Chapter V: Conclusions and Future Work
CHAPTER V
CONCLUSIONS AND FUTURE WORK
5.1 Conclusions
This research investigated the isolation of chromium tolerant microorganisms from
chromium contaminated sites. Two isolates one from solid waste and the other from liquid
effluent were considered to be chromium tolerant species and were sent for 16S rDNA
analysis, where it was found that the isolate from solid waste belonged to Bacillus sp. and
the isolate from liquid effluent was an Arthrobacter sp. Both the isolates could be
considered to be novel, as they were only 98% (Bacillus sp.) and 96% (Arthrobacter sp.)
similar to their nearest homologs in the phylogenetic tree. The biochemical characteristics
were also evaluated and both the isolates were found to exhibit catalyse and oxidase
activities.
The toxicity effect of chromium was evaluated for these chromium-tolerant species
by enriching the isolates sequentially with increasing concentration of chromium (added
in logarithmic phase). It was found that Arthrobacter sp. was more tolerant to Cr when
compared to Bacillus sp. The experiments demonstrated the adaptation capacity of an
organism towards chromium tolerance. It was found that Arthrobacter sp. adapts more to
the metal contaminated environment when compared to Bacillus sp.
During the chromium removal assay, it was found that both the isolates were able
to remove more than 50% of chromium in 3-5 hours and more than 95% in 24 hours at
concentration ranging from 2 ppm to 40 ppm of chromium. The intracellular
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compartmentalization of chromium by the two isolates showed that Bacillus sp. was able
to sequester/uptake 85.6% of the initial 40 ppm of chromium, whereas Arthrobacter sp..
sequestered only 76.5% of the initial 40 ppm of chromium. Transmission electron
microscopy results showed that higher intracellular uptake (and compartmentalization) of
chromium occurred for Bacillus sp. when compared to Arthrobacter sp. A higher
accumulation was found in the cytoplasmic region of Bacillus sp. whereas it was more in
periplasmic region in the case of Arthrobacter sp.
FTIR studies indicated that the main adsorption sites for chromium were the amino
and carboxylate groups. XRD analysis showed that chromium was associated with
phosphoryl, hydroxyl, caboxyl and carbonyl ligands. XPS analysis showed the speciation
of the chromium adsorbed by the cells. It was found that the intensity of the peaks were
more in the case of Bacillus sp. (S1) than Arthrobacter sp. (L4), indicating higher
adsorption.
The characterization of the exo-polysaccharides showed that L4 was more tolerant
towards chromium when compared to S1. Arthrobacter sp. is known to produce greater
amount of EPS. It was found that the EPS in treated sample mainly contained increased
amount of proteins when compared to carbohydrates. This again suggests that the metal
adhesion to the EPS is mainly to carboxyl and amino groups.
AFM analysis showed that L4 was the more tolerant organism. L4 again showed a
greater production of EPS when compared to S1. The roughness of Bacillus sp. (S1)
increased more (on chromium exposure) when compared to Arthrobacter sp. (L4),
possibly due to Bacillus sp. being less tolerant and hence undergoing lysis and forming
organic granules with the metal and thus causing greater roughness.
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SEM and EDX analysis showed the formation of cage-like structure in the case of
S1 (Bacillus sp.) that increased the capacity of the metal to bind to the cells. L4
(Arthrobacter sp.) was the more tolerant species and no deformities of the cells were
evident. There is formation of agglomeration of cells due to the greater production of EPS.
EDX analysis showed the percentage of sodium and potassium decreased, which was
attributed to the exchange of cations by chromium.
5.2 Future Work Recommendations
This area of research can further be investigated in the following directions:
It was found that L4 (Arthrobacter sp.) was more tolerant towards chromium by
production of a greater amount of EPS. The mechanism behind the production of
EPS can be elucidated in the future research. Future work can be directed in the
evaluation of enzymes or mediated paths that enhance this production of EPS
when the cells are exposed to high metal concentrations.
The genetic basis responsible for tolerance of the heavy metal towards
microorganism can be identified. It can be elucidated whether plasmid resistance
gene is responsible for this tolerance or not.
Isolation of plasmid DNA and transformation into the competent E.coli cells could
be an initial step. Then plasmid cured organism as well as E.coli (containing metal
tolerating plasmid) could be analysed for chromium uptake. In this way, whether
plasmid was responsible for chromium tolerance and uptake can be determined.
In view of possible commercial application, the bench scale work can be scaled up
and the parameter for maximum chromium removal can be optimized.
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The cells can be immobilized onto some supporting material to determine its
capacity/efficiency in the industry.
Instead of working on pure cultures, a consortium of several strains having high
metal tolerance can be used to determine enhanced removal or accumulation
capacity.
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... Conclusions and Future Work compartmentalization of chromium by the two isolates showed that Bacillus sp was able to sequester/uptake 85. 6% of the initial 40 ppm of chromium, whereas Arthrobacter. .. more (on chromium exposure) when compared to Arthrobacter sp (L4), possibly due to Bacillus sp being less tolerant and hence undergoing lysis and forming organic granules with the metal and thus... cations by chromium 5. 2 Future Work Recommendations This area of research can further be investigated in the following directions: It was found that L4 (Arthrobacter sp.) was more tolerant towards chromium