Ecotoxicology and Environmental Safety 238 (2022) 113577

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Ecotoxicology and Environmental Safety

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

Efficient removal of azo dyes by Enterococcus faecalis R1107 and its

application in simulated textile effluent treatment
Rui Wang a, b, Huanan Li a, b, Yanfang Liu d, Jianhui Chen a, b, Fang Peng a, c, Zhengbing Jiang a, b,
Jiashu Liu a, b, *, Huiting Song a, c, *
a
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei University, Wuhan 430062, PR China
b
Hubei Key Laboratory of Industrial Biotechnology, School of Life Science, Hubei University, Wuhan 430062, PR China
c
Hubei Key Laboratory of Regional Development and Environmental Response, Faculty of Resources and Environmental Science, Hubei University, Wuhan 430062, PR
China
d
Hubei Academy of Scientific and Technical Information, Wuhan 430071, PR China

A R T I C L E I N F O A B S T R A C T

Edited by: Dr G Liu This study aimed to exploit the potential of Enterococcus faecalis R1107 in the bioremediation of azo dyes. The
maximal decolorization of Congo Red (CR), Reactive Black 5 (RB5), and Direct Black 38 (DB38) were 90.17%,
Keywords: 96.82%, and 81.95%, respectively, with the bacterial treatment for 48 h. 65.57% of CR and 72.64% of RB5 could
Enterococcus faecalis be decolorized by E. faecalis R1107 within 48 h when the concentration of azo dyes increased up to 1000 mg/L.
Azo dyes
FTIR analysis confirmed that E. faecalis R1107 could effectively break down the chemical structures of three azo
Transcriptomic analysis
dyes. E. faecalis R1107 alleviated the phytotoxicity of azo dyes and improved seed germination, which
Detoxification
Simulated textile effluent contributed to the increase in the lengths of roots, stems, and leaves of Vigna radiata seedlings. Transcriptomic
analysis suggested that the gene regulatory networks in E. faecalis R1107 synergistically improved the degra­
dation and detoxification of RB5, including the major metabolic pathways, the secondary metabolism, the
transport system, the amino acid metabolic pathways, and the signal transduction systems. Simulated textile
effluent (STE) was used to mimic real textile effluent to evaluate the bioremediation potential of E. faecalis
R1107, and 72.79% STE can be decolorized after E. faecalis R1107 treatment for 48 h. In summary, our study
demonstrated that E. faecalis R1107 might be well suitable for potential applications in the bioremediation of
textile effluent.

1. Introduction Mutagenic, genotoxic, and carcinogenic risks of textile effluent threaten

Azo dyes are the most frequently used synthetic dyes in many in­
dustries, such as textile, printing and dyeing, papermaking, pharma­
ceutical, etc (Liu et al., 2020; Zhu et al., 2018). Azo dyes generally
comprise one or more azo bonds (-N = N-) in the chemical structures (Li
et al., 2022). The structural stability and high chromaticity of azo dyes
makes it resistant to degradation (Chen et al., 2020). The rapid devel­
opment of the textile industry leads to a great demand for synthetic dyes.
In particular, the textile industry accounts for more than 50% of the
global use of synthetic dyes (Zhuang et al., 2020). The discharge of
textile effluent causes serious environmental problems since less than
half of the dyes used is not being fixed in the products and will be dis­
charged into environment together with the effluent, especially azo dyes
(Liu et al., 2020). They are toxic to plants, animals, and even humans.
the health of humans and eco-systems (Liu et al., 2021). Conventional
physicochemical remediation methods of textile effluents are generally
high cost and may cause secondary pollution to the environment (Chen
et al., 2021; Liu et al., 2021). Notably, bioremediation technologies have
emerged as alternatives to textile effluent treatment because they are
cost-effective, high-efficiency, and eco-friendly (Chen et al., 2021). At
present, fungi, bacteria, and algae are known to degrade and detoxify
synthetic dyes mainly through biological adsorption and catabolic
pathways. Bacteria are ideal candidates for bioremediation because they
can greatly adapt to the extreme conditions of the effluents (Mir-Tutu­
saus et al., 2018). Additionally, only a short growth period is required
for bacterial culture. Thus, the exploitation of the bioremediation po­
tential of bacteria from the natural environments is needed.
Biodegradation of azo dyes by bacteria occurs under both aerobic

* Corresponding authors at: State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei University, Wuhan 430062, PR China
E-mail addresses: jsliu@hubu.edu.cn (J. Liu), htsong@hubu.edu.cn (H. Song).

