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Bioresource Technology 132 (2013) 313319

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Detection of ionic liquid stable cellulase produced by the marine

bacterium Pseudoalteromonas sp. isolated from brown alga Sargassum
polycystum C. Agardh
Nitin Trivedi, Vishal Gupta, C.R.K. Reddy , Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" Characterized an ionic liquid stable

"

"

"

"

cellulase from marine

Pseudoalteromonas sp.
Activation and stabilization of
cellulase in the presence of ionic
liquids.
Retained catalytic activity even at
ionic liquid concentration of 20% (v/
v).
Enhanced activity and stability than
the commercial cellulase in ionic
liquids.
Offers a single step continuous
saccharication process in ionic
liquids.

a r t i c l e

i n f o

Article history:
Received 6 September 2012
Received in revised form 6 January 2013
Accepted 7 January 2013
Available online 22 January 2013
Keywords:
Cellulase
Cellulose
Energy
Enzyme stability
Ionic liquid

a b s t r a c t
An extracellular cellulase produced by marine bacterium Pseudoalteromonas sp. was studied for its activity and stability in six different ionic liquids (ILs) over a wide range of concentrations (120% v/v) and
compared with aqueous medium as control. Enzyme showed its optimal activity at 45 C and at pH 5
in control. Although the activity varied with the type of IL and its concentration used, the activity measured at 5% (v/v) was maximum with [EMIM]Br followed by [EMIM]Ac, [BMIM]Cl, [C2MIM][CH3SO3],
[BMIM][OTF] and [BMPL][OTF] with 115%, 104.7%, 102.2%, 98.33%, 93.84% and 92.67%, respectively,
and >80% activity at 15% (v/v) in all ILs. The enzyme stability at 5% (v/v) IL concentration for 36 h was
superior to commercial cellulase. The cellulase activity enhanced by 1.35- to 1.72-fold over control when
5% (v/v) IL based reaction medium with algal biomass was used and thus showed potentials for saccharication of biomass in a single step process.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Cellulose is the most abundant renewable carbon source available on the earth for production of feedstock chemicals of commercial value, if converted efciently to monomeric D-glucose units
(Klemm et al., 2005). Both chemical and enzymatic hydrolysis
Corresponding author. Tel.: +91 278 256 5801/256 3805x6140; fax: +91 278
256 6970/256 7562.
E-mail addresses: crkcsmcri@gmail.com, crk@csmcri.org (C.R.K. Reddy).
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.01.040

has been employed for effective conversion of cellulosic biomass

into fermentable sugar. The former method is currently being employed as most common means for producing mono-saccharides
from cellulose. Nevertheless, chemical hydrolysis often leads to
the accumulation of undesired non-sugar byproducts that poses
considerable problems in recovery of resultant products (Sasaki
et al., 1998). On the contrary, enzymatic hydrolysis overrides all
such hindrances and has been, thus, preferred over chemical
hydrolysis. However, the enzymatic hydrolysis of cellulose is largely regarded as counterproductive due to its lower solubility in

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N. Trivedi et al. / Bioresource Technology 132 (2013) 313319

aqueous medium. As a result, cellulose is being pre-treated with

various chemicals or solvent fractionations prior to enzymatic
hydrolysis.
Recently, ionic liquids (ILs) have been utilized as pre-treatment
solvent because of their higher cellulose dissolution potential over
other solvents. The recyclability of IL is another addendum to their
utility as pre-treatment solvent. Although ILs have better cellulose
dissolution potential, their inhibitory effects on enzyme activity
has been the major concern (Turner et al., 2003). Therefore, considerable efforts have been directed to explore the IL stable cellulolytic enzymes from various sources. Such enzymes not only allow
elimination of cellulose regeneration step but also enable to carry
out downstream conversion in a single-step continuous process. In
earlier studies, commercial cellulases were mainly investigated for
IL stability (Turner et al., 2003; Lozano et al., 2011; Wang et al.,
2011). Also, a few IL stable cellulases derived from metagenome
(Pottkamper et al., 2009) as well as from culturable microbes such
as Penicillium janthinellum (Adsul et al., 2009), Thermatoga maritime
and Pyrococcus horikoshii, (Datta et al., 2010), and Halorhabdus
utahensis (Zhang et al., 2011) have been reported. IL induced partitioning of protein from surrounding hydration shell and disrupts
the hydrogen and hydrophobic interactions leading to aggregation
of protein that inhibits the enzyme activity (Bose et al., 2010; Moniruzzaman et al., 2010; Constatinescu et al., 2010). The extracellular enzymes produced under high salt concentrations showed
modications such as excessive number of charged acidic amino
acids on their surface which prevent the formation of protein
aggregation through electrostatic repulsive charges at protein surface. Such adaptations are presumed to make the enzyme stable in
IL.
The present study was therefore undertaken to nd out an IL
stable cellulase from a marine bacterium isolated from brown seaweed Sargassum polycystum C. Agardh. The bacterial cellulase obtained was rst investigated for optimization of assay conditions
in aqueous medium and then tested for its activity and stability
in the presence of different ILs at various concentrations ranging
from 1% to 20% (v/v). Further, the salt tolerance of the enzyme
was also investigated under different concentration of NaCl ranging from 1% to 20% w/v. Subsequently, the activation and stabilization efciency of bacterial cellulase in ILs has also been validated
against commercial cellulase from Trichoderma viride (cellulase
Onozuka R-10, Yakult Pharmaceutical Ind. Co. Ltd, Japan).

