Environmental Sustainability

, Volume 1, Issue 3, pp 233–244 | Cite as

Laccase based de-inking of mixed office waste and evaluation of its impact on physico-optical properties of recycled fiber

  • Shiv Shankar
  • ShikhaEmail author
  • Chandra Bhan
  • Rajesh Chandra
  • Sanjay Tyagi
Original Article


The de-inking ability of the crude laccase (benzenediol: oxygen oxidoreductase, EC produced by a white rot fungus Peniophora sp. (NFCCI-2131) was tested using mixed office waste paper. In addition, the impact on physical and optical properties of the de-inked pulp, and the chemical load of the effluent generated during laccase based de-inking process was also analyzed. In the present study, 2.5 U/ml laccase was produced by the fungus under submerged culture conditions. Laccase (10 U/g of air dried pulp) was found to improve the tensile strength of recycled pulp by 1.7-fold, de-inking efficiency by 1.5-fold, opacity by 3.84%, and brightness by 5.83%. Scanning electron microscopy analysis revealed less rupturing of cellulose fibers and increased detachment of ink particles from the surface of the fibers in comparison to chemical de-inking. Fourier-transform infrared spectroscopy analysis of laccase treated pulp revealed degradation of the guaiacyl group and a high degree of deformation in methyl group of lignin. The biochemical and chemical oxygen demand (BOD and COD) were found to be reduced by 52 and 36% respectively, in the effluent generated after crude laccase based de-inking of mixed office waste.

Graphical abstract


Laccase Peniophora sp. Mixed office waste De-inking Guaiacyl group 


Globally, paper industry is facing the problem of raw material i.e., cellulosic pulp, due to shortage of forest-based raw materials. Waste paper recycling is the only way to minimize dependency on forests. Recycling of waste paper not only saves trees but also reduces environmental pollution and water and energy consumption (Bajpai 2012; Ibarra et al. 2012; Chutani and Sharma 2015; Liu et al. 2017).

Approximately, the production of 1-ton recycled paper saves approximately 2.5 barrels of oil, 4100-kWh of electricity, 4 cubic meters of landfill, 31,780 l of water and 17 trees (Bajpai 2014). Mixed office waste (photocopier waste papers and laser printed paper) is thus emerging as a promising raw material for recycling.

During the recycling of waste paper, printing inks are removed from the cellulose fiber in a process called de-inking (Lee et al. 2013). In traditional de-inking practices, large amount of chemicals like NaOH, Na2SiO3, Na2CO3, H2O2, chelating agents and surfactants are employed (Bajpai 2012; Pathak et al. 2014a). Use of huge amount of chemicals makes the process costly and damaging to the environment. Treatment of toxic effluents characterized with high chemical oxygen demand (COD) requires costly wastewater treatment processes (Zhang et al. 2008). Keeping in view the disadvantages of chemical de-inking methods, development of an alternative process is urgently required. Enzymatic de-inking as a potentially effective solution is a possible alternative to the chemical process (Singh et al. 2012; Ibarra et al. 2012). Over the past few years, enzymatic de-inking, as a cost-effective and eco-friendly technology has attracted a great deal of attention of the researchers (Pathak et al. 2014b; Desai and Iyer 2016; Kumar et al. 2018). As compared to the chemical de-inking, pulp treated with enzymes renders improved physical and optical properties like drainage rate, tensile strength, brightness, whiteness, opaqueness and lower residual ink. The enzymes act by altering the surface of the fiber or bonds near the ink particles facilitating the removal of the printing ink. The detached ink particles are removed by washing or floatation (Xu et al. 2011; Pathak 2014; Saxena and Singh Chauhan 2017). Different enzymes like hemicellulases, pectinases, amylases, ligninases, and cellulases have been reported by different researchers as safe de-inking agents (Bajpai 1999; Claus et al. 2002; Pala 2004). Although de-inking of waste paper using cellulase and hemicellulase has been carried out at industrial scale, the physical properties of the de-inked pulp were not found to be good as compared to chemically de-inked pulp (Zhang 2008; Bansal et al. 2018). It is, therefore, desirable to explore efficient enzymes for de-inking. Laccases (benzenediol: oxygen oxidoreductases, EC are enzymes that belong to the group of blue oxidases, and have abilities to de-ink waste paper. (Camarero et al. 2004; Kiiskinen et al. 2004; Mohandass et al. 2008; Kuhad et al. 2010; Rodriguez-couto 2014; Burgueño and Rodriguez-couto 2014; Bhamare et al. 2018; Naraian et al. 2018).

