Water Air Soil Pollut (2023) 234:359
https://doi.org/10.1007/s11270-023-06377-7
Newly Isolated Ligninolytic Bacteria and Its Applications
for Multiple Dye Degradation
Farida Rahayu · Irfan Mustafa · Marjani ·
Fatkhur Rochman · Raina Aman Qazi ·
Khan Zeb · Nabi Ullah
Received: 20 December 2022 / Accepted: 17 May 2023
© The Author(s) 2023
Abstract The textile industry is expanding globally
and is considered the backbone of the world’s largest
source of foreign exchange. The development of the
textile industry has caused environmental contamina-
tion due to its dye waste, which is complex and very
difficult to resolve with chemical and physical treat-
ments. Azo dye is one of the most widely used dyes
in textile and other industries. It is one of the signifi-
cantly toxic dyes, and when released in water bodies,
it causes a serious threat to the environment. A bac-
terial strain having the potential to degrade a variety
of azo dyes such as Congo red (CR), methylene blue
(MB), Alizarin Red S (AR), and Remazol Brilliant
Blue R (RBBR) was isolated from soil samples in
the wood weathering area and further identified and
characterized. Ligninolytic microorganisms produce
laccase enzymes, lignin peroxidase, manganese per-
oxidase, and other enzymes that can decolorize dye
F. Rahayu (*)
Research Center for Applied Microbiology, National
Research and Innovation Agency, Bogor 16911, Indonesia
e-mail: fari021@brin.go.id
I. Mustafa
Department of Biology, Faculty of Mathematics
and Natural Sciences, Brawijaya University, Jl. Veteran,
Ketawanggede, Lowokwaru, Malang, East Java 65145,
Indonesia
Marjani · F. Rochman
Research Center for Horticultural and Plantation, National
Research and Innovation Agency, Bogor 16911, Indonesia
waste from the textile industry. The research phase
was qualitative and quantitative tests of ligninolytic
bacteria in the decolorization process using several
selected synthetic dyes, antagonism tests, and iden-
tification of potential bacteria based on 16S rDNA
gene sequences. The L11 isolate showed high per-
formance on CR dye of 82.79%, L1 isolate on dye
AR of 40.51%, L7 isolate on dye MB of 38.69%, and
L8 isolate on RBBR dye of 30.34%. The L11 isolate
with the highest potency was identified as Bacillus
paramycoides K7.2 with a similarity of 99.71%. After
7 days of incubation, the quantitative test findings
are the same as the qualitative test results, with iso-
late L11 having the largest clear zone on CR, AR, and
RBBR dyes.
Keywords Azo-dye · Anaerobic · Biodegradation ·
Lignin
R. A. Qazi
Department of Chemistry, Shaheed Benazir Bhutto
Women University, Larama Peshawar 25120, Pakistan
K. Zeb
Department of Chemistry, Abdul Wali Khan University
Mardan, Mardan 23200, Pakistan
N. Ullah (*)
Department of Inorganic and Analytical Chemistry,
Faculty of Chemistry, University of Lodz, Tamka 12,
90‑403 Lodz, Poland
e-mail: nabi.ullah@chemia.uni.lodz.pl
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1 Introduction
The textile industry is one of the essential contribu-
tors to the world economy, but it is also involved in
many industrial hazardous chemical effluents. Lim-
ited usage of natural dyes and growing textile demand
have significantly increased the usage of synthetic
dyes, which contributed to dye wastewater and has
become a substantial source of severe environmen-
tal concerns in modern times. The synthetic dye
waste generated is supposed to damage the environ-
ment because they seem to be insoluble in water and
harmful to aquatic living organisms such as plants,
animals, and microbes, and used in the batik tex-
tile industry, producing substantial long-term health
impacts (Dewi et al., 2018). The dye waste in the
water reduces the appeal, prevents sunshine from
penetrating the water, disrupting aquatic organisms’
photosynthetic activity, decreasing oxygen availabil-
ity in the water, and triggering anaerobic exercise,
which generates undesirable odor compounds (Dafale
et al., 2008). Concerning volume and effluent compo-
sition, textile industry wastewater is the most pollut-
ing of all industrial sectors because the textile indus-
