Bioresource Technology 155 (2014) 177–181

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Bioresource Technology

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Potential halophilic cellulases for in situ enzymatic saccharificationof ionic liquids pretreated lignocelluloses

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.12.101

⇑ Corresponding author. Tel.: +60 146646022; fax: +60 49798755.E-mail address: dachyar@unimap.edu.my (D. Arbain).

Ahmad Anas Nagoor Gunny a, Dachyar Arbain a,⇑, Rizo Edwin Gumba a, Bor Chyan Jong b, Parveen Jamal c

a School of Bioprocess Engineering, Universiti Malaysia Perlis, Kompleks Pusat Pengajian Jejawi 3, 02600 Arau, Perlis, Malaysiab Agrotechnology and Biosciences Division, Malaysian Nuclear Agency (Nuclear Malaysia), Ministry of Science, Technology and Innovation, Bangi, 43000 Kajang, Selangor, Malaysiac Bioenvironmental Engineering Research Center (BERC), Department of Biotechnology Engineering, Faculty of Engineering, International Islamic University Malaysia, 50728 Gombak,Kuala Lumpur, Malaysia

h i g h l i g h t s

� Aspergillus terreus UniMAP AA-6: a newly isolated halophilic cellulases-producing strain.� Thermotolerant properties of halophilic cellulases are improved with the increment of salt concentration.� Halophilic cellulases exhibits compatibility with ionic liquids.� Halophilic cellulases of A. terreus UniMAP AA-6 promise a single pot system for saccharification of ionic liquid pretreated lignocelluloses.

a r t i c l e i n f o

Article history:Received 14 November 2013Received in revised form 18 December 2013Accepted 22 December 2013Available online 2 January 2014

Keywords:Ionic liquidsCellulasesLignocellulosesSaccharificationHalophiles

a b s t r a c t

Ionic liquids (ILs) have been used as an alternative green solvent for lignocelluloses pretreatment.However, being a salt, ILs exhibit an inhibitory effect on cellulases activity, thus making the subsequentsaccharification inefficient. The aim of the present study is to produce salt-tolerant cellulases, with therationale that the enzyme also tolerant to the presence of ILs. The enzyme was produced from a locallyisolated halophilic strain and was characterized and assessed for its tolerance to different types of ionicliquids. The results showed that halophilic cellulases produced from Aspergillus terreus UniMAP AA-6exhibited higher tolerance to ILs and enhanced thermo stability in the presence of high saline conditions.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

A substantial amount of lignocellulosic materials such as agri-cultural residues, agricultural by-products, woody biomass areproduced annually worldwide. These materials are essentiallypotential raw materials for the production of various renewableproducts such are biofuel, biodegradable plastics, biosurfactants,enzymes, etc. (Pandey et al., 2000).

In general, the production of the renewable products fromlignocellulose biomass requires three steps; first, the pretreatmentsteps for removal of lignin and other waxy materials to exposecellulose. Second, the hydrolysis of the exposed cellulose to simplesugar by cellulases. Finally, the fermentation of simple sugar tovarious renewable products.

The pretreatment step is normally performed by using a strongacid or alkaline solution. However, they are not preferable due tocost and environmental issues. Ionic liquids (ILs), the liquid formof salt at room temperature have been recognized as an alternativegreen solvent for lignocelluloses pretreatment process (Mäki-Arvelaet al., 2010; Wang et al., 2012; Gunny and Arbain, 2013). This typeof salt has been shown to be effective in the liberation of cellulosefrom the complex structure of lignocellulose materials (Li et al.,2009; Xu et al., 2012; Moniruzzaman and Ono, 2012). While, ILsare effective for breaking down lignocelluloses, they can alsoinhibit cellulase enzymes used in the subsequent saccharification,thereby making the overall saccharification of cellulose inefficient.The inhibition might be related to the high salinity of ILs, whichhas the capacity to inactivate the enzymes by interfering withthe polypeptides folding of the enzymes (Turner et al., 2003; Zhaoet al., 2009; Salvador et al., 2010).

Due to the inhibition effect of ILs on enzyme activity, a washingprocess is required to remove residual ILs after the pretreatmentprocess. Large scale washing requires extra energy, thus incurring

178 A.A.N. Gunny et al. / Bioresource Technology 155 (2014) 177–181

more processing cost (Engel et al., 2010; Zhang et al., 2011). To dealwith this, some authors (Kamiya et al., 2008) have introduced theidea of in situ saccharification of cellulose in aqueous-ionic liquidmedia, whereby the pretreatment and saccharification process isperformed in a single-pot, thus skipping the expensive celluloseregeneration step. This work can be effectively performed if cellu-lases tolerant to ILs are used.