The morphological and biochemical characterization of the

strain was performed and compared with the Bergeys Manual of
Systematic Bacteriology. The molecular identication of the bacterial strain was carried out by 16S rDNA sequencing following the
method described by Trivedi et al. (2011).

2.2. Cellulase production and activity assay

A bacterial suspension (2  107 CFU/mL) was inoculated in the
enzyme production medium containing, Articial Seawater Salt
(ASW): 3.5% w/v; CMC-Na: 2% w/v; and Yeast extract: 0.5% w/v.
The ask was incubated at 30 2 C for 72 h in a shaker incubator
with shaking speed of 150 rpm. Following incubation, the bacterial
culture medium was centrifuged at 12,000g for 20 min at 4 C. The
obtained clear supernatant was considered as crude enzyme mix
and used for cellulolytic assay. Cellulase activity was determined
by 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The assay
mixture contained enzyme: substrate CMC-Na (1%) (1:2) and pH of
the mix was adjusted to 5.0 using sodium acetate buffer (50 mM).
The reaction mixture was incubated at 45 C for 1 h. After incubation equal volume of DNS reagent was added and the mixture was
heated to 99 C for 15 min in a boiling water bath. The release of
reducing sugar was estimated spectrophotometrically by measuring the absorbance at 546 nm. One unit of enzymatic activity
was dened as the amount of enzyme that released 1 lmol of
reducing sugars per minute in glucose equivalents. The protein
content was estimated by Bradford method using BSA as standard
(Bradford, 1976).
Total cellulase activity was also determined by FPase assay
using lter paper (Whatman No. 1) as substrate. Cellulolytic activity of the crude enzyme mix was further conrmed by monitoring
the change in molecular weight of the enzymatically hydrolysed
CMC-Na using Gel permeation chromatography (GPC) (Water Allaince, model 2695) equipped with GPC column ultra hydrogel 120
and 500 and refractive index detector (Waters 2414). The molecular mass distribution of hydrolysed product was also determined
by mass spectrometry (Waters Q-Tof) equipped with an electrospray ionization interface, MCP detector and Waters MassLynx
software (version 4). The mass spectrometer was run employing
direct ow injection technique and the mass fragmentation was recorded on ESI positive mode (ESI+).

2.3. Effect of pH and temperature on cellulase activity and stability

2. Methods
2.1. Isolation, screening and identication of cellulose degrading
bacteria
Bacterial strains were isolated from partially deteriorated marine macrophytic alga S. polycystum C. Agardh collected from Veraval coast (N 20 54.870 ; E 70 20.830 ), India in the month of
February 2011. The deteriorated thallus was rinsed with sterile distilled water and the wash-off water was spread on Zobell marine
agar plates (Zobell marine agar 2216, Hi-media Labs Pvt. Ltd., India). Petri plates were then incubated at 30 2 C for 48 h and different bacterial colonies obtained were maintained as pure
cultures. Cellulolytic potential of the isolates were determined by
streaking each strain on agar plates enriched with 2% carboxymethyl cellulose (CMC-Na) as a sole carbon source. After an incubation of 48 h at 30 2 C, plates were stained with Lugols
iodine solution and the zone of clearance was considered as qualitative measure of extracellular cellulase activity (Kasana et al.,
2008). The strain showing highest zone of clearance on CMC-agar
plates was considered for further analysis.

