Environmenta l Challenge s 7 (2022 ) 10045 9

Contents
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Environmental Challenges
journalhomepage:www.elsevier.com/locate/env
c

Adsorption-microbial fermentation based multi-step approach to dye remediation for

safe and environment compatible waste water treatment
Shalini Singh ∗, Kayode Tolulope Adeyemi, Shweta Vernwal
Lovely Professional University, Punjab, India 144411

articleinfo abstract
Keywords: Environmental pollution is a major problem with increasing human population and correspondingly increasing industrial
Industrial dyes activities. Chemical industrial dyes can cause allergies, cancers, etc., in humans and adversely affect aquatic life when dye rich
Lignocellulosic waste effluents are released into water bodies. Though natural and safe dyes are being explored yet the bulk of industrial dyes is
Solid state fermentation chemical based. The current study explores cheap lignocellulosics for dye decolorization and use of resulting dye-adsorbed
Recycling biomaterials as substrate in microbial fermentation (SSF). Further, we evaluated a reuse of such dye-laden lignocelluloses for 2
Environment sustainability nd
dye adsorption-microbial fermentation cycle to potentially enhance economic viability of the process. Rice husk and Wheat
bran showed above 90% dye adsorption while the others gave 85-90% dye removal. All the dye adsorbed substrates were
successfully fermented by Phanerochaete chryosporium to produce significant laccase activity. A 2 nd cycle of dye adsorption
yielded improved dye decolorization for some substrates while some loss (not more than ∼23%), was reported for other
substrates. The findings are very encouraging as the comparative evaluation of various lignocellulosics as adsorbents and
fermentation substrates provide promising alternative to dye pollution mitigation. The reuse of such agri-residues highlights
eco-friendliness & economic efficiency of the process for removing dyes from wastewater.

1. Introduction
Various dyes have been used throughout human history in different
applications. The textiles were dyed using locally available materials using
natural sources, majorly plants. The discovery of synthetic dyes (exhibiting
lesser cost and better binding properties than natural dyes) late in the 19th century
adversely affected large-scale market for natural dyes ( Yusuf et al., 2017 ) for
use in textile, food, paper making, color paper printing, leather, and cosmetic
industries ( Slampova et al., 2001 , Vishwakarma et al., 2012 ). Dyes impart their
color onto other objects, and are variedly classified ( Gupta, 2009 , Mani et al.,
2019 ).
The growing use of chemical industrial dyes significantly pollutes water
bodies raising serious concerns about their large scale use. Though with
increasing public awareness and stringent regulations, demand for natural dyes is
been developed ( Cardoso et al., 2011 , Walker et al., 2003 ) for the same.
However, many of these industrial dyes by-pass most of these conventional
treatments and due to their high stability against light, temperature and oxidizing
agents, they persist in the environ- ment for long time. Furthermore, these
conventional methods are rela- tively expensive and generally produce secondary
pollutants ( Lade et al., 2012 ). Physical adsorption and microbial degradation are
important ex- amples of methods which have great potential to mitigate dye
pollution in water ( Vishwakarma et al., 2012 , Gupta, 2009 ).
Lignocellulosics are a rich source of biomass for production of in- dustrially
important substances like, biofuel and microbial enzymes ( Keasling et al., 2021 ,
Singh et al., 2014 ). The use of such natu- ral, abundantly available
lignocellulosic waste as adsorbents holds im- mense potential in reducing dye
related pollution in water bodies ( Vishwakarma et al., 2012 , Gupta, 2009 ).
Wheat bran, wheat straw, mango bark, orange and banana peel, neem leaf

∗
Corresponding author.
E-mail address: shalinisingh.iit@gmail.com (S. Singh).

