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Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Customizing the spent coffee for Trichoderma reesei cellulase

immobilization by modification with activating agents
Aneta Buntić a , Marija Pavlović a,∗ , Dušan Antonović a , Vladimir Pavlović b ,
Dragana Vrućinić a , Slavica Šiler-Marinković a , Suzana Dimitrijević-Branković a
a
Faculty of Technology and Metallurgy, University of Belgrade, Department of Biochemical Engineering and Biotechnology, Karnegijeva 4, 11000 Belgrade,
Serbia
b
Faculty of Agriculture, University of Belgrade, Department of Agricultural Engineering, Nemanjina 6, 11080, Belgrade, Zemun, Serbia

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

Article history: This study has explored the possibility to reuse the waste, spent coffee material for the cellulase enzyme
Received 1 February 2017 immobilization. By the coffee surface modification with different activating agents, it was attempted to
Received in revised form 28 August 2017 develop the convenient method for creation of a capable porous carrier for this purpose. Among the most
Accepted 10 October 2017
common activating agents, glutaraldehyde, chlorine dioxide and hydrogen peroxide provided the most
Available online xxx
acceptable choice for the coffee surface modification. The changes that occurred on the coffee surface
due to agents’ treatment exposure were recorded by using of the FTIR spectra and SEM micrographs.
Keywords:
The highest immobilization yield (55%) and immobilization efficiency (45%) were attained during 30 min
Cellulase
Spent coffee
of the treatment time, by employing of 30% chlorine dioxide aqueous solution within 6 mL/g activa-
Immobilization tor/carrier ratio. The kinetic process was found to be predicted by the pseudo-second-order model. The
cellulase immobilization onto the coffee surface provides an excellent base for increasing the enzyme
availability to the substrate and enhancing the enzyme productivity, by offering the new perspectives to
the industrial sector.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction cellulases have been mainly engaged in pharmaceutical and med-

The agro-industrial waste materials and other waste bio-
material from the restaurant and food industry represent one of
the environmental problems associated with their disposal. These
materials may be of special interest because of their numerous
advantages, such as availability, technical feasibility, engineering
usable, cost effectiveness, for a requisite waste management strat-
egy by recycling into useful products. Therefore, managing of these
residues as raw material for adsorption and immobilization of
bioactive compounds, provides a value-added material [1–3].
Cellulases belong to the class of bio-catalytic enzymes respon-
sible for the conversion of cellulose into the soluble sugars [4–7].
Owning to such a bio-technological conversion potential, cellulases
provide opportunities to develop novel bioprocesses and prod-
ucts [7]. Nowadays, the usage of cellulases has been expanded
in research development of different industries such as agricul-
ture, brewery and wine, bio – ethanol, textile, food processing,
paper and pulp, olive oil extraction etc. On the other hand free
∗ Corresponding author.
E-mail address: marija.pavlovic@tmf.bg.ac.rs (M. Pavlović).
ical sciences and genetic and environmental engineering [7–10].
Some drawbacks of free-cellulases utilization (such as relative
stability, reusability, low specific activity, as well as additional
costs), have been of a major concern for their industrial applica-
tion [10–12]. Also, the free enzymes are unable to be recycled [12].
In order to solve these problems, an immobilization of the cellulase
enzyme on solid porous supports has been recognized as a viable
solution [10,12,13]. Another advantage of this purpose is that the
process enables the use of very low amounts of cellulase [10].
Cellulase has already been immobilized on a number of various
commercial materials which may be both insoluble or soluble car-
riers, as well as nano-scale materials [13–16]. The process of porous
carriers synthesis and its recovery may require additional costs
[4,14,17,18]. By contrast, the advantage of employment of cost-
effective adsorption materials is all the more prominent, especially,
if the used materials are sourced from recycled waste.
The spent coffee waste material discharged after coffee bev-
erage preparation, is being used for different purposes, such as
animal feeds, biodiesel and fertilizers production [19,20]. To the
greatest extent, this waste material contains hemicellulose, cel-
lulose, protein, total sugars, minerals [21,22]. Such a composition
makes it attractive per serving as a potential carrier for the cellulase

https://doi.org/10.1016/j.ijbiomac.2017.10.060
0141-8130/© 2017 Elsevier B.V. All rights reserved.

