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. 2023 Jan 26;13(2):61. doi: 10.1007/s13205-023-03484-4

Production of eco-friendly and better-quality sugarcane bagasse paper using crude xylanase and pectinase biopulping strategy

Divya Sharma 1,2, Sharad Agrawal 3, Raksha Nagpal 1, O P Mishra 4, Nishikant Bhardwaj 4, Ritu Mahajan 1,✉
PMCID: PMC9880078  PMID: 36714548

Abstract

This research aimed to investigate the efficiency of crude xylanase–pectinase in pulping of sugarcane bagasse. Optimum biopulping was obtained, using xylanase–pectinase dose 200–60 IU/g, bagasse/liquid ratio 1:10 and 1.0% Tween 80 concentration at 55 °C temperature, pH 8.5 and period of treatment 180 min. Treatment of sugarcane bagasse samples with these enzymes generated pulp with lower rejections (58.76%), total solids (12.64%), kappa number (47.77%), higher screened pulp yield (10.66%), along with enhanced optical and physical properties, in comparison with a chemical pulp. Bagasse biopulping resulted in a 13% decrease in alkali dose to obtain the optical and physical properties similar to those achieved under the 100% alkali dose. The breaking length, burst factor, tear index, double fold, gurley porosity and viscosity were improved by 15.19, 37.64, 2.47, 37.77, 35 and 23.17%, respectively, after bleaching treatment of biopulped samples. Thus, enzymatic pulping is an eco-friendly environmentally sustainable approach, since it reduces the use of pulping chemicals and simultaneously improves the paper quality. This is the first report, showing pulping of sugarcane bagasse, with crude xylanase–pectinase, produced by an isolate.

Keywords: Soda-AQ pulping, Sugarcane bagasse, Bleaching, Biopulping, Xylan–pectinolytic enzymes, Alkali dose

Introduction

Increased environmental awareness and forest conservation have focused studies on exploring sustainable, new fibrous materials and simultaneously less toxic methods of pulping. Wood is the principal raw material used in paper pulp manufacturing. However, in recent years, the use of agricultural residues such as sugarcane bagasse, cereal straws, reeds and bamboo for the paper and pulp production has increased steadily. Bagasse is a source of reliable raw material for paper industry in countries that have inadequate supplies of timber and produce large quantities of bagasse. In addition, bagasse has many benefits in comparison with agro-based residues (wheat and rice straws) due to the lower ash content.

Sugarcane is an important crop in South America, Africa and Asia (Cardona et al. 2010). Bagasse of sugarcane contains cellulose (41.0–55.0%), xylan (22.4%), lignin (18.0–26.3%), pectin and ash (Mokhena et al. 2017; Sporck et al. 2017). More than 500 million t of sugarcane bagasse is believed to be generated annually (Chambon et al. 2018).

Regulatory and environmental pressures also compelled the paper and pulp industry to change their pulping, bleaching, and effluent disposal techniques to minimize the effect on the environment of effluent discharged from mills (Hubbe et al. 2016). These pressures also led to the need for cleaner technologies for improving the pulping method by lowering the lignin content in the pulp, before reaching the bleaching plant.

Soda-AQ pulping generates pulp with better characteristics, however also has certain disadvantages, like heavy chemical load in paper making, and the ultimate environmental pollution culprit (Ates et al. 2008). A possible alternative for reducing chemical usage during paper making would be biopulping (Liu et al. 2017), decreasing environmental pollution and enhancing pulp strength characteristics (Martin-Sampedro et al. 2011).

Over the past few years, greater attention has been given to implement green biotechnology in pulping procedures to boost pulp quality and reduce pollution. The use of lignin-degrading white rot fungi, Ceriporiopsis subvermispora (Messner and Srebotnik 1994; Ferraz et al. 2007), Phanerochaete chrysosporiurn (Messner and Srebotnik 1994) and Physisporinus rivulosus (Maijala et al. 2008) has been mentioned in the literature for the biopulping of wood. The microbe, Ceriporiopsis subvermispora, has also been used for biochemical pulping of agricultural residues (Yaghoubi et al. 2008). The biopulping efficiency of Ceriporiopsis subvermispora, Trametes versicolor and Phanerochaete chrysosporium has also been checked for wood or other raw material (Kumar et al. 2020).

