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International Biodeterioration & Biodegradation 97 (2015) 143e150

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International Biodeterioration & Biodegradation

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

Biopulping of bagasse by Cryptococcus albidus under partiallysterilized conditions

Anjali Singhal a, Prashant Kumar Jaiswal a, b, Indu Shekhar Thakur a, *

a School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110 067, Indiab Department of Surgery, Montreal General Hospital, Montreal, Canada

a r t i c l e i n f o

Article history:Received 23 August 2014Received in revised form13 October 2014Accepted 15 October 2014Available online

Keywords:BagasseBiopulpingCryptococcus albidusFT-IRPartial sterilizationSEM

* Corresponding author. Tel.: þ91 11 26704321, þ26717586.

E-mail addresses: indushekhart@gmail.com(I.S. Thakur).

http://dx.doi.org/10.1016/j.ibiod.2014.10.0110964-8305/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Cryptococcus albidus was used for biopulping bagasse under partially sterilized conditions to access itspotential industrial application in the pulp and paper industry. Enzyme analysis of C. albidus treatedbagasse samples was carried out at different time periods (days 15, 30, and 60). Another set of bagassewas maintained in similar culture conditions without C. albidus inoculum (control) to assess the effect ofC. albidus treatment. The proportion of cellulose degrading enzymes was found to be much lower (3.0e3.5%) as compared to the control samples (21e56%). Scanning electron microscopy (SEM) clearlydemonstrated surface colonization and pit formation. Fourier-transformation infrared spectroscopy (FT-IR) indicated the chemical modification of bagasse. The signature peak for cellulose was found to beprominent in C. albidus treated samples. Denaturing gradient gel electrophoresis (DGGE) confirmed thepresence of C. albidus throughout the experiments. It was evident that C. albidus was able to suppress thegrowth of a native population. After 60 days both control and treated bagasse were given a kraft pulpingtreatment. The kappa number of C. albidus treated bagasse decreased by 42% while in control samples itwas found to be decreased by 39% only. There was increase in viscosity/kappa number ratio aftertreatment.

© 2014 Elsevier Ltd. All rights reserved.

Introduction

The pulp and paper sector represents one of the energy inten-sive and highly polluting sectors of the Indian economy. To keep upwith the increasing demand for pulp and paper and to meetstringent environmental regulations, the industry has beenconstantly looking towards technological improvements. Pollutionload can be reduced by modifying production process and by usingcellulose rich agricultural waste as raw material. Process modifi-cation involves changes in manufacturing processes viz. priortreatment of raw materials that lead to decreased consumption ofwater as well as energy and finally led to the formation of less toxicmaterial in the effluents. Biopulping and biobleaching are suchprocess modifications that involve the treatment of raw materialwith a fungus having lignolytic enzymes. Subsequently, the pro-cessing of material take place by mechanical or chemical pulpingand bleaching, thereby reducing the chemical, energy and water

91 11 26191370; fax: þ91 11

, isthakur@hotmail.com

requirements. The treatment of rawmaterial by fungus or enzymescan be solid state or liquid state fermentation (Pandey, 2003).

The concept of biopulping and biobleaching is based on theability of fungi to colonize and degrade lignin selectively in woodthereby leaving cellulose relatively intact. Some fungal speciesremove lignin more efficiently than other wood components; suchdegradation pattern is known as selective lignin degradation ordelignification. The delignification of wood facilitates its softeningin a subsequent pulping process (Ferraz et al., 2008). Wood cellwalls are made upmostly of cellulose, hemicellulose and lignin. Thetensile strength of wood fibers is primarily determined by celluloseand hemicelluloses, while lignin mediates adhesion among the fi-bers (Levin et al., 2007). Lignin is an extremely complex, threedimensional heteropolymer made up primarily of phenyl propaneunits. Due to large size of the polymer, degradation should takeplace in an extracellular fashion. Lignin requires aerobic conditionsfor degradation as carbonecarbon and ether bonds joining thesubunits together must be cleaved via an oxidative mechanism.Thus a very complex but non-specific enzyme system is requiredfor lignin degradation (Breen and Singleton, 1999). The ligninolyticenzyme system of most of the fungi is comprised of lignin peroxi-dase, manganese peroxidase, laccase and xylanase (Shah et al.,

A. Singhal et al. / International Biodeterioration & Biodegradation 97 (2015) 143e150144

2005; Vicentim and Ferraz, 2007; Singhal et al., 2013). In this studyCryptococcus albidus, a well known wood degrader under naturalconditions was used. The strain of C. albidus used here is known toproduce xylanase, laccase and manganese peroxidase in solid statefermentation (SSF) (Singhal et al., 2013).