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immobilization. In addition, excellent adsorption capabilities of

adsorbents prepared from spent coffee residues, for removal of
metal ions and dyes from aqueous solutions, have been previously
shown [23–26]. The possibility of using the spent coffee residues as
a porous carrier for cellulase immobilization has not been explored
yet. Under the chemical modification conditions, the performance
of coffee residues for the immobilization process may be facilitated,
considering that activating agents may contribute to the carriers
sorption capabilities improvement [27].
This research aims to investigate the possibility to utilize
the spent coffee by modification with specific activation agents,
named glutaraldehyde (GluA), chlorine dioxide (ClO2 ) and hydro-
gen peroxide (H2 O2 ), for preparing the solid carrier for commercial
cellulase (originated from Trichoderma reesei) immobilization. The
success of the immobilization process was evaluated by measuring
the cellulose activity, expressed by the immobilization yield (%) and
the immobilization efficiency (%). The most efficient modification
agent was further subjected to research, whereby the influence of
different experimental conditions, such as, activator concentration,
treatment time and activator/carrier ratio, have been evaluated. In
addition to this, the usefulness of pseudo-first- order and second-
order models for the adsorption kinetics of enzyme were also Fig. 1. FT-IR spectra of the coffee carrier: (a) native, (b) GluA-treated, (c) ClO2 -
discussed. treated and (d) H2 O2 -treated.

enzyme immobilization. The samples were dried, coated with gold

2. Material and methods and observed with the microscope at 20 kV and 700X magnification.

2.1. Enzyme and chemicals working solutions

2.3.2. Infrared spectroscopy
The characterization of modified coffee carriers was analyzed by
A cellulase enzyme from Trichoderma reesei ATCC 26921 was
® FT-IR spectrometry (BOMEM MB100, Canada). The samples were
purchased from Sigma-Aldrich , Denmark. The working cellulase
dried and further prepared in a form of KBr pellets (0.6 mg of
solutions concentrations of 1 mg/mL and 1% Avicel (w/v) (pur-
the sample and 200 mg of KBr). The measurements were carried
chased from Merck, Germany) were prepared fresh in pH 4.8
out with a 4 cm−1 resolution in the 400–4000 cm−1 wave number
tri-sodium citrate buffer (Na3 C6 H5 O7 ). The others, GluA, ClO2 and
range.
H2 O2 working solutions were made in distilled water. ClO2 solu-
tion has been prepared by using 27% sodium chlorite (NaClO2 ) and
2.4. Cellulase immobilization procedure
10% citric acid (C6 H8 O7 ) in ratio NaClO2 : C6 H8 O7 : dH2 O = 1:5:34
and they were purchased from ACRŌS ORGANIC, New Jersey, USA
The immobilization of 20 mL of the cellulase solution onto 1 g of
and Lachema, Czech Republic, respectively. The solution obtained
determined coffee carrier was done in a 100 mL round-bottomed
in that manner present 15% ClO2 solution.
flask. The mixture was placed on a translatory shaker with 150 rpm
for 2 h, at 40 ◦ C. Thereafter, enzyme immobilized carriers were col-
2.2. Preparation of coffee carrier lected by filtration and were washed by distilled water. Cellulase
activity was measured in triplicate using UV/VIS spectrophotome-
The spent coffee residues, remained after espresso beverage ter (Ultrospec 3300 pro, Amersham Biosciences, USA) at 540 nm.
preparation at a local cafe, were collected and dried for overnight The success of cellulase immobilization onto the coffee carrier
in a thermostat at 40 ◦ C. The material has been subjected to the has been determined by evaluating the following two terms: the
process of polyphenols extracting (for bioactive compounds iso- immobilization yield (%) and the immobilization efficiency (%), and
lation, according to the previously experiments of spent coffee calculated by Eqs. (1) and (2), respectively [29–31]:
exploitations) [28]. 2 g of spent coffee residues, remained after
Yield (%) = immobilized activity/starting activity ∗ 100 (1)
polyphenols extraction, have been treated with 30 mL of 2.5% GluA,
15% ClO2 and 15% H2 O2 , respectively, whereas those the activating Efficiency (%) = observed activity/immobilized activity ∗ 100 (2)
agent solutions have been prepared according to the preliminary
The correctly determination of the immobilized enzyme activ-
investigation results (data not shown). Thus, the immobilization of
ity was conducted by measuring the total activity (units, i.e.
cellulase enzyme was performed onto four different coffee orig-
␮mol/min) from the starting enzyme solution subtracting the total
inated carriers (Fig. 1). After 20 min of shaking on a translatory
residual enzyme activity that was left in the enzyme solution
shaker (IKA – KS 4000i control, Staufen, Germany) with 150 rpm,
after immobilization process [29]. The immobilization yield (%)
mixture was filtrated, washed with distilled water and then dried
described the percentage of total cellulase activity from the free
in an oven at 105 ◦ C. Dry material was crushed and powdered for
cellulase solution that is immobilized onto the carrier, whereas the
obtaining a particle size less than 500 ␮m.
immobilization efficiency (%) represents the percentage of bound
enzyme activity that was observed in the immobilizate [29].
2.3. Characterization of the carriers
2.5. Measurement of cellulase activity
2.3.1. Scanning electron microscopy (SEM)
SEM (JEOL JSM-6390LV, USA) analyses were used to understand Cellulase activities of the free and the immobilized cellulase
the surface modification of the porous adsorbent before and after were determined by the 3,5-dinitrosalicylic acid reagent (DNS)