The pH and temperature optima for enzyme activity was assayed at different pH ranging from 2 and 3 (50 mM GlycineHCl
buffer), 4, 5 and 6 (50 mM Sodium acetate buffer), to 7 and 8
(50 mM Phosphate buffer) and at different temperatures ranging
from 15 to 75 C with increments of 15 degrees unit. Further, the
stability of enzyme was investigated by estimating the residual enzyme activity after pre-incubation of 1 h at aforementioned pH and
temperature ranges.

2.4. Effect of various additives on enzyme activity

The effect of various additives on the enzyme activity was
determined in the presence of metal ions and other reagents. The
additives used in this study were CoCl2, PbCl2, FeCl2, MgCl2, MnCl2,
ZnCl2, EDTA and PMSF (5 mM each) and relative activity (%) was
estimated against control (without additive). Further, salt tolerance of enzyme was investigated by estimating the residual activity in the presence of different concentrations of NaCl ranging from
1% to 20% w/v against control (without NaCl) under optimized assay conditions.

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N. Trivedi et al. / Bioresource Technology 132 (2013) 313319

2.5. Effect of ionic liquids on cellulase enzyme

The ILs used in this study includes 1-ethyl-3-methylimidazolium methanesulfonate (IL1); 1-ethyl-3-methylimidazolium bromide (IL2); 1-ethyl-3-methylimidazolium acetate (IL3); 1-butyl1-methylpyrrolidinium triuromethanesulfonate (IL4); 1-butyl-3methylimidazolium triuoromethanesulfonate (IL5); 1-butyl-3methylimidazolium chloride (IL6). The residual enzyme activity
was determined at different ILs concentrations (1%, 3%, 5%, 10%,
15% and 20% v/v) under optimized assay conditions and was compared with the control reaction carried out in aqueous medium.
The ILs did not affect the pH values in the enzyme assay mix and
retained to pH around 5.0. The IL tolerance of the bacterial cellulase was further validated against the commercial cellulase derived
from T. viride (Cellulase Onozuka R-10, Yakult, Japan). Both bacterial and commercial cellulase were pre-incubated in ILs (5% v/v) for
different time intervals ranging from 12 to 48 h. Thereafter, the
residual activities for both the enzymes were determined under
the optimized assay conditions.
The ILs in which enzyme showed the highest activity and stability were then investigated for their cellulose dissolution potential
according to the method described by Dadi et al. (2006). Cellulose
(50 mg) was mixed with 450 ll of each IL in a separate glass vials
and incubated for 1 h at 45 C in inert atmosphere of nitrogen to
prevent the water uptake by IL. After incubation, cellulose was precipitated by washing the reaction medium with water as an antisolvent. The regenerated cellulose was then analyzed for loosening
of crystalline structure by Scanning Electron Microscope (LEO
1430VP, UK).
The thermal stability of enzyme in the presence of ILs (5% v/v),
wherein highest tolerance was observed, was investigated with a
pre-incubation of 1 h at different temperatures ranging from 15
to 60 C with increments of 15 degrees unit under optimized assay
conditions.
The effect of various additives such as PMSF, ZnCl2, CoCl2, and
FeCl2 (5 mM each) on the stability of enzyme was carried out with
a pre-incubation of 1 h in the same set of ILs (5% v/v) under optimized assay conditions.
The enzyme activity in the presence of different ILs was also assayed against cellulose (cotton linter) and natural algal biomass
(Ulva lactuca) in addition to CMC-Na.
2.6. Cellulase production using different substrates
Fresh bacterial culture was inoculated into the enzyme production medium containing 0.5% w/v Yeast extract, 3.5% w/v ASW and
different carbon source (1.5% w/v) viz green seaweed (U. lactuca),
Brown seaweed (S. polycystum) and CMC-Na. The medium was
incubated at 30 2 C for 5 days in a shaker incubator at150 rpm.
The supernatant from the production medium was taken periodically after every 12 h and assayed for cellulase activity using DNS
method under optimized assay conditions.

