Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=lwct20
Journal of Wood Chemistry and Technology
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lwct20
Ultrasound pretreatment and enzymatic deinking by using bacterial cellulases from rice husk
Saharman Gea, Rumondang Bulan, Emma Zaidar, Averroes Fazlurrahman Piliang, Noni Oktari, Sri Rahayu, Yasir Arafat Hutapea & Reka Mustika Sari
To cite this article: Saharman Gea, Rumondang Bulan, Emma Zaidar, Averroes Fazlurrahman Piliang, Noni Oktari, Sri Rahayu, Yasir Arafat Hutapea & Reka Mustika Sari (2023): Ultrasound pretreatment and enzymatic deinking by using bacterial cellulases from rice husk, Journal of Wood Chemistry and Technology, DOI: 10.1080/02773813.2023.2190594
To link to this article: https://doi.org/10.1080/02773813.2023.2190594
View supplementary material
Published online: 24 Mar 2023.
Submit your article to this journal
View related articles
View Crossmark data
Ultrasound pretreatment and enzymatic deinking by using bacterial cellulases from rice husk
Saharman Geaa,b, Rumondang Bulanb, Emma Zaidarb, Averroes Fazlurrahman Pilianga,c, Noni Oktaria,b, Sri Rahayua,b, Yasir Arafat Hutapeaa,d and Reka Mustika Saria,e
aCellulosic and functional materials research Centre, universitas sumatera utara, medan, indonesia; bdepartment of Chemistry, faculty of mathematics and natural sciences, universitas sumatera utara, medan, indonesia; cdepartment of Physics, faculty of mathematics and natural sciences, universitas sumatera utara, medan, indonesia; ddepartment of hydrogen energy systems, graduate school of engineering, Kyushu university, fukuoka, Japan; eresearch Center for food technology and Processing, national research and innovation agency, gunungkidul, yogyakarta, indonesia
ABSTRACT
Ultrasonic pretreatment could improve the accessibility of enzymes to cellulosic fibers, hence expanding the access of enzymes to effectively remove ink from paper. In this study, enzymes were isolated from paddy rice husks and utilized for biodeinking process. The characterizations of deinking from the pulp filtrate and paper samples were carried out to understand the effect of bacterial cellulases and ultrasonic pretreatment with time variables. The results showed that cellulases from bacterial isolates had the ability to hydrolyze cellulose at 1.78 U/
mL enzyme activity. Ink content measurement showed 2310 ppm of ink was removed in the paper samples with 15 min of ultrasound pretreatment and 2% cellulases. Meanwhile, morphological analysis showed significant differences in the surface areas between treated and untreated paper samples. The highest crystallinity index achieved was 70.9%, and thermal resistance was 21.0%. Other paper qualities such as stain decrease, and brightness level improvement were observed. The combination of 2% cellulases with optimum ultrasonication duration resulted in exceptional wastepaper recycling process.
Introduction
Paper waste, mainly printed paper, has been increasing with its daily consumption in offices and as other stationery. Paper recycle process is an alternative, yet a promising way for the sustainability of paper pro- duction process with limited raw material, such as woods.[1] However, the main issue in paper recycling is the process of ink removal from the paper fibers, commonly known as deinking process. To date, the attempts of ink removal process by using chemical substances are environmentally harmful.[2–4]
Conventional deinking methods that mainly involving chemicals can cause damage to the environment.
However, applying enzymatic method also known as biodeinking process, can prevent environmental dam- ages from conventional deinking.
Enzymatic deinking process provides some advan- tages, such as the requirement of neutral pH suspen- sion, reduction in chemical consumption and cost of
energy, as well as the improvement in fiber recycling process.[5] Enzymes can be obtained from bacterial isolates or cellulolytic fungi, which are commonly found in rice husks. Rice husks are one of the most abundant biomaterial waste from rice processing stage (600 million tonnes annually generated from rice-producing nations globally) in many countries.[6]
Although, rice husks are not edible, they can be used as bedding in animal cages, additives for poultry feed, and fertilizers in soil. The utilization of rice husks as the source of cellulase is a potential alternative to commercial enzymes synthesized by industries, which can reduce the inhibitory effect during fermentation process due to the presence of preservatives and sta- bilizers used in commercial enzyme industries.
Furthermore, the cost of production can be reduced by utilizing waste as the source of cellulase.[7,8]
The isolation of cellulolytic fungi from rice husks to produce cellulase for biodeinking process had been reported by Roushdy et al., where the use of cellulase
© 2023 taylor & francis group, llC
CONTACT saharman gea [email protected] Cellulosic and functional materials research Centre, universitas sumatera utara, Jl. Bioteknologi no.
1, medan 20155, indonesia
supplemental data for this article can be accessed online at https://doi.org/10.1080/02773813.2023.2190594.
https://doi.org/10.1080/02773813.2023.2190594
KEYWORDS
Cellulases; enzymatic deinking;
paper recycling; ultrasound pretreatment
in ink removal from printed papers provided excep- tional results, even better for industrial use.[9] However, the only drawback was longer deinking process dura- tion that took around 9 h. Thus, to reduce the deink- ing time, pretreatment such as ultrasound could be applied to reduce the size of ink particles and allowed these particles to be deinked more effectively from the fibers.[10] The ultrasound approach aimed to increase enzymatic modification via cavitation effect.
These changes appeared to have improved enzymes accessibility into the fibers, as well as hastened fibril- lation.[11] Some attempts of ultrasound pretreatment have been implemented to recycle waste paper.[12,13]
Virk et al. reported the use of xylanase and laccase enzymes together with sonication pretreatment led to swelling effect on the fibers that increased the contact between the fibers and enzymes on the surface area.[14]
Nonetheless, there are only a few reports about the combination of ultrasonication pretreatment and enzy- matic deinking. This study was carried out to inves- tigate deinking process of paper waste by using cellulases from rice husks and followed by ultrason- ication pretreatment at different lengths of time.