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method. The method is based on the determination of reducing
sugar these were released from the Avicel (1% w/v, suspended in
a tri-sodium citrate buffer pH 4.8) during 30 min of incubation at
50 ◦ C and under shaking conditions. Enzyme activity unit is defined
as the amount of enzyme producing 1 ␮mol of glucose equivalents
per minute. The protein concentration in solution was measured
by the Bradford protein assay using BSA (bovine serum albumin)
as standard and concentrations were calculated using appropriate
standard curves [32].
2.6. Adsorption kinetic studies
Adsorption kinetics experiments were conducted by 5 g/L of the
coffee carrier in Erlenmeyer flasks containing 200 mL of the cellu-
lase solution on a translatory shaker (150 rpm). The initial cellulase
concentration was fixed at 3 g/L, while the reaction temperature
was varied (30, 35, 40 and 45 ◦ C). The samples were collected at pre-
determined time intervals and analyzed spectro-photometrically
for the cellulase adsorption capacity.
The equilibrium adsorption capacity of cellulase on the coffee
carrier, qe (U/g) was calculated according to Eq. (3):
qe = (C0 − Ce ) /m ∗ V
where C0 (U/mL) and Ce (U/mL) are the initial and the equilibrium
concentration of cellulase solution, respectively, V (mL) is the vol-
ume of cellulase solution, and m (g) is the mass of the coffee carrier
[9].
A simple kinetic analysis of adsorption, based on most widely
used pseudo-first and pseudo-second order models, were further
employed to fit the experimental data. Both kinetic models assume
that the rate of change of the enzyme solute removed with time is
directly proportional to the difference in saturation concentration
and the amount of solid removed by carrier. Unlike pseudo-first-
order kinetic model, the pseudo-second-order kinetic model can
predict the behavior of the system on whole investigated time
range [33,34].
The linearized form of pseudo-first (Eq. (4)) and pseudo-second
order kinetic model (Eq. (5)) are presented as follows:
log (qe − qt ) = logqe − k1 /2.303 ∗ t
t/qt = 1/(k2 ∗ q2e ) + 1/qe ∗t
where k1 (1/min) and k2 (g/U min) are the rate constants of the
pseudo-first-order and pseudo-second-order adsorption kinetics,
respectively, qe is the ratio of enzyme adsorbed on the surface of
the adsorbent at equilibrium (U/g) and qt , is the ratio of enzyme
adsorbed any time (U/g) [9].
3. Results and discussion
3.1. The immobilization of cellulase onto four modified coffee
carriers
The three types of activators had been particularly selected for
coffee carrier surface modification because of their earlier appli-
cations [27]. The coffee modification with GluA has already been
used as a successful immobilizer for ˇ-glucosidase [35], while the
other two activating agents were employed to modify the natural
material through their strong oxidative capabilities [27,36]. In order
to understand the degree of coffee surface modification after the
evaluated agent’s treatment and to identify the functional groups
present on the carriers’ surface, infrared spectroscopy and scanning
electron microscopy were used.
FTIR spectra of the native and modified spent coffee carriers
have been presented in Fig. 1. It was observed that Glu-A-treated
coffee carrier has experienced inconsiderable surface changes.
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3
More noticeable surface variations have been found on H2 O2 -
treated coffee carrier, whereas the strongest signal drafts and
intensifications have been noted on ClO2 -treated coffee carrier. In
particular, the main changes, in all FTIR spectra bands occurred at
regions between 3200 cm−1 and 3600 cm−1 and between 900 cm−1
and 1700 cm−1 . Some of them increased or reduced or disappeared
by the influence of appropriate agents. The increasing of the signal
surface (3200–3600 cm−1 ) in FTIR spectra of the ClO2 -treated cof-
fee carrier suggests the stretching vibrations of hydrogen-bonded
hydroxyl groups  (OH) [37,38]. The signal surface increase of
1535% for  (OH) has been calculated on the basis of carbonyl
groups. On contrary, the picks at 2887 cm−1 and 2811 cm−1 have
been drastically diminished. The reduced picks are referred to C H
asymmetric and symmetric stretch of the methylene and methyl
groups [38,39]. The distinct peak at 1640.7 cm−1 observed on a
native coffee sample (this may be attributed to C O stretching
vibration of carboxylate ( COO )) has been even lost [17]. The
