International Biodeterioration & Biodegradation 98 (2015) 73e80

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International Biodeterioration & Biodegradation

journalhomepage:www.elsevier.com/locate/ibiod

Isolation of cellulolytic bacteria from the gastro-intestinal tract of Achatina

fulica (Gastropoda: Pulmonata) and their evaluation for cellulose
biodegradation
Mudasir A. Dar a, Kiran D. Pawar b, *, Jyoti P. Jadhav b, Radhakrishna S. Pandit a
a Department of Zoology, University of Pune, Ganeshkhind, Pune 411007, Maharashtra, India
b
Department of Biotechnology, Shivaji University, Vidyanagar, Kolhapur 416004, Maharashtra, India

article info abstract

Article history: Gastrointestinal tract of Giant African snail, Achatina fulica was investigated as a source for isolation of cellulose degrading
Received 17 July 2014 bacteria. The cellulose degrading bacteria from different gastrointestinal tract re-gions such as esophagus, crop, stomach,
Received in revised form 24 intestine and rectum were enriched in carboxymethyl cellulose and identified. Thirty two cellulose degrading bacteria belonging
November 2014
to two major phyla namely Proteo-bacteria and Firmicutes were enriched, isolated and identified by 16S rDNA amplification
Accepted 24 November 2014
and sequencing. Based on plate based assay, 18 of these isolates displayed cellulase activity and were iden-tified as the members
Available online
of Bacillus, Achromobacter, Ochrobactrum and Klebsiella. Among the 18 isolates, 5 isolates with high activity were further
studied for various enzyme activities such as endoglucanase, exoglucanase and xylanase on different lignocellulosic substrates.
Keywords:
Achatina fulica
Isolate identified as Ochrobactrum sp. K38 exhibited the highest CMCase activity (501.75 IU/ml extract) after 14 days of
Gastro-intestinal tract incubation. The highest avicelase activity (3116.92 IU/ml extract) was shown by Bacillus subtilis Cf60 on Filter paper as
Bacteria substrate after 10 days of incubation whereas all other isolates showed a low xylanase activity on all tested substrates except
CMC filter paper. The present study demonstrates the utility of snail gut as a rich source for isolation of cellulose degrading bacteria
16S rDNA that can have many industrial applications.
Cellulases

© 2014 Elsevier Ltd. All rights reserved.

Introduction
Interest in bioenergy has been sharply increasing from the onset of 21st
century due to the necessity of sustainable economies and clean environments
(Lynd et al., 1991, 2008; Sun and Scharf, 2010). Moreover drawbacks of fossil
fuels like global warming and non-renewability are motivating research into
optimizing new sus-tainable sources of energy from biological origin (Stern,
2006; Vertes et al., 2006; Escobar et al., 2009). Currently, the second
generation biofuels such as bioethanol, biodiesel, bio-hydrogen and methane
etc., are increasingly been produced from lignocellulosic wastes (LCW) rather
than from energy crops such as Jatropha, Switchgrass, hybrid Poplar and
Willow. This is because of the foodefuel conflicts related to energy crops (Mtui,
2009).
* Corresponding author. Tel.: þ91 (0) 2312609365; fax: þ91 (0) 2311691533. E-mail
addresses: muddar7@gmail.com (M.A. Dar), kdp308965@yahoo.co.in,
pawarkiran1912@gmail.com (K.D. Pawar), jpj_biochem@unishivaji.ac.in
(J.P. Jadhav), rspandit@unipune.ac.in (R.S. Pandit).
Furthermore, the idea of producing biofuels from bio-wastes comes naturally
as a promising alternative solution for problems related to much-feared energy
crisis, waste disposal management problems and will thus be helpful in the
upliftment of the rural population.
