microorganisms

Article
Optimization of the Decolorization of the Reactive Black 5 by a
Laccase-like Active Cell-Free Supernatant from
Coriolopsis gallica
Amal Ben Ayed 1,2, *, Bilel Hadrich 3 , Giuliano Sciara 2 , Anne Lomascolo 2 , Emmanuel Bertrand 2 ,
Craig B. Faulds 2 , Héla Zouari-Mechichi 1 , Eric Record 2 and Tahar Mechichi 1, *

1 Laboratory of Biochemistry and Enzymatic Engineering of Lipases, Ecole Nationale d’Ingénieurs de

Sfax (ENIS), University of Sfax, Sfax 3038, Tunisia; [Link]@[Link]
2 UMR1163, Biodiversité et Biotechnologie Fongiques, Aix-Marseille Université, INRAE, 13288 Marseille,
France; [Link]@[Link] (G.S.); [Link]@[Link] (A.L.);
[Link]@[Link] (E.B.); [Link]@[Link] (C.B.F.); [Link]@[Link] (E.R.)
3 Laboratory of Enzyme Engineering and Microbiology, Ecole Nationale d’Ingénieurs de Sfax (ENIS),
University of Sfax, Sfax 3038, Tunisia; [Link]@[Link]
* Correspondence: [Link]@[Link] (A.B.A.); [Link]@[Link] (T.M.)

Abstract: The textile industry generates huge volumes of colored wastewater that require multiple
treatments to remove persistent toxic and carcinogenic dyes. Here we studied the decolorization of a
recalcitrant azo dye, Reactive Black 5, using laccase-like active cell-free supernatant from Coriolopsis
gallica. Decolorization was optimized in a 1 mL reaction mixture using the response surface method-
ology (RSM) to test the influence of five variables, i.e., laccase-like activity, dye concentration, redox
mediator (HBT) concentration, pH, and temperature, on dye decolorization. Statistical tests were used
Citation: Ben Ayed, A.; Hadrich, B.; to determine regression coefficients and the quality of the models used, as well as significant factors
Sciara, G.; Lomascolo, A.; Bertrand, and/or factor interactions. Maximum decolorization was achieved at 120 min (82 ± 0.6%) with the
E.; Faulds, C.B.; Zouari-Mechichi, H.; optimized protocol, i.e., laccase-like activity at 0.5 U mL−1 , dye at 25 mg L−1 , HBT at 4.5 mM, pH
Record, E.; Mechichi, T. Optimization
at 4.2 and temperature at 55 ◦ C. The model proved significant (ANOVA test with p < 0.001): coefficient
of the Decolorization of the Reactive
of determination (R2 ) was 89.78%, adjusted coefficient of determination (R2 A ) was 87.85%, and root
Black 5 by a Laccase-like Active
mean square error (RMSE) was 10.48%. The reaction conditions yielding maximum decolorization
Cell-Free Supernatant from
were tested in a larger volume of 500 mL reaction mixture. Under these conditions, the decolorization
Coriolopsis gallica. Microorganisms
2022, 10, 1137. [Link]
rate reached 77.6 ± 0.4%, which was in good agreement with the value found on the 1 mL scale. RB5
10.3390/microorganisms10061137 decolorization was further evaluated using the UV-visible spectra of the treated and untreated dyes.

Academic Editor: Hongzhi Tang

Keywords: RB5 decolorization; Coriolopsis gallica; laccase-like activity; optimization; response sur-
Received: 19 April 2022 face methodology
Accepted: 30 May 2022
Published: 31 May 2022

Publisher’s Note: MDPI stays neutral

1. Introduction
with regard to jurisdictional claims in
published maps and institutional affil- Water is essential for all forms of life, making it the most important resource on Earth.
iations. However, water decline and depletion is an escalating problem driven by the increasing
global population [1,2]. This increase in population also increases pollution, regardless
of whether wastewater streams come from domestic, industrial, agricultural, or other
origins [3]. A telling example is the use of pesticide-contaminated water for plant irrigation,
Copyright: © 2022 by the authors. which leads to the spread of carcinogenic chemicals in soil and, successively, all along the
Licensee MDPI, Basel, Switzerland. food chain [4].
This article is an open access article The textile industry is the oldest sector in the world’s economy [5] and is still the
distributed under the terms and
world’s largest industry, employing huge numbers of workers worldwide (1.5 million in
conditions of the Creative Commons
Brazil and 8 million in China) and producing huge amounts of textiles (1.3 million tons in
Attribution (CC BY) license (https://
Brazil, and 79.29 million tons in China) [6–8]. The sector continues to grow with the growing
[Link]/licenses/by/
global population, increasingly productive machinery, and a wider variety of colors [9]. The
4.0/).

Microorganisms 2022, 10, 1137. [Link] [Link]

Microorganisms 2022, 10, 1137 2 of 19

textile industry uses a large number of dyes that are classified according to their chemical
structure (chemical classification) or application method (tinctorial classification) [10,11].
Most of the dyes used today are synthetic unsaturated organic or aromatic compounds,
which have a complex structure and are applied in a range of domains from cosmetics
and pharmaceuticals to paper and food [12,13]. The best-known class of synthetic dyes
are azo dyes, which possess an azo linkage (-N=N-) close to the aromatic rings that confer
their high resistance to oxidizing agents and photocatalytic stability [14,15]. Reactive
Black 5 (RB5) is a non-biodegradable azo dye known to be highly resistant to chemicals and
light [13,16], and it is one of the more common reactive dyes used in the textile industry [17].
Textile industries employ three major processes, i.e., washing, dyeing, and finishing, that
together use 50 to 100 L of water per kg of wet fiber produced [18,19]. These textile-industry
processes also produce effluents that are loaded with toxic and carcinogenic chemicals and
dyes (0.01 to 7 g L−1 of dyes) [20,21]. These effluents cause extensive water pollution and
thus severe problems for fauna and flora as well as toxicity for humans [22–24]. Therefore,
this alarming situation necessitates efforts to treat these effluents before they are released
back into the environment. The textile industry faces a major challenge to address this
damage and the allied water pollution crisis [19].
Wastewater treatment spans a series of processes designed to remove organic and
inorganic pollutants from water before it is reused or returned to the natural environment.
These wastewater treatments can employ chemical, physical and/or biological treatment
processes in various combinations and configurations [24,25]. Physical unit processes in-
clude filtration, coagulation/flocculation, and ion exchange. Chemical treatment processes
revolve around chemical reduction, advanced chemical oxidation, and electrolysis. Bio-
logical unit processes typically include aerobic, anaerobic or combined processes [26–28].
Physicochemical treatments have proven expensive and relatively inefficient as they fail to
curb the spread of dangerous chemicals and lead to potential ecological risks [28]. Conse-
quently, biological approaches emerge as the best candidate solutions to remove dyes and
preserve clean water resources [27,29]. Biological processes use the enzymatic machinery
of bacteria or fungi as their main tools, which make them non-toxic, eco-friendly, and
low-energy solutions [30].
White-rot fungi are the most intensively-studied heterogeneous organisms in the
literature for their ability to degrade lignin and other aromatic components, in addition to
their potential application in the textile industry to remove dyes from wastewater. Research
has pursued white-rot fungi mainly because they mobilize an extracellular enzyme system
composed of peroxidases and laccases [31,32]. For instance, Pycnoporus sanguineus, P.
coccineus, Trametes versicolor, Trametes hirsuta, and Phanerochaete chrysosporium, are fungal
strains that widely show efficiency for decolorizing textile dyes [31,33–35].
Coriolopsis gallica is an example of a white-rot basidiomycete that produces high levels
of extracellular enzymes such as laccase [36]. Studies have reported the ability of Cori-
olopsis gallica to degrade dyes such as Remazol Brilliant Blue R (RBBR), an anthraquinone
dye [37], Reactive Blue 198 [38], an azo dye, and Lanaset Grey G, a metal textile dye [39], as
well as phenolic compounds [40] and polycyclic aromatic compounds such as carbazole,
dibenzothiophene, and dibenzofuran [41].
Laccases (benzenediol, oxidoreductase, EC [Link]) are multi-copper enzymes that
catalyze the oxidation of various substrates, such as phenols, aromatic thiols, aniline, and
some inorganic ions via the reduction of O2 to H2 O [42,43]. These enzymes are found
in plants [44], bacteria [45], insects [46], and fungi [43,47]. Fungal laccases, especially
those from white-rot fungi, are among the best-described and widely-used enzymes in
various biotechnological applications from pharmaceuticals [48] to cosmetics [49], pulp
bleaches [50], food [51], bio-detergents [52,53] and textile dye decolorization [54–56]. How-
ever, laccases are unable to directly oxidize complex compounds that have a higher redox
potential than laccases or steric hindrance preventing access to the catalytic site of the
enzyme [57,58]. These cases demand the use of low-molecular-weight redox mediators
that act as electron shuttles to help oxidize non-phenolic compounds [59,60]. There are
Microorganisms 2022, 10, 1137 3 of 19