https://doi.org/10.1016/j.envc.2022.100459
Received 29 August 2021; Received in revised form 17 January 2022; Accepted 21 January 2022
2667-0100/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (
http://creativecommons.org/licenses/by-nc-nd/4.0/ )
rising yet, the chemical dyes still form bulk of industrial dyes, which when
released into wastewaters ( Couto, 2007 , Couto, 2009 ) pose health and
environment hazards. Such contaminated waters ex- hibit toxicity to plants &
animals and cause many human health hazards ( Lellis et al., 2019 , Lade et al.,
2012 ).
The removal of such harmful dyes from industrial wastewaters is thus,
important ( Salehi et al., 2010 ) and various physical-chemical methods have
powder, pine apple peel powder, guava leaf powder, sugarcane pulp, coconut
pulp, hyacinth and rice husk are few such industrially relevant residues which can
be ex- plored as dye adsorbents ( Lade et al., 2012 , Das et al., 2019 , Singh et al.,
2011 , Singh and Tolupe, 2017 , Parshetti et al., 2010 , Kalyani et al., 2009 ,
Waghmode et al., 2011 , Vishwakarma et al., 2012 ).
Microbial systems have been used for industrial dye removal for ef- fective,
natural and environment friendly solutions to the chemical dye
S. Singh, K.T. Adeyemi and S. Vernwal
problem ( Ozmen and Yesilada, 2012 ). Further, growing demands for industrial
processes that comply with the principles of circular econ- omy, minimize
quantity of waste disposed into the environment, or sig- nificantly reduce
industrial waste related toxicity, has made reutiliza- tion/recycling of waste
constituents an attractive option to deal with environment pollution.
The present study investigates agricultural residues as biosorbents for a
representative azo dye, methylene blue, and subsequently evalu- ates recycyling
potential of the used substrates so as to minimize harmful waste discharge into
the environment. For this, the dye-adsorbed ligno- cellulosic residue was
evaluated as a substrate for laccase production by Phanerochaete chrysosporium
( P. chrysosporium ) under solid state fer- mentation. The spent ligno-fungal
biomass thus obtained was further subjected to 2 nd cycle of adsorption-microbial
degradation to evaluate its use in multi-application cycles, to make the process as
economically efficient as possible.
Adsorption and microbial fermentation for dye decolorization can be
effectively integrated. The use of white rot fungi for dye removal using liquid
state fermentation have been reported ( Ozmen and Yesi- lada, 2012 ). Also, use
of other dye adsorbed substate as solid state fermentation substrate has been
reported but not for methylene blue ( Nigam et al., 2000 ). Further, few studies on
direct degradation of lignocellulosics like, rice straw by Phanerochaete
chrysosporium using semisolid fermentation, and further recycling experiments
of gener- ated biomass ( Zeng et al., 2015 ) are reported. But, to the best of our
knowledge, a comparative evaluation of different agricultural residues in multi-
cycle-reuse waste treatment system, comprising of a combina- torial physical-
microbiological methylene blue mitigation system, have not been carried out
with lignocellulosics & Phanerochaete chrysospo- rium.
2. Materials and methods
2.1. Materials and microbial cultures
All chemical reagents and microbiological culture media used were of
analytical grade from Loba-Chemie Pvt. Ltd., India, SD fine chemi- cals Limited,
India (Syringaldazine) and HiMedia Pvt. Ltd., India. Wheat Bran (WB), Rice
husk (RH) and Wheat straw (WS), were procured from local market of Phagwara,
Punjab, India.
The lignocellulosic residues were washed with warm water (2 to 3 times),
sundried, and dried in a hot air oven at 60°C for 4 days ( Moldes et al., 2012 ).
Further, WS and RH were milled. Subsequently, the milled (RH & WS) unmilled
(WB) substrates were sieved to collect 1 mm size particles, which were stored in
sterile ziplock bags at room temperature for subsequent use.
Lyophilized Phanerochaete chrysosporium MTCC787 was procured from
Microbial Culture Collection Center, Institute of Microbial Tech- nology
Chandigarh, India. It was revived by mixing the fungal mycelium in sterile Malt
extract broth, and transferring it onto sterile Malt extract agar (MEA) plates using
a sterile Pasteur pipette. The inoculated plates were incubated at 37 ˚C for 5 days
and then stored at 4 ˚C ( Urra et al., 2006 , Hossain and Anantharaman, 2008 ).
For long term preservation, 15% of glycerol stocks of the fungus were stored at -
5°C ( Carletto et al., 2008 ).
2.2. Evaluation of biosorptive ability of lignocellulosic-residues for methylene
blue
The effect of temperature and incubation duration on the decoloriza- tion of
Methylene blue by lignocellulosic substrates, singly (RH, WS, WB), and in
binary (1:1) combinations (RHWS, WBWS), were studied by incubating the
substrate-dye solution mixture (50mg/L) in a ratio of 1:10 in sterile 100 mL
Erlenmeyer flasks, at variable incubation temperatures (10°C, 20°C, 30°C, 40°C,
2
Environmental Challenges 7 (2022) 100459
50°C and 60°C) for different incubation time (30, 60, 90, 120, 150 and 180
minutes) ( Hossain and Ananthara- man, 2008 ). The initial pH of the dye solution
(before addition to the adsorbent) was maintained at 6.0. Similarly, the influence
of dye con- centration on % dye decolorization for substrates was determined
using (mg/L), (40, 50, 60, 70 and 80) of the methylene blue mixed with agri-
residues at a ratio of 10:1 ( Carletto et al., 2008 ) and initial dye pH at 6.0. The
influence of pH on dye decolorization was also tested as explained above by
varying pH (2.0-9.0).
Finally, the dye solution samples were withdrawn after desired in- cubation
time, and spectrophotometrically analyzed at 665nm. The % dye decolorization (
Hamdaoui and Chiha, 2007 , Jalandoni-Buan et al., 2009 ) was: % Dye adsorbed
= [(Initial – Final)/Initial] X 100 where, “Initial ” is absorbance of dye solution
without substrate.
“Final ” is absorbance of dye solution with substrate (after appropri- ate
incubation period).
2.3. Dye adsorption under optimized conditions for preparation of solid
substrate for microbial fermentation
The bio-sorptive ability of WB, RH, WS, singly and, in combina- tions was
determined under optimized conditions of temperature, time, pH and dye
concentration and the % dye decolorization was deter- mined ( Hamdaoui and
Chiha, 2007 , Jalandoni-Buan et al., 2009 ). Subse- quently, the dye-adsorbed
lignocellulosic biomass were crumbled, dried at 60°C for 4 days, collected and
stored for subsequent use.
2.4. Evaluation of dye adsorbed lignocellulosic residues as solid substrate in
microbial fermentation for laccase production
The dye-adsorbed substrates, were evaluated as solid substrates for laccase
production by Phanerochaete chrysosporium MTCC787 under solid state
fermentation (SSF) using classical approach of optimization of enzyme
production.
2.4.1. Effect of incubation period, temperature, inoculum size and initial medium
pH
Each of the dried dye-adsorbed substrates (5gm), obtained as above, was
moistened with a nutrient salt solution (initial pH 6.0, 15 mL), con- taining (NH 4
) 2 SO 4 —0.14%, KH 2 PO 4 —0.2%, MgSO 4 .7H 2 O —0.03%, CaCl 2 —0.03%,
FeSO 4 .7H 2 O —0.002%, ZnSO 4 .7H 2 O, —0.002%, MnSO 4 .7H 2 O —
0.002%, CoCl 2 —0.002% ( Stoilova et al., 2010 ) in sterile 250 mL Erlenmeyer
flasks. The flasks so prepared, were au- toclaved, cooled, inoculated with 2
fungal mycelium discs, each of 0.5cm diameter, obtained from appropriately
grown fungal culture ( Singh et al., 2009 ) cultivated on Malt extract agar (MEA)
plates. The inoculated flasks were incubated at 37°C ( Carletto et al., 2008 ) for 1
to 12 days.
The effect of incubation temperature on laccase production was de- termined
by inoculating the substrates as above and incubating inocu- lated flasks at
