Journal of Environmental Management 323 (2022) 116235

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Journal of Environmental Management

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

Research article

Evaluating the potential of thermo-alkaliphilic microbial consortia for azo

dye biodegradation under anaerobic-aerobic conditions: Optimization and
microbial diversity analysis
Samson Tizazu a, Getaneh Tesfaye a, Berhanu Andualem b, Aijie Wang c, Awoke Guadie a, c, *
a
Arba Minch University, College of Natural and Computational Sciences, Department of Biology, Biotechnology Stream, Arba Minch 21, Ethiopia
b
Gondar University, Institute of Biotechnology, Department of Industrial Biotechnology, Gondar, 196, Ethiopia
c
Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, PR China

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

Keywords: Wastewaters in textile industry are mainly characterized by higher pH, color, salt and chemical oxygen demand
Consortia (COD) values, which are environmentally undesirable. Among these textile effluent characteristics, color removal
Halotolerant is the most challenging task. In this study, the potential of Rift Valley halotolerant and thermo-alkaliphilic mi­
Reactive red 141
crobial consortia (collected from Shala hot spring located in Ethiopia) for azo dye biodegradation under
Rift valley
Shala hot spring
anaerobic-aerobic conditions were evaluated. Optimization and microbial diversity analysis were done using
Thermo-alkaliphilic Reactive Red 141. Under optimum conditions of pH (9), temperature (55 ◦ C), salinity (0.5%), and nutrients,
microbial consortia can remove >98% color and 92.7 ± 7.3% COD under anaerobic and aerobic conditions,
respectively. In addition, the consortia was capable of decolorizing initial dye concentrations of 100–1000 mg/L,
and various dye types including Everzol Blue LX, RY 84, RR 239, RB 198 and RY 700. The 16S rRNA gene
sequence results showed that Bacteroidetes (25.3%) > Proteobacteria (21.0%) > Chloroflexi (18.5%) > Hal­
obacterota (6.2%) dominant phyla. Based on the findings, non-color effluent adapted Rift Valley halotolerant and
thermo-alkaliphilic bacterial consortia can be a potential candidate for bioremediation of textile and other in­
dustries characterized by higher salinity, temperature and pH.

1. Introduction
A significant group of dyes, especially azo dyes are used in textile
industry (Zhang et al., 2019). They are categorized as reactive, direct,
basic, acid, dispersion, vat, sulfur, and metal complex dyes and distin­
guished by the existence of one or more N– – N linkages. Among these azo
dye groups, reactive azo dyes are the most widely used (30% of the
market share) in textile industry (Pearce et al., 2003).
Wastewaters in textile industry are mainly characterized by higher
pH, color, salt and chemical oxygen demand (COD) values. Among these
textile effluent natures, color removal is the most challenging task (Asad
et al., 2007). Dyes applied to offer color in textile industry are synthe­
sized to be stable both photolytically and chemically, making them to
exist longer time in the environment (Guadie et al., 2017, 2018; Saratale
et al., 2011).
So far, physical, chemical, and biological treatment approaches have
tried to treat textile contaminants (Guadie et al., 2017). Although
physical and chemical treatments efficiently remove color and other
environmental contaminates from textile effluent, they have drawbacks
related to cost, production of secondary contaminant, and high energy
demand (Asad et al., 2007; Guadie et al., 2018; Zhang et al., 2019). As a
sustainable approach, biological treatment is a good choice to alleviate
the limitations of physical and chemical textile wastewater treatment
methods. However, the challenge for biological treatment is finding
microbes that adapt and fit the nature of textile industry effluent. The
particular nature of textile wastewater is elevated temperature
(50–80 ◦ C), high pH (7–11), and high salt (15–20%) concentration (Guo
et al., 2020a, 2020b), which inhibit biodecolorization efficiency of mi­
croorganisms that have mesophilic, neutrophilic and non-halophilc na­
ture (Ali et al., 2014; Asad et al., 2007; Guo et al., 2020a, 2020b).
Extremophiles, which are active in extreme conditions, can adapt and
function physiologically in extreme situations, are the best options for
degradation of azo dyes in extreme environments (Guadie et al., 2017,
2018). Previous research studies reported halo-thermophilic bacterial

* Corresponding author. Department of Biology, College of Natural and Computational Sciences, Arba Minch University, Arba Minch 21, Ethiopia.
E-mail address: awokeg@yahoo.com (A. Guadie).

https://doi.org/10.1016/j.jenvman.2022.116235
Received 18 July 2022; Received in revised form 30 August 2022; Accepted 7 September 2022
Available online 13 September 2022
0301-4797/© 2022 Elsevier Ltd. All rights reserved.
S. Tizazu et al.
consortium (Guo et al., 2021); halophilic thermo-alkaliphilic bacterial
consortium (Guo et al., 2020a); thermophilic microflora (Chen et al.,
2018); and halotolerant bacterial consortium (Guo et al., 2020b) as an
effective means of azo dye decolorization. Although many studies car­
ried out to treat various wastewater natures (salinity, alkaline, uranium,
recalcitrant and hydrocarbons) using extremophiles, they have consid­
ered mostly a single (Kargi and Dincer, 2000) and only few more than
one (Guo et al., 2020a, 2021) extreme conditions during their
enrichment.
In the present study, the potential of Rift Valley halotolerant and
thermo-alkalophilic microbial consortia for azo dye biodegradation
were evaluated under anaerobic-aerobic conditions. The specific ob­
jectives are to answer (i) Does dye-uncontaminated microbial consortia
collected from hot spring adapt and enhance dye removal from textile
effluent? (ii) What are the optimum environmental conditions that the
bacterial consortia adapt and function best to remove azo dyes? (iii)
Which microbes are involved in the biodegradation of azo dyes? This
research gives insight into the use of halotolerant and thermo-
alkaliphilic bacterial consortia as a viable technology to treat textile
wastewater in the future.
2. Materials and methods
2.1. Dyes and reagents
The dye types used in this study were obtained from Ayka Addis
Textile investment group, Addis Ababa, Ethiopia. These dyes include
Everzol Blue LX, Reactive Red 141 (RR 141), Reactive Yellow 84 (RY
84), Everzol Red 3BS (RR 239), Evercion Blue HEGN (RB 198) and
Reactive Yellow 700 (RY 700) (Table S1). Among these azo dye types,
RR 141 was used as a model dye in this study (Fig. S1).
2.2. Inoculum source and culturing media
Great Rift Valley hot spring located in Ethiopia was the target sam­
pling site. Sediment and water samples were collected from Shala hot
spring (Fig. S2) located in Abijatta-Shala National Park (7◦ 28′ 59.99′′ N
38◦ 31’ 59.99” E), which is found at 200 km south of Addis Ababa
(Fig. 1). The hot springs are assembled along the eastern and south­
eastern shore of Lake Shala. With a depth of 266 m, Shala is ranked the
Fig. 1. Location map of sampling site. (A) Ethiopia, (B) Oromia, and (C) Shala-Billa Kebele.
2
Journal of Environmental Management 323 (2022) 116235
deepest lake among Ethiopian Rift Valley (Abijatta, Chamo, Abaya,
Hawassa, Langano, and Ziway) lakes. Due to anaerobic bacteria con­
verting sulfur from SO4 to H2S, the hot spring has smell like rotten eggs.
Although no textile industries are around the Shala hot spring, the
anaerobic microbial consortia can be the potential source of dye removal
under anaerobic conditions. Physicochemical parameters of the hot
spring, including pH, dissolved oxygen (DO), temperature, salinity,
conductivity and total dissolved solid (TDS) were measured in situ and
results were found to be 8.93 ± 0.7, 1.0–5.2 mg/L, 82.4 ± 1.2 ◦ C, 5.4 ±
0.1%, 9.6 ± 0.5 mS/cm and 5.3 ± 0.9 g/L, respectively. Microbes which
adapt these hot spring physicochemical parameters can easily acclima­
tize/adapt and function on textile wastewater, which have higher pH,
temperature, and salinity values.
To culture the hot spring microbial sample, thermus agar medium
(ATCC medium 697) was employed, which contains NaCl (5.0 g/L),
yeast extract (2.0 g/L), peptone (5.0 g/L) and agar (30.0 g/L). One molar
sodium hydroxide (NaOH) has been used to adjust the medium pH to
9.0. For COD removal, nitrogen source effect and carbon source effect
determination experiments, the media used was Mineral Salts Medium
(MSM) which was used by Guadie et al. (2018) (Table S2).
2.3. Inoculation, acclimatization and culturing conditions
The hot spring sediment (10 mg) sample was added to a sterilized
thermus medium containing 100 mg dye to make 1 L final volume. For
synthetic textile wastewater color and nutrient removals, anaerobic
(Gnanapragasam et al., 2011) and aerobic batch reactors were designed
(Fig. 2). To obtain acclimatized and stable microbial consortia under
anaerobic conditions, the bacterial consortia were maintained by
transferring into fresh ATCC medium 697 with dye (100 mg/L) in 3–5
day intervals for three months. The acclimatization was performed by
gradually exposing consortia to increasing dye concentrations up to
1000 mg/L (Kalyani et al., 2008). The anaerobic reactors performed
with an airtight lid to provide oxygen stress environment. Once the
acclimatization done, optimization of various (temperature, pH, nutri­
ents and salinity) parameters and removal efficiencies were evaluated
using anaerobically acclimatized microbial consortia and azo (RR 141)
dye. Further wastewater contaminant removal efficiencies were also
evaluated under aerobic condition. The aerobic reactor was inoculated
with 10% (v/v) aerobically acclimatized culture, cotton plugged and
S. Tizazu et al. Journal of Environmental Management 323 (2022) 116235