Materials and method Materials
Rice husks as the main source of bacterial isolates were supplied from local farms in Martubung area, Medan Labuhan sub-district, North Sumatera, Indonesia. Wastepaper for deinking process was a commercial A4 sized paper (Paper-One 70 gr) with both sides of paper printed. All the chemical reagents (NaCl, CMC, Yeast extract, Peptone, K2HPO4, MgSO4, Agar, (NH4)2SO4, and di-sodium hydrogen phosphate/
potassium dihydrogen phosphate 0.05 M used as a buffer solution pH 7, cellulase, glucose, ethanol, car- bon black, and distilled water) used were analytical grades, and all of them were purchased from Sigma-Aldrich.
Cellulolytic bacteria isolation and purification The dilution and spread plate techniques were used to isolate cellulose-degrading bacteria. Cellulolytic bacteria were isolated by immersing rice husks in 0.85% NaCl solution (1:10 w/v ratio), as described in a previous study.[15] After incubating for 14 days, the mixture was diluted to 106 times with 0.85% NaCl.
Bacterial isolates were grown in CYPE-agar media with composition of 1% CMC, 0.5% yeast extract, peptone, and NaCl, 0.1% K2HPO4, 0.02% MgSO4, and
2% agar (% w/v) at pH 7.[15] These samples were sterilized in an autoclave at 121 °C for 20 min. Bacterial isolates were grown in sterile condition and observed for a week before morphological analysis.
After 7 days of observation, the isolated bacteria were transferred into a new CYPE-agar medium for purification. This isolation procedure was carried out at room temperature for 24 h. The newly isolated bac- teria were used to determine the cellulolytic index and biochemical measurements, which were then plot- ted to show the bacteria’s growth curve.[15] Equation (1) below was used to measure cellulolytic index:
Cellulolyticindex diameter of transparent zones diameter of colo
= nnies (1)
Cellulase production
The production of cellulase was firstly done by select- ing certain amounts of purified bacterial isolates that have been rejuvenated into a new CYPE-agar media at incubated room for 24 h. After incubation, around 10 mL of bacterial suspences were set up by using sterilized distilled water, in which the optical density (OD) value reached 0.5 from UV–vis spectrophotom- eter measurement at λ = 600 nm. Then, the bacterial suspences were inoculated in 2% liquid-CYPE media to obtain bacterial cultures. Next, they were incubated in room temperature at 100 rpm of shaking for certain times according to the optimum time of bacterial growth. After that, the bacterial cultures were centrif- ugated at 10,000 rpm for 10 minutes at 4 °C, and con- tinuously decantated in cold condition. Finally, the crude enzymes were obtained and stored in cooling storage system at 4 °C until being used. The crude enzymes were collected to determine the characteristic of enzyme activity by using Nelson Somogy method (Equation (2)):
Enzymaticactivities U ml glucoseconcentration ppm
gluc
oosemolecular weight minute
1000 1
T (2)
Cellulosic purification
The purification of crude enzymes obtained with pre- cipitation method. First, crude enzyme samples were kept cold for 24 h before being centrifuged at 10,000 rpm at 4 °C for 20 min. The precipitates were
then collected and dialyzed using membranes sized 12–14 KDa. The dialysis was performed with a buffer solution (pH 7) that was replaced at least three times.
Finally, enzymatic activities were determined.
Wastepaper pulp preparation
Wastepaper pulp samples for deinking process were prepared by following a previous study[16] with a bit of modification. First, paper samples were cut in 2–3 cm in dimension. Then, 7 g of cut papers were soaked in 300 ml buffer solution (pH 7) for 24 h.
Then, paper samples were crushed into pulp by using a mechanical stirrer at 2600 rpm for 5 min to reach 2.3% consistency.
Deinking procedure
The deinking process was carried out by adding 2%
of cellulase (compared to initial mass of paper pieces which is 7 g) to paper pulp samples in an Erlenmeyer.
This process was carried out in an incubated shaker at 30 °C under constant shaking (150 rpm) for an hour.
After that, the Erlenmeyer was placed into boiling water for 15 min to deactivate the enzymatic reaction.
Next, the mixture was filtered and the filtrate was characterized to measure ink content (carbon black) removal by using UV–vis spectrophotometer. In order to investigate the effect of enzymatic reaction, deink- ing process conducted using ultrasonication pretreat- ment was carried out (37 kHz of frequency) for 15, 30 and 45 min. Meanwhile, as comparisons, paper pulp sample without biodeinking process and ultra- sound, as well as deinked sample with enzymes with- out ultrasound were also prepared.
Paper sheet fabrication
Deinked paper pulp samples under various processes were washed prior to molding them into paper sheets.
Paper mold (size 150 T) was placed in a container that had been filled with water to cover the surface area.
Next, the mold was performed in an open air under direct sun-ray. After drying, paper samples were removed from the mold and cut to A4 sizes. Next, paper sheets were pressed by using heating treatment (100 °C) for one minute and the characterizations were carried out.
Paper characterizations
Paper sheet samples were analyzed to observe their characteristics in accordance to certain standards, such
as brightness level (SNI ISO 2470:2014), stain index (TAPPI T 213), grammature (SNI ISO 536:2010), ten- sile strength (SNI ISO 1924-2:2010), color test by colorimeter (Amtast AMT 507), morphological anal- ysis by using scanning electron microscopy (SEM), crystallinity index by X-ray diffraction (XRD), and thermal analysis by thermogravimetric analysis (TGA).
The crystallinity index was measured by the fol- lowing Equation (3):
Xc
I I I
002 002
am 100% (3)
where: I002 is the 2θ intensity at 22° (crystalline phase) and Iam is the 2θ baseline intensity at 16° (amor- phous phase)
Results and discussion
Isolated and purified cellulolytic bacteria
There were seven different bacteria species obtained from rice husks. Purified bacterial isolates were mea- sured on their abilities to produce cellulolytic enzymes by using Congo red indicators. The clear zone in CMC agar was observed to determine the isolate’s ability to degrade cellulose. Cellulolytic activity was determined by growing the isolated bacteria on CMC agar medium in a point method. Inoculants were incubated at room temperature for 5 days. Following that, the plate was stained with 1% Congo red and washed with 1 M NaCl for 15 min.[17] Isolates with cellulolytic activity showed a distinct zone on the media. The ability to degrade cellulose was calculated using the clear zone diameter to colony diameter ratio.[18] The cellulolytic bacterial isolates are depicted in Figure 1a, b.