In nature, lignocellulosic (LC) biomass is degraded with the cooperation of
many microorganisms, mainly including diverse fungal and bacterial genera
producing a variety of cellulolytic and hemi-cellulolytic enzymes under aerobic
and anaerobic conditions (Kumar et al., 2008). Over the years, cellulase-
producing bacteria have been isolated from a wide variety of sources such as
com-posting plants, decaying plant material from forestry or agricultural waste,
the feces of ruminants, soils and organic matter (Lynd et al., 1991) and guts of
animals etc.
Herbivorous animals lack the capacity to degrade down ligno-cellulose
themselves and instead rely upon the gut microbial community having this
repertoire. The invasive Giant African Snail (GAS), Achatina fulica is one of
such animals which specifically feed on lignocellulosic plant material. A. fulica
nd
is most destructive pest, ranked 2 among the 100 worst alien invasive species
(Anon., 2013). Despite the economic and ecological importance of snails

http://dx.doi.org/10.1016/j.ibiod.2014.11.016 0964-8305/©
2014 Elsevier Ltd. All rights reserved.
74 M.A. Dar et al. / International Biodeterioration & Biodegradation 98 (2015) 73e80
in general, and A. fulica in particular, little is known about the di-versity and
community structure of cellulolytic bacteria in its Gastro-Intestinal (GI) tract.
In addition, not much is known about the potential role of bacteria in cellulose
digestion in Snail's GI tract.
Therefore, in the present study, we investigated the different GI regions of
the GAS as potential source for the isolation of cellulo-lytic bacteria. Members
of cellulolytic bacterial communities from different GI regions were enriched
in Carboxy Methyl Cellulose (CMC), grown and then identified by 16S rDNA
amplification and sequencing. Further, the potential of five promising isolates
to degrade various commercially available substrates such as CMC, Avicel,
Xylan, filter paper and agro-residues such as grass straw and wheat husk were
tested.
Materials and methods
Dissection of snails and enrichment of cellulolytic bacteria
Adult snails (n ¼ 3) in active state were collected from the premises of
Govt. Polytechnic College Pune, Maharashtra, India and immediately brought
to the laboratory prior to dissection. The shells were broken opened carefully
with the bone cutter and the animals were surface sterilized with 70% ethanol
(v/v) under sterile conditions in a laminar air flow. Animals were then
aseptically dissected with sterile forceps to remove the whole guts. The whole
GI tracts (n ¼ 3) were divided into different gut regions such as esophagus,
crop, stomach, intestine and rectum. Each GI region (n ¼ 3) was then
suspended in 2 ml of sterile Phosphate Buffer Saline (PBS) solution and
homogenized. One milliliter of GI region homogenate was aseptically
inoculated in enrichment culture flask containing 10 ml of Berg Minimal Salt
medium (BMS) which comprised of NaNO3, 2 g; K2HPO4, 0.5 g;
MgSO4$7H2O, 0.02 g; MnSO4$7H 2O, 0.02 g; FeSO4$7H2O, 0.02 g;
CaCl2$2H2O, 5 g per liter of solution supplemented with 0.5% CMC. The
inoculated medium was then agitated on a shaker at 120 rpm, 37 C for 2 days.
After two days, 1 ml of the culture of each region was sub-cultivated to 9 ml of
fresh BMS medium with 0.5% (w/v) CMC and remaining 9 ml cul-ture was
used to monitor the progress of enrichment by enumer-ation of total viable
count (TVC), isolation and identification of bacteria. This procedure of
sampling, sub-cultivation, enumeration of TVC was continued for two weeks.
Enumeration of total viable count and isolation of cellulolytic bacteria
From the 9 ml of culture remained after sub-cultivation, 100 ml was serially
8
diluted up to 10 and 100 ml of each dilution was spread on BMS agar plates
supplemented with 0.5% (w/v) CMC. Plates were incubated at 37 C for 48e72
h and then observed for bacterial growth and enumeration of TVC. Based on
visual inspec-tion of colony morphologies such as color, shape, elevation,
margin 2e3 colonies with similar morphology were picked and transferred to
BMS agar medium with CMC as well as Luria Bertani (LB) Agar media. Each
colony was made into pure culture by repeated streaking, then codified and
preserved for further use.
