506 BIODEGRADATION OF COTTON SEED SOAPSTOCKS BY NOVEL INDIGENOUS
BACILLUS SP.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Gayatriben B. Patel and Kamlesh R. Shah
in degradation of oil (Veerabagu et al., 2014). Research
in bacterial lipases is of great demand now because of
value added potential industrial application (Sirisha
et al., 2010). Industries are seeking lipase producing
strains of bacteria which contain excellent properties
using cost effective methods on large scale production
(Bharathi et al., 2018).
Lipase (triacyl glycerol acylhydrolases, EC 3.1.1.3)
catalyzes the hydrolysis of triacyl glycerol to glycerol
and long chain fatty acids at oil water interface (Pualsa
et al., 2013). Research can be done toward lipases from
plant and animal origin but lipases from microbial ori-
gin are receiving much attention with the rapid develop-
ment of enzyme technology. Lipase act as biocatalysts
constitute the signi cant important role for biotechno-
logical applications (Hasan et al., 2006, Saxena et al.,
1999). Microbial lipases constitute much application
such as in the detergent industry, food industry, paper
and pulp industry, organic synthesis, bioconversion in
aqueous media, resolution of racemic acids and alco-
hols, regioselectiveacylations, ester synthesis, oleochem-
ical industry and lipases in medical application (Sharma
et al., 2001, Verma et al., 2012, Mauti et al., 2016, Sar-
aswat et al., 2017)
This study was conducted to isolate lipase producing
bacteria which were screened on tributyrin agar plates.
They were further analyzed for cellulase and protease
production by plate assay. The bacterial genus Bacil-
lus were identi ed on the basis of biochemical tests
and molecular 16s r DNA Partial Gene sequencing ana-
lyzes. Quantitative analysis of lipase production was
done spectrophotometrically using p-NPP as substrate.
Further study will conducted on enzymatic degrada-
tion.
MATERIAL AND METHODS
Soapstock samples were collected from two different
cotton oil re nery industries nearby Kadi (North Guja-
rat), India. At the starting season of cotton (November),
Soapstock samples were collected from the owing
stock at Washer discharge end of the pipe in a sterile
and air tight container. B/H (Bushnell–Haas) medium
was selected for enrichment of cotton seed oil soap-
stocks for microbial growth (Guru et al., 2013).10 gram
of cotton seed oil soapstock samples were added to 100
ml of B/H mediums and incubated at 37C in static con-
dition for 5 days. From each sample, 1ml of enriched
samples were transferred to the 100 ml of Tributyrin
broth medium incubated at 37 C, in shaking condition
at 100rpm for 48 hours. Enrichment was performed over
a 7 days of incubation.Enriched Soapstock samples were
serially diluted
.
Diluted samples were spread on to Tribu-
tyrin agar medium for isolation of Bacteria .TBA Plates
were incubated at 37C for 2days. Isolated colonies
were puri ed on same medium by streak plate method.
Pure cultures isolate were preserved at low temperature
in Nutrient agar slants for screening and further use.
Lipase-producing strains were screened by qualitative
plate assay according to Lokre et al., 2014. Isolates were
spot inoculated on tributyrin agar plates and incubated
at 37°C for 2 days. Zone of clearance was observed due
to hydrolysis of tributyrin by lipase enzyme.
Cellulase and Protease activity were done by qualita-
tive agar plate assay in nutrient agar media containing
respective substrates. Culture was spot inoculated and
incubated at 37 °C for 2 days. Check for the zone of
clearance around the colonies due to utilization of the
particular substrate.
Culture was grown in medium containing 1% car-
boxy methyl cellulose (Dabhi et al., 2014). After incu-
bation the CMC plates were ooded with 0.1 % congo
red staining, after 5 min stain was discarded and the
plates were distained by 1M NaCl solution with con-
tinuous stirring for 15-20 min. The clear zone around
colonies indicated cellulose hydrolysis.Protease activ-
ity was checked in medium containing 1% skim milk
as substrate (Prabavathi et al., 2012). Spot inoculated
cultures were incubated at 37 °C for 2 days and observed
for clearance zone around colonies.
Selected Bacterial cultures that show Positive lipase
production in plate assay, which were subjected fur-
ther for Quantitative estimation. 2 days old bacterial
cultures grown on TBA medium were used for inocula-
tion. One loopfull culture was inoculated into 100 ml
of inoculum medium containing: peptone 0.5%, Yeast
extract 0.5%, NaCl 0.5% and cotton seed Oil 1%. Cul-
tures were incubated at 37C and 100 rpm for 4hrs. 5%
inoculum medium was further inoculated into 100 ml
of same medium (as mentioned above) for lipase pro-
duction and incubated at 37C and 100 rpm for 5 days.
Enzyme assay was performed according to the method
by Winkler et al., 1997 with some modi cation. The cul-
ture ltrate (production medium) was removed at 24 hr
interval from each ask & centrifuged at 10,000 rpm for
10 min at 4C. Supernatant was used for enzyme assay.
Lipase activity was determined by a spectrophotometric
assay using p-nitrophenyl palmitate (pNPP) as substrate.
P-NPP was hydrolysed by lipase to give p-NP which
gave yellow color, absorbance of which was measured
spectrophotometrically at 410 nm against enzyme free
blank. Statistical Analysis were done in Microsoft word
excel data analysis of lipase production.
The isolates showing maximum zone of clearance
were selected for further analysis. Morphological and
biochemical characteristics of the isolates were stud-
ied for the identi cation of the potent Bacterial isolate.
Molecular characterization of potent Bacterial strains