strate and toxic substances in the influent, etc. could affect the activities
of key enzymes in anaerobic granular sludge. Lin et al. (2020) showed
that the addition of PES and bisphenol A affected protease, acetate ki-
nase, and coenzyme F420 activities in the anaerobic granular sludge.
Aromatic compounds are a kind of important organic pollutants and
the structural components of macromolecules in the environment
(Wu et al., 2018). The size and shape of aromatic compounds can also
affect their physical and chemical properties (Wu et al., 2018).
However, the effects of microplastics and different structures aromatic
carboxylic acids on the performance, characteristics and microbial com-
munities of anaerobic granular sludge have not been systematically
documented.
This study mainly explored the influencing mechanism of different
structures aromatic carboxylic acids on the anaerobic granular sludge
under PES microplastics exposure. Accordingly, this work investigated
the effects of PES microplastics and aromatic carboxylic acids on
(1) the relative activity changes of acetate kinase, coenzyme F420 and
electronic transfer system (ETS) in the anaerobic granular sludge;
(2) the functional groups and extracellular polymeric substances
(EPS) of the anaerobic granular sludge using three-dimensional
excitation-emission matrix (3-D EEM); and (3) the microbial commu-
nity and KEGG (Kyoto Encyclopedia of Genes and Genomes) function
prediction using high-throughput sequencing technology.
2. Materials and methods
2.1. Inoculating sludge and wastewater composition
The anaerobic granular sludge used in this batch experiment was ob-
tained from an anaerobic fermentation tank of the Guilin Liquan Brew-
ery, China. The suspended solid (SS value was 24 g/L and a volatile
suspended solid (VSS): SS ratio of the anaerobic granular sludge was
0.70. Polyether sulfone (PES) microplastic used in the exposure experi-
ment was purchased from China Jinyunlai Co., LTD. and had a particle
size of 0.25 mm. Benzoic acid (BA), phthalic acid (PA), hemimellitic
acid (HA), and 1-naphthoic acid (1-NA) were chosen as model contam-
inants, all of which were purchased from Shanghai Alighting Biochemical
Technology Co., LTD. (for detailed information, see Table S1). During the
exposure experiment, synthetic wastewater was configured as influent,
with the following composition: 3000 mg/L glucose, 200 mg/L NH4Cl,
44 mg/L KH2PO4; 1 mL/L of a trace element solution was added (Lin
et al., 2020). Meanwhile, the pH value of the synthetic wastewater was
adjusted between 7.0 and 7.5 with sodium bicarbonate.
2.2. Experimental equipment and operation
For the sequential batch test, five 250-mL conical bottles were used.
The amount of anaerobic granular sludge in each conical flask was 100
mL, and daily, 150 mL of synthetic wastewater were added. The bottles
were labeled as follows: A-0.5 g/L PES; B-0.5 g/L PES and 40 mg/L BA; C-
0.5 g/L PES and 40 mg/L PA; D-0.5 g/L PES and 40 mg/L HA; E-0.5 g/L PES
and 40 mg/L 1-NA (Wu et al., 2018; Ma et al., 2019). During the expo-
sure experiment, the conical bottles were flushed with nitrogen for 10
min, sealed, and were placed in a water bath oscillator for micro-
oscillation at a temperature of 30–35 °C. The entire sequential batch
R. Qin, X. Lin, Z. Chen et al.
experiment lasted for 40 days, and the synthetic wastewater as the in-
fluent was changed daily. The influent and effluent COD concentrations
were also determined daily. To analyze the activity of anaerobic granu-
lar sludge, INT-ETS and TTC-ETS were measured on days 20, 30, and 40,
respectively. In addition, the activity of acetate kinase and coenzyme
F420 were analyzed on days 0, 20, and 40. At the end of the experimental
period, the structure of the anaerobic granular sludge was analyzed by
X-ray photoelectron spectroscopy (XPS), and the EPS component of
the sludge was determined by using EEM spectra. In addition, the mi-
crobial communities of the different experimental groups were ana-
lyzed via high-throughput sequencing technology.
To determine the components of pollutions leached from PES
microplastics, PES microplastics were respectively added in the six
serum bottles with the mixture of the anaerobic granular and different
aromatic carboxylic acids each. The details were as follows: deionized
water+0.5 g/L PES (a1), deionized water+0.5 g/L PES + anaerobic
granular sludge (a2), deionized water+0.5 g/L PES + anaerobic granu-
lar sludge+BA (b), deionized water+0.5 g/L PES + anaerobic granular
sludge+PA (c), deionized water+0.5 g/L PES + anaerobic granular
sludge+HA (d), and deionized water+0.5 g/L PES + anaerobic granular
sludge+1-NA (e). Serum bottles received 100 mL of the anaerobic gran-
ular sludge and 150 mL of deionized water. Each bottle was sealed by a
rubber stopper and placed in a water bath thermostatic shaker for
experiment (Equipment model: SHA-CA, Changzhou Putian Instru-
ment Manufacturing Co., Ltd., Jiangsu Province, China; Oscillation
frequency: 120 r/min, Operating temperature:30–35 °C). At day 5,
the components of waste in the serum bottles were measured by
LC-MS.
2.3. Analytical methods
The COD concentration was measured according to the standard
method (APHA et al., 2012). The evaluation of the ETS activity in the an-
aerobic granular sludge was based on the measurement of 2-para (iodo-
phenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) and
2,3,5-triphenyltetrazolium chloride (TTC) (Feng et al., 2014). The activ-
ities of acetate kinase (AK) and coenzyme F420 in the anaerobic granular
sludge were determined by potassium acetate spectrophotometry and