The pulp and paper industry requireswood as rawmaterials andthus has high pressure on natural resources. The cutting of trees canbe reduced by substituting wood with cellulose rich agriculturalwasteas rawmaterial. India ranks second in sugarcaneproduction inworld (Yadav and Singh, 2011). The Indian Sugar Mills Association(ISMA) has projected sugar production for year 2013e14 around 25million tonnes for India (Jai, 2013). Bagasse is the by-product ofsugarcane industry. For each tonne of sugarcane crushed, about300 kg of bagasse is retrieved (Yadav and Singh, 2011). Thus theestimated bagasse production for year 2013e14might be around 7.5million tonne. In India a number of pulp and paper mills are usingbagasse for paper production. Tamil Nadu Newsprint and PapersLimited (TNPL) is the largest bagasse based Paper Mill in the worldconsuming about one million tonne of bagasse every year and pro-ducing 2,45,000 tonne paper per annum (tpa). (http://www.tnpl.com/DisplayPage.aspx?file¼about_us.htm).

The current study is focused on the use of C. albidus a goodproducer of ligninolytic enzymes, for biopulping of bagasse. Theproduction of ligninolytic enzymes, substrate colonization (SEM)and chemical modifications (FT-IR) were analyzed to study theprocess of biopulping. Biopulp was subjected to kraft pulping andthe effectiveness of biopulping was judged by estimating the kappanumber and viscosity. Changes in microbial community were alsostudied as the biopulping was carried out under partially sterilizedconditions. Finally, the presence of C. albidus was tracked by DGGEanalysis during biopulping.

Material and methods

Microorganism and culture conditions

The C. albidus was isolated from the sediments of Century pulpand paper mill, Lalkuan, India (Singhal and Thakur, 2009a). Potatodextrose broth was used for growing the fungus at 30 �C and125 revolutions per minute (rpm) at pH 5.0 for 4 days.

Pretreatment of bagasse for biopulping

Bagasse 600 g was collected from Century pulp and paper mill,Lalkuan, India. It was divided into six equal parts (100 g each). Ex-perimentswere set up in triplicate. Threeparts of 100geach (treatedbagasse), was steamed for 10 min to inactivate the native microbialpopulation. This helps in establishment of C. albidus. Steaming waspreferredover the sterilization to reduce the operational costs (Scottet al., 1997; Hunt et al., 2001). Steam treatment was given usingsteam cooker at atmospheric pressure. After steaming, bagasse wasallowed to cool. It was then inoculated with 10% fungal inoculum(solid state) alongwith the culturemedium. A study has shown thatthe growth of fungi is linear with time when culture media is notadded however the growth becomes exponential if culture media isadded along with the fungi (Wall et al., 1993). Other three parts(100 g each) of bagasse was kept as a control. Control bagasse wasalso steamed for 10 min, cooled and was incubated with equalamount of culture media (uninoculated) to maintain similar condi-tions. The flasks were kept at 30 �C with 60% moisture content.Sampling was done on 15th, 30th and 60th day for enzyme esti-mation and DGGE. Scanning electronmicroscopy (SEM) and Fouriertransformation infrared spectroscopy (FT-IR) were conducted on60th day. The treated/control bagasse was used for kraft pulping.Kappa number and viscosity were determined.

