Hydrocarbon Degradation Potential of Halotolerant Bacteria

Catechol Metabolizing Enzymes

by Assistant Professor Vijay Kothari (Author) Meera Panchal (Author) Namrata Srivastava (Author)

Scientific Study 2013 57 Pages

Biology - Micro- and Molecular Biology



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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Hydrocarbon contamination is one of the major problems faced by the world that is constantly affecting the natural environment. Effective removal of these contaminants with the help of microorganisms is the beneficial solution. Certain organisms are known to degrade these HCs. We examined our isolates for their HC tolerating capacities, their antimicrobial activity, presence of certain catechol metabolizing enzymes and also their biofilm forming ability. We worked with four out of five halophilic isolates as they were able to tolerate HCs. we concentrated on V.salarius for detecting the presence of catechol metabolizing enzymes. The enzyme activity was then compared with that of P. oleovorans . P. oleovorans was used for the comparison as they were also been isolated form saline environment and are known for BTEX degradation (Zhou et al., 2011), and also due to its availability in our laboratory.

Catechol is the reaction intermediate in the microbial metabolism of phenol, benzoic acid, toluate, naphthalene, salicylate (Dagley et al., 1960) and substituted catechols are intermediates in the catabolism of methylated and chlorinated derivatives of these compounds. There are various pathways for catechol metabolism. In some species of Pseudomonas the benzene nucleus of catechol is cleaved by chlorocatechol 1,2 dioxygenase to give cis-cis muconic acid (Evans et al., 1951; Hayaishi et al., 1957; Gawa et al., 1963; Taniuchi et al., 1964). Whereas in other species of Pseudomonas catechol is oxidized to 2-hydroxy muconic semialdehyde by an enzyme that was designated as catechol 2,3 dioxygenase by Dagley et al.,(1960) and metapyrocatechase by Kojima et al., (1961).


- To study the hydrocarbon tolerating capacity of four halotolerant organisms isolated from the saline soil of Khambhat, Gujarat, India.
- To detect the presence of catechol metabolizing enzymes in Virgibacillus salarius and its comparison with Pseudomonas oleovorans.
- To check the effect of microwave on the enzyme activity.
- To evaluate the biofilm forming potential of Virgibacillus salarius and Pseudomonas oleovorans.


Hydrocarbon (HC) group of compounds consist of hydrogen and carbon in their structure. As petrochemical industries are flourishing worldwide, HC contamination has become one of the major environmental problems faced globally. Environment is particularly being contaminated with accidental releases of petroleum products. Some of the HC compounds can prove carcinogenic and neurotoxic to different life forms. Bioremediation is a promising approach for the treatment of HC contaminated locations as it is cost effective and can lead to complete mineralization. Bioremediation strategy exploits the metabolic pathways of living organisms (mainly microorganisms) for biodegradation of organic pollutants, leading to their partial or complete mineralization into carbon dioxide, water, and inorganic compounds. Degrading organisms may use the pollutant molecules as an energy source and for deriving building blocks for synthesis of their cellular components. In the process they transform the complex organic contaminants to simpler (may be less toxic) forms, which can further be utilized by other organisms. HC degradation often requires the presence of oxygen as the initial degradation occurs by the action of oxygenase enzymes (Atlas, 1991), however in subsequent steps nitrate or sulphate may serve as a terminal electron acceptor (Bartha, 1986).Various industries and different daily life processes use petroleum based products as the major source of energy. Leaks and accidental spills (Table 1.1) occur frequently during transportation, production, exploration, refining, and storage of petroleum and its derivatives. The amount of natural crude oil seepage was estimated to be 600,000 metric tons per year with an uncertainty of 200,000 metric tons per year (Kvenvolden and Cooper, 2003). The success of bioremediation efforts in the cleanup of the oil tanker Exxon Valdez oil spill of 1989 in Prince William Sound and the Gulf of Alaska created tremendous interest in the potential of biodegradation and bioremediation technology (Atlas, and Bartha, 1998). Release of HC into marine environment can lead to major water contamination causing extensive damage to biodiversity. The accumulation of these pollutants in marine animal and plant tissues may cause death or mutation. Further when these animals and plants become a part of food chain they may lead to more harmful effects through biomagnification.

There have been many examples of experimental success but there have also been many notable failures suggesting that although microorganisms have the primary catalytic role in bioremediation our knowledge of the alteration occurring in the microbial communities remains limited and the microbial community is still treated as a “black box” (Iwamoto and Nasu 2001). But on a positive side, bioremediation still remains a developing field because it has traditionally been carried out in a natural environment where many of the organisms are uncharacterized and because no two environmental projects are identical (Watanabe 2001; Verstraete 2002).

Table 1.1 Major oil spills in history

(http://www.mnn.com/earth-matters/wilderness-resources/stories/the-13-largest-oil-spills-in-history; last accessed on 20.3.2013)

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ISBN (eBook)
ISBN (Book)
File size
894 KB
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Institution / College
Nirma University – Institute of Science
Catechol; Salinity; Halotolerant; Marine Oil Spill; Bioremediation




Title: Hydrocarbon Degradation Potential of Halotolerant Bacteria