In presence of redox mediators, the so-called laccase-mediator system, fungal laccases are very efficient in the delignification of pulps, resulting in improvement of pulp properties (Bourbonnais et al. 1997; Bajpai 1999; Camarero et al. 2004). However, fungal laccases have not been extensively used for the treatment of waste papers. The most common laccase producers are wood-rotting fungi, such as Trametes versicolor, Trametes hirsuta, Pleurotus eryngii and Peniophora sp. (Bourbonnais et al. 1997; Bajpai 1999; Camarero et al. 2004; Shankar and Shikha 2012).

In the backdrop of aforesaid context, the present study was carried out to de-ink mixed office waste paper using crude laccase alone, and in combination with de-inking chemicals. Evaluation of effects of laccase on physical and optical properties of recycled pulp and chemical load on the effluent generated during the de-inking process was also done.

Materials and methods

All the reagents as well analytical grade chemicals used in the present study were procured from Sd Fine-Chem Ltd. (India), Merck (India) and Loba Chemie (India). Mixed office waste used in the study was collected from the office and laboratory of Department of Environmental Science, Babasaheb Bhimrao Ambedkar University Lucknow, Uttar Pradesh, India.

Laccase production under submerged culture conditions

White rot fungus Peniophora sp. (NFCCI-2131), isolated from pulp and paper mill effluent was used as a source of enzyme laccase (Shankar and Shikha 2012). It was maintained on malt extract agar medium (MEA; 20 g/l malt extract; 20 g/l glucose; 1 g/l peptone and 15 g/l agar) at 30 °C and pH 4.6.

MEB medium (MEA without agar; pH 4.6) was used for laccase production (Shankar and Shikha 2015). The 250 ml Erlenmeyer flasks containing 100 ml MEB were inoculated with 5 × 5 mm colonized agar discs and incubated for 22 days at 30 °C under static condition. Laccase activity (96 h) was assayed in the filtered supernatant obtained by centrifugation of culture broth at 10,000×g for 10 min at 4 °C followed by filtration.

Laccase assay

Laccase (EC activity was checked by monitoring the oxidation (at 465 nm) of 10 mM guaiacol to tetraguaiacol, in 50 mM sodium acetate buffer (pH 4.6) at 37 °C (Ademakinwa and Agboola 2016). One enzyme unit (EU) was defined as the amount of enzyme that forms 1 μmol product per min in the assay conditions (Jahangiri et al. 2018).

Pulp preparation and de-inking sequence

The de-inking efficiency and the physical and optical properties of the mixed office waste paper by the treatment with the crude laccase were evaluated. The mixed office waste paper was manually cut into small pieces (1.5–2.5 cm2) and soaked overnight in tap water at room temperature. The soaked papers were washed several times and disintegrated using a grinder to obtain soft cottony pulp (Virk et al. 2013). The pulp was squeezed to remove absorbed water and oven dried at 50 °C for 60 min. The oven dried pulp (ODP) was used for further experiments (Singh et al. 2012).

A standard de-inking sequence of (1) pulping process (P) and (2) chemical treatment (C) (Singh et al. 2012) was done. Conditions of P and C are described in Table 1. The standard enzymatic de-inking sequence was performed in 250 ml Erlenmeyer flasks containing 10 g of pulp at 6% consistency under both standard and modified conditions, pH 5.5 and at 55 °C for 45 min and 150 rpm as follows: (1) pulping process (P), (2) chemical treatment, followed by the enzymatic treatment with different amounts of the crude laccase (L; as described in Table 1).
Table 1

Conditions used for laccase based deinking of mixed office waste paper

De-inking stages

Pulping process (P)

 Pulping consistency


 Pulping time

5 min

Enzymatic treatment (L)


30 °C



 Pulp concentration


 Enzyme dosages

(1.0–2.5 EU)

 Contact time

45 min


55 °C


150 rpm

Chemical treatment stage (C)

 Sodium hydroxide


 Sodium silicate




 Pulp consistency


 Diethylenetriaminepentaacetate (DTPA)


 Tween 80


 Triton X-100




 Floatation temperature

50 °C


200 (rpm)

 Floatation time

15 min

In chemical treatment stage C, pulp at 6% consistency was treated with sodium hydroxide (2%), sodium silicate (2.0%), DTPA (diethylenetriaminepentaacetate, 0.5%), tween 80 (0.2%), Triton X-100 (1.2%) and H2O2 (1%) at 50 °C and agitation of 200 rpm for 15 min. During enzymatic treatments, H2O2 was not incorporated into the mixture with objective to prevent activity of other substrate specific enzymes like lignin peroxidase and manganese peroxidase.