In view of the above discussion, it is important to produce cel-lulases which are compatible with the saline condition of ILs. Theenzyme can be produced from halophilic microorganisms relyingon their capability to secrete enzymes which are active in a highsaline environment (Oren, 2010). Due to the above facts, the aimof the present study is to produce cellulases from halophilic micro-organism which are compatible with ionic liquids. In this study,1-ethyl-3-methylimidazolium acetate [EMIM][Ac], 1-butyl-3-methylimidazolium acetate [BMIM][Ac] and 1-butyl-3-methylimi-dazolium chloride [BMIM][Cl] were used for preliminaryexperiments on the compatibility of halophilic cellulases with ionicliquids.

The approach taken in the present study comprises of four ma-jor steps, first, the isolation and screening of cellulase producingmicroorganism from coastal areas. Second, on evaluation of thecapability of salt-tolerant microbes for producing salt-tolerantcellulases. Third, characterization of the enzyme focusing on thestability at different salt and temperature conditions. Finally, oninvestigation of enzyme stability in the presence of differentconcentrations of ILs. These studies were conducted with thehypothesis that the newly-isolated halophilic strain wouldproduce ILs-tolerant cellulases.

2. Methods

2.1. Isolation and screening of cellulases-producing microbes

Cellulases-producing microbes were isolated using filter paperas carbon source in a modified way as explained by Shahriarinouret al. (2011). The positive microbes were screened for plate screen-ing using Gram’s Iodine agar media (Kasana et al., 2008). Coloniesshowing discoloration upon addition of Gram’s Iodine were takenas positive cellulases-producing strains and selected for furtherstudies.

2.2. Molecular identification of fungi

Fungal samples were identified by the sequencing of theinternal transcribed spacer (ITS) regions. To determine theidentity of the two fungal samples, the amplified ITS PCRproducts obtained from total genomic DNA using primer setITS1-F (50-CTTGGTCATTTAGAGGAAGTAA-30) Gardes and Bruns(1993) and ITS4 (50-TC CTCCGCTTATTGATATGC-30) White et al.(1990) were sequenced. The sequences obtained were compared tosequences in the GenBank database (http://www.ncbi.nlm.nih.gov).A homology search was performed using bioinformatics toolsavailable online; BLAST (www.ncbi.nlm.nih.gov/BLAST) (Altschulet al., 1997). The ITS sequence data for fungal sample AA-3 andAA-6 was deposited into the GenBank database under theaccession numbers of KF364667 and KF364668, respectively.

2.3. Capability evaluation of salt-tolerant microbes for producing salt-tolerant cellulases

The positive microbial strains were isolated and inoculatedseparately on agar medium containing 0–30% (w/v) salt concentra-tions (NaCl). The ability of the microbial strains to degradecellulose under different salt concentrations were qualitatively

estimated using the hydrolysis capacity (HC) value, i.e. the diame-ter ratio of the clearing zone and colony/sample (Taechapoempolet al., 2011). Trichoderma reesei RUT C-30 and cellulases (commer-cial cellulases purified from Aspergillus niger) were used as apositive control. The best cellulases-producing microbe was cho-sen on the basis of highest salt tolerance activity.

2.4. Growth media and conditions

The best isolates were grown at pH 7.1, temperature 30 �C and arotation speed of 150 rpm using the media compositions in g/l:KH2PO4,13.61; KOH, 4.21; Yeast extract, 1.98; MgSO4�7H2O, 0.25;FeSO4�7H2O, 0.0017; NaCl, 30 and Peptone, 1.98. The carbon sourcein the medium was CMC (5 g/l) (Shivanand et al., 2012). Crude en-zyme was prepared through removal of the cell by centrifugationat 10,000 rpm for 10 min at 4 �C. The harvested supernatant wasassayed for cellulase activity.

2.5. Cellulase assay

The total cellulase activity was determined by filter paper assay(FPase) using Whatman No. 1 filter paper strip with dimensions1.0 � 6.0 cm equivalent to 50 mg as a substrate. They were assayedaccording to standard International Union of Pure and AppliedChemistry (IUPAC) procedures recommended by Ghose (1987)and expressed as international unit (IU). One FPA is the concentra-tion of cellulase that can release 2.0 mg of glucose from 50 mg ofcellulose over a 60 min period. One unit of enzyme activity was de-fined as the amount of enzyme capable of releasing 1 lmol ofreducing sugar per minute under the assay conditions. The amountof reducing sugar was determined by dinitrosalicylic acid (DNS)according to the standard method (Miller et al., 1960), and glucosewas used as a standard.