UV–vis spectrophotometry, respectively (Yang et al., 2019; Zhang
et al., 2020c; Zhao et al., 2021).
The EPS compositions in the anaerobic granular sludge of different
experimental groups were investigated by EEM fluorescence spectrom-
etry (F-7000, Hitachi, Japan). The excitation wavelength (Ex) increased
with a range from 220 to 500 nm, and the emission wavelength (Em)
increased with a range from 220 to 550 nm. The excitation and emis-
sion slits were maintained at 5 nm, the scanning speed was set at
2400 nm/min, and the photomultiplier tube (PMT) voltage was
400 V. The XPS analysis of the anaerobic granular sludge was con-
ducted with the ESCALAB250 model multi-functional surface analy-
sis system produced by Thermo VG, and the sludge was analyzed
with high-resolution monochrome AlK radiation (h = 1486.6 eV).
Step size was 0.1 eV, and the energy recording spectrum was
50.0 eV; data analysis was conducted with the XPS data processing
software Avantage (Cañas et al., 2020).
2.4. Liquid chromatography combined with mass spectrometry (LC-MS)
In order to analyze and detect the medium in the supernatant, the
sample suspension in the six serum bottles was collected, filtered by a
0.22 μm filter membrane and sealed in a glass bottle. The Agilent
6545Q-TOF LC/MS (Agilent Technologies, America) was used for detec-
tion, Agilent ZORBAX Eclipse Plus C18 column was used. The mobile
phases were distilled water and methanol. The flow rate was 0.300
mL/min, the column temperature was 35 °C, and the injection volume
was 1.00 μL. Electrospray ion source (ESI), positive ion scanning and
3
Science of the Total Environment 792 (2021) 148361
multi-reaction monitoring mode were used for mass spectrometry
detection.
2.5. Microbial community analysis
On day 40, anaerobic granular sludge was collected from five reac-
tion units, and genomic DNA was extracted from sludge samples
(Chen and Chang, 2017) to determine DNA concentration and purity.
High-throughput sequencing of 16S rRNA and gene amplification in
the targeted region were performed by Sangon Biotech (Shanghai)
Co., Ltd. The sequencing platform was Miseq 2 × 300 bp. The V3 and
V4 hypervariable regions of the 16S rRNA gene were amplified by PCR,
using the universal bacterial primers 341F and 805R. In addition, PCR
amplification targeting archaea was performed with the primers 349F
and 806R to analyze the horizontal distribution of bacteria and archaea
(Ma et al., 2020). The KEGG (Kyoto Encyclopedia of Genes and
Genomes) function prediction of the PICRUSt gene was also performed
(Xu et al., 2019).
3. Results and discussion
3.1. Influence of PES and aromatic carboxylic acids on COD removal
Based on Fig. 1, when 0.5 g/L PES microplastics were used as control
group, the average COD removal rate was 90.0% during the 40-day ex-
perimental period. After adding 40 mg/L BA, PA, HA, and 1-NA, the aver-
age COD removal efficiencies were 90.1, 89.9, 87.7, and 84.6%,
respectively. It can be seen from the figure that the two experimental
groups with 40 mg/L HA and 40 mg/L 1-NA showed COD removal inhi-
bition. The type of influent pollutants, the concentration of anaerobic
granular sludge and the activity of microorganisms may affect the
COD removal efficiency of anaerobic biological treatment technology.
The more hydrogen on the benzene ring was replaced by a carboxyl
functional group, the lower the COD removal rate by the anaerobic gran-
ular sludge. Wu et al. (2018) showed that the adsorption affinity of ar-
omatic carboxylic acids decreased with the increase of the number of
aromatic rings, following the order BA > NA. In addition, -COOH substi-
tution can weaken the adsorption affinity, resulting in the order BA > PA
> HA, which was consistent with the COD removal effect. More interest-
ingly, 1-NA has higher octanol-water distribution coefficient and lower
water solubility than BA, so the presence of 1-NA is more likely to inter-
fere with the removal of COD by microorganisms in anaerobic sludge
than BA. Furthermore, the more carboxyl substituted aromatic com-
pounds, the weaker their conjugated ability with benzene ring, so
they were more likely to produce free radical intermediates or anhy-
dride, which would cause certain inhibition to the removal of COD
(Artok and Schobert, 2000).
3.2. Influences of PES and aromatic carboxylic acids on the activity ETS
Sludge activity reflects the ability of microorganisms to degrade sub-
strates, making it an effective index to test the performance of the
sludge system. The electronic transfer system of sludge is one of the
most widely used sludge activity detection and evaluation methods
and it is also an efficient indicator of the impact of environmental con-
ditions on sludge microorganisms (Feng et al., 2014). In this sense, it
is feasible to characterize the biological activity of sludge via the sludge
electronic transfer system (ETS) activity (Wang et al., 2013). Since the
microbial electronic transport system (ETS) is associated with respira-
tory activity, the biological activity of the sludge, including TTC-ETS
and INT-ETS activity, can be indicated by the electron transport rate of
the microbial respiratory chain (Wang et al., 2013). In the experimental
study, at day 0, the activities of TTC-ETS and INT-ETS were 10.14 mg
TF·g TSS·h−1 and 16.04 mg TF·g TSS·h−1, respectively. Based on the
TTC-ETS activity analysis (Fig. 2), compared with the control group
with 0.5 g/L PES, the TTC-ETS activity in each group was generally
R. Qin, X. Lin, Z. Chen et al. Science of the Total Environment 792 (2021) 148361