Ligno-cellulolytic enzymes analysis

For enzyme analysis, enzymes were extracted from the controland fungus treated bagasse. Two types of buffers were used forextraction of enzymes: 50 mM sodium citrate buffer, pH 5 and50 mM sodium acetate buffer, pH 4 having Tween 60 (0.1 g/L). Atotal of 10 g substrate was removed aseptically and extraction wascarried out using 50 ml buffer. The mixture was shaken at 75 rpmand 4 �C for 4 h (Heidorne et al., 2006). Sodium citrate buffer wasfiltered throughWhatman grade 1 filter paper and filtrate was usedfor estimating cellulose and hemicellulose degrading enzymes:xylanase (Xyl; hemicellulase), carboxymethyl cellulase (CMCase;endoglucanase) and filter paper activity (FPase; total cellulose ac-tivity) (Adsul et al., 2004). For estimation of xylanase, CMCase andFPase sugar estimation was done by DNS method. The substrate forxylanase was oat meal xylan, CMCase was carboxymethyl celluloseand for FPase was filter paper (Adsul et al., 2004). Sodium acetatebuffer was filtered through Whatman no 1 filter paper and filtratewas used to estimate the activity of lignin degrading enzymes:lignin peroxidase (LiP) (Tien and Kirk, 1983), Mn-dependentperoxidase (MnP) (Glenn et al., 1986) and laccase (Lac) (Niku-Paavola et al., 1988). The substrate for LiP was vertryl alcohol, forMnP was Mn malonate and for Lac was ABTS. For estimating LiPactivity standard curvewasmade using veratryaldehyde. Themolarextinction coefficient of Mn (III) malonate at 270 nm is11,590 M�1 cm�1 (Wariishi et al., 1992) and for ABTS2þ ions at436 nm is 29,300 M�1 cm�1 (Mansur et al., 2003). Enzyme activityfor all the enzymes studied was calculated as mM change per min insubstrate concentration by the enzyme produced per g of bagasse.

SEM analysis

The bagasse from control and treated flasks was washed with0.1 M phosphate buffer saline and shaken slowly with buffer for10 min to remove any precipitate or adhering particles. They werefixed overnight using 0.1 M phosphate buffer saline having 1%glutaraldehyde and 2% para-formaldehyde at 4 �C. Further, bagassewas dehydrated in a series of ethanol-water solution (30, 50, 70,and 90% ethanol, 5 min each), and at critical point it was dried inCO2 atmosphere for 20 min. Mounting was done on aluminumstuds provided with adhesive disk, a line of silver paint was appliedon two sides of each sample. A thin layer of gold was coated usingBio-rad Polaron Gold/Silver Sputter coating unit for 30 min. Coatedwood chips were viewed at EHT 15 kV with scanning electronmicroscope (ZEISS Scanning Electron Microscope EVO 50). Aworking distance of 10/10.5 mm was maintained. Rate of scanningand magnification was varied as required (Srivastava and Thakur,2006).

FT-IR analysis

The bagasse from both control and treated flasks was rinsedwith autoclaved double distilled water and dried at 60 �C to have aconstant weight. The bagasse was grounded and sieved through a4 mm sieve. The powder was then mixed with spectroscopy gradepotassium bromide (KBr) in a ratio of 1:99 (w/w). Spectra wereacquired in transmission mode. Scan was performed from 450 nmto 4000 nm with Perkin-Elmer 1600 series FTIR Spectrometer(Nujol, KBr disks) (Singhal et al., 2013).

DGGE analysis for tracking C. albidus

Control and treated bagasse samples were analyzed on 15th,30th and 60th day. Two gram substrate was removed asepticallyand inoculated in potato dextrose broth (50 ml) for 7 day at 30 �C

A. Singhal et al. / International Biodeterioration & Biodegradation 97 (2015) 143e150 145

and 125 rpm. The sample was centrifuged at 8000 rpm for 20 min.The supernatant was discarded and pellet was used for analysis.

