Quantification of autolysis inPenicillium chrysogenumby semiautomated image analysis

Abstract

An image analysis method is described for the characterization of empty (autolyzed and inactive) regions within the mycelia of filamentous fungi. It extends a previous method that characterized only regions filled with cytoplasm or vacuoles (i.e., the active biomass). The method is semiautomatic, requiring some manual editing before automated measurements. When the method was used for samples from a batch fermentation of an industrial strain of Penicillium chrysogenum, the empty regions were observed to constitute up to 15% (by projected area) of the biomass during the growth phase. After nutrient exhaustion, however, the proportion of empty regions rose rapidly, eventually representing more than 50% of the biomass by the end of fermentation. The increase in the percentage of empty regions coincided with a decrease in biomass (as measured by dry cell weight) and a fall in penicillin titre. Further morphological analysis revealed that fragmentation of mycelia, particularly clumps, coincided with increases in the levels of empty regions. This new image analysis method gave additional information on hyphal differentiation and a measure of autolysis. It was also a useful indicator of the processes leading to autolysis.

Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Growth yields and morphological characterization

“…Despite the presence of two phases, the percentage of vacuoles decreased as the dilution rate increased over the range of dilution rates studied (Table III). This is in agreement with results reported by Paul et al (1994) and McIntyre et al (2001). The appearance of vacuoles represents the progression of a differentiation process that eventually results in the appearance of empty regions and subsequently autolysis (McIntyre et al, 1998).…”

“…It has also been reported that an increase in the percentage of empty regions within hyphae could make the structure weaker due to the ongoing process of autolysis. Highly differentiated hyphae are therefore more susceptible to mechanical shear, which results in breakage and fragmentation (McIntyre et al, 2001). The level of vacuoles in strain DS36079 measured in this work was rather low, indicating that the process of differentiation was beginning to proceed through the culture.…”

“…In addition to detailed characterization of the metabolism of penicillin-producing strains, some studies have focused on the relationship between morphological development and productivity (McIntyre et al, 2001;Paul et al, 1994;Paul and Thomas, 1996). Paul and Thomas (1996) proposed a structured kinetic model describing growth, differentiation, and penicillin production in submerged P. chrysogenum culture.…”

“…They divided the hyphae into four distinct regions on the basis of the activity and structure of the cells: (1) actively growing (mainly apical) regions; (2) nongrowing cytoplasm, where small vacuoles can be formed; (3) degenerated or metabolically inactive regions; and (4) autolysed regions. The degree of vacuolation has generally been used as an indicator for the advancement of the differentiation process Thomas, 1994, 1996), and may be used to predict detrimental effects of cell degeneration on the penicillin production process (McIntyre et al, 1998(McIntyre et al, , 2001.…”

Integrated Approaches for Assessment of Cellular Performance in Industrially Relevant Filamentous Fungi

“…32,33 This leads to heterogeneity in terms of the morphological forms present and the cell (compartment) types that contribute to the overall performance of the culture. 34 The challenge is to balance cellular potential, process design, and economic feasibility. High broth viscosities, and thus low oxygen mass transfer, are the main physical factors governing protein productivity in filamentous fungal cultivations.…”