https://doi.org/10.1016/j.ecoenv.2022.113577
Received 24 February 2022; Received in revised form 14 April 2022; Accepted 27 April 2022
Available online 5 May 2022
0147-6513/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
R. Wang et al.
and anaerobic conditions. The bacterial enzymatic systems can aerobi­
cally break the benzene ring, such as monooxygenase and dioxygenase
(Chen et al., 2021). The degradation products of azo dyes can be served
as nutrient sources with the help of other substrates and be further
mineralized. On the other hand, the anaerobic degradation of azo dyes
by bacteria starts with the cleavage of the azo bonds and then forms the
toxic intermediates, such as aromatic amines. Azo reductase is
frequently reported that involved in the anaerobic degradation of azo
dyes by bacteria (Cheng et al., 2021; Wang et al., 2021). The targeted
pollutants accept the electron from the electron donor, thereby
improving the complete degradation of azo dyes (Wang et al., 2021). In
practical applications, except for the synthetic dyes, the real textile ef­
fluents also contain a large number of heavy metals, salts, and auxiliary
chemicals (Prabhakar et al., 2021). The alkaline environment of the
textile effluent limits the degrading efficiency of microbes. Microor­
ganisms are generally considered less versatile in the presence of a
mixture of pollutants. The harsh environment inhibits the growth of
microbes and interrupts the biodegradation of synthetic dyes. However,
bacteria are recently found that can decolorize and detoxify synthetic
dyes effectively even in raw textile effluents (Rathour et al., 2021).
Therefore, a better understanding of the adaptive response of bacteria to
toxic dyes is necessary for advancing bioremediation to practical
applications.
Enterococcus faecalis is a facultative anaerobic bacterium, which can
catabolize different pollutants effectively (Chen et al., 2018; Eslami
et al., 2019; Sahasrabudhe et al., 2014). The enzymatic systems in
E. faecalis have been proven that exhibit great potential in the biore­
mediation of azo dyes, such as azo reductase and nitroreductase (Cha­
lansonnet et al., 2017). However, the global gene expression profiles of
E. faecalis in response to azo dyes are still unclear. The remediate po­
tential of E. faecalis on textile effluent needs to be exploited. In our study,
a dye-degrading bacterium isolated from the textile effluent was iden­
tified and named E. faecalis R1107. The potential of E. faecalis R1107 to
degrade and detoxify three azo dyes, including Congo Red (CR), Reac­
tive Black 5 (RB5), and Direct Black 38 (DB38), were evaluated. The
biodegradation of these azo dyes was characterized by UV-Vis spec­
troscopy analysis, Fourier transform infrared spectroscopy (FTIR)
analysis, and degradation kinetics. The toxicity of the degradation
products of azo dyes was examined by phytotoxicity study. The tran­
scriptomic sequencing was performed to illuminate the mechanism
underlying the efficient removal of RB5 by E. faecalis R1107. Thereafter,
we further investigated the potential of E. faecalis R1107 on simulated
textile effluent treatment.
2. Material and methods
2.1. Strain, media, and culture condition
Enterococcus faecalis R1107 was isolated from the textile effluent and
preserved in our laboratory. Luria-Bertani (LB) medium was used for the
preservation of E. faecalis R1107. The compositions of LB medium were
yeast extract 5 g/L, tryptone 10 g/L, NaCl 10 g/L. Brain Heart Infusion
(BHI) medium was used for the subsequent experiments related to textile
dyes decolorization. The compositions of BHI medium were yeast
extract 10 g/L, K2HPO4 0.075 mg/L, KH2PO4 0.175 mg/L, (NH4)2SO4
0.45 mg/L, NaCl 0.045 mg/L, MgSO4 0.09 mg/L, CaCl2 0.015 mg/L,
hemin 0.002 mg/L, Na2CO3 400 mg/L, and L-cysteine HCl 600 mg/L
(Zahran et al., 2019). E. faecalis R1107 was pre-cultured in BHI medium
at 37 ◦ C with shaking at 150 rpm to OD600 of approximately 0.6–0.8.
Then the culture was transferred into the fresh BHI medium (10% of
inoculum size (v/v)). The different concentrations of azo dyes were
added into the inoculated BHI medium simultaneously. The cultures
were proceeded under the same conditions as described above.
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Ecotoxicology and Environmental Safety 238 (2022) 113577
2.2. 16S rRNA sequencing
The bacterial pellets were collected after incubated in LB medium at
37 ◦ C with shaking at 150 rpm for 12 h. Genomic DNA was isolated and
then used as template for 16S rRNA sequencing. The amplified specific
DNA fragment was then aligned with known sequences using Basic Local
Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.
cgi). The specie of this bacterium was identified based on the results
of BLAST.
2.3. Decolorization analysis of different azo dyes by E. faecalis R1107
Three types of azo dyes, including Congo Red (CR), Reactive Black 5
(RB5), and Direct Black 38 (DB38), were used to investigate the capacity
of E. faecalis R1107 on dye decolorization (Table S1). The different
concentrations (50 mg/L, 100 mg/L, 200 mg/L, 500 mg/L, 1000 mg/L)
of CR, RB5, and DB38 were added into the inoculated BHI medium,
respectively. After continuous incubation at 37 ◦ C with shaking at 150
rpm, the supernatants were collected at different time intervals. Decol­
orization of azo dyes was assessed by measuring the maximal absor­
bance of CR at 497 nm, RB5 at 597 nm, and DB38 at 585 nm,
respectively (Zhu et al., 2018). UV–visible spectral scanning of azo dyes
(50 mg/L) was carried out after E. faecalis R1107 treatment for 0, 2, and
6 h. The decolorization (%) of azo dyes was calculated by the following
formula:
Decolorization (%) = [(A0 - A)/A0]×100 (1)
where A0 and A represent the initial and final absorbance of azo dyes,
respectively.
Additionally, adsorption analysis of azo dyes (48 h) was conducted
by treating the pellets with equivalent volume of methanol (70% v/v).
The desorbed dyes were measured at the corresponding absorbance (Yin
et al., 2021). All measurements were performed in triplicates.
2.4. FTIR analysis
The samples used for FTIR analysis were prepared based on E. faecalis
R1107 treated 50 mg/L of CR, RB5, and DB38 in BHI medium at 37 ◦ C
for 12 h. The supernatant was centrifuged and then extracted with
equivalent volume of ethyl acetate for three times. The organic layer was
completely dehydrated with anhydrous sodium sulfate and then evap­
orated using the rotary vacuum evaporator. The samples were re-
dissolved in methanol (HPLC grade) and filtrated by 0.22 µm micropo­
rous membrane filter. Thereafter, the samples were mixed with potas­
sium bromide and then pressed into transparent pellets. The changes of
chemical structures in azo dyes were analyzed using Thermo Scientific
Nicolet iS10 FTIR Spectrometer. The transmittance (%) was observed
from 400 to 4000 cm− 1 (Chen et al., 2021).
2.5. Phytotoxicity study
To evaluate the potential of E. faecalis R1107 for azo dyes detoxifi­
cation, Vigna radiata (mung bean) seed germination and seedling growth
experiments were conducted. 50 mg/L of CR, RB5, and DB38 were
added into the inoculated BHI medium, respectively. The incubation
proceeded at 37 ◦ C for 12 h with shaking at 150 rpm. The supernatants
used for the phytotoxicity study were collected after centrifugation.
Seed germination and seedling growth experiments were conducted in
Petri dishes (diameter 90 mm) as previously reported (Ameenudeen
et al., 2021; Haq et al., 2018). Briefly, 10 mL of E. faecalis R1107-treated
azo dyes were added to the Petri dishes, respectively. Ten seeds of
V. radiata were soaked in each Petri dish and incubated at 25 ◦ C for 5
days. The seeds treated with sterile water, raw azo dyes, and the
un-inoculated BHI medium were set as the control. The seed germina­
tion was observed after different treatments for 5 days. Thereafter, five
R. Wang et al. Ecotoxicology and Environmental Safety 238 (2022) 113577