Fig. 2. Experimental design to treat textile wastewater (A) azo dye (RR 141) containing synthetic wastewater, (B) anaerobic treatment, and (C) aerobic treatment.

incubated on shaking (adjusted 150 rpm) water bath for 48 h (Fig. 2).
Table 1 shows the microbial consortia operating under anaerobic and
aerobic conditions, the reactor volume and inoculum size were main­
tained constant. Under anaerobic conditions, the pH (3, 7, 8, 9 and 11)
and temperature (30, 45, 55, 65 and 80 ◦ C) values were investigated.
The effect of carbon sources on bacterial activity was also determined
using (0.5 g/L for each) glucose, maltose, and starch, while the effect of
nitrogen sources was studied using 0.01 g/L organic (peptone, yeast
extract) and 1 g/L inorganic (ammonium sulfate, sodium nitrate, and
sodium nitrite) nitrogen sources. Furthermore, a range of dye
(100–1000 mg/L), and salt (0.5–30.0%) concentrations effects were also
studied under anaerobic conditions. The consortia were also exposed to
100 mg/L of various dye types including Everzol Blue LX, RR 141, RY
84, RR 239, RB 198 and RY 700 to determine the effect of dye types on
bacterial consortia decolorization activities. The optimization experi­
ments were all conducted in triplicate sets and the mean and standard
deviations were reported. For aerobic treatment, the optimum condi­
tions identified during anaerobic treatment were directly used (Table 1).
Biodegradation of the synthetic textile wastewater was studied by
measuring the absorbance at the pre-determined maximum absorbance
(λmax = 544 nm) using a UV–visible spectrophotometer. The color
removal efficiency is calculated based on Eq. (1):
Ainitial ​ − ​ Afinal
Color ​ removal ​ (%) ​ = ​ ( )∗100
Ainitial
where, Ainitial and Afinal are the initial and final absorbance, respectively.
The COD content was determined according to a standard procedure
(APHA, 1999) shown in Eq. (2):
initial ​ COD − ​ COD(t)
COD ​ removal ​ (%) = ( )∗100
initial ​ COD
The nitrate content was determined by cadmium reduction method
which is a colorimetric method using DR 900 HACH instrument. Kjel­
dahl procedure (Bremner and Mulvaney, 1982) was used to determine
the total nitrogen content.
2.4. Metagenomic analysis
Metagenomic DNA from the aerobic and anaerobic reactors were
collected and extraction was carried out using Fast DNA Spin Kit (MP
Biomedicals, Santa Ana, California, USA), in compliance with the
manufacturer’s protocols. The DNA extract was checked on 1% agarose
gel, and DNA concentration and purity were determined with Nanodrop
2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, USA).
Primers 515F (5′ -TGTGTAGCGGTGAAATGCG-3′ ) and 806R (5′ -
CATCGTTTACGGCGTGGAC-3′ ) were used for amplifying the hyper
variable region V3–V4 of 16S rRNA using polymerase chain reaction
(PCR) thermocycler (ABI GeneAmp®9700 USA). The PCR amplification
of 16S rRNA gene sequence was done as follows: Initial denaturation at
94 ◦ C for 5 min, followed by 40 cycles of denaturing at 94 ◦ C for 30 s,
annealing at 56 ◦ C for 30 s and extension at 72 ◦ C for 40 s, and single
extension at 72 ◦ C for 10 min, and end at 4 ◦ C. A total of 25 μL PCR
mixtures containing 4 μL master mix, 2.5 mM dNTPs (2 μL), 5 μM for­
ward and reverse primers (0.8 μL each), DNA polymerase 0.4 μL, tem­
plate DNA (2 μL), and finally 15 μL distilled water was used. The control
(1) reaction with no DNA template was also performed and all the PCR
reactions were performed in triplicate sets.
Illumina Miseq throughput sequencing (Shanghai Majorbio
Biotechnology Co. Ltd., China) was used to sequence the extracted and
purified DNA. High-throughput result of 16S rRNA gene sequences were
analyzed using the free online platform exists on Majorbio Cloud. Se­
(2) quences were clustered into operational taxonomic units (OTUs), using
97% similarity cut values. Shannon-Wiener, Simpson, Chao1 and
coverage, are bacterial diversity indexes calculated.

2.5. Statistical analysis

Table 1
Value of different operating parameters in anaerobic and aerobic conditions.
All experiments were carried out in triplicates, and the results were
Operating conditions Unit Anaerobic Aerobic
expressed as mean ± standard error. Statistical significance between
Reactor volume mL 1000 1000 mean values were determined using one-way analysis of variance
Inoculum size % 10 10
(ANOVA) with Tukey post hoc test. For principal component analysis,
Temperature ◦
C 30–80 55
pH - 3–11 9
bacterial abundance at phylum level was used. If p ≤ 0.05, the difference
Analysis time h 3–9 48–120 was considered significant.
Carbon sources g/L C-sourcesa Glucose
Nitrogen sources g/L N-sourcesb Yeast extract and peptone 3. Results and discussion
Dye type - Dye typesc RR 141
Dye concentration mg/L 100–1000 100
Salt concentration % 0.5–30 0.5 3.1. Optimization of various parameters
Shaking rpm 30 150
a
0.5 g/L each carbon sources (glucose, maltose, starch). 3.1.1. pH
b
1 g/L each nitrogen sources (peptone, yeast extract, NaNO3, NaNO2, Azo dyes are bound to cotton fibers at high pH (7–11), which needs
(NH4)2SO4). microbial consortia adapting these conditions (Aksu, 2003; Ali et al.,
c
100 mg/L each dye type (Everzol Blue LX, RR 239, RY 84, RY 700, RR 141, 2014; Guo et al., 2021; Imran et al., 2014; Samuchiwal et al., 2021). In
RB 198). this study, the pH values were adjusted at a range of 3–11 and, color