two broad classes of redox mediators: synthetic and natural [61]. Among the synthetic
redox mediators, 1-hydroxybenzotriazole (HBT) is a well-known N-OH-type mediator [59].
Other examples of N-OH-type mediators include N-hydroxyphthalimide (HPI), violuric
acid (VIO), N-hydroxyacetanilide (NHA), 2,2,6,6-tetramethylpiperidin-1-yloxyl (TEMPO),
and 10-(3-dimethylaminopropyl) phenothiazine (Promazine) [59,62]. Natural mediators
include syringaldehyde, vanillin, acetosyringone, acetovanillone, and p-coumaric acid [62].
This study aims to evaluate the potential of a crude Coriolopsis gallica laccase for the
decolorization of the recalcitrant azo dye RB5. The fungal strain used in this study had
been isolated from decayed acacia wood in northwest Tunisia [37]. Statistical optimization
was performed using Response Surface Methodology (RSM) (coupled screening and Box–
Behnken designs) to determine optimized conditions for multifactorial experimentation
and the interactions between variables. The optimized conditions for decolorization were
first verified at a small scale (1 mL) and then tested in a larger volume of 500-mL. In
addition, the UV-visible spectra of treated and untreated dye were analyzed.

2. Materials and Methods

2.1. Fungal Strain and Culture Conditions
This study used Coriolopsis gallica strain CLBE55 [ON340792] for laccase production.
Coriolopsis gallica strain CLBE55 was deposited at the culture collection “Centre International
de Ressources Microbiennes” (CIRM) under accession number BRFM 3473.
Solid cultures of C. gallica were performed on PDA media that contained 39 g of
dehydrated media (Accumix) suspended in 1000 mL of distilled water sterilized by auto-
claving at 120 ◦ C for 30 min. Liquid pre-cultures were performed in 25 mL of Malt Extract
medium (Sigma–Aldrich, St. Louis, MI, USA) containing 30 g Malt Extract per L at pH 5.5
sterilized by autoclaving at 120 ◦ C for 30 min. The pre-cultures were inoculated with 3 agar
plugs (6 mm in diameter) cut from the growing edge of a plate stock culture and incubated
at 30 ◦ C for 3 days at 160 rpm. Mycelia from these three-day precultures were then partially
ground down using glass beads (0.6 mm). The mycelial mixture obtained was used to
inoculate 500-mL Erlenmeyer flasks containing 100 mL of M7 medium [27,63]. The basal
medium contained (in g L−1 ) glucose 10, peptone 5, yeast extract 1, ammonium tartrate 2,
KH2 PO4 1, MgSO4 , 7H2 O 0.5, KCl 0.5, and 1 mL of trace element solution. Composition
of the trace-element solution was (in g L−1 ): B4 O7 Na2 , 10H2 O 0.1, CuSO4 , 5H2 O 0.01,
FeSO4 , 7H2 O 0.05, MnSO4 , 7H2 O 0.01, ZnSO4 , 7H2 O 0.07, (NH4 )6 Mo7 O24 , 4H2 O 0.01. pH
was adjusted to 5.5, and cultures were incubated in a rotary shaker for 7 days at 30 ◦ C
and 160 rpm. On day 3 of incubation, 300 µM CuSO4 was added as a laccase inducer.

2.2. Laccase-like Activity Assays of the Cell-Free Supernatant from Coriolopsis gallica
The supernatant of a 7-day culture of C. gallica was filtered on a Miracloth membrane
(Merck, Fontenay-sous-Bois, France) and centrifuged at 30 ◦ C for 5 min at 10,000× g prior
to use. The laccase-like activity of the cell-free supernatant was assayed by monitoring
the oxidation of 5 mM 2,6-dimethoxyphenol (DMP) in 50 mM citrate buffer, pH 5 (469 nm,
ε469 = 27,500 M−1 cm−1 ) in the presence of 50 µL of supernatant. The assay was carried
out at 30 ◦ C for 1 min. One unit of DMP-oxidizing activity was defined as the amount of
enzyme oxidizing 1 µmol of substrate per minute.

2.3. Experimental Design and Data Analysis

An experimental design was used to optimize the enzymatic decolorization of the
recalcitrant azo dye Reactive Black 5 (RB5) (Aldrich Chemical Co., St. Louis, MO, USA)
by using the culture supernatant of C. gallica. The chemical properties of the RB5 dye are
reported in Table 1.
 Microorganisms
Microorganisms 2022,
2022, 10, 10, 1137
1137 4 of 194 of 19

TableTable 1. Properties
1. Properties of Reactive
of Reactive Black Black
5 dye.5 dye.
Properties
Properties Reactive Black
Reactive Black 55
CAS number 17095-24-8
CAS number 17095-24-8
Molecular weight (gmol−1) 991.82
Molecular weight (gmol−1 ) 991.82
EC Number 241-164-5
EC Number 241-164-5
CI
CI 20505
20505
Dye content
Dye content ≥≥50%
50%
Chemical formula 26 21 N55Na44O
CC H
26 H 21N O1919SS6 6
max (nm)
λλmax (nm) 598