seedlings were randomly selected from each Petri dish to measure the
lengths of roots, stems, and leaves, respectively. All experiments were
performed in triplicates.
2.6. Study of the mechanism underlying RB5 degradation by E. faecalis
R1107
2.6.1. The kinetic study of RB5 decolorization by E. faecalis R1107
The decolorization kinetic study of azo dye was performed based on
the calculation of decolorization ratio of different concentration of RB5
by E. faecalis R1107. The removal rate constant (k) of azo dyes was
calculated according to the first-order kinetic model:
lnCt = lnC0 – kt (2)
− 1
where k is the constant of removal rate (h ), t is the removal time (h) of
azo dyes, C0 is the initial concentration of azo dyes, and Ct is the residual
azo dyes at different time points.
The half-life of azo dyes removal (t1/2) was calculated as follows:
3. Results and discussion
3.1. Decolorization analysis of azo dyes in different concentrations by
E. faecalis R1107
A dye-degrading bacterium, E. faecalis R1107, isolated from the
textile effluent was identified via 16S rRNA sequencing. The perfor­
mance of E. faecalis R1107 for azo dyes decolorization, including CR,
RB5, and DB38, was then investigated. Three different azo dyes could be
effectively decolorized by E. faecalis R1107 within 48 h (Fig. S1). As
shown in Fig. 1, when the dye concentration was 50 mg/L, the decol­
orization ratio of CR, RB5, and DB38 rapidly increased up to 87.61%,
83.08%, and 69.77% within 6 h, respectively. Notably, the maximal
decolorization of CR, RB5, and DB38 was 90.17%, 96.82%, and 81.95%,
respectively. The increase of azo dye concentration decreased the
decolorization of azo dyes by E. faecalis R1107, however, E. faecalis
R1107 exhibited a good decolorization performance on a high concen­
tration of CR and RB5. When the dye concentration increased up to

t1/2 = ln2/k (3)

− 1
where k is the constant of removal rate (h ).