Amplification of DNADNA was extracted using the method described by Doyle and

Doyle (1987). Internal transcribed spacer regions, ITS1, 5.8S andITS2, were amplified using forward primer 50 TCCGTAGGT-GAACCTGCGG 30 (ITS1) and reverse primer 50 TCCTCCGCTTATT-GATATGC 30 (ITS4) (Hortal et al., 2006). Reaction mix (25 ml) wasprepared using buffer with MgCl2 (2.5 mM), dNTP (1 mM each),ITS1 (forward primer 10 mM), ITS4 (reverse primer 10 mM), Taqpolymerase (5 U/ml), DNA (20e50 ng) and the final volume wasmade up by water. The amplification was carried out at 95 �C for3 min, followed by 35 cycles of amplification at 95 �C for 1 min,55 �C for 1 min and 72 �C for 1 min and a final extension of 5 min at72 �C. Finally the product was stored at 4 �C until analysis. Further,ITS1 region was amplified for DGGE analysis. For this, nested PCRwas performed using the template amplified by ITS1 and ITS4primers that included ITS1 region. For amplification of ITS1 region,forward primer (ITS1) 50-TCCGTAGGTGAACCTGCGG-30 and reverseprimer (ITS2) 50-GCTGCGTTCTTCATCGATGC-30 were used (Renet al., 2004). A 40 bp GC clamp was attached to the ITS1 primer(Muyzer et al., 1993). The reaction mix was prepared as shownabove using these primers. The amplification was carried out at95 �C for 3 min, followed by 25 cycles of amplification at 95 �C for30s, 59 �C for 1 min and 72 �C for 30 s and a final extension of 5 minat 72 �C. The amplified products were run on 0.7% agarose gelshaving EtBr (1 mg/ml) (Singhal and Thakur, 2009a).

DGGE analysisDGGE was performed according to the method used by

Michaelsen et al. (2006). Briefly, 0.5 � TAE (20 mM Tris, 10 mMacetate, 0.5 mM Na2EDTA; pH 7.8) with 8% (w/v) acrylamide gelcontaining a gradient of denaturants (7M urea and 40% formamide)was used in a Bio-Rad DCode™ system. The gradient of denaturantswas 30%e50% in the direction of electrophoresis. Electrophoresiswas performed for 2 h at 150 V. After completion of electrophoresis,gels were stained in EtBr (1 mg/ml) and documented with Geldocumentation system, Model AITM 26 (Alpha Innotech Corpora-tion) (Sahni et al., 2011).

Kraft pulping, estimation of kappa number and viscosity

Kraft pulping was performed for both control and treatedbagasse samples. The kraft cooking liquor contained 16% activealkali and 25% sulfidity. The bagasse to cooking liquor ratio was1:10. The pulping temperature was ambient to 175 �C for 105 minand maintained at 175 �C for 60 min. Pulp tests for kappa numberand viscosity were performed according to the methods given byTechnical association of the pulp and paper industry (TAAPI,Atlanta, USA): Kappa number T 236 cm-85 (Tappi, 1985) and vis-cosity T230 om-89 (Tappi, 2004).

Statistical analysis

The data of enzyme production during biopulping and changesin kappa number and viscosity during kraft pulping were main-tained in Microsoft Excel 2002 spreadsheets (Microsoft Corp.,Redmond, WA). Statistical analysis was done using SPSS 10.0 (SPSS,Inc., Chicago, IL). ANNOVA was done at 95% confidence limit.Graphs were prepared by SigmaPlot 2001 (SPSS, Inc., Chicago, IL)(Singhal and Thakur, 2009b).

Results and discussion

Ligno-cellulolytic enzymes analysis

Bagasse was inoculated with C. albidus after treatment withsteam for 10 min to reduce the population of indigenous microbes.The production of enzymes was found to be much lower in controlsamples as compared with treated samples indicating suppressedgrowth of indigenousmicrobes (Fig.1). Bagasse is mainly composedof cellulose (32e48%), hemicellulose (19e24%) and lignin (23e32%)(Reddy and Yang, 2005). Enzymes produced in control as well astreated samples could degrade the cellulose (endoglucanase, totalcellulose activity), hemicellulose (xylanase) and lignin (Ligninperoxidase, manganese peroxidase and laccase). Since, the paper ismade up of cellulose, the aim was to achieve selective delignifica-tionwhere hemicellulose and lignin should be degraded more thancellulose. In C. albidus treated samples the proportion of cellulosedegrading enzymes was found to be much lower (3.0e3.5%) ascompared to the control samples (21e56%) (Fig. 1). Laccase enzymeandmelanin production is the characteristic feature of Cryptococcusspecies (Ikeda et al., 2002). Production of xylanases by C. albiduswas reported in 1970's (Notario et al., 1976). A study in 1980 re-ported that the C. albidus produces extracellular/intracellularinducible xyalanse and is unable to grow on cellulose or cellobiose(Biely et al., 1980).

