HPLC analysis revealed the retention time of authentic standards
of DMP, MMP and PA to be 8.86, 6.84, and 5.12 min respectively.
During incubation of X. indica, DMP concentration declined
after initial lag phase of 3 days and highest degradation (98.5%) was
observed at 9th day, further reduction was noticed up to 12 days
(Fig. 3). In control, no degradation of DMP was observed confirming
the role of X. indica in bioremediation of DMP. During the course of
degradation other metabolites (MMP and PA) were also detected.
MMP started accumulating after 3 days and concentration reached
140 mg l1 by 6th day. PA was also detected during this period and
gradually increased till 9th day. No peak of MMP or PAwas detected
throughout the experiment in uninoculated sterile mineral medium
supplemented with DMP.
In separate experiments, where MMP and PA were used as
carbon source, 85% and 55% degradationwas observed respectively
after 12 days. Finally, after 15 days MMP was not detected but 35%
of PA remain unutilized.
The bacterial growth in terms of total cell pellet protein and
esterase activity was measured to assess the relationship between
these parameters and degradation of different phthalates. The
growth of X. indica peaked (263.75 mg/ml) by 9th day along with
degradation of DMP. Growth of X. indica exhibited highly significant
positive correlation (r ¼ 0.70) with degradation of DMP as well as
formation of MMP (r ¼ 0.80) and PA (r ¼ 0.92). Maximum growth of
X. indica was observed (284.5 mg/ml) on 12th day, when MMP was
used as sole carbon source in the medium (Fig. 4).
The esterase activity of the culture supernatant also increased
and correlated well with the degradation of DMP (r ¼ 0.887). DMP
and MMP was used as sole carbon source in minimal media.
Maximum activity of esterase (46.94 IU/ml) was recorded at 9th
Day (Fig. 4). Aerobic degradation of DMP is mainly due to breakdown
of ester bond by esterases to form MMP and PA subsequently.
To confirm the role of esterase in DMP degradation another
experiment was carried out with purified Xenorhabdus esterase
enzyme, which resulted in >25.6% degradation of DMP within 24 h
along with formation of MMP and PA. In uninoculated control, no
degradation of DMP was observed.
Substrate specificity analysis of culture supernatant and purified
enzyme showed that p-nitrophenyl octanoate and p-nitrophenyl
palmitate were virtually inert substrate. The esterase enzyme
showed a strong preference for short chain esters i.e., p-nitrophenyl
butyrate and p-nitrophenyl deconoate
HPLC analysis revealed the retention time of authentic standardsof DMP, MMP and PA to be 8.86, 6.84, and 5.12 min respectively.During incubation of X. indica, DMP concentration declinedafter initial lag phase of 3 days and highest degradation (98.5%) wasobserved at 9th day, further reduction was noticed up to 12 days(Fig. 3). In control, no degradation of DMP was observed confirmingthe role of X. indica in bioremediation of DMP. During the course ofdegradation other metabolites (MMP and PA) were also detected.MMP started accumulating after 3 days and concentration reached140 mg l1 by 6th day. PA was also detected during this period andgradually increased till 9th day. No peak of MMP or PAwas detectedthroughout the experiment in uninoculated sterile mineral mediumsupplemented with DMP.In separate experiments, where MMP and PA were used ascarbon source, 85% and 55% degradationwas observed respectivelyafter 12 days. Finally, after 15 days MMP was not detected but 35%of PA remain unutilized.The bacterial growth in terms of total cell pellet protein andesterase activity was measured to assess the relationship betweenthese parameters and degradation of different phthalates. Thegrowth of X. indica peaked (263.75 mg/ml) by 9th day along withdegradation of DMP. Growth of X. indica exhibited highly significantpositive correlation (r ¼ 0.70) with degradation of DMP as well asformation of MMP (r ¼ 0.80) and PA (r ¼ 0.92). Maximum growth ofX. indica was observed (284.5 mg/ml) on 12th day, when MMP wasused as sole carbon source in the medium (Fig. 4).The esterase activity of the culture supernatant also increasedand correlated well with the degradation of DMP (r ¼ 0.887). DMPand MMP was used as sole carbon source in minimal media.Maximum activity of esterase (46.94 IU/ml) was recorded at 9thDay (Fig. 4). Aerobic degradation of DMP is mainly due to breakdownof ester bond by esterases to form MMP and PA subsequently.To confirm the role of esterase in DMP degradation anotherexperiment was carried out with purified Xenorhabdus esteraseenzyme, which resulted in >25.6% degradation of DMP within 24 halong with formation of MMP and PA. In uninoculated control, nodegradation of DMP was observed.Substrate specificity analysis of culture supernatant and purifiedenzyme showed that p-nitrophenyl octanoate and p-nitrophenylpalmitate were virtually inert substrate. The esterase enzymeshowed a strong preference for short chain esters i.e., p-nitrophenylbutyrate and p-nitrophenyl deconoate
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