Molecular structure

2.4. Plackett–Burman
2.4. Plackett–Burman Design
Design
Plackett–Burman
Plackett–Burman experimental
experimental designs designs are useful
are useful first-step
first-step screening
screening designsdesigns
for for
identifying
identifying the most
the most significant
significant factors
factors and weeding
and weeding out uninfluential
out uninfluential factors
factors for further
for further
experimentation [64]. Here we applied a 15-run Plackett–Burman
experimentation [64]. Here we applied a 15-run Plackett–Burman design, with 3 replicates design, with 3 replicates
including the center points, on five independent factors to determine their influence on on
including the center points, on five independent factors to determine their influence
RB5 decolorization.
RB5 decolorization. The The
fivefive factors
factors were were laccase-like
laccase-like activity
activity (x1(x ), initial
),1initial dyeconcentra-
dye concentration
(x2 ), HBT concentration (x3), pH (x 4 ), and temperature (x 5 ).
tion (x2 ), HBT concentration (x3 ), pH (x4 ), and temperature (x5 ). The center point The center point tested the lin-
tested
earity ofofthe
the linearity theexperimental
experimental points
points(external
(external versus
versus center
center point.
[Link] Theexperimental
experimental design
designadopted
adopted required
requiredthree levels,
three i.e., i.e.,
levels, lowlow (coded −1), medium
(coded −1), medium (coded 0), and
(coded 0),high
and(coded
high +1)
(coded(Table 2). Table
+1) (Table 3 reports
2). Table the Plackett–Burman
3 reports the Plackett–Burman design used used
design and the andpercentage
the percentagedecolori-
zations achieved at 120 min. The reaction took a total volume
decolorizations achieved at 120 min. The reaction took a total volume of 1 mL using 50 mM of 1 mL using 50 mM citrate
buffer. All experiments
citrate buffer. All experiments were were performed in triplicate. Results are presented
in triplicate. Results are presented as means as means ±
standard
± standard deviation
deviation (Table
(Table 3). The
3). The first-order
first-order form form of equation
of the the equation adopted
adopted in part
in this this part
of of
the study
the study is (Equation
is [65] [65] (Equation
(1)): (1)):

ŷ = ŷ=β
β0 +0 ∑  i ( i=1
+ βiβ·xi i⋅(xi = . k)k )
1 . . (1) (1)
i i

where
where ŷ is ŷthe
is the fitted
fitted response
response (% (% decolorization
decolorization at 120at 120 β0𝛽and
min),
min), β𝛽
0 and 𝑖 are the intercept and
i are the intercept
and linear
linear coefficient
coefficient of ofthe
themodel,
model,respectively,
respectively, xxi iisislevel-coded
level-codedfactor
factorvariable,
variable,andandkkisisnum-
ber of factors.
number of factors.

TableTable 2. Levels
2. Levels of factors
of factors testedtested
in thein the screening
screening design.
design.

Symbol
Symbol Coded
Coded and and Uncoded
Uncoded Levels Levels
FactorFactor Unit Unit
Code
Code −1 0
−1 0+1 +1
x1 x1 Laccase-like activityactivityU mL−1
Laccase-like U mL −1
0.02 0.02
0.26 0.26
0.5 0.5
x2 x2 InitialInitial
dye concentration
dye concentration mg L−1 mg L25−1 75
25 125
75 125
x3 x3 HBT concentration
HBT concentration mM mM 0.5 2.5
0.5 4.5
2.5 4.5
x4 pH - 3.0 4.5 6.0
x5
x4 Temperature
pH ◦C - 25 3.0
40
4.555
6.0
x5 Temperature °C 25 40 55
Microorganisms 2022, 10, 1137 5 of 19

Table 3. Plackett–Burman screening-plan runs and response as percent decolorization at 120 min.

Symbol Code of Factors

Run Decolorization (%)
x1 x2 x3 x4 x5
1 +1 −1 +1 −1 −1 74.8 ± 0.4
2 +1 +1 −1 +1 −1 1.0 ± 0.2
3 −1 +1 +1 −1 +1 1.3 ± 0.6
4 +1 −1 +1 +1 −1 5.2 ± 0.3
5 +1 +1 −1 +1 +1 1.3 ± 0.6
6 +1 +1 +1 −1 +1 55.8 ± 1.2
7 −1 +1 +1 +1 −1 1.3 ± 0.4
8 −1 −1 +1 +1 +1 6.0 ± 0.7
9 −1 −1 −1 +1 +1 3.1 ± 0.1
10 +1 −1 −1 −1 +1 65.7 ± 3.2
11 −1 +1 −1 −1 −1 1.2 ± 0.4
12 −1 −1 −1 −1 −1 3.7 ± 1.1
13 0 0 0 0 0 62.0 ± 0.2
14 0 0 0 0 0 62.3 ± 0.3
15 0 0 0 0 0 63.4 ± 0.2
x1 , x2, x3, x4, x5: coded variables for laccase-like activity, initial dye concentration, HBT concentration, pH, and
temperature, respectively.

Decolorization was measured every 30 min over a 2-h incubation period by tracking the
decrease in absorbance of the RB5 dye (598 nm). Percentage decolorization was calculated
using the following formula (Equation (2)):

Ai − At
Decolorization(%) = × 100 (2)
Ai

where Ai is initial absorbance of the dye at the maximum wavelength before incubation
with the enzyme, and At is dye absorbance after 2 h of incubation. All experiments were
done in a total volume of 1 mL reaction mixture in 50 mM citrate buffer.

2.5. Response Surface Methodology Using a Box–Behnken Design

The best condition for RB5 decolorization was determined using RSM based on a Box–
Behnken design with influential variables obtained previously via the Plackett–Burman
design. A total of 46 runs (Table 4) were performed in triplicate with the same 5 variables
and factor levels as described above. Decolorization was adjusted using a second-order
polynomial equation, and multiple regression was performed on the data to obtain an
empirical template associated with the factors. The general form of the second-order
polynomial equation (Equation (3)) is:

ŷ = β0 + ∑ βi ·xi + ∑ ∑ βij ·xi ·xj + ∑ βii ·x2i (i, j = 1, . . . , k) (3)

i i i6 =j i

where ŷ is the response (percent decolorization at 120 min), β0 , βi , βij and βii are the
model’s intercepts, linear, interactions and quadratic coefficients, respectively, xi is level-
coded factor variable, and k is 5 in our experimental setup.
Microorganisms 2022, 10, 1137 6 of 19

Table 4. The Box–Behnken design and data on percent decolorization at 120 min.

