Environmental Challenges 7 (2022) 100459

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Environmental Challenges
journal homepage: www.elsevier.com/locate/envc

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

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

Keywords: Environmental pollution is a major problem with increasing human population and correspondingly increasing
Industrial dyes industrial activities. Chemical industrial dyes can cause allergies, cancers, etc., in humans and adversely affect
Lignocellulosic waste aquatic life when dye rich effluents are released into water bodies. Though natural and safe dyes are being
Solid state fermentation
explored yet the bulk of industrial dyes is chemical based. The current study explores cheap lignocellulosics for
Recycling
dye decolorization and use of resulting dye-adsorbed biomaterials as substrate in microbial fermentation (SSF).
Environment sustainability
Further, we evaluated a reuse of such dye-laden lignocelluloses for 2nd 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 2nd 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 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 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 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

∗
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/)
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 2nd 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 temper-
atures (10°C, 20°C, 30°C, 40°C, 50°C and 60°C) for different incubation
2
Environmental Challenges 7 (2022) 100459
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 (NH4 )2 SO4 —0.14%, KH2 PO4 —0.2%, MgSO4 .7H2 O—0.03%,
CaCl2 —0.03%, FeSO4 .7H2 O—0.002%, ZnSO4 .7H2 O, —0.002%,
MnSO4 .7H2 O—0.002%, CoCl2 —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 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 lac-
S. Singh, K.T. Adeyemi and S. Vernwal
case 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 2nd
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.
3.2. 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 op-
timized conditions of incubation (Fig. 2). WB closely followed RH in
dye removal efficiency with only 0.4% (almost negligible) lesser dye re-
moval than that observed for RH. Even the other adsorbents maintained
more than 85% dye decolorization.
3
Environmental Challenges 7 (2022) 100459
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 9th day of incubation while lowest laccase activity was observed, as
expected, on 1st day of incubation (50U/mL). A decrease of almost 28%
in laccase activity was observed on 10th day of incubation for RH. Both
WS and WB as substrates, encouraged the maximum enzyme production
by the test fungus on 5th 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 4th 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 4th 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 2nd 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.
3.5. Potential reuse of spent fungal-agri-biomass in dye decolorization and
laccase production
An evaluation of test lignocellulosic materials for a 2nd 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 sat-
isfactory dye removal in the 2nd cycle, with 71.9, 76.7, 79.2% dye de-
colorization for RH, WB and WBWS, respectively.
When laccase activity was checked in the 2nd cycle of microbial fer-
mentation of the agri-residues, RH yielded a slight increase in enzyme
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

Table 1
Influence of incubation temperature & time on dye decolorization.

Incubation time, min Incubation temperature, o C Adsorbents

Dye decolorization, %
RH 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
40 88.2 83.5 81.4 82.2 71.6
50 78.3 84.5 81.2 81.3 69.9
60 77.2 83.7 80.0 79.8 69.2

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 1st cycle of
treatment. Other substrates on the other hand, recorded lesser laccase
activities in comparison to the 1st 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 adsorp-
tion 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 decol-
orization 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

4
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

100 RH WS WB RHWS WBWS

80
D y e R e mo v a l ( %)

60

40

20

0
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
pH
Fig. 1. Effect of pH on % dye decolorization.
Fig. 2. Decolorization of Methylene blue by agri-
100 residues under optimized conditions.
Dye Removal (%)

80

60

40

20

0
B
S

S
H

S
W

BW
W
R

H
R

Type of Agri-residue
(Moenian 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.
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).

5
S. Singh, K.T. Adeyemi and S. Vernwal Environmental Challenges 7 (2022) 100459

Fig. 3. Laccase activity of P. chrysosporium MTCC787

800
LACCASE ACTIVITY (U/mL)
using agri-residues as substrate under optimized fer-
mentation conditions.

600

400

200

0
B

S
S
H

S
W
W

BW
W
R

H
R

Type of Agri-residue W

Table 2 Table 3
Influence of dye concentration on dye decolorization. Effect of incubation period on laccase production.