2.6.2. Transcriptomic sequencing

Total RNA of pellets were extracted from the bacterial cultures
treated with 50 mg/L RB5. The untreated sample was set as the control.
The quantity and quality of RNA were assessed using the RNA Nano
6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies,
CA, USA). mRNA was purified from the total RNA and used for cDNA
strand synthesis. The library preparation was conducted based on the
fragmented cDNA. The transcriptomic sequencing was performed using
the Illumina NovaSeq 6000 (Novogene, Beijing, China). The raw data
was subsequently filtered to remove adapters and low-quality reads. The
clean data obtained from the raw data were mapped to the reference
genome using Bowtie2 (Langmead and Salzberg, 2012), and then
assembled into transcripts using Rockhopper (McClure et al., 2013). The
p-values were adjusted using Benjamini and Hochberg’s approach for
controlling the false discovery rate. Log2 fold change ≥ 1 and adjusted
p-value ≤ 0.05 were set as the threshold for significantly differential
expression. Gene expression levels were quantified before annotation.
Thereafter, the identification of differentially expressed genes, gene
ontology (GO) enrichment analysis, and KEGG pathway analysis were
carried out.

2.7. Bioremediation of simulated textile effluent by E. faecalis R1107

The simulated textile effluent was designed based on the literatures

with a slight modification (Kaushik and Malik, 2009; Kurade et al.,
2017; Osma et al., 2010; Prabhakar et al., 2021). The simulated textile
effluent was composed of 15 g/L NaCl, 5 g/L Na2CO3, 0.05 mM Cr(VI),
0.05 mM Cu(II), 0.05 mM Ni(II), 0.05 mM Pb(II), 0.05 mM Zn(II), 0.05
mM Mg(II), 50 mg/L CR, 50 mg/L RB5, and 50 mg/L DB38. To maintain
the basal activity of E. faecalis R1107, a small amount of yeast extract
(1%) was supplemented in simulated textile effluent (Prabhakar et al.,
2021). The actively growing bacterial culture was added into the
simulated textile effluent (10% v/v of the total volume of the effluent).
The decolorization analysis was performed at 37 ◦ C for 48 h with
shaking at 150 rpm. The samples were filtered through 0.22 µm pore size
filters to remove suspended matter before analysis. Color of the simu­
lated textile effluent was measured by dilution multiple method as
previously reported (Liang et al., 2018; Wang et al., 2018). The distilled
water was set as the control.

Fig. 1. Time-course analysis of azo dye decolorization at different concentra­

tions by E. faecalis R1107, including (A) Congo Red, (B) Reactive Black 5, and
(C) Direct Black 38. The decolorization percentage of azo dyes was estimated by
taking the corresponding untreated dye as the control (100%).

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R. Wang et al. Ecotoxicology and Environmental Safety 238 (2022) 113577

1000 mg/L, the decolorization of CR (47.51%) and RB5 (65.07%)

increased rapidly within 6 h, while only 12% of DB38 was decolorized
by E. faecalis R1107. After treatment for 48 h, the decolorization of CR,
RB5, and DB38 increased up to 65.57%, 72.64%, and 23.39%,
respectively.
The results of UV–visible scanning indicated that the characteristic
peaks of azo dyes obviously decreased after bacterial treatment (Fig. S2).
To date, approximately 60% of synthetic dyes used in the textile industry
belong to azo dyes, indicating that a large amount of azo dyes have been
discharged into the wastewater (Liu et al., 2020). An improvement of
synthetic dyes decolorization at a high concentration is needed to deal
with the dye treatment effectively. The comparative analysis of dye
decolorization at 1000 mg/L is performed in Table S2. E. faecalis R1107
exhibited a good potential in CR and RB5 decolorization within a short
period of time but not for DB38. Most of the other microbes exhibited a
certain degree of dye decolorization at high concentration as previously
reported, however, a long time was required compared with the per­
formance of E. faecalis R1107 in the present study. The azo bonds formed
in azo dyes are stable and recalcitrant against degradation (Ameenudeen
et al., 2021). CR and RB5 were classified as diazo dye while DB38 was
defined as triazo dye. Therefore, the increase of azo bonds in synthetic
dyes might reduce the decolorization efficiency of azo dyes by E. faecalis
R1107.
Adsorption of dyes may occur on microbial biomass during the
decolorization (Khan et al., 2013). To assess the role of adsorption in azo
dye decolorization, we measured the desorbed dyes from the bacterial
pellets at the corresponding absorbance (Table S3). Consequently,
adsorption (> 42%) became an important factor in CR decolorization
when the dye concentration increased up to 1000 mg/L. On the con­
trary, the adsorption ratio of DB38 increased by over 28% when the dye
concentration decreased to 200 mg/L. Notably, the adsorption effect
(less than 8%) was negligible on RB5 decolorization by E. faecalis R1107.
RB5 is toxic to ecosystems and RB5 at concentration up to 100 mg/L can
induce 100% toxicity to Daphnia magna (Chang et al., 2010). In our
study, E. faecalis R1107 capable of degrading RB5 ranging from 50 to
1000 mg/L efficiently, implying that this bacterium was well-suited for
azo dye degradation, especially RB5.