Symbol Code of Factors

Decolorization (%)
Run x1 x2 x3 x4 x5
1 −1 −1 0 0 0 77.6 ± 1.3
2 +1 −1 0 0 0 80.9 ± 0.7
3 −1 +1 0 0 0 1.3 ± 0.3
4 +1 +1 0 0 0 43.7 ± 1.8
5 0 0 −1 −1 0 0.8 ± 0.3
6 0 0 +1 −1 0 60.9 ± 0.3
7 0 0 −1 +1 0 1.7 ± 0.2
8 0 0 +1 +1 0 2.0 ± 1.0
9 0 −1 0 0 −1 75.6 ± 0.1
10 0 +1 0 0 −1 29.0 ± 1.9
11 0 −1 0 0 +1 85.4 ± 2.1
12 0 +1 0 0 +1 56.2 ± 4.1
13 −1 0 −1 0 0 0.8 ± 0.3
14 +1 0 −1 0 0 53.5 ± 1.0
15 −1 0 +1 0 0 0.6 ± 0.1
16 +1 0 +1 0 0 69.9 ± 1.8
17 0 0 0 −1 −1 52.6 ± 1.3
18 0 0 0 +1 −1 1.6 ± 0.3
19 0 0 0 −1 +1 63.7 ± 3.7
20 0 0 0 +1 +1 1.5 ± 0.2
21 0 −1 −1 0 0 74.7 ± 0.4
22 0 +1 −1 0 0 23.9 ± 2.8
23 0 −1 +1 0 0 82.8±0.8
24 0 +1 +1 0 0 37.0 ± 7.7
25 −1 0 0 −1 0 0.6 ± 0.0
26 +1 0 0 −1 0 56.6 ± 0.3
27 −1 0 0 +1 0 0.9 ± 0.2
28 +1 0 0 +1 0 0.3 ± 0.1
29 0 0 −1 0 −1 43.8 ± 1.5
30 0 0 +1 0 −1 15.0 ± 1.8
31 0 0 −1 0 +1 62.9 ± 5.1
32 0 0 +1 0 +1 76.4 ± 4.3
33 −1 0 0 0 −1 1.4 ± 0.2
34 +1 0 0 0 −1 55.1 ± 2.9
35 −1 0 0 0 +1 8.6 ± 5.0
36 +1 0 0 0 +1 74.9 ± 4.4
37 0 −1 0 −1 0 76.2 ± 0.2
38 0 +1 0 −1 0 31.5 ± 1.9
39 0 −1 0 +1 0 4.3 ± 0.5
40 0 +1 0 +1 0 0.8 ± 0.3
41 0 0 0 0 0 62.3 ± 0.7
42 0 0 0 0 0 64.0 ± 1.7
43 0 0 0 0 0 65.2 ± 0.4
44 0 0 0 0 0 64.2 ± 1.0
45 0 0 0 0 0 64.5 ± 1.0
46 0 0 0 0 0 64.2 ± 1.4
x1 , x2, x3, x4, x5: coded variables for laccase-like activity, initial dye concentration, HBT concentration, pH, and
temperature, respectively.

2.6. Design and Statistical Analysis

Minitab 16 Statistical Software (Minitab Inc., State College, PA, USA) was used for
the experimental designs and statistical analysis. Factor coefficients were determined
using the least-squares method. Analysis of variance (ANOVA) and a Student’s t-test were
used to identify the level of significance of both the fitted model and the factors and their
interactions. The probability (p < 0.05) and high F-values (Fisher’s test) demonstrated that
the model and factors were significant. The quality of the model was analyzed using a
Microorganisms 2022, 10, 1137 7 of 19

coefficient of determination (R2 ), adjusted coefficient of determination (R2 adj), and root
mean square error (RMSE).

2.7. Decolorization Assay in 500 mL Volume

The optimal conditions determined for RB5 decolorization in a reaction volume
of 1 mL were then tested on a larger scale (500 mL). Experiments were performed in dupli-
cate in 1 L Erlenmeyer flasks containing 500 mL of the following reaction mixture: 50 mM
citrate buffer pH 4.2, 25 mg L−1 RB5 dye, 4.5 mM HBT, and 0.5 U mL−1 crude C. gallica
laccase. The reaction mixture was incubated at 55 ◦ C for two hours, and 1-mL samples
were taken every 30 min over the 2-h incubation period. Percentage rates of decolorization
were calculated following Equation (2).

2.8. RB5 Spectrum Analysis

The UV-visible spectrum of RB5 was analyzed to visualize its color and absorbance
peak shift. Decolorization was carried out for 24 h in reference conditions described in
Table S1. After 24 h, UV-visible spectra were measured on a Genesys 50 UV-VIS spec-
trophotometer (Thermo Fisher Scientific, Waltham, MA, USA) running in the 200–800 nm
wavelength range at 2-nm increment steps.

3. Results
3.1. Screening Design
Preliminary experiments were performed to fix the levels of factors. With a view
to potential scale-up for use in textile industry applications, reaction time was set at 2 h.
Note that we ran tests at several reaction times (>2 h) and found no change in the final
decolorization of the dye. In addition, a previous study had demonstrated that the main
laccase of C. gallica was active and stable at acidic pH (4–7) for 24 h of incubation retaining
from 40 to 80% of its initial activity and the same high temperature (up to 50–55 ◦ C) as
tested here [37] and that HBT was a powerful mediator for dye removal at up to 5 mM [37].
Based on these results, we thus performed the experimental design in these conditions.
Table 3 shows the experimental percentages of RB5 decolorization for all 15 runs tested
(Plackett–Burman design). All response values (percentage decolorization) presented
very low standard deviations (from 0.1 to 3.2% units of decolorization) (Table 3), thus
demonstrating good repeatability of all the experiments done in the different conditions.
The Pareto chart of the standardized effects (Figure S1) shows that 4 of the 5 variables
had significant effect on RB5 decolorization (tfactor > tstudent (p = 0.05) = 2.024; p < 0.05)
and that enzyme concentration was the most effective variable (p < 0.001) compared to
pH (p < 0.001), initial dye concentration (p < 0.001) and HBT concentration which was the
least effective variable (p < 0.01). Temperature was found to be a non-significant variable
but was kept for the second step of the RSM-based optimization protocol for two reasons:
(1) its p-value (0.073) was almost ruled in as significant (i.e., very close to the 0.05 limit of
significance); (2) temperature was found to be insignificant as the responses at extreme
points (−1; 1) were very close to each other, but the center point was very significantly
different to these values (Figure 1; p < 0.001).
Statistical results obtained from Minitab 16 gave the model coefficient, and the fitted
form of the equation was then represented as follows (Equation (4)):

ŷ = 18.36 + 15.61·x1 − 8.03·x2 + 5.71·x3 − 15.39·x4 + 3.84·x5 (4)

A positive-signed main effect of a factor indicates that using higher levels gave the
most effective response whereas a negative-signed main effect indicates that using lower
levels was more effective. Thus, according to Equation (4), laccase-like activity (x1 ), HBT
(x3 ) concentrations, and temperature (x5 ) were positive-signed whereas dye concentration
(x2 ) and pH (x4 ) were negative-signed. Moreover, the coefficient of determination (R2 ), the
adjusted coefficient of determination (R2 (adj)), and the root mean square error (RMSE) were
 Microorganisms 2022, 10, 1137 8 of 19

, 10, 1137 8 of 19
85.62%, 83.35%, and 12.5% (units of RB5 decolorization), respectively, thus demonstrating
the goodness-of-fit of the adopted model (Equation (4)).