S. No. Substrate Dye Concentration, mg/L Dye decolorization, % Incubation period Laccase activity (U/mL)
(Days)
1. RH 40 91.9 RH WS WB RHWS WBWS
50 90.4
1 50.0±3.0 275.8±3.4 196.0±1.7 292.0±1.2 315.8±2.7
60 86.7
2 72.0±1.1 300.6±1.1 234.0±1.5 303.0±0.9 325.6±1.6
70 85.5
3 110.0±0.5 309.1± 1.2 468.0±3.5 342.0±1.1 330.0±1.4
80 85.1
4 132.8±1.0 315.0±2.0 513.0±1.9 374.0±1.3 341.0±1.8
2. WS 40 87.0
5 170.0±3.0 339.0±3.1 635.0±1.3 359.0±1.6 322.3±1.4
50 85.8
6 220.5±1.3 300.0±2.3 421.0±1.8 344.0±2.0 295.2±2.0
60 83.2
7 253.6±2.4 291.3±1.0 402.2±3.0 - -
70 82.8
8 284.9±1.2 - - - -
80 82.2
9 378.1± 2.2 - - - -
3. WB 40 91.7
10 271.0±3.2 - - - -
50 89.0
11 243.0±1.5 - - - -
60 83.6
12 - - - - -
70 81.7
80 81.5 Incubation conditions:
4. RHWS 40 81.4
Inoculum size: 2 fungal discs of 0.5 cm each
50 84.0
60 83.4
Initial medium pH: 6.0
70 84.1 Substrate: Moistening agent: 1:3
80 82.0 Incubation temperature, °C: 37
5. WBWS 40 85.4 ±: means Standard deviation from mean
50 84.1
60 84.2
70 79.2
80 76.7 mentation probably due to much high lignin and silica content, which
Incubation conditions:
might lead to longer incubation times to fermentation (Wu et al., 2018).
Incubation temperature, °C: RH (20), WS and WBWS (50), WB (30), Interestingly, WS showed best laccase activity in much lesser time,
RHWS (40) probably because beyond a point, the fungus was not able to assimilate
Incubation duration, mins: RH(60), WS and WBWS (150), WB (90), nutrients from the complex structure. WB showed the highest activity
RHWS(30) amongst the substrates tested, indicating that the nutrient complex and
Substrate: Dye: 1:10 the kind of nutrient forms were more easily used up by the fungus under
pH: 6.0 the given set of conditions.
The laccase activity, in response to variable incubation temperature,
increased with time upto the maximum. The encouraging performance
As reported above, RH needed the longest incubation time for max- of substrates in binary combinations might be due to better availabil-
imum laccase production in comparison to other substrates tested. This ity of nutrients in a complex nutritional environment with two sub-
is most likely due to restricted availability of nutrients from a complex strates mixed together as in case of WB mixed with WS (WBWS) or
substrate like RH. The fungus thus, required more time to mineralize even RHWS, which was also better substrate than the substrates used
and assimilate. Studies also indicate recalcitrant nature of RH to fer- singly, except in case of singly used WB. P.chrysosporium grows best be-

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

Table 4 Table 6
Effect of incubation temperature on laccase production. Effect of initial medium pH on laccase production.

S.No Substrate Temperature (°C) Laccase activity (U/mL) S.No Substrate pH of Medium Laccase activity (U/mL)