trial effluents have high chemical oxygen demand
(COD), biochemical oxygen demand (BOD), color,
pH, and metal ions, making it difficult to treat such
effluents (Mezohegyi et al., 2009; Senan & Abraham,
n.d.). As a result, textile industries produce millions
of liters of untreated wastewater per day, discharged
directly into chugging water resources like rivers and
lakes. In addition, the changes in the pH raise the
BOD and COD levels and produce intense coloration
(Telke et al., 2010). Most of the artificial dyes used
in the textile industry are azo-based dyes accounting
for the most significant chunk. The discharge of these
dyes into the environment is indeed a public health
concern. Additionally, textile dyes reduce the aes-
thetic appeal of water bodies by raising the biochemi-
cal and chemical oxygen demand, which reduces pho-
tosynthesis, stunts plant development, enters the food
chain, causes recalcitrance and bioaccumulation, and
may even be poisonous, mutagenic, and carcinogenic
(Mudhoo et al., 2020). The azo group accounts for
approximately 60–70% of synthetic colors manufac-
tured worldwide. As a result, the azo dye class is the
most often utilized artificial dye class in the indus-
trial sector. In addition, azo dyes are more commonly
employed in staining because they are easier to get,
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Water Air Soil Pollut (2023) 234:359
offer a broader range of hues, and are less expensive
than natural dyes. However, their presence in a water
body is full of health risks due to their mutagenic and
carcinogenic behavior, and they are challenging to
handle chemically or photolytically (Sudiana et al.,
2018). Diazotized amine, an amine/phenol, and one
or more azo links are the main component of azo
dyes. Approximately 300 distinct azo dyes are exten-
sively used in the textile, paper, food, cosmetics, and
pharmaceutical sectors. Previous research has looked
at the impact of pH, temperature, the nature and
amount of oxygen substrate, and oxygen consumption
on the rate of biological reduction of a range of azo
dyes (Wuhrmann et al., 1980).
Treatment of synthetic dye-containing wastewa-
ter has traditionally been carried out using appropri-
ate conventional techniques (physical or chemical)
such as flocculation, coagulation, adsorption, mem-
brane filtration, precipitation, irradiation, ozoniza-
tion, and Fenton oxidation. However, it may produce
large quantities of chemical sludge, whose removal
in a secure landfill increases process cost (Rajeswari,
2014; Lodha & Chaudhari, 2007; Kumar et al., 2006).
As a result, novel and environmentally friendly
wastewater treatment methods are desirable. Several
published studies have described the decolorization
of untreated wastewater by ligninolytic microorgan-
isms such as Shewanella decoloration is MBTD16,
Bacillus amyloliquefaciens, and Paenibacillus glu-
canolyticus SLM1 representing oxidative enzymes
such as laccases and lignin peroxidases (Babu et al.,
2013; Lončar et al., 2014; Mathews et al., 2016). The
obtained data from these works confirmed the usage
of ligninolytic bacteria is one of the most appealing
remediation options.
Bacteria secrete enzymes that can degrade lignin
and organic compounds, such as lignin peroxidase,
lignin manganese, and laccase enzymes, and cer-
tain microorganisms, such as ligninolytic bacteria,
act as bioremediation agents. The ligninolytic bac-
teria as bioconversion agents are applied in various
issues. As a result, this novel bacterium is applied
in this research to determine its potential in waste
management, specifically azo-dye decolorization,
a competent organism to resolve these textile waste
problems. The primary goal is to find high-potency
ligninolytic bacteria strains that can approach azo-dye
treated wastewater and act as bioremediation agents.
In this regard, the ligninolytic bacteria as functional
Water Air Soil Pollut (2023) 234:359
bioremediation agents are isolated and characterized
for remediation application. In the experiment, the
activity of color-degrading enzymes in isolated bac-
teria is evaluated spectrophotometrically by spectro-
