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Chemosphere 83 (2011) 706712

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Technical Note

Solvent tolerant marine bacterium Bacillus aquimaris secreting organic solvent

stable alkaline cellulase
Nitin Trivedi, Vishal Gupta, Manoj Kumar, Puja Kumari, C.R.K. Reddy , Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientic and Industrial Research (CSIR), Bhavnagar 364 021, India

a r t i c l e

i n f o

Article history:
Received 19 July 2010
Received in revised form 1 February 2011
Accepted 1 February 2011

Keywords:
Solvent tolerance
Bacillus aquimaris
Fatty acid
Cellulase
Ionic liquids

a b s t r a c t
The organic solvent tolerant bacteria with their physiological abilities to decontaminate the organic pollutants have potentials to secrete extracellular enzymes of commercial importance. Of the 19 marine bacterial isolates examined for their solvent tolerance at 10 vol.% concentration, one had the signicant
tolerance and showed a relative growth yield of 86% for acetone, 71% for methanol, 52% for benzene,
35% for heptane, 24% for toluene and 19% for ethylacetate. The phylogenetic analysis of this strain using
16S rDNA sequence revealed 99% homology with Bacillus aquimaris. The cellulase enzyme secreted by this
strain under normal conditions showed an optimum activity at pH 11 and 45 C. The enzyme did show
functional stability even at higher pH (12) and temperature (75 C) with residual activity of 85% and
95% respectively. The enzyme activity in the presence of different additives were in the following order:
Co+2 > Fe+2 > NaOCl2 > CuSO4 > KCl > NaCl. The enzyme stability in the presence of solvents at 20 vol.%
concentration was highest in benzene with 122% followed by methanol (85%), acetone (75%), toluene
(73%) and heptane (42%). The pre-incubation of enzyme in ionic liquids such as 1-ethyl-3-methylimidazolium methanesulfonate and 1-ethyl-3-methylimidazolium bromide increased its activity to 150% and
155% respectively. The change in fatty acid prole with different solvents further elucidated the physiological adaptations of the strain to tolerate such extreme conditions.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
The environment nowadays is increasingly contaminated with
highly toxic monocyclic aromatic hydrocarbons (benzene, toluene,
ethyl benzene and xylenes) as a consequence of rapidly developing
petrochemical and power generating industries worldwide (Wang
et al., 2008). These hydrocarbons are collectively grouped under
BTEX and reported to be carcinogens and pose serious concern to
human health and other kinds of life (Fang et al., 2004). The marine
environment receives about 700 m3 of oil and a variety of hydrocarbons annually from shipping activities, accidental fuel spills,
and petrochemical based industries (Islam and Tanaka, 2004).
Organic solvents are extremely toxic to cells by virtue of their
ability to disrupt the normal functioning of biological membranes.
The toxicity of solvents is classied on the basis of the measured
values of their partition coefcient in n-octanol and water and
estimated in terms of log Pow value. Solvents with a low log Pow
(1.54.0) are considered extremely toxic, while others with a higher log Pow are less toxic (Inoue et al., 1991). The toxicity level of
organic solvents leads to the cell death by dissolving and disorga Corresponding author. Tel.: +91 278 256 5801/3805x614; fax: +91 278 256
6970/7562.
E-mail address: crk@csmcri.org (C.R.K. Reddy).
0045-6535/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2011.02.006