Identification and phylogenetic analysis
The isolates were grown overnight in LB broth at 37 C and then DNA from
each isolate was extracted by using the HipurA™ 96 Bacterial Genomic DNA
purification Kit MB505-250 PR (HiMedia Pvt Ltd, Mumbai, India). For
amplification 16S rRNA gene, PCR was performed with the bacteria-specific
primer pair 27F (50-AGAGTTTGATYMTGGCTCAG-30) and 907R (50-
CCGTCAATTCMTTT-GAGTTT-30) (Weisburg et al., 1991). The 50 ml PCR
reaction consisted
of 4 ml (10 ng/ml) of template DNA, 5 ml of Taq buffer (Bangalore Genei,
India), 0.2 mmol/l dNTPs (Bangalore Genei, India), 10 pmol of each of
primers, and 0.5 U of Taq polymerase (Bangalore Genei, India). The PCR
conditions consisted of initial denaturation at 95 C for 5 min, 35 amplification
cycles of primary denaturation at 94 C for 1 min, annealing at 55 C for 1 min
and extension at 72 C for 1 min, and a final extension step at 72 C of 7 min. All
PCR reactions were carried out in triplicates and products were checked for
size and purity on 1% (w/v) agarose gels.
The sequencing of 16S rDNA amplicon of each isolate was per-formed
using the ABI Big-Dye version 3.1 sequencing kit as per the manufacturer's
instructions, and using 27F and 907R primers. The generated sequences were
analyzed using ChromasPro software
(http://www.technelysium.com.au/ChromasPro.html). The se-quences
representing each isolate were compared with NCBI database using BLASTN
with GenBank (http://www.ncbi.nlm.nih. gov) and the closest matches to
bacterial isolates were obtained. For phylogenetic analysis, multiple sequence
alignments of 16S rRNA gene sequences were performed with ClustalX
(Thompson et al., 1997) and were edited manually using DAMBE (Xia and
Xie, 2001) to obtain an unambiguous sequence alignment. A bootstrap
Neighbor Joining (NJ) method was used for the construction of phylogenetic
trees using MEGA 5.0 program (Tamura et al., 2011).
Nucleotide accession numbers
The 16S rDNA sequences generated from bacterial isolates have been
deposited to NCBI database under accession numbers KJ669189eKJ669218.
Screening of cellulolytic activity of bacterial isolates
Screening for the cellulolytic activity of isolates was carried out by using
plate base technique. All the bacterial isolates obtained from enriched cultures
were patched and grown (patch size not more than 0.5 mm) on BMS Agar plates
supplemented with 0.5% CMC (w/v). After an incubation of 48e72 h at 37 C
and proper growth of patched isolates, the plates were flooded with 5 ml of
Gram's Iodine (Kasana et al., 2008) for 5 min. The excess stain was drained
and plates were observed for zone of clearance around the colonies hereafter
referred as “Halos”. Based on the diameter of halos (cm), cellulolytic activities
of isolates were identified as low (0.5e1.9), medium (2.0e3.9) and high (above
4). Cellulose-degrading potential of the positive isolates was also semi-
quantitatively estimated by calculating hydrolysis capacity (HC), that is, the
ratio of the diameter of zone of clearance to the diameter of colony (Hendricks
et al., 1995). The isolates showing higher ac-tivities were preserved and
selected for further studies.
Enzyme assays
The cellulolytic potential of promising isolates was tested by estimating
various enzyme activities such as endoglucanase, exo-glucanase and Xylanase.