3
S. Tizazu et al.
removal activities of the bacterial consortia were evaluated. The 7 h
decolorization efficiencies were 35.7 ± 0.9, 44.7 ± 0.9, 96.6 ± 1, 99.9
± 0.0 and 64.8 ± 1.0% for reactor adjusted to pH values of 3, 7, 8, 9, and
11, respectively (Fig. 3a and Fig. S3). This result shows that decolor­
ization rate was significantly low under acidic and at pH 11, but
decolorization efficiencies were significantly increased in pH 8 and 9.
This significant decolorization variation could be due to alteration of
bacterial cell surface charge that has negative effect on enzymes
involved in dye decolorization (Guo et al., 2021). In the present study,
pH value of 9 was investigated to be the optimum pH for bacterial
consortia to decolorize RR 141. Similarly, Ayed et al. (2011) reported
99.6% decolorization efficiency result for the azo dye Methyl Red.
Mohanty & Kumar (2021) also reported that the degradation of Indan­
threne Blue RS by microbial consortia was higher in alkaline pH and
significantly reduced in acidic pH values. According to Lalnunhlimi and
Krishnaswamy (2016) the maximum decolorization of DB 151 and DR
31 was observed at a pH value of 9.5.
3.1.2. Temperature
Temperature of the textile effluent from dye baths and rinsing pro­
cesses is usually 50–80 ◦ C (Guo et al., 2021), which could limit the use of
mesophilic microbes for treatment of textile wastewater (Zhang et al.,
2019). The use of thermophilic microbial consortia to decolorize
dye-containing textile wastewater is considered as an effective degra­
dation strategy (Aksu, 2003; Ali et al., 2014; Boonyakamol et al., 2009;
Chen and Chang, 2007; Imran et al., 2014; Samuchiwal et al., 2021). In
the present study, incubation temperature (30–80 ◦ C) showed a signif­
icant (p < 0.05) effect on dye decolorization. The decolorization rate
significantly (p < 0.05) increased with increasing temperatures from
30 ◦ C (41.1 ± 1.0%) to 55 ◦ C (99.9 ± 0.0%), and the temperature above
55 ◦ C resulted in a significant reduction (below 60% at 80 ◦ C) in RR 141
decolorization efficiency (Fig. 3b and Fig. S4). From this result, optimum
incubation temperature for decolorization of RR 141 was observed at
55 ◦ C. Another study by Dos Santos et al. (2005) also reported that the
anthraquinone dyes’ color removal rates were enhanced at 55 ◦ C as
compared with 30 ◦ C. Increased temperatures inhibited bacterial
growth, resulting in a low decolorization rate (Guo et al., 2020a, 2021;
Saratale et al., 2011) or it may lead to the cell viability loss. Another
previous research work by Chen et al. (2020) reported that 55 ◦ C was
found to be the optimum temperature for the decolorization studies. Guo
et al. (2021) also reported that increasing temperature from 30 to 50 ◦ C
enhanced decolorization efficiency, but further increasing reduced the
rate of decolorization, which could be stated due to cell viability loss or
inhibition of the enzymes involved for decolorization. As stated by
Willetts et al. (2000), a high temperature system appears to be superior
for treating dye waste streams, which support the bacterial consortia of
this study could efficiently be applied for bioremediation of actual
textile effluent with increased temperature.
3.1.3. Carbon source
Carbon sources including glucose, maltose, and starch, were used to
test their effect, and one reactor was set as carbon-free (control). The
decolorization of RR 141 was 100% with glucose as a carbon source in 5
h, followed by 94.4 ± 0.6, 76.3 ± 1.00, and 52.8 ± 1.1% with maltose,
starch, and carbon-free reactors, respectively. The best dye decoloriza­
tion efficiency was recorded in glucose provided reactor, suggesting that
glucose was discovered to be the best carbon source. Several prior
studies have identified glucose as the optimum co-substrate for dye
decolorization (Guo et al., 2021; Jiling et al., 2019; Mohanty and Kumar,
2021). Guo et al. (2020b) identified glucose as one of the best carbon
sources for Metanil Yellow G decolorization, which generate NADH that
may serve as an immediate electron donor for azo dye reduction (Guo
et al., 2021). Microorganisms use complex carbon molecules like
starches only when other simple carbon sources are scarce in their sur­
roundings, as seen by the poor decolorization outcome in the starch
supplemented reactor. The carbon-free reactor had the lowest
4
Journal of Environmental Management 323 (2022) 116235
decolorization efficiency, indicating that availability of electron donors
is essential for better dye decolorization. The difference between
carbon-free and starch-provided reactors in 3 h incubation time was not
statistically significant (p > 0.05) (Fig. 3c). This is due to the fact that
bacteria take longer time to convert the complex starch molecule into a
simple and useable form of carbon. When cells are inoculated into new
medium, they may lack necessary enzymes, vitamins, or cofactors,
among other things, that must be generated in order to utilize available
resources. The difference between maltose and glucose provided re­
actors were also not statistically significant (p > 0.05) after 7 h of in­
cubation, suggesting that 7 h incubation is long enough to convert the
disaccharide maltose into two glucose molecules.
3.1.4. Nitrogen source
To figure out the effect of different nitrogen source on dye removal,
the experimental reactors were supplied with organic (yeast extract,
peptone) and inorganic, (sodium nitrate, sodium nitrite, and ammonium
sulfate) nitrogen sources (Fig. 3d). Mineral salt medium without nitro­
gen sources was employed as a control. The decolorization efficiencies
were 27.8 ± 1.0% for NaNO3 (minimum) and 93.7 ± 1.4% for yeast
extract (maximum) after 5 h of incubation. Several previous studies
came up with similar findings that inorganic nitrogen sources signifi­
cantly affect dye removal efficiency than organic sources (Asad et al.,
2007; Guadie et al., 2017; Khehra et al., 2005; Lalnunhlimi and Krish­
naswamy, 2016; Liao et al., 2013). For instance, Khehra et al. (2005)
reported that decolorization of azo dyes requires co-substrates like
peptone, yeast extract, or carbohydrates. Another study by Mathew and
Madamwar (2004) also reported that the use of 0.1% yeast extract for
the decolorization of ranocid fast blue dye showed highest color removal
efficiency than other nitrogen sources. Organic nitrogen sources such as
yeast extract produce NADH, which serves as an electron donor for the
reduction of azo dyes by microorganisms, and thus serve as an essential
medium supplement (Guadie et al., 2017; Guo et al., 2021; Lalnunhlimi
and Krishnaswamy, 2016; Rohit et al., 2019). The decolorization per­
formance of reactors supplied with inorganic nitrogen sources (NaNO3
and NaNO2) showed lower decolorization efficiency than even the
nitrogen-free reactor. The availability of oxygen in NO3 and NO2 com­
pounds may explain the low decolorization performance in inorganic
nitrogen supplemented reactors. As a result, under anaerobic condition
microorganisms most preferentially choose NO3 and NO2 to serve as
final electron acceptor than dyes. With no preferential electron accep­
tors (O2, NO2, and NO3), dye can be used as final electron accepter.
Except for peptone and yeast extract, the remaining nitrogen sources did
not show significant changes in dye decolorization (Fig. 3d). From the
results obtained, it is possible to conclude that the nitrogen sources
supplied to the mineral salts medium had a significant impact on the
bacterial consortia to achieve efficient dye decolorization.
3.1.5. Salt concentration
The effects of different salt concentrations (0.5–30%) on dye decol­
orization performance were studied. The salt concentration of 0.5% was
found to be the optimum concentration for color removal activity of the
consortia. Even with the maximum salt concentration (30%), the con­
sortia were able to decolorize 61.3 ± 2.1% of the dye (Fig. 3e) in 6 h.
Under salinity levels of 1–10%, Guo et al. (2020a) reported that halo­
philic bacterial consortia can decolorize Metanil Yellow G (MYG).
Recently, Guo et al. (2021) also discovered halo-thermophilic bacterial
consortia that can decolorize azo dye at a salt concentration of 10%.
Khalid et al. (2008) stated the possibility of decolorizing two azo dyes in
a salt concentration of 50 g/L. Different strains of Halomonas that
decolorized Remazol Black B under 10–40 g/L NaCl concentration were
discovered (Asad et al., 2007). In the current study, there is no decol­
orization difference between salt concentration of 10 and 15% (p =
1.000), 15 and 20% (p = 0.627), and 10 and 20% (p = 0.612) (Fig. 3e).
However, the dye decolorization activity of the bacterial consortia was
significantly different at its optimal salt concentration (0.5%). Most
S. Tizazu et al. Journal of Environmental Management 323 (2022) 116235

Fig. 3. Optimization of (a) pH, (b) temperature, (c) carbon source, (d) nitrogen source, (e) salt concentration, (f) dye concentration, and (g) dye type.