In L stage, different laccase doses viz. 0, 1, 1.5, 2.0, 2.5 EU laccase/g of pulp on oven dry basis (L0, L1, L1.5, L2.0, and L2.5) were employed for the treatment of the mixed office waste pulp. Similarly in C stage (chemical de-inking), different proportion of de-inking chemicals at varying concentration (25–100%) were used. The de-inking was carried out under standard P–C sequence and modified treatment stages viz. Treatment T1 (T1: P-L2.5-C0) involved pulping stage P, enzymatic treatment stage L (2.5 EU laccase/g of pulp) and no chemical bleaching. The treatment T2 (P-L2.0-C25) involved pulping stage P, enzymatic treatment stage L with 2.0 EU laccase/g of pulp and Chemical bleaching stage C performed with 25% de-inking chemicals. The treatment T3 (P-L1.5-C50) involved pulping stage P, enzymatic treatment stage L with 1.5 EU laccase/g of pulp and chemical bleaching stage C with 50% deinking chemicals. The treatment T4 (P-L1.0-C75) comprised of pulping stage P followed by enzymatic treatment stage L performed with 1 EU/g of pulp followed by chemical bleaching stage C performed with 75% de-inking chemicals. The treatment T 5 (P-L0-C100) involved pulping stage P, devoid of enzymatic treatment stage L and followed chemical bleaching stage containing 100% de-inking chemicals. Mixed office waste pulp without treatment with laccase and de-inking chemicals was kept as control (C: P-L0-C0). The reaction of laccase and de-inking chemicals was carried out in 250 ml Erlenmeyer flasks containing 10 g of pulp at 6% consistency under both standards and modified conditions at pH 5.5 and 55 °C for 45 min with consistent agitation (150 rpm).

Preparation of hand sheets of chemical and enzyme-treated pulp

The hand sheets of treated pulp of mixed office waste were prepared as per the protocol of Technical Association of the Pulp and Paper Industry (TAPPI), TAPPI Test Method T218 om-91 (TAPPI Test Methods 1996; Kumar et al. 2016). This method employs Buchner funnel for preparing specimen sheets. Enzyme and chemical treated pulps were washed thrice with de-ionized water. A suspension of the aforesaid treated pulp was prepared at 6% consistency in distilled water with pH adjusted to 6.5 ± 0.5. The slurry was carefully poured into Buchner funnel (150 mm) equipped with the mesh having pore size 1 mm. The sheets formed were dried at room temperature for the analysis of physical and optical properties.

Evaluation of physical and optical properties of the pulp

Physical and optical properties of the paper were evaluated by following Central Pulp and Paper Research Institute, (CPPRI) standard methods (CPPRI 2001a). Tensile strength was measured as per standard method TM II-A-42, CPPRI using tensile tester (Model: Elrepho- LNW, Sweden). The brightness of the chemical and enzyme-treated hand sheet was analyzed in accordance with standard method ISO-2471 using brightness tester (Model: Elrepho ZB-A, Sweden). The opacity of hand sheets of chemical and enzyme-treated mixed office waste pulp was determined as per the protocol suggested by CPPRI (CPPRI 2001b) (Test method TM II-B2, 2001; ISO 2471).

X-ray diffraction analysis of MOW treated with laccase and chemicals

X-ray diffraction analysis of chemical and laccase de-inked mixed office pulp was carried out with Rigaku Ultima IV, X-ray diffractometer (USA) to determine the extent of crystallinity of cellulose which has a profound effect on hardness, density, and transparency of cellulose. After freeze-drying, samples were analyzed using the X-ray diffractometer with X-ray generator from 2 to 50 of 2 theta (scattering angle). The crystalline index of cellulose (Xc) was calculated from the X-ray diffraction patterns by the following equation (Heinze and Liebert 2001).
$${\text{Xc}} = {\text{I}}00 2 {\text{ - IamX1}}00/{\text{I}}00 2.$$

The peak intensity from (002) lattice plane (2 Theta = 22.6 u) represents the crystalline cellulose, while peak intensity of amorphous phase occurs at 2 Theta = 19 u.

