Extremophiles

DOI 10.1007/s00792-017-0958-7

ORIGINAL PAPER

Delignification and detoxification of peanut shell bio‑waste using

an extremely halophilic laccase from an Aquisalibacillus elongatus
isolate
Rezvan Rezaie1 · Shahla Rezaei1 · Nasrin Jafari1 · Hamid Forootanfar2 ·
Mohammad Reza Khoshayand3 · Mohammad Ali Faramarzi1 

Received: 10 June 2017 / Accepted: 21 August 2017

© Springer Japan KK 2017

Abstract  Lignocellulose bioconversion is a harsh process
requiring the use of surfactants and organic solvents. Conse-
quently, the incorporation of laccases in this bioconversion
requires the bioprospecting of enzymes that can remain stable
under extreme conditions. An extracellular, highly stable lac-
case was produced by the halophilic isolate Aquisalibacillus
elongatus in submerged liquid culture fermentation. Statistical
and non-statistical strategies gave the highest enzymatic activity
(8.02 U mL−1) following addition of glucose (1.7 g L−1), cop-
per sulfate (0.8 g L−1), urea (15 g L­ −1), and ­CaCl2 (0.8 g L−1).
The enzyme, after purification using a synthetic affinity sup-
port, delignified a peanut shell substrate by 45%. A pH of 8.0
and a temperature of 35 °C were optimal for delignification of
this bio-waste material. Addition of ­[Bmim][PF6], 1,4-dioxane,
acetone, and HBT promoted this bio-waste delignification. Bio-
treatment in the presence of 50% ­[Bmim][PF6] gave a maximal
lignin removal of 87%. The surfactants tested had no significant
effects on the delignification yield. The laccase also detoxified
the toxic phenols found in peanut shell waste. The high catalytic
efficiency of this enzyme against a lignocellulosic sample under
extreme conditions suggests the suitability of this laccase for
industrial applications.
Keywords  Laccase · Halophile · Optimization ·
Aquisalibacillus elongatus · Delignification · Peanut shell

Communicated by L. Huang. Introduction

Mohammad Reza Khoshayand is the correspondance statistical Lignin degradation and detoxification by laccases has received
experimental design. considerable attention as a means of reducing the accumu-
Electronic supplementary material  The online version of this
lation of lignocellulosic bio-wastes (Virk et al. 2012). Lac-
article (doi:10.1007/s00792-017-0958-7) contains supplementary case digestion provides a milder, cleaner, and more efficient
material, which is available to authorized users. treatment method for bio-delignification of lignocellulose,
without damaging the cellulose (Asina et al. 2016; Sindhu
* Mohammad Ali Faramarzi
et al. 2016). The use of ligninolytic enzymes is very effective
faramarz@tums.ac.ir
when combined with a solvent or surfactant that enhances the
Mohammad Reza Khoshayand
accessibility of the lignin to the enzyme (Rezaei et al. 2017).
khoshayand@tums.ac.ir
Recent studies have demonstrated that halophilic enzymes
1
Department of Pharmaceutical Biotechnology, Faculty are very selective in terms of their activity in the presence
of Pharmacy and Biotechnology Research Center, Tehran of ionic liquids, which makes them ecofriendly alternatives
University of Medical Sciences, P.O. Box 14155‑6451,
to co-solvents, organic solvents, and surfactants (Oren 2010;
1417614411 Tehran, Iran
2
de Lourdes Moreno et al. 2013). Consequently, the use of
Department of Pharmaceutical Biotechnology, Faculty
a halophilic ligninolytic enzyme would seem to represent a
of Pharmacy, Kerman University of Medical Sciences,
Kerman, Iran very attractive technology for bio-delignifying natural wastes.
3 The production of ligninolytic enzymes needs to be
Department of Drug and Food Control, Faculty
of Pharmacy, Tehran University of Medical Sciences, studied in various species with different ecological back-
P.O. Box 14155‑6451, 1417614411 Tehran, Iran grounds. Bacteria can degrade lignocellulose effectively,