From seven purified isolates obtained, SP3 and SP6 bacterial colonies had demonstrated quick growth, where they have almost covered all the surface area of the media. The assessment of cellulolytic enzyme production ability by congo red indicator showed 3 species of bacteria with the ability to produce enzymes, indicated from the presence of clear zones within the surrounding bacterial colonies, such as bacterial iso- late SP1, SP3, and SP6. The displays the cellulolytic index, which is the ability of bacterial isolates to pro- duce cellulose (Figure 2, supplementary material).
Only SP1, SP3, and SP6 bacterial isolates proceeded to the next stages of research.
Cellulolytic index was measured from the ratio between clear zone diameters and bacterial colony diameters. SP3 bacteria had the highest index level
at 1.97, with 11.8 mm clear zone. Meanwhile, bacterial isolate SP6 and SP1 had 1.78 and 1.23 index level, respectively.
These three bacterial isolates, with the potential abilities to produce cellulase, were tested to deter- mine their metabolic activities via biochemical test- ing, such as starch hydrolysis, gelatin hydrolysis, citrate test, hydrogen sulfide test, motility test, and catalase test. The results of biochemical tests for SP1, SP3, and SP6 bacterial isolates (Table 1, Supplementary material).
SP1 and SP3 bacterial isolates had the ability to hydrolize starch, whereas SP6 showed no such ability.
This means that SP1 and SP3 colonies could produce amylase enzymes. There were three tests which showed negative biochemical tests, such as gelatin hydrolysis, citrate, and catalase tests. A negative result in gelatin hydrolysis into amino acid implied the isolates’ inability to produce gelatinase enzymes.
Whereas in citrate test, negative results indicated that the bacteria have no ability to metabolize citrate as the source of carbon for energy harvesting.
Furthermore, negative catalase test results indicated that the bacterial isolates have no catalase enzymes to convert hydrogen peroxide into water and oxygen.
Meanwhile, hydrogen sulfide tests were performed in Slant–Butt media, where red color in Slant and Butt media indicated no fermentation of sugar and for- mation of gas of hydrogen sulfide (H2S) during fer- mentation, yellow color indicated the presence of glucose. SP1 bacterial isolates showed no sugar fer- mentation and gas formation, whereas SP3 and SP6 bacterial isolates had formed glucose. As for motility test, one positive result which indicates the ability of bacterial isolates movements/motile via the growth in the area of ose was observed in SP6 isolates. Shape observation of the medium inoculated by bacterial culture, such as the puncture area, showed that the bacterial isolates may have simple motion system like flagella or cilia.
Morphological characteristic of cellulolytic bacteria
Morphological characteristics of SP1, SP3, and SP 6 isolates were carried out by observing single colonies of each bacterium. The following Table 2 showed the observation results of the bacterial colonies.
The colonial shapes of SP1 and SP3 isolates were circular, while SP6 was irregular. They had different edges, such undulate, entire, and lobate for SP1, SP3, and SP6, respectively. All isolates has the same flat elevation. As for colors, SP1 was yellow, while SP3 and SP6 were white.
Figure 1. (a) the growth of bacterial isolates from rice husks in CyPe-agar media; (b) seven various of purified bacterial isolate species based on their single colony with naming system sP1 to sp7.
Figure 2. Cellulolytic index of bacterial isolates from rice husks.
Bacterial isolate growth curves
The growth curve was plotted to investigate growth phases of the isolates, so optimum production of cel- lulase could be achieved. Data collection was done for every 4 h. The following Figure 3 shows the growth curve of three selected bacterial isolates of SP1, SP3, and SP6.
The increase in enzyme production was related to the increase in cell growth, where cellulose was used with regard to time, the isolates were exponentially growing for more than 48 h. SP1 had optimum growth for 56 h, whereas the SP6 demonstrated optimum growth for 60 h and SP7 had 56 h for optimum growth. After the exponential phase has passed, stationary phase con- tinued and eventually ended into death phase. SP1 and SP6 isolates, however, had increases in absorbance values after the stationary phase, which may have implied some contamination in liquid media. During the study, bac- terial cultures had to be repeatedly exposed, so that higher chance of contamination may have occurred.
Cellulase production from cellulolytic bacteria Bacterial isolate selected to proceed to the next stage of study was based on the cellulolytic index, which was SP3 bacteria with the highest cellulolytic index.
SP3 bacteria was grown in CYPE-liquid media and incubated accordingly to the optimum growth time, which is 56 h, to produce crude enzyme. Cellulase production was determined from the amount of glu- cose successfully produced from cellulose hydrolysis by the enzymes.
Cellulose hydrolysis by enzymes was carried out under three variations of phosphate buffer with pH 6, 7, and 8. The relationship between optimized pH and cellulase activities, in which pH levels
significantly affected enzyme activities (Figure 4, Supplementary material). Enzymes effectively operated in neutral pH. However, at higher or lower pH, enzyme activities were at higher level. The study con- ducted by Prasad reported cellulase from Streptomyces griseorubens with the highest activities at pH 7.[19] Lee also reported that optimum results of cellulase activ- ities, isolated from soil microorganisms, were observed in pH 7.[20] The highest absorbance among the crude enzymes was found in 0.0632 U/mL in phosphate buf- fer pH 7. However, in regard of different buffer (glu- cose solution), lower amount of glucose reduction was resulted from the degradation of cellulose by cellulase.
This result may be caused by cold storage storing for 2 weeks before use. The crude enzymes were suspected to decreased in activities.
Cellulase purification
During crude enzyme purification process, the use of 60% ammonium sulfate led to precipitation with more frequent buffer solution replacement (4 times) within 24 h of dialysis. This condition aimed to purify the enzymes from contaminants. The dialysis process used higher concentration of sulfate compounds in the outer system, causing the buffer solution to enter into the membrane. Purified enzymes were obtained after equilibrium condition was achieved, indicated by the exit of salts. Next, the enzymes were analyzed to mea- sure the enzymatic activities (Table 2, Supplementary material).
