Analysis of Fungal Pellets by UV-Visible Spectrum Diffuse Reflectance Spectroscopy

Abstract

The application of the UV-visible spectrum diffuse reflectance spectroscopy for the determination of intracellular pH in vivo, for determination of cytochrome content, and for the noninvasive in vivo detection of the redox state of fungal mitochondrial cytochromes in filamentous fungi is introduced. The time course of the intracellular pH values, mitochondrial cytochromes, and CO-binding pigments content and the correlations between the actual redox state of cytochrome aa3 and saturation of growth medium with oxygen in pellets of the basidiomycete Phanerochaete chrysosporium were determined. As the test microorganism, the yeast Saccharomyces cerevisiae was used. UV-visible spectrum diffuse reflectance spectroscopy proved to be a promising method for the quick and simple analysis of light-impermeable biological structures for which the classical transmittance spectrophotometric methods are difficult to implement.

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium

“…Lestan et al (22) described the measurement system based on diffuse reflectance spectroscopy for in vivo monitoring of the redox state of mitochondrial cytochromes in pellets of P. chrysosporium suspended in growth medium. The reflectance of cytochrome spectra increases as the growth medium oxygenation decreases (22). Since mitochondrial cytochromes absorb light only in the reduced state, the increased reflectance indicates the higher reduction level.…”

“…The identities of the mitochondrial cytochromes in pellets of filamentous fungi can be easily determined by diffuse reflectance spectroscopy (22). Mitochondrial cytochromes are coupled to oxidative phosphorylation, most important in the energy production of the living, aerobic cell.…”

“…Measurements of intracellular pH. The intracellular pH of P. chrysosporium pellets was determined by the spectrophotometric technique, as described previously (32) and adapted to diffuse reflectance spectrophotometry (22). The dye 6-carboxyfluorescein was used as the intracellular pH indicator.…”

“…In vivo determination of the redox state of mitochondrial cytochromes. On-line measurements of the redox state of mitochondrial cytochromes of P. chrysosporium, as a function of the saturation of the growth medium with oxygen, were performed with the measurement system presented elsewhere (22). The growth medium oxygen saturation was detected with a polarographic type of 02 electrode.…”

“…These methods were used to determine the intracellular pH, cytochrome content, and intracellular lignin peroxidase activity of P. chrysosporium. A measurement system was also designed for quantitative, continuous, and in vivo determination of the redox state of respiratory cytochromes in fungal pellets (22).…”