An Insight into the Celluloytic Potential of Three Strains of Bacillus Spp. Isolated from Benthic Soil of Aquaculture Farms in East Kolkata Wetlands, India

1Department of Zoology, Vidyasagar College, Block CL, Plot 3-8 & 44-50, Sector II, Salt Lake, Kolkata, West Bengal700 091, India. 2Department of Life Sciences, Presidency University, 86/1, College Street, Kolkata, West Bengal 700073, India. 3UGC – HRDC, Jadavpur University, Block LB, Plot No 8, Sector III, Salt Lake City, Kolkata, West Bengal 700098 Kolkata 700098, India. 4University of Burdwan, Rajbati, Burdwan, West Bengal 713104 , India.

The process of photosynthesis converts light energy into chemical energy and thus results in the production of plant biomass with cellulose as a major component 1 . Thus as one of the most abundant materials on Earth, cellulose plays a key role in the material flow of this planet. Cellulose is one of the major renewable resources and is considerably available in agricultural and municipal wastes, however, most practiced disposal method of this waste is by burning which not only eliminates the possibility of recycling but also pollutes the environment. 1,2 . Hence, research has been directed towards the study of the natural process of cellulose decomposition. The biogeochemical cycle of carbon completes with the degradation of the cellulose by microorganisms of the soil and guts of animals. Cellulase, an inducible enzyme is synthesized and secreted extracellularly by a diversity of microorganisms, including bacteria and fungi during their growth in cellulosic materials 3 . Cellulose is decomposed by the microorganisms with a multi-enzyme system which converts it into its monomeric form of glucose. The enzyme system comprises Endob -1,4 glucanase which randomly cleaves b 1,4 linkage and extracts elementary fibrils from cellulose crystals, followed by Exob -1,4 glucanase which cleaves the non-reducing end of the cellulose fibrils and converts it into Cellobiose or Cellodextrose which is finally hydrolysed to glucose by b -Glucosidases 4 . In the present study, the focus is drawn towards the prokaryotic genera only. There are numerous references of cellulose degrading bacteria from soil sources such as manure compost 5 , tea garden soils 6 , the soil of paddy field 7 , etc. These studies report several genera of bacteria like Bacillus, Acinetobacter, Clostridium, Paenibacillus, Trichonympha, Actinomycetes, etc. 7,8 with various degradation ability and enzymatic activity. Cellulose degrading bacteria have also been isolated from animal guts such as insect caterpillar and snail 1 , termites 9 and grass carp 10 .
T h i s p h e n o m e n o n o f c e l l u l o s e decomposition is important not only ecologically but also economically from the human perspective. Deeper insight into the cellulolytic microcosm can provide plausible remedies in the management of pollution caused by Solid Municipal Wastes 11 . On the other hand, depletion of non-renewable sources of energy is a matter of utmost concern to humanity. To solve this problem, plant biomass is the only foreseeable source that has a sustainable future. Cellulosic materials in this context are enticing because of its low cost and plentiful supply. Apart from these, the cellulolytic microorganisms are also found convenient in their industrial application where cellulose is used as a raw material. The most important of the industrial application may be the prospective use of lignocellulosic biofuels, where cellulosic materials would be used as a raw material for the production of sugars that can be converted into ethanol and other liquid fuels. The whole process would incorporate the use of microorganisms especially the prokaryotes to facilitate this conversion 12 . Other notable industrial application of the cellulose degrading bacteria are found in the composting, paper industry, breweries and food industries 13 However, data of the prokaryotes with cellulolytic potential from the benthic soil of aquatic ecosystems are scanty. The only related study has been done on mangrove soils of Mahanadi river delta, India which reports genera of bacteria like Micrococcus spp., Bacillus spp., Pseudomonas spp., Xanthomonas spp. and Brucella spp. as potential cellulolytic strains 14 . The study of the prokaryotic microcosm of the benthic soil of aquatic ecosystems is of utmost importance as they play a wide array of roles in the maintenance of the ecosystem. Recent advancements in the development of the aquaculture systems promote the idea of microbial-based culture systems over traditional culture methods. The Microbial-based culture system involves the study of the benthic soil which can lead to a better understanding of the roles of the members of the prokaryotic community in the microcosm of the aquaculture system 15 . Banking on this idea, this study investigates the benthic soil of two aquaculture farms in East Kolkata Wetlands in search of prokaryotic strains with cellulolytic potentials which can be used in the designing of a sustainable aquaculture system.
(Data in this paper are from first author's thesis to be submitted shortly for the partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Department of Zoology, The University of Burdwan, West Bengal, India.)