newly formed pick at 1552.7 cm−1 on a ClO2 -treated coffee sample
represents amino groups stretching vibrations [40]. These amino
groups stretching vibrations bands have been superimposed on the
side of the hydroxyl group band at 3500–3300 cm−1 [40]. Also, the
visible changes occurred in the fingerprint region. The changes in a
(3) characteristic set of bands between 900 cm−1 and 1600 cm−1 may
be assigned to the C C stretching mode and the C H deformation
mode in the aromatic ring plane [41].
Generally, the hemicellulose, cellulose, proteins and lignin are
a group of target biopolymers those are expected to be function-
alized after the spent coffee activation treatment. The introducing
of hydroxyl, amino and aldehyde groups is expected to enable a
favorable microenvironment to the immobilized enzymes. Other
researchers proved that the ease of accessibility of cellulases to
cellulose was the main parameter for driving the enzyme adsorp-
tion [42]. On the other hand, lignin may be limiting factor in the
interactions between the carrier and enzyme. There is a group of
researchers that found the differences in the underlying mecha-
nism of the enzymes adsorption onto lignins from diverse types
of lignocellulosic biomass, due to diverse lignin composition and
structural features. It has been generally established for functional
groups, which affect lignin-enzyme interactions, that a low car-
(4) boxylic acid group content and a higher phenolic hydroxyl content
(5) in lignins could result in the increased protein adsorption capac-
ity [42–44]. There is a favorable circumstance that spent coffee
grounds contain lignin in much lesser extent in comparison to other
lignocellulosic biomass. Bearing in mind that spent coffee is a by-
product of coffee-industry, many processing steps of coffee cherry
influence the lost lignin, thus has been retained just in a small
proportion (0.05 ± 0.05%) [21].
Scanning electron micrographs of surface structure of the cof-
fee carriers, before and after modification, were presented in Fig. 2.
It was found that modification treatments evidently impacted the
porosity of the coffee carrier. The most explicit porosity has been
obtained by using of ClO2 (Fig. 2(c)) and H2 O2 (Fig. 2(d)) acti-
vating agents, but numerous macropores and mesopores have
been developed after the GluA treatment (Fig. 2(a)), as well. The
ClO2 activating agent obviously formed a dominantly macroporous
and mesoporous network with a sufficient pore size diameter for
ensuring the passage of the enzyme macromolecule to the inte-
rior surface of the coffee carrier, which provided an increased
enzyme immobilization (Fig. 3). Porous material with dominance
of mesopores also allows a higher cellulase enzyme mobility and
flexibility within the cavities. It leads to higher cellulase activ-
ity, which has been revealed in previous literature reports within
the actual research area [13,45,46]. Besides the H2 O2 -treated cof-
fee carrier expressed large surface area (Fig. 2(d)), the amount of
immobilized enzymes was quite low, probably because of pres-
ence of many micropore channels, these disable the free enter of
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Fig. 2. SEM images of the coffee carrier: (a) native, (b) GluA-treated, (c) ClO2 -treated and (d) H2 O2 -treated.
Fig. 3. The cellulase immobilization onto coffee carriers (coffee carrier: 1 g, cellulase
solution: 20 mL, under shaking for 2 h).
the cellulase molecules and their efficient adsorption (Fig. 3). In
addition, the absence of an appropriate active groups on the car-
rier surface may lead to the immobilization efficiency decrease, too.
On the other hand, the GluA-treated carrier (Fig. 2(b)) showed less
porosity, but the relatively good ability to immobilize the cellu-
lase, which may probably be related with the presence of favorable
functional groups on the carrier surface (Fig. 3). Considering the
immobilization yield, the results of this study indicate the note-
worthy potential of spent coffee to immobilize the cellulase (Fig. 3).
The values were ranged up to about 27% in favor of the ClO2 -treated
coffee carrier. On the other hand, the corresponding immobiliza-
tion efficiencies were slightly lower and these values were ranged
from 10 to 24%. Probably, some chemical groups existing on car-
rier influenced negatively the immobilized enzymes to manifest
their actual activity [47]. The interaction with certain compounds
