BIP - A new method: measurement of microleakage volume using human, dog and bovine permanent teeth

Several algae are useful sources of nutraceuticals due to their rich content of essential nutrients not commonly available from other nutritional sources. For example, Spriulina platensis and Chlorella minutissima are rich in polyunsaturated fatty acids. Attempt to modulate their content was explored by studying the response of these organisms to the herbicide SAN 9785, an inhibitor of an enzyme involved in processing of fatty acids. It was observed that fatty acid synthesis and their desaturation were regulated differently in these organisms as a consequence of their response to the herbicide. These studies could be taken forward to understand the processes involved in fatty acid metabolism in these algae and to develop industrially useful strains.

BIP Article

The health benefits of polyunsaturated fatty acids (PUFA) have spurred interest in their commercial production. Microalgae have been an attractive source of PUFA (Benemann et al. 1987). Cyanobacterium Spirulina is rich in γ-linolenic acid (GLA) (Mahajan and Kamat, 1995) while Chlorella minutissima is an eukaryotic alga with a fast growth rate and high PUFA content (Seto et al. 1984). Since these algae could be relatively easily cultivated at different stress conditions, they offer the prospect of a good source of PUFA for the nutraceutical market.

Murphy et al. (1985) had shown that SAN 9785 inhibits the desaturation of long chain fatty acids, and this finding has been used for obtaining strains overproducing PUFA (Cohen et al. 1993). We have compared the response of S. platensis and C. minutissima to SAN 9785.

Materials and Methods

Spirulina platensis was obtained from Ballarpur Industries Limited, New Delhi, and Chlorella minutissima from Sammlung von Algenkulturen, Göttingen. SAN 9785, developed by BASF, was obtained from the laboratory of Dr. J. St. John, US Department of Agriculture, Beltsville, MD. Herbicide tolerant cultures were obtained by repeated subculture at 4 d interval in growth medium supplemented with SAN 9785 at 0.2 mM concentration. Free fatty acid was extracted as described by Jayaraman (1981) and analyzed by gas chromatography.

Results and Discussion

S. platensis exposed to 0.2 mM SAN 9785 exhibited a prolonged lag phase, while cells exposed to 0.4 mM SAN 9785 did not grow for more than one month. The surviving cells grew slower than cultures not exposed to the herbicide, suggesting that the herbicide interfered with the normal growth of S. platensis. C. minutissima was more tolerant to SAN 9785 as compared to S. platensis, acclimatized more readily. This suggested that the metabolic processes affected by SAN 9785 were less influential on the overall growth of C. minutissima, as compared with the case in S. platnensis.

S. platensis growing in 0.2 mM SAN 9785 showed only 52% palmitic acid (16:0) as in control. Upon removal of the herbicide however, palmitic acid content reverted to a level seen in untreated cells. In contrast, stearic acid was not normally detectable, but upon exposure became detectable. The amount of the C₁₈unsaturated fatty acids - oleic (18:1), linoleic (18:2) and linolenic (18:3) acids - increased and became a substantial fraction of the free fatty acids when grown in the presence of the herbicide. Upon removal of the herbicide the fatty acid profile reverted to that seen in untreated cultures.In C. minutissima growing in 0.2 mM SAN 9785, there was no difference in the levels of saturated fatty acids palmitic acid and stearic acid. The amount of linolenic acid, increased about five times to that found in control culture. However, upon removal of the herbicide the linolenic acid reduced to the control level. Thus, in the presence of the herbicide, the tolerant cells of both the algae overcompensated with enhanced accumulation of linolenic acid.

These results indicate the difference in the regulation of fatty acid synthesis but not the desaturase activity between S. platensis and C. minutissima. In S. platensis there appeared to be no compensation of palmitic acid while its utilization for the synthesis of the unsaturated fatty acids had increased upon exposure to the herbicide, in C. minutissima the increased consumption of the saturated fatty acids was compensated. This suggests that in C. minutissima there was an increased de novo fatty acid synthesis. While in C. minutissima the amount of saturated fatty acids, palmitic and stearic acid, were stringently maintained at amounts observed in untreated cultures, in S. platensis their amounts were greatly influenced by their conversion to the desaturated fatty acids.

Concluding Remarks

These studies suggest that S. platensis responds to SAN 9785 by driving the fatty acid desaturation pathway without compensating for the enhanced conversion of the precursor palmitic acid, while C. minutissima responds by adequate compensation of the fatty acid precursor pool, possibly through enhanced de novo synthesis. Perhaps due to this robustness of C. minutissima, it was able to tolerate higher doses of SAN 9785.

References

BENEMANN, John R.; TILLETT, David M. and WEISSMAN, Joseph C. Microalgae biotechnology. Trends in Biotechnology, February 1987, vol. 5, no. 2, p. 47-53. [CrossRef]

COHEN, Zvi; REUNGJITCHACHAWALI, Marasri; SIANGDUNG, Wipawan; TANTICHAROEN, Morakot and HEIMER, Yair M. Herbicide-resistant lines of microalgae: Growth and fatty acid composition. Phytochemistry, November 1993, vol. 34, no. 4, p. 973-978. [CrossRef]

JAYARAMAN, J. Laboratory Manual in Biochemistry. New Delhi, New Age Publishers, 1981, 180 p. ISBN 0852264283.

MAHAJAN, G. and KAMAT, M. γ-Linolenic production from Spirulina platensis. Applied Microbiology and Biotechnology, July 1995, vol. 43, no. 3, p.466-469. [CrossRef]

MURPHY, Denis J.; HARWOOD, John N.; LEE, Kevin A.; ROBERTO, Francisco; STUMPF, Paul K. and ST. JOHN, Judith B. Differential responses of a range of photosynthetic tissues to a substituted pyridazinone, Sandoz 9785. Specific effects on fatty acid desaturation. Phytochemistry, September 1985, vol. 24, no. 9, p. 1923-1929. [CrossRef]

SETO, Akira; WANG, H.L. and HESSELTINE, C.W. Culture conditions affect eicosapentaenoic acid content of Chlorella minutissima. Journal of the American Oil Chemists Society, May 1984, vol. 61, no. 5, p.892-894.