cellulose , a structural carbohydrate of the plant cell wall , is an abundant and ubiquitous polymer .
the use of cellulose for the second generation biofuel production involves the hydrolysis of cellulosic biomass , that is , saccharification , to form simple sugar monomers for the fermentation into bioethanol [ 13 ] .
cellulases are the group of enzymes involved in the conversion of cellulosic substrates to fermentable sugars
. main members of this group include endoglucanase ( ec 3.2.1.4 ) , exoglucanase or cellobiohydrolase ( ec 3.2.1.91 ) , and -glucosidase ( ec 3.2.1.21 ) .
the endoglucanase hydrolyzes -1,4 bonds in cellulose molecule , whereas exoglucanase cleaves the ends to release cellobiose , and -glucosidase converts cellobiose to glucose .
several cellulase producing fungi such as aspergillus , rhizopus , and trichoderma species [ 6 , 7 ] and bacteria such as bacillus , clostridium , cellulomonas , thermomonospora , ruminococcus , bacteroides , erwinia , and acetivibrio species [ 810 ] have been identified . however , the isolation and characterization of novel cellulose hydrolyzing enzymes from bacteria are still a highly active research area , because bacteria have a higher growth rate than fungi , leading to greater production of enzymes .
also , the habitat of bacteria covers different environmental niches , which favors the existence of versatile strains such as thermophiles , psychrophiles , alkaliphiles , and acidophiles .
the culturable cellulase producing bacteria have been isolated from the variety of sources such as composting heaps , decaying agricultural wastes , the feces of cow   and elephant , gastrointestinal tract of buffalo and horse , soil , and extreme environments like hot - springs .
varga and kovler   have reported that the feed fibers were not completely converted to animal product in intensive animal farming , and 2070% undigested cellulose was carried out with feces .
some studies have explained that the crude fiber degradation in gut is not optimal , and the fiber content of feces is still high , which can be utilized efficiently by microbes present in the feces of the herbivores .
rhinoceros are presumed to have an efficient system for cellulose digestion , as its main food wild grass primarily consists of cellulose . in this study
, the dung of the pachyderm from kaziranga national park , assam , india , has been used as the source of cellulolytic bacteria .
carboxymethylcellulose ( cmc ) ( low viscosity , 50200  cp ) was procured from sigma aldrich ( st . louis , mo , usa ) .
medium components and congo red ( analytical grade ) were procured from hi - media pvt . ltd . ,
the dung sample of one - horned indian rhinoceros ( rhinoceros unicornis ) was collected from its natural habitat , kaziranga national park , assam , india .
presterilized spatula and plastic bags were used for sample collection , and before bacterial isolation the samples were stored at 4c in ice box for approximately 12  h. dung sample ( 0.5  g ) was suspended with 50  ml 0.85% ( w / v ) sterile nacl solution in a 250  ml conical flask , which was shaken at 180  rpm for 1  h at 37c .
an aliquot of 100  l of each dilution was spread plated onto bushnell haas medium ( bhm )   agar plates amended with carboxymethylcellulose ( cmc ) ( ph 7.0 ) containing ( g / l ) cmc ( 10.0 ) , k2hpo4 ( 1.0 ) , kh2po4 ( 1.0 ) , mgso47h2o ( 0.2 ) , nh4no3 ( 1.0 ) , fecl36h2o ( 0.05 ) , cacl2 ( 0.02 ) , and agar ( 20.0 ) [ 19 , 20 ] .
the plates were incubated at 37c for 96  h. morphologically dissimilar and discrete colonies were picked from different dilution plates and streaked on separate bhm - cmc plate with grids drawn over it and incubated at 37c for 96  h. the replica plates were also prepared separately for staining .
the stain was poured off , and the plates were washed with 1  m nacl .
the isolates showing clear zone around the colonies were picked from master plate and further used for the enzyme production in liquid medium .
the selected cultures were maintained on nutrient agar slants containing ( g / l ) peptone ( 5.0 ) , beef extract ( 1.0 ) , yeast extract ( 2.0 ) , nacl ( 5.0 ) , and agar ( 20.0 ) .
the isolates , selected on the basis of plate staining method , were grown in 50  ml enzyme production medium ( at ph 7.0 ) containing the following components ( g / l ) : cmc ( 10.0 ) , k2hpo4 ( 1.0 ) , kh2po4 ( 1.0 ) , mgso47h2o ( 0.2 ) , nh4no3 ( 1.0 ) , fecl36h2o ( 0.05 ) , cacl2 ( 0.02 ) , and yeast extract ( 5.0 ) .
this medium is the same as the previously used medium during isolation , with the only difference of addition of yeast extract .
