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Using Hyperspectral Imaging to Quantify Phototrophic Biofilms on Granite

A. Ramil1, D. Vázquez-Nion2, J. S. Pozo-Antonio3,*, P. Sanmartín2, and B. Prieto2

  1. Laboratorio de Aplicacións Industriais do Láser, Centro de Investigacions Tecnoloxicas (CIT), Departamento de Enxeñaría Naval e Industrial, Escola Politécnica Superior, Universidade de A Coruña (UDC), Campus Ferrol, 15403-Ferrol, Spain
  2. Departamento de Edafoloxía e Química Agrícola. Facultade de Farmacia. Universidade de Santiago de Compostela, 15782 - Santiago de Compostela, Spain
  3. Departamento de Enxeñaría dos Recursos Naturais e Medio Ambiente, Escola de Enxeñaría de Minas e Enerxía, Universidade de Vigo, 36310-Vigo, Spain

*Corresponding author. Tel.: 00 34 986130211; fax: + 34 986 811 924. E-mail address: (J. S. Pozo-Antonio).


This paper reports a non-invasive method for in situ biofilm quantification based on the use of hyperspectral imaging to analyze a chromatically and texturally heterogeneous substrate as granite colonized by green algae and cyanobacteria. Biofilm-forming microorganisms were inoculated on granite blocks and incubated under laboratory conditions for 21 days. The intensity of the green stains formed on the granite surfaces differed depending on the initial concentration of microorganisms used. A biofilm quantification (BQ) index was computed to determine the level of colonization on the surfaces. The index was obtained by comparing the quotient of the reflectance of the green (G) and red (R) bands of each pixel of the hyperimage against a threshold value, fth. The optimal value of the threshold was determined by examining the linear correlation between the BQ index and the chlorophyll a extracted. The BQ results were then compared with the F0 and ΔE*ab parameters yielded by colour spectrophotometry and PAM fluorometry techniques and used as reference methodologies to quantify greening on stone surfaces. The BQ index showed a high level of consistency with all of the other parameters; it was linearly correlated with the chlorophyll a concentration, F0 andΔE*ab, with high coefficients of determination (r2 > 0.92) for the range from 1.87 to 5.69 μg chl The strength of the BQ index lies in its double use as a biomarker of quantity and percentage cover, as well as its good performance under different conditions, ranging from initial phototrophic colonization and thin (young) biofilms covering 21.19% of the surface to profuse biological colonization covering 67.36% of the surface. Moreover, the proposed BQ index may be able to be used with other types of rock with less heterogeneous surfaces than granite.

Keywords: biofilms, chlorophyll fluorescence, colour spectrophotometry, hyperspectral imaging, non-invasive technique

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