Fungal treatment has been shown to enhance the diffusion of NaOH in woods/woodchips and also improves the pulp consistency (Woldesenbet et al. 2012). However, the major drawbacks of fungal pretreatment are sluggish growth of fungus (around 2–4 weeks) and yield loss, due to polysaccharides and lignin degradation during fungal treatment. In order to counter such problems, crude enzymes isolated from industrially compatible microbial sources may be the best solution. The benefit of enzymatic pretreatment is the very less treatment time in comparison with the many days taken for fungal pretreatment, which renders enzymatic pulping viable for industries. The mechanism of biopulping has been studied primarily in the last decade only (Zhao et al. 2002; Liu et al. 2017; Varghese et al. 2020; Nagpal et al. 2021). Very few reports are available in the literature, on the biopulping using enzymes.

This study has been conducted with two objectives—first, to valorize sugarcane agrowaste and second, to reduce environmental pollution using eco-friendly enzymatic approach. There is also a great pressure on the paper industries to adopt cleaner technologies for the generation of paper. In this study, the efficacy of crude xylan–pectinolytic pretreatment (produced by a bacterial isolate, Bacillus pumilus AJK), in the pulping of bagasse samples, has been evaluated, with an aim to reduce the environmental pollution and simultaneously to improve the paper quality.

Materials and methods

Materials

Both xylanase and pectinase enzymes used in this study were produced using Bacillus pumilus AJK (MTCC 10414). Xylose and galacturonic acid, xylan and pectin were purchased from Sigma-Aldrich, USA, and all other chemicals used were from HiMedia, India. Sugarcane bagasse samples were obtained from a nearby village of Kurukshetra, Haryana, India. Bagasse samples were depithed manually, dried and kept in plastic bags till further use.

Xylan–pectinolytic enzymes production and their estimation

Enzymes were produced under submerged fermentation conditions, as stated by Kaur et al. (2017). Flasks (250 mL) with medium 50 mL, pH 7.0 (washed, dried, agro-wastes—2% citrus peel—2% wheat bran; peptone, 5.0 g/L; magnesium sulfate-10 mM) was inoculated with 2% of 21-h-old culture and kept at 37 °C for 60 h and at 200 rpm, in an incubator (Remi India, model CIS-24 Plus). The batch fermentation was terminated by centrifugation (Remi India, model C-24 Plus) of the harvested medium at 8000 rpm for 10 min. Crude enzymes were obtained in the supernatant and their activity was estimated, as stated by Kaur et al. (2011), and the reducing sugar content after enzyme–substrate reaction was measured by the method, given by Miller (1959). The batch of enzymes used for this study, contained 300 IU of xylanase and 90 IU of pectinase per mL of the supernatant, obtained above after centrifugation.

Biopulping condition optimization and evaluation

Bagasse samples were pretreated with crude enzymes before chemical pulping. Pretreatment conditions were optimized with bagasse-to-liquid ratio ranging from 1:5 to 1:20 g/mL and pH of liquid varied from 7.5 to 10.0. Various enzyme dosages of xylanase (50–500 IU/g) and pectinase (15–150 IU/g) enzymes were also tested. A second shot of optimized enzymes dose was given. Similarly to select the best conditions for biopulping treatment, temperature from 45 to 65 °C and time of treatment from 60 to 360 min were also checked. To improve the biopulping efficacy of xylan–pectinolytic enzymes further, optimization of Tween-80, using various concentrations ranging from 0.1 to 2.0%, was also carried out. The samples (control) were also processed under the same conditions, but without the application of enzymes. After treatment, washing of these pretreated sugarcane bagasse samples was given thoroughly with tap water and after that dried at 45 °C. All experiments were repeated six times.

Assessment of the biopulping conditions was performed by taking biopulp-free filtrate samples and estimated the reducing sugar content, by the method, given by Miller (1959). The release of other impurities in filtrates was evaluated by noting the optical density in UV range for lignin (Nissen et al. 1992; Khandeparkar and Bhosle 2007) for hydrophobic compounds at 465 nm (Patel et al. 1993) and for phenolic compounds at 237 nm (Gupta et al. 2000; Khandeparkar and Bhosle 2007). Microscopic pictures were also taken to check the cells of sugarcane bagasse after biopulping.

Chemical pulping

All sugarcane bagasse samples were further processed with soda-AQ chemical pulping. The conditions of cooking in an oil bath rotating digester were: liquid-to-bagasse ratio 5:1 (v/w); alkali content (13.6–16%) and anthraquinone content 0.05%, temperature and time at treatment temperature—168 °C and 45 min, time was taken to reach treatment temperature was 90 min. The pH of spent liquor, total solids, Kappa number, and residual alkali content were checked after chemical pulping. Other factors like unscreened and screened pulp yield, brightness and rejects were also checked. Washing of pulp samples was first done thoroughly with hot water, after that with cold water and further used for experiments. All experiments were repeated six times.