nizing the cell membrane and nally causes the loss of lipids and
proteins. The microbes dwelling in such toxic organic solvent
habitats can effectively be utilized for bioremediation and various
biotransformation processes of industrial efuents (Inoue et al.,
1991; Ramos et al., 1995; Na et al., 2005; Fang et al., 2006; Chen
et al., 2009). Micro-organisms develop versatile mechanisms such
as rigidication and alteration of cell membrane, or by forming
vesicles to resist the entry of organic solvents (Heipieper et al.,
2007; Segura et al., 2008). The cistrans isomerization of fatty acids
(FAs) has also been reported as adaptive mechanism in Pseudomonas and Vibrio (Ramos et al., 1997; Heipieper et al., 2003). The other
adaptive mechanisms include modication of lipopolysaccharide
or the porines, active export of the solvent and transformation of
the solvent (Isken and de Bont, 1998). The organic solvent tolerating microbes particularly the BTEX degraders have been investigated from terrestrial environments that includes largely from
the soil, sewage and also from anaerobic habitat (Botton and
Parsons, 2006). However, only a few micro-organisms from marine
habitat have been investigated for solvent tolerance. Marine
microbes are often exposed to diverse conditions ranging from
extreme temperature, pressure, salinity, pH, photo-radiations, heavy metals, organic solvents and hydrocarbons. In addition to physiological adaptive characteristics, synthesis of exozymes is one of
the notable adaptive catabolic mechanisms of micro-organisms

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N. Trivedi et al. / Chemosphere 83 (2011) 706712

that facilitate their sustenance in such natural habitats (Voget

et al., 2006). Thus, it would be of great interest to investigate the
microbes living under adverse conditions and characterization of
their extracellular enzymes with functional capacity in such environmental conditions.
Further, the enzymatic hydrolysis of biomaterials is the foremost demand of biomass based economy for efcient utilization
of biofuels, ber, ne chemical and plastic industry (Heinze and
Liebert, 2001). Cellulose will play a crucial role in emerging biomass based economy due to its large annual production through
photosynthesis. Many efforts are currently underway towards the
conversion of cellulose into both fuels and glucose as platform
molecules. Also, the enzymatic hydrolysis of cellulose at industrial
scale has great demand for highly stable cellulases with their functioning at extreme environmental conditions such as pH, temperature, salinity and tolerant to various organic solvents.
Thus the present study aimed at the isolation and characterization of marine bacteria tolerant to various toxic solvents along with
their evaluation for secreting highly stable and industrially important extracellular cellulase and its properties.
2. Materials and methods
2.1. Isolation and screening of organic solvent tolerant marine bacteria
Bacteria were isolated from deteriorated seaweed Ulva, cultured
and maintained in the laboratory. The degraded thallus was spreaded over Zobell marine agar plates. Further, the marine habitat
of the strain was checked by their growth measurement on 3.5%
articial seawater (ASW) media containing following salts (in
g L 1): NaCl 24.6, KCl 0.67, CaCl2 2H2O 1.36, MgSO4 7H2O 6.29,
MgCl2 6H2O 4.66, NaHCO3 0.16, supplemented with 0.5% yeast extract powder and incubated at 30 2 C for 23 d.
The initial screening for their solvent tolerance with a broad
range of log Pow towards benzene (2.0), toluene (2.73), acetone
( 0.25), heptane (4.39), ethylacetate (0.86) and methanol ( 0.82)
was examined by supplementing the ASW media with 10 vol.% of
individual solvents as carbon source along with the control. Subsequently, the growth performance of bacteria in the presence of
10 vol.% ionic liquids (Green solvents) namely, 1-ethyl-3-methylimidazolium methanesulfonate and 1-ethyl-3-methylimidazolium
bromide was also examined. The solvent tolerance was further
conrmed by spreading of the bacterial culture on the agar media
plate of ASW salt and further overlaid with the solvent (10 vol.%)
for which the strain has shown tolerance and incubated for 34 d
at 30 2 C (Nielsen et al., 2005).