Ink (carbon black) content analysis
In the deinking process, carbon black acted as ink pigment indicator that needed to be removed from Table 1. Biochemical tests for sP1, sP2, and sP6.
no. Biochemical tests
isolates
sP1 sP3 sP6
1. starch hydrolysis + + −
2. gelatin hydrolysis − − −
3. Citrate test − − −
4. hydrogen sulfide tests
slant red yellow yellow
Butt red red red
5. motility test − − +
6. Catalase test − − −
Table 2. enzymatic activities during isolation and purification stages.
no. isolation and purification stages glucose reduction (ppm) enzymatic activities (u/ml)
1. Crude enzymes 8.83 1.64
2. Precipitation (nh4)2so4 10.22 1.89
3. dialysis 9.63 1.78
the paper pulp. Ink content was measured by using UV–vis spectrophotometer. The UV-spectra of the filtrate paper pulp samples with different treatment are summarized in Figure 5 in supplementary material.
The concentration of carbon black deinked from pulp filtrate samples with different treatment (Table 3, Supplementary material). Samples with no treat- ment only had 1286 ppm ink removed. Meanwhile, pulp paper samples with 2% of cellulase treatment showed 1761 ppm ink removed. An important contri- bution of cellulase was also attacking the irregular thin fibrils on the surface and caused swelling con- dition, loosened the fibers, and removed ink particles.[21]
Sample with and ultrasonication pretreatment for 15 min and 2% cellulase showed the highest amount of ink removed (2310 ppm). Ultrasound had improved the deinking process, particularly by modifying the fibrils. At the beginning stage of ultrasound treatment, Figure 3. the growth curve of sP1, sP3, and sP6 bacterial isolates.
Figure 4. Cellulase activities in optimized ph.
Figure 5. uV-spectra of paper pulp samples; (a) ultrasonication with various treatments; and (b) ultrasonication without adding cellulase (control).
Table 3. Carbon black content in pulp filtrates with various treatment.
no samples Carbon black content
in the filtrate (ppm)
1 no treatment 1286
2 2% cellulase 1761
3 15 min ultrasound + 2% cellulase 2310 4 30 min ultrasound + 2% cellulase 1846 5 45 min ultrasound + 2% cellulase 1599
6 ultrasound 1032
the mechanical force from the ultrasound vibration allowed the fibrillation on the surface of cellulosic fibers and assisted the removal of ink particles. This is supported by the research of Anne et al., who have studied the ultrasound treatment of deinking on paper for 5–30 min resulting in the effect of time, namely the longer the ultrasound treatment given, the higher the ink removal on the paper.[22] However, longer ultrasound treatment damaged the surface of fibrils in samples due to the degradation of cellulosic struc- ture.[13] In this study, the ultrasonication pretreatment for 30 and 45 minutes damaged cellulosic structure in samples. The amount of ink removed decreased in samples that received ultrasound for longer than 15 min.
Morphological analysis
Morphological analysis was carried out by SEM to paper samples pre- and post biodeinking and ultra- sound treatment. The morphological analysis is dis- played in the following Figure 6.
Figure 6a shows the morphology of paper surface without enzyme and ultrasound treatment. This sam- ple had ink particles and contaminants on the surface of fibers despite no indication of them from UV–vis spectrophotometer analysis. Significant differences could be observed in samples with enzymatic treat- ments (Figure 6b, f), where no ink particles and con- taminants observed. Figure 6c–e are the morphological
images of paper samples with biodeinking (cellulase) and ultrasound treatment for 15, 30, and 45 min, respectively. The fibers of the sample with 15 min ultrasound and cellulase treatment had smoother sur- face compared to samples without treatment. There were no significant differences among the three sam- ples with ultrasonication treatment for different time length. Darjana et al. reported that continued soni- cation time from 20 to 30 min resulted in lower max- imum adsorption capacities for cellulase, which became an ineffective way to degrade substrates as well as no relation to the enzymatic adsorption.[23]
Ultrasound affected reactions with cavitation con- dition. In the pretreatment of lignocellulosic sub- stances, ultrasound significantly allowed the opening of surface area in solid substrate. During ultrasoni- cation, cavitation appeared due to physical and chem- ical forces, which provided an acceptable condition for enzymes to perform enzymatic hydrolysis.[23]
Enzymatic hydrolysis was indicated by the presence of smooth fibers on the surface. Jiang et al. found that xylanase treatment accompanied with ultrasoni- cation increased fibrillation on fibers based on their observation in morphological structures.[11] Increase in fibrillation implied the presence of delignifica- tion.[24] Meanwhile cellulose treatment made two changes in the pulp fibers, such as internal structure modification and rough surface condition.[25] All these changes occurred on the surface area improve deink- ing process where trapped ink was removed.[26]
Figure 6. the morphological photographic images of paper samples with 5K magnification (a) sample without treatment, (b) sample with 2% cellulase, (c) sample with 15 min ultrasound + 2% cellulase, (d) sample 30 min ultrasound + 2% cellulase, (e) sample 45 min ultrasound + 2% cellulase, and (f) ultrasound without adding cellulase (control).
Brightness degree analysis
One of the most important factors in paper character- istics is the brightness degree that indicated successful deinking process on recycled paper. Sample with no treatment showed the lowest degree of brightness (54.47%). The highest degree of brightness, 58.74%, was found in sample with 2% cellulase treatment. Whereas samples with ultrasonication treatment at various time length had brightness degree between 55% and 57%
(Table 4, Supplementary material). Higher brightness level was suggested as the effect of enzymatic reaction to reduce the amount of remaining ink.[27] Meanwhile, ultrasonication pretreatment for 15 min appeared to be most optimum treatment and consistent with previous research in which a longer treatment time with ultra- sound resulted in a higher brightness level.[22]
Ultrasound played the same role as phase transfer catalyst (PTC), which supported the reaction between solid (pulp) and liquid (enzymes) phases. Ultrasonic system was assumed to accelerate heterogenic reac- tions, where sample characteristics were altered in terms of the occurrence of cavitation from greater interaction between solid and liquid interfacial on the surface area. This small gap allowed cavitation into non-spherical shape, forced ink to the surface, and caused damages on the surface, which led to frag- mentation of materials, in this case was ink from recycled paper.[12]
Stain index analysis
Stain is considered as alien constituents attached on the surface of paper sheets. The measurement of stain
is determined on black areas within 0.04 mm2 in min- imum area. The amount of stains on recycled paper represented the cleanse level of the fibers, which implied no interaction among the fibers.[28] In Table 5, samples with 2% cellulase, ultrasound and 2% cel- lulase + 15 min ultrasound had no stain detected.