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apparently affected the folding or stability of the enzyme [48]. Also,
if they had been immobilized in great amounts, many of them could
remain inactivated. Tejirian and Xu [48] pointed out the possibil-
ity that chemical compounds affecting cellulase activity, among
others, may be phenolics as well, these undoubtedly remained in
spent coffee in small portions. But, it has been perceived that a
rich mineral content consisting in coffee, particularly the amounts
of potassium, magnesium and calcium, undoubtedly contributes
to the immobilized enzyme stabilization and manifestation of its
favorable functional properties [22]. In a case of the GluA agent
application, a certain quantity of the cellulase were immobilized
(according to the yield); however, the lower enzyme activity has
been expressed which may be due to the reticulation among the
enzyme molecules. This phenomenon may be due to formation of
layers between fibrous polymer chains on the carrier and immobi-
lized enzyme molecules, resulting in a sticky web like coating over
the particle surface. Thus, these layers may prevent the diffusion
of the large substrate molecules (like Avicel) to the inner layer of
the polymer brushes [49]. Besides GluA activation technique is one
of the most popular for enzyme immobilization, it has not been
selected as an appropriate activator for further study, mainly due
to its eco-toxicity [50,51]. On the other hand, chlorine dioxide was
very efficient in making the biocompatible environment for the
cellulase immobilization, by disarming the negative influence of
certain surface agents those may affect the enzyme activity. Thus,
the cellulase immobilization process has been improved. This out-
come has been particularly good, because of the carrier material
has been activated in a non-toxic manner and, thus, may allow the
safe further product application [52]. Thus, the best combination
of both evaluated parameters, the immobilization yield (%) and
the immobilization efficiency (%), was attained in the case of the
immobilization cellulase onto coffee residues activated with chlo-
rine dioxide. A quarter of the total amount of cellulase enzyme from
starting solution was attached, and of which more than one-fifth
had demonstrated the activity.
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Fig. 4. FT-IR spectra of the ClO2 -treated coffee carrier after enzyme immobilization.
Table 1
Factors and levels of single factor experiment using the chlorine dioxide as an acti-
vator of coffee carrier for the cellulase immobilization.
Factors Level
Chlorine dioxide concentration (%) 20 25* 30 35 40
Treatment time (min) 20* 30 40 50 60
Ratio (chlorine dioxide/coffee) (mL/g) 6* 8 10 12 14
*
The levels kept constant when other factors were investigated.
As a result of previous investigation steps, the chlorine dioxide
activating agent has been adopted for further coffee carrier modi-
fication in order to conduct a more detailed evaluation of cellulase
enzyme immobilization.
3.2. The properties of cellulase immobilized on the ClO2 -treated
coffee carrier
The FTIR spectrum of the ClO2 -treated coffee carrier after the
enzyme immobilization has been presented in Fig. 4 in order to con-
firm the presence of immobilized enzyme onto carrier. The peaks of
CH and C O groups were intensified and slightly broader in com-
parison with the same peaks recorded for the ClO2 -treated coffee
carrier before immobilization (Fig. 1). The most important changes
were observed in an amide group peak (O C NH) corresponding
to cellulase enzyme at 1639 cm−1 , which was slightly shifted from
1641.3 cm−1 and intensified [53]. Also, NH2 group can be associ-
ated with NH stretch at 3471.6 cm−1 . This peak was shifted from
3456.1 cm−1 and intensified, as well, probably due to the presence
of water retained in the sample [11,17]. It may be hypothesized
that hydrogen bonding forces and electrostatic forces establish
the connection between the enzyme and the pre-treated matrix.
The coupling reaction probably took place between the free amino
groups of an enzyme (i.e., lysine residues, considering that most
proteins contain many lysine residues, usually located on the pro-
tein surface) and polar groups of the activated carrier. In addition,
hydrophobic interactions were speculated to occur between cellu-
lases and lignin. Similar assertions have been previously reported
by other researchers [6,43,49,54,55].