50  ml medium ( containing 2% inoculum ) was taken in 250  ml erlenmeyer flask and incubated at 37c at 180  rpm for 72  h. after every 6  h , the culture was centrifuged at 12000  g for 20  min at 4c .
the cell - free culture broth containing the crude enzyme was used for estimation of cmcase activity .
based on the higher cmcase activity ( as described later ) , an isolate ss35 ( named after its colony number ) was selected for further characterization and identification .
the enzyme production by the isolate ss35 was monitored with cell growth at 600  nm using uv - visible spectrophotometer ( perkin elmer , model lambda-45 ) .
the cmcase activity ( u / ml ) was measured by estimation of reducing sugars liberated from cmc .
the enzyme assay was carried out by incubating the enzyme with cmc for 15  min at 37c . the reaction mixture
( 100  l ) contained 50  l of enzyme and 1.0% ( w / v ) final concentration of cmc in 50  mm phosphate buffer ( ph 7.0 ) .
the reducing sugar was estimated by the method of nelson and somogyi [ 23 , 24 ] .
the absorbance was measured at 500  nm using a uv - visible spectrophotometer ( perkin elmer , model lambda-45 ) against a blank with d - glucose as standard .
one unit ( u ) of cellulase activity is defined as the amount of enzyme that liberates 1  mol of reducing sugar ( glucose ) in 1  min at 37c and ph 7.0 .
morphological and biochemical properties of the isolate were identified , evaluated , and compared , as described in bergey 's manual of systematic bacteriology .
the cell morphology of the selected isolate was observed under scanning electron microscope ( leo 1430 vp , leo electron microscopy ltd . ,
gram staining , endospore staining , and urease test were done as per standard protocol .
the catalase activity was determined adding few drops of 3% ( v / v ) h2o2 to 5  ml of 18  h grown culture .
the nitrate agar slants ( m072 , himedia ) were used to test nitrate reducing property of the isolate ss35 .
triple sugar iron ( tsi ) slants ( m021i , himedia ) containing three sugars , namely , glucose , lactose , and sucrose , were used for acid and h2s production test .
acid production after carbohydrate fermentation was detected by the visible change in color from red to yellow .
the temperature tolerance test was performed by growing the isolate in nutrient broth and incubating at the temperatures ranging 2050c .
16s rdna and partial gyrase a gene sequencing of bacterial culture were done in xcelris labs limited , ahmedabad , india .
the genomic dna of the isolate ss35 was extracted using qiagen dna extraction kit and purified by qiaamp dna purification kit ( qiagen ) for nucleotide sequence analysis .
the universal 16s rdna primer 8f ( 5 agagtttgatcctggctcag 3 ) and 1492r ( 5 acggctaccttgttacgactt 3 ) were used for amplification of genomic dna by polymerase chain reaction ( pcr ) .
the gyra region was amplified using the primers , p - gyra - f ( 5 cagtcaggaaatgcgtacgtcctt 3 ) and p - gyra - r ( 5 caaggtaatgctccaggcattgct 3 ) .
the concentration of each primer in 25  l pcr reaction mixture was 10  pmol and 1x pcr master mix ( mbi fermentas ) .
the pcr reaction was run for 30 cycles in a thermal cycler ( eppendorf ) , and the thermal profile used for the pcr was as follows : initial denaturation at 95c for 2  min , final denaturation at 94c for 30  s , primer annealing at 52c for 30  s , and extension at 72c for 90  s. full extension of the products was ensured by running a final cycle that included extension for 10  min at 72c .
pcr product of 5  l from each tube was mixed with 1  l of 6x gel loading dye , and this mixture was subjected to electrophoresis on 1.2% agarose gel to confirm the targeted pcr amplification .
the amplified product was excised from the gel and purified using qiaamp dna purification kit ( qiagen ) .
the concentration of the purified dna was determined , and it was subjected to automated dna sequencing on abi 3730xl genetic analyzer ( applied biosystems , usa ) .
the cycle sequencing was carried out using bigdye terminator v3.1 cycle sequencing kit following manufacturer 's instructions .
the cycle sequencing was carried out in a final reaction volume of 20  l using 200  l capacity pcr tube .
the cycling protocol was designed for 25 cycles as follows : denaturation at 96c for 10  s , annealing at 52c for 5  s , and extension at 60c for 4  min .
after cycling , the extension products were purified and mixed well in 10  l of hi - di formamide .
eluted products were placed in a sample plate , heated at 95c for 5  min , chilled , and loaded into autosampler of the instrument .
both the ends of the sequences were verified with the chromatogram file , and the resulted consensus sequences were used to carry out basic local alignment search tool ( blast ) with nr database of ncbi genbank using megablast algorithm .
multiple sequence alignment was performed by using clustal w , and evolutionary history was inferred using the neighbor - joining method .
the evolutionary distances were computed using the kimura 2-parameters method , and phylogenetic analysis was carried out with mega4 .
among 36 isolates , 9 cellulose hydrolyzing microorganisms were screened on the basis of plate staining method . the isolates ( no .
21 , 24 , 25 , 28 , 31 , 32 , 34 , 35 , and 36 ) showed clear zone around colonies after staining the plates with congo red and destaining with 1  m nacl as shown in figure 1 .
however , plate - screening method is not quantitative because of poor correlation between enzyme activity and colony to clear zone ratio .
the colonies showing yellow - colored halo zones were picked from replica plate and further screened on the basis of cmcase production in liquid medium . out of 9 isolates , isolate no .