different temperatures. For evaluating the effect of inocu- lum size, on laccase
production, the flasks were prepared and inoculated with different number of
fungal discs (1, 2, 3, 4) of 0.5 cm size each, cut from the periphery of
appropriately grown plate culture. The effect of initial medium pH (5 to 8) was
studied by adjusting the pH of moisten- ing agent to variable pH before
autoclaving (No further maintenance of medium pH was done). The inoculated
flasks were incubated under spe- cific fermentation conditions ( Stoilova et al.,
2010 , Singh et al., 2009 ).
Appropriate controls inoculated with agar plugs cut from the surface of
uninoculated MEA plates were also prepared. At the end of desired incubation
time, 15.0 mL of distilled water was poured into the Erlen- meyer flasks and their
S. Singh, K.T. Adeyemi and S. Vernwal
content was crushed with a glass rod, and then shaken on a rotary shaker (10
minutes, 200 rpm). The substrates were then filtered through 2 layers of cheese
cloth and the extracts were cen- trifuged (15 min,15000 rpm, 27 ˚C). The
supernatant (crude enzyme) ( Singh et al., 2009 , Muthezhilan et al., 2007), used
for determining laccase activity ( Ride, 1980 ). The laccase activity was
expressed as U/mL of the sample.
2.5. Microbial fermentation under optimized incubation conditions for
preparation of substrates for 2nd adsorption-laccase cycle
Phanerochaete chrysosporium MTCC787 was subjected to SSF for each dye-
adsorbed substrate by adding 50 gms of each dye-adsorbed substrate in sterile
Erlenmeyer flasks with 150 mL of nutrient salt so- lution (1:3 ratio) under
optimized conditions of temperature, time, ini- tial fermentation medium pH, and
inoculum size. The crude enzyme was subsequently harvested to determine
laccase activity ( Ride, 1980 ) and the resulting spent ligno-fungal biomass (for
each substrate) was collected (fungal biomass not separated from the agri-
biomass). The biomass was air-dried and subsequently dried in a hot air oven at
60 °C. After 4 days, the biomass samples were crumbled, collected and stored in
sterile ziploc bags.
2.6. Evalution of potential reuse of spent fungal-agri-biomass in dye
decolorization and laccase production
The spent fungal-agri-biomass, obtained as above, was used for a 2 nd cycle of
dye adsorption-microbial fermentation. The % methylene blue decolorization
and laccase activity under SSF using already optimized conditions were
respectively determined, as explained above.
2.7. Statistical analysis
All experiments were done in triplicates and the results for dye de-
colorization were represented as an average of % dye decolorization. The laccase
activity was reported as mean ± standard deviation of the values.
3. Results
3.1. Evaluation of biosorptive ability of selected lignocellulosic-residues for
methylene blue
All the tested lignocellulosic materials were found to adsorb signifi- cant
amount of the dye from its aqueous solution ( Table 1 ), with the least adsorption
shown by WBWS (75.3%) at 50°C, 150 mins, and RH & WB showing 90% (90
for RH and 90.2% for WB) dye adsorption. Still, WB required more time (30
mins) and higher temperature (higher by 10°C) for achieving similar
decolorization as RH. RHWS showed 83.1 (30 min, 40°C) % decolorization
while WS was slightly better than RHWS with 86.4 (150 mins, 50°C) %
decolorization.
All the adsorbents except one (RHWS), showed maximum dye re- moval for
the least concentrated (40 mg/mL) dye solution ( Table 2 ), out of all the dye
concentrations tested, indicting better dye removal ef- ficiency of the adsorbents
in lesser concentrated dye solutions. RHWS on the other hand, showed best
decolorization at much higher dye concen- tration (70 mg/mL) than 40 mg/mL.
In response to varied dye concen- tration, 87, 85.4 and 84.1% dye removal was
seen for WS, WBWS, and RHWS, respectively, while RH and WB removed dye
more than 90%.
Methylene blue was best adsorbed by all the adsorbents at pH 6.0 ( Fig. 1 ).
The adsorbents can be arranged in the following decending order of dye
decolorization ability, WB > RH > WS > WBWS > RHWS.
3
Environmental Challenges 7 (2022) 100459
3.2. Dye adsorption under optimized conditions for preparation of solid
substrate for microbial fermentation
RH was found to be the best dye decolorizer (93.2)% under optimized
conditions of incubation ( Fig. 2 ). WB closely followed RH in dye removal
efficiency with only 0.4% (almost negligible) lesser dye removal than that
observed for RH. Even the other adsorbents maintained more than 85% dye
decolorization.
3.3. Evaluation of dye adsorbed lignocellulosic residues as solid substrate for
microbial fermentation
In general, for each substrate used, laccase production increased upto a
maximum as incubation time increased upto an optimum ( Table 3 ) Be- yond the
optimum, enzyme activity gradually declined. RH encouraged the maximum
production of laccase (378.1 U/mL) by the test fungus on 9 th day of incubation
while lowest laccase activity was observed, as expected, on 1 st day of incubation
(50U/mL). A decrease of almost 28% in laccase activity was observed on 10 th
day of incubation for RH. Both WS and WB as substrates, encouraged the
maximum enzyme production by the test fungus on 5 th day of incubation with
339 and 635 U/mL of laccase activity, respectively. The shortest optimum
incubation time for enzyme activity was found to be 4 th day when substrates were
used in combinations (RHWS and WBWS). RHWS was slightly better
(maximum laccase activity, 374 U/mL) than WBWS (8.8% lesser enzyme
activity) on 4 th day of incubation.
37 ± 2 °C temperature ( Table 4 ) was found to be the most suitable for laccase
production by test fungal strain for all the substrates used except, that for RHWS
(33°C ± 2, 394 U/mL) under desired incubations condi- tions. WB produced the
highest (650 U/mL) of laccase activity under the given set of incubation
conditions. WBWS was only slightly better (almost negligible) than ( ∼0.8%
higher laccase activity).
Inoculum size ( Table 5 ) was also found to be an important pa- rameter for
laccase production with variable enzyme activity observed for different inoculum
size in case of different substrates. The sub- strates responded in following
descending order of laccase production WB > RH > RHWS > WBWS > WS. Both
WB and RH best produced laccases with 3 fungal discs of 0.5 cm each, but the
combination-substrates (RHWS and WBWS) produced highest enzyme activity
with only 2 fun- gal discs of 0.5 cm each. WS on the other hand required the
highest inoculum size for maximum laccase production (355.3 U/mL) by the
fungus.
Interestingly, the test fungus showed varied preference to the medium pH to
show best laccase activity while using different sub- strates. As can be seen (
Table 6 ), while the test fungus produced highest laccase activity with RH (570.7
U/mL) at 6.5, WS (363 U/mL) at 5.5, it produced 705 and 440 U/mL of laccase
with WB and RHWS, respec- tively, at pH 6.0. Interestingly, WBWS (776
U/mL) showed significant rise in laccase activity (a rise of more than 50% in
laccase activity) at pH 7.0, than that observed at pH 6.0, while recording the best
laccase production amongst the substrates tested.
3.4. Microbial fermentation under optimized incubation conditions for
preparation of substrates for 2 nd adsorption-laccase cycle
Finally, the test fungus was subjected to optimized conditions of incubation
(time, temperature, inoculum size and pH) under SSF for each substrate tested (
Fig. 3 ). The combination of WBWS was found to be the best substrate (780
U/mL) for laccase production by the test fungus, followed by WB (714 U/mL),
RH (574.7 U/mL), RHWS (442.3 U/mL) and WS (366.7 U/mL) in the decreasing
order of enzyme production.
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