Laccase-like activity Dye concentration HBT concentration

60

45

30

15
Mean

0
-1 0 1 -1 0 1 -1 0 1
pH Temperature
60

45
Point Type
Corner
30 Center
15

0
-1 0 1 -1 0 1
1. Main
Figure plot
Figure 1. Main effects effects
for RB5plotdecolorization
for RB5 decolorization
afterafter
120120min
min of
ofreaction as function
reaction of coded value
as function of coded
of factors. Red squares are center points and black diamonds are corner points.
value of factors. Red squares are center points and black diamonds are corner points.
Figure 1 illustrates the main effects plot of the five tested variables, which shows
Statistical results obtained
that no linear fromcould
correlation Minitab 16between
be found gave the model
center pointscoefficient, and the
and extreme points. Thisfitted
nonlinearity, which was very significant (p < 0.001), indicated a second-order model was
form of the equation was then represented as follows (Equation (4)):
able to fit decolorization as a function of all factors. We consequently applied a second
response-surface experimental design for the same variables in our protocol optimization
ŷ = 18.36 + 15.61·x1 − 8.03·x2 + 5.71·x3 − 15.39·x4 + 3.84·x5
step.
(4)
A positive-signed main Design
3.2. Box–Behnken effect of a factor indicates that using higher levels gave the
most effective response whereas
Response surfaceamethodology
negative-signedbased onmain effect indicates
a Box–Behnken design was that using to
performed lower
levels was moreoptimize (via Thus,
effective. a second-order
accordingpolynomial model) the(4),
to Equation conditions of RB5 decolorization
laccase-like activity (x1), byHBT
crude laccase obtained from C. gallica. The Box–Behnken design also statistically predicted
(x3) concentrations, and temperature (x5) were positive-signed whereas dye concentration
the same 5 variables as previously tested by the Plackett–Burman design. Table 4 reports the
(x2) and pH (x4) were negative-signed.
percentage decolorization with Moreover, the coefficient
standard deviations ofconditions.
for all tested determination (R²), the
The standard
adjusted coefficient of determination (R²(adj)), and the root mean square error (RMSE)
deviations were relatively low (from 0.1 to 7.7% in percentage units of decolorization). R2
2
was 89.78%, R (adj) was 87.85%, and RMSE was 10.48% (in percentage units of decoloriza-
were 85.62%, 83.35%, and 12.5% (units of RB5 decolorization), respectively, thus demon-
tion), thus indicating good agreement between experimental and predicted values using
strating the goodness-of-fit of the adopted model (Equation (4)).
the proposed model.
Figure 1 illustrates the main effects
The second-order polynomial plot of theindicating
equation, five tested variables,
the main which shows
effects, interactions among that
variables, and the quadratic effect with coefficients obtained from statistical analysis are
no linear correlation could be found between center points and extreme points. This nonlin-
represented as follows (Equation (5)):
earity, which was very significant (p < 0.001), indicated a second-order model was able to fit
ŷ = 64.07 + 20.61·xas
decolorization 1 −a20.69 ·x2 + 4.69
function of·xall
3 −factors.
21.10·x4 +We 10.16 ·x5 − 18.57·x1 2 +applied
consequently 1.54·x2 2 −a11.72 ·x3 2 −response-sur-
second
2 2
33.40·x4 − 3.60 x5 + 9.78·x1 ·x2 + 4.04·x1 ·x3 − 12.88·x1 ·x4 + 2.45·x1 ·x5 + 1.94·x2 ·x3 + 10.41·x2 ·x4 + (5)
face experimental design 4.33·x2for
·x5 −the same
13.17 ·x3 ·x4variables
+ 10.57·x3 ·xin
5 −our
1.71 protocol
· x ·
4 5 x optimization step.

3.2. Box–Behnken Design

Response surface methodology based on a Box–Behnken design was performed to
optimize (via a second-order polynomial model) the conditions of RB5 decolorization by
Microorganisms 2022, 10, 1137 9 of 19

According to Equation (5), laccase-like activity (x1 ), initial dye concentration (x2 ), and
pH (x4 ) were the most effective variables with the highest coefficients in a linear regression
with positive (x1 ) and negative (x2 , x4 ) effects. Moreover, as illustrated in Table 5, ANOVA
on RB5 decolorization confirmed that p-values were less than 0.001 and 0.01 for linear
regression (factors without their interactions), thus making all factors highly significant. Re-
garding the quadratic effect of factors, some of them were statistically non-significant with
p-values over 0.05, such as initial dye concentration (p = 0.465) and temperature (p = 0.089),
whereas the rest of the quadratic-term factors (laccase-like activity, HBT concentration,
and pH) had a significant influence (p < 0.001). Regarding the interactions between the
five variables, 5 among the 10 were significant. The highest p-values found were for pH ×
temperature (p = 0.592) and dye × HBT concentration (p = 0.561). pH × laccase-like activity
and pH × HBT concentration were the most significant and valuable interactions (with
the highest negative coefficients; Equation (5)) and had a significant influence on RB5
decolorization (p < 0.001). Moreover, the initial dye concentration × pH interaction was
found to be significant with a positive-signed effect and a p-value < 0.01.

Table 5. Analysis of variance for RB5 decolorization.

Source Degree of Freedom Sum of Squares Mean Square F-Value p-Value Probability > F
Regression 20 102,350 5117.5 46.6 <0.001
x1 1 17,064 17,063.8 155.2 <0.001
x2 1 19,526 19,525.6 177.7 <0.001
x3 1 931 931.0 8.5 0.004
x4 1 19,003 19,003.2 172.9 <0.001
x5 1 4585 4584.6 41.7 <0.001
x1 2 1 8115 8115.4 73.8 <0.001
x2 2 1 59 59.1 0.5 0.465
x3 2 1 3340 3339.9 30.4 <0.001
x4 2 1 27,057 27,056.6 246.2 <0.001
x5 2 1 323 323.3 2.9 0.089
x1 × x2 1 1148 1148.1 10.5 0.002
x1 × x3 1 162 161.6 1.5 0.228
x1 × x4 1 1477 1477.5 13.4 <0.001
x1 × x5 1 59 59.4 0.5 0.464
x2 × x3 1 37 37.4 0.3 0.561
x2 × x4 1 1300 1300.1 11.8 0.001
x2 × x5 1 225 225.4 2.1 0.155
x3 × x4 1 1891 1890.7 17.2 <0.001
x3 × x5 1 1341 1340.9 12.2 0.001
x4 × x5 1 32 31.8 0.3 0.592
Residual Error 106 11,649 109.9
Total 126 114,000

Residual plots (Figure 2) produced using the Box–Behnken design showed that all
points were aligned on the normal line, confirming that the model used (Equation (5)) fits
the experimental values (Figure 2A). This is also confirmed for the residual vs. fit and
residual vs. order plots, which demonstrated that all experimental points were randomly
distributed within the whole domain (Figure 2B,C). Figure 2D presenting the frequency of
residuals clearly shows that the highest frequency (40%) was for the zero residuals. The
second-highest frequency (at about 25%) was for the residual equal to 5 (in percentage
units of decolorization), and all the remaining values of residuals had a frequency of less
than 10%. Based on these findings, the adopted model showed a good fit.
Microorganisms 2022, 10, 1137

Microorganisms 2022, 10, 1137

units of decolorization), and all the remaining values of residuals had 10
a of
frequency
19
than 10%. Based on these findings, the adopted model showed a good fit.