5
S. Tizazu et al. Journal of Environmental Management 323 (2022) 116235

probably, this is due to the acclimation process which was carried out for of azo bonds results in lower rate of decolorization) available in various
3 months with 0.5% salt concentration in the media which helped the dyes also mentioned to be a reason of variation in the rate of decolor­
microbial consortia to easily adapt and perform best. Microorganisms ization (Guo et al., 2020a, 2021). The Rift Valley hot spring is an
tend to reduce their activity when they are exposed to new environ­ inoculum source for potential candidates for colored wastewater treat­
mental conditions or new nutrient media supplements. Despite having ment because it has been discovered that the thermo-alkaliphilic mi­
the best decolorization results in the lowest salt concentration, the crobial consortia from the hot spring have been found to decolorize
bacterial consortia used in this study had good decolorization activity various commonly used textile dye types.
even at the maximum salt concentration (30%). Textile wastewater is
known for its high salt (15–20%) concentration, thus the bacterial
3.2. Anaerobic-aerobic dye degradation under optimum conditions
consortia of this work could be a promising choice for textile wastewater
bioremediation.
Synthetic textile wastewater was prepared as explained in section 2.3
and incubated under anaerobic conditions and then by aerobic condi­
3.1.6. Dye concentration
tions (Fig. 2), aiming to treat color, and further mineralize aromatic
The bacterial consortia were tested for decolorization of various
amines, respectively. The optimum conditions (temperature = 55 ◦ C,
initial dye concentrations (100–1000 mg/L) (Fig. 3f and Fig. S5). The
pH = 9, carbon = glucose and nitrogen = yeast extract sources) obtained
Rift Valley halotolerant and thermo-alkaliphilic bacterial communities
in section 3.1 were used for biodegradation analysis.
can efficiently decolorize (e.g., 83.3 ± 0.9% at 48 h) the initial dye
Table 2 shows the various physicochemical parameters of untreated
concentrations up to 1000 mg/L. The removal efficiency of all dye
and treated (anaerobically and aerobically) synthetic textile wastewa­
concentrations showed statistically significant (p < 0.05) variation. But,
ters. The dissolved oxygen concentrations were found to be below 0.1
the rate of decolorization was shown to be inversely proportional to the
mg/L for anaerobic and 1.5–3.5 mg/L for aerobic conditions. After
dye concentration. After 6 h incubation period, decolorization efficiency
treatment (anaerobic and aerobic), salinity and pH results have shown a
was 98 ± 1.0 and 9.7 ± 1.1% for reactors incubated with 100 and 1000
significant increasing and decreasing values (Table 2), suggesting that
mg/L initial dye concentrations, respectively. According to Khehra et al.
RR 141 degradation release inorganic components from the structure
(2005), an increased dye concentration leads to a drop in color removal
that increase salinity and produce smaller organic acids that reduce the
percentages. Another previous study by Donkadokula et al. (2020) also
initial pH (initial > anaerobic > aerobic). A significantly lower pH value
stated that the decolorization of Brilliant Green dye reduced as the
observed under aerobic condition also confirmed that more degradation
concentration of the dye in the stock solution increased. Similarly, other
process has been occurred and produced more acidic conditions than
studies by Hossen et al. (2019) and Vaigan et al. (2009) found that an
anaerobic treatments. The concentration of RR 141 and COD were also
increasing dye concentrations lowered dye removal efficiencies. In the
significantly reduced after anaerobic and aerobic treatments (Table 2).
current study, highest decolorization efficiencies were observed after
As shown in Fig. 4a, the thermo-alkaliphilic bacterial consortia under
longer incubation time for high dye concentrations. This could be owing
optimum conditions achieved over 98% for RR 141 in 6 h, while the
to the toxicity of higher dye concentrations, the formation of inorganic
COD removal efficiency were below 5%. Although complete decolor­
ions that impede dye decolorization, or the inhibition of azoreductase
ization was achieved in shorter times under anaerobic condition, the
enzyme active sites that might be adapted through longer exposure time
COD removals were comparatively slow and reached >90% after 3 days
(Khehra et al., 2005; Rauf et al., 2011). According to Mohanty & Kumar
under aerobic condition (Fig. 4a). This result suggests that using thermo-
(2021), the loss of decolorization efficiency at increased dye concen­
alkaliphilic microbial consortia, dye and COD treatment were efficient
trations can be related to the dye’s toxicity on the bacterial consortia or
under anaerobic and aerobic conditions, respectively. For efficient dye
lack of enough biomass concentration that decolorize higher dye con­
removal under anaerobic condition, the most probable reason could be
centrations. In the current study, the bacterial communities that can
due to azo dye used as final electron acceptor under oxygen stress
decolorize initial dye concentrations up to 1000 mg/L within 48 h in­
condition (Guadie et al., 2017; Jadhav et al., 2010). The poor COD
cubation period suggests the potential of Rift Valley hot spring hal­
removal efficiency observed under anaerobic treatment might be due to
otolerant and thermo-alkaliphilic microbial consortia to be applied for
the absence of oxygen in the anaerobic reactor that inhibits the activities
textile wastewater treatment.
of heterotrophic microorganisms which plays an important role in the
removal of COD from the wastewater. Compared to anaerobic treat­
3.1.7. Dye type
ment, aerobic treatment is the major approach for COD removal as the
In addition to the model azo dye (RR 141) investigated, experiments
aerobic bacteria use dissolved oxygen as final electron acceptor to
were carried out to understand the response of Rift Valley hot spring
metabolize organic pollutants.
(Shala) halotolerant and thermo-alkaliphilic microbial consortia to
Nitrate and total nitrogen concentration showed a decreasing trend
other five (Everzol Blue LX, RY 84, RR 239, RB 198, and RY 700)
in the first 24 h under anaerobic condition, while their formation and
commonly used textile dyes (Fig. 3g and Fig. S6a). Maximum and
increments were observed during further aerobic culturing condition.
minimum color removal efficiencies within 7 h incubation periods were
For example, total nitrogen content was 36.9 ± 2.1 mg/L in 24 h
observed for reactors supplemented with RR 239 (99.9 ± 0.0%) and RB
anaerobic treatment, which was increased to 49.2 ± 4.5 mg/L after 48 h
198 (75.9 ± 1%) (Fig. S6b). Indeed, RR 239 has a molecular weight of
1136.32 g/mol, while the molecular weight of RR198 is 1304.80 g/mol
(Table S1), which could be the reason for the observed variation in Table 2
Parameters of untreated, anaerobically and aerobically treated RR 141 con­
decolorization efficiency. The statistical analysis result showed that
taining wastewaters.
removal efficiencies were insignificantly different between Everzol Blue
LX and RB 198 (p = 0.79) and RR 239 and RR 141 (p = 1.000), sug­ Parameter Untreated Anaerobic Aerobic
gesting that these dyes may share similarity in their chemical structure, Retention time (h) 0 3–24 48–120
or they may have comparable number of azo bonds. Previous studies Dissolved oxygen (mg/L) 2.0–4.0 0.05–0.08 1.5–3.5
also mentioned that variation in the rate of decolorization might be due pH 9.0 ± 0.1 8.2 ± 0.3 7.8 ± 0.1
Total dissolved solid (g/L) 5.1 ± 0.4 7.3 ± 0.1 6.8 ± 0.1
to structural variation among the dyes (Guo et al., 2021; Hossen et al., Conductivity (mS/cm) 7.9 ± 0.4 13.2 ± 0.9 10.7 ± 1.5
2019; Khehra et al., 2005). Low molecular weight and structurally NO3–N (mg/L) 158.3 ± 2.5 21.6 ± 0.6 38.1 ± 1.9
simple dyes exhibit higher decolorization rates, while higher molecular Total nitrogen (mg/L) 206.9 ± 9.7 36.9 ± 2.1 49.2 ± 4.5
weight and highly substituted dyes exhibit lower decolorization rates Chemical oxygen demand (mg/L) 1244.3 ± 52.1 1033.4 ± 47.7 290.0 ± 4
Dye concentration (mg/L) 100.00 1.0–7.0 0.0–0.1
(Pearce et al., 2003). Furthermore, number of azo bonds (higher number