Culture Media
All reagents used in the study were of analytical grades procured from HIMEDIA (India) and Sigma Aldrich Chemical (USA). Minimal salt media (MSM) was used for culture of bacterial strains as a source of essential inorganic nutrients. Regular Culture and subculture were done with nutrient agar medium having Peptone 5 g, NaCl 5 g, Beef extract 1.5 g, Yeast extract 1.5 g, Agar 2%, pH 7.4±0.2 (per litre) and Luria agar having Casein enzymic hydrolysate 10g, Yeast extract 5g, Sodium chloride 5g, Agar 2%, pH 7.0±0.2 (Per litre) used after autoclaving (120°C temperature and 20 psi pressure, 15 minutes).

Collection
Soil (~5g) was collected from two Aquaculture farms in East Kolkata Wetlands (22.5528° N, 88.4501° E). Soil samples were collected from the top layer of the benthic soil under 60 cm of water . Soils were aseptically transferred to sterile containers. The containers were labelled and taken to laboratory for further processing.

Isolation
Bacterial isolation was done following the protocol of Gupta et al., 2012, 1 with minor modifications. 2g of soil was inoculated in 50 ml Minimal salt media (MSM) with 2 % w/v Carboxymethylcellulose (CMC) as the sole carbon source. The culture was maintained for 7 days at 37°C with constant shaking at 120 rpm.
The culture media was then subjected to serial dilution up to 10 6 times and was spread on Congo Red agar media KH 2 PO 4 0.5 g, MgSO 4 0.25 g, CMC 20 g, agar 15 g, Congo-Red 0.2 g, and gelatin 2 g; distilled water 1 L and at pH 6.8-7.2. This media is a fast process to test the cellulolytic activity via discoloration of Congo red around the colonies as halos. After incubation for 12 hours at 37°C, single colonies of the isolates were streaked in Congo red agar media. The colony size and diameter of the halo are noted. The Hydrolysing Capacity (HC) of the isolates were calculated using the ratio of the diameter of the halo and diameter of the colony. This method was used to determine the best isolates with considerable cellulolytic potential and were selected for further studies. In this study three strains outperformed other isolates and were selected for further enzymatic analyses.
The three selected isolates were cultured in an enzyme production media composed of MSM supplemented with sterilised Filter paper (@ 0.05g / 20 ml, cut into 1cm 2 pieces) for Exo-b -1,4 glucanase assay and the same media containing CMC (0.5 g / 20 ml), at pH 6.8-7.2 for Endob -1,4 glucanase assay, and the cultures were incubated for 96 hours at 37°C in a shaker incubator at 120 rpm.
After the incubation period, the cultures were subjected to centrifugation at 8000 rpm for 10 mins at 4°C. The supernatant was collected, filter sterilised and was stored at 4°C for future enzymatic assays. The pellet of the media supplemented with Filter paper was collected to analyze the degradation percentage by Gravimetric Analysis 1 .