SEM analysis of MOW pulp treated with enzyme and chemicals

Scanning electron microscopy (SEM) was carried out (MIRA 3, Tescan USA) to analyze the surface morphology of the MOW pulp hand sheets treated under different laccase doses and de-inking chemicals. Properly washed fibers of the treated pulp were dehydrated by employing 20–90% acetone and eventually suspended into 100% acetone. Air dried small piece of fibers were placed on stubs, mounted with silver tape and examined at 10 kV.

Fourier-transform infrared spectroscopy (FTIR) analysis of pulp treated with enzymes and chemicals

FTIR spectra of laccase and chemically de-inked MOW pulp hand sheets were recorded with FTIR spectrometer (NICOLET TM 6700, Thermo Scientific USA). FTIR spectra for pulp samples were recorded with a resolution of 400 cm−1 over the wave number range of 4000–400 cm−1, using 32 scans per sample.

Evaluation of effluent characteristics after de-inking process

The total phenolic contents in effluent generated during enzymatic and chemical deinking were determined according to the method described by Justino et al. (2009), taking gallic acid as a standard. The released reducing sugars were measured by the dinitrosalicylic acid (DNS) method (Miller 1959), using glucose as standard molecule. BOD and COD estimation were carried out in accordance with the standard method of American Public Health Association (Rice 2012).

Results and discussion

Laccase production

A white rot fungus isolated from the effluent of pulp and paper industry which was identified as a novel Peniophora sp. (NFCCI-2131) by Agharkar Research Institute Pune was used as a source of enzyme in the present study (Shankar and Shikha 2012). Results showed that maximum laccase production was obtained after 16 days growth at pH 4.6 and 30 °C (data not shown).

Measurement of de-inking efficiency of crude laccase

The paper de-inking efficiency by laccase was analyzed as described by Lee (2011). Results (Table 2) revealed that the de-inking efficiency of MOW pulp was higher for T2 treatments (2.0 UE/g of pulp and 25% de-inking chemicals) and T3 (1.5 UE/g of pulp and 50% de-inking chemicals) rather than treatments T1, T4 and T5, suggesting that the mix of both laccase and chemicals may improve the de-inking efficiency. The ability of laccases for pulp brightening has been previously reported (Eugenio et al. 2010; Fillat and Roncero 2010; Andreu and Vidal 2011; Ravalason et al. 2012; Chen et al. 2012; Kumar et al. 2018). The crude laccase produced in our work showed a better performance compared with the conventional chemicals used for deinking.
Table 2

Physico-optical properties of the mixed office waste paper pulp and chemical load of the effluent generated during laccase based de-inking of mixed office waste paper pulp








De-inking efficiency (%)

8.5 ± 0.4

13.3 ± 0.8

12.6 ± 0.3

9.3 ± 0.6

8.8 ± 0.2

5.0 ± 0.2

ISO brightness (%)

76.8 ± 1.5

80.8 ± 3.0

80.2 ± 2.4

78.8 ± 3.1

75.0 ± 4.1

70 ± 5.1

Tensile strength (N/m)

870.0 ± 26.1

847.0 ± 35.5

703.0 ± 35.1

601.0 ± 24.0

509.0 ± 12.7

856.0 ± 25.6

Whiteness (%)

97.7 ± 4.8

106.8 ± 3.2

104.0 ± 2.6

100.0 ± 5.0

97.9 ± 1.9

97.7 ± 3.2

Opacity (%)

80.0 ± 1.9

85.25 ± 3.6

82.0 ± 3.1

72.0 ± 3.8

81.0 ± 2.4

75.0 ± 3.9

Crystallinity index (%)

65.9 ± 2.2

64.0 ± 1.7

58.0 ± 2.0

51.0 ± 0.8

16.0 ± 0.5

64.0 ± 3.5

Total phenol (mg/l)

630.0 ± 2.2

490.0 ± 1.7

410.0 ± 2.0

315.0 ± 0.86

530.0 ± 0.56

115.0 ± 3.5

Total reducing sugars (mg/l)

9.43 ± 0.19

11.2 ± 0.39

23.4 ± 0.96

32.6 ± 1.6

43.8 ± 1.9

6.0 ± 0.2

BOD (mg/l)

47.3 + 2.1

59.2 + 1.8

64.5 + 1.5

53.3 + 2.6

88.8 + 1.3

35.5 + 1.6

COD (mg/l)

440.0 ± 15.8

320.0 ± 16.6

400.0 ± 9.2

920.0 ± 46.7

1220.0 + 54.4

150.0 + 4.5

Data shown is mean of three observations (Standard deviation ± 5%)