On 60th day, xylanase (6.15 IU/g of bagasse) was found to be thedominant enzyme, followed by LiP (5.82 IU/g of bagasse), CMCase(4.13 IU/g of bagasse), FPase (2.74 IU/g of bagasse), laccase (2.13 IU/gof bagasse) and MnP (2.07 IU/g of bagasse) in the control samples(Fig. 2). However, in treated sample laccase (60.99 IU/g of bagasse)was found to be a dominant enzyme, followed by xylanase(33.74 IU/g of bagasse) on 60th day (Fig. 2). Production of MnPstarted after 15th day (5.60 IU/g of bagasse) (Fig. 2b). Activity ofCMCase and FPasewere observed to be 1.51 and 1.98 IU/g of bagassein treated samples, respectively (Fig. 2c). In C. albidus treatedbagasse samples, production of laccase and xylanase was found tobe 22 and 3 times higher than control. The increase in laccase(F(1,4) ¼ 681.54, p � 0.001)and xylanase (F(1,4) ¼ 406.46,p � 0.001) was statistically significant. At the same time, produc-tion of CMCase and FPase was found to be less than half. Thedecrease in CMCase (F(1,4) ¼ 1211.36, p � 0.001) and FPase(F(1,4) ¼ 22.53, p � 0.009) was statistically significant. The differ-ence in enzyme production in control and treated bagasse samplewas significant (p � 0.05) throughout the experiment (Fig. 2). Theproduction of enzymes varies with the microorganisms. Laccaseproduction as low as 1 U/g of bagasse by Lentinus polychrous L�ev. to90 U/g of ethanol treated bagasse by Pycnoporus cinnabarinus hasbeen reported by Meza et al. (2006) and Sarnthima et al. (2009).Similarly xylanase production 4 U/g of bagasse by Ganodermalucidum to 1597 U/g of bagasse in forced aeration bioreactor byThermoascus aurantiacus has been reported (Malarvizhi et al., 2003;Milagres et al., 2004).

Production of enzyme also depends on the type of substrateused. In a previous study, C. albidus was used for biopulping ofeucalyptus chips where maximum laccase productionwas found tobe 16.07 IU/g wood on 60th day (Singhal et al., 2013). Laccaseproduction in case of bagasse was almost four times higher thaneucalyptus. Studies have reported that bagasse can act as aninducer for laccase production (Hossain and Anantharaman, 2006).In a separate study eleven different inducers were screened forlaccase production by C. albidus in submerged fermentation wherebagasse was observed to be the most suitable inducer (219 IU/mgprotein) (Singhal et al., 2009).

The duration for biopulping can vary from few days to months.In control samples, the total enzyme production was highest on

Fig. 1. Production of lignin þ hemicelluloses (xylanase, laccase, LiP and MnP) and cellulose (CMCase and Fpase) degrading enzymes during biopulping of bagasse by Cryptococcusalbidus under partially sterilized conditions on 15th, 30th and 60th day.

A. Singhal et al. / International Biodeterioration & Biodegradation 97 (2015) 143e150146

30th day while in C. albidus treated samples it was observed on60th day (Fig. 1). In a study by Koshy and Nambisan, (2011) paddystraw was biopulped by Pleurotus sp. Maximum production oflaccase and LiP was observed on 42nd day. Longer duration of 90days were observed when Ceriporiopsis subvermisporawas used forbiopulping Pinus taeda (De Souza-Cruz et al., 2004).

