Characterization of Penicillium chrysogenum physiology in submerged cultures by color and monochrome image analysis

Abstract

Although filamentous microorganisms are widely used in industrial fermentation processes, their growth and differentiation are not yet fully understood, because their biomass is structured, and therefore difficult to describe and to quantify. This lack of appropriate tools can hinder the optimization and control of the fermentation. A quantitative image analysis method was therefore developed for characterizing the physiology of the penicillin-producing mold Penicillium chrysogenum. This method is based on a differential staining procedure showing six physiological states: growing material, three differentiated states characterized by an increasing granulation, a highly vacuolized state, and dead segments having lost their cytoplasm. The image analysis software, with versions written for monochrome and color images, consisted of a semiautomatic binary mask computation step and a fully automatic segmentation step based on a fuzzy classification.

Growth and product formation ofAspergillus oryzae during submerged cultivations: Verification of a morphologically structured model using fluorescent probes

“…An exception is the work of Paul and Thomas (1996) in which image analysis was used to characterize the cellular differentiation of P. chrysogenum for use in the estimation of model parameters. Vanhoutte et al (1995) demonstrated the potential of automated image analysis by using multiple staining of hyphal elements to distinguish between six different hyphal zones in P. chrysogenum, ranging from the active apical regions to the most distal dead regions, but the results obtained were not used for any modeling purposes.…”

Biomass Quantification by Image Analysis

“…Two thresholds are defined to detect the three regions of interest, that enable to define three physiological states: growing, non-growing (cytoplasm with vacuoles < 30 mm 3 ), degenerated (cytoplasm with vacuoles > 30 mm 3 ). A simple monochrome CCD-camera can be used, which was not the case for the staining procedure developed for the same microorganism by Vanhoutte et al [169]: a mixture of Methylene Blue and Ziehl Fuschin takes various hues (orange, gray, purple), the subtle changes of which can be picked up correctly only by a 3-CCD color camera. Six physiological states were defined: growing, four states with increased degree of differentiation, dead, and the physiological status of hyphae could be monitored along the culture.…”

Characterization of Fungal Morphology using Digital Image Analysis Techniques

“…Fluorescent stains coupled with image analysis have been used in the works of Vanhoutte et al [39], Agger et al [40], Wongwicharn et al [41], Hamanaka et al [42] and Amanullah et al [13]. Vanhoutte et al [39] using a differential staining procedure and color image analysis in studies on the growth and differentiation of P. chrysogenum showed and quantified six physiological states: growing material (zone 1), three differentiated states characterized by increased granulation (zones 2,3,4), a highly vacuolated state (zone 5) and dead segments empty of cytoplasm (zone 6). Ager et al [40] used a double staining procedure (calcofluor and DiOC6), fluorescence microscopy and automatic image analysis in studies on the morphology of A. oryzae.…”