T1 (P-L2.5-C0), laccase (2.5 EU laccase/g of pulp) without de-inking chemicals

T2 (P-L2.0-C25), laccase (2.0 EU laccase/g of pulp) with 25% de-inking chemicals

T3 (P-L1.5-C50), laccase (1.5 EU laccase/g of pulp) with 50% de-inking chemicals

T4 (P-L1.0-C75), laccase (1.0 EU laccase/g of pulp) with 75% de-inking chemicals

T5 (P-L0-C100), without laccase with 100% de-inking chemicals

C control without laccase and de-inking chemicals

Evaluation of physical and optical properties of the pulp

Tensile strength

Tensile strength was found to be highest (856 N/m) in case of control (P-L0-C0). The tensile strength in case of MOW pulps treated under P-L2.5-C0, P-L2.0-C25: P-L1.5-C50 and P-L1-C75 (T1, T2, T3 and T4) were found to be 870, 847, 703 and 601 N/m, respectively. The tensile strength of mixed office waste treated with highest doses of de-inking chemicals in P-L0-C100 treatments (T5) was found to be lowest with 509 N/m (Table 2).

Results indicated that laccase at highest doses improved maximum tensile strength (870 N/m) of paper as evidenced in P-L2.5-C0 treatments (T1) but with the decrease in concentration of enzyme, it resulted in a decrease of tensile strength. It was also observed that with increase in the concentration of de-inking chemicals, substantial decrease in tensile strength of the hand sheets took place. Use of highest concentration of de-inking chemicals may have reduced the tensile strength due to high degree of depolymerization of cellulose content of the fibers. Our results indicate that laccase based de-inking does not result in loss of the strength of the fiber as in the case of chemical treatment.

Our results are in accordance with Pala et al. (2004), who reported the improvement of tensile strength of the mixed office waste after treatment with commercial enzymes like celluclast, and buzzme (Pala et al. 2004). Generally, the hydrolytic activity exerted by de-inking chemicals is frequently associated with the reduction in paper’s mechanical properties due to increased de-polymerization of cellulose (Pala et al. 2004). Cellulose is arranged in the form of long bundles of cellulose polymer chains known as microfibrils, which includes amorphous zone and crystalline zone. Amorphous zone is comprised of a random chain of cellulose which is confined between cellulose crystallites (Nishiyama et al. 2003). In crystalline zone, cellulose chains form plains where equatorial hydroxyl groups in pyranose rings are linked with hydrogen. Chemical de-inking caused a substantial decrease in crystalline cellulose peak as well as destroys crystalline microfibril core thereby leading to alteration in crystalline surface of the fiber. Laccase remains adsorbed on fiber surface after the enzymatic treatment (Kiiskinen et al. 2004). Laccase modifies the surface of the fiber but has no effect on micro fibril core which remained crystalline. Recently, laccase has gained significant attention for improving paper properties in general and strength in particular. Several workers have also reported the improvement in tensile strength of the cellulose fibers after laccase treatment of the pulp (Chandra et al. 2008; Aracri et al. 2011; Ibarra et al. 2012; Gupta et al. 2015). The present study proposes the use of laccase as a promising supplement of de-inking and paper recycling on the grounds of improving the strength of the fiber which is required for quality paper products.

Brightness of the MOW treated with laccase and chemicals

Brightness is the intrinsic reflectance factor measured at an effective wavelength of 457 nm with a reflectometer. The brightness of the MOW treated with chemicals and the different amount of laccase was measured. The results revealed that the brightness increased after all the treatments, except in the control (70%). Treatment of MOW pulp under treatment conditions T2, T3 and T4 increased the brightness to 80.84, 80.26 and 78.84%, respectively (Table 2). On the contrary, under the conditions described in T1 and T5, brightness showed lower values of 76.81 and 75.01%, respectively, suggesting that higher doses of laccase (2.5 U/g) and de-inking chemicals (100%) do not favor brightness. Several investigators have studied the effect of laccase on brightness, and reported an improved brightness of the pulp by enzymatic treatment. Ibarra et al. (2012) reported a 3% increase of brightness of the old news paper pulp (ONP) treated with the laccase-mediator system (Ibarra et al. 2012). Gupta et al. (2015) reported increased brightness (11.8%) of kraft pulp de-inked with the combination of laccase and xylanase. Generally, enzymes like laccases act directly on cellulose fibrils and break the fiber-ink bonding regions, peel off the fibers and degrade ink thereby improving the brightness of the pulp (Gupta et al. 2015). Results of the present study are in accordance with the findings of other researchers showing an increase in the brightness of the MOW pulp attributed to enzymatic action (Vyas and Lachke 2003; Lee et al. 2013).

