Bioethics in India: Proceedings of the International Bioethics Workshop in Madras: Biomanagement of Biogeoresources, 16-19 Jan. 1997, University of Madras; Editors: Jayapaul Azariah, Hilda Azariah, & Darryl R.J. Macer, Copyright Eubios Ethics Institute 1997.
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84. Hydrolysis of Oils and Marine Environmental Ethics

N.S. Kavitha, A. Hilda, S.Gopinath & K. Latha
Centre for Advanced Studies in Botany, Guindy Campus, University of Madras, Chennai 600 025.


Abstract

In recent years there has been an increase in the awareness of potentially harmful effects of world-wide oil spillage in both salt and fresh waters. Oil pollution is a serious obstacle to photosynthesis, a fundamental life process in plant kingdom, which affects the food chain and productivity of the sea. Oil is also absorbed by fish eventually to reach and endanger human life. The lipolytic activity of physiologically diverse micro organisms can be used to degrade oil spills in marine environment. In the present study, the fungus Acremonium alternata exhibited varied efficiency in hydrolyzing the various oils. It degraded olive and ground nut oils to a maximum efficiency. Therefore, it is suggested that a bio-remedial procedure using Acremonium alternata may be helpful to maintain a sustainable development both in the coastal zone and in the industrial area.

Introduction

In recent years there has been an increase in the awareness of potentially harmful effects of world wide oil spillage in both salt and fresh water bodies (Kawahara, 1969; Kawahara and Fiutem, 1983). Oil pollution is a serious obstacle to photosynthesis which in turn affects the food chain and productivity of the sea. Oil is also absorbed by fish, eventually to reach and endanger human life. Therefore, biodegradation of compounds of hydrocarbon is absolutely essential. Biodegradation of hydrocarbon compounds has been well demonstrated for a considerable number of physiologically diverse micro-organisms, including many taxa of yeast and moulds. Emphasis on the development of microbial protein from petroleum has stimulated considerable research into the species of Candida lipolytica and its mode of attack on the varied constituents of petroleum. The ability of the fungus to exist in any environment would depend largely on their ability to produce enzymes that are capable of breaking down the various complex organic materials (Oso, 1978). Fungi, which are ubiquitous in their distribution, are involved in the biodeterioration and biodegradation of various materials under different environmental conditions.

The fungi are useful in protecting the marine environment. The lipolytic activity of physiologically diverse microorganism can be used to degrade oil spills in the marine environment. It is significant to isolate microbes of higher potential for the biodegradation of oil. Therefore, in this study, an attempt has been made to assess the bio-potentials of a few fungal species with reference to their ability to degrade lipid from oil seeds under laboratory conditions.

Isolation Of Fungi

The present investigation aims to isolate the fungal species associated with a few oil seeds and commercially available vegetable oils. The associated mycoflora were isolated: 1. Acremonium alternata; 2. Aspergillus flavus; 3. Aspergillus niger; 4. Aspergillus terreus; 5. Aspergillus versicolor; 6. Cladosporium cladosporioides; 7. Cladosporium sp.; 8. Cunninghamella sp.; 9. Curvularia lunata; 10. Fusarium solani; 11. Fusarium sp.; 12. Mucor sp.; 13. Penicillium citrinum; 14. Penicillium sp.; 15. Rhizopus stolonifer; 16. Syncephalastrum racemosum.

To detect fungi for lipolytic activity, the Rhodomine B method was used in the present investigation. Initially, all the fungi isolated from a few oil seeds were screened for hydrolysis of lipids. Acremonium alternata was selected to be the best organism.

Direct Bio Assay For Lipase Activity Of Acremonium Alternata On Few Vegetable Oils By Rhodamine-B Method (Kouker and Jaeger, 1987):

NaCl 4.0 g; Agar 10.0 g; Yeast extract-Peptone medium 500 ml; Vegetable oil 31.3 ml; Rhodamine B (0.001% w/v) 10.0 ml; pH 7.0

Data on the extracellular lipase activity of the fungus, Acremonium alternata, on different vegetable oils, as substrates in the presence of a fluorescent dye, Rhodamine-B, which served as an indicator in the Yeast-extract-Peptone nutrient medium, are given in Table I. The hydrolysis of various oils resulted in the formation of fluorescent zones around the fungal colonies which were visible upon UV irradiation.