photometry to assess the movement of color-degrada-
tion enzymes in isolated bacteria. In addition, the rate
of color removal efficiency is used to determine the
capability of bacterial lignin peroxidases in decolor-
izing azo-dye-containing wastewater. This study pro-
vides a milestone for low-cost and environmentally
friendly solutions for the textile industry and essential
information on the ability of ligninolytic bacteria to
be used as bioremediation agents in dye biodegrada-
tion for high efficiency.
2 Materials and Methods
2.1 Screening Test of Ligninolytic Bacteria
Measuring the clear zone index created on the lignin
agar medium may provide a semi-quantitative assess-
ment of bacteria’s ability to break down lignin. This
experiment was carried out on the surface of a lignin
agar plate utilizing blank disk diffusion. A lignino-
lytic bacterial isolate was inoculated into a culture
vial containing 5 mL of liquid lignin medium, and
then incubated for 5 days at 30 °C. A spectrophotom-
eter with a wavelength of 600 nm was used to deter-
mine the absorbance value of each isolate’s starting
culture. Each starting culture was equalized using
a liquid lignin medium and its cell density value. A
sterile blank disk was filled with 30 mL of lignino-
lytic bacterial culture. The blank disk was placed on
the surface of the lignin agar medium and incubated
for 5 days at 30 °C after it had been entirely absorbed.
The formation of a clear zone around the colony
shows the potential for bacteria. A 10-min immersion
aided in assessing the clear zone in the 0.1% Congo
red dye medium. Solution of 0.1 M NaCl rinsed the
medium discarding the residual Congo red.
In this study, to calculate the ligninolytic activity,
the ligninolytic index used the ratio of the diameter
of the clear zone to the colony (Rahayu et al. 2010).
By breaking or degrading 1,4 glycosidic connections
in lignin and releasing Congo red, ligninolytic bac-
teria can hydrolyze the lignin medium, allowing the
hydrolyzed medium to bind Congo red and produce
a clear zone. As a result, the semi-quantitative tests
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were repeated three times. Following that, the isolate
with the largest clear zone has a good chance of being
used in the next test.
2.2 Potential Bacterial Isolates for Synthetic Dye
Decolorization
Inoculating ligninolytic bacteria on an agar dye
medium with 100 mg/L dye concentrations for meth-
ylene blue (MB), Congo red (CR), Alizarin Red S
(AR), and Remazol Brilliant Blue R (RBBR) were
used for screening. For 7 days, the bacterial isolates
were cultured at 30 °C. Synthetic dyes decolorize the
colony according to a clear zone around it, and these
experiments were repeated three times.
2.2.1 Dye Decolorization Test
This bacterial isolate was chosen using a medium
containing (in grams per liter): 2.5 N ­aNO3, 1
­KH2PO4, 0.01 C ­ aCl2•2H2O, 0.3 M ­ gSO4•7H2O, 0.1
NaCl, 0.01 F ­ eCl3•6H2O, one yeast extract, 15 bac-
teriological agar, and synthetic colors with graded
concentrations (multiples of 100 mg/L). The isolates
were then firmly streaked into four-quadrant serving
as duplicates and cultured at 30 °C for 7 days. Next,
bacterial isolates with a clear zone were streaked
again onto a synthetic dye medium carrying a greater
concentration of synthetic dye (Bandounas et al.,
2011). The bacteria would be chosen for the next test
when creating the largest clear zone with a high dye
concentration in the medium.
The decolorization test on the liquid medium used
the modified Alalewi & Jiang (2012) method with
three replications. The test used four synthetic dyes:
Congo red (CR), methylene blue (MB), Alizarin
Red S (AR), and Remazol Brilliant Blue R in Spec
160% (RBBR). The bacterial isolates were injected
in 50 mL of 25 mg/L liquid dye medium and cul-
tured on a rotary shaker at 150 rpm and 30 °C until
they reached the exponential phase. After equaliz-
ing the cell density to ­108 CFU/ml, 10 mL of culture
was inoculated into 100 mL of simple liquid min-
eral with 1 g/L yeast extract. It had the most signifi-
cant quantity of synthetic dye from the previous test