3.5. Potential reuse of spent fungal-agri-biomass in dye decolorization and

laccase production

An evaluation of test lignocellulosic materials for a 2 nd cycle of dye

adsorption-microbial fermentation yielded very encouraging results ( Table 7 )
with WS and RHWS showing visible enhancement (4.9 and 2.7% increase,
respectively) in dye adsorption ability, as compared to that for virgin adsorbent (
Fig. 2 ). The other substrates too showed satisfactory dye removal in the 2 nd
cycle, with 71.9, 76.7, 79.2% dye decolorization for RH, WB and WBWS,
respectively.
When laccase activity was checked in the 2 nd cycle of microbial fermentation
of the agri-residues, RH yielded a slight increase in enzyme

4
 Table 1
Influence of incubation temperature & time on dye decolorization.
Incubation time, min Incubation temperature, o C Adsorbents
Dye decolorization, %
S. Singh, K.T. Adeyemi and S. Vernwal RH Environmental Challenges 7 (2022) 100459
WS WB RHWS WBWS
0 10 32.0 30.2 30.1 40.2 32.1
20 34.9 31.7 32.5 45.6 33.3

30 33.8 32.0 34.9 49.9 32.5

40 41.7 34.9 35.1 52.8 34.1

50 42.3 35.6 35.8 53.1 34.4

60 43.4 36.9 37.0 72.0 35.3

30 10 51.2 37.6 47.9 75.5 36.1

20 62.1 39.5 49.1 77.8 37.4

30 73.1 41.6 57.4 80.6 39.4

40 78.4 42.7 60.8 83.1 41.5

50 82.9 43.1 63.6 82.2 43.0

60 84.7 45.9 65.5 81.5 44.6

60 10 87.3 47.2 69.4 80.1 46.7

20 90.0 48.6 73.8 82.3 48.0

30 88.9 50.1 76.0 81.1 50.9

40 88.0 50.9 79.2 82.4 53.0

50 87.3 48.9 80.1 83.0 53.7

60 86.4 47.1 83.2 81.8 55.8

90 10 87.3 46.3 83.8 82.1 57.8

20 90.2 45.2 85.0 80.5 59.0

30 89.5 49.9 90.2 81.9 60.9

40 89.4 51.4 89.7 80.8 61.0

50 88.0 54.0 89.1 79.5 62.9

60 88.1 54.8 88.2 79.7 63.1

120 10 86.9 57.9 88.4 78.2 64.0

20 89.1 60.9 87.6 79.5 64.8

30 88.6 63.0 88.1 80.6 65.6

40 88.7 69.8 87.5 81.0 66.1

50 88.0 71.4 87.6 80.9 68.4

60 86.7 72.7 87.0 79.1 69.0

150 10 87.5 74.8 86.3 79.6 70.2

20 88.9 78.1 86.1 80.1 71.0

30 89.8 81.1 84. 3 88.7 72.4

40 86.8 82.2 82.0 77.4 73.2

50 88.9 86.4 83.1 78.6 75.3

60 89.5 83.2 81.9 80.5 72.8

180 10 87.0 83.7 81.5 81.2 73.4

20 90.1 82.5 82.0 80.4 74.0

30 89.4 82.8 80.9 81.9 73.1

5
40 88.2 83.5 81.4 82.2 71.6

50 78.3 84.5 81.2 81.3 69.9

S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

Incubation conditions:
Dye concentration: 50 mg/ml
Substrate: Dye solution: 1:10 pH of
the dye solution: 6.0

activity (597.5 U/mL), compared to dye adsorbed RH in the 1 st cycle of

treatment. Other substrates on the other hand, recorded lesser laccase activities in
comparison to the 1 st cycle.

4. Discussion

Dye decolorization was significant for all the substrates tested, in re- sponse
to variable time and temperature conditions. The dye decoloriza- tion efficiency
of WS (86.4%) at 150 mins and 50°C, was better than that reported earlier (
Nigam et al., 2000 ), where 70-75% adsorption was reported for WS under
specific conditions. Overall, all adsorbents, irrespective of the incubation
temperature, reported higher dye decol- orization with longer incubation
duration, upto a maximum. Further, dye decolorization attained a plateau with
almost similar or slightly less decolorization than maximum. Usually, with a rise
in incubation tem- perature, dye ions diffuse better upto a, point which enhances
the rate of adsorption by the adsorbent ( Benguella and Benaissa, 2002 ). Beyond
this, a state of equilibrium is reached due to adaptation of biosorbent ( Khalaf,
2008 ). Similarly, as the incubation time increases, dye adsorption improves till
saturation is reached (due to rapid adsorption on dye binding sites and
accumulation of dye molecules on the surface of the adsorbent), while restricting
further dye movement within the adsor- bent ( Das et al., 2019 ). The effect of the
incubation temperature on the dye decolorization capacity for each substrate
varied from each other. Similar trend of decolorization in response to variable
incubation time and temperature were seen by Hamdaoui and Chiha and Carletto
et al. ( Carletto et al., 2008 , Hamdaoui and Chiha, 2007 ).
As can be observed above, the concentration of the dye plays a cru- cial role in
determining the dye decolorization and our findings indi- cated that majorly the
deye decolorization was higher at lower concen- tration than at higher
concentrations and a similar trend was reported by Sumanjit et al., and Carletto et
al. ( Sumanjit and Kaur, 2007 , Carletto et al., 2008 ) where, as the concentration
of the dye increased, the decol- orization activity decreased. This might be due to
hinderance created for the dye particles at the surface of the adsorbent due to
initial build up of dye ions ( Tiwari et al., 2017 ). In contrast, RHWS showed
better decolorization at higher dye concentration in comparison to other
substrates tested and such cases where, as the dye concentration increased, the
dye adsorption by the test adsorbent increased ( Tiwari et al., 2017 ), has also
been reported. Rice husk silica used for methylene blue adsorption

6
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

(
M
o
e
n
i
Dy e Re m o v a l ( %)

a
n

pH
Fig. 1. Effect of pH on % dye decolorization.
Dye Removal (%)

Fig. 2.
Decolorization of Methylene blue by agri-

Type of Agri-residue
and Mehidinia, 2019 ) showed maximum removal efficiency at 10mg/L
indicating much better response of substrates tested in the given study in terms of
higher dye adsorption at higher dye concentra- tion than 10mg/L.

7
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

The medium pH influences dye ionization and the surface fea- tures of the
adsorbent, which, directly affects the rate of sorption ( Akazdam et al., 2017 ) and
preference to acidic pH for methylene blue decolorization has been reported by
others too. Corn husk was also found to remove 90% of MB at pH 6.2 ( Malik et
al., 2016 ). Hameed et al. ( Hameed et al., 2009 ), while working on pine apple
stem for adsorption of methylene blue, also suggested that the acidic pH is
favorable for its adsorption.
The use of brewers’ spent grain for removal of methylene blue from its
aqueous solution yielded 70-90% dye removal efficiency, indicating better
efficiency of residues tested in our study for removal of methylene blue ( Kezerle
et al., 2018 ).
LACCASE ACTIVITY (U/mL)

Fig. 3. Laccase activity of P. chrysosporium MTCC787

Type of Agri-residue
Table 2 60 83.6
Influence of dye concentration on dye decolorization.
S. No. Substrate Dye Concentration, mg/L Dye decolorization, % 70 81.7

1. RH 40 91.9 80 81.5
50 90.4
4. RHWS 40 81.4
60 86.7 50 84.0

70 85.5 60 83.4

80 85.1 70 84.1

2. WS 40 87.0 80 82.0
50 85.8
5. WBWS 40 85.4
60 83.2 50 84.1

70 82.8 60 84.2

80 82.2 70 79.2

3. WB 40 91.7 80 76.7
50 89.0
Incubation conditions:

8
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

Incubation temperature, °C: RH (20), WS and WBWS (50), WB (30), RHWS (40) 33 ± 2 317.0 ± 1.1
Incubation duration, mins: RH(60), WS and WBWS (150), WB (90),
RHWS(30) 37 ± 2 347.3 ± 1.3
Substrate: Dye: 1:10 pH: 6.0
40 ± 2 262.0 ± 1.4