6
S. Tizazu et al. Journal of Environmental Management 323 (2022) 116235

Fig. 4. Anaerobic followed by aerobic treatment of Reactive red 141 (a) color and COD removal efficiencies, (b) concentration of NO3–N and total nitrogen. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

under aerobic treatment for RR 141 (Fig. 4b), suggesting that the azo
dye (RR 141) further degraded under aerobic condition. Indeed, nitro­
gen containing aromatic compounds can be mineralized under aerobic
condition and increase the various nitrogen species (like ammonia, ni­
trate and nitrite) concentration (Guadie et al., 2021).
In general, this finding shows that the bacterial consortia used in this
study is capable of decolorizing textile dyes at high pH, temperature,
and salt concentrations (Figs. 3 and 4). This indicates that the microbial
consortia is characterized by alkaliphilic, thermophilic, and halophilic
natures, which makes this report interesting to use Rift Valley hot spring
microbial consortia to degrade color containing wastewater at high pH,
temperature, and salinity conditions. According to Guo et al. (2021)
investigation, a halo-thermophilic bacterial consortium enriched at
50 ◦ C and 10% salinity degraded 92.1% of Metanil Yellow G after 36 h.
Chen et al. (2018) revealed that a newly isolated thermophilic micro­
flora decolorized 97% of Direct Black G textile dye in 8 h. Guo et al.
(2020a) described the ability of the bacterial consortium named GG-1 to
decolorize azo dyes at salinities ranging from 1 to 10%.
The decolorization efficiency of the bacterial consortia in this study
was compared with other previous researches to learn more about hal­
otolerant and thermo-alkaline environmental sample inoculum for
textile dye treatment (Table 3). The halotolerant and thermo-alkaliphilic
bacterial consortia collected from dye-uncontaminated Rift Valley hot
spring (Shala) showed complete decolorization in 6 h, compared to
92.1–100.0% decolorization in 9–48 h for microorganisms from textile
wastewater sludge (Table 3). Textile waste contaminated soil, on the
other hand, showed relatively efficient (94.8%) dye decolorization at
shorter (5 h) incubation period (Guo et al., 2020a). Lalnunhlimi and
Krishnaswamy (2016) isolated four bacterial strains from soil samples of
saline environments and used them as a moderately alkaliphilic bacte­
rial consortium for decolorization of DB 151 and DR 31. Compared with
the consortium of Lalnunhlimi and Krishnaswamy (2016), which
decolorized 87% of the dyes in 120 h, the bacterial consortium of this
study decolorized 99.9 ± 0.0% of RR 141 dye in 6 h. Similarly, Cinar
et al. (2008) also reported that 72% dye and 75% COD removal effi­
ciencies within 24 h using anaerobic sequencing batch reactor.
Compared to this report, the result of the current study that achieved
complete color removal in 7 h and a total of 93% COD removal were
found to be by far more efficient. This is because the consortia of this
study might consist of more abundant and diverse bacterial strains
which works synergistically and achieved greater efficiency. Thus, the
halotolerant and thermo-alkaliphilic bacterial consortia from Shala hot
spring in this study are the best options for degradation of azo dyes in
extreme environments of textile wastewater, compared to mesophilic
microorganisms, whose decolorization efficiency was limited due to the
harsh characteristics of textile wastewater (Ali et al., 2014; Asad et al.,
2007).

Table 3
Comparison of textile dye removal efficiency of this study and other previous studies.
Inoculum source Dye type (Conc. mg/L) Treatment type Temperature Salinity pH Time Decolor References
(◦ C) (%) (h) (%)

Hot spring sediment RR 141 (100) Anaerobic/ 55 0.5–30 9 6 99.9 This study
aerobic
Textile wastewater sludge MYG (100) Anaerobic 50 10 8 36 92.1 Guo et al. (2021)
Waste contaminated soil DBG (600) Microaerobic 55 5 8 48 97.0 Chen et al. (2018)
Waste contaminated soil Metanil yellow (100) Microaerobic - 5 6 5 94.8 Guo et al. (2020a)
Textile wastewater sludge MYG (100) Anaerobic 50 1 10 48 93.3 Guo et al. (2020b)
Textile wastewater sludge AR 88 (20) Anaerobic 35 - 7 24 99.9 Khehra et al. (2005)
Dye contaminated soil Actual (100) Anaerobic 30 - 7 20 99.9 Phugare et al. (2011)
Dye contaminated soil Blue RS (100) Aerobic 35 - 10 9 99.9 Mohanty & Kumar (2021)
Dye contaminated soil RO 16 (100) Anaerobic 30 - 7 48 99.9 Jadhav et al. (2010)
Alkaline lake sediment RR 239 (100) Anaerobic 30 - 10 24 99.9 Guadie et al. (2017)
Dye contaminated effluent RO 16 (50) Aerobic 37 - 11 24 99.9 Saha & Rao (2020)
Dye contaminated soil Scarlet R (50) Anoxic 37 - 7 30 89.8 Saratale et al. (2009)
Saline soil sample DB 151/DR 31 (100) Aerobic 36 - 9.5 120 87.0 Lalnunhlimi & Krishnaswamy
Wastewater treatment Remazol brilliant Anaerobic/ 25 - 7.2 24 72 (2016)
plant violet 5R Aerobic 70 - 7 14 90 (Cinar et al., 2008)
Thermophilic reactor Acid Orange 7 Anaerobic 35 - 7 24 100 Zhang et al. (2019)
Sludge samples Acid Red 88 (20) Aerobic 35 - 8 48 73 Khehra et al. (2005)
Textile effluent Textile effluent (98 Anaerobic Samuchiwal et al. (2021)
mL)
-
= Not considered, A = Acid Red, DB = Direct Blue, DBG = Direct Black G, D = Direct Red, MYG = Metanil Yellow G, RO = Reactive Orange, RR = Reactive Red