results, which bacteria could never decolorize. As a
control, a medium without inoculum was employed.
The medium was incubated on a rotary shaker at
30 °C, 150 rpm for 7 days, then took 5 mL on 0, 1,
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3, and 7 days. A spectrophotometer with a wave-
length of 600 nm was used to quantify cell density
in the samples. The bacterial cells were separated by
centrifugation at 10,000 rpm for 10 min at 20 °C. A
UV–Vis spectrophotometer with wavelengths of MB
(λ 520 nm), AR (λ 427 nm), CR (λ 470 nm), and
RBBR (λ 595 nm) was used to investigate dye decol-
orization (595 nm). The proportion of decolorization
was calculated using an algorithm devised by López
et al. (2006).
(OD0−ODT)
Decolorization (%) = OD0
× 100%
Noted:
OD0 = Day 0 absorbance ODT = Day 7 absorbance
2.3 Identification of the Decolorization Bacteria
Potency by Using 16S rDNA Sequences
The Quick-DNATM Fungal/Bacterial Miniprep Kit
was used to extract genomic DNA molecules from
bacterial cultures potentially. The 16S rDNA gene
was amplified using forward primer 27F (5′-AGA​
GTT​TGA​TCC​TGG​CTC​AG-3’) and reverse primer
1492R (5′-GGT​TAC​CTT​ACC​TTG​TTA​CGA​CTT​
-3′) that were introduced into a PCR master mix that
included 2 L template DNA, 25 L GoTaq®Green
Master Mix, 2 L of forwarding primer and 2 L of
reverse primer, and 19 L of sterile distilled water.
The PCR process was conducted at pre-denatured
settings of 95 °C for 300 s for one cycle, then dena-
tured at 95 °C for 30 s, annealing at 52 °C for 45 s,
and extension at 72 °C for 90 s for 35 cycles each,
followed by an extension period of 300 s at 72 °C.
Electrophoresis was used to confirm the 16S rDNA
sequence amplicon, and the quantity of DNA recov-
ered was quantified using a Nano-Drop Spectropho-
tometer. The agarose concentration was 1.5%, and
running electrophoresis was performed for 30 min at
100 V. The buffer solution used is Tris–borate EDTA
(TBE) 1X. The DNA bands were recorded with Gel
Doc, and the PCR product was purified with the PCR
Purification Kit. Samples are sent to First Base Pte.
Malaysia for sequencing (Rupaedah et al., 2019).
With the Bioedit application, the 16S rDNA
sequence was contiguous. The contig findings were
evaluated by online Blast and matched on GenBank
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Water Air Soil Pollut (2023) 234:359
for nucleotide sequences. The neighbor-joining tree
technique is used in the MEGA11 software to gener-
ate a phylogenetic tree using Bootstrap 1000.
2.4 Data Analysis
Each test was carried out in 3 replications, and the
mean value and standard deviation were shown
(Karim, 2018). The data obtained were analyzed
statistically using one-way analysis of variance
(ANOVA) with p 0.05, which has previously been
tested for normality of the data; if the results were
significantly different, further tests were performed.
Data analysis was calculated by SPSS 16.0 for Win-
dows software.
3 Result
3.1 The Potency of the Isolate in Degrading Lignin
The blank disk diffusion technique was used on a
specific agar medium to examine bacteria’s capac-
ity to break down lignin. After 7 days of incubation,
colonies on lignin agar media for ligninolytic bacteria
reached an apparent zone size, with typically equal
growth rates amongst isolates. This study performed
on 11 distinct ligninolytic bacteria isolates (L1, L2,
L3, L4, L5, L6, L7, L8, L9, L10, and L11). The pres-
ence of lignin breakdown by isolates is represented
by producing a clear zone surrounding the colony.
The diameter of the clear zone differed sig-
nificantly (α 0.05) across the tested isolates; three
isolates demonstrated the capacity to break down
lignin, including isolates L5, L6, and L8, which had
clear zones of 1.65 mm, 2.11 mm, and 1.96 mm,
respectively (Fig. 1). The cleared zone index is
comparable to Seesatat et al. (2021) work using
bacteria isolated from soil and a cleared zone index
of 2 to 3 mm. The ability of bacteria to degrade
lignin varies depending on the type of enzyme pre-
sent in each bacterium.
The ability of isolates to break down lignin was