To accomplish this, isolates were grown on commercially available cellulosic
substrates such as CMC, Avicel, Xylan; alkali treated agro residues viz., Grass
straw, Wheat husk, filter paper and then activities were estimated. Isolates were
cultured in BMS liquid medium with 0.5% (w/v) cellulosic sub-strates, agitated
at 120 rpm, 37 C for 14 days. To optimize optimum incubation period and
monitor how enzymes activity varies over the incubation period of 14 days,
sampling was done on every alternate day by taking small aliquotes of cultures
(~5 ml). Aliquotes were centrifuged at 10,000 rpm for 10 min at RT and
supernatants were used for estimation of enzyme activities. Endoglucanase
(CMCase) activity was determined according to the method
 M.A. Dar et al. / International Biodeterioration & Biodegradation 98 (2015) 73e80
Table 1
Distribution and cellulolytic activity of bacterial isolates enriched and grown from different regions of GI tract.
Gut region Total no. of No. showing No. showing highest
isolates activity & (%) activity & (%)
Esophagus 23 3 (13.04) 0 2 (8.69)
Crop 19 6 (31.58) 0 0
Stomach 13 5 (38.46) 0 0
Intestine 19 5 (26.32) 3 (15.78)
Rectum 18 13 (72.22) 9 (50)
Total 92 32 (34.78) 12 (13.04)
Values are represented as no. of isolates followed by percentage of total no. of isolates in parenthesis.
described by Nitisinprasert and Temmes (1991), with few modifi-cations using
a reaction mixture containing 1 ml of enzyme solu-tion with 1 ml of 1% CMC
in sodium citrate buffer (pH 5.4) and incubated at 50 C for 15 min.
Exoglucanase (avicelase) activity was assayed by following the Nitisinprasert
and Temmes (1991) method. In this assay method, the reaction mixture
containing 250 ml enzyme extract with 250 ml of 1% avicel cellulose in
sodium citrate buffer (pH 5.4) was incubated at 40 C for 2 h. The reaction was
terminated by addition of 750 ml of 3, 5-dinitrosalicylic acid (DNSA) reagent.
Extensive care was taken during this assay to prevent the avicel from mixing
with the enzyme extract after centrifugation. In these tests, reducing sugars
were estimated spectrophotometrically at 540 nm with DNSA (Miller, 1959)
method, using glucose as standard (Kumar et al., 2008). Xylanase activity was
determined according to Nitisinprasert and Temmes (1991), using a reaction
mixture containing 250 ml of enzyme so-lution appropriately diluted in sodium
citrate buffer (pH 5.4) with 250 ml of aqueous suspension of 1% (w/v) xylan
at 50 C for 10 min. The reaction was terminated by adding 750 ml of DNSA
reagent and heating in boiling water bath for 5 min. The amount of reducing
sugars released was determined using D-xylose as standard.
Enzyme activity was determined in international units (IU) where 1 IU of
activity is defined as the amount of enzyme required to liberate 1 mmol of
glucose equivalent per min under the assay conditions. The remaining
supernatant was used for the estimation of proteins by Lowry method (Lowry,
1990). A standard curve was generated using solutions containing 10 mg/ml
Bovine Serum Al-bumin (BSA). Absorbances were measured in triplicate at
660 nm after 30 min of incubation at room temperature.
Results
Determination of TVC, isolation and identification of cellulolytic
bacteria
The progress of enrichment over the incubation period of two weeks was
monitored by estimating the TVC on every alternate day. The determination of
TVC in enrichment cultures from all GI re-gions under study indicated that
TVC increased up to the 8th day and then decreased slightly. Except in crop,
TVCs in cultures from all other GI regions under study were highest on 8th
9
day; whereas highest bacterial load (20.8 10 CFU/ml) in culture from crop
was observed on the 12th day of incubation (Fig. S1).
Initially, a total of 114 isolates were purified and obtained from enriched
cultures of all GI regions under study. Subsequently, based on size, color and
morphology, 22 isolates were removed due to similar size, color and
morphological characteristics. Thus, from the entire GI regions, total 92
isolates were obtained, Maximum 23 isolates from esophagus followed by 19
isolates each from intestine and crop, 18 isolates from rectum and 13 isolates
from stomach were obtained (Table 1).