Fig. 2. Enzyme activity on days (a) 15th, (b) 30th, and (c) 60th: CMCase, FPase, xyla-nase, lignin peroxidase, LiP manganese peroxidase (MnP), and laccase. Bars showmeanvalue along with standard deviation. * shows statistically significant changes inenzyme production as compared with control samples.

SEM analysis for surface colonization of bagasse

The sugarcane bagasse was viewed at higher magnificationsusing SEM to obtain insight into structural changes caused by C.albidus treatment after 60 days. In control samples, fungal myce-lium growing on the surface was observed (Fig. 3a, b and c). Thesurface of control samples appeared compact, smooth and unal-tered. In treated samples, the growth of microorganisms was morewidespread (Fig. 3d, e and f). The surface of bagasse fibers wereeroded by microbes forming pits. This surface modification mightbe due to loss of lignin and hemicellulose that resulted inincreased porosity. Such changes can help in reducing chemicaland energy requirements during chemical and mechanical pulp-ing, respectively. Similar results were also observed in Panustigrinus treated sugarcane bagasse and composted agave bagasse(Costa et al., 2005; Iniguez et al., 2011). Disruption of bagasse fibersurface by formation of pits had been reported with use of acidand alkaline treatments as well (De Rocha et al., 2011; Muhammadet al., 2011).

FT-IR analysis for chemical modification of bagasse

The FT-IR spectra of bagasse (Fig. 4) clearly demonstrated thecharacteristic peak of lignocellulose biomass in percent trans-mittance. Increase in a peak (i.e., transmittance) signifies decreasein the substance represented by that peak. The bagasse spectrumwas influenced by the spectra of its three main biopolymers; lignin,hemicellulose, and a-cellulose (Adel, 2007). The broad absorptionaround 3340e3412 cm�1 relates to stretching of H-bonded OHgroups and at 895 cm�1 relates to b linkages of cellulose (Binodet al., 2012). There was decrease in broad adsorption band at3340e3412 cm�1. In control sample, the peak at 895 cm�1 wasmasked with hemicellulose. After fungal treatment, this peak wasclearly evident in treated sample indicating clear disruption ofhemicelluloses. Similar results were observed when sugarcanebagasse was treated with dilute sulfuric acid (Chen et al., 2011). Thepeak at 1737 cm�1 was related to the stretching of C]O in hemi-celluloses (Zhang et al., 2011). The transmittance increased at thispeak. The band near 1430 cm�1 showed aromatic skeletal vibra-tions combined with CeH in plane deformation for lignin. Therewas increase in transmittance in treated samples whichmay be dueto ring cleavage (Adel, 2007). Other signature peaks for lignin were1512 cm�1 (stretching of phenyl ring), 1636 cm�1 (stretching of C]O), 1370 cm�1 (Zhang et al., 2011). In treated samples, trans-mittance increased for these peaks. FT-IR analysis showed thatbagasse was chemically modified.

Fig. 3. Scanning electron microscopy (SEM) of control (a,b,c) and Cryptococcus albidus treated (d, e, f) sugarcane bagasse at different magnifications on 60th day. White arrow showspit formation. Black arrow shows different types of microorganisms growing on bagasse surface.

A. Singhal et al. / International Biodeterioration & Biodegradation 97 (2015) 143e150 147

DGGE analysis for tracking of C. albidus

It has been observed that the microbial strains performing verywell in laboratory conditions fail to perform in same manner underfield conditions. Even a slight contamination can lead to the failureof whole process. In most of the studies that have used steam topartially sterilize the raw material, growth of the fungi was notmonitored. According to studies by Ferraz and co-workers (Masarinand Ferraz, 2007; Fernando et al., 2009), Ceriporiopsis sub-vermispora and Phanerochaete chrysosporium were used for bio-pulping of eucalyptus under non-aseptic conditions. Ceriporiopsissubvermisporawas unsuccessful, while P. chrysosporiumwas able toestablish itself successfully. Thus it is very important tomonitor themicrobe during the whole process.