707

spectrometric determination (absorbance at 546 nm) for generating reducing sugars by 3,5-dinitrosalicylic acid (DNS) method
(Miller, 1959).
One unit of enzymatic activity was dened as the amount of enzyme that released 1 lmol of reducing sugar min 1. The protein
content was estimated by Bradford method using BSA as standard
(Bradford, 1976).
2.4. Inuence of pH, temperature on cellulase activity and stability
The residual enzyme activity was measured at different pH as 8
(50 mM Phosphate buffer), 9, 10, 11 and 12 (50 mM glycineNaOH
buffer) and temperature at 1575 C. The stability of the crude enzyme was measured at different temperatures (1575 C) and pH
(612) after incubation for 30 min.
2.5. Enzyme stability in solvents and effect of various additives on
enzyme activity
For solvent stability, enzyme was pre-incubated for 30 min in
different solvents (20 vol.%) as well as in ionic liquids (20 vol.%).
Thereafter residual activity was determined under optimized assay
conditions considering control as 100%. The effect of various additives on the crude cellulase activity was determined by the presence of metal ions and other reagents. The additives used in this
study were KCl, CoCl2, HgCl2, PbCl2, FeCl2, NaCl, CuSO4, CdCl2, HgCl2,
NaOCl2, EDTA and PMSF and detergents (10 vol.%) including sodium dodecyl sulphate, Triton X-100, Tween-20 and Tween-80.
The concentration of each additive was 5 mM. The reaction mixtures with various additives were incubated for 50 min at 45 C
and the cellulase activity was assayed by DNS method.
2.6. Identication and molecular characterization of solvent tolerating
bacteria
The morphological and biochemical characterization of the
strain was performed and compared to the Bergey,s Manual of Systematic Bacteriology. The electron micrograph of bacterial surface
structure was taken on scanning electron microscopy LEO 1430VP
(UK) system.
The molecular identication of the bacterial strain was carried
out by 16S rDNA sequencing following the method described by
Trivedi et al. (2011). Multiple sequence alignment was carried
out with CLUSTAL W and compared with sequence in nucleotide
database (National Center for Biotechnology Information) using
the BLAST algorithm. The neighbor-joining phylogenetic analysis
was carried out with MEGA software (Tamura et al., 2007).

2.2. Growth rate determination

2.7. Fatty acid proling
Fresh bacterial inoculum was prepared in articial seawater
broth and incubated overnight at temperature 30 2 C. Thereafter
it was inoculated to the broth containing solvents (10 vol.%). The
initial inoculum added was 2.01  109 CFU mL 1. The growth was
measured by estimating the CFU mL 1 for every 4 h interval up
to 96 h.

The physiological adaptability in terms of FA change of the

strain towards the different solvents was analyzed. Cultures in different solvents were grown to their stationary phase and cells were
harvested and FAs were extracted according to Dionisi et al. (1999)
and analyzed in GCMS (Shimadzu, GC-2010).

2.3. Screening for cellulase production, optimization and activity assay

3. Results

The solvent tolerating strain was spread and cultured on the

agar plate supplemented with 2 vol.% carboxymethylcellulose
(CMC). The zone of clearance as qualitative measure of extracellular cellulase activity was screened after ooding the plates with Lugols iodine solution (Kasana et al., 2008). Fresh bacterial inoculum
was prepared and inoculated in the production media (ASW + 2%
CMC). The media was thereafter incubated at 30 2 C with shaking at 150 rpm for 24 h. The cellulolytic activity was quantied by

3.1. Isolation and screening of organic solvent tolerant marine bacteria

Among the 19 marine bacterial isolates investigated, the bacterial strain NT1 was found to be solvent tolerant by its growth in the
culture media supplemented with 10 vol.% solvents studied. The
solvent tolerance of this strain was further conrmed with its colonization abilities on the solid media plate overlaid with different
solvents.

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3.2. Determination of growth rate