75
No. showing medium No. showing lowest No. showing no
activity & (%) activity & (%) activity & (%)
1 (4.34) 20 (86.96)
6 (31.57) 13 (68.42)
5 (38.46) 8 (61.54)
0 2 (10.52) 14 (73.68)
0 4 (22.22) 5 (27.78)
2 (2.17) 18 (19.56) 60 (65.22)
Based on 16S rDNA amplification and sequencing, many isolates were
found common among different GI regions. Two third (56) of the total number
of isolates obtained were the members of Gamma proteobacteria while
Firmicutes accounted for 1/9 (12) of the total number of isolates from the GI
tract. Alpha (1) and Beta-proteo-bacteria (1) were represented by single genus
each such as Ochro-bactrum and Achromobacter respectively. The members of
Gammaproteobacteria identified included the species of genera Klebsiella,
Enterobacter, and Shigella, which are usually found in soil, plants, and the
intestines of animals (Park et al., 2007). The mem-bers of Klebsiella (35.86%),
Enterobacter (23%), un-cultured bacte-rium clone (19%) and Bacillus (12%)
dominated the entire gut with 33, 21, 18 and 11 isolates, respectively (Fig. S2).
Screening for cellulolytic activity
Although all bacteria were isolated from cultures enriched in CMC, not all
of them showed cellulolytic activity when tested using plate based assay. The
plate based screening (Fig. 1) indicated that 32 (34.78%) isolates showed
cellulolytic activity amongst which 18, 2 and 12 isolates exhibited low, medium
and maximum cellulolytic activities respectively (Table 1). Isolates from only
two GI regions such as rectum and intestine displayed highest activity. In com-
parison to other GI regions, rectum was found to harbor highest load of
cellulolytic bacteria among which 50% showed highest ac-tivity. Only 13% of
isolates from esophagus were found with either moderate or lowest activities.
The hydrolytic capacity which is a measure of the ratio of diameter of zone of
clearance to the colony diameter ranged from 1.4 to 17 (Fig. S3) among all
cellulolytic iso-lates, with 59.3% of the isolates showing high CMC-degrading
ac-tivity (ratio 3). This demonstrated that multiple bacterial isolates from the
snail gut possess the ability to produce CMCase. The cellulolytic activity of the
isolates from crop and stomach was relatively poor. This pattern of activity
shown by the isolates from different regions showed a positive correlation with
the physiology of digestion indicating that the digestion of the ingested material
occurs mainly in the hindgut region of the GI tract.
The phylogenetic analysis of cellulolytic isolates and their closest related
strains from the GenBank indicated that the cellu-lolytic bacteria were the
members of two major phyla namely Fir-micutes and the Proteobacteria (Fig.
2). However, cellulolytic members of Proteobacteria exhibited low or medium
cellulolytic activity as compared to members of Firmicutes. Among the Pro-
teobacteria, isolates showing closest database matches to Ochro-bactrum sp.
K38 and Achromobacter xylosidans were the only strains that showed highest
activity. Besides these two isolates with high cellulolytic potential, four
Bacillus subtilis strains also showed high cellulolytic potential when tested
using cellulose agar plate based assay. Therefore, based on plate base assay,
four isolates identified as strains of B. subtilis namely B. subtilis AN5, B.
subtilis EHFS1, B. subtilis Cf60, B. subtilis A2 and one Ochrobactrum sp. K38
were chosen for further various cellulolytic enzyme assays.
76 M.A. Dar et al. / International Biodeterioration & Biodegradation 98 (2015) 73e80
Fig. 1. Plate based cellulolytic activity screening of isolates. A: Isolates patched and grown on CMC agar plate (before activity staining); B: isolates showing zones of clearance around the colonies
after activity staining with Gram's Iodine solution.
Cellulolytic enzyme assays
Endoglucanase activity
To study the lignocellulolytic potential of selected five isolates, their
various enzyme activities on different substrates were stud-ied. To achieve this,
the selected isolates were grown for 14 days on CMC, various agro-residues
such as grass straw, wheat husk, filter paper and activities were determined on
every alternate day. Using CMC as substrate, all tested isolates except B.
subtilis A2 showed increased endoglucanase activity over the period of 14 days.