0.5g/L PES & 0.5g/L PES & 0.5g/L PES & 40mg/L 0.5g/L PES &40mg/L
0.5g/L PES 40mg/L Benzoic acid 40mg/L Phthalic acid Hemimellitic acid 1-Naphthoic acid
3500
110
Influent Effluent Removal efficiency
3000 100

COD concentrations (mg/L)

90

Removal efficiency (%)

2500
80

70
2000
60

1500 50

40
1000
30

20
500
10

0 0
0 20 40 0 20 40 0 20 40 0 20 40 0 20 40
Time (d)

Fig. 1. Effect of PES and aromatic carboxylic acids on the COD removal efficiency.

inhibited, indicating that the addition of aromatic carboxylic acids lim- amplitude reduction rate reached 57.7%, indicating that in groups D
ited the TTC-ETS activity and reduced the ETS activity in the sludge. In and E, the electronic transfer system of the sludge was significantly
groups D and E, the TTC-ETS activity values of sludge were 28.1 mg inhibited, which resulted in an impeded electron transfer rate and, con-
TF·g TSS·h−1 and 16.3 mg TF·g TSS·h−1, respectively, at day 40. In ad- sequently, in lower microbial growth and respiration; the most pro-
dition, the TTC-ETS activity of sludge in groups A, B, C, and D all in- nounced effect was observed for the group E.
creased over time, while the group E showed a decreasing trend. It Overall, aromatic carboxylic acids addition had a more significant in-
was indicated that the group E could not combine with electrons better hibitory effect on the TTC-ETS activity than on the INT-ETS activity,
due to the complex addition of 1-NA functional group and its weak abil- most likely because the TTC-ETS has a higher redox potential (+490
ity to compete with oxygen for electrons. Possibly, the adsorption affin- mV), a lower electron affinity, and a less electron competition ability
ity decreased from BA to 1-NA, and π electron delocalization into an with oxygen. In contrast, the INT-ETS redox potential is lower (+90
additional ring led to a decrease in the electron deficiency of the 1-NA mV) and electron affinity and oxygen competition are stronger. To receive
aromatic ring and in the specific interaction (Wu et al., 2018). However, electrons, TTC-ETS must undergo a complete transfer process in the mi-
aromatic compounds with a single ring, such as BA, lack electrons more crobial oxidative respiratory chain (Yin et al., 2005). Interference with
easily and tend to combine with electrons in the microbial respiration any part of the electron transfer process will affect the TTC-ETS activity,
chain. In Fig. 2, compared with the control group A, the activity of INT- making it more vulnerable to inhibition. However, in previous studies,
ETS in groups B and C showed the same growth trend as that in the con- the activity of INT-ETS was only inhibited when the pre-cytochrome B
trol group, and the activity of INT-ETS in the group B was higher than transfer process was blocked (Cheng et al., 2011; Baños et al., 2020;
that in the control group A, reaching 27.3 mg TF·g TSS·h−1 at day 40. Nachlas et al., 1960). In addition, it was reported that high organic content
However, groups D and E showed a decreasing trend, which had the will cause the INT-ETS activity value in the system to be much lower than
highest downward trend for the group E. At day 40, the activity of the TTC-ETS activity value (Cheng et al., 2011), which was consistent with
INT-ETS in the group E was only 6.9 mg TF·g TSS·h−1, and the the results in Fig. 2, indicating that the activity of TTC-ETS was more easily
affected by toxins than that of INT-ETS. Therefore, compared with INT-ETS
analysis, TTC-ETS analysis may more effectively reflect the inhibitory ef-
A (TTC-ETS activity)
B (TTC-ETS activity) 70
fect of aromatic carboxylic acids migrated out of microplastics on sludge
70
C (TTC-ETS activity) activity, and with a higher amount of hydrogen on the aromatic benzene
TTC-ETS activity (mg TF·(g TSS·h)-1)

INT-ETS activity (mg TF·(g TSS·h)-1)

D (TTC-ETS activity)
E (TTC-ETS activity) 60 ring of aromatic carboxylic acids replaced by carboxylic acids, its inhibi-
60 A (INT-ETS activity)
B (INT-ETS activity) tory effect on sludge is stronger, consistent with the results of previous
C
D
(INT-ETS activity)
(INT-ETS activity) 50 studies on PES and bisphenol A (Lin et al., 2020).
50
E (INT-ETS activity)

40 3.3. Influences of PES and aromatic carboxylic acids on the enzyme activity
40

30 Anaerobic reaction is a complex biological process controlled by a

30
variety of enzymes. Anaerobic digestion of sludge mainly involves
20 three steps: hydrolysis, acidification and methanation of sludge.
20
Fig. S1 showed metabolic pathways related to methane production
10
from anaerobic degradation (Wei et al., 2019a). AK and coenzyme F420
10
are two key enzymes involved in the formation of anaerobic methane.
0
While the first is essential for converting acetyl-CoA into acetic acid dur-
20 30 40
ing the acidification step, the latter can be used for methanation during
Time (d)
anaerobic digestion. To further explore the potential influences of aro-
Fig. 2. Effect of PES and aromatic carboxylic acids on the activity of ETS (A: adding 0.5 g/L
matic carboxylic acids on the anaerobic granular sludge during this pro-
PES; B: adding 0.5 g/L PES and 40 mg/L BA; C: adding 0.5 g/L PES and 40 mg/L PA; D: cess, the activity of key enzymes in different experimental groups was
adding 0.5 g/L PES and 40 mg/L HA; E: adding 0.5 g/L PES and 40 mg/L 1-NA). evaluated. In this study, the enzyme activity of the control group on