However, samples with 2% cellulase + 30 and 45 min of ultrasound had stain index of 2.7 mm2/m2 and 4.0 mm2/m2, respectively.
Tensile and grammature analysis
Tensile strength in paper is a mechanical characteristic that determines the maximum stress in a piece of paper.[2] There was a decrease in tensile strength in samples with cellulase and ultrasound treatment. From Table 4, sample with no treatment and ultrasound treat- ment had tensile strength of 0.60 and 0.33 kN/m, respec- tively. Whereas samples with cellulase treatment had 0.49 kN/m tensile strength. Meanwhile, samples with enzymes and ultrasound treatment experienced decreases in tensile strength, where the lowest value was obtained from samples with 2% cellulase + 45 min ultrasound.
Deinking process that involved enzymatic deinking has been reported to result in positive and negative impacts on the physical characteristics of samples, which differed accordingly based on the types of enzymes, paper, and the percentages of enzymes as well as time interaction between enzymes and subtrates.[29]
In this study, the use of 2% cellulase as well as ultrasonic pretreatment decreased the tensile strength in paper samples. Unlike the study conducted by Gea et al. that combined ultrasound and chemical treat- ments, where longer ultrasound at 90 min had increased the mechanical properties of cellulosic com- posites compared to samples with 30 and 60 minutes of ultrasound treatment.[30] Similarly, Manfredi et al.
also reported that ultrasonication treatment led to the improvement in tensile strength of paper sample;[31]
the only difference that can be found in both studies was in alkali conditions, while in this study the con- dition was at neutral pH.
Lee et al. used Penicillum rolfsii c3-2(1) IBRL microorganisms to remove ink in recycled paper.
Table 4. Characteristic of paper samples with various treatment.
no. samples
Parameter
grammature (g/m2) Brightness (%) stain index (mm2/m2) tensile strength (kn/m)
1. no treatment 73.0 54.57 0.1 0.60
2. 2% cellulase 64.0 58.74 0.0 0.49
3. 15 min ultrasound + 2% cellulase 62.8 57.17 0.0 0.31
4. 30 min ultrasound + 2% cellulase 64.6 55.61 2.7 0.31
5. 45 min ultrasound + 2% cellulase 60.3 56.30 4.0 0.23
6. ultrasound 63.6 56.15 0.0 0.33
Table 5. Color results of paper sample with various treatment.
no. sample L* a* b*
1. untreated 79.95 2.65 0.66
2. 2% cellulase 80.95 2.04 −1.14
3. 15 min ultrasound
+ 2% cellulase 81.65 2.52 2.19
4. 30 min ultrasound
+ 2% cellulase 82.77 3.03 2.90
5. 45 min ultrasound
+ 2% cellulase 80.27 2.39 2.21
6. ultrasound 98.50 −1.30 9.6
Crude enzymes were reported to be able to decrease paper mechanical properties as the main compositions of paper were cellulosic. The decrease in tensile strength was related to the degradation of cellulose by cellulase during the deinking process.[1] Table 2 shows the enzymatic activities in hydrolyzing cellulose.
On the other hand, ultrasound vibration is assumed to damage mechanical properties of the paper samples due to the attacks on cellulosic structure.
Another observable characteristic was the gramma- ture value of paper samples displayed in Table 4. All paper samples were fabricated with the same compo- sitions; however, grammature levels were different for each sample. Sample without any treatment given showed grammature level of 73 g/m2. However, after biodeinking process with 2% cellulase and ultrasoni- cation pretreatment, grammature levels decreased to as low as 60.3 g/m2 in samples with 2% cellulase + 45 min ultrasound treatment. The reduction was assumed to be removed during the deinking process that took place in different containers. Moreover, the ultrasonication treatment decreased the ink particle s ize[32] and led to mass reduction during paper fabrication. Different grammature levels were also presumed to have effects in the decrease in tensile strength.
Color analysis
Color analysis quantitatively identified different colors on paper samples. This analysis was required to stan- dardize the samples. CIE L* a* b* color test is gen- erally used to describe colors. L was related to brightness, in which equals to 100 indicated white and 0 means black color. While a* value showed red to green (high to low) color scale and b* value showed color gradient from blue to yellow (low to high).[33]
The following Figure 7 shows the color measurement of paper samples.
The L* level became slightly higher after the treat- ment with both ultrasonication and 2% cellulose (Table 5, Supplementary material). This indicated that the treatment resulted in better brightness level.
However, red color level (indicated by a*) has changed from 2.65 in untreated sample to −1.30 and 2.04 in sample with ultrasound and 2% cellulase. This showed a lower level of red color on the paper.[34] The treat- ment with 2% cellulase was able to reduce red color on paper, meanwhile b* level also reduced to −1.14, from 0.66. This treatment had the lowest a* and b*
value among all samples. The use of 2% of cellulase on paper samples tend to yield on blue color.
A combination of 15 minutes ultrasound and 2% of cellulose resulted in sample with L* 81.65, a* 2.52, and b* 2.19. The highest L* level was obtained in sample with ultrasound treatment. At 45 minutes of ultrasound, the sample had lower values of all parameters compared to level all parameter compared to the 30 min of treat- ment. This showed that ultrasound changed yellow and red colors in paper samples, and this may occur due to the ultrasonication which is a vibrational energy.[35]
The above data suggested the highest L* level is 98.50 which was found in sample with ultrasound treatment, and for the samples with 45 min of treatment, the L*
level was lower which nearly reached to level of sample without ultrasonic treatment. Thus, it suggested that, ultrasonic treatment with more than 30 min is ineffec- tive in the improvement of paper brightness property.