3.2. Degradation analysis of azo dyes by E. faecalis R1107

The FTIR analysis was conducted to assess the capacity of E. faecalis

R1107 on azo dye degradation. The FTIR spectra are shown in Fig. 2. As
expected, E. faecalis R1107 could effectively break the structures within
azo dyes. The FTIR spectrum of untreated CR exhibited N-H stretching
vibration at 3400 cm− 1, -C-H stretching vibration on the benzene ring at
2900 cm− 1 to 3200 cm− 1, azo vibration absorption peak at 1600 cm− 1,
C–– C stretch in benzene at 1450 cm− 1, the stretching vibration sulfonic
group at 1050 cm− 1 to 1200 cm− 1, and C-N stretching vibration at
1000 cm− 1 (Bao et al., 2019; Bessaha et al., 2019; Setyawati et al.,
2020). The spectrum of CR after E. faecalis R1107 treatment showed a
significant difference in comparison with the untreated CR. The peaks at
2900 cm− 1 to 3200 cm− 1 and 1450 cm− 1 disappeared in the FTIR
spectrum after E. faecalis R1107 treatment, implying that the benzene
rings in CR were broken. Notably, the cleavage of azo bonds was
confirmed due to the peak corresponding to azo vibration at 1600 cm− 1
decreased. The band at 3400 cm− 1disappeared after E. faecalis R1107 Fig. 2. FTIR spectra of untreated azo dyes and E. faecalis R1107 treated azo
treatment, implying that the degradation intermediates, aromatic dyes. (A) Congo Red, (B) Reactive Black 5, and (C) Direct Black 38.
amines, might be further broken down by E. faecalis R1107. The FTIR
spectrum of untreated RB5 exhibited N-H stretching at 3450 cm− 1, O-H degradation products, implying that the cleavage of the benzene ring
stretching at 3360 cm− 1, C-H stretching vibration at 2930 cm− 1, the and azo bonds might occur after treatment with E. faecalis R1107. The
overlap between the azo bond vibration and the benzene ring skeleton decrease in the intensities of the bands at 3450 cm− 1 suggested that the
vibration at 1400 cm− 1 to 1600 cm− 1, C-N stretching at 1230 cm− 1, and degradation products of RB5 might not form toxic amine. The sulfonic
-SO stretching at 1100 cm− 1 (Chen et al., 2021; Cheng et al., 2021). The group might be broken during RB5 degradation due to the peak at
changes in FTIR spectrum of RB5 were similar to that of the spectrum of 1100 cm− 1 disappeared. The peaks of DB38 at 3350 cm− 1, 2900 cm− 1,
CR after E. faecalis R1107 treatment. The stretching vibrational bands at 1680 cm− 1, and 1300 cm− 1 reflect OH stretching, C-H stretching
1400 cm− 1 to 1600 cm− 1 almost disappeared in the spectrum of RB5