4
R. Qin, X. Lin, Z. Chen et al.
day 0 was set to 100%, and the enzyme activity results of the experimen-
tal group adding microplastics and aromatic carboxylic acids with dif-
ferent structures in different culture days were expressed as relative
values of the control group for analysis. Based on the changes in the rel-
ative activities of AK and coenzyme F420 in the anaerobic granular
sludge with the addition of different types of aromatic carboxylic
acids, the relative activities of AK and coenzyme F420 continued to de-
cline, and their activities were inhibited (Fig. 3). At day 40, the relative
activity of AK had decreased to 83.9, 63.6, 55.5, 46.5, and 44.0%, respec-
tively, while that of coenzyme F420 had decreased to 81.7, 71.3, 61.9,
44.4, and 33.7%, respectively. During anaerobic acidification and meth-
ane production, AK is indispensable in the conversion of acetyl-coA to
acetic acid, and inhibition of this process leads to a decrease in acetic
acid production. Acetic acid is the most important intermediate com-
pound of organic anaerobic degradation under methane-producing
conditions, and coenzyme F420 also plays a key role in this process. In
this sense, the addition of aromatic carboxylic acids can inhibit sludge
anaerobic acidification and methanogenesis. This finding was consistent
with the inhibitory effect of additives released by microplastics on an-
aerobic digestion, reported by Zhang et al. (2020d) and adding higher
levels of PE Microplastics (i.e., 100 and 200 Particles/g Ts), reported by
Wei et al. (2019c). In addition, the addition of 40 mg/L 1-NA to group
E had the greatest effect on the key enzyme activity of anaerobic sludge
digestion, followed by 40 mg/L HA (three carboxyl groups), 40 mg/L PA
(two carboxyl groups), and 40 mg/L BA (one carboxyl group), contain-
ing a benzene ring structure. This leads us to infer that any changes in
the number of benzene rings and carboxyl groups in aromatic carbox-
ylic acids affect the anaerobic digestion of sludge. The larger the number
of benzene rings, the more obvious the influence, and with a higher
number of carboxyl functional groups, this influence also gradually in-
creased. It has been reported that microplastics can induce the produc-
tion of reactive oxygen species (ROS), thus causing the loss of cell
vitality (Wei et al., 2019c).With the increase of culture days, the activity
of acetic acid kinase and coenzyme F420 decreased, so it can be inferred
that microplastics-induced ROS may be one of the key factors to inhibit
the performance of anaerobic digestion. Moreover, even in the anaero-
bic system, through respiration, there are trace concentrations of oxy-
gen in the reactor, PES microplastic after adding aromatic carboxylic
acids, make the surface more electron donors on the active site interact
with molecular oxygen (O2) can lead to electronic was captured by the
O2 (Degli Esposti and McLennan, 1998), which may lead to generate
more ROS, it further explains the microbial enzyme activity continues
to decline after adding aromatic carboxylic acids. In subsequent EPS
studies, although EPS has a protective effect, the attachment of
microplastics and their aromatic carboxylic acid additives to cell surface
Fig. 3. Effect of PES and aromatic carboxylic acids on the relative activity changes of acetate kinase (a) and coenzyme F420 (b) in anaerobic granular sludge (A: adding 0.5 g/L PES; B: adding
0.5 g/L PES and 40 mg/L BA; C: adding 0.5 g/L PES and 40 mg/L PA; D: adding 0.5 g/L PES and 40 mg/L HA; E: adding 0.5 g/L PES and 40 mg/L 1-NA).
5
Science of the Total Environment 792 (2021) 148361
biomolecules increases the chance of contact with key enzymes, which
may further reduce enzyme activity and inactivation, reducing the pro-
ductivity and potential of methane.
3.4. Influences of PES and aromatic carboxylic acids on the EPS
Extracellular polymeric substances (EPS) are composed of microbe
secretions, polymers generated by cell lysis, and organic compounds
adsorbed from wastewater (Zhang and Chen, 2020). These substances
contribute to biofilm stability, enabling it to withstand greater shear
force, and provide a thicker and more complete protective layer for mi-
croorganisms, making them more resistant to adverse environmental
conditions. EPS bind to cells through complex interactions, forming a
huge network structure which protects the cells against dehydration
and toxic substances. According to the degree of binding to the cell sur-
face, EPS can be divided into TB-EPS, with the inner layer tightly bound
to the cell surface, and LB-EPS, with the outer layer loosely bound
(Sheng et al., 2010). EPS contain an aromatic structure with fluores-
cence characteristics, providing important information about the com-
position and functional groups of sludge EPS. As shown in Fig. 4, the
EEM spectra of EPS extracted from the anaerobic granular sludge sam-
ples showed three distinct characteristic peaks of LB-EPS, among
which the two peaks located at Ex/Em = 225–237 nm/320–360 nm
and Ex/Em = 275–290 nm/335–370 nm were fluorescence peaks of ar-
omatic proteins, which could be identified as tryptophan-like aro-
matic amino acids (Zhang et al., 2017). The third fluorescence peak
was Ex/Em = 408–430 nm/465–495 nm and belonged to coenzyme
F 420 . In addition, when D and E were added to the experimental
group, a fluorescence peak at Ex/Em = 260 nm/470 nm appeared,
belonging to the fulvic-like acid group. This may be because LB-
EPS, as a loose mucus layer, is more likely to absorb organic pollut-
ants. Moreover, it shows that HA and 1-NA have stronger biological
toxicity. As additives, they may be discharged into the organism
from the matrix of MPs and cause tissue erosion. These substances
may have endocrine disrupting effects, and even very low levels
will interfere (Hamidian et al., 2021). Therefore, in order to protect
microorganisms from being exposed to higher levels of these
potentially harmful substances, and even aggravate their toxicity,
the microorganisms' own stress response will cause them to secrete
more EPS substances to protect themselves. In Fig. 4, there is an
obvious fluorescence peak in TB-EPS, with a value of Ex/Em =
290–320 nm/360–395 nm, indicating humic acid. When the sub-
stances of BA and PA were added to the anaerobic granular sludge,
a fluorescence peak at Ex/Em = 415 nm/485 nm also appeared,
indicating humic acid (Zhang et al., 2017). When HA and 1-NA
R. Qin, X. Lin, Z. Chen et al. Science of the Total Environment 792 (2021) 148361

500 500 500

0.000 0.000 0.000
LB-EPS ( A ) LB-EPS ( B ) 17.19
LB-EPS ( C ) 16.88
29.06
450 450 450
34.38 33.75
58.13
51.56 50.63
77.50
400 400 68.75 400 67.50
96.88
85.94 84.38

