Polyhydroxyalkanoate granules quantification in mixed microbial cultures using image analysis: Sudan Black B versus Nile Blue A staining.
Polyhydroxyalkanoates (PHA) can be produced and intracellularly accumulated as inclusions by mixed microbial cultures (MMC) for bioplastic production and in enhanced biological phosphorus removal (EBPR) systems. Classical methods for PHA quantification use a digestion step prior to chromatography analysis, rendering them labor intensive and time-consuming. The present work investigates the use of two quantitative image analysis (QIA) procedures specifically developed for PHA inclusions identification and quantification. MMC obtained from an EBPR system were visualized by bright-field and fluorescence microscopy for PHA inclusions detection, upon Sudan Black B (SBB) and Nile Blue A (NBA) staining, respectively. The captured color images were processed by QIA techniques and the image analysis data were further treated using multivariate statistical analysis. Partial least squares (PLS) regression coefficients of 0.90 and 0.86 were obtained between QIA parameters and PHA concentrations using SBB and NBA, respectively. It was found that both staining procedures might be seen as alternative methodologies to classical PHA determination.
CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Daniela P
DP
Mesquita
CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal; Instituto Politécnico de Coimbra, ISEC, DEQB, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra, Portugal.
A Luís
AL
Amaral
Instituto Politécnico de Coimbra, ISEC, DEQB, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra, Portugal.
Cristiano
C
Leal
CQFB/REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
Adrian
A
Oehmen
CQFB/REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
Maria A M
MA
Reis
CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Electronic address: ecferreira@deb.uminho.pt.
Eugénio C
EC
Ferreira
D001391:Azo Compounds / Q000737:chemistry*; D015169:Colony Count, Microbial / Q000379:methods*; D005453:Fluorescence; D016018:Least-Squares Analysis; D009281:Naphthalenes / Q000737:chemistry*; D010078:Oxazines / Q000737:chemistry*; D054813:Polyhydroxyalkanoates / Q000032:analysis* / Q000096:biosynthesis; D013194:Staining and Labeling
D001391:Azo Compounds; D009281:Naphthalenes; D010078:Oxazines; D054813:Polyhydroxyalkanoates; C008619:Nile Blue; C016118:Sudan Black B
Enhanced biological phosphorus removal (EBPR) systems; Image analysis; Mixed microbial cultures (MMC); Nile Blue A (NBA); Polyhydroxyalkanoates (PHA); Sudan Black B (SBB)