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Problems in extraction and spectrophotometric determination of chlorophyll from epilithic microbial biofilms: towards a standard method

Published online by Cambridge University Press:  04 April 2001

R.C. Thompson
Affiliation:
School of Biological Sciences, University of Southampton, Southampton, SO16 7PXr.c.thompson@soton.ac.uk
M.L. Tobin
Affiliation:
Port Erin Marine Laboratory, Department of Environmental and Evolutionary Biology, University of Liverpool, Port Erin, Isle of Man, IM9 6JA
S.J. Hawkins
Affiliation:
School of Biological Sciences, University of Southampton, Southampton, SO16 7PXr.c.thompson@soton.ac.uk
T.A. Norton
Affiliation:
Port Erin Marine Laboratory, Department of Environmental and Evolutionary Biology, University of Liverpool, Port Erin, Isle of Man, IM9 6JA

Abstract

A variety of methods are available to extract chlorophyll from epilithic biofilms using solvents. The relative efficiency of these has not been determined simultaneously and there is no recognized standard procedure. In this paper techniques for sample collection, storage, preparation and extraction are reviewed and compared experimentally.

Extraction of chlorophyll was incomplete unless biofilms were fully hydrated. This factor was highly significant for all the solvents tested, with at least three times more pigment being extracted from hydrated samples than from dry ones. Methanol was the most efficient solvent, releasing over 96% of the total chlorophyll during a single extraction; hot ethanol extracted 86%, while acetone extracted less than 50%. Sonicating samples during extraction did not release any additional pigment. Centrifuging to remove suspended material did not alter estimates and was not advantageous. Rugose rock surfaces released more chlorophyll than smooth ones. However, a simple method to quantify surface rugosity at an appropriate scale was not available.

Based on these observations, a standard method for chlorophyll extractions from epilithic biofilms using 100% methanol at room temperature (20°C) is proposed. This technique requires considerably less supervision than previously preferred methods and gave a chlorophyll extract which was stable for 15 h.

Type
Research Article
Copyright
© 1999 Marine Biological Association of the United Kingdom

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