Ex/nm
116.3

Ex/nm

Ex/nm
103.1 101.3
350 135.6 350 350
120.3 118.1

155.0 137.5 135.0

300 300 300

250 250 250

250 300 350 400 450 500 550 250 300 350 400 450 500 550 250 300 350 400 450 500 550

Em/nm Em/nm En/nm

500 500
0.000
LB-EPS ( D ) 0.000
LB-EPS ( E ) 15.25
25.50
450 450
30.50
51.00
45.75
76.50
400 61.00
400 102.0
76.25
127.5

Ex/nm
Ex/nm

91.50
153.0
350 350
106.8
178.5
122.0
204.0

300 300

250
250

250 300 350 400 450 500 550

250 300 350 400 450 500 550

Em/nm Em/nm

500 500
500
0.000
TB-EPS ( A ) 0.000
TB-EPS ( B ) 38.25
TB-EPS ( C ) 0.000

44.13
56.44
450 450
450 112.9 76.50 88.25

150.5 114.8 132.4

225.8 153.0
400 400 400 176.5
301.0 191.3 220.6
376.3
Ex/nm

Ex/nm
Ex/nm

229.5 264.8
451.5 350 350
350 267.8 308.9
526.8
306.0 353.0
602.0

300 300 300

250 250 250

250 300 350 400 450 500 550 250 300 350 400 450 500 550 250 300 350 400 450 500 550

Em/nm Em/nm Em/nm

500 500
0.000 0.000
TB-EPS ( D ) 38.50
TB-EPS ( E ) 39.38
450 450
77.00 78.75

115.5 118.1

400 154.0 400 157.5

192.5 196.9
Ex/nm

Ex/nm

231.0 236.3
350 350
269.5 275.6

308.0 315.0

300 300

250 250

250 300 350 400 450 500 550 250 300 350 400 450 500 550

Em/nm Em/nm

Fig. 4. EEM spectra of LB-EPS and TB-EPS of anaerobic granular sludge under the influence of PES and aromatic carboxylic acids (A: adding 0.5 g/L PES; B: adding 0.5 g/L PES and 40 mg/L BA;
C: adding 0.5 g/L PES and 40 mg/L PA; D: adding 0.5 g/L PES and 40 mg/L HA; E: adding 0.5 g/L PES and 40 mg/L 1-NA).

substances were added, there was a fluorescence peak at Ex/Em = to carboxyl groups in aromatic groups, thus reducing the damage
230 nm/315 nm, indicating an aromatic tyrosine-like protein. TB- to sludge.
EPS was closely bound to the cell surface, and the increasing
secretion of substances was more conducive to the protection of 3.5. XPS analysis of anaerobic granular sludge
cells. Based on the EEM spectra of LB-EPS and TB-EPS, with the addi-
tion of different kinds of aromatic carboxylic acids, the outer layer of By analyzing the XPS spectra, the surface structure characteristics of
LB-EPS started to decay. The impact of environmental change on LB- anaerobic granular sludge in presence of different aromatic carboxylic
EPS was greater than that on TB-EPS (Tian et al., 2019). LB-EPS be- acids were investigated. As indicated by the C1s spectra in Fig. 5, XPS
longs to the loose outer layer of EPS, and organic pollutants such as analysis, C1s all showed the same type of functional groups in the
microplastics and aromatic carboxylic acids would first contact with LB- sludge, but the percentage of atomic content changed slightly. The
EPS. Therefore, the addition of aromatic carboxylic acids with different main functional groups in anaerobic granular sludge were C-C/C-H, C-
structures was more likely to bind to the binding sites of the outer biofilm O-H, and C_O. Compared with the control group A, with the addition
and attach to the surface of LB-EPS, leading to change of its composition. of different types of aromatic carboxylic acids, the C-C/C-H atom per-
When BA and PA were added, the inner layer of TB-EPS started to centage increased to some extent, while for groups B, C, D, and E, the
decay, possibly due to the addition of aromatic compounds. Overall, atom percentage increased by 15.8, 19.2, 0.07, and 17.3%, respectively.
HA and 1-NA were most toxic to anaerobic granular sludge microor- This suggests that with the addition of aromatic carboxylic acids to
ganisms. This was mainly related to the carboxylic acid functional PES, microplastic chain fracture may occur on the surface of the polymer
groups and the number of benzene rings in the added aromatic car- material, along with the formation of unsaturated bonds, resulting in
boxylic acids. Previous studies have shown that EPS easily bind to or- the migration of aromatic carboxylic acids to the microplastic surface
ganic pollutants, which is mainly attributed to the presence of (Zhang et al., 2013). Such a transfer would lead to changes in anaerobic
hydrophobic areas, functional groups, and binding sites (Shi et al., granular sludge performance. In addition, compared with the control
2017). The amino functional groups of amino acids in proteins bind group, the atomic content percentages of functional groups C-O-H

6
R. Qin, X. Lin, Z. Chen et al. Science of the Total Environment 792 (2021) 148361

(A) C1s (A) O1s

40000 80000
C-O-H
C-C/C-H (90.09%)
(62.91%)

Intensity(a.u.)
60000

Intensity(a.u.)
30000

C-O-H 40000
20000
(20.57%)
O=C-OH
C=O (1.85%) C=O
(16.52%) (8.06%)
10000 20000

0
35000 800000

(B) C1s (B) O1s

70000
30000 C-C/C-H
C-O-H
(78.73%)
(92.87%)
60000
25000

Intensity(a.u.)
Intensity(a.u.)

50000

20000

40000

15000 C-O-H
(8.59%) 30000

O=C-OH C=O
10000 C=O 20000 (1.90%) (5.23%)
(12.68%)

5000 10000

40000
0 0

(C) C1s 70000

(C) O1s
35000

60000 C-O-H
C-C/C-H
30000 (82.08%) (89.62%)

Intensity(a.u.)
50000
Intensity(a.u.)

