Quantification of phototrophic biomass on rocks: optimization of chlorophyll-a extraction by response surface methodology.

Biological colonization of rock surfaces constitutes an important problem for maintenance of buildings and monuments. In this work, we aim to establish an efficient extraction protocol for chlorophyll-a specific for rock materials, as this is one of the most commonly used biomarkers for quantifying phototrophic biomass. For this purpose, rock samples were cut into blocks, and three different mechanical treatments were tested, prior to extraction in dimethyl sulfoxide (DMSO). To evaluate the influence of the experimental factors (1) extractant-to-sample ratio, (2) temperature, and (3) time of incubation, on chlorophyll-a recovery (response variable), incomplete factorial designs of experiments were followed. Temperature of incubation was the most relevant variable for chlorophyll-a extraction. The experimental data obtained were analyzed following a response surface methodology, which allowed the development of empirical models describing the interrelationship between the considered response and experimental variables. The optimal extraction conditions for chlorophyll-a were estimated, and the expected yields were calculated. Based on these results, we propose a method involving application of ultrasound directly to intact sample, followed by incubation in 0.43 ml DMSO/cm(2) sample at 63°C for 40 min. Confirmation experiments were performed at the predicted optimal conditions, allowing chlorophyll-a recovery of 84.4 ± 11.6% (90% was expected), which implies a substantial improvement with respect to the expected recovery using previous methods (68%). This method will enable detection of small amounts of photosynthetic microorganisms and quantification of the extent of biocolonization of stone surfaces.