Enzyme Assay
The end product of the cellulose digestion is reducing sugar 4 , thus, total cellulase activity was estimated by measuring the amount of reducing sugar formed from CMC and filter paper. The enzyme activity was determined according to the methods recommended by the International Union of Pure and Applied Chemistry (IUPAC) Commission on biotechnology following the protocol given by Rajoka and Malik, 1994 16 with minor modifications. The Exo-b -1,4 glucanase activity of the bacterial isolates were determined by incubating 0.5ml of the sterilised supernatant collected as mentioned earlier, with 1ml of 0.1M citrate buffer pH-4.8 and sterilised Whatman no1 filter paper (50mg) in the form of 1cm 2 pieces for 1hour at 50°C. The reaction was stopped by adding 3 ml of the 3,5-dinitrosalicylic acid (DNS) reagent to the reaction mixture. DNS added reaction mixture was boiled for 15 minutes and 40% sodium potassium tartrate was added to it. After cooling to room temperature, absorbance was measured at 550 nm (Systronics R2202 Double Beam Spectrophotometer) against blank with same constituents as above except the supernatant which was replaced with sterile triple distilled water of same volume.
Endo-b-1,4 glucanase activity was assayed by measuring the amount of reducing sugar from Carboxymethylcellulose and was determined by a similar process as that of Exo-b-1,4 glucanase, except the absorbance, was measured at 540 nm against blank 16 . The OD value thus obtained was then put into the equation as given below to obtain the activity of the enzyme in IU ml -1 min -1 . Enzyme Activity (IU ml -1 min -1 ) = Absorbance x Standard factor A standard estimation was done using known concentration gradient of glucose (0.1 -1.0 mg ml -1 , 10 samples at 0.1 mg ml -1 interval) and following the same principle as stated above. The OD value thus obtained was used to calculate the standard factor. The same procedure was followed once for Endo-b -1,4 glucanase at 540nm and for Exo-b-1,4 glucanase at 550 nm. The Standard factor was calculated according to the following formula and finally, the mean value of the Standard factor was taken for enzyme activity estimation.  Standard Factor = Concentration of Glucose standard (mg ml -1 ) / Absorbance (at 540 or 550nm) pH and Temperature stability Cell-free supernatants containing the crude enzymes were tested for their optimal pH and thermostability. The same process of enzymatic analysis was done by varying the pH of the enzyme production media and temperature of the enzyme prior to incubation. The pH was tested within a range of 4 -10 (7 samples with an interval of 1 pH) and temperature were tested from 20°C to 70°C (6 samples with 10°C interval).

Biochemical Characterisation
Biochemical characterisation of the bacterial isolates were done by an array of tests including MR, VP, Motility, Gram's Staining, Citrate, Amylase, Oxidase, Catalase etc. were done to characterize the strains according to Bergey's Manual of Systematic Bacteriology 17 .

Identification and Phylogenetic analyses
The Sequences obtained were analyzed using BLASTn in the NCBI database (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). The results thus obtained were screened and ten nearest neighbors were chosen based on the Highest Max Score and Total Score. The aligned sequence files were downloaded and further alignment and phylogenetic analyses were done in MEGA7 18 . The alignment of the sequences with their nearest neighbors was done using CLUSTALW. To eliminate the possibility of incorporation of potential chimeras, the sequences were checked using online webtool DECIPHER 19 . The phylogenetic tree was constructed based on the evolutionary distances calculated from the number of base substitutions per site of all the three strains by the Neighbor-joining method 20 using Maximum Likelihood Composite model 21 . The difference in composition bias was considered in evolutionary comparisons during the construction of the Phylogenetic tree 22 .

Statistical analysis
All experiments were performed at least in triplicate and values are expressed as mean values ± standard deviation (S.D.)]. Statistical analyses were done and means were compared by Student's t-test using SPSS 17.0 (SPSS Inc., Chicago, USA). Differences between means at (P ≤ 0.05) level were considered significant.

Characterisation of Cellulose degrading Strains
The three isolates were names as strains SWA4, SWA13a, and SWO6. Results of morphological characterization of these strains like colony shape, cellular morphology, gram character and detailed biochemical characteristics are provided in Table 1. The Results showed that SWO4 and SWO13a as a Gram-negative, nonmotile strain whereas SWA6a is a gram-positive, motile strain. All the three strains were capable of producing enzymes like gelatinase, amylase, urease, citratase, catalase and oxidase other than cellulase. The strains were capable of fermenting sugars with varying potentiality.

Molecular Characterisation
BLASTn analysis results are depicted in Table 2. Isolates SWO13a and SWA6a were identified to be as Bacillus cereus with 97% and 99% homology respectively. Isolate SWO4 was Bacillus flexus with 98% sequence homology. The Phylogenetic tree constructed is depicted in Figure  3. The analysis of the tree shows that the isolates are in separate lineages and are different from one another with SWO4 in a separate lineage altogether.