25000

40000
20000

C-O-H 30000
15000 (6.42%)
C=O
20000 (10.38%)
10000 C=O
(11.50%)
10000
5000

0 60000
0

35000 (D) C1s (D) O1s

50000
C-C/C-H
30000 C-O-H
(62.98%)
(89.01%)
Intensity(a.u.)

25000 40000
Intensity(a.u.)

20000
30000
C-O-H
(21.02%)
15000
20000 O=C-OH
(1.47%) C=O
10000 C=O (9.52%)
(16.00%)
10000
5000

140000
100000

(E) C1s (E) O1s

12000
C-O-H
8000
(91.72%)
10000
Intensity(a.u.)

C-C/C-H
Intensity(a.u.)

(80.20%)
6000 8000

6000
4000 C-O-H
(7.58%)
4000 O=C-OH
C=O (1.63%) C=O
(12.22%) (6.65%)
2000
2000

0
0
292 290 288 286 284 282 538 536 534 532 530 528

Binding Energy (eV) Binding Energy (eV)

Fig. 5. XPS analysis of anaerobic sludge under the influence of PES and aromatic carboxylic acids (A: adding 0.5 g/L PES; B: adding 0.5 g/L PES and 40 mg/L BA; C: adding 0.5 g/L PES and 40
mg/L PA; D: adding 0.5 g/L PES and 40 mg/L HA; E: adding 0.5 g/L PES and 40 mg/L 1-NA).

and C_O showed a decreasing trend, indicating that chemical reac- the migration and leaching into the environment, the second was
tions such as substitution occurred after the addition of aromatic car- to add biphenyl formate the group D, the last was the 1-NA group
boxylic acids, which destroyed the chemical structure of aromatic E, due to its fused ring structure was more complex and leaching
carboxylic acids. The functional group C-O-H had the largest content speed was relatively slow.
in O 1s XPS spectra. Based on the atomic content composition of C-O-
H and O=C-OH functional groups, the atomic content percentage of 3.6. LC-MS analysis under the influence of PES and aromatic carboxylic acids
the group B increased by 2.78% and 0.05%, respectively, further indi-
cating that benzoic acid was the most stable aromatic carboxylic acid The components of pollutants leached from PES microplastics were
(Dunn et al., 2003). The disappear of O=C-OH in the group C sug- analyzed by LC-MS (Fig. 6). Polyether sulfone resin with the relative mo-
gested that PA was added to the anaerobic granular sludge, the lecular weight of 232 monomers was precipitated after reaction. In addi-
chemical structure was damaged and were more likely to happen tion, the coordination bonds between sulfur and oxygen in the sulfone

7
R. Qin, X. Lin, Z. Chen et al. Science of the Total Environment 792 (2021) 148361

Fig. 6. Mass spectrograms of effects with PES and aromatic carboxylic acids on anaerobic granular sludge (a1: distilled water + 0.5 g/L PES; a2: anaerobic sludge + 0.5 g/L PES + distilled
water; b: anaerobic sludge + 0.5 g/L PES + distilled water + 40 mg/L BA; c: anaerobic sludge + 0.5 g/L PES + distilled water + 40 mg/L PA; d: anaerobic sludge+0.5 g/L PES + distilled
water + 40 mg/L HA; e: anaerobic sludge + 0.5 g/L PES + distilled water + 40 mg/L 1-NA).

group were broken and two oxygen atoms were lost, resulting in a mate-
rial with a mass-charge ratio (m/z) of 200 (Athira et al., 2020). In addition,
the molecular ion peak of S-CH2-CH2-CH=CH-CH2-CH2-O substance
existed at the m/z 130.15, which may be the substance formed after PES
further fractured benzene ring rearrangement. It could be found from
the mass spectrometry (b) that a [M + H]+ peak was formed at the m/z
125, which was formed when molecular ions captured a H in the ion
source. Hence, M may be the substance formed after PES sulfone group
fractured and benzene ring lost. Meanwhile, BA were not detected in
the (b) mass spectrometry, indicating that BA had been degraded. The

8
R. Qin, X. Lin, Z. Chen et al. Science of the Total Environment 792 (2021) 148361

substance with m/z 158 was found in the (c) mass spectrogram, which
was speculated to be the aromatic ring in PA that was broken and the car-
boxyl group was reduced to C8H14O3. After the aromatic ring was broken,
its molecular ion stability became lower, so it was more easily degraded.
In addition, it could be found from the (e) mass spectrometry that there
was a [M + H]+ peak with m/z 173, and the M substance was 1-NA,
which indicated that 1-NA was still in the anaerobic reaction device and
had not been completely degraded, so it will cause toxic effect on the mi-
croorganisms of anaerobic granular sludge. It was illustrated that PES
microplastics leaching of pollutants under the condition of the coexis-
tence of different structure aromatic carboxylic acids would affect the
characteristics of anaerobic granular sludge, and thus affect the methana-
tion process to a certain content.
3.7. Microbial community structure as influenced by PES and aromatic
carboxylic acids
3.7.1. Bacterial community structure and function
Fig. 7a showed the influence of aromatic carboxylic acids on the mi-
crobial community structure at the phylum level. The dominant bacte-
rial groups at the phylum level were Proteobacteria, Actinobacteria,
Firmicutes, Chloroflexi, and Bacteroidetes; they play an important role
in anaerobic digestion (Liu et al., 2016; Venkiteshwaran et al., 2017;
Yang et al., 2016). Proteobacteria were most abundant, with relative
abundances of 20.41, 47.17, 43.28, 78.46, and 24.53% in groups A, B, C,
D, and E, respectively. Compared with the control group A, the relative
abundance of Proteobacteria increased after the addition of aromatic
carboxylic acids, especially in the group D, with an increase of 58.05%;
this indicated that the addition of aromatic carboxylic acids stimulated
the growth of Proteobacteria, which may degrade target pollutants
(Ma et al., 2020). Firmicutes and Chloroflexi are resistant to high organic
loads and degrade numerous organic compounds, indicating that their
presence is more conducive to the degradation of aromatic carboxylic
acids and other toxic chemicals leached from microplastics (Xu et al.,
2018). Previous studies have also shown that Firmicutes can degrade or-
ganic acids and provide short-chain fatty acids or hydrogen for end-
degradants such as hydrotrophic and acetic acid-trophic methanogens
(Wang et al., 2017; Zhen et al., 2021).
Fig. 7b shows the relative abundances of KEGG functional categories
after the addition of different aromatic carboxylic acids. Among all
functional categories, five functional types stand out: Metabolic
Metabolism (49.09%), Genetic Information Processing (15.33%),