7
S. Tizazu et al. Journal of Environmental Management 323 (2022) 116235

3.3. Metagenomic analysis

To evaluate the community structure of the bacterial consortia from

aerobic and anaerobic reactors, high-throughput sequencing was used to
amplify the V3–V4 hypervariable regions of the 16S rRNA gene. At 97%
similarity cutoff value, all samples have coverage above 0.98 and total
OTUs number of 3005. The initial sample showed higher OUTs number
(2181) than anaerobic (2135) and aerobic (1844) culturing conditions
(Table 4), suggesting that the initial microbial community collected
from Shala hot spring sediment sample was influenced by the acclima­
tization conditions, and the characteristics of the synthetic wastewater.
The microbial structure richness in the system was evaluated using
Chao1 index and it was observed in ranges from 2478 to 2708. The
Shannon index showed that highest bacterial diversity (7.4) was recor­
ded for initial sample followed by An-RR 141 (5.2) and Ar-RR 141 (6.4).
Shala hot spring microorganisms which have no any contact with dye
wastewater surprisingly adapted the dye contaminated environment
with higher diversity during aerobic culturing condition than anaerobic
one. However, the acclimatization and treatment conditions reduced
some bacterial species in the initial sample; specifically, the anaerobic
conditions much more significantly reduced the thermo-alkaliphilic Fig. 5. Principal component analysis.
microorganisms than the aerobic conditions.
Fig. 5 shows the principal component analysis of the bacterial di­
versity, and it indicates that the aerobic (Ar-RR 141) and anaerobic (An-
RR 141) treatments showed a separate cluster with initial samples,
suggesting that the treatment or acclimatization conditions determined
the bacterial diversity structure. The presence of the aerobic, anaerobic
and initial samples in three separate clusters clearly shows the effect of
the different environmental conditions on shaping bacterial community
structure. For example, microorganisms in anaerobic reactor are those
which are capable of existing under stressed dissolved oxygen condition
(Fig. 5). The physicochemical results of pH, COD and TN values
observed also contribute in shaping the initial microbial community
structures in aerobic and anaerobic conditions.
The microbial community structure of anaerobic, aerobic and initial
samples at phylum and class levels were shown in Fig. 6. Fourteen
bacterial phyla covered 93.2% of all the sequences, while others (50
phyla) accounted only 6.8% (Fig. 6a). Anaerobic reactor was dominated
by ten bacterial phyla (88.6%), while the aerobic and initial samples
were dominated by seven (86.5%) and six (77.6%) phyla, respectively.
Bacteroidota represented the most dominant followed by Proteobac­
teria, Chloroflexi and Halobacterota among anaerobic, aerobic and
initial samples. Particularly, Proteobacteria (25.4%) was found to be
dominant for aerobic treatment, while Bacteroidota (25.3%) and
Chloroflexi (20.5%) accounted higher abundance in anaerobic treat­
ments (Fig. 6a), suggesting that the treatment types shaped microbial
community structure. This finding is consistent with another study that
reported Bacteroidota (31.1%), Proteobacteria (9.6%) and Chloroflexi
(7.7%) abundance in constructed wetland designed for textile dye
wastewater treatment (Patel et al., 2021). Similarly, Zhao et al. (2018)
reported Proteobacteria, Chloroflexi, and Bacteroidota with abundance
ranges of 12.3–58.5, 2.8–37.7, and 0.7–19.2% in different wastewater
treatment plants, respectively. Guadie et al. (2021) also reported Pro­ Fig. 6. Microbial community structure before and after treatment (a) phylum
teobacteria, Chloroflexi and Bacteroidota as the dominant three level, and (b) class level.

bacterial phyla identified from wastewater treated under anaerobic,

Table 4 aerobic and microaerobic conditions.
Microbial diversity of Shala hot spring before and after treatment. Bacteroidota represented the most dominant (23–27%) phylum of
Sample Tag OTU Index at 97% Coverage the total sequences for initial, anaerobic and aerobic treatments. Bac­
code number number
Shannon Simpson Chao1 teroidia, which is a member of phylum Bacteroidota was found to be the
most dominant (23.3%) class in all the treatments. Furthermore, Bac­
Initial 54,021 2181 7.4 0.01471 2699 0.98891
An-RR 54,991 1844 5.2 0.01959 2708 0.98907
teroidetes vadinHA17 was the most dominant family under phylum
141 Bacteroidota (Fig. S7). This family of bacteria is more dominant in
Ar-RR 53,603 2135 6.4 0.0144 2478 0.98937 anaerobic reactor (14.8%) than that of aerobic reactor (4.2%). Previ­
141 ously, the Bacteroidetes vadinHA17 has been reported as proteins and
An = Anaerobic, Ar = Aerobic, RR 141 = Reactive Red 141. complex sugar polymer degraders under anaerobic condition (Mei et al.,