13
Vol.:(0123456789)
 Extremophiles
with laccases being the most important members of
the ligninolytic system (Sondhi et al. 2015; Asina et al.
2016). Laccases are currently viewed as highly interest-
ing enzymes for industrial purposes because of their wide
variety of potential substrates (Forootanfar and Faramarzi
2015). Therefore, a broad study of microorganisms with
laccase activity is necessary for the identification of suit-
able laccases from various strains. Many fungal laccases
have been isolated and characterized in the past years (Lu
et al. 2010; Knežević et al. 2013; Ghorbani et al. 2015;
Karp et al. 2015), and substantial effort is being made to
identify highly specific enzymes that can be tailored to
particular industrial processes.
Enzymes used in industrial processes are frequently
required to function in harsh conditions, so highly desir-
able characteristics include the ability to retain activity at
extreme pH and temperature and to tolerate high salin-
ity, metal ions, and organic solvents (Ventosa et al. 2004;
Moshfegh et al. 2013; Raddadi et al. 2015). Of the laccase
producers, halophilic bacteria have attracted much atten-
tion as their laccases seem to have potential industrial
applications (Rezaei et al. 2017). The pre-treatment of
lignocellulosic feedstocks also generates a number of by-
products that can inhibit the fermentation of lignocellulosic
hydrolysates. Many studies also have shown the potential
of laccase for detoxification of the treated lignocelluloses
or similar substrates (Rezaei et al. 2015; Vithanage et al.
2015). A method for facile removal of these inhibitory bio-
waste compounds would, therefore, aid pre-treatment and
improve the overall enzymatic hydrolysis and subsequent
fermentation processes.
The present work reports the production of a highly sta-
ble laccase by an isolate of Aquisalibacillus elongates, an
extremely halophilic bacterium, and optimization of this
enzyme production. The laccase was confirmed to have clear
potential for industrial application based on its ability to
delignify and detoxify a sample high-lignin biomass, pea-
nut shell (~30% lignin), under diverse operating conditions.
Peanut shell is an important agro-residue generated in high
amounts by related industries around the world (Bharthare
et al. 2014; Anike et al. 2016; Yamoum and Magaraphan
2017).
Materials and methods
Chemicals
Laccase substrates were obtained from Sigma-Aldrich (St.
Louis, MO, USA). Buffers and culture media components
were purchased from Merck (Darmstadt, Germany). All
other chemicals and reagents were of analytical grade and
obtained from commercial sources, unless otherwise stated.
13
Strain, culture conditions, and submerged fermentation
The isolate used in this study was Aquisalibacillus elonga‑
tus SR-073 Halo, a stock culture from the Department of
Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran (GeneBank
Accession No. KU587812, www.ncbi.nlm.nih.org/ avail-
able June 10, 2017). The strain was transferred monthly to
Luria–Bertani agar (LBA) media, which included 1% pep-
tone, 0.5% yeast, 1.5% agar, 11.7% NaCl, 0.006% C ­ uSO4,
and was stored at 4  °C. An inoculum was produced by
removing one loop-full of stock culture from LBA plates
and inoculating it into 250 mL flasks containing 50 mL of
LB broth (LBB). After 2 days of incubation, 1 mL of the
pre-culture was inoculated into Erlenmeyer flasks (250 mL)
containing 50 mL of fermentation M9 basal medium, and
culture was continued. The basal medium used for the exper-
iments contained (g L−1): glucose (1.0); ­Na2HPO4 (12.8);
­KH2PO4 (3.0); ­MgSO4.7H2O (0.1); ­NH4Cl (1.0); NaCl
(116.9), and C­ uSO4 (0.062). The initial medium pH was
adjusted to 7.0. Cultures were incubated at 37 °C on a rotary
shaker (150 rpm) for laccase production. Upon conclusion of
the fermentation process, the liquid cultures were harvested
and pooled. Cells were removed by centrifugation at 8000×g
for 15 min at 4 °C and the obtained supernatant was used as
the source of the crude enzyme.
Laccase assay and protein estimation
Laccase activity was assayed at 40 °C using 2,6-dimethoxy-
phenol (2,6-DMP) as the substrate. The oxidation of 2,6-
DMP was followed by measuring the absorbance increase at
468 nm after 1 min using a spectrophotometer. The reaction
solution volume was 2 mL, and consisted of 1 mL of 4 mM
2,6-DMP in phosphate buffer (50 mM, pH 7.5) and 1 mL of
the enzyme solution. The enzyme activity was expressed as
international units (IU), where 1 IU represents the amount of
enzyme that forms 1 μmol of product per minute. All assays
were carried out in triplicate. The protein concentration was
estimated by the Bradford’s method with a bovine serum
albumin standard (Bradford 1976).
Optimization of laccase production process
and experimental designs
The point of maximal growth and laccase production was
determined by measuring laccase activities over a 5-day
period. The effect of carbon and nitrogen sources, trace ele-
ments, salts, inducers, culture pH, and culture temperature
on laccase production by A. elongatus was estimated using
the one-factor-at-a-time (OFAT) method.
Ten effective factors (urea, pyrogallol, glucose, ­CaCl2,
­KH2PO4, ­Na2HPO4, ­CaCO3, ­FeSO4, ­CuSO4, and M ­ nSO4)
Extremophiles
were selected by OFAT. Two additional steps were then
required for the optimization of laccase production. First,
the effective medium composition variables (selected pre-
viously by preliminary tests) that had significant impact on
laccase production were identified using the application of
Plackett–Burman design (PBD) (screening study) (Aghaie-
Khouzani et al. 2012; Samaei-Nouroozi et al., 2015). The
second step involved the use of central composite design to
determine the optimum values for the selected variables to
optimize laccase production (optimization study) (Aghaie-
Khouzani et al. 2012; Samaei-Nouroozi et al. 2015) (Tables
S1‒S4).
Optimization of laccase production process
by one‑factor‑at‑a‑time (OFAT)
The effects of various carbon sources (glucose, fructose,
galactose, lactose, sucrose, maltose, and soluble starch, each
at 1% w/v) were determined by replacing glucose in the basal
medium. Various nitrogen sources (peptone, yeast extract,
skim milk, gelatin, and urea) were added individually (1%
w/v) to the fermentation medium. The cultures were incu-
bated at 37 °C and pH 8.0 and laccase activity was deter-
mined after cultivation for 96 h.
The effects of culture pH and temperature were investi-
gated by culturing at 25‒45 °C and pH 4.0‒10.0. Laccase