Measurement of whiteness of the MOW pulp treated with laccase and chemicals

Whiteness is the measurement of the light reflected by the paper across the visible (daylight) spectrum. The results of the analysis of whiteness are shown in Table 2. MOW pulp undergone treatments T2, T1, T5 and control (untreated pulp) showed values of 106.85, 97.76, 97.66 and 97.76% whiteness, respectively. Laccases have been well reported as prolific bleaching agents (Valls and Roncero 2009; Thakur et al. 2012; Sharma et al. 2014), thus the increase of whiteness may be due to the bleaching effect of the enzyme.

Opacity of the MOW treated with enzyme and chemical

Opacity is the ratio between the luminous reflectance factor in a black background and the intrinsic luminous reflectance factor of the sample. The opacity was measured with a reflectometer. Results (Table 2) show that the highest opacity (85.25%) was obtained when samples underwent treatment T2, followed by T3, T5, T1, control and T4. Results suggest the use of 2.0 U/g and 25% of chemicals slightly contributed to opacity, probably due the combination of both enzymatic and chemical treatments. Traces of lignin within fibers may produce a darkness appearance of MOW, which may result in lower reflectivity of the paper and thus causes an increase in the paper opacity. Vyas and Lachke (2003) have also reported about 1.3% increment in opacity, after enzymatic de-inking of MOW.

Estimation of total phenols and reducing sugars in effluent generated during de-inking of MOW

Results showed that the total phenol contents in the effluent were highest (630 mg/l) in case of T1 followed by T5 and (530 mg/l). According to results obtained, both chemicals and laccase when used at highest concentrations resulted in the maximum release of phenols from the pulp. However, phenolic contents were found to be lowest (115 mg/l) in effluent obtained in case of control (P-L0-C0) (Table 2). The high phenolic content in effluent generated during P-L2.5-C0 and P-L0-C100 treatments (T1 and T5) may be due to dissociation of residual lignin and sugars under the effect of laccase and chemicals. Laccase has been very well reported to degrade lignin (Shankar and Shikha 2012; Ai et al. 2014; Rich et al. 2016). The reducing sugars were found to be least (6.01%) in the effluent generated in case of control. The rate of release of reducing sugars in case of effluent generated from the treatment of MOW under T1, T2 and T3 treatments was found to be increased. The reducing sugars under these treatments were found to be 9.43, 11.2 and 23.45 mg/l, respectively. Results also indicated that amount of reducing sugars released was highest (42.34 mg/l) in case of effluent generated during P-L0-C100 treatments (T5). The reducing sugars were found to be least (6.01%) in case of effluent generated in case of control. The increased rate of reducing sugars in case of elevated concentration of de-inking chemicals may be due to dissociation of sugars from pulp fibers (Virk et al. 2013).

X-ray diffraction analysis of MOW treated with laccase and chemicals

X-ray diffraction analysis of chemical and laccase de-inked mixed office pulp was carried out to determine the extent of crystallinity of cellulose which has a profound effect on hardness, density, and transparency of cellulose. The peak intensity from (002) lattice plane (2 Theta = 22.6 u) represents the crystalline cellulose, while peak intensity of amorphous phase occurs at 2 Theta = 19 u. Results (Fig. 1a) revealed that the I002 intensities at 2θ = 22.60 were 14566.7 (untreated MOW pulp, T0) while the peak intensity at 2Θ = 190 was recorded to be 5237.5 in case of control (C). The crystallinity index of control was found to be 72% (Table 2) which was highest among all the treatments. In case of pulp treated under T1 conditions, the I002 intensities at 2Θ = 22.60 and the peak intensity at 2Θ = 190 were found to be 20391.7 and 6950.6, respectively (Fig. 1b). The crystallinity index for this treatment was found to be 65% which was the second highest among all the treatments (Table 2). In case of MOW pulp treated under T2 conditions, the I002 intensities at 2Θ = 22.60 and the peak intensity at 2Θ = 190 were found to be 13712.5 and 4925.1, respectively (Fig. 1c). The crystallinity index for this treatment was recorded to be 64% (Table 2) which was similar to T5 treatment.
Fig. 1

X-ray diffractograms of MOW pulp treated under different conditions. a Control: P-L0-C0, b T1: P-L2.5-C0, c T2: P-L2.0-C25, d T3: P-L1.5-C50, e T4: P-L1-C75, f T5: P-L0-C100