4
R. Wang et al.
vibration, C– – C stretching vibration, and C-N vibration, respectively
(Junejo et al., 2020). Comparably, the decrease in the intensity of the
peak at 1680 cm− 1 implied that the benzene ring was opened by
E. faecalis R1107 during DB38 degradation. Therefore, E. faecalis R1107
is capable of degrading azo dyes through catabolic pathways, such as
benzene ring opening, azo bond cleavage, and deamination.
3.3. Toxicity assessment of biodegradation products of azo dyes through
phytotoxicity study
The toxicological risk of azo dyes threatens the health of ecosystems
(Liu et al., 2021). Whether the toxicological risk of azo dyes could be
alleviated after E. faecalis R1107 treatment was evaluated via phyto­
toxicity study. 100% germination was observed in seeds soaked in
bacterial treated azo dyes. Compared with the seeds treated with raw
azo dyes, the seed germination and seedling growth could be improved
by adding bacterial-treated azo dyes, implying that the toxicity of azo
dyes was significantly reduced after E. faecalis R1107 treatment. The
lengths of roots, stems, and leaves in V. radiata seedlings treated with CR
degradation products increased by 3.27-, 2.73-, and 1.65-folds than that
of the untreated CR, respectively (Fig. 3A). The lengths of roots, stems,
and leaves in V. radiata seedlings exposed to RB5 degradation products
increased by1.77-, 2.91-, and 3.26-folds than that of the untreated RB5,
respectively (Fig. 3B). The lengths of roots, stems, and leaves in
Fig. 3. Phytotoxicity studies of azo dyes treated and untreated with E. faecalis R1107 for 5 days. (A) Congo Red (CR), (B) Reactive Black 5 (RB5), and (C) Direct Black
38 (DB38).
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Ecotoxicology and Environmental Safety 238 (2022) 113577
V. radiata seedlings treated with DB38 degradation products increased
by 5.92-, 2.63-, and 3.95-folds than that of the untreated DB38,
respectively (Fig. 3C). Both BHI medium and azo dyes showed an
inhibitory effect on seed germination and seedling growth of V. radiata.
However, the germination and growth of V. radiata seeds were recov­
ered after consumption of the medium and degradation of azo dyes by
E. faecalis R1107.
As previous studies described, azo dyes are generally recalcitrant and
toxic aromatic compounds (Haq et al., 2018; Liu et al., 2020). In our
study, the exposure of V. radiata seeds to CR, RB5, and DB38 signifi­
cantly inhibited seed germination and reduced the lengths of roots,
stems, and leaves in seedlings. In many cases, azo dyes can be degraded
into low-toxic compounds and even be mineralized to carbon dioxide
and water by microbes (Liu et al., 2021; Rodrigues de Almeida et al.,
2019). Consistent with the previous report (Liu et al., 2021), the toxicity
of azo dyes could be alleviated by treating with E. faecalis R1107 in our
study. Additionally, the un-inoculated BHI medium might be harmful to
V. radiata seeds germination, which leads to growth inhibition of
V. radiata seedlings. The incubation of E. faecalis R1107 in BHI medium
containing azo dyes not only degraded azo dyes but also alleviated the
inhibitory effect of the medium on seedling growth. Overall, the
degradation products of azo dyes exhibited lower phytotoxicity, indi­
cating that E. faecalis R1107 has a good potential in the detoxification of
textile dyes.
R. Wang et al.
3.4. Kinetics of RB5 decolorization in different concentrations
In the present study, E. faecalis R1107 showed a good decolorization
capacity on RB5 among three azo dyes. Before the transcriptomic
analysis, the degradation kinetic analysis of RB5 was performed. As
shown in Table S4, the degradation of RB5 by E. faecalis R1107 was well
fit to the first-order kinetics with high correlation coefficients (R2). The
increase of RB5 concentration delayed RB5 decolorization by E. faecalis
R1107, which resulted in the increase of t1/2 from 2.16 to 3.164 h. A
relatively high decolorization activity of E. faecalis R1107 was observed
when lowered the RB5 concentration. Previous study indicates that the
bio-decolorization efficiency of RB5 decreased along with the increase of
the dye concentration up to 1000 mg/L (Wang et al., 2009). Although
the toxicity of RB5 inhibited the bioactivity of E. faecalis R1107, this
bacterium still showed high decolorization performance even in a high
RB5 concentration. Thereafter, the transcriptomic analysis was con­
ducted based on the result of the kinetic study of 50 mg/L RB5
decolorization.
3.5. Transcriptomic analysis revealed gene expression profiles of
E. faecalis R1107 during RB5 degradation
Transcriptomic analysis was performed to elucidate the mechanism
underlying RB5 degradation by E. faecalis R1107. A total of 2393
unigenes were detected. With the RB5 exposure, 109 differentially
expressed genes (DEGs) were up-regulated and 28 of DEGs were down-
regulated (Fig. S3). Based on the GO enrichment analysis, a total of 109
differentially expressed genes were enriched in GO terms (Fig. S4). In
Biological Process terms, most DEGs were enriched in three categories,
including transport, localization, and establishment of localization. In
Molecular Function terms, organic cyclic compound binding and het­
erocyclic compound binding were the two categories enriched with 10
DEGs. Cellular Component analysis revealed that the most of DEGs were
distributed in the membrane category and cellular component category.
The presence of aromatic compounds may evoke the gene and protein
expression related to catabolism (Kumar et al., 2018). Our results of GO
analysis implied that E. faecalis R1107 could utilize RB5 due to its