activity was determined every 24 h, and the highest activities
at each culture pH and temperature were noted. Triplicate
experiments were conducted for each treatment.
The effect of removal of different trace elements
­(KH2PO4, ­CaCl2, ­CaCO3, ­Na2HPO4, and M ­ gSO4) from the
basal medium or addition of others (­ MnSO4, ­FeSO4, ­ZnSO4,
and ­CuSO4) at concentrations of 0.01% w/v, and the effects
of salts (NaCl, KCl, and LiCl; 1‒5 M) were also studied.
The effects of inducers, such as 2,6-DMP, caffeine, catechol,
gallic acid, guaiacol, pyrogallol, o-toluidine, tyrosine, vanil-
lin, veratryl alcohol, and 2,5-xylidine, on laccase production
were also determined at three concentrations between 1 and
10 mM. Except when determining the effects of salt on the
enzyme production, the salt concentration in other OFAT
experiments was 3 M.
Screening study using the Plackett–Burman design
(PBD)
We used the Plackett–Burman design to identify and select
significant factors, since this method decreases the number
of runs with suitable resolution. Nevertheless, only a frac-
tion of all possible levels of each variable were run, due to
limitations of time and money. In this study, a resolution
of III was interpreted as indicating no confounding relation
between main effects. In this design, the assumption is made
that no interaction exists between the main factors. Hence,
a first-order multiple regression can appropriately model
the data. The levels of each factor in the Plackett–Burman
design are listed in Table S1. A 17-run experiment was gen-
erated by Design Expert (version 7.0.0, Stat-Ease, Inc., Min-
neapolis, MN, USA) at high (+1) and low (−1) levels, with
1 dummy variable and related response (Table S2). The use
of a dummy variable allowed the estimation of the variance
(experimental error) in the experiment and confirmation of
the adequacy of the first-order model. An F test was used
to show the statistical significance of the first-order model.
All experiments were conducted in triplicate. Data were
pooled and analyzed by Design Expert 7.0. Factors signifi-
cant at the 5% level (p < 0.05) were considered to have a
statistically significant effect on laccase production.
Optimization of the laccase production process
by central composite design (CCD)
Response surface methodology (RSM) was applied as the
statistical method to determine the optimal level of each fac-
tor. Central composite design (CCD) is the most popular
RSM method and was applied to optimize the laccase pro-
duction. It consists of a two-level fractional factorial and two
other types of points: central and axial. Uniform precision
and orthogonality are introduced as two prominent proper-
ties of this design. The factors and respective coded and
actual levels in the central composite design are given in
Table S3. A 30-run experiment generated by Design Expert
7.0 was carried out with 16 factorial points, 8 axial points,
and 6 trials at the center point (Table S4). All experiments
were conducted in triplicate. The mean value of laccase
activity (U m­ L−1) was taken as the response. A multiple
regression analysis was applied to the data obtained. The
behavior of the system was explained by the following quad-
ratic equation:
Y = 𝛽0 + 𝛽1 A + 𝛽2 B + 𝛽3 C + 𝛽4 D + 𝛽12 AB + 𝛽13 AC + 𝛽14 AD
+ 𝛽23 BC + 𝛽24 BD + 𝛽34 CD + 𝛽11 A2 + 𝛽22 B2 + 𝛽33 C2 + 𝛽44 D2 ,
where Y is the enzyme activity (U m ­ L−1), β0 is the intercept,
β1, β2, β3, and β4 are linear coefficients, β11, β22, and β33
are quadratic coefficients, β12, β13, β14, β23, β24, and β34 are
interactive coefficients, and A, B, C, and D are the coded
independent factors.
Design expert was used to perform the statistical analy-
sis using regression modeling and response surface graphs.
ANOVA was also used through Fisher’s test to find the
effect of the independent variables on the response, and
a p value < 0.05 was considered statistically significant.
The fitness of the second-order polynomial model equa-
tion was indicated qualitatively by the multiple correlation
coefficients (R2) and adjusted R2. Contour plots and three-
dimensional surface plots illustrated the relationship and
13
 Extremophiles
interaction between coded variables and the responses. The
equation from the final quadratic model and a grid search
of an RSM plot were used to determine the optimal points.
Enzyme purification
The enzyme was produced and purified using the method
described by Rezaei et al. (2017). Briefly, the enzyme was
produced using a 2% inoculum in a 2000-mL Erlenmeyer
flask containing a working volume of 500 mL of the fermen-
tation medium, which consisted of the optimized medium
with a pH of 8.0, and incubating at 37 °C on a rotary shaker
(150 rpm). After 72 h of incubation, the cell-free supernatant
was recovered by centrifugation (8000×g, 15 min) of the
harvested culture at 4 °C and subjected to subsequent steps
of concentration and purification to yield the crude laccase
preparation. The clear supernatant was precipitated using
10% polyethylene glycol saturation. The precipitate was then
recovered by centrifugation at 18,000×g for 15 min, re-sus-
pended in 20 mL of phosphate buffer containing 3 M NaCl,
and dialyzed overnight against repeated changes of the same
buffer, followed by centrifugation at 8,000×g for 10 min.
The SBA-15-vanillyl affinity column (Rezaei et al. 2017)
was prepared by synthesizing SBA-15 by a typical method
and then using a post-synthesis modification method to
activate the resultant mesoporous SBA-15. The functional-
ized support was then coupled to vanillin by reacting the
aminated support (1 g) with vanillin (1 g dissolved in 2 mL
ethanol) in 50 mL of toluene alkalinized with trimethyl-
amine (1 mL) at room temperature for 24 h with slow stir-
ring (500 rpm). After the coupling step, the product was
washed several times with water and acetone and a reduction
reaction was performed at 4 °C on the produced Schiff base
using a solution of 50 mg of sodium borohydride ­(NaBH4)
in methanol.
The concentrate was loaded and applied to the affinity
column (10 × 100 mm) previously prepared and pre-equili-
brated with the above buffer. The column was then washed
with about 50 mL of phosphate buffer and eluted with a lin-
ear gradient of pH, urea, (­ NH4)2SO4, and C
­ uSO4. Fractions
were concentrated, dialyzed against 50 mM phosphate buffer
(pH 8.0), analyzed for laccase activity and protein concen-
tration, and stored at −20 °C for subsequent experiments.
Delignification studies
Laccase‑assisted pretreatment of peanut shell
The purified laccase was used to conduct a delignification
