639148
Isolation and
characterization of
cellulolytic bacteria from the
Stain Lake House
- Sterilized
cellulose bait
- Different depths
- Equidistant point
- Provided matrices for colonizing bacteria
- Transfer into Falcon tubes
containing freshwater,
same lake
- Isolated vigorously
- Remove biofilm
- Inoculated on tryptic soy agar
plates
- Incubated for 7 days at 18 degree
celcius
- Individual Colonies - purified and
stored in 20% glycerol at 18 degree
celcius
- Bacteria recovered from cellulose
baits were plated in TSA , Incubated
at 18 degree celcius for 3-7 days
- agar medium flooded with an
aqueous solution of congo red foe
30 min
- Performed in triplicate
- Performed in triplicate
- agar medium flooded with an
aqueous solution of congo red foe
30 min
- Bacteria recovered from cellulose
baits were plated in TSA , Incubated
at 18 degree celcius for 3-7 days
- Individual Colonies - purified and
stored in 20% glycerol at 18 degree
celcius
- Incubated for 7 days at 18 degree
celcius
- Remove biofilm
- Isolated vigorously
- Transfer into Falcon tubes
containing freshwater,
same lake
- Different depths
- classification
- CMAA 1185
- closely related to Bacillus saffensis
- shared 16S rRNA similarity of 98.9%
- higher activity pH 3
- higher activity pH 3
- shared 16S rRNA similarity of 98.9%
- closely related to Bacillus saffensis
- CMAA 1184
- Form distinct phyletic line
- associated with Bacillus subtilis
- closest with B. subtilis subsp.inaquosorum
- shared 16s rRNA gene similarity of 99.4 %
- produce alkaline cellulase
- optimum pH 10
- optimum pH 10
- produce alkaline cellulase
- shared 16s rRNA gene similarity of 99.4 %
- closest with B. subtilis subsp.inaquosorum
- associated with Bacillus subtilis
- Form distinct phyletic line
- CMAA 1185
- Identification
- Genomic DNA was extracted
- PureLink genomic kit
(Invitrogen)
manufacturer protocol
- PCR ampification, Zucchi et al
method
- Almost complete 16s rRNA gene
sequence aligned manually using
MEGA version 5 software
- Phylogenetic and bootrap were inferred
- Further evaluated by Fatty Acid
Methyl ester
- Peak were identified by Sherlock
Microbial Identification (MIDI)
- Inoculating selected isolates
to tryptic soy broth
- With wheat bran and
incubated at 20 degree
celcius for 1 day
- Enzyme was sampled for
centrifugation & Filtration
- Enzyme extracted and incubated at
20 degree celcius for 30 min
- Cellulase activity was also assayed
using Dixtrin, microcrystallline cellulose,
and xylan as subtrates
- Reaction interrupted with
addition of 3,5-dinitrosalicyclic
acid and heated at 100 degree
celcius for 5 min prior to
quantification by
spectrophotometer at 540 nm
- Cellulase activity was also assayed
using Dixtrin, microcrystallline cellulose,
and xylan as subtrates
- Enzyme extracted and incubated at
20 degree celcius for 30 min
- With wheat bran and
incubated at 20 degree
celcius for 1 day
- TSB version 6 database
- Inoculating selected isolates
to tryptic soy broth
- 7683 Series injector, Flame
ionization detector, HP-5
capillary column
- Peak were identified by Sherlock
Microbial Identification (MIDI)
- Further evaluated by Fatty Acid
Methyl ester
- Phylogenetic and bootrap were inferred
- Almost complete 16s rRNA gene
sequence aligned manually using
MEGA version 5 software
- PureLink genomic kit
(Invitrogen)
manufacturer protocol
- Genomic DNA was extracted
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