Thermal analysis
Based on TGA curve of the paper samples (Figure 8), all five samples had almost similar degradation steps.
Figure 7. Color measurement of paper samples with various treatment.
In the beginning, dehydration process occurred at 100 °C, where mass reduction was less than 10%. Next mass reduction appeared at between 300 and 400 °C.
Stevulova et al. reported second step of mass reduction of cellulosic fibers appeared at 206–400 °C and the max- imum mass reduction may have taken place within this range.[36] Whilst, Lengowski et al. reported that the first degradation phase of cellulose pulp occurred at 275 °C.[37]
T50 and T70 levels which are the mass reduction temperature to 50% and 70%, respectively (Table 6, Supplementary material). The untreated sample had mass reduction by half at 394 °C, whereas sample with ultrasound treatment and 2% cellulase experienced 50% loss of mass at 363.29 and 389 °C. In previous research, the presence of cellulose during the enzy- matic treatment typically results in an increase in the residue percentage. The addition of 2% cellulase improved the thermal stability of the newspaper pulp with value of 22.36%. While pulp from a conventional method has reduced residual value and obtain a least residual amount of 5.48%.[16]
On the other hand, samples with ultrasound treat- ment had decline in T50 temperature at 363.29. At 15 and 30 min of ultrasonication had increased in T50 was at 394 °C and 398 °C respectively. The residual percentage in sample with 45 min which was around 12.8% has indicated the higher time of ultrasonication may reduce the thermal properties of paper samples.
The temperature for mass loss at 70% was initiated at approximately 400 °C. Paper sample that degraded to 70% and had the least residual amount (11.4%) was sample with 2% cellulase treatment. Sample with ultrasound treatment had the residual amount (15.4%).
Sample with 30 minutes of ultrasonication had the highest amount of residual (20.6%) which was almost the same percentage as the untreated sample. This
implied that biodeinking with 2% of cellulase and 30 minutes of ultrasonication of treatment provided better effect on thermal characteristic. However, in sample with 45 min of ultrasonication, the residual amount appears to be the highest among them, which was 12.8%, indicating poor thermal resistancy.
Crystallinity analysis
The crystallinity of cellulose was related to physical and chemical characteristics, as the crystallinity index defined the relative density and stability.[13] Figure 9. is the X-ray diffractogram of paper samples. The crystal- line phase of cellulose was confirmed at 2θ in between 22° and 23°, whereas the amorphous phase appeared at 2θ around 15° and 16°. The addition of 2% cellulase and ultrasound treatment influenced the degree of crys- tallinity of the samples obtained. According to Wu et al., the enzymatic hydrolysis process destroyed a small frac- tion of the crystallized cellulose.[38]
The untreated sample had 62.5% crystallinity index.
After being treated with 2% of cellulase, the crystallinity index became higher at 65.6% (Table 7, Supplementary material). The study by Gea et al. reported an increase in crystallinity index with the addition of cellulase.[16] For sample with 15 min of ultrasonication, the crystal- linity index was the highest at 70.9%. Apparently, ultra- sound, 30 and 45 min ultrasonication samples showed lower index values which were almost similar to the Figure 8. thermogram curves of paper samples with various treatment.
Table 6. t50, t70 and residual percentage data of paper samples with various treatment.
no. samples t50 (°C) t70 (°C) % residual at 600 °C
1. untreated 394.32 417.01 21.0
2. 2% Cellulase 389.13 402.65 11.4
3. 15 min ultrasound + 2% cellulase 394.32 409.96 17.8
4. 30 min ultrasound + 2% cellulase 398.05 414.54 20.6
5. 45 min ultrasound + 2% cellulase 393.79 408.97 12.8
6. ultrasound 363.29 374.95 15.4
Table 7. Crystallinity index of paper samples with various treatment.
no. sample Crystallinity index (%)
1. no treatment 62.5
2. 2% cellulase 65.6
3. 15 min ultrasound + 2% cellulase 70.9 4. 30 min ultrasound + 2% cellulase 61.3 5. 45 min ultrasound + 2% cellulase 62.6
6. ultrasound 68.3
untreated sample, such as 68.3%, 61.3%, and 62.6%
respectively. As reported by Xu et al., the crystallinity of fibers increased at the first 10 minutes of pretreatment and then it would reduce with the increase in pretreat- ment time due to the cavitation effects and several chemicals used.[39] In this study, the combination of ultrasound and cellulose enzyme showed similar results.
The presence of cavitation effects allowed several holes to emerge on the surface of fibers, which may result in crystalline damages in the intact part of crystalline zones.
Thus, the crystallinity index was reduced.[39]
Conclusion
Rice husks were used as the source of cellulolytic bac- terial isolates, where SP3 sample had the best perfor- mance in producing cellulase. The cellulase ability within 56 h with pH 7 at room temperature was able to hydrolyze cellulose with 1.78 U/mL enzyme activity.
Sample with 2% cellulase and 15 min of ultrasonication pretreatment resulted in the highest ink removal (2310) ppm during biodeinking process. Furthermore, this paper sample had an exceptional brightness level due to the reduction of stain residual as well as higher crystalline parts and thermal properties. Ultrasound pretreatment that lasted longer than 15 min had reverse effects and decreased paper quality particularly in grammature and tensile strength, whereas with 2% of cellulase and ultrasonication pretreatment had decreased the grammature level as well as tensile strength.
Acknowledgments
The authors would like to thank the rector of Universitas Sumatera Utara, Medan for the research fund via Talenta Program 2019 Universitas Sumatera Utara with given con- tract No. 4167/UN5.1.R/PPM/2019.
Reference
1. Lee, K. C.; Tong, W. Y.; Ibrahim, D.; Arai, T.; Murata, Y.; Mori, Y.; Kosugi, A. Evaluation of Enzymatic Deinking of Non-Impact Ink Laser-Printed Paper Using Crude Enzyme from Penicillium rolfsii C3-2(1) IBRL. Appl. Biochem. Biotechnol. 2017, 181, 451–463.
DOI: 10.1007/s12010-016-2223-4.
2. Rahmah, S. A.; Aisya, N.; Fithri, L.; Pratama, A.;
Darmokoesoema, H.; Rohman, A.; Puspaningsih, N. N.