Bleaching of bio and chemically treated sugarcane bagasse pulps

After pulping, biopulped samples (with 87% alkali dose) and 100% chemically treated samples were bleached parallelly, using sequence, i.e., DoEPD1D2, where EP is sodium hydroxide and hydrogen peroxide treatments and Do, D1, and D2 are ClO2 treatment stages. The ClO2 treatment dose was decided based on the Hise equation (Hise 1996). Washing of pulp samples was done thoroughly before analyzing their optical properties. All experiments were repeated six times.

Formation of handsheets and testing of bleached and unbleached pulps

The control (100% chemically treated) bleached and unbleached pulp samples, biopulped (87% alkali dose) bleached and unbleached samples were washed and used further for the handsheet preparation, using the TAPPI method. These handsheets were used further for testing physical, strength-related, and optical properties, using various TAPPI methods (optical properties like brightness, whiteness and yellowness, method T217 wd-77 (2004); physical properties such as burst index, method T403 om-10 (2010); breaking length, T494 om-01 (2001); tear index, T414 om-04 (2004); viscosity, T 230 om-99 (1976); double fold number, T 511 om-02 (2002); pulp freeness (SCAN-C 19:65) and Gurley porosity T 460 om-02 (2002).

Statistical analysis

Each experiment was repeated six times, and, in each repeat, each sample in triplicate was tested to assess the effect of various biopulping conditions on sugarcane bagasse and also to find out the significant difference between control and test. Results are the mean of each experiment (average and standard deviation values are from triplicates of six repeats, as mentioned above).

Results and discussion

Assessment of biopulping parameters

Essential factors that influence the effectiveness of sugarcane bagasse biopulping have been examined such as bagasse with liquid ratio, enzyme dosage, pH, treatment time, temperature and surfactant used during pulping. Evaluation of biopulping was performed by checking the release of hydrophobic compounds, lignin, and phenolic compounds by calculating the reducing sugar content released in the filtrates. Biopulping capacity was found to be optimal at 10% consistency of sugarcane bagasse (Fig. 1, i). Similarly, in the case of pulping wheat and rice straw, pulp consistency of 1:10 (g/mL) was found to be optimum (Varghese et al. 2020; Nagpal et al. 2021). The highest sugar release was obtained at pH 8.5 (Fig. 1, ii). Pulping efficiency was also maximum at pH 8.5 (Varghese et al. 2020; Nagpal et al. 2021). Biopulping results indicated that the xylanase–pectinase dosage of the 200–60 IU/g sugarcane bagasse sample was enough for effectual treatment (Fig. 1,iii). A booster dose of enzymes, as a second shot, could not enhance the effectiveness of biopulping. In the case of wheat and rice straw, a crude pectinase–xylanase dosage of 120–400 IU/g was found to be optimum (Varghese et al. 2020; Nagpal et al. 2021). A temperature of 55 °C and a processing period of 180 min were optimum for sugarcane bagasse with crude xylan–pectinolytic enzymes (Fig. 1, iv, v). Similar treatment time and temperature ranges have been reported in case of wheat and rice straw (Varghese et al. 2020; Nagpal et al. 2021). Biopulping of sugarcane bagasse has been done at a pulp consistency of 10% (v/v), pectinase dosage of 60 IU/g, time 60 min, 55 °C, pH 9.0 (Liu et al. 2017). Tween 80 concentration of 1.0% (v/v) was sufficient to extract maximal reducing sugar from sugarcane bagasse (Fig. 1, vi). In case of wheat and rice straw, Tween 80 concentration of 0.75% and 1% was best for optimal biopulping (Varghese et al. 2020; Nagpal et al. 2021). The enzymatic pretreatment released 84.841 ± 3 mg/g of reducing sugars from bagasse samples. The optical density profile for the release of various impurities (lignin, phenolic and hydrophobic compounds) under various biopulping conditions is shown in Fig. 2. This also confirms the efficacy of xylan–pectinolytic enzymes.

Fig. 1.

Fig. 1

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Effect of bagasse-to-liquid ratio (i), pH (ii), enzyme dose (iii), temperature (iv), retention time (v) and Tween 80 concentration (vi) on sugarcane bagasse biopulping

Fig. 2.

Fig. 2

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Optical density (O.D.) profile of bagasse fiber-free filtrates obtained after biopulping, with respect to control

Microscopic analysis of biopulped and raw bagasse samples also supports the effectiveness of enzymes. In the case of biopulped samples, the cell walls were damaged, indicating that xylan and pectin have been broken by enzymes, whereas the surface of raw samples was not damaged and was smooth (Fig. 3).

Fig. 3.