The strain NT1 was further investigated for determination of
growth rate in medium supplemented with 10 vol.% solvent. These
growth experiments revealed substantial tolerance of NT1 strain to
acetone and benzene as compared to other solvents examined. The
exponential growth phase of this strain in acetone was observed at
6 h and benzene at 12 h and continued up to 85 and 80 h respectively. Conversely, for other solvents, prolonged lag phase of more
than 30 h was recorded with accompanying exponential phase of
30 h supplementary material (SM), Fig. SM-1. The relative growth
yield of NT1 strain in different solvent systems was in the following
order: acetone > methanol > benzene > heptane > toluene > ethylacetate (Fig. 1). In comparison to hazardous solvents, bacterial
growth was found to be luxuriant in the presence of ionic liquid
(10 vol.%) compared to other organic solvents studied. The lag phase
of the strain in ionic liquid 1-ethyl-3-methylimidazoliumbromide
was only of 46 h and the optimum growth was observed during
1418 h after incubation while no growth was observed in medium
with ionic liquid 1-ethyl-3-methylimidazolium methanesulfonate.
3.3. Biochemical and molecular characterization
The biochemical characterization of the solvent stable NT1 strain
revealed the organism as Gram-positive, aerobic and motile bacte-

ria (Table SM-1). The electron micrograph conrmed it as short

rod in shape (Fig. 2a). The homology match of 16S rDNA nucleotide
sequences in the NCBI Gene Bank conrmed the strain as Bacillus.
Based on the evolution distance and the phylogenetic tree resulting
from partial sequencing and the neighbor-joining method this
strain showed 99% homology with Bacillus aquimaris (Fig. 2b).
3.4. Fatty acid proling
The FA analysis showed that B. aquimaris contained 66% of
branched chain FAs of the total fatty acid contents (TFAs). The FAs
like 12- and 13-methyl tetradecanoic acids (i.e. iso and anteisopentadecanoic acids respectively) accounted for 50% in the control.
The total contents of predominant branched chain FAs decreased sharply in response to all the solvents studied with highest
decline in acetone and heptane with 3-fold, followed by benzene
(2-fold), methanol (1.7-fold) and toluene (1.5-fold). In contrast,
no branched FAs were detected in bacteria grown in ethylacetate.
This decrease in branched chain FAs was accompanied by a concomitant increase in monounsaturated fatty acids (MUFAs) and
polyunsaturated fatty acids (PUFAs) in all the solvents except benzene and toluene (Table 1). Interestingly some long chain C18 and
C20 PUFAs were also detected that were totally absent in control.
In methanol and acetone, the contents of MUFAs were almost
equal to PUFAs, while heptane caused accumulation of exceptionally high amount of PUFAs accounting to 43% of TFAs which was
mainly due to linolenic acid (37% of TFAs). Moreover, long-chain
FA arachidonic acid (C20:4, n-6) was also detected in methanol,
acetone and heptane (Table 1), along with other C20 unsaturates
particularly in acetone indicating the activation of downstream
PUFA biosynthetic pathway and their respective enzymes.
Although the contents of both total saturated fatty acids (SFAs)
including branched FAs of the bacteria decreased with all the solvents studied, the content of palmitic acid (C16:0) showed an
exceptional increase. This increase was about 5-fold in apolar solvents like benzene, toluene and heptane and 79-fold in relatively
polar solvents like methanol and acetone (Table 1). The increase in
palmitic acid content may be a step to combat the altered uidity
of the membrane to maintain its integrity for bacterial sustenance.
Further, an increase in straight chain SFAs to anteiso-FAs was also
observed from 1.1 in control to a maximum of 7.6 in benzene.
3.5. Screening for cellulase production, optimization and activity assay

Fig. 1. Relative growth yields of B. aquimaris in presence of different solvents. E.

acetate stands for ethylacetate.

The cellulase activity of the strain was conrmed with the

appearance of zone of clearance after ooding the solid media plate

Fig. 2. (a) Electron-micrograph showing rod shaped bacilli (Scale-3 lm). (b) Phylogenetic tree of B. aquimaris associated with other members of the genus Bacillus using 16S
rDNA sequence. Number at each clade refers to bootstrap values and Bayesian posterior probability. Clades with bootstrap values less than 50% were omitted from the gure.

