6
S. Chatterjee et al.
on CMC agar plate was deposited and sequenced at Microbial Type Culture Collection and Gene Bank (MTCC), CSIR-IMTECH, Chandigarh, India. The sequence
obtained was compared using BLASTN program and the members of the closely
related genera were retrieved from EzTaxon server (Chun et al. 2007) and aligned
using the MEGA software version 6.0 (Tamura et al. 2013). Phylogenetic trees were
constructed using the neighbor-joining as well as maximum parsimony algorithms.
Bootstrap analysis was performed to assess the confidence limits of the branching.
2.6 Production of Bioethanol
The isolated CDB strain was grown in two sets, one in CMC media and another in
basal salt media composed of KH 2 PO 4 , 2 g; MgSO 4 , 0.2 g; NaCl, 0.2 g; NaNO 3 , 2.5 g;
CaCl 3 ·6H 2 O, 0.1 g; Whatman No. 1 filter paper, 2.5 g; distilled water, 1 L; pH 6.8–7.2,
for 4 days at 37 °C with constant shaking of 150 rpm. The supernatant obtained from
the first set of culture was used as the source of enzymes for saccharification process,
where as the broth obtained from the second culture was used for fermentation
using Saccharomyces cerevisiae (NCIM Accession No: 3594, NCL, Pune, India).
Simultaneous saccharification and fermentation processes were carried out at 27 °C
for 6 days as described by Jain et al. 2014. Preparation of yeast growth medium was
done using yeast extract 5.0 g, peptone 5.0 g, diluted saccharified slurry to attain an
overall sugar percentage of 1%, volume made up to 1000 ml with pH 5.0.
Post fermentation, iodoform test was done to confirm the presence of ethanol. The
fermented mash was distilled and alcohol percentage was estimated (Ghosal et al.
2013).
2.7 Iodoform Test
The presence of ethanol in the culture broth was confirmed by iodoform test (Lieben
1870). Around 10 drops of methanol, ethanol, and the culture broth were taken
in three separate test tubes. The methanol and ethanol were taken as negative and
positive control, respectively. Around 10 drops of diluted NaOH solution were added
to each test tube along with 30 drops of iodine solution. The color developed was
later analyzed.
3 Results and Discussion
3.1 Isolation of Cellulose-Degrading Bacteria
Cellulose-degrading bacteria were isolated from the soil sample on CMC agar
medium using serial dilution. Serial dilution ranging from 10
−1 to 10
−3 under sterile
S. Chatterjee et al.
on CMC agar plate was deposited and sequenced at Microbial Type Culture Collection and Gene Bank (MTCC), CSIR-IMTECH, Chandigarh, India. The sequence
obtained was compared using BLASTN program and the members of the closely
related genera were retrieved from EzTaxon server (Chun et al. 2007) and aligned
using the MEGA software version 6.0 (Tamura et al. 2013). Phylogenetic trees were
constructed using the neighbor-joining as well as maximum parsimony algorithms.
Bootstrap analysis was performed to assess the confidence limits of the branching.
2.6 Production of Bioethanol
The isolated CDB strain was grown in two sets, one in CMC media and another in
basal salt media composed of KH 2 PO 4 , 2 g; MgSO 4 , 0.2 g; NaCl, 0.2 g; NaNO 3 , 2.5 g;
CaCl 3 ·6H 2 O, 0.1 g; Whatman No. 1 filter paper, 2.5 g; distilled water, 1 L; pH 6.8–7.2,
for 4 days at 37 °C with constant shaking of 150 rpm. The supernatant obtained from
the first set of culture was used as the source of enzymes for saccharification process,
where as the broth obtained from the second culture was used for fermentation
using Saccharomyces cerevisiae (NCIM Accession No: 3594, NCL, Pune, India).
Simultaneous saccharification and fermentation processes were carried out at 27 °C
for 6 days as described by Jain et al. 2014. Preparation of yeast growth medium was
done using yeast extract 5.0 g, peptone 5.0 g, diluted saccharified slurry to attain an
overall sugar percentage of 1%, volume made up to 1000 ml with pH 5.0.
Post fermentation, iodoform test was done to confirm the presence of ethanol. The
fermented mash was distilled and alcohol percentage was estimated (Ghosal et al.
2013).
2.7 Iodoform Test
The presence of ethanol in the culture broth was confirmed by iodoform test (Lieben
1870). Around 10 drops of methanol, ethanol, and the culture broth were taken
in three separate test tubes. The methanol and ethanol were taken as negative and
positive control, respectively. Around 10 drops of diluted NaOH solution were added
to each test tube along with 30 drops of iodine solution. The color developed was
later analyzed.
3 Results and Discussion
3.1 Isolation of Cellulose-Degrading Bacteria
Cellulose-degrading bacteria were isolated from the soil sample on CMC agar
medium using serial dilution. Serial dilution ranging from 10
−1 to 10
−3 under sterile