regulated by incubation time, lignin concentration,
and pH in the medium. At this point, ligninolytic
bacterial isolates were chosen based on the diameter
index of the generated clear zone. The cleared zone
index of the isolates was higher than 1.5 mm, with an
L6 isolate exhibited the highest clear zone index.
Water Air Soil Pollut (2023) 234:359
Fig. 1  Diameter of clear
zona of ligninolytic bacteria
(different notations above
Diameter of clear zone (mm)
the histogram indicate the 2.5
clear zone of the medium
was significantly different
between isolates (p > 0.05))
1.5
0.5
3.2 Screening of Maximum Tolerance Concentration
(MTC) on Varieties Concentration of Azo Dye
In this study, azo dyes such as Congo red (CR) and
methylene blue (MB) were utilized, as well as anth-
raquinone dyes such as Alizarin Red S (AR) and
Remazol Brilliant Blue R Spec 160% (RBBR) were
also subjected to bacteria. The intensity of its appli-
cation in the textile sector and the structural resem-
blance to lignin influence the color selection.
The tight scratch method was used to select pro-
spective bacterial isolates by analyzing the growth
rate and capacity to generate a clear zone after seven
days of incubation. After 24 h of incubation at 30 °C,
almost all bacterial isolates were able to grow on
media with RBBR, AR, CR, and MB dyes at the con-
centration of 100 mg/L, which shows the presence of
the clear zone around the colony, suggesting its abil-
ity to decolorize the dye. Bacterial isolates L4 and L5
were unable to grow on all mediums with all colors
and were subsequently not used.
After obtaining pure isolates, screening is car-
ried out to determine potential bacteria used for fur-
ther tests. The manufacture of the dye medium in
the screening test was carried out by following the
method of Tian et al. (2016). First, except for the syn-
thetic dyes used, all medium components were steri-
lized using an autoclave. Then, the synthetic dye was
added aseptically to the medium after being fixed and
conditioned at 80 °C for 1 h with occasional shak-
ing; the concentration of the synthetic dyes used in
this screening process was 100 mg/L. The screening
Page 5 of 11 359
3
c
bc
ab
2 abc ab ab
a a a a a
0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11
Ligninolytic bacteria
was performed using the streak plate method with
four replications and incubated for 7 days at 30 °C.
The required isolates for further testing were chosen
based on their ability to develop in the dyes medium
and decolorize the additional synthetic dyes; a clear
zone showed the ability to degrade around the colony.
Eleven bacterial isolates were used to degrade four
dyes (MB, CR, AR, and RBBR).
The cleared zone formation indicated that the test
isolates could decolorize the synthetic dyes added to
the agar medium. The bacterial colonies can consume
the supplied dyes as nutrition for their growth (Ban-
dounas et al., 2011). The ability of bacteria to degrade
synthetic dyes is presented in Table 1. Almost of bac-
teria can grow in media with synthetic dyes after 24 h
of incubation at 30 °C. In contrast, the isolates L4
and L5 may not grow up in each media with synthetic
dyes (Table 1).
The MTC test was performed using a dense streak
approach to one quadrant and incubated for 7 days at
30 °C with varying doses to test all ligninolytic bac-
teria, as indicated in Table 1. The clear zone created
by the test isolates in each color at 200 mg/L was not
significantly different from the 100 mg/L concentra-
tion. Based on the results of this test, the L1, L6, L7,
L8, and L11 bacterial isolates have significant poten-
tial for dye degradation. Table 1 shows that increas-
ing the concentration of synthetic dyes tested can
reduce the percentage of decolorization efficiency
and reduce the growth of almost all the tested bacteria
due to the dye’s toxic effect, which blocks the active
site of the azo-reductase enzyme with dyes molecules
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Table 1  Screening of maximum tolerance concentration (MTC) on varieties concentration of azo dyes
Isolate MB (mg/L) CR (mg/L) AR (mg/L) RBBR (mg/L)