315

otide sequences homology match within the NCBI GenBank. The

DNA sequence for the isolated strain was submitted to NCBI GenBank with Accession No. HQ156241.
3.2. Effect of pH and temperature on cellulase activity and stability
The enzyme showed optimum activity at pH 5 and temperature
45 C. The cellulase (CMCase) and total cellulase (FPase) activity in
the crude enzyme mix was found to be 6.04 and 2.11 U/mL, respectively. The GPC spectrum for enzymatic hydrolyzed products conrmed the degradation of polymeric cellulose into low molecular
weight fractions. The GPC chromatogram showed peaks corresponding to molecular weights of 1049 and 156 indicative for oligosaccharide and monosaccharide, respectively (Supplementary
Fig. 2). Further, LCMS analysis conrmed that the liberated monosaccharide was glucose. The peaks obtained at 223.19, 245.03 and
269.09 have matched well with the calculated m/z peaks 224.03
[(D-Glucose) + 2Na+], 246.01 [(D-Glucose) + 3Na+] and 267.99 [(DGlucose) + 4Na+]. Hence the formation of monomeric unit (Glucose) is conrmed in the hydrolysate. These results provide clear
evidence for the existence of functionally active cellulase mixture
of all the three units in crude enzyme mix.
Interestingly, the enzyme was found to be active over a broad
range of temperatures from 15 to 75 C with marginal decline in
activity by 2% at 60 C and 10% at 75 C. The thermal stability analysis of enzyme showed 100% activity at 4 C whereas activity at 15,
30 and 45 C was found to be 90%, 86% and 80%, respectively, after
a pre-incubation of 1 h. Higher temperatures are generally detrimental to enzyme activity but the enzyme in this study retained
its activity even at 60 and 75 C with 66% and 57% activity, respectively. The thermal stability of cellulase in this study was found
similar to the cellulolytic activity (57%) reported from Geobacillus
sp. T1 at 80 C (Assareh et al., 2012). However, cellulase from
Aspergillus fumigates Z5 has been shown to have an enzyme activity
<50% at 70 C (Liu et al., 2012). It has been reported that cellulases
originated from Bacillus strains were stable at 050 C while at
higher temperature (>70 C) the enzyme activity declined to
<50% (Lin et al., 2012). The bacterial cellulase with higher thermal
stability as described in this study would nd wider applications
particularly in the bio-reneries and food processing industries.
The enzyme activity showed linear increase with increasing pH
from 2.0 to 5.0 and found to be optimum at pH 5.0 indicating the
acidic nature of the enzyme. These results are in accordance with
those reported from the genus Bacillus and Aspergillus having an
optimum enzyme activity in acidic range (pH 3.56.5) and at temperature 4060 C (Assareh et al., 2012: Lin et al., 2012: Liu et al.,
2012). Nevertheless, the enzyme activity marginally declined with
increasing pH from 6.0 to 8.0 and ranged between 83% and 85%.
The ndings of pH stability experiments showed retention of more
than 80% of the enzyme activity at pH 6.08.0. In contrast, the cellulase from bacterial strain Geobacillus sp. T1 (Assareh et al., 2012)
and fungal strain Chrysosporium lucknow (Dotsenko et al., 2012)
showed decreased enzyme activity of about 60% at pH >7 and
50% at pH >5, respectively.

3. Results and discussion

3.3. Effects of additives on enzyme activity
3.1. Screening and identication of cellulose degrading bacteria
Of the total 12 bacterial strains isolated from S. polycystum, four
isolates were positive for cellulase activity with Lugols iodine test.
Among these four isolates, the one showed highest zone of clearance on CMC agar plate was selected for further studies (Supplementary Fig. 1). The morphological and biochemical analysis
revealed that the strain with maximum cellulase activity was a
gram negative, aerobic and short rods. The identity of the strain
was conrmed as Pseudoalteromonas sp. based on 16S rDNA nucle-

The inuence of various metal ions and additives on cellulase

activity was studied at 5 mM concentration each. The enzyme
activity increased to 139%, 150% and 157% in the presence of
Zn+2, Co+2 and Fe+2, respectively. However, slight decline in the enzyme activity was found in the presence of Mg+2 (95%), PMSF (93%),
Pb+2 (82%), Mn+2 (78%) and metal chelator EDTA (75%). Li and Yu,
2012 also reported inhibitory effect of EDTA, PMSF and Mn+2 on
cellulase isolated from halotolerant Bacillus sp. L1. In another study
on soil metagenome-derived endoglucanase Cel5A, the inhibitory

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N. Trivedi et al. / Bioresource Technology 132 (2013) 313319

Fig. 1. Effect of different ionic liquids on cellulase (a) activity, (b) stability at 5% IL concentration and (c) stability of commercial cellulase at 5% IL concentration.

effect of Zn+2, Co+2, Fe+2 has been reported (Voget et al., 2006). In
addition to metal ions, effect of salt on enzyme activity has also
been investigated. Enzyme could retain 95.46% and 88.45% activity
at salt concentration 10% and 20% w/v, respectively. Such salt tolerance revealed for the halophilic nature of the studied enzyme.