pretreatment of peanut shell obtained from a local store in
Iran. The peanut shell was washed with distilled water and
dried at 100 °C to ensure low moisture content prior to sub-
jecting to the other stages and treatments, It was then ground
13
in a knife mill, and the resulting particles were sieved (mesh
size 60), autoclaved, and dried in an oven to a constant
weight before subjecting the material to the next steps. The
delignification pre-treatment of the prepared peanut shell
was performed by the purified laccase in a reaction mixture
containing potassium phosphate buffer (50 mM, pH 8.0), the
purified laccase (~50 U), and peanut shell (final concentra-
tion 5% w/v) at 35 °C with orbital shaking at 150 rpm for
48 h. Control samples without the enzyme were processed
in parallel with the test samples. After the enzymatic del-
ignification process, the detoxification of peanut shell was
also determined.
Kappa number, brightness, viscosity, and delignification
efficiency measurements
Delignification activity was determined by measuring the
decrease in kappa number and the change in chemical char-
acteristics of the bio-waste. The chemical properties of pea-
nut shell were analyzed at the end of delignification process
according to the Tappi Test Methods of T 236 om-94, T
236 om-99, and T 452 (Tappi Standard Procedures 2012)
for viscosity, kappa number, and brightness measurement,
respectively. Delignification efficiency was determined as
follows: % delignification efficiency = (initial kappa num-
ber − final kappa number)/initial kappa number.
Effect of pH, temperature, surfactants, and solvents
on bio‑delignification
The effect of pH, temperature, surfactants, and organic sol-
vents on the enzymatic delignification was assessed by run-
ning reactions containing 5% peanut shell and 50 U of the
purified enzyme at 150 rpm agitation at a range of pH and
temperature of 6.0‒9.0 and 35‒55 °C, respectively, and in
the presence of 50% organic solvents ­([Bmim][PF6], acetone,
1,4-dioxane, DMSO, ethanol, or methanol), 1% surfactants
(CTAB, SDS, triton X-100, or tween-80), or 5 mM HBT.
After 24 h of incubation, the solid fraction was withdrawn
and the kappa number was measured.
Detoxification analysis
An acute toxicity test using Escherichia coli, Staphylococ‑
cus aureus, Pseudomonas aeruginosa, and Staphylococcus
epidermidis strains was performed to evaluate the cytotoxic-
ity, where toxicity was expressed in terms of inhibition of
bacterial growth. The strains were grown at 37 °C in Muel-
ler–Hinton agar (MHA) medium supplemented with samples
taken at the beginning and end of delignification process at
concentrations of 0.12‒2.00 mg mL−1, and a serial dilution
of ciprofloxacin solution from 6.25‒100.00 μg mL−1 as a
control.
Extremophiles
Statistical analysis
All experiments were performed in triplicate and the
reported data were presented as mean ± standard devia-
tion. One-way and two-way ANOVA tests were conducted
to calculate the statistical significance between the mean
values (SigmaPlot version 12.0, Systat Software, Inc., San
Jose, CA, USA). Probability values <0.05 were considered
statistically significant.
Results and discussion
Optimization of laccase production
OFAT
Laccase was secreted by the isolate in a submerged culture
containing 2 M NaCl and 0.25 mM C ­ uSO4. Extracellular
laccase activity appeared at the beginning of the exponential
phase of growth (24 h) and reached a maximal production
of approximately 0.5 U ­mL−1 at the end of this phase (48 h)
under non-optimized conditions. This pattern of secretion
was similar to that reported in other studies, in which lac-
case achieved maximal production in the stationary phase
(Uthandi et al. 2010; Sondhi et al. 2015).
Laccase production by the A. elongatus SR-073 Halo
strain used here was growth dependent. This suggests that
laccase production requires an adequate mass of metaboli-
cally active cells. When compared with the result of the pre-
sent study, the maximal production of bacterial laccase from
Haloferax volcanii (Uthandi et al. 2010) and Streptomyces
psammoticus (Niladevi and Prema 2008) was 0.17 and 5.90
U  mL−1, respectively, under non-optimized conditions.
(a)
1.0 1M
2M
3M * 1.0
4M
0.8
5M
Cell growth (OD 600 nm)
0.6
0.4
0.2
0.0
0 24 48 72
Time (h)
Fig. 1  Growth (a) and laccase production (b) of Aquisalibacillus elongatus in different concentrations of NaCl (1‒5 M) after 4 days of incuba-
tion at 37 °C in a basal medium containing C
­ uSO4 (0.25 mM), pH 8.0. Error bars represent the standard errors of three replicates
Therefore, the enzyme activity in different bacteria appears
to vary considerably and is associated with the fermentation
conditions and the bacterial species.
The effects of salts indicated that highest growth and lac-
case production (1.06 U mL−1) were observed for NaCl at
3 M (Fig. 1a, b), whereas the enzyme production was only
0.23 and 0.30 U m ­ L−1 in the presence of 2 M LiCl and 3 M
KCl, respectively (Fig. S1). The pH-temperature profile for
the enzyme production was also similar to profiles observed
for laccases from other bacteria. The laccase production
showed an optimal pH at 8.0 (Fig. 2a, b). The temperature
profile showed a maximal production at 35 °C and more than
80% production at temperatures between 30 and 40 °C, but
less than 20% of the enzyme production at 25 °C (Fig. 2c,
d). Similarly, Brander et al. (2014) and Niladevi and Prema
(2008) reported the same ranges of pH and temperature for
CotA laccase production by Bacillus clausii and S. psam‑
moticus, respectively.
An appropriate inducer can enhance laccase produc-
tion and is necessary for effective large-scale production.
In bacteria, many different inducers have been widely used
to stimulate extracellular laccase production. The effects of
various inducers on laccase production are shown in Fig. 3a.
The highest increase in laccase production (fourfold) was
observed with pyrogallol on the second day, followed by
caffeine (3.8-fold), 2,6-DMP (2.8-fold), and l-tyrosine (2.8-
fold), when compared to the control without inducers. No
appreciable induction was observed after the addition of
o-toluidine and guaiacol. Extracellular laccase production
by A. elongatus was also stimulated by addition of catechol,
veratryl alcohol, vanillin, and 2,5-xylidine.
In agreement with the results of this study, Niladevi
and Prema (2008) demonstrated that pyrogallol and
p-anisidine were the most effective inducers of laccase
(b)
1.2 1M
*
2M
3M
4M
5M
Enzyme activity (U mL )
-1
0.8
0.6
0.4
0.2
0.0
96 0 24 48 72 96
Time (h)
13
 Extremophiles