Fig. 3

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Microscopic pictures of (i) bagasse sample and (ii) bagasse sample after biopulping

Comparison of biopulped and chemically pulped samples

Both enzyme-treated (test) and non-enzyme-treated (control) samples were chemically pulped using the same conditions, to assess the additional effect of biopulping (Table 1). Kappa number of sugarcane bagasse (pectinase-treated) was decreased to 17.8% after biopulping (Liu et al. 2017). The unused active alkali content in black liquor was also improved from 1.12 to 2.40 g/L, brightness increased by 23.18% and total solids decreased to 12.64% in biopulped samples, in comparison with non-biopulped (100% chemically treated) samples. Biopulping increased the yield of screened pulp by 10.66% and rejects decreased by 58.76% (Table 1).

Table 1.

Effect on alkali dose after biopulping of sugarcane bagasse

Conditions Chemical Enzyme-treated bagasse
100 100% 95% 93% 90% 87% 85%
Alkali dose (%) 16 16 15.2 14.8 14.4 13.92 13.6
Unscreened pulp yield (%) 55 60.2 60.4 60.7 60.9 61.13 61.41
Rejects (%) 0.97 0.40 0.46 0.56 0.61 0.67 0.78
Screened pulp yield (%) 54.03 59.79 59.93 60.13 60.29 60.46 60.63
Pulp brightness (%ISO) 33.26 40.97 38.92 36.75 35.91 34.71 33.14
Kappa no. 20.93 10.93 12.8 15.4 18.6 20.87 22.33
Unused alkali (g/l) 1.12 2.40 2.14 1.79 1.46 1.20 1.09
Total solids (%) 11.79 10.30 10.26 10.21 10.18 10.14 10.11
pH of black liquor 11.04 12.21 12.01 11.82 11.55 11.27 11.00
Physical parameters
 °SR 15 ± 0.20 13 + 0.22
 GSM 60.4 ± 0.72 59.7 + 0.73
 Bulk 1.78 ± 0.01 1.75 + 0.02
 Gurley porosity (s) 40 ± 0.41 47 + 0.45
 Burst index (kN/g) 2.95 ± 0.02 3.97 + 0.03
 Double fold 41 ± 1 53 + 1
 Tear index (mNm2/g) 7.58 ± 0.06 8.16 + 0.07
 Viscosity (cP) 17.4 ± 0.16 19.0 + 0.22
 Breaking length (m) 3044 + 3.2 3386 + 4.3
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Bold values indicates that, results are better with 87% alkali dose

In order to get the decline in alkali content after biopulping, the different concentrations of alkali, 95%, 93%, 90%, 87% and 85%, were tried to the biopulped samples, which resulted in 13% reduction of alkali dose and also higher yield of pulp was obtained (Table 1). Varghese et al. (2020) and Nagpal et al. (2021) have observed 12% and 10% decrease in the consumption of alkali dose in biopulped samples. Liu et al. (2017) recorded a 1% reduction in alkali load, relative to control in chemical pulping of pectinase-treated bagasse of sugarcane. This method of biopulping using enzymes renders it environment friendly. The explanation for reduced alkali usage (87%) is the concurrent presence of pectinase and xylanase, breaking both pectin and xylan, present in bagasse, thereby increasing the penetration of the pulping chemicals into lignin sheet of sugarcane bagasse. The biopulping method improved the delignification and increase the dissemination of chemicals into the bagasse, removed the degraded lignin and ultimately resulted in less chemical demand.

Table 1 clearly displays the result of biopulping treatment on various properties of unbleached pulps. Considerable increase in tear factor (7.65%), breaking length (11.23%), double fold (29.26%), burst factor (34.57%), viscosity (9.19%) and gurley porosity (17.50%) values of unbleached biopulped samples was seen in comparison with control samples (Table 1).

After pulping, bleaching of both enzymes plus chemically treated pulp (87% chemical used) and chemical pulp (100% chemical used) was done. The biotreated samples showed an enhancement in brightness (7.53%) and less consumption of ClO2 after the bleaching stage (Table 2). In bleaching stage EP, brightness and whiteness values increased by 2.42% and 9.16%, and a decrease in yellowness and kappa number of biopulped samples by nearly 17.15% and 16.27%, respectively, were observed as compared to control samples. Similarly, an enhancement in brightness and whiteness and reduction in yellowness (8.54%) values was observed after the bleaching stage, D1, in the case of biopulped samples (Table 2). After the completion of the D0EPD1D2 bleaching phases, the yellowness value decreased by 22.61% in biopulped-bleached samples, in comparison with the chemically pulped-bleached bagasse samples. The consumption of ClO2 was also reduced by 9.2% in biopulped samples, along with no requirement for the bleaching stage, D2. In the case of rice straw, the D2 stage of bleaching is also not required in case of biopulped samples (Nagpal et al. 2021). An increase in brightness was also seen in pectinase-treated sugarcane bagasse (Liu et al. 2017).