up-regulated genes might result in intensify of transport, organic cyclic
compound binding, and heterocyclic compound binding. The proteins
located in membrane and cellular component might play an important
role in RB5 degradation.
To elucidate the biological function of DEGs in RB5 biodegradation,
Fig. 4. KEGG enrichment analysis of DEGs of E. faecalis R1107 treated by RB5. Bar chart shows the number of up-and-down regulated genes grouped in relevant
KEGG categories.
6
Ecotoxicology and Environmental Safety 238 (2022) 113577
KEGG pathway analysis of DEGs was performed. As shown in Fig. 4,
DEGs were classified into 25 KEGG categories. Except for two down-
regulated genes enriched to ribosome pathway, the DEGs enriched in
other 24 of KEGG pathway were up-regulated. The detailed gene in­
formation is listed in Table S5. In the presence of RB5, the metabolic
activity of E. faecalis R1107 was activated due to a total of 24 genes
involved in the metabolic pathway showing up-regulated. The up-
regulated genes enriched in metabolic pathway also play a key role in
other pathways. For example, dihydrolipoyl dehydrogenase is involved
in Glycolysis, Propanoate metabolism, Pyruvate metabolism, Glyoxylate
and dicarboxylate metabolism, Glycine, serine and threonine meta­
bolism, Carbon metabolism, Biosynthesis of secondary metabolites,
Microbial metabolism in diverse environments, and Biosynthesis of co­
factors. Dihydrolipoyl dehydrogenase is a part of the pyruvate dehy­
drogenase multi-enzyme complex in gram-negative bacteria (de Kok
et al., 1998). This enzymatic complex can transform pyruvate into
acetyl-CoA, a central intermediate related to many downstream path­
ways. Thus, the resistance of E. faecalis R1107 against RB5 might be
enhanced.
Followed by Metabolic pathways, Purine metabolism and Biosyn­
thesis of secondary metabolites were the second and third largest cate­
gories containing up-regulated genes, respectively. As previous study
mentioned, the activation of purine metabolism can improve bacterial
survival and proliferation by providing the cofactors and energy (Li
et al., 2022). The microbial activity of E. faecalis R1107 might be
maintained and then improve the degradation of RB5. Meanwhile, the
genes related to secondary metabolism showed up-regulation, implying
that the enzymatic system responsible for dye degradation was
activated.
The major metabolic pathways in bacterial cells were also activated
due to a large number of up-regulated genes being enriched in these
pathways, such as Carbon metabolism, Oxidative phosphorylation, Py­
ruvate metabolism, and Glycolysis (Fig. 4). For instance, the gene
expression related to oxidative phosphorylation, encoding NAD(P)H-
dependent oxidoreductase subunit E and NADH-ubiquinone oxidore­
ductase-F iron-sulfur binding region increased by 3.37- and 1.46-folds,
respectively, compared with the untreated control. The major meta­
bolic pathways are closely related to the TCA cycle (Chen et al., 2020;
Wu et al., 2017), which may influence the biological process of
biodegradation. In general, the stable chemical structure of azo dyes
leads to its recalcitrant properties against degradation (Liu et al., 2021).
It indicates that azo dyes could not be utilized as the nutrient sources by
R. Wang et al.
microbes directly (Chen et al., 2020). In the present study, the activated
major metabolic pathways might provide sufficient energy for RB5
degradation by E. faecalis R1107, such as glycolysis, pyruvate meta­
bolism, and oxidative phosphorylation. Co-metabolism of RB5 by
E. faecalis R1107 might be occurred to improve RB5 degradation and
alleviate the toxicity of RB5. The oxidative phosphorylation together
with the electron transfer could be inhibited with the toxicity of azo
dyes. However, the physiological activity of E. faecalis R1107 showed
strong resistance against RB5 toxicity.
The up-regulated genes related to the transport system in E. faecalis
R1107 were identified. These genes were enriched in two KEGG cate­
gories, including ABC transporter and Phosphotransferase system. ABC
transporters are able to export the toxic compounds from microbial cells
with the assistant of ATP (Husada et al., 2018; Xu et al., 2016). The
phosphotransferase system in prokaryotes is generally conserved that
responsible for carbon nutrient uptake (Rom et al., 2021). Therefore,
with the exposure of E. faecalis R1107 to RB5, a highly-effective trans­
port system might benefit for the efflux of toxic compounds and the
enhancement of major metabolic pathways.
Importantly, three amino acid metabolic pathways, including
Alanine, aspartate and glutamate metabolism, Arginine and proline
metabolism, and Glycine, serine and threonine metabolism, showed up-
regulation as well. Accordingly, metabolism of aspartate, glutamate,
arginine, and proline can improve succinic acid synthesis and cell entry
into the TCA cycle (Qiao et al., 2018). Glycine, serine and threonine
metabolism play a key role in elevating bacterial sensitivity to stress (Ye
et al., 2018). Thus, highly expressed amino acid metabolism in E. faecalis
R1107 exposed to RB5 might enhance the bacterial defense and the
sensitivity towards synthetic dye.
In response to the environmental stress, the signal transduction
systems in prokaryotes receive external signals and impact the internal
regulatory networks (van Hoek et al., 2019). In our study, a total of five
genes involved in the quorum sensing system and two-component sys­
tem were up-regulated. In the presence of RB5, the log2 fold change of
winged-helix domain-containing protein, APC family permease,
