Chlorophyll extraction methods for the quantification of green microalgae colonizing building facades

Abstract The occurrence of microalgae on man-made surfaces such as roof tiles, concrete, building facades and other artificial surfaces in urban areas, where it causes aesthetically unacceptable discolouration of the surfaces, represents a conspicuous problem because of its contribution to biodeterioration and accelerated weathering. So far, microbial colonization has been recorded mainly macroscopically, and hence semi-quantitatively evaluated by the degree of discolouration, and the visual data assigned to a colour scale for algal infestation intensity. However, these assessments lack inter-calibration and suffer from colour modifications because of co-occurring fungi or background properties. By using chlorophyll a as a specific biomarker of aeroterrestrial microalgae, an extraction method was therefore developed to quantify biomass. Two green microalgae, Stichococcus sp. and Chlorella sp., were isolated from facades of buildings, established as monocultures and used in the present study. Algal samples are best preserved by freezing in liquid nitrogen followed by lyophilisation and subsequent storage at −18 °C. Dimethyl formamide (DMF) proved to be the best extraction solvent. Mechanical homogenisation improved extraction by up to 20%. However, chlorophyll a extraction efficiency was strongly species-specific and further influenced by the growth conditions, i.e. whether cells were in logarithmic or stationary growth phase. To achieve maximum values for chlorophyll a from samples of unknown algal species composition and physiological state it was found necessary to make at least two 24-h extractions in DMF. If the second extraction step contributed > 20 % to the total chlorophyll a concentration, a third DMF extraction was applied. Using this biomarker assay, up to 313 mg chlorophyll a m−2 was obtained from building facades, equivalent to c. 100 g algal fresh weight, which represents a high organic load.

[1]  Takashi Ishimaru,et al.  An improved method for the determination of phytoplankton chlorophyll using N, N-dimethylformamide , 1990 .

[2]  J. Neveux.  Extraction of chlorophylls from marine phytoplankton: With 4 tables in the text , 1988 .

[3]  Richard J. Geider,et al.  LIGHT AND TEMPERATURE DEPENDENCE OF THE CARBON TO CHLOROPHYLL a RATIO IN MICROALGAE AND CYANOBACTERIA: IMPLICATIONS FOR PHYSIOLOGY AND GROWTH OF PHYTOPLANKTON , 1987 .

[4]  D. Arnon.  COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. , 1949, Plant physiology.

[5]  Piero Tiano,et al.  Biodiversity of photosynthetic micro-organisms dwelling on stone monuments , 2000 .

[6]  H. Viles.  Blue-green algae and terrestrial limestone weathering on Aldabra Atoll: An S.E.M. and light microscope study , 1987 .

[7]  Christine C. Gaylarde,et al.  Deteriogenic biofilms on buildings and their control: A review , 1999 .

[8]  S. Blackburn,et al.  The effect of growth phase on the lipid class, fatty acid and sterol composition in the marine dinoflagellate, Gymnodinium sp. in batch culture. , 2003, Phytochemistry.

[9]  S. Jørgensen.  Handbook of Ecological Parameters and Ecotoxicology , 1991 .

[10]  R. J. Porra,et al.  Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy , 1989 .

[11]  Cesáreo Sáiz-Jiménez,et al.  Factors affecting the weathering and colonization of monuments by phototrophic microorganisms , 1995 .

[12]  R. Foy.  The phycocyanin to chlorophyll α ratio and other cell components as indicators of nutrient limitation in two planktonic cyanobacteria subjected to low-light exposures , 1993 .

[13]  R. Highsmith.  Lime-boring algae in hermatypic coral skeletons , 1981 .

[14]  A. Hursthouse,et al.  A Preliminary Study of the Phycological Degradation of Natural Stone Masonry , 2003, Environmental geochemistry and health.

[15]  R. Porra.  Recent advances and re-assessments in chlorophyll extraction and assay procedures for terrestrial, aquatic, and marine organisms, including recalcitrant algae , 1991 .

[16]  S. Wright,et al.  Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods , 1997 .

[17]  S. Wright,et al.  Rapid extraction and high-performance liquid chromatography of chlorophylls and carotenoids from marine phytoplankton , 1984 .

[18]  G. F. Humphrey,et al.  New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton , 1975 .

[19]  Richard C. Starr,et al.  UTEX—THE CULTURE COLLECTION OF ALGAE AT THE UNIVERSITY OF TEXAS AT AUSTIN 1993 LIST OF CULTURES 1 , 1993 .

[20]  N. Bishop,et al.  PHOTOSYNTHETIC EFFICIENCY OF A PHYCOCYANIN‐LESS MUTANT OF CYANIDIUM * , 1968 .

[21]  R. C. Starr.  THE CULTURE COLLEVTION OF ALGAE AT THE UNIVERSITY OF TEXAS ATAUSTIN 1 2 3 , 1978 .

[22]  Sutomo,et al.  MONSOONAL ALTERNATION OF A MIXED AND A LAYERED STRUCTURE IN THE PHYTOPLANKTON OF THE EUPHOTIC ZONE OF THE BANDA SEA (INDONESIA) - A MATHEMATICAL-ANALYSIS OF ALGAL PIGMENT FINGERPRINTS , 1988 .

[23]  D. Rentsch,et al.  Seston particles along a eutrophication gradient in coastal waters of the Southern Baltic Sea: significance of detritus and transparent mucoid material , 2001 .

[24]  D. Allsopp,et al.  Microbial deterioration of building stone - a review. , 1993 .

[25]  J. U. Grobbelaar,et al.  Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis , 1984, Hydrobiologia.

[26]  U. Karsten,et al.  Seasonality in UV-absorbing compounds of cyanobacterial mat communities from an intertidal mangrove flat , 1998 .

[27]  C. O'kelly,et al.  Phylogenetic relationships of green algae assigned to the genus Planophila (Chlorophyta): evidence from 18S rDNA sequence data and ultrastructure , 2002 .

[28]  P. Khanna,et al.  Allergic bronchopulmonary aspergillosis without clinical asthma caused by Aspergillus niger , 2004, Allergy.