Table 2.

Properties of soda-Anthraquinone pulp after biopulping and bleaching stages

Conditions and optical parameters Pulping alkali dose (100%) Biopulping + 87% pulping alkali dose
Kappa no. 20.93 20.87
Do (ClO2%) 2.066 2.066
ClO2 utilized (%) 1.849 1.692
Brightness (% ISO) 56.92 61.21
Alkali (%) 2.44 2.44
H2O2 (%) 0.6 0.6
K. no. 4.3 3.6
Brightness (% ISO) 81.59 83.57
Whiteness (CIE) 60.36 65.89
Yellowness (ASTM) 11.54 9.56
D-1 (ClO2%) 0.7 0.7
ClO2 utilized (%) 0.62 0.55
Brightness (% ISO) 87.72 88.96
Whiteness (CIE) 78.97 81.35
Yellowness (ASTM) 4.80 4.39
D-2 (ClO2%) 0.3 0.3
ClO2 utilized (%) 0.22 0.12
Brightness (% ISO) 88.71 89.04
Whiteness (CIE) 81.74 82.46
Yellowness (ASTM) 3.98 3.08
Physical properties
 °SR 18 ± 0.17 18 ± 0.16
 GSM 60.7 ± 0.70 60.06 ± 0.71
 Bulk 1.76 ± 0.02 1.77 ± 0.03
 Gurley porosity (s) 40 ± 0.41 54 ± 0.46
 Tear index (mNm2/g) 8.50 ± 0.05 8.71 ± 0.06
 Burst index (kN/g) 2.55 ± 0.04 3.51 ± 0.03
 Double fold 45 ± 1 62 ± 2
 Breaking length (m) 3982 ± 3.8 4587 ± 4.5
 Viscosity (cP) 8.2 ± 0.11 10.1 ± 0.12
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The biopulped samples (with 87% alkali dose) had a higher tear index (2.47%), breaking length (15.19%), double fold (37.77%), gurley porosity increased by 35%, along with higher viscosity (23.17%) and higher burst index (37.64%), under the conventional bleaching conditions, in comparison with chemically pulped samples with 100% alkali dose (Table 2). Strength properties of the sugarcane pulps were enhanced, breaking length, burst factor and tear factor by 17.1%, 16.5%, 7.0%, respectively, after pectinase pretreatment (Liu et al. 2017). Similarly, the strength-related and optical properties also improved in the case of wheat and rice straw using xylanase–pectinase enzymes (Varghese et al. 2020; Nagpal et al. 2021).

A summary of the production of eco-friendly and better-quality sugarcane bagasse paper using crude xylanase and pectinase biopulping strategy is depicted in Fig. 4.

Fig. 4.

Fig. 4

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Production of eco-friendly and better-quality sugarcane bagasse paper using crude xylanase and pectinase biopulping strategy

Conclusion

This is the first study, showing the potential of crude xylan–pectinolytic enzymes in pulping of sugarcane bagasse. Xylan–pectinolytic pretreatment can make bagasse soda-AQ pulping more effective and environmentally safe, together with the production of better-quality paper. Moreover, enzymes simultaneously produced from a bacterial isolate also decrease production cost and ultimately the application cost. Thus, biopulping of sugarcane bagasse using xylan–pectinolytic treatment is a feasible, pollution-reducing approach for the paper industry.

Acknowledgements

The corresponding author is thankful to ACIRD, Yamunanagar, India, for utilizing their laboratory facilities.

Author contributions

RM (Corresponding Author): idea of concept, planning and designing of various experiments done for this manuscript. Experimental work done by the first author for this manuscript under the corresponding author’s supervision. DS (First Author): all experimental work mentioned in this manuscript done, at Research laboratory level, and also written the whole manuscript. SA: microscopic analysis of samples done by him and also confirmed the results of first author. RN: work done at paper industry. OPM & NKB: research work done, by Raksha Nagpal, at Paper Industry under their supervision.

Funding

The corresponding author is thankful to Department of Biotechnology, Government of India, for providing financial support (Grant number: BT/PR 20438/BCE/8/1220/2016 for 4 years).

Data availability

Not applicable

Declarations

Conflict of interest

There is no conflict of interest.

Research involving human participants and/or animals

No.

Informed consent

NA.

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