The crystallinity index of T3 and T4 were recorded to be 58 and 51%, respectively (Table 2). Generally, the crystallinity has an effect on the mechanical properties of cellulose fibers. Mechanical strength increases and flexibility decreases with increasing crystallinity. Cellulose is an aggregate wherein several units of glucose are arranged more or less parallel to each other and stabilized in the lateral direction by hydrogen bonds opposite to hydroxyl group and is crystalline in nature. Generally, crystallinity index of cellulose ranges from 50 to 90%. Higher crystallinity index indicates the well-organized arrangement of the polymer. Results (Table 2), clearly indicated that the degree of crystallinity index kept on increasing with the increase in the proportion of laccase in the mixture and reached to highest when 100% enzyme was incorporated in the mixture. On the other hands, the crystallinity index was found to be decreased with increase in the concentration of chemicals in the mixture. Results thus suggested that cellulose fibers lost their strength more when treated with chemicals in comparison to when treated with laccase. High crystallinity indicates a decrease in amorphous cellulose and increase in the crystalline cellulose of the pulp (Park et al. 2010). The increase in crystallinity might be due to less de-polymerization of cellulose. Our results are in agreement with the work of Virk et al. (2013) who also reported an increase in crystallinity index of old news paper (ONP) from 29.16% (untreated) to 65.47% after treatment with laccase.

SEM analysis of MOW pulp treated with enzyme and chemicals

SEM analysis was carried out to analyze the surface morphology of the MOW pulp hand-sheets treated under different laccase doses and de-inking chemicals. Results revealed that the fiber surface turned rough, with conspicuous fibers on the surface (Fig. 2a–f), when MOW pulp was treated with different doses of chemicals (25–100%). The laccase alone did not result in rupturing of the fibers. However, in MOW pulp fibers treated under T2, rupturing started (Fig. 2b) which kept on increasing with increase in the concentration of the chemicals. Maximum rupturing was obtained when pulp was treated under T5 conditions (Fig. 2e), indicating that higher concentration of de-inking chemicals deforms the structural integrity of the cellulose fibers. SEM observation showed that the surface of the MOW pulp hand sheet in case of control control was intact and smooth with visible ink particles (Fig. 2f). In the present study, it is clearly evident that the detachment of the ink particles from the surface of the fibers was more prominent in case of pulp treated with laccase as compared to treatment with de-inking chemicals. Rupturing and fibrillation may be due to de-lignification on fiber surface which can release fibrils (Virk et al. 2013). SEM analysis results clearly revealed that laccase does not deform the structure of the cellulose fibers.
Fig. 2

SEM micrograph of mixed office waste pulp subjected to different treatments. a T1: P-L2.5-C0, b T2: P-L2.0-C25, c T3: P-L1.5-C50, d T4: P-L1-C75, e T5: P-L0-C100, f Control: P-L0-C0

FTIR analysis of pulp treated with enzymes and chemicals

FTIR spectra of laccase and chemically de-inked MOW pulp hand sheets were recorded. Pulp treated under different conditions revealed different characteristics and prominent changes (Fig. 3). MOW treated under different conditions revealed transmittance structures similar to P-C0-L0 but with different intensities. The FTIR spectra of untreated MOW pulp (control) exhibited well-defined bands at approximately 3424.2, 2923.2, 2874.2, 2515, 2361.2 1797, 1423.6 1033.4, 914.9, 876 and 756.9 cm−1. Generally, the broad bands in the 3600–3100 cm−1 region are due to the OH-stretching vibration. The intensity of the peak 3424.2 cm−1 was found to be decreased when the MOW pulp was subjected to T1, T2, T3 and T4 treatments. Between the region, 2950–2923 cm−1, several peaks at 2925.4, 2949.4, 2927.1 and 2950 cm−1 were obtained in MOW pulp treated under T1, T2, T3 and T5 treatments indicating C–H asymmetrical stretching vibration in CH3, CH2, CH in cellulose (Virk et al. 2013; Khalil et al. 2014). The intensity of band was found to increase with an increase in the concentration of laccase indicating increased degradation of aliphatic side chains in cellulose. In treatments, T1, T2, T3 and T4, bands at 1425.4, 1427.4, 1442 and 1451 cm−1 occurred, respectively due to stretching vibrations combined with –CH3 in-plane deformations, showing that some methoxyl groups were removed during the enzymatic treatment. However, in control, the band at 1423 cm−1 occurred at low intensities. The intensity of this band increased with increase in the concentration of chemicals and enzymes indicating the high degree of deformation in methyl group. The peaks appearing at 1093.5 cm−1 in case of control and 1094.1 cm−1 in case of T1 treatment, are due to the C–O–C stretching vibrations of the 1,4-glycosidic ring linkages between the d-glucose units in cellulose (Parker 1971). This band was completely removed in case of T2, T3 and T4 treatments indicating distortion of 1,4-glycosidic bonds. The bands at 1033 and 1060 cm−1 match the vibration of deformation of C–O–C bonds in the cellulose and hemicelluloses (Chutani and Sharma 2015).
Fig. 3