T. Exploration and Analysis of a Lignocellulase Consortium for Deinking of Recycled Paper in the Eco-Paper Industry.
J. Chem. Technol. Metall. 2020, 55, 389–396.
3. Shankar, S.; Shikha; Bhan, C.; Chandra, R.; Tyagi, S.
Laccase Based De-Inking of Mixed Office Waste and Evaluation of Its Impact on Physico-Optical Properties of Recycled Fiber. Environ. Sustain. 2018, 1, 233–244.
DOI: 10.1007/s42398-018-0021-3.
4. Kamali, M.; Khodaparast, Z. Review on Recent Developments on Pulp and Paper Mill Wastewater Treatment. Ecotoxicol. Environ. Saf. 2015, 114, 326–
342. DOI: 10.1016/j.ecoenv.2014.05.005.
5. Lasheva, V.; Todorova, D.; Kotlarova, S.; Kamburov, M.
Deinking of Waste Offset Printed Paper by the Use of Enzymes. Sci. Proc. Int. Sci. Conf. High Technol.
Business Soc. 2016, I, 40–42.
6. Onwuka, F. O.; Orji, F. A.; Uzeh, R. E.; Ugoji, E. O.
Properties of Bacterial Cellulases and Secondary Metabolites of Cellulolytic Bacterial Fermentation of Rice Husks. Asian J. Biotechnol. Bioresour. Technol.
2017, 2, 1–11. DOI: 10.9734/AJB2T/2017/35196.
7. Oliveros, D. F.; Guarnizo, N.; Perea, E. M.; Arango, W.
M. Cellulase Activity of Filamentous Fungi Induced by Rice Husk. Afr. J. Biotechnol. 2014, 13, 4236–4245.
DOI: 10..5897/A.
8. Zheng, G. J.; Zhou, Y. J.; Zhang, J.; Cheng, K. K.; Zhao, X. B.; Zhang, T.; Liu, D. H. Pretreatment of Rice Hulls for Cellulase Production by Solid Substrate Fermentation. J. Wood Chem. Technol. 2007, 27, 65–71.
DOI: 10.1080/02773810701486675.
9. Roushdy, M. M. Biodeinking of Photocopier Waste Paper Effluent by Fungal Cellulase under Solid State Fermentation. J. Adv. Biol. Biotechnol. 2015, 2, 190–
199. DOI: 10.9734/JABB/2015/15378.
10. Tatsumi, D.; Higashihara, T.; Kawamura, S.; Matsumoto, T. Ultrasonic Treatment to Improve the Quality of Recycled Pulp Fiber. Japan Wood Res. Soc. 2000, 46, 405–409. DOI: 10.1007/BF00776405.
11. Jiang, Q.; Yang, G.; Wang, Q.; Sun, Q.; Lucia, L. A.;
Chen, J. Ultrasound-Assisted Xylanase Treatment of Chemi-Mechanical Poplar Pulp. BioResources 2016, 11, 4104–4112. DOI: 10.15376/biores.11.2.4104-4112.
12. Xing, M.; Yao, S.; Zhou, S. K.; Zhao, Q.; Lin, J. H.; Pu, J. W. The Influence of Ultrasonic Treatment on the Bleaching of CMP Revealed by Surface and Chemical Structural Analyses. BioResources 2010, 5(3), 1353–1365.
13. Guo, X.; Jiang, Z.; Li, H.; Li, W. Production of Recycled Cellulose Fibers from Waste Paper via Ultrasonic Wave Processing. J. Appl. Polym. Sci. 2015, 132, 1–9. DOI:
10.1002/app.41962.
14. Virk, A. P.; Puri, M.; Gupta, V.; Capalash, N.; Sharma, P. Combined Enzymatic and Physical Deinking Methodology for Efficient Eco-Friendly Recycling of Figure 9. X-ray diffractogram of paper samples with various
treatment.
Old Newsprint. PLoS One 2013, 8, e72346. DOI:
10.1371/journal.pone.0072346.
15. Jannah, A.; Aulanniam, Ardyati.; T.; Suharjono.
Isolation, Cellulase Activity Test and Molecular Identification of Selected Cellulolytic Bacteria Indigenous Rice Bran. Indones. J. Chem. 2018, 18, 514–521. DOI: 10.22146/ijc.26783.
16. Gea, S.; Oktari, N.; Andriayani, A.; Rahayu, S.; Piliang, A. F. Comparative Optimization of Cellulase and Laccase Enzymes in Deinking Process of Used Newspapers. J. Kim. Sains Dan Appl. 2020, 23, 353–
359. DOI: 10.14710/jksa.23.10.353-359.
17. Teather, R. M.; Wood, P. J. Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rumen. Appl. Environ. Microbiol. 1982, 43, 777–780. DOI: 10.1128/aem.43.4.777-780.1982.
18. Dewiyanti, I.; Darmawi, D.; Muchlisin, Z. A.; Helmi, T.
Z.; Arisa, I. I.; Rahmiati, R.; Destri, E. Cellulase Enzyme Activity of the Bacteria Isolated from Mangrove Ecosystem in Aceh Besar and Banda Aceh.
IOP Conf. Series EarthEnviron. Sci. 2022, 951, 012113.
DOI: 10.1088/1755-1315/951/1/012113.
19. Prasad, P.; Singh, T.; Bedi, S. Characterization of the Cellulolytic Enzyme Produced by Streptomyces gris- eorubens (Accession No. AB184139) Isolated from Indian Soil. J. King Saud Univ. - Sci 2013, 25, 245–250.
DOI: 10.1016/j.jksus.2013.03.003.
20. Lee, Y. J.; Kim, B. K.; Lee, B. H.; Jo, K. I.; Lee, N. K.;
Chung, C. H.; Lee, Y. C.; Lee, J. W. Purification and Characterization of Cellulase Produced by Bacillus Amyoliquefaciens DL-3 Utilizing Rice Hull. Bioresour.
Technol. 2008, 99, 378–386. DOI: 10.1016/j.
biortech.2006.12.013.