3 WB 28 ± 2 442.0 ± 1.4
As reported above, RH needed the longest incubation time for max- imum
33 ± 2 621.2 ± 1.9
laccase production in comparison to other substrates tested. This is most likely
due to restricted availability of nutrients from a complex substrate like RH. The 37 ± 2 650.0 ± 2.3
fungus thus, required more time to mineralize and assimilate. Studies also
indicate recalcitrant nature of RH to fer- 40 ± 2 554.4 ± 1.7
Table 3
Effect of incubation period on laccase production. 4 RHWS 28 ± 2 284.7 ± 1.4
Incubation period Laccase activity (U/mL ) 33 ± 2 394.3 ± 1.2
(Days )
RH WS WB RHWS WBWS 37 ± 2 386.0 ± 0.9
1 50.0 ± 3.0 275.8 ± 3.4 196.0 ± 1.7 292.0 ± 1.2 315.8 ± 2.7
2 72.0 ± 1.1 300.6 ± 1.1 234.0 ± 1.5 303.0 ± 0.9 325.6 ± 1.6 3 110.0 ± 0.5 40 ± 2 207.7 ± 0.5
309.1 ± 1.2 468.0 ± 3.5 342.0 ± 1.1 330.0 ± 1.4 4 132.8 ± 1.0 315.0 ± 2.0 513.0 ± 1.9 374.0
± 1.3 341.0 ± 1.8 5 WBWS 28 ± 2 240.0 ± 2.7
5 170.0 ± 3.0 339.0 ± 3.1 635.0 ± 1.3 359.0 ± 1.6 322.3 ± 1.4 33 ± 2 326.0 ± 3.0
6 220.5 ± 1.3 300.0 ± 2.3 421.0 ± 1.8 344.0 ± 2.0 295.2 ± 2.0
7 253.6 ± 2.4 291.3 ± 1.0 402.2 ± 3.0 - - 37 ± 2 350.0 ± 1.6
8 284.9 ± 1.2 - - - -
9 378.1 ± 2.2 - - - - 40 ± 2 224.5 ± 1.5
10 271.0 ± 3.2 - - - -
11 243.0 ± 1.5 - - - -
Incubation conditions:
12 - - - - -
Inoculum size: fungal discs of 0.5 cm each
Initial medium pH: 6.0
Incubation conditions: Substrate: Moistening agent: 1:3
Inoculum size: 2 fungal discs of 0.5 cm each Incubation period: RH (9 th day of incubation),WS and WB (5 th day of
Initial medium pH: 6.0
incubation), RHWS and WBWS (4 th day of incubation)
Substrate: Moistening agent: 1:3
Incubation temperature, °C: 37 ± : means ± : means Standard deviation from mean
Standard deviation from mean
Table 5
Effect of inoculum size on the laccase production.
S.No Substrate Number of Disc (s) inoculated Laccase activity (U/mL)
mentation probably due to much high lignin and silica content, which might lead
1 RH 1 230.7 ± 3.7
to longer incubation times to fermentation ( Wu et al., 2018 ).
Interestingly, WS showed best laccase activity in much lesser time, probably 2 396.0 ± 0.4

because beyond a point, the fungus was not able to assimilate nutrients from the
3 409.3 ± 0.6
complex structure. WB showed the highest activity amongst the substrates tested,
indicating that the nutrient complex and the kind of nutrient forms were more 4 385.0 ± 2.2
easily used up by the fungus under the given set of conditions.
The laccase activity, in response to variable incubation temperature, 2 WS 1 113.7 ± 1.2
increased with time upto the maximum. The encouraging performance of 2 303.0 ± 1.7
substrates in binary combinations might be due to better availabil- ity of nutrients
in a complex nutritional environment with two sub- strates mixed together as in 3 335.0 ± 1.3
case of WB mixed with WS (WBWS) or even RHWS, which was also better
substrate than the substrates used singly, except in case of singly used WB. 4 355.3 ± 1.4

P.chrysosporium grows best be-

3 WB 1 306.4 ± 1.9
Table 4
2 622.0 ± 1.3
Effect of incubation temperature on laccase production.
S.No Substrate Temperature (°C) Laccase activity (U/mL)
3 695.0 ± 1.1
1 RH 28 ± 2 125.0 ± 0.7
33 ± 2 134.7 ± 0.8 4 422.0 ± 1.2

37 ± 2 386.3 ± 1.5 4 RHWS 1 316.0 ± 1.2

2 404.7 ± 0.9
40 ± 2 155.3 ± 2.2
3 266.7 ± 0.7
2 WS 28 ± 2 221.7 ± 1.5

9
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

4 124.7 ± 1.1 5.5 178.0 ± 0.9

5 WBWS 1 288.5 ± 2.1 6.0 518.0 ± 1.2

2 368.0 ± 3.1
6.5 570.7 ± 0.8
3 286.0 ± 2.6
7.0 411.3 ± 0.9
4 222.1 ± 1.8
7.5 301.0 ± 1.5
Incubation conditions:
Initial medium pH: 6.0 8.0 198.9 ± 0.9
Substrate: Moistening agent: 1:3
Incubation period, days: RH (9 th day of incubation),WS and WB (5 th day of incubation), 2 WS 5.0 322.8 ± 1.2
5.5 363.0 ± 0.7
RHWS and WBWS (4 day of incubation)
th

Incubation temperature, ˚C: 37 (WB, WS, WB + WS) 6.0 351.0 ± 0.5

± : means Standard deviation from mean
6.5 253.0 ± 0.7

tween 20-50°C. At higher temperatures (above 40°C), death rate exceeds growth 7.0 208.7 ± 0.8
rate while at lower temperatures, metabolic ativities are highly reduced ( Hossain
and Anantharaman, 2008 ). 7.5 198.0 ± 2.1
Inoculum size ( Table 5 ) was also found to be an important parameter for
8.0 195.2 ± 0.9
laccase production with variable enzyme activity observed for differ- ent
inoculum size in case of different substrates. The findings indicated that probably
3 WB 5.0 542.1 ± 1.0
both RHWS and WBWS though produced lesser laccase activity than the best 5.5 564.0 ± 0.9
performer (WB), yet these two substrates were in-
Table 6 6.0 705.0 ± 2.1
Effect of initial medium pH on laccase production.
S.No Substrate pH of Medium Laccase activity (U/mL) 6.5 650.0 ± 1.0
1 RH 5.0 125.3 ± 1.2
7.0 610.0 ± 1.2