8
S. Tizazu et al.
2020; Patel et al., 2021; Zhang et al., 2018). According to Mei et al.
(2020), Bacteroidota populations that have lineages to the family Bac­
teroidetes vadinHA17 revealed as the dominant proteolytic amino acid
degraders in anaerobic digesters. According to Zhao et al. (2018), the
Bacteroidetes vadinHA17 family stated their function linked to pro­
teinaceous compound breakdown in wastewater treatments. In this
study, Bacteroidota particularly Bacteroidetes vadinHA17 are believed
to play a significant role in nitrogen and COD removal activities from
textile dye containing effluent. Patel et al. (2021) also reported that
Bacteroidota, in their treatment system played a crucial role in biore­
mediation of dyestuff containing wastewater.
The second dominant phylum was Proteobacteria (17–25%) for
aerobic and anaerobic treatments. In this phylum, the two dominant
classes are Gammaproteobacteria (14–21%) and Alphaproteobacteria
(2–5%) (Fig. 6b). In biological wastewater treatments, Proteobacteria
plays an important role in biodegradation and floccule formation (Xin
et al., 2008). Some groups of Proteobacteria are commonly known as
ammonia-oxidizing Proteobacteria, which play an important role in
wastewater treatments, where they get rid of excess ammonia by con­
verting it to nitrite, which is then converted to nitrate by other bacteria.
Thus, this group of bacteria might also be responsible for the nitrogen
removal (Ajibade et al., 2021) task of this study. Gammaproteobacteria
is responsible for degradation of ammonia from the wastewater (Guadie
et al., 2021).
Chloroflexi was the third dominant phylum detected in the waste­
water samples with a proportion of 20.5% and 16.5% for anaerobic and
aerobic reactors, respectively (Fig. 6a). In bacterial classification,
phylum Chloroflexi consisted of six classes such as Chloroflexia, Ther­
momicrobia, Dehalococcoidetes, Anaerolineae, Caldilineae and Ktedo­
nobacteria. Among these, Anaerolineae and Caldilineae were found to
be the dominant classes of phylum Chloroflexi obtained in this study
(Fig. 6b). Proportion of Anaerolineae was 19.1 and 15.0% in anaerobic
and aerobic reactor, respectively. Anaerolineae was the second most
dominant class (17.1%) when considering all the samples. At family
level, Anaerolineaceae was the most dominant group under phylum
Chloroflexi and the second most dominant (9.1%) family when consid­
ering all the samples (Fig. S7). Chloroflexi play an important environ­
mental role in the degradation of carbohydrates and cellular materials
(Hu et al., 2012). All the species in the Anaerolineaceae family are
chemoheterotrophs, which are either mesophilic or thermophilic.
Anaerolineaceae was reported for its role in removing nitrogen from
wastewaters (Tian et al., 2019). Caldilineaceae also belongs to phylum
Chloroflexi, and they are thermophilic, growing in both aerobic and
anaerobic environments. They are all chemoheterotrophs, which can
thrive on a variety of sugars and proteinaceous substances. Caldilinea­
ceae are closely linked to wastewater treatment systems, and their role
in wastewater treatment is reducing the level of COD and eliminating
nitrogen-containing chemicals such as ammonia (Guadie et al., 2021).
Caldilineaceae were discovered in thermophilic environments like hot
springs and aquifers, which is evident in our treatment cultured at 55 ◦ C.
Ajibade et al. (2021) investigated Caldilineaceae as the most dominant
(0.73–8.6%) family in secondary wastewater effluent.
Three harsh environmental conditions were fitted to the microbial
consortia derived from Shala hot spring, including elevated pH, tem­
perature, and salt concentration, all of which are features of textile
effluent. Thus, the bacterial consortia’s excellent color and COD removal
performance at elevated temperature, pH, and salt concentration was
positively correlated with the Rift Valley hot spring environmental
conditions including high pH (8.93 ± 0.7), temperature (82.4 ± 1.2 ◦ C),
salt (5.4 ± 0.1%), and conductivity (9.6 ± 0.5 mS/cm) values. Peptone
water, yeast extract, and beef extract are all plausible sources of
nitrogen-containing compounds in the ATCC 697 medium employed in
this study. In addition, aromatic amines formed from azo dye (RR 141)
during anaerobic treatment most probably further mineralized and used
as a nitrogen source. The prevalence of nitrogen compound degrader
bacteria such as Bacteroidetes vadinHA17, Anaerolineaceae,
9
Journal of Environmental Management 323 (2022) 116235
Methanosarcinacea, and Caldilineaceae is most likely due to these fac­
tors. Sugar-containing compounds may be formed as a result of the pure
sugar (glucose) in the MSM medium and the yeast extract used, and
these constituents may be responsible for the presence of Chloroflexi and
Proteobacteria, which are carbohydrate and sugar-containing com­
pound degraders and thus, remove COD from the wastewater (Fig. 4a).
The salt content of the ATCC 697 medium is also derived from peptone
water and sodium chloride, and this salt composition is responsible for
the occurrence of Halobacterota as the major bacterial phylum (Fig. 6a).
4. Conclusions
Under optimum operating conditions of pH (9), temperature (55 ◦ C)
and salinity (0.5%), Rift Valley hot spring microbial consortia used in
this study effectively remove color (anaerobic) and COD (aerobic). The
ability of the consortia to decolorize six structurally different reactive
dyes and dye concentration ranges of 100–1000 mg/L RR 141 also
indicated the potential of Rift Valley hot spring microbial consortia for
textile wastewater treatment. Bacteroidota, Chloroflexi, Proteobacteria,
and Halobacterota were the four most prevalent phyla. Bioremediation
of textile effluent with high alkalinity, temperature, and salt content
seems to be an excellent fit for these halotolerant and thermo-
alkaliphilic Rift Valley hot spring bacterial consortia.
Author contributions staement
Samson Tizazu: Conceptualization; Methodology; Formal analysis;
Investigation; Data Curation; Writing-Original Draft. Getaneh Tesfaye:
Funding acquisition; Resources; Supervision. Berhanu Andualem:
Methodology; Writing-Review & Editing; Supervision. Aijie Wang:
Software; Writing-Review & editing; Validation. Awoke Guadie:
Conceptualization; Methodology; Formal analysis; Investigation;
Writing-Review & Editing; Supervision.
Declaration of competing interest
The authors declare that they have no known competing finantncial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
The data that has been used is confidential.
Acknowledgement
This work was supported by Arba Minch University (AMU/CNS/
371/04/01/6223) and Chinese Academy of Sciences-President’s Inter­
national Fellowship Initiative (2019PE0033).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jenvman.2022.116235.
References
Ajibade, F.O., Wang, H.-C., Guadie, A., Ajibade, T.F., Fang, Y.-K., Sharif, H.M.A., Liu, W.-
Z., Wang, A.-J., 2021. Total nitrogen removal in biochar amended non-aerated
vertical flow constructed wetlands for secondary wastewater effluent with low C/N
ratio: microbial community structure and dissolved organic carbon release
conditions Bioresour. Technol. 322, 124430.
Aksu, Z., 2003. Reactive dye bioaccumulation by Saccharomyces cerevisiae. Process
Biochem. 38, 1437–1444.
Ali, S., Khatri, Z., Khatri, A., Tanwari, A., 2014. Integrated desizingebleachingereactive
dyeing process for cotton towel using glucose oxidase enzyme. J. Clean. Prod. 66,
562–567.
S. Tizazu et al.
APHA, 1999. American Public Health Association. Standard Methods for the
Examination of Water and Wastewater. American Public Health Association,
Washington, D.C, USA.
Asad, S., Amoozegar, M.A., Pourbabaee, A.A., Sarbolouki, M.N., Dastgheib, S.M., 2007.
Decolorization of textile azo dyes by newly isolated halophilic and halotolerant
bacteria. Bioresour. Technol. 98, 2082–2088.
Ayed, L., Mahdhi, A., Cheref, A., Bakhrouf, A., 2011. Decolorization and degradation of
azo dye Methyl Red by an isolated Sphingomonas paucimobilis: biotoxicity and
metabolites characterization. Desalination 274, 272–277.
Boonyakamol, A., Imai, T., Chairattanamanokorn, P., 2009. Key factors regarding
decolorization of synthetic anthraquinone and azo dyes. Appl. Biochem. Biotechnol.
158, 180–191.
Bremner, J.M., Mulvaney, C.S., 1982. Nitrogen-total. In: A, L., Miller, R.H., Keeney, D.R.
(Eds.), Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties,
Page, American Society of Agronomy. Soil Science Society of America, Madison,
Wisconsin, pp. 595–624.
Chen, B.-Y., Chang, J.-S., 2007. Assessment upon species evolution of mixed consortia for