2. Residual
Figure 2.
Figure Residualplots for RB5
plots for decolorization using theusing
RB5 decolorization Box–Behnken design. (A) Normal
the Box–Behnken probabil-
design. (A) Normal
ity plot. (B) Versus fits. (C) Histogram. (D) Versus order.
bility plot. (B) Versus fits. (C) Histogram. (D) Versus order.
Iso-responses plots (contour curves) were analyzed to visualize the interaction between
Iso-responses
factors. Figure 3A showingplotsRB5(contour curves)
decolorization were as
computed analyzed
a functiontoof visualize the interacti
dye concentration
and HBT concentration (all other factors were held at center level) indicates
tween factors. Figure 3A showing RB5 decolorization computed as a function of dy that the increase
in dye concentration
centration and HBT from 25 mg L−1 (level
concentration (all−other
1) to 125 mg L−were
factors
1 (level +1) decreased RB5
held at center level) ind
decolorization from 80% to 30%. Increasing HBT concentration to 2.5 mM (center of the
that the increase in dye concentration from 25 mg L−1 (level −1) to 125 mg L−1 (le
experimental domain—level 0) improved RB5 decolorization compared to the condition
decreased
with 0.5 mM RB5 decolorization
of HBT. Contour plotsfromfor the80% to 30%.activity
laccase-like Increasing HBT concentration
× temperature interaction to 2
(center
(Figure of3B)the experimental
showed domain—level
that increasing 0) improved
both factors improved RB5 decolorization
decolorization percentages compa
the condition with 0.5 mM of HBT. Contour plots for the laccase-likeinteraction
from 20% to more than 70%. Contour plots for the laccase-like activity × pH activity× tempe
(Figure 3C) showed that maximum percentage decolorization, at more than 75%, happened
interaction (Figure 3B) showed that increasing both factors improved decolorizatio
at levels +0.75 and ~−0.5, respectively. Moreover, percentage decolorization as a function
centages from 20% (Figure
of pH × temperature to more 3D)than
and pH70%. Contour
× HBT plots for
concentration the laccase-like
(Figure activity ×
3E) reached more
teraction
than 68% (Figure 3C) showed
in both cases. However, that
the maximum
lowest levelspercentage
of factors fordecolorization,
both the pH andat more tha
dye
concentration
happened at variables together
levels +0.75 andare likelyrespectively.
~−0.5, to exceed 85% RB5 decolorization
Moreover, (Figure 3F).
percentage decoloriza
a3.3.
function ofOptimization
Conditions pH × temperature (Figure 3D) and pH × HBT concentration (Figu
reached more
Minitab 16 than 68%
statistics in both
software wascases. However,
used to identify thethe lowestconditions
optimized levels offorfactors
RB5 for bo
pH and dye concentration
decolorization. variables
The special condition together
combining are likely
all 5 variables to exceed
included 85%activity
laccase-like RB5 decolor
−1 —high level (+1)), dye concentration (25 mg L−1 —low level (−1)), HBT con-
(Figure 3F).
(0.5 U mL
centration (4.5 mM—high level (+1)), pH (4.2—close to level 0 (−0.203)), and temperature
(55 ◦ C—high level (+1)). The best values for each variable to obtain maximum decol-
orization as predicted by the Box–Behnken design are highlighted in red (Figure 4). The
optimized conditions obtained here were adopted and repeated four times experimentally,
reaching 82 ± 0.6% decolorization.
Microorganisms 2022, 10, 1137 11 of 19
Microorganisms 2022, 10, 1137 11 of 19

Figure 3. Iso-responses
Figure 3. Iso-responses plots
plotsofofRB5
RB5decolorization as function
decolorization as functionofofinteractions
interactions between
between factors. (A)
factors.
Dye(A)concentration × HBT×concentration
Dye concentration interaction.
HBT concentration (B) (B)
interaction. Laccase-like activity××Temperature
activity
Laccase-like Temperature interac-
[Link]. (C) Laccase-like
(C) Laccase-like activityactivity × pH interaction.
× pH interaction. (D) pH(D)×pH × Temperature
Temperature interaction.(E)
interaction. (E)HBT
HBT concen-
concentration × pH interaction. (F) pH × Dye concentration interaction. All other factors
tration × pH interaction. (F) pH × Dye concentration interaction. All other factors were held were heldat center
at center level
level for each case. for each case.

3.3. Conditions Optimization

Minitab 16 statistics software was used to identify the optimized conditions for RB5
decolorization. The special condition combining all 5 variables included laccase-like ac-
tivity (0.5 U mL−1—high level (+1)), dye concentration (25 mg L−1—low level (−1)), HBT
concentration (4.5 mM—high level (+1)), pH (4.2—close to level 0 (−0.203)), and tempera-
 Microorganisms 2022, 10, 1137 12 of 19

Microorganisms 2022, 10, 1137 12 of 19

optimized conditions obtained here were adopted and repeated four times experimen-
Microorganisms 2022, 10, 1137 tally, reaching 82 ± 0.6% decolorization. 12 of 19
optimized conditions obtained here were adopted and repeated four times experimen-
tally, reaching 82 ± 0.6% decolorization.

Figure 4. Optimized condition-set for RB5 decolorization obtained with Minitab 16 statistical soft-
ware. 4. Optimized condition-set for RB5 decolorization obtained with Minitab 16 statistical soft-
Figure
Figure 4. Optimized condition-set for RB5 decolorization obtained with Minitab 16 statistical soft-
ware.
ware.
3.4. Decolorization Process in 500 mL Volume
3.4.
3.4. Decolorization
The optimized
Decolorization Process in
in500500mL
condition-set
Process mLVolume
(laccase-like activity 0.5 U mL−1, dye concentration 25
Volume
mg The
L , HBT
The
−1 concentration
optimized
optimized 4.5 mM,
condition-set
condition-set pH 4.2, and
(laccase-like
(laccase-like temperature
activity
activity0.50.5
UU mL55−1°C)
mL −1obtained
, dye via the 25
concentration
, dye Box–
concentration
mgBehnken
25 mgL ,LHBT
−1 − 1 design was
concentration
, HBT tested
concentration in
4.5 mM,a total reaction
pH 4.2,
4.5 mM, pHand volume
4.2,temperature of 500
and temperature mL
55 °C) 55to test
obtained
◦ the conditions
via the via
C) obtained Box–thein
larger
Behnken
Box–Behnkenvolumes.
design Figure
was was
design 5
tested shows
in a in
tested the
total kinetics
reaction
a total of RB5
volume
reaction decolorization
volume of 500 mLmL
of 500 to toas a function
testtest
thethe of
conditionsincuba-
conditionsinin
tion
larger time,
[Link]
Figure 77.6
5 ±
shows0.4% decolorization
the kinetics of RB5within 2 h of
decolorization incubation
as
larger volumes. Figure 5 shows the kinetics of RB5 decolorization as a function of incubation a (73.3
function ±
of0.4% in
incuba- 30-
time, reaching 77.6 ± 0.4% decolorization within 2 h of incubation (73.3 ± 0.4% in 30-min±
min
tion time-steps),
time, reaching followed
77.6 ± 0.4% gradually by
decolorization a slight increase
within 2 h of in decolorization
incubation (73.3 ± to reach
0.4% in 84.7
30-
0.6%
min in 6 [Link]
time-steps),
time-steps), At 24 h of
followed thegradually
graduallyincubation
by aby period,
a slight
slight decolorization
increase
increase reachedto
in decolorization
in decolorization 86.4 to± reach
reach 0.4%. The
84.7 84.7 ±RB5
± 0.6%
color
0.6% in changed
6 h. At from
24 h ofdark
the blue to
incubation intense
period,yellow (Figure
decolorization S2) during
reached
in 6 h. At 24 h of the incubation period, decolorization reached 86.4 ± 0.4%. The RB5 color the
86.4 decolorization
± 0.4%. The RB5pro-
[Link]
color
changed from from
dark dark blue
blue to to intense
intense yellow yellow (Figure
(Figure S2) during
S2) during the decolorization
the decolorization pro-
process.
cess.