80
(a) A
70
B
C
Relative abundance (%)

60

50
D
E
40

30

20

10

cte
ria ria cutes fie
d exi detes tes aeota ria cetes
cte i ssi ofl i iste h cte
my
teoba inoba Firm ncla Chlor ctero nerg ryarc ariba
cto
t u Ba Sy h
Pro Ac Eu Sacc P lan
tus
ida
a nd
C

(b) Amino Acid Metabolism

Biosynthesis of Other Secondary Metabolites
Carbohydrate Metabolism
A
Energy Metabolism B
Enzyme Families C
Glycan Biosynthesis and Metabolism
Lipid Metabolism
D
Metabolism
Metabolism of Cofactors and Vitamins E
Metabolism of Other Amino Acids
Metabolism of Terpenoids and Polyketides
Nucleotide Metabolism
Xenobiotics Biodegradation and Metabolism
Genetic Folding, Sorting and Degradation
Information Replication and Repair
Processing Transcription
Translation
Environmental Membrane Transport
Information Signal Transduction
0.18

0.16 0.15
Processing
Relative abundance (%)

Signaling Molecules and Interaction 0.14

0.12 0.11
Cell Growth and Death 0.10 0.09
Cellular 0.08 0.07
0.06
Cell Motility 0.06

Processes Transport and Catabolism

0.04

0.02

Endocrine System 0.00

A B C D E
Organismal Environmental Adaptation Endocrine System
Systems
0 0.5 1 1.5 2 2.5 3 3.5 4
Relative abundance (%)

Fig. 7. Microbial community distribution and KEGG function prediction of PES and aromatic carboxylic acids in bacteria (a: Community distribution at the phylum level; b: Predicting
function of KEGG) (A: adding 0.5 g/L PES; B: adding 0.5 g/L PES and 40 mg/L BA; C: adding 0.5 g/L PES and 40 mg/L PA; D: adding 0.5 g/L PES and 40 mg/L HA; E: adding 0.5 g/L PES
and 40 mg/L 1-NA).

9
R. Qin, X. Lin, Z. Chen et al. Science of the Total Environment 792 (2021) 148361

Environmental Information Processing (15.99%), Cellular Processes
(4.47%), and Organismal Systems (0.61%). Amino Acid Metabolism
and Carbohydrate Metabolism were dominant in the Metabolism. Com-
pared with the control group A, the relative abundance of metabolism in
each group increased after the addition of different types of aromatic
carboxylic acids, indicating stimulated growth. Based on Genetic Infor-
mation Processing, Replication and Repair was the main way, the rela-
tive abundances were 1.14, 1.42, 1.34, 1.57, and 1.12%. Compared with
the control group, the abundance of Replication and Repair in groups
B, C, and D were increased, while its relative abundance in the group E
reduced. It was showed that adding 40 mg/L 1-NA was toxic to cells
and inhibit the Replication of Genetic Information. In Environmental
Information Processing, membrane transport occupied a large pro-
portion, with relative abundances of 1.94, 2.75, 2.71, 3.98, and
2.00%, respectively. Membrane transport was clearly promoted
with the addition of aromatic carboxylic acids. In the organic
systems, the Endocrine System was the main functional category,
with relative abundances of 0.07, 0.11, 0.09, 0.15, and 0.06%, respec-
tively. The addition of monocyclic aromatic carboxylic acids pro-
moted the endocrine activity of microorganisms, while the addition
of 1-NA (containing two benzene rings) inhibited the endocrine sys-
tem of microorganisms, which is in agreement with a previous study
(Wang et al., 2018b).
3.7.2. Archaeal community structure and function
Based on Fig. 8a, the main archaea were Methanothrix,
Methanobacterium, and Methanospirillum. The relative abundance of
Methanothrix was 76.25, 75.29, 61.01, 46.99, and 64.3%, respectively, in
the five groups. Methanothrix is an acetic acid methanogen and can
convert acetic acid into CH4 and CO2 (Li et al., 2018b; Zhang and Chen,
2020). Compared with control group A, the relative abundance of
Methanothrix was inhibited in groups B, C, D, and E after aromatic
carboxylic acids addition, indicating that the conversion of acetic acid
to CH4 was inhibited after the addition of the aromatic carboxylic
acids. Methanobacterium belongs to the order Methanobacteriales,
while Methanospirillum belongs to the order Methanomicrobiales; all
are hydrogen methanogens, using H2 and CO2 as substrate (Maus
et al., 2013). The relative abundances of Methanobacterium and
Methanospirillum were 21.50, 21.26, 35.43, 49.40, and 32.87%, respec-
tively. In addition to the group B, 40 mg/L BA was added to the changes
in the relative abundance of the flora was not obvious, the other group
increased significantly. From aromatic carboxylic acids added chemical
structured analysis, the easier it was replaced by dehydrogenation reac-
tion, the more obvious of its relative abundance increase. After dehydro-
genation to generate hydrogen ions, more conducive to hydrogen ions
used CO2 to produce methane. This indicated that methane production
process based on acetic acid decreased, which was consistent with the