7.5 581.1 ± 3.0

8.0 532.5 ± 1.1

4 RHWS 5.0 211.3 ± 1.2

5.5 278.0 ± 0.8

6.0 440.3 ± 1.3

6.5 236.7 ± 0.9

7.0 220.3 ± 1.2

7.5 201.5 ± .2.0

8.0 197.1 ± 3.1

5 WBWS 5.0 110.0 ± 0.8

5.5 172.3 ± 1.7

6.0 372.1 ± 2.3

6.5 400.5 ± 1.4

7.0 776.0 ± 1.8

7.5 743.3 ± 1.0

8.0 720.5 ± 2.1

Incubation conditions:
Substrate: Moistening Agent: 1:3

10
S. Singh, K.T. Adeyemi and S. Vernwal
Incubation temperature, ºC: RH(37°C), WS(37°C), RHWS (35°C)
Incubation period, days: RH (9days), WS (5days), RHWS (4days)
Inoculum size: 3 fungal discs (RH and WB), 4 fungal discs
(WS), 2 fungal discs (RHWS and WBWS)– of 0.5 cm each
± : means Standard deviation from mean
vaded faster and probably better by the test fungi, than WB. This further showed
that better growth support provided by combo-substrates than WB, a singly used
substrate. The need for largest inoculum size tested for WS can be attributed to
the complex physical and chemical composition of WS ( Singh et al., 2011 ).
As the ability of the microbes to assimilate nutrients is also influ- enced by
medium pH, which may enhance or reduce the microbes’ abil- ity to use nutrients
from even in a nutrient rich environment, initial fermentation medium pH was
also evaluated as a factor in laccase pro- duction in the given study. Interestingly,
but not surprisingly too, the test fungus showed varied preference to the medium
pH to show best laccase activity while using different substrates. The medium pH
also influences solubility (and hence, availability to fermentation) of media
components ( Singh et al., 2009 ).
As our findings indicate, dye-adsorbed substrates in all cases, were
successsully fermented by the test fungus. Though the laccase activities varied
for different substrates, yet the amount of the test enzyme pro- duced, was
significantly high. As also supported by other investigations ( Liu et al., 2016 ),
successful growth and laccase production thereof, us- ing dye adsorbed
substrates also might further modify the physical and chemical properties of the
dye adsorbed substrate, probably leading to breakdown of the complex residue
structure and making its degradation in the environment and also provide laccase
as a beneficial co-product.
Table 7
Recycling potential of dye adsorbed lignocellulosics for dye decolorization and laccase production.
S.No. Reuse potential of spent ligno-cellulosic biomass
Spent agri-fungal
biomass Dye decolorization (%) for recycled Efficiency as dye
increase (U/mL) for recycled substrate for laccase
1 RH 71.9
2 WS 92.4
3 WB 76.7
4 RHWS 88.1
5 WBWS 79.2
Incubation conditions for dye decolorization:
Incubation temperature, °C: RH (20), WS and WBWS (50), WB (30), RHWS (40)
Incubation duration, mins: RH(60), WS and WBWS (150), WB (90), RHWS(30)
Substrate: Dye: 1:10
Dye concentration, mg/L: RH, WB, WS and WBWS (40), RHWS (70) pH: 6.0
Incubation conditions for laccase production:
Substrate: Moistening Agent: 1:3
Incubation temperature, ºC: RH (37°C), WS (37°C), RHWS (35°C)
Incubation period, days: RH (9days), WS (5days), RHWS (4days)
Inoculum size: 3 fungal discs (RH and WB), 4 fungal discs (WS), 2 fungal discs (RHWS and WBWS)– of 0.5 cm each
Initial medium pH: RH(6.5), WS (5.5), WB (6.0), RHWS (6.5) WBWS (7.0)
Microbial fermentation of such dye adsorbed agricultural residues also
improves the surface area available, as lignin degradation by enzymes weaken
the integrity of the biomass structure ( Huang et al., 2008 ).
Significant dye decolorization as well as microbial fermentation yielding
laccases in a repeat sequence were very encouraging and clearly support
potential multi-step utilization of complex-structured adsor-
Environmental Challenges 7 (2022) 100459
bents/fermentation substrates. The microbial action on the substrate might
loosen and break the complex lignin-cellulose-hemicellulose structure of the
dye-laden lignocellulosics used as adsorbents, creating additional binding
sites or enhanced surface area available for improved dye adsorption in the
subsequent repeat-cycle. The increased dye decol- orization ability of the
spent substrate is thus, most likely due to the “biological pre-treatment ”
owing to the first cycle of laccase production. As no treatment, apart from
gentle crumbling & drying the dye-laden substrates, was done before a 2 nd
cycle of adsorption-fermentation was adopted, a decrease in dye
decolorization might be expected for some agri-residues, as the already bound
dye molecules, in such cases, might not allow further uptake/binding of the
dye molecule, thereby reducing the dye adsorption ability to some degree.
When laccase activity was checked in the 2 nd cycle of microbial
fermentation of the agri-residues, apart from RH, all other substrates recorded
lesser laccase activities in comparison to the 1 st cycle. It might be possible that
a higher amount of residual lignin was available for enzymatic breakdown
after 1 st cycle of enzyme treatment for RH than other substrates, due to which
laccase activity improved slightly in this case, but not for others.
Use of dye adsorbed substrate for fungal growth and enzyme produc- tion,
in multiple alternating adsorption-microbial fermentation cycles has also been
supported by others ( Hameed et al., 2009 ).
The use of solid state fermentation (aerobic cultures) using fungal cultures
have added advantage over the use of bacterial cultures under anaerobic
conditions as studies indicate that complete mineralization is possible under
such conditions. On the other hand, under anaerobic bacterial fermentations,
chances of formation of toxic amines upon dye decolorization are higher
Laccase production Efficiency as adsorbent adsorbent (%
↑ /decrease ↓ in comparison fermentation substrate production (% increase
to virgin adsorbents) ↑ /decrease ↓ in
comparison to virgin
fermentation substrate)
22.9 ↓ 597.5 ± 1.3 3.8 ↑
4.9 ↑ 326.0 ± 2.5 11.0 ↓
17.42 ↓ 412.0 ± 2.2 42.3 ↓
2.7 ↑ 510.0 ± 1.0 13.3 ↑
8.5 ↓ 564.0 ± 2.1 27.7 ↓
(Banat etl., 1996). Fermentation experiments using Phanerochaete
chrysosporium and Coriolus vesicolor and dye ad- sorbed substrates have
been reported where effect of such substrates on microbial growth and protein
production has been done ( Nigam et al., 2000 ). Appearance of fungal growth
on the dye adsorbed substrates is a good indication to show lack of any adverse
effect of dye adsorbed substrate on fungal growth but, our study further
enhances the value of the investigation by quantitatively evaluating the
laccase production.
11
S. Singh, K.T. Adeyemi and S. Vernwal
5. Conclusion
With industrial waste disposal becoming an ever increasing prob- lem due to
associated environment and health hazards, a well designed waste water
treatment strategy that can reduce the bulk and/or toxi- city of waste generated,
before its disposal into the environment, can go a long way in reducing
environmental load. Multi-level recyling of chemical-dye-adsorbed
lignocellulosic waste, as substrate for fermenta- tion (further an important source
of industrial enzymes), along with its use in multiple cycles of adsorption-
fermentation, greatly enhances the overall economy and industrial acceptance of
the process.
The ‘left-over’ agri-fungal biomass can subsequently be utilized for various
end uses like, as a soil conditioner where cultivation of at least, flowering plants
can be conveniently done. The uses of spent biomass for cultivation of plants has
been supported by other studies too. Further investigations might also attempt to
evaluate solid biomass as a source of industrially important substances like,
industrial enzymes, food fla- vors, etc., which would further make the process
highly cost efficient. The production of industrially relevant enzymes like,
laccases in the cur- rent case, can also be used in various processes directly
associated with enzyme applications. The use of laccases for example, can be in
further waste water treatment, apart from many other applications using lac-
cases. Such applications can potentially provide a closed-loop system for