200 300 400 500 200 300 400 500 200 300 400 500 200 300 400 500

L1 + + + + + + + + ++ ++ ++ ++ + + + ++

L2 - ND ND ND + + + + + + + + - ND ND ND

L3 + - ND ND + + + + ++ ++ + + + + + +

L4 - ND ND ND - ND ND ND - ND ND ND - ND ND ND

L5 - ND ND ND - ND ND ND - ND ND ND - ND ND ND

L6 + - - - ++ ++ ++ ++ + + + + + + + +

L7 ++ ++ ++ ++ + + + + + + + + + + + +

L8 ++ ++ ++ ++ + + + + + + + + ++ ++ ++ ++

L9 ++ ++ + + + + + + ++ ++ + + + + + +

L10 ++ + + - + + + + + + + + + + + -

L11 + + + - ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

Clear zone: + + (strong), + (medium), ND not determine

of different structures. Furthermore, a decline in
decolorization efficiency might be due to a low ratio
of cells in the medium to break down all colors trans-
ported across the cell membrane (Tony et al., 2009).
Based on this qualitative test, it was discovered
that isolate L1 could decolorize AR dye, isolate L6
had potency in decolorizing CR dye, isolate L7 had
a single potential as bacteria to degrade MB dye, iso-
late L8 could decolorize MB and RBRR dyes, and
isolate L11 had triple potency in decolorizing AR,
CR, and RBRR dyes (Table 1). The creation of the
clear zone indicated that the test isolate could decol-
orize the synthetic dyes introduced to the medium.
The bacterial colonies could use the supplied dyes as
nutrition for their growth. No additional experiments
were performed on several other bacterial isolates
since the bacteria did not thrive at low doses, imply-
ing that these bacteria could not decolorize colors.
Furthermore, bacteria with the most significant
capacity for degrading dyes to the highest concen-
trations are evaluated in a liquid medium. The liquid
medium decolorization test was performed statically
to simulate the original environmental conditions of
industrial textile waste. The dyes degradation test
yielded a variety of results. As seen in Table 2, each
bacterial isolate has a unique capacity to break down
azo dyes.
The statistical analysis of the decolorization test
results for several azo dyes, namely MB, CR, AR, and
RBBR, demonstrates a homogeneity in their results.
Furthermore, there was a significant difference in
sampling times. Nonetheless, there was no significant
difference between the isolates used and no interac-
tion between the isolates and the sampling time. On
the seventh day of incubation, the maximum decolor-
ization percentage in a medium containing synthetic
dyes MB, CR, AR, and RBBR was 38.96%, 82.79%,

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Table 2  The highest potency of ligninolytic bacteria as azo temperature for the incubation of CR dye decoloriza-
decolorization tion. Another study found that after 72 h of incuba-
Isolate Days Decolorization (%) tion with a dye concentration of 10 mg/100 mL, the
MB CR AR RBBR
bacteria Lysinibacillus sphaericus decolorized the
synthetic dye RBBR by 58% (Chantarasiri & Boon-
L1 0 - - 16.06 - tanom, 2017). The temperature of incubation in this
1 - - 34.81 - study was 30 °C. In contrast, according to Illakkiam
3 - - 36.81 - et al. (2016), the optimal incubation temperature for
7 - - 40.51 - the AR decolorization process was 37 °C, with the
L6 0 - 66.36 - - percentage decolorization reaching 67% using Pseu-
1 - 77.45 - - domonas sp. after 48 h of incubation. That is why
3 - 80.04 - - the rate of decolorization in this study was low. He
7 - 70.6 - - also stated that pH affects the Alizarin Red (AR) dye
L7 0 11.43 - - - decolorization process, and the effective pH used is
1 23.37 - - - pH 7.0. According to the results of this study, the iso-
3 34.28 - - - lates L1 and L11 generated the highest percentage of
7 38.96 - - - decolorization on the seventh day of sampling, with a
L8 0 8.87 - - 23.16 pH of the medium near pH 7, precisely 6.84 and 6.85,
1 17.58 - - 12.7 respectively. The change in pH seen in this study was
3 31.7 - - 9.99 most likely caused by the dye’s high number of azo
7 35.15 - - 30.34 linkages, which degraded to generate aromatic amine
L11 0 - 65.02 10.3 2.58 metabolites, which are more alkaline than the original
1 - 78.75 22.34 9.18 azo dyes (Hanis et al., 2020).
3 - 81.69 33.65 4.19 Under anaerobic conditions, the decolorization
7 - 82.79 37.64 26.27 of azo dyes (MB and CR) can begin by reducing the
-N = N- link to produce colorless aromatic amines,
which can be degraded aerobically or anaerobically
40.51%, and 30.34% on isolate L7, L8, L1, and L11, (Sh Alabdraba et al., 2014). According to Saratale
respectively. The L11 was the most promising iso- et al. (2011), the azoreductase enzyme transfers four
late in the decolorization process, with the highest electrons (reducing equivalents) throughout two
percentage of decolorization in CR dye of 82.79%. It transfer phases, with two electrons transferred to the
also showed a significant response to the degradation azo dyes functioning as the final electron acceptor
of other dyes, such as AR and RBBR. After 7 days of form a colorless or decolorizing solution. The anaero-
incubation, the quantitative test findings are the same bic decolorization of azo dyes is generally regarded
as the qualitative test results, with isolate L11 having as a straightforward and non-specific procedure, as
the largest clear zone on CR, AR, and RBBR dyes seen in the CR dye decolorization process, which has
and isolate L7 having the most extensive clear zone a more significant percentage of decolorization in
on MB dye at a concentration of 500 mg/L. anaerobic circumstances than other dyes decoloriza-
According to Bandounas et al. (2011), the per- tion procedures. Meanwhile, because it is challenging
centage of MB dye decolorization by Bacillus sp. to validate the breakdown route, the decolorization
after 25 h, MB showed excellent performance for the of anthraquinone dyes has not precisely clarified the
degradation of 53% at a concentration of 25 mg/L decolorization mechanism under anaerobic circum-
lower than the dose applied in this study, which was stances (Routoula & Patwardhan, 2020). Accord-
100 mg/L. Sarim et al. (2019) conducted research ing to Li et al. (2019), bacteria’s degeradation of an
for the CR decolorization procedure, achieving a anthraquinone dye is a complicated process combin-
decolorization percentage of 84.5% using a bacte- ing adsorption, degradation, and enzyme catalysis.
rial isolate of Bacillus subtilis strain HAU-KK01. Adsorption happens when the dyes adhere to the
The incubation temperature employed in this inves- surface of the bacterial cell and causes the bacterial
tigation was 30 °C, while 35 °C was recommended cell to become more concentrated or change color