2.6. Characterization of halophilic cellulases

2.6.1. Effect of salt concentration on cellulase activityFor the study of halostability, the cellulase was pre-incubated in

0.05 M citrate buffer (pH 4.8) with different concentrations of NaCl(0–20%, w/v) at 30 �C. Cellulase activity was determined for 1 h and24 h incubation times respectively.

2.6.2. Effect of temperature on cellulase activityFor the study of thermal stability, the enzyme was pre-incu-

bated at a different temperature (4–90 �C) in saline conditions(1–3 M). Cellulase activity was determined after a 1 h incubationperiod.

2.6.3. Stability of halophilic cellulases in the presence of ionic liquidsCellulase from the newly-isolated strain was used to check its

stability in ionic liquids. The relative enzyme activity was deter-mined at a different ILs concentration (0–20%, v/v) in 0.05 M citratebuffer (pH 4.8). The mixtures were incubated at 30 �C for 1 h.

3. Results and discussion

3.1. Isolation and screening of cellulases-producing microbes

Cellulases-producing microbes were enriched and isolated byusing filter paper as carbon sources. The two cultured samplesshowed positive results as the medium turned cloudy and the filterpaper became degraded. The positive microbial strains were iso-lated and screened using Gram’s Iodine agar media. The strainsformed a hydrolysis zone around their colonies indicating theirability to secrete extracellular cellulases. Based on the morphology

Fig. 2. Effect of different salt concentrations on the HC value of each sample.

Fig. 3. Relative cellulase activity of A. terreus UniMAP AA-6 at different saltconcentrations after 1 h and 24 h.

A.A.N. Gunny et al. / Bioresource Technology 155 (2014) 177–181 179

and growth pattern, these strains were identified as fungi andlabelled as UniMAP AA-3 and UniMAP AA-6.

3.2. Molecular identification of fungi

Phylogenetic analysis revealed that fungal sample UniMAPAA-3 and UniMAP AA-6 showed a strong resemblance to Penicilliumsp. PSF39 (HQ850362, 98%) and Aspergillus terreus strain C1-11(JQ717316, 99%), respectively (Fig. 1).

3.3. Capability evaluation of salt-tolerant microbes for producing salt-tolerant cellulases

The effect of salt concentration (NaCl) on the ability of the iso-lated strains to secrete cellulases was evaluated using the hydroly-sis capacity (HC) value as presented in Fig. 2. Both A. terreusUniMAP AA-6 and Penicillium sp. UniMAP AA-3 strains exhibitedhigher HC values compared to T. reesei RUT C-30 (commercialstrain) and cellulases from A. niger (commercial cellulases) withinthe studied range of NaCl concentrations. This shows the halophiliccharacteristic of the strains which favour both growth and activityin saline conditions (Enache and Kamekura, 2010). In addition, thedegradation of cellulose at elevated NaCl concentrations indicatesthe capability of both strains to secrete salt-tolerant cellulases inan extracellular manner. A. terreus UniMAP AA-6 was selected forfurther studies since this strain had the highest HC value at allelevated NaCl concentrations.

3.4. Characterization of halophilic cellulases

3.4.1. Effect of salt concentration on cellulase activityThe effect of salt concentration on cellulase activity was ana-

lyzed by exposing the enzyme to different concentrations of NaClfor a period of time. It was found that high halotolerant stabilityof the cellulases was observed from A. terreus UniMAP AA-6, as itretained more than 80% over a broad range of NaCl concentrations(5–20%, w/v), even after 24 h incubation (Fig. 3). This observationcan be attributed to the presence of excess acidic amino acids onthe cellulases which provide a greater charge on the enzymesurface and resist any aggregation effect upon contact with saltmolecules caused by interaction with each molecules (Dansonand Hough, 1997; Begemann et al., 2011). The enzyme showedoptimal at 15% NaCl concentration. This concentration is slightlyhigher than the previous report on the marine halophilic cellulaseswhich showed optimal response at 10% NaCl concentrations

Uni Penicilliu

Penic Pe83

57

65

55

100

8

0.01

Fig. 1. Neighbor joining phylogenetic tree showing the position of fungal samples Uninumbers for the sequences used are shown in parentheses after the strain designationanalysis of 1000 replicates. The scale bar indicates 0.01 substitutions per site.