azo dye decolorization. J. Chin. Inst. Chem. Eng. 38, 259–266.
Chen, G., An, X., Feng, L., Xia, X., Zhang, Q., 2020. Genome and transcriptome analysis
of a newly isolated azo dye degrading thermophilic strain Anoxybacillus sp.
Ecotoxicol. Environ. Saf. 203, 111047.
Chen, Y., Feng, L., Li, H., Wang, Y., Chen, G., Zhang, Q., 2018. Biodegradation and
detoxification of Direct Black G textile dye by a newly isolated thermophilic
microflora. Bioresour. Technol. 250, 650–657.
Cinar, O, Yasar, S, Kertmen, M, Demiroz, K, Yigit, N.O, Kitis, M, 2008. Effect of cycle time
on biodegradation of azo dye in sequencing batch reactor. Process Saf. Environ. Prot.
86, 455–460.
Donkadokula, N.Y., Kola, A.K., Saroj, D., 2020. Modelling and optimization studies on
decolorization of brilliant green dye using integrated nanofiltration and
photocatalysis. Sustain. Environ. Res. 30.
Dos Santos, A.B., Bisschops, I.A.E., Cervantes, F.J., Van Lier, J.B., 2005. The
transformation and toxicity of anthraquinone dyes during thermophilic (55 ◦ C) and
mesophilic (30 ◦ C) anaerobic treatments. J. Biotechnol. 115, 345–353.
Gnanapragasam, G., Senthilkumar, M., Arutchelvan, V., Velayutham, T., Nagarajan, S.,
2011. Bio-kinetic analysis on treatment of textile dye wastewater using anaerobic
batch reactor. Bioresour. Technol. 102, 627–632.
Guadie, A., Gessesse, A., Xia, S., 2018. Halomonas sp. strain A55, a novel dye decolorizing
bacterium from dye-uncontaminated Rift Valley Soda lake. Chemosphere 206,
59–69.
Guadie, A., Han, J.L., Liu, W., Ding, Y.C., Minale, M., Ajibade, F.O., Zhai, S., Wang, H.C.,
Cheng, H., Ren, N., Wang, A., 2021. Evaluating the effect of fenton pretreated
pyridine wastewater under different biological conditions: microbial diversity and
biotransformation pathways. J. Environ. Manag. 287, 112297.
Guadie, A., Tizazu, S., Melese, M., Guo, W., Ngo, H.H., Xia, S., 2017. Biodecolorization of
textile azo dye using Bacillus sp. strain CH12 isolated from alkaline lake. Biotechnol.
Rep. 15, 92–100.
Guo, G., Hao, J., Tian, F., Liu, C., Ding, K., Zhang, C., Yang, F., Xu, J., 2020a.
Decolorization of Metanil Yellow G by a halophilic alkalithermophilic bacterial
consortium. Bioresour. Technol. 316, 123923.
Guo, G., Li, X., Tian, F., Liu, T., Yang, F., Ding, K., Liu, C., Chen, J., Wang, C., 2020b. Azo
dye decolorization by a halotolerant consortium under microaerophilic conditions.
Chemosphere 244, 125510.
Guo, G., Liu, C., Hao, J., Tian, F., Ding, K., Zhang, C., Yang, F., Liu, T., Xu, J., Guan, Z.,
2021. Development and characterization of a halo-thermophilic bacterial
consortium for decolorization of azo dye. Chemosphere 272, 129916.
Hossen, M.Z., Hussain, M.E., Hakim, A., Islam, K., Uddin, M.N., Azad, A.K., 2019.
Biodegradation of reactive textile dye Novacron Super Black G by free cells of newly
isolated Alcaligenes faecalis AZ26 and Bacillus spp obtained from textile effluents.
Heliyon 5, e02068.
Hu, M., Wang, X., Wen, X., Xia, Y., 2012. Microbial community structures in different
wastewater treatment plants as revealed by 454-pyrosequencing analysis. Bioresour.
Technol. 117, 72–79.
Imran, M., Crowley, D.E., Khalid, A., Hussain, S., Mumtaz, M.W., Arshad, M., 2014.
Microbial biotechnology for decolorization of textile wastewaters. Rev. Environ. Sci.
Biotechnol. 14, 73–92.
Jadhav, P., Kalyani, C., Telke, A., Phugare, S., Govindwar, P., 2010. Evaluation of the
efficacy of a bacterial consortium for the removal of color, reduction of heavy
metals, and toxicity from textile dye effluent. Bioresour. Technol. 101, 165–173.
Jiling, C., Edmond, S., Wenhua, L., Wei, Z., Ying, L., 2019. Decolorization and
detoxification of Direct Blue 2B by indigenous bacterial consortium. J. Environ.
Manag. 242, 229–237.
10
Journal of Environmental Management 323 (2022) 116235
Kalyani, D.C., Patil, P.S., Jadhav, J.P., Govindwar, S.P., 2008. Biodegradation of reactive
textile dye Red BLI by an isolated bacterium Pseudomonas sp. SUK1. Bioresour.
Technol. 99, 4635–4641.
Kargi, F., Dincer, A.R., 2000. Use of halophilic bacteria in biological treatment of saline
wastewater by fed-batch operation. Water Environ. Res. 72, 170–174.
Khalid, A., Arshad, M., Crowley, D.E., 2008. Decolorization of azo dyes by Shewanella sp.
under saline conditions. Appl. Microbiol. Biotechnol. 79, 1053–1059.
Khehra, M., Saini, H., Sharma, D., Chadha, B., Chimni, S., 2005. Decolorization of
various azo dyes by bacterial consortium. Dyes Pigments 67, 55–61.
Lalnunhlimi, S., Krishnaswamy, V., 2016. Decolorization of azo dyes (Direct Blue 151
and Direct Red 31) by moderately alkaliphilic bacterial consortium. Braz. J.
Microbiol. 47, 39–46.
Liao, C.-S., Hung, C.-H., Chao, S.-L., 2013. Decolorization of azo dye Reactive Black B by
Bacillus cereus strain HJ-1. Chemosphere 90, 2109–2114.
Mathew, S., Madamwar, D., 2004. Decolorization of ranocid fast blue dye by bacterial
consortium SV5. Appl. Biochem. Biotechnol. 118, 371–381.
Mei, R., Nobu, M.K., Narihiro, T., Liu, W.-T., 2020. Metagenomic and metatranscriptomic
analyses revealed uncultured bacteroidales populations as the dominant proteolytic
amino acid degraders in anaerobic digesters. Front. Microbiol. 11, 593006.
Mohanty, S., Kumar, A., 2021. Enhanced degradation of anthraquinone dyes by
microbial monoculture and developed consortium through the production of specific
enzymes. Sci. Rep. 11, 7678.
Patel, D., Bapodra, S.L., Madamwar, D., Desai, C., 2021. Electroactive bacterial
community augmentation enhances the performance of a pilot scale constructed
wetland microbial fuel cell for treatment of textile dye wastewater. Bioresour.
Technol. 332, 125088.
Pearce, C., Lloyd, J., Guthrie, J., 2003. The removal of colour from textile wastewater
using whole bacterial cells: a review. Dyes Pigments 58, 179–196.
Phugare, S., Kalyani, C., Surwase, N., Jadhav, P., 2011. Ecofriendly degradation,
decolorization and detoxification of textile effluent by a developed bacterial
consortium. Ecotoxicol. Environ. Saf. 74, 1288–1296.
Rauf, M.A., Meetani, M.A., Hisaindee, S., 2011. An overview on the photocatalytic
degradation of azo dyes in the presence of TiO2 doped with selective transition
metals. Desalination 276, 13–27.
Rohit, R., Kunal, J., Datta, M., Chirayu, D., 2019. Microaerophilic biodegradation of raw
textile effluent by synergistic activity of bacterial community DR4. J. Environ.
Manag. 250, 109549.
Saha, P., Rao, B., 2020. Biotransformation of Reactive Orange 16 by alkaliphilic
bacterium Bacillus fexus VITSP6 and toxicity assessment of biotransformed
metabolites. Int. J. Environ. Sci. Technol. 17, 99–114.
Samuchiwal, S., Gola, D., Malik, A., 2021. Decolourization of textile effluent using native
microbial consortium enriched from textile industry effluent. J. Hazard Mater. 402,
123835.
Saratale, R.G., Saratale, G.D., Chang, J.S., Govindwar, S.P., 2011. Bacterial
decolorization and degradation of azo dyes: a review. J. Taiwan Inst. Chem. Eng. 42,
138–157.
Saratale, R.G., Saratale, G.D., Kalyani, D.C., Chang, J.S., Govindwar, S.P., 2009.
Enhanced decolorization and biodegradation of textile azo dye Scarlet R by using
developed microbial consortium-GR. Bioresour. Technol. 100, 2493–2500.
Tian, T., Zhou, K., Xuan, L., Zhang, J.-X., Li, Y.-S., Liu, D.-F., Yu, H.-Q., 2019. Exclusive
microbially driven autotrophic iron-dependent denitrification in a reactor inoculated
with activated sludge. Water Res. 170, 115300.
Vaigan, A., Alavi Moghaddam, R., Hashemi, H., 2009. Effect of dye concentration on
sequencing batch reactor performance Iran. J. Environ. Health. Sci. Eng. 6, 11–16.
Willetts, M.R., Ashbolt, J.N., Moosbrugger, E.R., Aslam, R.M., 2000. The use of a
thermophilic anaerobic system for pretreatment of textile dye wastewater. Water Sci.
Technol. 42, 309–316.
Xin, J., Mingchao, M., Jun, L., Anhuai, L., Zuoshen, Z., 2008. Bacterial diversity of active
sludge in wastewater treatment plant Earth. Sci. Frontier 15, 163–168.
Zhang, B., Xu, X., Zhu, L., 2018. Activated sludge bacterial communities of typical
wastewater treatment plants: distinct genera identifcation and metabolic potential
diferential analysis. Amb. Express 8, 184.
Zhang, F., Guo, X., Qian, D.K., Sun, T., Zhang, W., Dai, K., Zeng, R.J., 2019.
Decolorization of Acid Orange 7 by extreme-thermophilic mixed culture. Bioresour.
Technol. 291, 121875.
Zhao, J, Feng, C, Tong, S, Chen, N, Dong, S, Peng, T, Jin, S, 2018. Denitrification
behavior and microbial community spatial distribution inside woodchip-based solid-
phase denitrification (W-SPD) bioreactor for nitrate contaminated water treatment.
Bioresour. Technol. 249, 869–879.