3.4. Effect of ionic liquids on cellulase enzyme

The enzyme activity and stability was found to vary across
the investigated IL types and their concentrations. The enzymatic
activity was found >90% for all the ILs when used at concentration 5% v/v. The maximum activity was measured with [EMIM]Br
(IL2) followed by [EMIM]Ac (IL3), [BMIM]Cl (IL6), [C2MIM][CH3SO3] (IL1), [BMIM][OTF] (IL5) and [BMPL][OTF] (IL4) with 115%,
104.7%, 102.2%, 98.33%, 93.84% and 92.67%, respectively. The
enzyme activity at 20% (v/v) IL concentration was in the following order of IL3 (94.37%) > IL4 (80.2%) > IL5 (74.69%) > IL6
(73.2%) > IL2 (67%) > IL1 (59%) (Fig. 1a). The cellulase activity in
[EMIM]Ac, regarded as gold standard for biomass treatment, at
15% v/v was 97.22% and was substantially higher than that of
50% reported previously for recombinant cellulase (Tma Cel5A)
from bacterium T. maritime but was similar to the Pho EG from
P. horikoshii (Datta et al., 2010). Furthermore, the residual activity of the studied enzyme at higher concentration (20% v/v) of
[EMIM]Ac was 94.37% which was also superior to that of the
same reported as 55% and 43% from fungal strain Trichoderma
reesei (Zhang et al., 2011) and Aspergillus fumigatus (Singer
et al., 2011), respectively. With other ILs at higher concentration
of 20% v/v, the residual activity estimated was 80.2%, 74.69% and
73.2% for IL4, IL5 and IL6, respectively. These values were comparatively superior to that of the same reported from metagenome derived cellulases (pFosCelA2 and pFosCelA84) but
similar to pFosCelA3 (Ilmberger et al., 2012).

Fig. 2. Effect of (a) temperature and (b) additives on enzyme stability in the
presence of ionic liquids. The values represent averages from triplicate experiments.
Error bars represent the standard deviation.

In addition to functional activity of the enzyme, its stability

with ILs (5% v/v) was also studied for different pre-incubation

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N. Trivedi et al. / Bioresource Technology 132 (2013) 313319

Table 1
Recently reported ionic liquid stable cellulase from different sources.
Cellulase

Optimum
pH

Cellulase

5.0

CelA2

6.5

CelA3
CelA84

Optimum
Temperature
(C)
45

Specic
activity
(U/mg)
49.21

55

11.9

70
46

1.9
7.5

Cel5A

6.06.5

Hu-CBH1

9.5

37

5.0

50

4.8

45

Cellulase

4.5

37

11

Tma Cel5a

4.8

80

30

PhoEG

6.4

>95

1.9

CellA10

7.5

55

2.4

CelA24

55

23.3

Endoglucanase
Celluclast

7580

294

0.096

Rel. activity (%) in (%) IL

Rel. enzyme activity in

(%) IL (stability)

Organism

References

115% in 5% and 59% in 20%

[EMIM]Br
98% in 5% and 67% in 20%
[C2MIM] [CH3SO3]
105% in 5% and 94% in 20%
[EMIM]Ac
93% in 5% and 80.2% in 20%
[BMPL] [OTF]
94% in 5% and 74.69% in 20%
[BMIM] [OTF]
102% in 5% and 73.2% in 20%
[BMIM]Cl
54% in 30% [EMIM] [OTF]

92.16% (48 h) in 5%
[EMIM]Br
102% (48 h) in 5% [C2MIM]
[CH3SO3]
97% (48 h) in 5% [EMIM]Ac

Pdeudoalteromonas sp.