subsequent disposal of the solid waste generated.
On evaluation of the findings of the given study, we can thus confi- dently
state that the agri- residues can well be reused in dye decoloriza- tion and laccase
production (enzymatic fermentations) in a step-wise, multiple stage approach.
The application of dye-adsorbed substrate for fungal fermentations, more than
one time, further improves the overall cost & process efficiency. All the tested
lignocellulosics especially, rise husk and wheat bran, can well be used for
reduction of chemical dye related environmental pollution.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors are thankful to Lovely Professional University for pro- viding
facilties for conducting the study.
References
Akazdam, S., Chafi, M., Yassine, W., Gourich, B., 2017. Removal of acid orange 7 dye from
aqueous solution using the exchange resin amberlite FPA-98 as an efficient ad- sorbent:
Kinetics, Isotherms, and Thermodynamics study. J. Mat. Env. Sci. 8, 2993– 3012
https://www.jmaterenvironsci.com/Document/vol8/vol8_N8/319-JMES-2601- Akazdam.pdf
. Res. 77, 20–28. doi: 10.5897/AJMR.9000593 .
Benguella, B., Benaissa, H., 2002. Cadmium removal from aqueous solutions by chitin: kinetic
and equilibrium studies. Water Res. 36, 2463–2474. doi: 10.1016/s0043-1354(01)00459-6 .
Cardoso, N.F., Pinto, R.B., Lima, E.C., Calvete, T., Amavisca, C.V., Royer, B., Cunha, M.L.,
Fernandes, T.H.M., Pinto, I.S., 2011. Removal of remazol black B textile dye from aqueous
solution by adsorption. Desal 269 (1-3), 92–103. doi: 10.1016/j.desal.2010.10.047 .
Carletto, S., Chimirri, F., Bosco, F., Ferrero, F., 2008. Adsorption of congo red dye on hazel- nut
shells and degradation with Phanerochate chrysosporium. BioRes 3, 1146–1155
https://ojs.cnr.ncsu.edu/index.php/BioRes/article/view/BioRes_03_4_1146_Carletto_
CBF_Adsorp_Congo_Red_Hzelnut .
Couto, R.S., 2007. Decolouration of industrial azo dyes by crude laccase from Trametes hirsuta. J.
Hazard. Mater. 148 (3), 768–770. doi: 10.1016/j.jhazmat.2007.06.123 .
Couto, R.S., 2009. Dye removal by immobilised fungi. Biotechnol. Adv. 27 (3), 227–235. doi:
10.1016/j.biotechadv.2008.12.001 .
Das, S., Singh, S., Garg, S., 2019. Evaluation of wheat bran as a biosorbent for potential mitigation
of dye pollution in industrial wastewaters. Orient. J. Chem. 35 (5), 1565– 1573. doi:
10.13005/ojc/350514 .
Gupta, V.K., 2009. Suhas, Application of low-cost adsorbents for dye removal – A review. J.
Environ. Mgt. 90, 2313–2342. doi: 10.1016/j.jenvman.2008.11.017 .
Hamdaoui, O., Chiha, M., 2007. Removal of methylene blue from aqueous solutions by wheat
bran. Acta Chim. Slov. 54, 407–418
12
Environmental Challenges 7 (2022) 100459
https://www.researchgate.net/publication/224892540_Removal_of_Methylene_Blue_
from_Aqueous_Solutions_by_Wheat_Bran .
Hameed, B.H., Krishni, R.R., Sata, S.A., 2009. A novel agricultural waste adsorbent for the
removal of cationic dye from aqueous solutions. J. Hazard. Mater. 162, 305–311. doi:
10.1016/j.jhazmat.2008.05.036 .
Hossain, S.M., Anantharaman, N., 2008. Effect of wheat straw powder on enhancement of
lignolytic enzyme activity using P. chrysosporium. Indian J. Biotechnol. 7, 502–507
http://nopr.niscair.res.in/handle/123456789/2354 .
Huang, D-L., Zeng, G-M., Feng, C-L., Hu, S., Jiang, X-Y., Tang, L., Su, F-F., Zhang, Y., Zeng,
W., Liu, H-L., 2008. Degradation of lead-contaminated lignocellulosic waste by
Phanerochaete chrysosporium and the reduction of lead toxicity. Environ. Sci.Technol. 42,
4946–4951. doi: 10.1021/es800072c .
Jalandoni-Buan, A.C., Decena-Soliven, A.L.A., Cao, E.P., Barraquio, V.L., Barraquio, W.L.,
2009. Congo red decolorizing activity under microcosm and decolorization of other dyes by
congo red decolorizing bacteria. Philippine J. Sc. 138 (2), 125–132
https://philjournalsci.dost.gov.ph/images/pdf/pjs_pdf/vol138no2/pdfs/Conge_Red
_Deocolorizing.pdf .
Kalyani, D.C., Telke, A.A., Dhanve, R.S., Jadhav, A., 2009. Eco-friendly biodegradation and
detoxification of Reactive red 2 textile dye by newly isolated Pseudomonas sp., SUK1. J.
Hazard. Mater. 163 (2-3), 735–742. doi: 10.1016/j.jhazmat.2008.07.020 .
Keasling, J., Martin, H.G., Lee, T.S., Mukhopadhyay, A., Singer, S.W., Sundstrom, E.,
2021. Microbial production of advanced biofuels. Nature Rev. Microbiol. 19, 701– 715. doi:
10.1038/s41579-021-00577-w .
Kezerle, A., Velic, N., Kovacevic, D., 2018. Lignocellulosic materials as dye adsorbents:
adsorption of methylene blue and congo red on Brewer’s Spent Grain. Croat. Chem. Acta. 91
(1), 53–64. doi: 10.5562/cca3289 .
Khalaf, M.A., 2008. Biosorption of reactive dye from textile wastewater by non-viable biomass of
Aspergillus niger and Spirogyra sp. Biores. Technol. 99, 6631–6634. doi:
10.1016/j.biortech.2007.12.010 .
Lade, H.S., Waghmode, T.R., A.A.Kadam, S.P.Govindwar, 2012. Enhanced biodegrada- tion and
detoxification of disperse azo dye Rubine GFL and textile industry efflu- ent by defined
fungal-bacterial consortium. Int. Biodeterior. Biodegrad. 72, 94–e107. doi:
10.1016/j.ibiod.2012.06.001 .
Lellis, B., Favaro-Polonio, C.Z., Pamphile, J.A., Polonio, J.C., 2019. Effects of textile dyes on
health and the environment and bioremediation potential of living organisms. Biotechnol.
Res. Innov. 3 (2), 275–290. doi: 10.1016/j.biori.2019.09.001 .
Liu, J. , Li, E. , You, X. , Hu, C. , H, Q. , 2016. Adsorption of methylene blue on an agrowaste
oiltea shell with and without fungal treatment. Sci. Rep. Nat. 6, 38450
https://www.nature.com/articles/srep38450#:~:text = Without%20fungal%20 treatment%2C
%20the%20maximum,85.7%20mg%2Fg%2C%20respectively .
Malik, D.S., Jain, C.K., Yadav, A.K., Kothari, R., Pathak, V.V., 2016. Removal of methylene blue
dye in aqueous solution by agricultural waste. Int. Res. J. Engg. Technol. 3 (7), 864–880
https://www.irjet.net/archives/V3/i7/IRJET-V3I7160.pdf .
Mani, S., Chowdhary, P., Bhargava, R.N., 2019. Emerging and eco-friendly approaches for waste
management. In: Bhargava, R., Chowdhary, P. (Eds.), Textile Wastewater Dyes: Toxicity
Profile and Treatment Approaches. Springer, Singapore, pp. 219–244. doi: 10.1007/978-981-
10-8669-4_11 .
Moenian, K., Mehidinia, S.M., 2019. Removing Methylene Blue from Aqueous Solu- tions Using
Rice Husk Silica Adsorbent. Pol. J. Environ. Stud. 28 (4), 2281–2287. doi:
10.15244/pjoes/91044 .
Moldes, D., Ferna´ndez-Ferna´ndez, M., Sanroma´n, M.A., 2012. Role of laccase and low
molecular weight metabolites from Trametes versicolor in dye decolorization. The Scient.
World J. 398725. doi: 10.1100/2012/398725 , 9 pages .
Muthezhilan, R., Ashok, R., Jayalakshmi, S., 2007. Production and optimization of ther- mostable
alkaline xylanase by Penicillum oxalicum in solid state fermentation. African J. Microbiol.
Nigam, P., Armour, G., Banat, I.M., Singh, D., Marchant, R., 2000. Physical removal of textile
dyes from effluents and solid-state fermentation of dye-adsorbed agricultural residues. Biores.
Technol. 72, 219–226. doi: 10.1016/S0960-8524(99)00123-6 .
Ozmen, N. , Yesilada, O. , 2012. Valorization and of dye adsorbed on lignocellulosic using white
rot fungi. Bio. Res. 7 (2), 1656–1665 https://bioresources.cnr.ncsu.edu/
resources/valorization-and-biodecolorization-of-dye-adsorbed-on-lignocellulosics- using-
white-rot-fungi/ .
Parshetti, G.K., Telke, A.A., Kalyani, D.C., Govindwar, S.P., 2010. Decolorization and
detoxification of sulfonated azo dye methyl orange by Kocuria rosea MTCC 1532. J. Hazard.
Mater. 176, 503–509. doi: 10.1016/j.jhazmat.2009.11.058 .
Ride, J.P., 1980. The effect of induced lignification on the resistance of wheat cell walls to fungal
degradation. Physiol. Plant Pathol. 16, 187–196
https://www.cabdirect.org/cabdirect/abstract/19801364560 .
Salehi, R., Aramia, M., Mahmoodib, N.M, Bahramia, H., Khorramfara, S., 2010. Novel
biocompatible composite (Chitosan–zinc oxide nanoparticle): Preparation, char- acterization
and dye adsorption properties. Coll. Surf. B: Biointerf. 80, 86–93. doi:
10.1016/j.colsurfb.2010.05.039 .
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