Among the isolates tested, B. subtilis EHFS1 was estimated with maximum
activity of 230.86 IU/ml extract after 12 days of incubation. The endoglucanase
activities shown by remaining isolates were more or less in similar range (Fig.
3A).
On Grass straw, filter paper and wheat husk as substrate, Ochrobactrum sp.
K38 produced maximum endoglucanase activity than other four studied B.
subtilis strains. Ochrobactrum sp. K38 produced highest endoglucanase
activities of 502.75 IU/ml extract (14th day), and 347.65 IU/ml extract (4th
day) 112.68 IU/ml (6th day) on grass straw, wheat husk, and filter paper
respectively (Fig. 3BeD). Although the pattern of endoglucanase activity was
similar on Grass straw and filter paper as substrate, the level of endoglucanase
activity was more on grass straw than filter paper (Fig. 3B and D). When wheat
husk as substrate was used, Ochro-bactrum sp. K38 was noted with highest
endoglucanase activity of 347.65 IU/ml extract on fourth day which suddenly
decreased on sixth day (Fig. 3C).
Exoglucanase activity
The highest (2628.65 IU/ml extract) and lowest (200.57 IU/ml extract)
exoglucanase activities were revealed by B. subtilis AN5 after 12 and 8 days
of incubation respectively (Fig. 4A) while rest of the isolates showed a
moderate exoglucanase activity when grown on Avicel as substrate. When all
substrates tested were compared, it was found that highest (3777.61 IU/ml
extract) exoglucanase ac-tivity was produced on filter paper by B. subtilis AN5
on 10th day of incubation (Fig. 4AeD). This was 1.43 fold higher than activity
on
Avicel substrate by the same isolate. Moreover, the overall exo-glucanase
activity was 7 fold higher than the highest endogluca-nase activity recorded. In
case of wheat husk, maximum 2406.31 IU/ml extract exoglucanase activity was
produced by Ochrobactrum sp. K38 after 14 days of incubation (Fig. 4C).
On grass straw, B. subtilis EHFS1, B. subtilis Cf60, B. subtilis A2 and
Ochrobactrum sp. K38 showed similar pattern of increased exo-glucanase
activity over the period of 14 days whereas B. subtilis AN5 was estimated with
slight increase in activity on fourth day and then activity decreased till last day
of incubation (Fig. 4B). B. subtilis Cf60 was determined with maximum
(1838.77 IU/ml extract) exoglucanase activity among the isolates on 10th day
of incubation.
Xylanase activity
On xylan as substrate, the highest xylanase activity was exhibited by B.
subtilis AN5 on the 2nd day and then slightly decreased over the incubation
period of 14 days, whereas all other tested isolates showed slight increase in
activity (Fig. 5A). Likewise on grass straw and wheat husk, all isolates showed
a slight increase in xylanase activity from first to 14th day of incubation.
Maximum 82.03 IU/ml extract xylanase activity was produced by Ochrobac-
trum sp. K38 using grass straw on 14th day, whereas B. subtilis AN5 showed
a highest activity (60.22IU/ml extract) on wheat husk after 4 days of incubation
(Fig. 5B and C). However, on filter paper, only Ochrobactrum sp. K38 was able
to produce xylanase activity that too displayed a decreasing trend with a
maximum activity of 24.23 IU/ ml extract on 4th day of incubation. The overall
xylanase activity produced by all tested isolates on all substrates used was
compar-atively lower than endo- and exo-glucanase activities.
Discussion
The GAS, A. fulica, is known to feed primarily on vascular plants (Rauth
and Barker, 2002) and plant materials with high protein and calcium contents
(Iglesias and Castillejo, 1994; Chevalier et al., 2003). This snail also
participates, with other soil invertebrates, in the decomposition of leaf litter
(Hatziioannou et al., 1994).