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Table 1
Fatty acid composition of Bacillus aquimaris (w/w% of total FAME) in the presence of different organic solvents.
Fatty acids

Control

Methanol

Acetone

Heptane

Benzene

Ethylacetate

Toluene

Lauric acid (C12:0)

Tridecanoic acid (C13:0)
Myristic acid (C14:0)
Iso-pentadecanoic acid (iC15:0)
Anteiso-pentadecanoic acid (aiC15:0)
Pentadecanoic acid (C15:0)
2-Hydroxytetradecanoic acid (C14:0, 2 OH)
Anteiso-hexadecanoic acid (aiC16:0)
Palmitoleic acid (C16:1, n-7)
7-Hexadecenoic acid (C16:1, n-9)
Palmitic acid (C16:0)
Anteiso-heptadecanoic acid (aiC17:0)
C17:cyclopropane (cyc C17:0)
10-Heptadecenoic acid (C17:1, n-7)
Heptadecanoic acid (C17:0)
Stearidonic acid (C18:4, n-3)
Linolenic acid (C18:3, n-3)
Linoleic acid (C18:2, n-6)
Oleic acid (cis C18:1, n-9)
Elaidic acid (trans C18:1, n-9)
Stearic acid (C18:0)
Eicosanoic acid (C20:0)
Eicosapentaenoic acid (c20:5, n-3)
Arachidonic acid (C20:4, n-6)
8,11,14-Eicosatrienoic acid (C20:3, n-6)
11,14,17-Eicosatrienoic acid (C20:3, n-3)
11,14-Eicosadienoic acid (C20:2, n-6)
P
Straight chain FA
P
MUFA
P
PUFA
Straight chain/anteiso FA
Anteiso/iso FA

2.1
19.7
23.7
26.9
0.8
1.2
5.0
1.0

3.7
10.7
2.3

0.3

2.2

28.7
1.3

1.1
0.7

1.3
4.6
16.0
15.3

1.5
1.9
2.0

23.7
4.8
5.3

5.7
10.3
1.6

5.4

29.8
14.0
11.1
1.9
0.7

0.5
0.9
4.3
4.5
9.3
1.0
0.8
3.5
4.7

27.6
1.8
0.5
0.6
0.4

1.3
6.3
14.4
1.1
4.2
0.3
1.6
8.1
0.6
0.2
0.2
39.6
20.9
18.7
4.2
0.9

0.7
0.7
5.5
8.7
8.0
1.0
0.6
1.0
1.6
1.1
15.5
1.5
1.7
0.2
0.4
0.3
37.6
3.1
1.7
0.3
5.8

0.5
1.4

29.9
5.2
43.1
3.7
0.7

1.3
40.4
9.3
9.6
0.4
0.8
1.7
0.4

17.9
2.8
1.3

0.5

13.0

73.3
0.9

7.6
0.7

2.0

4.5

34.4

16.8
24.3
4.0
13.8

54.95
28.4
16.8

1.3
19.7
16.8
17.1
0.7
2.2
3.3
1.6
1.2
22.0
6.7
2.4

0.2

0.5
1.9
0.4
1.2

45.42
5.2
0.5
2.6
0.6

containing CMC (1.5 vol.%) with Lugols iodine (Fig. 3). The cell free
extract of the enzyme production media was considered as crude
enzyme preparation. The optimum temperature and pH for enzyme activity was found to be 45 C and 11 respectively. The enzyme activity showed linear increase with increase in pH from 9
to 11 and thereafter a decrease of 12 and 17% in the activity was
recorded at higher pH 12 and 13 respectively (Fig. 4a). The enzyme
showed sustainable activity in broad range of temperature from 15
to 75 C. A partial decline of 17 and 20% in enzyme activity was observed at higher temperature of 60 and 75 C respectively (Fig. 4b).