In the following investigation, the effects of the activator
(chlorine dioxide) concentration, modification treatment time and
activator/carrier ratio have been further examined. The single fac-
tor batch experiments were carried out and the conditions were
shown in Table 1. The success of the cellulase immobilization onto
the modified coffee carrier has been determined on the way that
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was mentioned above, by measuring the immobilization yield (%)
and the immobilization efficiency (%).
3.2.1. Effect of activator concentration
The effect of the chlorine dioxide concentration on the cellulase
immobilization onto the coffee carrier was investigated and pre-
sented on Fig. 5(a). This effect was studied in the range of 20 up to
40% of the chlorine dioxide working solution, whereas other vari-
ables: the time of modification (min) and the activator/carrier ratio
(mL/mg) were held constant (Table 1).
With increasing of the chlorine dioxide concentration up to 30%,
the immobilization yield and the immobilization efficiency were
increased, too. The immobilization yield reached 53%, while the
immobilization efficiency achieved 44%. After this value (30% of
chlorine dioxide solution concentration), the observed parameters
were decreased. The immobilization yield has been reduced for
about 23%, while the immobilization efficiency for about 25%. Prob-
ably, up to this critical point, chlorine dioxide successfully oxidized
the compounds these had negatively affected the cellulase immo-
bilization, and contributed to lower enzyme activities expressions.
After carrier modificaion with the strongest evaluated concentra-
tion of chlorine dioxide, the structural changes of the carrier surface
have been visually observed, as well. The material became some
kind of glutinous form. Probably, such a texture contributed to a
lower success of the immobilization yield and the immobilization
efficiency, because of reducing the number of available immobi-
lization sites for cellulase molecules. On the other hand, there
is another possibility for such a behavior, described by Ferreira
et al. [56] for protease immobilization and stabilization onto silica
derivatives, that cross-linking may occurred among the cellulase
molecules.
3.2.2. Effect of treatment time
The influence of the cellulase immobilization efficiency onto the
coffee carrier in relation to the treatment time of the carrier with
chlorine dioxide was shown on Fig. 5(b). The effect was studied in
a range of 20 up to 60 min of the treatment time, whereas other
variables: the activator concentration (%) and the activator/carrier
ratio (mL/mg) were held constant (Table 1).
With the short time of the modification process (from 20 to
40 min) the immobilization yield and the immobilization efficiency
were increased, while after 40 min of the treatment time, both
examined parameters have been decreased, that may be due to
the interaction among the immobilized cellulases which reduced
the expression of their activity [56]. More precisely, it may be
observed that immobilization yield was higher, while the process
efficiency was lower when the process was performed for 40 min.
The immobilization yield has reached the 52%, while the immobi-
lization efficiency has achieved the 40%. So, the best results were
attained with 30 min of the treatment time of coffee carrier with
25% of aqueous chloride dioxide solution, under the ratio (activa-
tor/carrier) of 6 mL/mg.
3.2.3. Effect of activator/carrier ratio
The effect of the chlorine dioxide/coffee carrier ratio on the
success of cellulase immobilization can be seen on Fig. 5(c). The
examined ratio of the chlorine dioxide (25%) solution and the
amount of coffee material has been in the range of 6 mL/mg up
to 14 mL/mg. The treatment time, of 20 min, was constant.
The immobilization yield was more or less decreased with
increasing of the ratio of the chlorine dioxide/coffee carrier, while
the immobilization efficiency was behaving similarly, resulted
between 41 to 38%. It may be observed that the ratio of chlo-
rine dioxide/coffee carrier between 6 and 10 mL/mg did not
demonstrate any considerable changes in the process of coffee
modifications for the cellulase immobilization, thus the ratio of
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Fig. 5. The influence of: (a) the activator concentration, (b) treatment time and (c) activator/carrier ratio on cellulase adsorption.