Singh, S. , Tolupe, K. , 2017. Lignocellulosic residues for physical removal of harmful dyes from
industrial waste water-availability, utilization and scope. Int. J. ChemTech. Res. 10 (2), 1005–
1020 https://www.researchgate.net/publication/338606813_
Lignocellulosic_Residues_for_Physical_Removal_of_Harmful_Dyes_from_Industrial_
Waste_Water-Availability_Utilization_and_Scope .
Singh, S., Tyagi, C.H., Dutt, D., Upadhyaya, J.S., 2009. Production of high level of cellulase-poor
xylanases by wild strains of white rot fungus Coprinellus disseminatus in solid state
fermentation. New Biotechnol. 26 ( ), 165–170. doi: 10.1016/j.nbt.2009.09.004 .
Singh, S., Dutt, D., Tyagi, C.H., 2011. Complete characterization of wheat straw (Triticum
aestivum PBW-343 L. Emend. Fiori & Paol.)–A renewable source of fibres for pulp and
paper making. BioRes 6 (1), 154–177. doi: 10.15376/biores.6.1.154-177 .
Singh, S., Singh, S., Bali, V., Sharma, L., Mangla, J., 2014. Production of Fungal Amylases using
cheap, readily available agri-residues for poten- tial application in Textile industry. BioMed.
Res. Int. 215748, 9 pages. https://www.hindawi.com/journals/bmri/2014/215748/ .
Slampova, A., Smela, D., Vondrackova, A., Jancarova, I., Kuban, V., 2001. De- termination of
synthetic colorants in foodstuffs. Chem. Listy. 95, 163–168
https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1745-4557.2005.00012.x .
Stoilova, I., Krastanov, A., Stanchev, A., 2010. Properties of crude laccase from Trametes
versicolor produced by solid-substrate fermentation. Adv. Biosc. Biotechnol. 1, 208– 215.
doi: 10.4236/abb.2010.13029 .
Sumanjit, T.P.S.W., Kaur, R., 2007. Removal of health hazards causing acidic dyes from aqueous
solutions by the process of adsorption. Onl. J. Health All. Scs. 6 (3), 1–10
http://cogprints.org/5929/1/2007-3-3.pdf .
Tiwari, M., Shukla, S.P., Mohan, D., 2017. Toxicity of Disperse Dyes and its removal from
wastewater using various adsorbents: a review. Res. J. Env. Toxicol. 11, 72–89. doi:
10.3923/rjet.2017.72.89 .
Urra, J., Sepulveda, L., Contreras, E., Panssa, C., 2006. Screening of static culture and comparison
of batch and continuous culture for textile dye by P. chrysosporium. Braz. J. Chem. Engg. 23
(3), 281–290. doi: 10.1590/S0104-66322006000300002 .
Vishwakarma, S., Singh, M.P., Srivastava, A.K., Pandey, V.K., 2012. Azo dye (direct blue 14)
decolorization by immobilized extracellular enzymes of Pleurotus species. Cell. Mol. Biol. 58
(1), 21–25 https://pubmed.ncbi.nlm.nih.gov/23273187/ .
Vishwakarma, S.K. , Singh, M.P. , Srivastava, A.K. , Pandey, V.K. , 2012. Azo dye (direct blue
14) decolorization by immobilized extracellular enzymes of Pleurotus species. Cell. Mol.
Biol. 58 (1), 21–25 10.1170/T916 .
Waghmode, T.R., Kurade, M.B., Khandare, R.V., Govindwar, S.P., 2011. A sequential
aerobic/microaerophilic decolorization of sulfonated mono azo dye Golden yel- low HER by
microbial consortium GG-BL. Int. Biodeter. Biodeg. 65, 1024–e1034. doi:
10.1016/j.ibiod.2011.08.002 .
Walker, G.M., Hansen, L., Hanna, J.A., Allen, S.J., 2003. Kinetics of a re- active dye adsorption
onto dolomitic sorbents. Water Res 37, 2081–2089. doi: 10.1016/S0043-1354(02)00540-7 .
Wu, J., Elliston, A., Gall, G.L., Colquhoun, I.J., Collins, S.R.A., Wood, I.P., Dicks, J., Roberts,
I.N., Waldron, K.W., 2018. Optimizing conditions for bioethanol production from rice husk
and rice straw: effects of pre-treatment on liquor composition and fer- mentation inhibitors.
Biotechnol. Biofuels. 11, 62. doi: 10.1186/s13068-018-1062-7 .
Yusuf, M., Shabbir, M., Mohammad, F., 2017. Natural colorants: historical, processing and
sustainable prospects. Nat. Prod. Bioprospect 7, 123–145. doi: 10.1007/s13659-017-0119-9 .
Zeng, G., Cheng, M., Huang, D., Lai, C., Xu, P., Wei, Z., Li, N., Zhang, C., He, X., He, Y., 2015.
Study of the degradation of methylene blue by semi-solid state fermentation of agricultural
residues with Phanerochaete chrysosporium and reutilization of fer- mented residues. Waste
Mgt 38, 424–430. doi: 10.1016/j.wasman.2015.01.012 .

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