Present study

Metagenome

Ilmberger et al.
(2012)

Thermoanaerobacter
tengcongensis
Halorhabdus utahensis

Liang et al.
(2011)
Zhang et al.
(2011)
Singer et al.
(2011)
Engel et al.
(2010)
Datta et al.
(2010)

68% in 30% [EMIM] [OTF]

8% in 30% [BMPL] [OTF]
65% in 20%
[BMIM]Cl
60% in 30% [Amim]Cl at 2 M
NaCl
43% in 20% [C2mim][OAc]
40% in 10%
[MMIM] [DMP]
40% in 5%
[EMIM]Ac
40% in 20%
[EMIM]Ac
95% in 20%
[EMIM]Ac
74% in 30%
[BMPL] [OTF]
2% in 30%
[BMPL] [OTF]

intervals from 12 to 48 h. An increase in enzyme activity was

measured for all the ILs after a pre-incubation of 24 h. The enzyme
activity after 24 h of pre-incubation was highest with IL2 (130.86%)
followed by IL1 (122.9%), IL6 (113%), IL3 (102%), IL4 and IL5 (both
101.7%). Further, an increase in pre-incubation period to 36 h the
resultant activity was in the order of IL2 (121%) > IL6 (119%) > IL1
(108%) > IL3 (100%) > IL5 (99.51%) > IL4 (98%) (Fig. 1b). The stability of enzyme was then evaluated against commercial cellulase.
The residual activity of commercial enzyme after 24 and 36 h were
lower than bacterial cellulase. The residual activity was found in
the following order of 101.46 (IL1) > 99.5% (IL5) > 95.75%
(IL3) > 94.22% (IL6) > 93.13% (IL4) > 91.84% (IL2) after 24 h and
82.56%
(IL2) > 91.38%
(IL6) > 91%
(IL1) > 96%
(IL3) > 96%
(IL5) > 92% (IL4) after 36 h (Fig. 1c).
This study further analyzed the thermal stability of the enzyme
with ILs. The ILs (IL1, IL2, IL3 and IL6) where enzyme showed comparatively higher activity were selected. The enzyme showed activity of 136% (IL1), 110% (IL2), 101.7% (IL6) and 97.2% (IL3) at 15 C
after a pre-incubation of 1 h. At higher temperature of 60 C, partial
decline in activity was observed with IL3 (82.93%) and IL6 (81.49%)
whereas activity got declined to 68% and 51% for IL1 and IL2,
respectively (Fig. 2a). The earlier studies reported that the activity
of cellulases of fungal origin has been highly inhibited by ILs than
the same from bacterial origin (Datta et al., 2010; Singer et al.,
2011; Zhang et al., 2011). The disruption of hydrogen and hydrophobic interactions of protein along with the partitioning of surrounding hydration shell could be some of the common
attributes for IL induced inhibition of enzyme activity (Bose
et al., 2010; Moniruzzaman et al., 2010; Constatinescu et al.,
2010). Similar disruption mechanisms have also been caused by

89% (48 h) in 5% [BMPL]

[OTF]
93% (48 h) in 5% [BMIM]
[OTF]
83% (38 h) in 5% [BMIM]Cl
11% (5 days) 60% [BMPL]
[OTF]
79% (4 days) 60% [BMIM]Cl
81% (4 days) 60% [BMPL]
[OTF]
80% (5 h) 40%
[BMIM]Cl
100% (1 h) in 20% [Amim]Cl
at 2 M Nacl
10% (12 h) in 10%
[C2mim][OAc]
40% (11 days) 10%
[MMIM] [DMP]
0% (15 h) 15%
[EMIM]Ac
44% (15 h) 15%
[EMIM]Ac
79% (15 h) 15%
[EMIM]Ac
0.8% (17 h) 60%
[BMPL] [OTF]
0.02% (17 h) 60%
[BMIM] [OTF]

Aspergillus fumigatus
JF1
Trichoderma reesei
Trichoderma viride
Thermotoga maritima
Pyrococcus horikoshii
Metagenome

Pottkamper
et al. (2009)

Metagenome

Fig. 3. Effect of different substrates (a) CMC (b) Ulva lactuca (c) Sargassum
polycystum on cellulase production.

high salt concentration as well. Microbes dwelling in marine environment (>3 M salt) develop adaptive mechanisms to survive in
high salt conditions. Proteins of such microbes contain an excessive number of charged acidic amino acids on their surface which
allow protein to get solubilise even in high salt concentration
either by forming a hydrated ion network with cations/anions or
by preventing the formation of protein aggregation through electrostatic repulsive charges at protein surface (Fukuchi et al.,
2003; Paul et al., 2008; Tadeo et al., 2009). The isolated bacterial
strain Pseudoalteromonas sp. in this study is also of marine origin
and produced a cellulase of high salt tolerance (up to 20% w/v)
thereby gave circumstantial evidence for aforementioned adaptations which led to IL tolerance.