6 mL/mg was adopted as optimal for further application of coffee

carrier for the cellulase immobilization. In addition, bearing in mind
the economical and eco-friendly side of the application of amount
of carrier, it should be opt for spending fewer amounts of carriers.

3.2.4. Adsorption kinetics

The effect of time on the adsorption capacity, during cellulase
immobilization on coffee carrier, observed on different tempera-
tures is presented in Fig. 6.
It was clear that temperature alterations affected the degree of
the adsorption capacity, but on the other hand, did not influence the
equilibrium attainment. As the reaction time rises up to 40 min, the
adsorbed enzymes have been increased for all tested temperatures,
too. The exponential increase in the capacity of adsorption ranged
from 0.7 U/g up to about 1.4 U/g for the temperature range from
30 to 40 ◦ C, while for the adsorption at 45 ◦ C, maximum adsorption
capacity after 40 min of the immobilization process was lower and
reached about 1 U/g. After this stage, the amount of enzymes being Fig. 6. Effect of contact time for cellulase adsorption onto coffee residues (adsorbent
mass: 5 g/L, cellulase concentration: 3 g/L, 150 rpm).
adsorbed onto the coffee carrier was more or less constant, because
the amount of enzymes being adsorbed onto the coffee carrier was
in a state of dynamic equilibrium with the amount of enzymes des- The corresponding kinetic parameters from the pseudo-first and
orbed from the carrier surface (Fig. 6) [57]. The amount of cellulases pseudo-second-order linear plots were obtained at three different
adsorbed at the equilibrium time reflected the maximum enzyme temperatures and were reported in Table 2. The validity of the
adsorption capacity of the coffee carrier under the evaluated exper- kinetic models was based on the regression coefficients and the
imental conditions [57]. According to the obtained results, it was agreement between experimental and predicted qe values. Bearing
observed that the reaction temperature of 40 ◦ C affected the high- that in mind, the immobilization of cellulase onto coffee residues
est adsorption capacities, which were chosen for further enzyme was found to follow the pseudo-second order kinetic model. Based
adsorption experiments. on a principle of pseudo-second order kinetics, the rate limit-

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G Model
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Table 2
Kinetic model parameters for cellulase adsorption on spent coffee at different temperatures (adsorbent mass 5 g/L, cellulase concentration 3 g/L, 150 rpm).
Parameter Pseudo-first order kinetic model
Temperature (◦ C) qe,exp (U/g) k1 (1/min) qe,cal (U/g)
30 1.2492 0.1046 0.3613
35 1.2895 0.0752 0.2062
40 1.3587 0.0723 0.2586
45 0.9899 0.0936 0.9689
ing step of the immobilization process may be chemical sorption.
Daoud et al. [9] also revealed that the kinetic of pseudo-second
order model served to describe the adsorption behavior of cellulase
from Aspergillus niger on a commercial activated carbon.
4. Conclusion
This research has explored a possibility to utilize spent coffee
for the cellulase enzyme immobilization and evaluate the con-
ditions for preparing the optimal carrier properties. The highest
values of the immobilization yield and immobilization efficiency
were attained by employing the chlorine dioxide as an activating
agent. The process conditions were found to be mild, not extreme,
nor complicated, providing the economically viable procedure, con-
ducted in an environmental friendly manner. The final product
represents a great potential for the concentration and stabilization
of the cellulase enzyme, that may be ready to be used for imme-
diate implementation in an animal feed formulation, providing a
safe, useful and profitable application.
Acknowledgement
This work was supported by the Ministry of Science and Educa-
tion of the Republic of Serbia (project TR 31035).
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