The enzyme was found to be stable in the broad range of pH (6

12). At higher alkaline pH enzyme retained 85% activity at incubation pH of 12 and reduced to 65% at pH 13 (Fig. 4c). Also, the enzyme showed functional stability at all temperature ranges
studied with only marginal decline of 2% and 5% after incubation
of 30 min at 60 and 75 C respectively (Fig. 4d).
3.6. Enzyme stability in solvents and effect of various additives on
enzyme activity
The enzyme showed 122% increase in activity in benzene, 85%
in methanol followed by acetone (75%) and toluene (73%). However, a sharp decline in the residual activity was observed in heptane (42%), while the enzyme was completely denatured in
ethylacetate (Fig. 5a). In the presence of both 1-ethyl-3-methylimidazolium methanesulfonate and 1-ethyl-3-methylimidazolium
bromide the enzyme showed signicant increase in residual activity of 150% and 155%. The residual activity increased to twofold
(210%) in the presence of Co+2 and Fe+2. The enzyme retained its
100% activity in the presence of NaClO2, CuSO4, KCl, NaCl whereas
the ratio of inhibition of the enzyme activity was 20% (CdCl2), 32%
(EDTA), 39% (HgCl2) and highest reduction of 45% was observed
with PMSF (Fig. 5b). The anionic and non-ionic detergents were
found as activators of the cellulase enzyme as the enzyme activity
increased with SDS (136%), Triton X-100 (124%), Tween-20 (109%)
and Tween-80 (103%) (Fig. 5c).
4. Discussion

Fig. 3. Screening for cellulase enzyme as zone of clearance on the agar plate ooded
with Lugols iodine.

Among the 19 marine bacterial isolates investigated for solvent

tolerance, B. aquimaris NT1 showed substantial tolerance against
the organic solvents selectively to acetone, methanol and benzene.
In addition, the strain also showed its potential to produce extracellular alkaline cellulase with functional stability at extremities

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N. Trivedi et al. / Chemosphere 83 (2011) 706712

Fig. 4. Effect of: (a) pH on enzyme activity, (b) temperature on enzyme activity, (c) pH on enzyme stability, and (d) temperature on enzyme stability.

of pH, solvents and detergents of both ionic and non-ionic in nature. The degree of solvent tolerance reliant on the strain and has
been demonstrated in the range of 1050 vol.% of solvents supplemented in the growth medium (Aono and Inoue, 1998). The isolated strain B. aquimaris can now be included in the list of other
solvent tolerant microbial taxa reported from marine environment
such as Pseudomonas, Moraxella, Pseudoalteromonas, Rhodococcus,
Exiguobacterium and Marinomonas (Inoue et al., 1991; Hun et al.,
2003; Segura et al., 2008; Wang et al., 2008). In addition, many
Bacillus sp. such as Bacillus pumilus, Bacillus sphericus 205y, Bacillus
sp. SB1, Bacillus sp. BC1 and Bacillus EEZMo-3 have also been previously accounted for their tolerance to benzene and toluene at
10 vol.% concentration (Kato et al., 1996; Lee et al., 1998; Sardessai
and Bhosle, 2002; Heipieper et al., 2007; Chen et al., 2009).
The solvent tolerance mechanisms of Gram-negative bacteria
have been extensively studied but the same is hardly been investigated for Gram-positive organisms. The reason could be that the
presence of an additional outer membrane in Gram-negative bacteria which was shown to be involved in promoting solvent tolerance. Moreover, ions such as Mg+2 or Ca+2 stabilize the organization
of the outer membrane resulting to higher solvent tolerance (Devi
et al., 2006). In the present study, B. aquimaris contained 50% of
TFAs as 12- and 13-methyl tetradecanoic acids (i.e. iso and anteiso-pentadecanoic acids respectively), similar to FA prole reported for B. aquimaris TF-12T isolated from seawater of a tidal
at of the Yellow Sea in Korea (Yoon et al., 2003). It has also been
reported that bacteria containing higher amounts of branched
chain FAs, have minor roles of unsaturated FAs in the membrane