(a) (b)
1.0 pH 4 1.0 pH 4
pH 5 * pH 5
*
pH 6 pH 6
pH 7 pH 7
0.8 0.8 pH 8
pH 8

Enzyme activity (U mL )
Cell growth (OD 600 nm)

pH 9 pH 9

-1
pH 10 pH 10
0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0

0 24 48 72 96 0 24 48 72 96

Time (h) Time (h)

(c)
1.0 25 °C
(d)
30 °C 0.5 25 °C
35 °C * 30 °C
40 °C 35 °C
0.8
45 °C
0.4
40 °C *
Cell growth (OD 600 nm)

_1
Enzyme activity (U ml ) 45 °C

0.6
0.3

0.4
0.2

0.2 0.1

0.0 0.0

0 24 48 72 96 0 24 48 72 96

Time (h) Time (h)

Fig. 2  Growth (a, c) and enzyme production (b, d) by an Aquisali‑ of ­CuSO4 and 3 M of NaCl; a pH range of 4.0‒10.0, and a tempera-
bacillus elongatus strain as a function of pH and temperature after ture range of 25‒45 °C. Error bars represent the standard errors from
4 days of incubation in the basal broth medium containing 0.25 mM three experiments

production. Addition of ­CuSO4 at 0.01% gave a high lac-
case yield of 1.2 U m ­ L −1, which was threefold greater
than that achieved in the basal medium when analyzing
the activities obtained on day 2 of fermentation (Fig. 3b).
Different studies have shown that laccase production is
regulated by metal ions, such as C­ u2+, ­Mn2+, and F
­ e3+, by
gene expression induction, or through positive changes in
the activity and stability of the enzyme (Soden and Dob-
son 2001). ­CuSO4 and ­MnSO4 stimulate laccase produc-
tion in many different strains (Sivakumar 2010; Uthandi
et al. 2010; Fang et al. 2011; Lu et al. 2013). Among the
carbon sources tested in this study, glucose enhanced the
laccase production due to the increase in bacterial growth
(Fig. 3c) (Periasamy and Palvannan 2010; Poojary and
Mugeraya 2012). The results in Fig. 3d show that laccase
production by A. elongatus was not affected by different
concentrations of glycine or (­ NH 4) 2SO 4 after 2 days of
fermentation. The expression of the enzyme was induced
to the greatest extent by peptone, followed by urea, yeast
extract, skim milk, ­N H 4NO 3, and gelatin, in agreement
with previous results (Poojary and Mugeraya 2012; do
Valle et al. 2014). The higher cell growth in response to
organic and inorganic nitrogen had an apparent positive
effect on the enzyme production.
PBD
A total of 10 variables were analyzed for their effects on lac-
case production using a Plackett–Burman design. The design
matrix selected for the screening of significant variables for
laccase production and the corresponding responses are
shown in Table S2.