35 exhibited maximum cmcase activity of 0.079  u / ml ( details given in table 1 ) .
this value was higher than activity of cmcase produced from some known natural isolates ( expressed per ml of cell - free culture broth ) , for example , cellulomonas sp .
( 0.0336  u / ml , isolated from coir retting effluents ) , micrococcus sp .
( 0.0152  u / ml , isolated from coir retting effluents ) , bacillus sp .
( 0.0197  u / ml , isolated from coir retting effluents ) , brevibacillus sp .
jxl ( 0.02  u / ml , isolated from swine waste ) , brevibacillus sp .
duselg12 ( 0.02  u / ml , isolated from gold mine ) , geobacillus sp .
duselr7 ( 0.058  u / ml , isolated from gold mine ) , geobacillus sp .
( 0.0113  u / ml , isolated from sugar refinery wastewater ) , and bacillus subtilis as3 ( 0.07  u / ml , isolated from cow dung ) [ 3337 ] .
have reported maximum cmcase activity of 0.079  u / ml by bacillus pumilus eb3 produced in a 2  l stirred tank reactor , which was equal to the cmcase activity of the isolate in this study .
35 was revealed to be an efficient cmcase producer species and was designated as ss35 .
the growth curve of ss35 along with cmcase production profile ( figure 2 ) revealed that the enzyme production was associated with cell growth and reached maximum at late log phase .
slight reduction in enzyme production after 48  h could be a consequence of instability of the enzyme at 37c or the activity of proteases present in the crude enzyme solution .
the isolate ss35 was found to be rod - shaped cells with a width and length of 0.50.6  m and 1.51.6  m , respectively , as observed under scanning electron microscope ( figure 3 ) .
the isolate was found to be a gram - positive , spore forming bacterium , and it gave positive test for catalase , nitrate reduction , and starch hydrolysis , whereas negative for urease and hydrogen sulfide production .
the absence of black precipitate at the base of the tube indicated that hydrogen sulfide was not produced .
the color of tsi agar slant was turned from red to yellow , which indicated that the bacterium was able to ferment the sugars glucose , lactose , and sucrose .
the temperature tolerance test revealed that the isolate was able to grow at a wide temperature range 2050c .
the phylogenetic tree generated using 16s rdna gene sequences of the isolate ss35 showed that the bacterium has the highest homology with bacillus amyloliquefaciens
ab679994.1 ) ( figure 4 ) . the bacterial identification using 16s rdna gene sequence is a widely practiced technique , although with limitations for the members of closely related taxa . to overcome this limitation ,
several studies have been done , which concluded that some protein - coding genes such as rna polymerase ( rpob ) gene , rna polymerase sigma factor ( rpod ) gene , gyrase b ( gyrb ) gene , and gyrase a ( gyra ) gene   can be used for the identification of closely related taxa , because the genetic variation in protein - coding genes are much higher .
chun and bae   demonstrated that the gyra sequences , code for dna gyrase subunit a , can be used for accurate identification of bacillus amyloliquefaciens and related taxa including bacillus subtilis , bacillus vallismortis , bacillus mojavensis , bacillus atrophaeus , and bacillus licheniformis .
therefore , in this study partial gyra gene sequences have been used for the confirmation of the result obtained from 16s rdna sequence analysis .
the phylogenetic analysis using partial gyra gene sequences also revealed that the isolate ss35 has the highest homology with bacillus amyloliquefaciens fzb45 ( genbank accession no . : fn662840.1 ) , as shown in figure 5 .
numbers at nodes of the tree are indications of the levels of bootstrap support based on a neighbor - joining analysis of 1,500 resampled datasets .
the 16s rdna and gyra gene sequences of the isolate b. amyloliquefaciens ss35 have been deposited in the ncbi nucleotide sequence database under the accession nos .
cellulose hydrolytic bacteria have been isolated from rhinoceros dung , and some potent cellulose degrading bacteria have been identified . among all cellulolytic bacteria ,
bacterium has been identified as bacillus amyloliquefaciens ss35 on the basis of 16s rdna and partial gyrase a gene sequence analyses .
the cmcase production has been observed to be associated with cell growth and has maxima at the late exponential phase of growth .
optimization of medium composition and fermentation parameters can further increase the cellulase production from b. amyloliquefaciens ss35 .
these attributes also make b. amyloliquefaciens ss35 a potential candidate in solid state fermentation for cmcase production using cellulosic biomass .
 cellulose hydrolyzing bacteria were isolated from rhinoceros dung and tested for clear zone formation around the colonies on the agar plates containing the medium amended with carboxymethylcellulose as a sole carbon source . 
 isolates were further screened on the basis of carboxymethylcellulase production in liquid medium . out of 36 isolates 
 , isolate no . 
 35 exhibited maximum enzyme activity of 0.079  u / ml and was selected for further identification by using conventional biochemical tests and phylogenetic analyses . 
 this was a gram - positive , spore forming bacterium with rod - shaped cells . 
 the isolate was identified as bacillus amyloliquefaciens ss35 based on nucleotide homology and phylogenetic analysis using 16s rdna and gyrase a gene sequences . 