In this study steam was used to partially sterilize the bagasse.Untreated bagasse was used as negative control. The results ofDGGE analysis (Fig. 5) showed a very low microbial diversitydemonstrating the effective steam treatment of bagasse. C. albidussuppressed the growth of native microbial population and was

dominant throughout the experiment. The C. albidus isolate used inthis study was isolated from the drain carrying pulp and paper milleffluent where bagasse is used as a raw material (Singhal andThakur, 2009a). It was found to be associated with the bagassepresent in nature (De Azeredo et al., 1998). This might be the reasonfor successful colonization of bagasse by C. albidus.

Estimation of Kappa number and viscosity

The control and C. albidus treated bagasse was washed toperform the kraft pulping. The kappa number and viscosity wasmeasured before and after the kraft pulping treatment (Table 1). A22% reduction in kappa number was observed after C. albidustreatment of bagasse. Kraft pulping further decreased the kappanumber by 20%. Thus there was a total reduction of 42% in kappanumber of bagasse after combined treatment of C. albidus and kraftpulping. The reduction in kappa number was statistically signifi-cant (F(1,4) ¼ 22.81, p � 0.009). On the other hand, chemicalpulping reduced the kappa number by 39%. Similar results were

Fig. 4. Fourier e transform infrared (FT-IR) spectra of (a) control and (b) Cryptococcus albidus treated sugarcane bagasse. The spectra are in transmission mode. They cover thewavelength range from 450 nm to 4000 nm.

Fig. 5. DGGE profile of control and Cryptococcus albidus treated sugarcane bagasse.Lane 1 shows DGGE band for Cryptococcus albidus. Lanes 2e4: DGGE profile of fungalcommunity of treated samples on days 15, 30, and 60, respectively. Lanes 5e7 showDGGE profile of fungal community of control samples on days 15, 30, and 60, days,respectively. The bands of the fungus occurring repeatedly on different days arenumbered.

A. Singhal et al. / International Biodeterioration & Biodegradation 97 (2015) 143e150148

reported during biopulping of bagasse using Curvularia lunata LW6and Klebsiella aerogenes NCIM 2098 (Narkhede and Vidhale, 2005;Jha and Patil, 2013). Another study reported 28% reduction intime required for cooking during kraft pulping using Ceriporiopsissubvermispora for biopulping bagasse for 30 days (Costa et al.,2005). In current study, viscosity/kappa number ratio increasedafter C. albidus treatment and kraft pulping as compared withcontrol samples. This indicated lignin degradationwhile preservingfibers (Goncalves et al., 1998).

Table 1Delignification of bagasse by Cryptococcus albidus.

Sample kappa number Viscosity (cP) Viscosity/kappa number

Beforekraftpulping

Afterkraftpulping

Beforekraftpulping

After kraftpulping

Beforekraftpulping

Afterkraftpulping

Controlbagasse

36 ± 1.97 22 ± 0.56a 12 ± 1.42 10 ± 1.62 0.33 0.45

C. albidustreatedbagasse

28 ± 2.13 21 ± 1.23a 11 ± 1.33 10 ± 2.14 0.39 0.48

a Shows statistically significant difference.

A. Singhal et al. / International Biodeterioration & Biodegradation 97 (2015) 143e150 149

Conclusion

C. albidus produced various lignin degrading (MnP and Laccase)and hemicellulose degrading (xylanase) enzymes. Production ofcellulose degrading (CMCase and FPase) enzymes was significantlylow in C. albidus treated samples. Steam treatment was effectiveand SEM analysis shows surface colonization of bagasse. FT-IRanalysis confirmed cellulose enrichment of C. albidus treatedbagasse samples. C. albidus was able to establish itself successfullyin partially sterilized conditions. The ability of C. albidus to performwell under partially sterilized conditions makes it a suitablecandidate for industrial applications after optimization.

Acknowledgments

The authors thank University Grants Commission, India, andCouncil of Scientific and Industrial Research, India for providingSenior Research/Post Doc Fellowships. Authors thank Indian Insti-tute of Technology, New Delhi and Central Drug Research Institute,Lucknow for SEM and FT-IR analysis respectively. Authors thankreviewers for their suggestions.

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