It is evident from Table I that the fungus, Acremonium alternata exhibited varied efficiency in hydrolyzing the various vegetable oils. Interestingly, the fungus exhibited minimum (low) growth on rice bran, cottonseed, mustard, gingelly, soybean, palm and neem oils. Moderate growth was reported in olive, groundnut, coconut and sunflower oils. Regarding extracellular lipolytic activity, minimum (low) activity was reported in castor, soybean, palm and neem oils. Moderate activity was exhibited on rice bran, cottonseed, mustard, gingelly and sunflower oils. In olive oil and groundnut oil the fungus exhibited maximum (high) activity.

Table I: Extracellular lipolytic activity of Acremonium alternata on vegetable oils - Rhodamine-B method

Oil Activity Remark

Olive +++ High

Rice bran ++ Moderate

Cotton seed ++ Moderate

Groundnut +++ High

Coconut ++ Moderate

Castor + Low

Mustard ++ Moderate

Gingelly ++ Moderate

Soybean + Low

Sunflower ++ Moderate

Palm + Low

Neem + Low

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+++ -> High activity ++ -> Moderate activity + -> Low activity

Effect of Different Oil Seed Powder on Lipolytic Activity of Acremonium Alternata

Dried powder of some oil seeds was added into the assay growth medium and the extracellular lipase released into the culture media was assayed using p-nitrophenyl palmitate. The p-nitrophenol liberated was measured at 410 nm in DU 40 Spectrophotometer.

Acremonium alternata produced oil degrading enzyme. It was stimulated in the media containing powdered oil seeds. It exhibited uniformly moderate levels of lipase production on all the oil seeds with high activity in the presence of peptone (Table II).

The fungi which could degrade the vegetable oils can also be used to protect the marine environment specially in the biodegradation of oils. Therefore, it is suggested that a bio-remedial procedure using Acremonium alternata may be helpful to maintain a sustainable environment both in the coastal zone and in the industrial area. It is significant to develop hyperproductive strain of fungi by cloning oil degrading genes from different fungi. However, these Genetically Modified Organisms, with higher potential to degrade oil, may pose a risk to human life, if their biosafety in the environment is not fully understood. Also there is no experimental evidence to assess the biocorrosion, if any, of metals in petrol tank if these cloned hyperactive fungi are present in the fuel supply systems of vehicles (petrol/diesel tank).

Table II: Lipolytic activity of Acremonium alternata on oil seed powder

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Sources of Enzyme Protein Specific

Oil seed Unit/ml mg/ml activity

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Control 0.20 0.126 1.59

Peptone 2.61 0.315 8.29

Castor 0.22 0.189 1.16

Cotton 2.01 0.387 5.19

Groundnut 1.47 0.338 4.35

Mustard 0.71 0.180 3.94

Sesamum 2.20 0.432 5.09

Soybean 1.76 0.351 5.01

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Acknowledgements

The authors thank Prof. A. Mahadevan, Director, Centre for Advanced Studies in Botany, University of Madras, Chennai for his encouragement. The financial assistance by UGC and DoEn are greatly acknowledged.

References
Kawahara, F. K. 1969. Identification and differentiation of heavy residual oil and asphalt pollutants in surface waters by comparative ratios of infra red absorbances. Environ. Sci. Technol. 150-153.
Kawahara, F. K. and R. A. Fiutem. 1988. Development of a novel method for monotoring oils in water. Analytica Chimica Acta 151: 315-327.
Kouker, G. and Jaeger, K. 1987. Specific and senisitive plate assay for bacterial lipases. Appl. Environ. Microbiol. 53: 211-213.
Oso, B. A. 1978. The lipase activity of Talaromyces emersonii. Canadian Journal of Botany. 56: 1840-1843.


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