13
Extremophiles

(a) (b)
1.4 1.6
** Laccae production Laccase production
Cell growth
1.2
** Cell growth
1.4

1.5
1.5 1.2

Cell groowth (OD 600 nm)

Cell groowth (OD 600 nm)

Enzyme activity (U mL )

Enzyme activity (U mL )
1.0
-1

-1
1.0
0.8
1.0
1.0 0.8
0.6
0.6

0.5 0.4
0.5 0.4

0.2 0.2

0.0 0.0 0.0 0.0

Vanillin

Gallic acid
Caffein
2,6-DMP

Veratryl alcohol
PYrogallol
Guaiacol

Catechol
M9

2,5-Xylidine

o-Toluidine

L-Tyrosine

KCl
M9

CuSO4

ZnSO4

FeSO4

MnSO4

CaCO3

MgSO4

CaCl2

Na2 HPO4

KH2 PO4
Trace element
Inducer

(c) (d)
1.4 2.0

*
Laccase production Laccase production

1.2
*
Cell growth
1.0
* Cell growth

1.0
1.5 * 1.5
Cell growth (OD 600 nm)
Enzyme activity (U mL )

Cell growth (OD 600 nm)

Enzyme activity (U mL )
-1

-1

0.8

0.8
1.0
1.0 0.6
0.6

0.4
0.4
0.5 0.5

0.2
0.2

0.0 0.0 0.0 0.0

Starch

Gelatin
M9

Yeast extract
M9

Urea

Skim milk
Glucose

Fructose

Sucrose

Lactose

Galactose

Maltose

Peptone

Glycine
Carbon source Nitrogen source

Fig. 3  Growth behavior of Aquisalibacillus elongatus and its laccase nitrogen sources (1% w/v) after 2  days of incubation at 35  °C in a
production profile in the presence of different a inducers (1‒10 mM), basal medium containing 3  M NaCl; pH 8.0. Data are expressed as
b trace elements (0.01% w/v), c carbon sources (1% w/v), and d mean ± standard errors (n = 3)

Statistical analysis by an F test and a normal percent CCD

probability plot as a graphical tool were employed to reveal
significant independent variables (Fig S2; Table S5). As As shown in Table S3, the optimal levels of the selected
shown in Fig. S2, ­CaCl2 was the most significant factor, independent variables for laccase production were deter-
followed by glucose, ­CuSO4, and urea, as laccase produc- mined using central composite design and response surface
tion deviated from linearity indicating a significant effect. methodology. Table S4 shows the significant factors dis-
As shown in Fig S2, all the variables exerted positive played in coded form and laccase production as the response.
effects on enzyme production. Further statistical analysis The F value acquired from the analysis of variance was used
of data using ANOVA confirmed that these four factors had to select the best model for the data. We determined that a
p value less than 0.05, indicating statistical significance quadratic second-order polynomial equation was a proper
(Table S5). choice for the data (Table S6). As shown in Table S6, the