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The additives which are inducers for cellulolytic activity in

aqueous medium were further studied for their effect on enzyme
stability in association with ILs. In the presence of PMSF, the enzyme could retain its functional activity with IL1 (108%) and IL3
(115%) whereas Zn+2 was found to induce activity in IL1 (105%),
IL2 (112%) and IL3 (103%). However, the enzyme was found active
only in IL3 (100%) and IL6 (102%) in the presence of Fe+2 and Co+2,
respectively (Fig. 2b).
The activation and stabilization of the cellulase in the presence
of conventionally dened cellulose dissolving ionic liquids (Freire
et al., 2011; Liu et al., 2011) offers its potential for efcient hydrolysis of cellulosic biomass in a one step continuous process. Consequently, the steps involved in regeneration of cellulose followed in
conventional strategies can be bypassed. Recently reported cellulases capable of tolerating ILs are summarized in Table 1.
Additionally, an attempt was made in this study to evaluated the
industrial applicability of the cellulase enzyme using the algal substrate (U. lactuca) and cellulose (Cotton linter) in a medium containing ILs (IL1, IL2, IL3 and IL6) (5% v/v). The specic activity of the
enzyme against cellulose (Cotton linter) was found in the following
order of IL1 (138.46 U/mg) > IL2 (102.44 U/mg) > IL6 (94.6 U/mg)
and IL3 (91.76 U/mg) while the same in aqueous medium was comparatively low as 61.11 U/mg. In the presence of natural biomass (U.
lactuca) as substrate, the specic activity was found in the order of
IL1 (112 U/mg) > IL6 (98 U/mg) > IL2 (91 U/mg) and IL3 (88 U/mg)
while in aqueous medium registered specic activity was as low
as 64.82 U/mg. The overall results showed that the activity of enzyme is being induced by ILs. The higher cellulolytic activity with
ILs is mostly due to increased accessibility of enzyme to cellulose because of its higher dissolution. The loosening of rigid crystalline
structure of cellulose is generally attributed to its solubility in ILs
and the same is conrmed through SEM analysis.
3.5. Cellulase production using different substrates
The effect of different naturally available carbon sources particularly algal biomass from marine origin on the production of cellulase
enzyme was also investigated to increase the scope of industrial
application. The marine macroalgal species belonging to chlorophyta (U. lactuca) and phaeophyta (S. polycystum), rich in cellulosic
content, were employed as a sole carbon source in enzyme production medium. The production medium supplemented with CMC-Na
as carbon source was considered as control. The maximum enzyme
production was recorded on second day for medium supplemented
with seaweed biomass and on third day with CMC. The specic activity was found to be highest with U. lactuca (73.84 U/mg) followed by
S. polycystum (49.21 U/mg) and CMC (42.82 U/mg) after their
respective optimal incubation period (Fig. 3). However in the earlier
report, highest cellulase production from Thermoascus aurantiacus
and Thielavia terrestris was found from articial substrate than the
natural substrate (McClendon et al., 2012).
4. Conclusion
This study reports the ionic liquid stable cellulase from marine
bacterium Pseudoalteromonas sp. The cellulase had activity over a
broad range of pH, temperature and salt concentrations. The activation and stabilization of cellulase in ILs including in [EMIM]Ac,
regarded as gold standard for biomass treatment, substantiate its
utility for simultaneous IL treatment (for cellulose solubilisation)
and saccharication of cellulose in a single step continuous process. This enzyme therefore holds merit for the development of
efcient biomass hydrolysis process provided the production of
the enzyme is substantially enhanced using the synthetic and systems biology approach.

Acknowledgements
The nancial support received from CSIR, New Delhi (Biomass
to chemicals) is gratefully acknowledged. Mr. Nitin Trivedi and
Mr. Vishal Gupta gratefully acknowledge the CSIR for the award
of Senior Research Fellowships.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2013.
01.040.

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