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

A variety of methods are available to extract chlorophyll from epilithic bi ofilms using solvents. The relative efficiency of these has not been determ ined 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 hydrate d. 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 extra ction did not release any additional pigment. Centrifuging to remove suspen ded material did not alter estimates and was not advantageous. Rugose rock surfaces released more chlorophyll than smooth ones. However, a simple meth od 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 degrees C) is proposed. This technique requires considerably less supervision than previously preferred methods and gave a chlorophyll extract which was stab le for 15 h.