FTIR spectrum of chemically and laccase treated pulp. Control: P-L0-C0; T1: P-L2.5-C0; T2: P-L2.0-C25; T3: P-L1.5-C50; T4: P-L1-C75; T5: P-L0-C100

Evaluation of effluent characteristics post de-inking

The effluent generated during chemical and enzymatic de-inking was analyzed in terms of BOD and COD. In comparison to alkaline de-inking process, the COD was lower in enzymatic de-inking; this reduces the load on wastewater treatment systems (Lee 2011). Results (Table 2), revealed that BOD and COD values obtained from wastewater effluent after de-inking with 2.5 U/g laccase (T1) were lower as compared to chemical de-inking process. About 53 and 36% reduction in BOD and COD values respectively, were recorded after enzymatic de-inking compared to chemical de-inking of mixed office waste. BOD and COD values of untreated MOW pulp were recorded to be 35.54 and 150 mg/l, respectively. Our results are in conformity with the earlier study. Lee (2011), reported about 47.1 and 33.8% reductions in BOD and COD value after enzymatic de-inking as compared to chemical de-inking. There also are other environmental advantages by avoiding the use of high alkalinity in the pulping stage (Jeffries and Viikari 1996; Bajpai 1999; Verma et al. 2011).


In the present study, a novel laccase producing white rot fungus Peniophora sp. (NFCCI-2131) was selected for the de-inking of mixed office waste paper. Laccase was found to de-ink MOW 1.5-fold higher than chemicals. Laccase obtained from the fungus was also found to improve tensile strength, brightness, whiteness, opacity, and crystallinity of fibers. Fiber surface of MOW pulp when treated with laccase did not rupture extensively as compared to the pulp treated with chemicals. In addition, the detachment of ink particles was seen more prominent in case of enzymatic treatments than chemical treatment. Laccase treated pulp showed several characteristics and prominent changes including degradation of the guaiacyl group and a high degree of deformation of methyl group in lignin. In effluent generated during laccase based de-inking, 52% reduction in BOD and 36% reduction in COD was recorded which indicates enzymatic de-inking reduces the chemical load in wastewater as compared to chemical process. Such results indicate that laccase can be employed as a promising agent of de-inking and paper recycling as it enhances the physical and optical properties of the paper. Laccase based de-inking is thus an efficient, green technology for the recycling of waste paper promoting environmental sustainability by reducing the use of harmful chemicals during pulp and paper production.



Young Scientist Fellowship (Award no: CST/242/, dated 12/05/2015, Council of Science and Technology Uttar Pradesh, Lucknow India) to first author Dr. Shiv Shankar is gratefully acknowledged. The authors acknowledge Dr. R. K. Jain, Director, Central Pulp, and Paper Research Institute Saharanpur, India and Ms. Prachi Kaushik, Senior Research Fellow, CPPRI, Saharanpur for their help in the analysis of physical and optical properties of the paper. The authors are thankful to Vice Chancellor, Babasaheb Bhimrao Ambedkar University, and Head, Department of Environmental Science, Babasaheb Bhimrao Ambedkar University (A Central University) for rendering necessary facilities.


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Copyright information

© Society for Environmental Sustainability 2018

Authors and Affiliations

  • Shiv Shankar
    • 1
  • Shikha
    • 1
    Email author
  • Chandra Bhan
    • 1
  • Rajesh Chandra
    • 2
  • Sanjay Tyagi
    • 3
  1. 1.Department of Environmental Science, School for Environmental SciencesBabasaheb Bhimrao Ambedkar University (A Central University)LucknowIndia
  2. 2.Department of Polymer and Process EngineeringIndian Institute of Technology (I.I.T.) Roorkee, IIT Roorkee CampusSaharanpurIndia
  3. 3.Paper Testing DivisionCentral Pulp and Paper Research Institute SaharanpurSaharanpurIndia

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