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depending on the absorbed color before further deg-
radation occurs.
The elements that contributed to the low decoloriza-
tion percentage were incubation temperature, medium
pH, oxygen and agitation, initial concentration of
added dyes, dye structure, incubation period, the den-
sity of inoculation cells, and bacteria species employed.
The activity medium in this study is a simple mineral
medium with 0.1% yeast extract added, which works
as a co-substrate in the co-metabolism of the synthetic
dye tested to regenerate NADH, which functions as an
electron donor in the reduction process by microorgan-
isms, allowing the effectiveness of decolorization to
be detected (Chang et al., 2000; Chantarasiri & Boon-
tanom, 2017; Illakkiam et al., 2016; Saratale et al.,
2011; Sarim et al., 2019). The bacterial isolates used in
this study were ligninolytic bacteria, which might also
generate enzymes such as lignin peroxidase (LiP), and
dye-decolorizing peroxidase (DyP), manganese per-
oxidase (MnP), catalase, and laccase, all of which are
used to boost the efficacy of decolorization. According
to Tian et al. (2016), the decolorization of RBBR and
MB cannot be accomplished by low redox potential
oxides like manganese peroxidase (MnPs) and laccase.
However, it can be accomplished by high redox poten-
tial agents like lignin peroxidase (LiP).
3.3 The Identification of Decolorization Bacteria by
Using 16S rDNA Sequences
The bacteria that will be detected molecularly have
the most significant capacity for lowering the concen-
tration of the synthetic dyes tested, which is defined
as a high percentage of decolorization in this study. In
this investigation, the isolate with the highest poten-
tial was L11. The isolate exhibited a 99.71% resem-
blance to the species Bacillus paramycoides strain
K7.2 as determined by BLAST-N using the 16S
rDNA sequencing. The phylogenetic tree was built
by comparing isolates L11 with the genus Bacillus
sp. as in groups, specifically Bacillus paramycoides
and E. coli species as outgroups (Fig. 2). Bacterial
isolates with a resemblance greater than 99% are con-
sidered as one strain. However, isolates with lower
than 95% similarity are said to represent a new genus
(novel genera) (Schlaberg et al., 2012). Identification
based on molecular characterization was reported and
revealed the existence of a strain belonging to the
genera of Bacillus sp. was isolated from soil samples
Vol:. (1234567890)
13
Water Air Soil Pollut (2023) 234:359
Fig. 2  Using the Tamura-Nei method, the phylogenetic tree of
L11 isolates and reference isolates is based on neighbor-join-
ing analysis
in the wood weathering area. In another research, the
genus Bacillus is commonly used to degrade azo dye
(Avci et al., 2023; Srivastava et al., 2022; Wu et al.,
2022).
According to Liu et al. (2017), Bacillus paramy-
coides is a facultative anaerobic bacterium isolate and
a form of a rod cell with a length of 1.8–2.2 m and
a width of 0.8–1.2 m. These gram-positive bacteria
are nonmotile, can generate endospores, and thrive
at temperatures ranging from 15 to 39 °C (optimum
at 30 °C), pH ranges from 5 to 9, and a salt concen-
tration of 0.5%. Bacterial colonies showed a waxy
appearance, were circular, non-permeable to light,
and 2–3 mm in diameter after 48 h at 32 °C, which
was consistent with the findings of this investigation.
In addition, Bacillus paramycoides performed well in
catalase and oxidase tests and were able to hydrolyze
starch, skim milk, and casein. Based on the results
of the API 20E test, Bacillus paramycoides can pro-
duce acetoin (Voges-Proskauer), gelatinase, and acid,