 M.A. Dar et al. / International Biodeterioration & Biodegradation 98 (2015) 73e80
Fig. 2. Phylogenetic tree of the 16S rRNA gene from cellulolytic bacterial isolates and their
closest related strains from the GenBank. The tree was generated by using the Neighbor Joining
method with Kimura 2 parameter distances in MEGA 5.0 software. Numbers at nodes indicate
percent bootstrap values calculated based on 1000 repli-cates. Activity of each isolate in
parenthesis is denoted as: þ, low activity; þþ, medium activity and þþþ, high activity given
besides.
Consequently, a large set of cellulose degrading bacteria might inhabit the GI
tract of this snail. Recently, we found that different regions of GI tract of this
snail harbor high bacterial diversity (Pawar et al., 2012). Also recent
metagenomic analysis of micro-biota of crop of this snail revealed an
abundance of sequences
77
coding for oligosaccharide-degrading enzymes and many novel cellulase and
hemicellulase coding sequences (Cardoso et al., 2012). None of these reports
revealed the composition and community structure of cellulose degrading
bacteria. The cellulolytic bacterial community structure in the gut of GAS is
not known yet. Keeping this in view, we investigated the composition and
community structure of cellulolytic bacteria in GI tract of this snail. To accom-
plish this, cellulolytic bacterial community was enriched using CMC as
cellulosic substrate and characterized. Since different GI regions such as
esophagus, crop, stomach, intestine and rectum are the specialized
compartments with specialized function, we hypothe-sized that each of these
regions might be harboring unique set of cellulolytic bacteria. To test this
hypothesis, the cellulolytic bacteria were enriched, isolated and identified from
each of these GI regions separately and compared. In congruence with our
previous obser-vations (Pawar et al., 2012), cellulolytic members in the GI tract
of GAS were dominated by members of Proteobacteria and Firmicutes. It was
interesting to note that cellulolytic members of Tenericutes were not isolated
in this study. Interestingly, bacteria with high cellulolytic potential were
isolated only from intestine and rectum (Table 1). The low abundance of
cellulolytic bacteria in GI regions such as stomach can be attributed to the
digestive physiology and pH milieu of the stomach. As reported by Misra and
Shrivastava (1994), the pH in stomach of gastropod molluscs is more acidic
than other GI tract regions. Moreover, digestion in stomach is car-ried out by
enzymes produced by digestive glands or gastric cecae. The absence or low
abundance of cellulolytic members in stomach may be as a result of acidic
environment and extracellular enzy-matic digestion.
The significant outcome of our study is the isolation and iden-tification of
B. subtilis strains with high lignocellulolytic potentials from the GI tract of
GAS. Moreover our study indicates that these isolates possess various
extracellular enzyme activities and have significant potential to degrade various
lignocellulosic substrates. When five isolates with high cellulolytic potential
obtained in this study were tested for their capabilities to degrade various ligno-
cellulosic substrates, it was observed that they were able to degrade all the
tested substrates to varying extend. In comparison, these tested isolates
degraded agro residue based substrates such as grass straw, wheat husk and
filter paper more efficiently than commer-cially available cellulosic substrates
such as CMC, Avicel and Xylan. Most of these tested isolates produced
maximum endoglucanase, exoglucanase and xylanase activities when grown
on agro residues like grass straw, filter paper and wheat husk respectively.
Bacillus strains have been widely reported as predominant aerobic cellu-lolytic
species isolated from soils and waste sites with high cellu-lose content (Ulrich
and Wirth, 1999; Pourcher et al., 2001). Many previous studies have also
indicated that B. subtilis shows prom-ising potential for the production of
several bio-based products from lignocellulosic feedstock, since, unlike many
other currently used industrial microbes, it naturally imports and metabolizes
cellobiose and C5 sugars (Tobisch et al., 1997; Stülke and Hillen, 2000).