13
 Extremophiles
ANOVA results indicate that this model is appropriate and
significant (p < 0.05). The computed model F value of 40.84
implies the model is significant and that only a 0.01% chance
existed that this large value of “model F value” could occur
due to noise. The very small “model p value” < 0.0001 from
the analysis of ANOVA and the suitable coefficient of deter-
mination (R2 = 0.962) and adjusted coefficient of determi-
nation (R2 adjusted = 0.927) confirmed that the modified
polynomial model was highly significant and sufficient to
represent the actual relationship between the response and
the significant variables. The predicted R 2 of 0.834 also
agreed with the adjusted-R2, indicating the good predictive
ability of the selected model. The value of the coefficient
of variation (CV % = 5.44), an estimate of the standard
deviation around the mean associated with the experiment,
also supported the reliability of the model. No trends were
revealed by the plot of Studentized residual versus the value
predicted by the model. This shows the homogeneity of vari-
ance in the data and the absence of outliers in the experimen-
tal runs (data not shown).
Thus, modeling the level of laccase production could be
achieved using the quadratic second-order polynomial equa-
tion, which was determined as adequate over the range of
independent variables in this study. The coefficient of the
model was determined using multiple regression analysis,
and the equation fitted to the data was as follows:
Y = 7.99 + 0.018A − 0.15B + 0.13C − 0.16D + 0.23BC
− 0.14CD − 0.77A2 − 0.72B2 − 0.69C2 − 0.59D2 ,
where Y is the predicted laccase activity (U ­mL−1) and A, B,
C, and D are independent variables of glucose, C­ aCl2, urea,
and ­CuSO4, respectively.
The optimal level of each variable and the effect of their
interactions on laccase production as a function of two
variables were studied by plotting the 3-D response surface
curves (while keeping the other variables at their optimum
points) (Fig. 4). The final optimized culture medium for
laccase production by A. elongatus was 3 M NaCl, 0.17%
glucose, 0.13% ­CuSO4, 1.5% urea, 0.08% ­CaCl2, 0.05%
­KH2PO4, 0.125% pyrogallol, 0.05% ­MnSO4, 0.125% ­FeSO4,
0.05% ­Na2HPO4 (see Tables S3, S4, and S6). After optimiz-
ing the conditions, an approximately 20-fold enhancement
of laccase production was achieved.
A literature search indicated that this study is appar-
ently the first to report on the optimized production of lac-
case from a halophilic bacterium. However, optimization
of the medium for laccase production, with highly differ-
ent increasing folds, has been widely reported for laccase
production in, for example, Phellinus noxius (Poojary and
Mugeraya 2012), Pleurotus ostreatus (Elsayed et al. 2012),
Paraconiothyrium variabile (Aghaie-Khouzani et al. 2012),
and Bacillus tequilensis (Sondhi et al. 2015). In addition,
comparison with other studies on laccase production by
13
strains of Streptomyces psammoticus (Niladevi and Prema
2008), Bacillus tequilensis (Sondhi et al. 2015), and Halofe‑
rax volcanii (Uthandi et al., 2010), with production yields
of 5.9, 5.7, and 0.17 U m­ L−1, respectively, confirm A. elon‑
gatus as an efficient laccase producer.
Validation of the model
Validation of the model requires that the fitted polynomial
quadratic equation also be able to predict the laccase pro-
duction in practice. This ability was demonstrated under
the optimal conditions determined for laccase production
by measuring the amount of laccase produced in five experi-
ments. Using a statistically optimized medium, the maxi-
mum response for laccase production was 7.96 ± 0.11 U
­mL−1, implying a strong similarity between observed and
predicted values and thereby confirming the validity and
precision of the model.
Enzyme purification
A 75 kDa protein with laccase activity was purified from the
optimized culture medium. The homogeneity of the purified
laccase was checked by SDS-PAGE under reducing condi-
tions and by protein-staining analysis, and a unique protein
band was obtained for the purified enzyme. The purified
enzyme showed a specific activity of about 500 U m ­ g−1
when 2,6-DMP was used as the substrate.
Delignification and detoxification by laccase
A previous study (Rezaei et al. 2017) showed that the puri-
fied laccase was very stable across a broad range of pH and
temperature, which makes this enzyme a suitable candi-
date for applications requiring alkaline pH and high tem-
peratures. Numerous organic solvents with different log p
values, and 1-butyl-3-methylimidazolium hexafluorophos-
phate ­([BMIM][PF6]) as a hydrophobic ionic liquid, also
had positive effects on the laccase activity, indicating that
the enzyme was an organic solvent tolerant laccase. The
enzyme also showed activity towards a wide range of sub-
strates, including phenolic and non-phenolic substrates. The
enzyme was also stable in the presence of triton X-100 and
CTAB, and its activity was strengthened in the presence of
SDS and tween-80. These features of the purified enzyme
were used to design subsequent experiments to determine
the factors affecting delignification yield.
One of the most promising industrial applications of lac-
cases is in the delignification of lignocellulosic bio-wastes
and detoxification of the produced phenols, because the
phenolic compounds in biomass hydrolysates are known
to inhibit saccharifying enzymes, enzyme production,
and alcohol fermentation. Multiple studies have shown
Extremophiles

Fig. 4  Response surface graph for the laccase enzyme activity from Aquisalibacillus elongatus as a function of a ­CaCl2 and glucose, b urea and
­CaCl2, c ­CuSO4 and ­CaCl2, d ­CuSO4 and glucose, e ­CuSO4 and urea, and f urea and glucose concentrations

the ability of culture filtrates of fungi to delignify and to reduce the lignin content and to remove the resultant
detoxify wastes, although few studies have focused on this phenolic compounds of peanut shell, as a sample biomass.
ability with respect to bacterial laccases. Here, we demon- The sample had an initial brightness of 32.1%, a kappa
strated the ability of the halophilic laccase of A. elongatus number of 22.5, and a viscosity of 1023 mL g−1. After 48 h