tagatose-bisphosphate aldolase, bacterial extracellular solute-binding
proteins, and peptide ABC transporter substrate-binding protein
increased by 3.37-, 4.78-, 1.04-, 1.17-, and 1.02- folds, respectively.
Previous studies suggest that the activation of quorum sensing and
Fig. 5. Putative mechanism underlying RB5 degradation by E. faecalis R1107.
7
Ecotoxicology and Environmental Safety 238 (2022) 113577
two-component system in the presence of synthetic dyes is essential for
the improvement of bacterial survival and pollutant remediation (Chen
et al., 2020; Wang et al., 2020). The adaptive response of E. faecalis
R1107 to azo dye might benefit for keeping health of bacterial cells in
harmful environments.
Overall, we proposed the putative mechanism underlying the high
effective degradation of RB5 by E. faecalis R1107 (Fig. 5). The central
metabolic pathways in E. faecalis R1107 were activated in response to
the stress caused by RB5. With the help of signal transduction and
transmembrane transport, many metabolic pathways related to electron
transfer and amino acids metabolism are involved in the degradation of
RB5 by E. faecalis R1107. As an electron acceptor, the cleavage of
linkage within RB5 was proceeded, which resulted in further degrada­
tion of azo dye.
3.6. Evaluation of the remediation potential of simulated textile effluent
by E. faecalis R1107
To further investigate the potential of E. faecalis R1107 in the prac­
tical applications of bioremediation, remediation of simulated textile
effluent by E. faecalis R1107 was performed. Notably, E. faecalis R1107
has a good potential for simulated textile effluent decolorization (Fig. 6).
After 48 h treatment with E. faecalis R1107, the chroma of the effluent
significantly decreased from 600◦ to 163.26◦ (Table S6). The pH was
slightly decreased from 9.82 to 9.22 after bacterial treatment. These
results indicated that 72.79% of simulated textile effluent could be
decolorized using E. faecalis R1107, together with an effect on the
neutralization of alkaline effluent. The real textile effluent generally
composes of a variety of different components, such as synthetic dyes,
heavy metals, and auxiliary chemicals (Kurade et al., 2017). The high
color intensity of textile effluent limits sunlight penetration to the
aquatic microorganisms, which may lead to the disorder of photosyn­
thesis in the aquatic ecosystem (Gita et al., 2019). The microbial
degradation efficiency of the mixture of synthetic dyes generally de­
clines due to the chemical structures of synthetic dyes are complicated
and recalcitrant (Zabłocka-Godlewska and Przystaś, 2020). The
co-existence of heavy metals and alkaline conditions in textile effluent
provides an extreme environment for microbes, which may lead to cell
apoptosis and death. In the present study, the chroma in effluent
R. Wang et al.
Fig. 6. Comparative analysis of chroma between raw simulated textile effluent
(STE) and E. faecalis R1107 treated STE (T-STE) for 48 h. The water was set as
the control.
decreased with E. faecalis R1107 treatment under an alkaline environ­
ment. The resistance of E. faecalis R1107 towards the hazardous chem­
icals and the salinity might benefit the further application of E. faecalis
R1107 in bioremediation of real textile effluent.
4. Conclusion
In our study, a highly-efficient azo dye-degrading strain E. faecalis
R1107 was isolated. CR, RB5, and DB38 could be effectively decolorized
by E. faecalis R1107 within 48 h. Moreover, the maximal decolorization
of CR and RB5 at 1000 mg/L reached 65.57% and 72.64%, respectively.
On the basis of the FTIR analysis, we confirmed that E. faecalis R1107
could effectively break the structures within azo dyes, including CR,
RB5, and DB38. The phytotoxicity analysis showed that the toxicity of
azo dyes treated by E. faecalis R1107 was significantly reduced. Tran­
scriptomic sequencing revealed that the major metabolic pathways, the
secondary metabolism, the transport system, the amino acid metabolic
pathways, and the signal transduction systems of E. faecalis R1107 were
activated in the presence of RB5. The synergistic effect of the regulatory
networks in E. faecalis R1107 improves the RB5 degradation and
detoxification. Furthermore, E. faecalis R1107 can treat simulated textile
effluent containing a mixture of azo dyes, heavy metals, and salts at
alkaline condition. 72.79% of the effluent could be effectively decolor­
ized by E. faecalis R1107 within 48 h. Overall, our findings provided a
theoretical basis and a potential candidate for the practical application
of bioremediation to textile effluent.
CRediT authorship contribution statement
Rui Wang: Data curation, Formal analysis, Software, Validation,
Writing – original draft. Huanan Li: Investigation, Resources. Yanfang
Liu: Formal analysis. Jianhui Chen: Conceptualization. Fang Peng:
Visualization. Zhengbing Jiang: Funding acquisition. Jiashu Liu:
8
Ecotoxicology and Environmental Safety 238 (2022) 113577
Methodology, Project administration, Supervision, Writing – review &
editing. Huiting Song: Methodology, Funding acquisition, Project
administration, Supervision.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by China National Key R&D Program
(2020YFA0908400, 2019YFA0905000, 2021YFC2100100), the Na­
tional Natural Science Foundation of China (32100099), the Science and
Technology Innovation Program of Hubei Province (2020BBA056), and
the Wuhan Science and Technology Plan (2019020701011496).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2022.113577.
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