Hydrocarbon contamination of marine ecosystems has been a major environmental concern. Hydrocarbon metabolizing capacity of four halotolerant bacteria (Bacillus atrophaeus, Halomonas shengliensis, Halomonas koreensis, and Virgibacillus salarius) isolated from saline soil of Khambhat, India was investigated. Presence of catechol metabolizing enzymes (catechol 2,3 dioxygenase, chlorocatechol 1,2 dioxygenase, and protocatechuate 3,4 dioxygenase) was checked in V. salarius, as only this among all the test organisms could grow on the hydrocarbon substrates used, and compared with Pseudomonas oleovorans. Effect of salinity of the growth medium on activity of catechol metabolizing enzymes was also studied. Catechol 2,3 dioxygenase activity in both the organisms was more susceptible to increase in salinity of the growth medium than chlorocatechol 1,2-dioxygenase activity. To the
best of our awareness, this is the first report of catechol metabolism in V. salarius. V. salarius was found to be capable of weak biofilm formation. As V. salarius is capable of growing at high salt concentration, alkaline pH, hydrocarbon degradation, and also of growth in presence of various metal ions, it can be an attractive candidate for bioremediation of marine oil spills. Organisms like V. salarius can also serve as a model for multiple stress tolerance in prokaryotes.
Index
1. Introduction
1.1 Objectives
1.2 Bioremediation
1.3 Hydrocarbon overview
1.4 Need for biodegradation
1.5 Approaches to biodegradation of hydrocarbons
1.6 Laboratory methods for studying hydrocarbon degradation
1.7 Microorganisms known to degrade hydrocarbons
1.8 Enzymes involved in hydrocarbon degradation
1.9 Factors affecting hydrocarbon degradation
1.10 Salinity and hydrocarbon degradation
1.11 Test organisms
2 Methodology
2.1 Materials
2.2 Instruments
2.3 Test organisms
2.4 Preliminary qualitative analysis
2.5 Cell lysis
2.6 Enzyme assay: catechol 2,3 dioxygenase
2.7 Enzyme assay: chlorocatechol 1,2-dioxygenase
2.8 Enzyme assay: protocatechuate 3,4-dioxygenase
2.9 Biofilm formation
2.10 Estimation of biofilm formation by crystal violet assay
2.11 Effect of microwave radiation on enzyme activity
3. Findings
3.1 Qualitative analysis
3.2 Enzyme activity
3.3 Effect of salinity on enzyme activity
3.4 Biofilm formation
4. Appendices
5. References
A. List of tables
Table 1.1 Major oil spills
Table 1.2 Halophiles known to degrade hydrocarbons
Table 1.3 Enzymes involved in biodegradation of petroleum
Table 2.1 Enzyme assay for catechol 2,3 dioxygenase
Table 2.2 Enzyme assay for chlorocatechol 1,2 dioxygenase
Table 2.3 Enzyme assay for protocatechuate 3,4-dioxygenase
Table 2.4 Classification of bacterial adhesion and biofilm formation
Table 3.1 Results of qualitative analysis
Table 3.2 Growth on combination of HC
Table 3.3 Activity of catechol 2,3 dioxygenase...
Table 3.4 Activity of chlorocatechol 1,2 dioxygenase
Table 3.5 Percent change in enzyme activity of V. salarius wrt P. oleovorans and their mixture
Table 3.6 Percent change in enzyme activity at different salt conc
Table 3.7 Results of biofilm formation
Table A1 Results of biofilm formation
B. List of figures
Fig 1.1 Catalytic cycle for intradiol cleavage
Fig 1.2 Catalytic cycle for extradiol cleavage
Fig 1.3 Ortho pathway for chlorocatechol degradation
Fig 1.4 General pathway for aromatic hydrocarbon degradation
Fig 3.1 Comparison of enzyme activity of catechol 2,3 dioxygenase at 6% salt concentration
Fig 3.2 Comparison of enzyme activity of chlorocatechol 1,2 dioxegenase at 6% salt concentration
Fig 3.3 Comparison of enzyme activity of catechol 2,3 dioxygenase at 10% salt concentration
Fig 3.4 Comparison of enzyme activity of chlorocatechol 1,2 dioxegenase at 10% salt concentration
Fig 3.5 Comparison of enzyme activity of V. salarius on different salt concentration
Fig3.6 Comparison of enzyme activity of P. oleovorans on different salt concentration
Fig 3.7 Comparison of enzyme activity mixture of V. salarius and P. oleovorans on different salt concentration
Fig 3.8 Comparison of enzyme activity of V. salarius on different salt concentration
Fig 3.9 Comparison of enzyme activity of P. oleovorans on different salt concentration
Fig 3.10 Comparison of enzyme activity mixture of V. salarius and P. oleovorans on different salt concentration34
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