[Link]
Figure KineticsofofRB5
RB5decolorization
decolorizationasasa afunction
functionofofincubation
incubationtime.
time.
Figure 5. Kinetics of RB5 decolorization as a function of incubation time.
3.5.
[Link]-VIS
UV-VISSpectrum
Spectrum
3.5. UV-VIS Spectrum
Spectrum
Spectrumanalysis
analysisshowed
showedthat thatthe
themaximum
maximumabsorbance
absorbancepeak
peakofofRB5
RB5was
wasatat598
598nm,
nm,
whichSpectrum
is in the analysis showed
visible-light that (Figure
region the maximum
S3). absorbance
Moreover, thepeak of RB5 was
absorbance of at 598 nm,
naphthalene
which is in the visible-light region (Figure S3). Moreover, the absorbance of naphthalene
which
and is in the visible-light region (Figure S3). Moreover, the absorbance of naphthalene
andbenzene
benzenerings
ringswas
wasobserved
observedininthetheUV
UVregion
regionatat254
254and
and310
310nm,
nm,respectively.
[Link]
test
and benzenecontaining
conditions rings was HBT,
observed
thereinwas
the aUV region at peak
significant 254 and 310
with nm, respectively.
a high In testin
absorbance value
the 280–310 nm range that was almost absent in the other conditions, which may indicate
the maximum absorbance of the HBT mediator. Compared to RB5 incubated solely with
the crude C. gallica laccase, the presence of HBT led to a 14% increase in decolorization (82%
Microorganisms 2022, 10, 1137 13 of 19

vs. 67%) accordingly a decrease in the peak absorbance of RB5 at 598 nm. Note that there
was no decrease in absorbance when RB5 was incubated with HBT but without enzyme.

4. Discussion
Textile manufacturing encompasses a complex cluster of various different technologies
and machinery [7] that ultimately produce vast amounts of colored clothing but use
vast amounts of water [66,67]. Textile-industry wastewater is discharged with various
contaminants such as dyes that are associated with toxicity and hazardous effects on both
health and the environment. The Reactive Black 5 studied here is a well-known azo dye
responsible for 70% of global demand [68]. RB5 has a well-defined chemical structure and
has been widely used in studies directed toward fungi-driven decolorization of dyestuffs
in textile manufacturing effluents [69,70]. Using a laccase-like active cell-free supernatant
of the basidiomycete C. gallica to decolorize RB5 could therefore be an efficient solution
to minimize RB5 concentrations in wastewater. The experimental design successfully
performed here used a screening plan that came with five factors and indicated that 4
of the 5 factor variables tested were significant (laccase-like activity, dye concentration,
HBT concentration, and pH). Moreover, for the fifth variable, which was temperature, we
choose to rule it as significant as it was very close to the p = 0.05 limit (p = 0.073). Daâssi
et al. (2012) [71] found that the same five factors as described here had significant effects
on RB5 decolorization using a crude laccase from T. trogii. However, whereas the Pareto
chart (Figure S1) indicated that laccase-like activity was the most effective factor for RB5
decolorization here, Daâssi et al., (2012) [71] found that with T. trogii, enzyme concentration
had a minor effect on RB5 removal. Likewise, the decolorization of two reactive dyes, i.e.,
Reactive Blue 114 (RB114) and Reactive Red 239 (RR239), was unaffected by the enzyme
concentration of a commercial laccase from Aspergillus sp. [72]. Furthermore, our results
showed that pH was the second important factor after laccase-like activity. pH has been
considered a relevant factor that mostly affects the decolorization reaction as it is a decisive
factor for optimum activity and stability of the enzyme [73,74]. For instance, the optimum
pH of the laccase of C. gallica is 3.0 (see Songulashvili et al. (2016) [75]) but the enzyme
has only demonstrated stability in a range of pH 4.0 to 7.0 [37]. Our analysis of the pH
effect found a negative slope that was very different from that found for the laccase-like
activity factor. Here, the increase in pH (from 3 to 6) decreased decolorization efficiency.
Dye removal by crude laccase from Trametes sp. was found to be sensitive to small pH
changes [71]. When using the whole fungus culture for the decolorization of RB5 by P.
eryngii F032, the optimum pH occurred at 3, affording a maximum of 94.56% dye removal,
whereas an increase in pH up to 10 decreased RB5 decolorization down to just 25.9% [76].
However, for free and immobilized cells of T. versicolor and Yarrowia lipolytica NBRC 1658,
increasing pH values increased the RB5 decolorization rates [77,78].
The Box–Behnken experimental design and RSM then served as a suitable approach
to facilitate the process modeling and determine the optimized conditions for improving
the decolorization rate. The contour plots indicated that increasing the initial dye concen-
tration has a negative effect on RB5 decolorization. Our results are consistent with data
from El Bouraie and El Din (2016) [79] and Khan et al. (2021) [80] demonstrating that a
high concentration of dye progressively decreases the efficiency of color removal. In a
similar way, Bonugli–Santos et al. (2016) [81] showed that increasing dye concentration
to over 200 mg L−1 negatively affected RB5 decolorization by Peniophora sp. CBMAI 1063,
possibly due to the inhibitory effect of the dye on laccase-like enzymes at high concentra-
tions [82,83]. To increase the efficiency of the laccase treatment, redox mediators are often
used [84]. Analysis of the contour curvature showed that the high concentration of HBT
increased the percentage rate of decolorization. Daâssi et al. (2013 a) [28], Murugesan et al.
(2007) [85] and Neifar et al. (2011) [86] all observed similar results. Despite the role it plays
in improving decolorization rates, HBT may inhibit the enzyme at HBT concentrations
over 5 mM [85]. HBT already has an inhibitory effect on the crude laccase from T. trogii and
laccase from Trametes sp. strain CLBE55 at up to 1 mM, [27,87].
Microorganisms 2022, 10, 1137 14 of 19