integrity (Kaneda, 1991). However, B. aquimaris in our study

adopted a dual strategy by synthesizing straight chain SFAs and
unsaturated FAs by the activity of FA desaturase. Similar ndings
have also been reported earlier for B. subtilis in response to cold
shock and synthetic buckminsterfullerene (C60) (Aguilar et al.,
1998) and Arthrobacter chlorophenolicus in the presence of phenol
(Unell et al., 2007). In the present study, B. aquimaris responded
to benzene and toluene by increasing the contents of straight chain
FAs for membrane rigidication as also reported in earlier studies
(de Carvalho et al., 2007; Duldhardt et al., 2010). The increase in
membrane unsaturation in the presence of methanol, acetone
and heptane was counterbalanced by 4.27.5 folds increase of
straight chain C16:0 for maintaining the membrane integrity. A
similar contrasting behaviour of B. subtilis has been reported by
Fang et al. (2007). These adaptive responses varied with the hydrophobicity (log Pow) of the solvents and thereby attribute for the
bacterial growth corresponding to the solvent employed. Theoretically solvents with a low log Pow (1.54.0) are extremely toxic,
while others are considered to be less toxic (Inoue et al., 1991; Heipieper et al., 2003). In contrast, ethylacetate (log Pow 0.86) was
observed to be extremely toxic for bacteria in this study. The FA
prole also showed disappearance of both the iso and anteisoFAs (not detected), accompanied by an increase in MUFAs contents
(21-fold) especially, oleic acid content that increased from 0.3% of
TFAs in control to 24.3% of TFAs and 16.8% of PUFAs contents. Such
mechanisms have earlier been reported by Fang et al. (2007) for
B. subtilis cells exposed to higher doses of nC60. Therefore unsaturated FAs play a more important role for adaptation of Gram

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711

Fig. 5. Effect of: (a) organic solvents and ionic liquids on cellulase stability, (b) additives on enzyme activity, and (c) detergents on enzyme stability.

positive bacteria towards organic solvents over Gram negative

ones however, these considerations need further investigation in
Bacillus sp.
The solvent tolerant B. aquimaris further signify its importance
by the secretion of an extracellular solvent stable cellulase. In
our study, the cellulase activity was found optimum at pH 11
and temperature 45 C. The increase in activity of enzyme pre-

incubated for 30 min with benzene (122%), 1-ethyl-3-methylimidazolium methanesulfonate (150%) and 1-ethyl-3-methylimidazolium bromide (155%) suggests the prospects of the enzyme for
various industrial applications. Voget et al. (2006) also reported a
cellulase with stability in the presence of the organic solvent
(15 vol.%) from uncultured bacteria. Recently, cellulases (CelA10,
CelA20 and CelA24) characterized from metagenomic approach

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from aquatic sources have also been tested for their functional stability against ionic liquids and different organic solvents (Pottkamper et al., 2009). They have also exhibited increased activity in the
presence of 5 vol.% detergent sodium dodecyl sulphate, Triton X100, Tween-20 and Tween-80. In the present study, B. aquimaris
also showed enhanced cellulase activity even at relatively higher
concentrations of detergent (10 vol.%), in addition to solvent stability. The activity of alkaline cellulase especially from Bacillus sp. has
been reported to vary with additives present in assay medium. The
increase in the activity of cellulase with Co+2, Fe+2, NaClO2 in the
present study has also been observed by Pottkamper et al.
(2009). Some metal ions were found to be ineffective for the enzyme activity as reported in previous studies on Bacillus sp. (Christakopoulos et al., 1999). The inducing effect of heavy metal Cd+2,
Co+2 along with NaOCl2 observed in the present study suggest
the unique characteristic of the cellulase from B. aquimaris.
Thus, B. aquimaris with its organic solvent tolerance and virtue
of being a source of highly stable alkaline cellulase can play a key
role in industrial processes involving biphasic organic-aqueous fermentation and bioremediation of hydrocarbon-saturated
environments.
Acknowledgements
The nancial support received from CSIR (RSP 0016), New Delhi
is gratefully acknowledged. Mr. Manoj Kumar and Miss. Puja Kumari gratefully acknowledge the CSIR for awarding the Senior
and Junior Research Fellowships respectively.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.chemosphere.2011.02.006.
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