Hitherto B. subtilis has been largely studied for its tremen-dous industrial and
other biotechnological interests. But most of
€
those strains were isolated from sources (Oztürk et al., 2008; Sarker et al., 2010)
other than from invertebrate gut. B. subtilis is also known to have
multicellulase-carrying minicellulosomes due to which it has been extensively
exploited in conversion of lignocel-lulosic biomass into bio products viz.
bioethanol, biohydrogen etc. Moreover, Bacillus strains are known for their
ability to enter into resting states (spores) and are good producers of secondary
me-tabolites such as antibiotics (Lynd et al., 2002). These strategies could
provide them with an additional advantage over competitors under aestivation
and conditions of slow growth on cellulosic substrates.
78 M.A. Dar et al. / International Biodeterioration & Biodegradation 98 (2015) 73e80

Fig. 3. Endoglucanase activity of tested isolates on various lignocellulosic substrates (A: CMC, B: Grass straw, C: Wheat Husk, D: Filter paper). Values are means ± SD (n ¼ 3).

Fig. 4. Exoglucanase activity of tested isolates on various lignocellulosic substrates (A: Avicel, B: Grass straw, C: Wheat Husk, D: Filter paper). Values are means ± SD (n ¼ 3).

Gastropods have not been the main focus of recent prospecting studies However, our findings suggest that the isolates obtained in this study possess
targeting the cellulolytic bacteria due to the initial suc-cesses with the insect higher cellulolytic activities than the isolates from the guts of these insect. In
families, most notably termites (Tokuda and Watanabe, 2007) and beetles comparison, the endoglucanase activity of our isolates was found many fold
(Cazemier et al., 1997, 2003). higher than the activity of
 M.A. Dar et al. / International Biodeterioration & Biodegradation 98 (2015) 73e80
Fig. 5. Xylanase activity of tested isolates on various lignocellulosic substrates (A: Xylan, B: Grass straw, C: Wheat Husk). Values are means ± SD (n ¼ 3).
Bacillus sp. (Cho et al., 2010), Pseudoxanthomonas sp., Micro-bacterium sp.
(Okeke and Lu, 2011) and Promicromonospora pach-nodae (Cazemier et al.,
2003) isolated from the gut of termite
Reticulitermes speratus, scarab beetle Pachnoda marginata and forest litter soil
respectively. Similarly, avicelase activity exhibited by our isolates was higher
than avicelase activity by Serratia sp., Chrys-eobacterium sp., and Bacillus sp.
isolated from the gut of termite R. speratus (Cho et al., 2010).
Although, cellulolytic bacteria have been isolated from many resources, the
present study is the first report to have evaluated the A fulica's GI tract as a
source for isolation of lignocellulolytic bac-teria. The evaluation of cellulolytic
potential of isolates using plate based and various enzyme assay have shown
that these isolates indeed posses cellulolytic potentials and may have good
applica-tions for production of various lignocellulolytic enzymes for biofuel
industry. However, we attempted the isolation by employing the enrichment
technique that used CMC as cellulosic substrates. In order to reveal the
complete potential of GI tract of GAS as a source for isolation of
lignocellulolytic bacteria, further studies would be needed to know the effects
on enrichment by using various lignocellulosic substrates. Also, for the present
study, snails in active state were collected from only one location in one season.
The effects of physiological states of snail such as active and aes-tivating,
season of collection and collection sites on enrichment, isolation and
identification would need to be revealed. In the view of fact that B. subtilis has
multicellulase-carrying minicellulosomes, our future research will focus on
purification, identification and analysis of such multicellulase-carrying
minicellulosomes from the b. subtilis strains and Ochrobactrum sp. K38
obtained in this study. In conclusion, we have demonstrated that different GI
tract regions of GAS can serve as a good source for isolation of lignocellulolytic
bacteria that can be used for purification of various cellulolytic enzymes for
bioethanol industry.
79
Conflict of interest
Declared none.
Acknowledgment
Authors are grateful to Dr. Yogesh Shouche for permission to use the
sequencing facility at NCCS, Pune, India. MD acknowledges University of
Pune for providing the stipend.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ibiod.2014.11.016.
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