(a)
100

90
Methanoregula
80 Methanosphaerula
Methanomethylovorans
Relative abundance (%)

70 Nitrososphaera
Methanolinea
60 Methanomassiliicoccus
unclassified
Methanospirillum
50
Methanobacterium
Methanothrix
40

30

20

10

A B C D E

(b) Amino Acid Metabolism

Biosynthesis of Other Secondary Metabolites
Carbohydrate Metabolism
Energy Metabolism
Enzyme Families
Metabolism Glycan Biosynthesis and Metabolism
Lipid Metabolism A
Metabolism of Cofactors and Vitamins B
Metabolism of Other Amino Acids
Metabolism of Terpenoids and Polyketides
C
Nucleotide Metabolism D
Xenobiotics Biodegradation and Metabolism E
Folding, Sorting and Degradation
Genetic
Replication and Repair
Information
Transcription
Processing
Translation
Environmental Membrane Transport
Information Signal Transduction
Processing Signaling Molecules and Interaction
Cell Growth and Death
Cellular Cell Motility
Processes Transport and Catabolism

0 0.5 1 1.5 2 2.5

Relative abundance (%)

Fig. 8. Microbial community distribution and function prediction of PES and aromatic carboxylic acids in archaea (a: Community distribution at the genus level; b: Predicting function of
KEGG) (A: adding 0.5 g/L PES; B: adding 0.5 g/L PES and 40 mg/L BA; C: adding 0.5 g/L PES and 40 mg/L PA; D: adding 0.5 g/L PES and 40 mg/L HA; E: adding 0.5 g/L PES and 40 mg/L 1-NA).

10
R. Qin, X. Lin, Z. Chen et al.
fact that methane production from acetic acid was inhibited by the re-
duction of AK and coenzyme F420 after the addition of aromatic carbox-
ylic acids. However, hydrogen-derived methanogens began to gradually
occupy a certain advantage.
As seen in Fig. 8b, 81.24% of the entire sequence were composed of
õMetabolism (49.33%), Genetic Information Processing (20.71%),
Environmental Information Processing (10.11%), and Cellular Processes
(1.09%). Among them, Amino Acid Metabolism, Carbohydrate
Metabolism, and Energy Metabolism were the main pathways. The rel-
ative abundance of Amino Acid Metabolism in each reaction unit was
2.21, 2.23, 2.31, 2.23, and 2.15%, respectively. Monocyclic aromatic car-
boxylic acids promoted the amino acid metabolism of sludge microor-
ganisms, while the addition of 40 mg/L 1-NA inhibited the amino acid
metabolism of microorganisms. 1-NA is a cyclic aromatic carboxylic
acid and has a potential toxic effect on the growth and metabolism of
sludge microorganisms. Carbohydrate metabolism and energy metabo-
lism were promoted after the addition of aromatic carboxylic acids com-
pared with the control group. In particular, with the addition of 40 mg/L
PA to the group C and 40 mg/L HA to the group D, the relative abun-
dance reached 1.95 and 2.03%. Energy Metabolism reached to 2.14 and
2.16%. In Genetic Information Processing, translation is the main
method, and the relative abundances were 1.75, 1.76, 1.84, 1.78, and
1.69%, respectively. Membrane Transport was the main method of
Environmental Information Processing, with relative abundances of
1.78, 1.78, 1.80, 1.91, and 1.88%, respectively. Therefore, based on our
findings, the addition of 40 mg/L PA and 40 mg/L HA would stimulate
the growth and development of various sludge microorganisms. In con-
trast, the addition of 40 mg/L 1-NA had a certain toxic effect on mi-
crobes, while the addition of 40 mg/L BA only slightly affected the
microbial community in the anaerobic granular sludge.
4. Conclusions
The addition of HA and 1-NA could inhibit acidification and meth-
ane production by anaerobic granular sludge. Due to the structural
characteristics of aromatic carboxylic acids, microorganisms in the
anaerobic granular sludge secreted more proteins to form a good
EPS protective layer. The XPS C 1s and O 1s spectra showed that the
chemical structure of the aromatic carboxylic acids was destroyed,
resulting in the migration of aromatic carboxylic acids to the
microplastic surface. As indicated by the microbial community struc-
ture, the addition of aromatic carboxylic acids inhibited the acetic
acid nutrient methanogen, and the hydrogen nutrient methanogens
Methanobacterium and Methanospirillum dominated. Function pre-
diction using the KEGG database further confirmed that 1-NA was
more toxic to the microbial community of the sludge than carboxylic
acid, which only contains one aromatic ring. In the presence of PES,
the degree of negative effects of different structures aromatic car-
boxylic acids on anaerobic granular sludge was as follows: 1-NA >
HA > PA > BA.
CRediT authorship contribution statement
Ronghua Qin: Writing - original draft, Writing - review & editing,
Data curation, Methodology. Xumeng Lin: Writing - original draft,
Writing - review & editing, Methodology. Zhenpeng Chen: Data
curation. Chengyuan Su: Writing - original draft, Writing - review
& editing, Methodology. Fenghua Zhu: Data curation. Wenjing
Yang: Data curation. Zhuxin Chen: Data curation. Pingping Lu:
Data curation.
Declaration of competing interest
The authors declare that we have no known competing financial in-
terests or personal relationships that could have appeared to influence
the work reported in this paper.
11
Science of the Total Environment 792 (2021) 148361
Acknowledgements
The authors acknowledge the research grant provided by the National
Natural Science Foundation of China (Grant No. 52060003), the Natural
Science Foundation of Guangxi (Grant No. 2017GXNSFAA198277) and
the Key Laboratory of Ecology of Rare and Endangered Species and
Environmental Protection (Guangxi Normal University), Ministry of
Education (ERESEP2021Z09) for providing financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2021.148361.
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