13
 Extremophiles
of the delignification reaction, the bio-waste was deligni-
fied up to a maximum of 47%, although 45% reduction in
the lignin content of peanut shell was achieved after 24 h
(Fig. 5a). After 24 and 48 h of enzymatic treatment, bright-
ness increased to 58.5 and 62.1%, respectively (Fig. 5b).
In addition, as shown in Fig. 5b, the viscosity decreased to
37.7 and 41.2% after 24 and 48 h incubation, respectively.
The effects of pH, temperature, ionic and non-ionic sur-
factants, and organic solvents on the delignification effi-
ciency were also tested. As shown in Fig. 6a, a pH of 8.0
and a temperature of 35 °C were optimal for delignifica-
tion of the bio-waste. A more marked effect on the delig-
nification was observed when the reaction was performed
in the presence of [­ Bmim][PF6], 1,4-dioxane, acetone, or
HBT (Fig. 6b). The treatment with 50 U of enzyme in the
presence of 50% of ­[Bmim][PF6] showed a maximal 87%
lignin removal after a 24 h incubation. With the exception
of CTAB and triton-X100, other additives increased the effi-
ciency of the delignification reaction.
Detoxification studies also showed that laccase from A.
elongatus was able to detoxify the toxic phenols of peanut
shell (Table 1). With the exception of S. aureus, the MICs
for a lignin solution after enzymatic treatment were sharply
increased. The toxicity is generally accepted to decrease
after laccase treatment, and the results of this study confirm
this hypothesis (e.g., a higher concentration of lignin solu-
tion was needed to inhibit the strains).
Many studies have shown that laccases are able to delig-
nify (Knežević et al. 2013; Sondhi et al. 2015) and detoxify
(a)
50
* 60
40
D e lignification e fficie ncy (% )
30
20
10
0
0 1 2 6 12 24
Time (h)
Fig. 5  Laccase-assisted a delignification efficiency and b increase in
brightness and decrease in viscosity in a reaction mixture containing
potassium phosphate buffer (50  mM, pH 8.0), the purified Aquisali‑
bacillus elongatus laccase (~50 U), and a peanut shell substrate (final
13
(Chandel et al. 2007; Jurado et al. 2009; Moreno et al. 2012,
2013) lignocellulosic biomaterials. The results presented
here confirmed that the enzyme detoxified lignin, probably
due to the removal of phenolic compounds liberated from
the lignin. These results are consistent with a previous study
by Sondhi et al. (2015), who reported a 28% delignifica-
tion in the absence of a mediator and a 47% delignification
in the presence of 1-hydroxybenzotriazole (HOBT), for a
bacterial laccase from B. tequilensis. Other studies (Gutiér-
rez et al. 2012; Knežević et al. 2013; Ghorbani et al. 2015)
showed that fungal laccases gave 30–97% delignification
yields. Similar results for changes in viscosity (Camarero
et al. 2007) and brightness (Addleman and Archibald 1993)
were reported for fungal laccase-assisted treatment of pulps
from different sources.
Two possible routes are available for lignin degradation:
simultaneous degradation of all pulp components and a more
selective degradation of lignin. Of these two, the latter case
is more suitable for papermaking. The processing of ligno-
cellulosic biomass is essential for the production of cellu-
losic pulp as a raw material for paper manufacture, and the
application of enzymes such as laccase for this purpose is an
option for those industries seeking to reduce the use of chemi-
cal bleaching agents as a way to minimize the environmental
impact of their processes (Woolridge 2014). A recent study
on delignification of almond shell bio-waste by a halophilic
laccase from Chromohalobacter salexigens (Jafari et al. 2017)
showed negligible sugar release after enzymatic treatment of
lignocellulosic waste in the presence of ­[Bmim][PF6], organic
(b)
Brightness
Viscosity
*
50
*
50
Incre as e in brightne s s (% )
D e cre as e in vis cos ity (% )
40
40
30
30
20
20
10 10
0 0
36 48 0 1 2 6 12 24 36 48
Time (h)
concentration 5% w/v) after orbital shaking at 150  rpm for 48  h at
35  °C. Control samples without the enzyme were processed in par-
allel with the test samples. Data are expressed as mean  ±  standard
errors from three repetitions
Extremophiles

(a) solvents, and surfactants. Therefore, the use of a combination

50 of ionic liquid with a halophilic bacterial laccase can extract
* 35 °C
40 °C
intact cellulose fibers. The stable laccase studied here, which
45 °C effectively delignified peanut shell under special conditions,
40
could represent a promising enzyme for application in ligno-
Delignification efficiency (%)

cellulosic biomaterial-based industries.

30

20
Conclusion

This study examined the optimal production of a novel halo-

10 philic laccase from an isolate of A. elongatus, the ability
of this laccase to delignify peanut shell under harsh condi-
0 tions, and the properties of laccase-treated bio-waste, with
6 7 8 9 the overall aim of applying this laccase in industrial lig-
pH nocellulosic bio-waste delignification. The high expression
(b) levels of the laccase under optimized conditions indicate
100
bright prospects for large-scale production. Treatment of
bio-waste with this laccase decreased the kappa number and
* viscosity while increasing the brightness. The removal of
80
Delignification efficiency (%)

lignin by the enzyme was enhanced under extreme condi-

tions, including the presence of an ionic liquid, a mediator,
60
surfactants, organic solvents, and a wide range of pH and
temperature, under which the enzyme remained stable. This
40
demonstration of a high efficiency of delignification by a
stable halophilic laccase under industrial-like conditions
20
demonstrates the great potential of halophilic laccases as
industrial enzymes for environmentally friendly delignifica-
0
tion of natural wastes.
DMSO
HBT

SDS

Triton-X100

Tween-80
CTAB
Acetone

1,4-Dioxane
[Bmim][PF6]

Ethanol

Methanol
Control

Acknowledgements  Research reported in this publication was sup-

ported by Elite Researcher Grant Committee under Award Number
Additives
943687 from the National Institutes for Medical Research Development
(NIMAD), Tehran, Iran to M.A.F.
Fig. 6  Delignification efficiency of Aquisalibacillus elongatus lac-
case a at a range of pH and temperature (pH 6.0‒9.0 and 25‒45 °C)
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