1 RH 28±2 125.0±0.7 1 RH 5.0 125.3±1.2

33±2 134.7±0.8 5.5 178.0±0.9
37±2 386.3±1.5 6.0 518.0± 1.2
40±2 155.3±2.2 6.5 570.7± 0.8
2 WS 28±2 221.7±1.5 7.0 411.3±0.9
33±2 317.0±1.1 7.5 301.0±1.5
37±2 347.3±1.3 8.0 198.9±0.9
40±2 262.0±1.4 2 WS 5.0 322.8±1.2
3 WB 28±2 442.0±1.4 5.5 363.0±0.7
33±2 621.2±1.9 6.0 351.0±0.5
37±2 650.0±2.3 6.5 253.0±0.7
40±2 554.4±1.7 7.0 208.7±0.8
4 RHWS 28±2 284.7±1.4 7.5 198.0±2.1
33±2 394.3± 1.2 8.0 195.2±0.9
37±2 386.0±0.9 3 WB 5.0 542.1±1.0
40±2 207.7±0.5 5.5 564.0±0.9
5 WBWS 28±2 240.0±2.7 6.0 705.0 ± 2.1
33±2 326.0±3.0 6.5 650.0±1.0
37±2 350.0±1.6 7.0 610.0±1.2
40±2 224.5±1.5 7.5 581.1±3.0
8.0 532.5±1.1
Incubation conditions: 4 RHWS 5.0 211.3±1.2
Inoculum size: fungal discs of 0.5 cm each 5.5 278.0±0.8
Initial medium pH: 6.0 6.0 440.3±1.3
Substrate: Moistening agent: 1:3 6.5 236.7±0.9
Incubation period: RH (9th day of incubation),WS and WB (5th 7.0 220.3±1.2
day of incubation), RHWS and WBWS (4th day of incubation) 7.5 201.5±.2.0
8.0 197.1±3.1
±: means Standard deviation from mean
5 WBWS 5.0 110.0± 0.8
5.5 172.3±1.7
Table 5 6.0 372.1 ± 2.3
Effect of inoculum size on the laccase production. 6.5 400.5±1.4
7.0 776.0±1.8
S.No Substrate Number of Disc (s) inoculated Laccase activity (U/mL) 7.5 743.3±1.0
8.0 720.5±2.1
1 RH 1 230.7±3.7
2 396.0± 0.4 Incubation conditions:
3 409.3±0.6
Substrate: Moistening Agent: 1:3
4 385.0±2.2
Incubation temperature, ºC: RH(37°C), WS(37°C), RHWS
2 WS 1 113.7±1.2
2 303.0±1.7 (35°C)
3 335.0±1.3 Incubation period, days: RH (9days), WS (5days), RHWS
4 355.3±1.4 (4days)
3 WB 1 306.4±1.9 Inoculum size: 3 fungal discs (RH and WB), 4 fungal discs
2 622.0±1.3 (WS), 2 fungal discs (RHWS and WBWS)– of 0.5 cm each
3 695.0± 1.1 ±: means Standard deviation from mean
4 422.0 ±1.2
4 RHWS 1 316.0± 1.2
2 404.7± 0.9
3 266.7±0.7 vaded faster and probably better by the test fungi, than WB. This further
4 124.7±1.1 showed that better growth support provided by combo-substrates than
5 WBWS 1 288.5±2.1 WB, a singly used substrate. The need for largest inoculum size tested for
2 368.0±3.1
WS can be attributed to the complex physical and chemical composition
3 286.0±2.6
4 222.1±1.8
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-
Incubation conditions:
ity to use nutrients from even in a nutrient rich environment, initial
Initial medium pH: 6.0
fermentation medium pH was also evaluated as a factor in laccase pro-
Substrate: Moistening agent: 1:3
Incubation period, days: RH (9th day of incubation),WS and WB (5th day of duction in the given study. Interestingly, but not surprisingly too, the
incubation), RHWS and WBWS (4th day of incubation) test fungus showed varied preference to the medium pH to show best
Incubation temperature, ˚C: 37 (WB, WS, WB+WS) laccase activity while using different substrates. The medium pH also
±: means Standard deviation from mean 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
tween 20-50°C. At higher temperatures (above 40°C), death rate exceeds successsully fermented by the test fungus. Though the laccase activities
growth rate while at lower temperatures, metabolic ativities are highly varied for different substrates, yet the amount of the test enzyme pro-
reduced (Hossain and Anantharaman, 2008). duced, was significantly high. As also supported by other investigations
Inoculum size (Table 5) was also found to be an important parameter (Liu et al., 2016), successful growth and laccase production thereof, us-
for laccase production with variable enzyme activity observed for differ- ing dye adsorbed substrates also might further modify the physical and
ent inoculum size in case of different substrates. The findings indicated chemical properties of the dye adsorbed substrate, probably leading to
that probably both RHWS and WBWS though produced lesser laccase breakdown of the complex residue structure and making its degradation
activity than the best performer (WB), yet these two substrates were in- in the environment and also provide laccase as a beneficial co-product.

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

Table 7
Recycling potential of dye adsorbed lignocellulosics for dye decolorization and laccase production.

S.No. Spent Reuse potential of spent ligno-cellulosic biomass

agri-fungal
Dye decolorization (%) for recycled Efficiency as dye Laccase production Efficiency as
biomass
adsorbent adsorbent (% increase (U/mL) for recycled substrate for laccase
↑/decrease ↓ in fermentation production (%
comparison to virgin substrate increase ↑/decrease
adsorbents) ↓ in comparison to
virgin fermentation
substrate)

1 RH 71.9 22.9 ↓ 597.5±1.3 3.8 ↑

2 WS 92.4 4.9 ↑ 326.0±2.5 11.0 ↓
3 WB 76.7 17.42 ↓ 412.0±2.2 42.3 ↓
4 RHWS 88.1 2.7 ↑ 510.0±1.0 13.3 ↑
5 WBWS 79.2 8.5 ↓ 564.0±2.1 27.7 ↓

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-
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 2nd 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 2nd cycle of microbial
fermentation of the agri-residues, apart from RH, all other substrates
recorded lesser laccase activities in comparison to the 1st cycle. It might
be possible that a higher amount of residual lignin was available for
enzymatic breakdown after 1st 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 (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.
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.

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

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