while based on the results of the API 50CHB test, the
bacteria can produce acid from D-ribose, D-galac-
tose, D-glucose, D-fructose, D-mannose, N-acetyl-
glucosamine, amygdalin, arbutin, aesculin ferric cit-
rate, cellobiose, maltose, trehalose, and starch.
Bacillus paramycoides may also generate
enzymes such as oxidase, catalase, amylase, cel-
lulase, urease, proteases, and caseinase, which can
be employed in the industry. In addition, Bacil-
lus paramycoides is a ligninolytic bacterium capa-
ble of degrading lignin and is hypothesized to
be capable of producing enzymes required in the
Water Air Soil Pollut (2023) 234:359
bio-delignification process, such as Li-P (lignin
peroxidase) and Mn-P (manganese peroxidase)
(Çağlayan, 2021; Rupaedah, 2019). Bacillus para-
mycoides were employed as immobilized bacteria in
nanofibers in the decolorization process for process-
ing methylene blue (MB) colored trash.
4 Conclusion
Four isolates of ligninolytic bacteria have a spe-
cific ability to decrease BOD5 in leachate water and
decolorize azo dyes. L1, L7, L8, and L11 are effi-
cient candidates of biodegradation agents because
of their capability to break down and decolorize azo
dyes in the textile dyeing industry’s final effluent.
Bacterial isolate L8 was considered the most poten-
tial to reduce BOD5 values up to 95.87% on day ten,
and the maximum decolorization percentage in a
medium containing synthetic dyes MB, CR, AR, and
RBBR was 38.96%, 82.79%, 40.51%, and 30.34% on
isolates L7, L8, L1, and L11, respectively. Isolate
L11 exhibited a 99.71% resemblance to the species
Bacillus paramycoides strain K7.2 as determined by
BLAST-N using the 16S rDNA sequencing was the
most promising isolate in the decolorization process,
with the highest percentage of decolorization in CR
dye of 82.79%, and it was also capable of decolor-
izing other dyes (AR and RBBR). The isolate L7
has the most extensive clear zone on MB dye at a
concentration of 500 mg/L. The obtained results are
comparatively high and good. Thus, it can be used as
a candidate for the degradation of pollutants (dyes)
to reduce water pollution.
Data Availability Not applicable.
Declarations
Conflict of Interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Com-
mons Attribution 4.0 International License, which permits
use, sharing, adaptation, distribution and reproduction in any
medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Crea-
tive Commons licence, and indicate if changes were made. The
images or other third party material in this article are included
in the article’s Creative Commons licence, unless indicated
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the permitted use, you will need to obtain permission directly
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 PERNYATAAN CONTRIBUTORSHIP KARYA TULIS ILMIAH

Karya Tulis Ilmiah (KTI) berjudul “Newly Isolated Ligninolytic Bacteria and Its Applications
for Multiple Dye Degradation” yang diterbitkan di Water Air Soil Pollut 234:359 Tahun 2023,
https://doi.org/10.1007/s11270-023-06377-7 ditulis oleh 7 orang dengan kontribusi masing-
masing penulis sebagaimana tercantum di bawah ini :

No Nama Kontributor Tanda Tangan Keterangan

dalam KTI
1 Farida Rahayu Utama BRIN

2 Irfan Mustafa Utama Departemen Biologi,

FMIPA, Universitas
Brawijaya

3 Marjani Utama BRIN

4 Fatkhur Rochman Utama BRIN

5 Raina Aman Qazi Utama Department of

Chemistry, Shaheed
Benazir Bhutto
Women University,
Pakistan
6 Khan Zeb Utama Abdul Wali Khan
University, Pakistan

7 Nabi Ullah Utama University of Lodz,

Polland

Demikian surat pernyataan ini dibuat untuk dipergunakan sesuai keperluan.