21. Akbarpour, I.; Ghasemian, A.; Resalati, H.; Saraeian, A.
Biodeinking of Mixed ONP and OMG Waste Papers with Cellulase. Cellulose 2018, 25, 1265–1280. DOI:
10.1007/s10570-017-1641-y.
22. Gaquere-Parker, A. C.; Ahmed, A.; Isola, T.; Marong, B.; Shacklady, C.; Tchoua, P. Temperature Effect on an Ultrasound-Assisted Paper de-Inking Process. Ultrason.
Sonochem. 2009, 16, 698–703. DOI: 10.1016/j.ult- sonch.2009.01.004.
23. Darjana, Z.; Ivetic; Omorjan, R. P.; Dordevic, T. R.; Antov, M. G. The Impact of Ultrasound Pretreatment on the Enzymatic Hydrolysis of Cellulose from Sugar Beet Shreds:
Modeling of the Experimental Results. Environ. Prog.
Sustain. Energy 2017, 36, 1164–1172. DOI: 10.1002/ep.
24. Xu, Q. H.; Wang, Y. P.; Qin, M. H.; Fu, Y. J.; Li, Z.
Q.; Zhang, F. S.; Li, J. H. Surface Characterizatition of Old Newsprint Pulp Deinking by Combining Hemicellulace with Laccase Mediator System. Bioresour.
Technol. 2011, 102, 6536–6540. DOI: 10.1016/j.
biortech.2011.03.051.
25. Efrati, Z.; Talaeipour, M.; Khakifirouz, A.; Bazyar, B.
Impact of Cellulase Enzyme Treatment on Strength, Morphology and Crystallinity of Deinked Pulp. Cellul.
Chem. Technol. 2013, 47, 547–551.
26. Chutani, P.; Sharma, K. K. Concomitant Production of Xylanases and Cellulases from Trichoderma longibra- chiatum MDU-6 Selected for the Deinking of Paper
Waste. Bioprocess Biosyst. Eng. 2016, 39, 747–758. DOI:
10.1007/s00449-016-1555-3.
27. Lee, C. K.; Ibrahim, D.; Omar, I. C.; Rosli, W. D. W.
Enzymatic and Chemical Deinking of Mixed Office Wastepaper and Old Newspaper: Paper Quality and Effluent Characteristics. BioResources 2011, 6, 3809–3823.
28. Campano, C.; Lopez-Exposito, P.; Blanco, A.; Negro, C.;
van de Ven, T. G. M. Hairy Cationic Nanocrystalline Cellulose as Retention Additive in Recycled Paper.
Cellulose 2019, 26, 6275–6289. DOI: 10.1007/
s10570-019-02494-x.
29. Liu, M.; Yang, S.; Long, L.; Wu, S.; Ding, S. The Enzymatic Deinking of Waste Papers by Engineered Bifunctional Chimeric Neutral Lipase – Endoglucanase.
BioResources 2017, 12, 6812–6831. DOI: 10.15376/
biores.12.3.6812-6831.
30. Gea, S.; Rahayu, S.; Andriayani, A.; Piliang, A. F. R.;
Oktari, N. Mechanical and Morphological Characteristic Investigations of Deinked Used Newsprint Paper via Ultra-Sonochemistry Method. J. Nat. 2020, 20, 49–55.
DOI: 10.24815/jn.v20i2.16649.
31. Manfredi, M.; de Oliveira, R. C.; Reyes, R. I. Q.; da Silva, J. C. PAPER PHYSICS: Ultrasonic Treatment of Secondary Fibers to Improve Paper Properties. Nord.
Pulp Pap. Res. J. 2013, 28, 297–301. DOI: 10.3183/
npprj-2013-28-02-p297-301.
32. Saxena, A.; Singh Chauhan, P. Role of Various Enzymes for Deinking Paper: A Review. Crit. Rev. Biotechnol.
2017, 37, 598–612. DOI: 10.1080/07388551.2016.1207594.
33. Mauchauffé, R.; Lee, S. J.; Han, I.; Kim, S. H.; Moon, S. Y. Improved De-Inking of Inkjet-Printed Paper Using Environmentally Friendly Atmospheric Pressure Low Temperature Plasma for Paper Recycling. Sci. Rep.
2019, 9, 1–11. DOI: 10.1038/s41598-019-50495-4.
34. Imamoglu, S.; Karademir, A.; Pesman, E.; Aydemir, C.;
Atik, C. Effects of Flotation Deinking on the Removal of Main Colors of Oil-Based Inks from Uncoated and Coated Office Papers. BioResources 2013, 8, 45–58.
DOI: 10.15376/biores.8.1.45-58.
35. Ramírez Casillas, R.; Ramírez Valdovinos, E.; Carrillo Parra, A.; Dávalos Olivares, F.; Navarro Arzate, F.
Deinking of Laser Printing with Ultrasound of Intensive Action to Obtain High Purity Cellulose. Rev.
Mex. Ciencias For. 2018, 9, 80–101.
36. Stevulova, N.; Hospodarova, V.; Estokova, A. Study of Thermal Analysis of Selected Cellulose Fibres. Geosci.
Eng. 2016, 62, 18–21. DOI: 10.1515/gse-2016-0020.
37. Lengowski, E. C.; Muñiz, G. I. B. D.; Andrade, A. S.
D.; Simon, L. C.; Nisgoski, S. Morphological, Physical and Thermal Characterization of Microfibrillated Cellulose. Rev. Árvore 2018, 42, e420113. DOI:
10.1590/1806-90882018000100013.
38. Wu, S.; Zhang, Y.; Jiang, X.; Wang, S.; Liu, J.; Wu, S.
Changes in Supramolecular Structure and Improvement in Reactivity of Dissolving Pulp via Enzymatic Pretreatment with Processive Endoglucanase EG1 from Volvaria Volvacea. J. Wood Chem. Technol. 2020, 40, 163–171. DOI: 10.1080/02773813.2020.1722700.
39. Xu, Y.; Yan, Y.; Yue, X.; Zhu, Z.; Zhang, D.; Hou, G.
Effects of Ultrasonic Wave Pretreatment on the Fibrillation of Cellulose Fiber. Tappi J. 2014, 13, 37–43.