Increasing the temperature parameter improved dye decolorization up to 55 ◦ C. This

result is in good agreement with the optimum temperature profile of the C. gallica laccase,
which showed a correlation between the increase in temperature and laccase activity in
the range of 20–60 ◦ C [75]. Using the commercial laccase of Aspergillus oryzae, Wang et al.
(2011) [88] reported that changing the temperature from 20 ◦ C to 50 ◦ C had no effect on
the decolorization rate, but no explanation was provided for these results. Fernandez
et al. (2020) [74] reached a similar conclusion with a crude secretome of Pleurotus sajor-caju,
where increasing the temperature led to high levels of dye decolorization, with a maximum
found at 35–40 ◦ C. However, a study using cauliflower (Brassica oleracea) bud peroxidase to
remove dyes (Reactive Red 2, Reactive Black 5, Reactive Blue 4, Disperse Orange 25, and
Disperse Black 9) found that the decolorization rate was maximal at 40 ◦ C and decreased at
temperatures above 70 ◦ C, probably due to the thermal denaturation of the enzyme [89].
The optimized conditions obtained here using RSM for laccase-like activity, dye concen-
tration, HBT concentration, pH, and temperature were 0.5 U mL−1 , 25 mg L−1 , 4.5 mM, 4.2,
and 55 ◦ C, respectively, yielding 82 ± 0.6% color removal after 2 h of incubation. This result
was in the same range for the decolorization of Tubantin direct dye bath for pH (4) and
enzyme concentration (0.6 U mL−1 of laccase-active cell-free supernatant from Trametes
sp.) leading to 88% decolorization [87]. However, the reaction parameters were held at
different values, i.e., 75 mg L−1 of Tubantin bath dye, 1 mM HBT, 30 ◦ C, and 20 h [87].
The laccase-active cell-free supernatant of Trichoderma asperellum was also reported to
decolorize 90% of 50 mg L−1 RB5 within 24 h in the presence of an HBT mediator [90].
Complete decolorization was achieved on 50 mg L−1 of RB5 within 24 h using purified
NADH-dichlorophenol indophenol (NADH-DCIP) reductase and lignin peroxidase (LiP)
obtained from Sterigmatomyces halophilus SSA-1575 [91]. In conclusion, the result obtained
in this work can be considered very competitive compared to others, especially given that
the process used is short (2 h) and does not require any purification steps.
Tests were run at a larger scale (500 mL reaction volume) to increase the total quantity
of dyes with the best condition-set and achieved 77.6 ± 0.4% decolorization within 2 h and
compared to controlled bioreactor processes. Mohorčič et al. (2004) [12] reported that the
immobilized mycelium of Bjerkandera adusta removed 95% of the azo dye RB5 (0.2 g L−1 )
in a 5 L aerated stirred tank bioreactor within 20 days. Pavko (2011) [92] reported that the
white-rot fungus T. versicolor was capable of decolorizing 97% of the synthetic dye Orange
G in a 1.5 L bioreactor within 20 h. In a 3 L-volume bioreactor, Pseudomonas putida SKG-1
strain was able to decolorize 98% of the reactive orange 4 dye within 60 h [93]. This shows
the value of our results obtained with a 2-h process in non-controlled flask conditions
and the reproducibility of our results between the 1-mL lab-scale and the larger 500-mL
scale-up.
The UV-visible spectrum of RB5, in the range of 200 to 800 nm, indicated peak ab-
sorbance of RB5 at 598 nm together with the presence of benzene and naphthalene ring
signatures at 254 and 310 nm, respectively, [94,95]. Spectra analysis showed that the
addition of HBT decreased the absorbance peak of RB5 dye only in the presence of the
laccase-like active cell-free supernatant of C. gallica, demonstrating that the laccase mediator
system efficiently decolorizes RB5. Our results confirmed those of Tavares et al. (2008) [96]
who showed that the presence of the 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonique)
mediator reduced the maximum absorbance peak of the dye and that the decolorization
occurred when both the mediator and the enzyme were added to the reaction mixture.

5. Conclusions
The statistical design-of-experiments approach employed here showed that 4 factors
among the 5 studied were key factors for the degradation of the RB5 dye. Among all these
parameters, the main factor was the concentration of laccase activity used in the process.
This is a critical point, as the price of the enzyme dictates the economic viability of the
process. The second most critical factor was pH, probably due to the biochemical properties
of the enzymes (pH optimum and pH stability). However, the present study confirmed
Microorganisms 2022, 10, 1137 15 of 19

that the laccase cell-free supernatant of C. gallica was active on RB5 dye using the mediator
HBT, and that an enzymatic treatment of 2 h was sufficient to achieve up to 82 ± 0.6%
decolorization in a lab-scale reaction volume of 500 mL Further scale-up experiments are
required to demonstrate that this treatment can be made amenable to pilot scale and to
future industrial applications. This work provides proof-of-concept for the biodegradation
of textile-industry dyes as a promising alternative route to physicochemical treatments.
Further experiments could usefully measure the biochemical oxygen demand (BOD) and
chemical oxygen demand (COD) of the treated dye solution to evaluate the toxicity of the
residual dye. Processes using cheaper and eco-friendly alternative plant-origin mediators
should be tested in an effort to engineer a more sustainable system.

Supplementary Materials: The following are available at [Link]

microorganisms10061137/s1. Figure S1: Pareto Chart of the standardized effects for RB5 decoloriza-
tion (after 120 min of reaction) as function of coded value of factors. Figure S2: Shift of RB5 color
from dark blue to intense yellow after 2 h of incubation. Untreated dye: 25 mg L−1 RB5 dye, 4.5 mM
HBT, pH 4.2 and temperature 55 ◦ C. Treated dye: 0.5 U mL−1 laccase-like activity, 25 mg L−1 RB5
dye, 4.5 mM HBT, pH 4.2 and temperature 55 ◦ C. Figure S3: UV–VIS spectrum of RB5 showing origi-
nal dye (25 mg L−1 ) (dark blue), RB5 added with 4.5 mM HBT (red), RB5 added with the laccase-like
cell free supernatant from C. gallica (green), RB5 added with both 4.5 mM HBT and the laccase-like
cell free supernatant from C. gallica (purple), HBT with the laccase-like cell free supernatant from C.
gallica (light blue) and 4.5 mM HBT (orange) after a 24 h incubation period. Table S1: Decolorization
conditions.
Author Contributions: Conceptualization, A.B.A., B.H., E.R., T.M.; Methodology, A.B.A., B.H., H.Z.-
M., T.M.; Software, B.H.; Validation, A.B.A., B.H., H.Z.-M., E.R., T.M.; Formal Analysis, A.B.A., B.H.;
Investigation, A.B.A.; Resources, A.B.A., B.H.; Data Curation, A.B.A., B.H., E.R., T.M.; Original Draft
Preparation, A.B.A., B.H., E.R., T.M.; Review and Editing, A.B.A., B.H., G.S., A.L., E.B., C.B.F., H.Z.-M.,
E.R., T.M.; Supervision, B.H., E.R., T.M.; Project Administration, E.R., T.M.; Funding Acquisition, E.R.,
T.M. All authors have read and agreed to the published version of the manuscript.
Funding: Tunisian ministry of higher education and scientific research: no grant number. INRAE
(TRANSFORM division): no grant number.
Acknowledgments: The authors are grateful to Tunisian ministry of higher education and scientific
research for providing part of the financial support to ABA. The authors extend their appreciation to
financial support by INRAE (TRANSFORM division).
Conflicts of Interest: The authors declare no conflict of interest.

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