(Annamalai et al., 2011). Halophilic enzyme usually works opti-mally at high salt concentration conditions due to stimulation oftranscription and translation activities of the enzyme (Averhoffand Müller 2010). These studies confirm the moderate halophilicnature of the fungus strain and its capability to tolerate differentlevels of salinity.

3.4.2. Effect of temperature on cellulase activityThe heat stability of halophilic cellulases in different salt

concentrations is presented in Fig. 4. The results showed thathalophilic cellulases are stable at a wide range of temperatures(from 4 �C to 40 �C). There was a gradual decrease in the relativeactivity of the halophilic enzyme when the temperature was

Aspergillus terreus ATCC 10690 (KF278453) Aspergillus terreus DSM 5770 (KF154418) Aspergillus terreus ATCC 1012 (AY373871) Aspergillus sp. 7 BRO-2013 (KF367546) Aspergillus terreus C1-11 (JQ717316)UniMAP AA-6 (KF364668)

Penicillium sp. MAB-2010a (HQ829057) Penicillium sp. BE (GU566206)

MAP AA-3 (KF364667)m janthinellum GZU-BCECD8 (GU565141)

illium sp. PSF39 (HQ850362)nicillium janthinellum P11.16 (EU833221)

9

MAP AA-3 and UniMAP AA-6 relative to members of related genus. The accession. Bootstrap values are shown for each node that had >50% support in a bootstrap

Fig. 4. Stability of halophilic cellulases at various temperatures in different saltconcentrations.

180 A.A.N. Gunny et al. / Bioresource Technology 155 (2014) 177–181

increased from 40 �C to 80 �C. The fact that the enzyme retainedmore than 55% of activity at 70 �C showed the thermophilic char-acteristic of the enzyme. These results correlate to the previousreports (Oren, 2003; Mesbah and Wiegel, 2005), which were high-lighting that some halophilics enzymes are able to exhibit thermo-philic characteristics. Furthermore, the results demonstrate thatthe halophilic enzyme appeared to be more stable at elevated NaClconcentrations. These findings suggest that the addition of saltimproves the stability of halophilic cellulases at high temperatures.This may be due to an increase in the surface charge, ionic interac-tion and changes in cytoplasmic membrane which adapt to hightemperature conditions (Karan et al., 2012).

3.5. Stability of halophilic cellulases in the presence of ionic liquids

Halophilic cellulases are further characterized by their stabilityin the presence of ILs. Fig. 5 shows that after 1 h of incubation, thecellulase activity was found to be highest at 10% (v/v) ILs concen-tration for all the types of ILs. This result is comparable to the workreported by Trivedi et al. (2013), who found that salt-tolerant cel-lulases produced from isolated marine bacteria were stable at 5%(v/v) ILs concentration. The results demonstrate the stability ofhalophilic cellulases in elevated ILs saline conditions. Among theILs tested, [EMIM][Ac] showed the best compatibility with halo-philic cellulases as compared to the other types of ILs. This maybe due to the low viscosity of [EMIM][Ac] which have a lowerimpact on the interaction between the enzyme and the substrate(Romero et al., 2008; Samayam and Schall, 2010). On the otherhand, [BMIM][Ac], which is more viscose than [EMIM][Ac], reducesthe diffusion rate of the enzyme, thus resulting in lower cellulaseactivity (Bose et al., 2012).

Fig. 5. Effect on cellulases activity incubate at different concentrations on differenttypes of ionic liquids.

In addition, the halophilic enzyme showed a tolerance of up to20% (v/v) of [BMIM][Cl] with more than 50% of relative activity,which resulted in an improvement compared to the previous study(Turner et al., 2003) which reported that the Cl-ion of [BMIM][Cl]induced an inactivation of cellulases from T. reesei. This resultdemonstrates that the halophilic enzyme exhibits special charac-teristics which enable the enzyme to thrive in the high salineconditions of ILs.

4. Conclusion

The present study has shown that cellulases from halophiles A.terreus UniMAP AA-6 exhibit excellent halotolerant properties;moreover, the halophilic enzyme shows thermotolerant character-istics. Interestingly, the thermotolerant properties are increasedwith salt concentration. As expected, the enzyme showed stabilityand compatibility in the presence of different types of ILs, wherebyup to 10% (w/w) of ILs can be tolerated. This compatibility betweenhalophilic cellulases can be exploited for the in situ saccharificationof ionic liquid pretreated lignocellulose biomass. It is anticipatedthat both halophilic cellulases and ILs will prove effective for ligno-cellulose saccharification and its subsequent products.

Acknowledgement

The authors thank to School of Bioprocess Engineering, Univer-sity Malaysia Perlis for their support in this research.

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