Screening test results
The Screening test result performed in Congo Red agar media revealed a glimpse of the  3. Evolutionary relationships of the three strains. The Genbank Accession Numbers for each strains used in this analysis is mentioned in parenthesis. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.10455038 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The differences in the composition bias among sequences were considered in evolutionary comparisons. The analysis involved 30 nucleotide sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 829 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 comparative account of cellulolytic potential of the three strains involved and is given in Table 3. The highest HC value was found in Isolate SWA6a and the lowest was given by SWO4. However, it is mention worthy that these three strains excelled other strains that were subjected to the same test and hence was selected for the enzymatic assay.

Enzyme Assay
The enzyme assay confirms the result obtained in the screening test and is given in Table  4. Isolate SWA6a scores the best enzymatic activity for both Exo -b,1-4glucanase and Endo -b,1-4-glucanase with an activity of 0.08214 ± 0.00412 and 0.11263 ± 0.00478 IU ml -1 min -1 respectively. The Overall enzyme activity ranged from 0.06112 to 0.08214 IU ml -1 min -1 for Exo -b,1-4glucanase and 0.10018 to 0.11263 IU ml -1 min -1 for Endo -b,1-4-glucanase. Gravimetric analysis revealed that isolates SWO4, SWO13a, and SWA6a were able to degrade 55.1%, 61.9% and 63.4% of raw cellulose given in the form of filter paper respectively, in a span of 96 hours under the given conditions.
Results of pH and Temperature stability estimation of both the enzymes of the three isolates are depicted graphically in Figure 1 and 2 respectively. Both the analyses showed a common pattern for both the enzymes. For pH, the maximum activity of the enzymes obtained from the strains showed the highest activity within a range of 6 -8 and diminishing at either extremities. In case of temperature the highest activity was recorded within 30 -40°C with the same trend of diminishing activity with either increase or decrease of the temperature. The isolate SWA6a not only showed the highest activity under optimum pH and temperature conditions but also was more temperature and pH tolerant when compared to the other two isolates.

DISCUSSION
Cellulose is indeed considered as a viable option for the supply of raw materials for several industries. Several research indicates its potentiality in this regard. However, the most environment friendly way to decompose and utilise this complex biomolecule is to degrade it using microorganisms like bacteria as a tool 23 . The past references showed varied degree of cellulolytic potential from unidentified strains with ranges of 0.012 to 0.196 IU ml -1 min -1 for Exo -b,1-4glucanase and 0.1622 to 0.400 IU ml -1 min -1 for Endo -b,1-4glucanase was estimated from bacteria inhabiting the gut of cellulose -feeding organisms 1 . Another study demonstrated the cellulolytic potential of Bacillus licheniformis isolated from Indian Hot springs to be 0.542 IU ml -1 min -1 for Exo -b,1-4glucanase and 0.120 IU ml -1 min -1 for Endo -b,1-4glucanase 24 .These two results correspond to the data obtained in the study. On the other hand, present findings do not correspond to the observations of Behera et al. who described a very high cellulolytic potential (2.471 to 98.253 IU ml -1 min -1 for Endo -b,1-4glucanase) produced by several genera of bacteria like Micrococcus spp., Bacillus spp., Pseudomonas spp., Xanthomonas spp. and Brucella spp. isolated from the benthic soil of Mahanadi river 14 .
The results from various experimental protocols explored revealed that all the three isolates were capable of utilizing cellulose as a sole carbon source and their efficacy in the breakdown of cellulose is also mention worthy. Similar results were also obtained in the estimation of cellulolytic potential by the earlier workers 1,24 . The data seems to be the first report of prokaryotes with cellulolytic potential from the benthic soil of Aquaculture farms of East Kolkata Wetlands. It can be said that these bacteria are efficient in the decomposition of cellulosic materials in the aquatic ecosystem and hence maintains the ecological balance of the aquaculture ponds by maintaining a proper biogeochemical cycle of carbon providing an ecologically healthy environment for fish culture. This study also illuminates the prospective use of these strains in the cellulose dependent industry and that these strains have the potential efficacy. They can also be used in Solid Waste Management via composting method 11 . From the perspective of aquaculture, identification of such strains with cellulolytic potential paves the way for future implementation for microbial -based culture methods. This strains can also be bioaugmented in the natural aquaculture ponds with high level of plant debris and will ultimately lead to the sustenance of the pond.