Online Biomass Monitoring Enables Characterization of the Growth Pattern of Aspergillus fumigatus in Liquid Shake Conditions

Abstract

Numerous filamentous fungal species are extensively studied due to their role as model organisms, workhorses in biotechnology, or as pathogens for plants, animals, and humans. Growth studies are mainly carried out on solid media. However, studies concerning gene expression, biochemistry, or metabolism are carried out usually in liquid shake conditions, which do not correspond to the growth pattern on solid media. The reason for this practice is the problem of on-line growth monitoring of filamentous fungal species, which usually form pellets in liquid shake cultures. Here, we compared the time-consuming and tedious process of dry-weight determination of the mold Aspergillus fumigatus with online monitoring of biomass in liquid shake culture by the parallelizable CGQ ("cell growth quantifier"), which implements dynamic biomass determination by backscattered light measurement. The results revealed a strong correlation of CGQ-mediated growth monitoring and classical biomass measurement of A. fumigatus grown over a time course. Moreover, CGQ-mediated growth monitoring displayed the difference in growth of A. fumigatus in response to the limitation of iron or nitrogen as well as the growth defects of previously reported mutant strains (ΔhapX, ΔsrbA). Furthermore, the frequently used wild-type strain Af293 showed largely decreased and delayed growth in liquid shake cultures compared to other strains (AfS77, A1160p+, AfS35). Taken together, the CGQ allows for robust, automated biomass monitoring of A. fumigatus during liquid shake conditions, which largely facilitates the characterization of the growth pattern of filamentous fungal species.

Keywords: Aspergillus fumigatus; backscatter; biomass monitoring; bioprocess automation; flask culture; fungi; liquid shake culture; molds; online monitoring.

Figure 1

The CGQ-measurement principle. (a) CGQ sensors are mounted underneath the shake flasks for non-invasive measurements through the glass wall of the vessel. The sensors are connected to a base station that communicates the data to a computer outside of the incubator. (b) Biomass measurements are mediated via backscattered light detection. An LED emits light into the medium, which is scattered by cells/pellets/particles within the culture. A portion of the scattered light is detected as backscatter by a photodiode that is part of the CGQ sensor. With higher cell densities the backscattered light intensity is higher compared to lower cell densities.

Figure 2

CGQ-mediated growth monitoring of A. fumigatus AfS77 during 14–72 h liquid shake culturing under +Fe and −Fe conditions. A. fumigatus AfS77 was cultured for 14 h, 17 h, 20 h, 24 h, 48 h, and 72 h in liquid shake cultures under −Fe and +Fe conditions in biological triplicates and biomass was monitored with the CGQ. The different colors discriminate the cultivations conducted for different incubation times. Thin lines display growth dynamics of individual runs and shaded areas show the variance within the parallels of each experiment. Thick lines represent a smooth curve fitted by local polynomial regression using the LOESS algorithm with a span value of a = 0.3.

Figure 3

Protease activity is detectable in culture supernatants of +Fe cultures at 48 h and to a lesser extent in 72 h but not in those of younger (24 h) +Fe or in −Fe cultures. Supernatants from cultures were spotted onto an unprocessed X-ray film to test for the presence of proteolytic activities that hydrolyze the gelatin-containing light-sensitive layer (visible as bright circles). Two-fold dilution series (1:1–1:16) in PBS is indicated on top of the picture. The decrease of proteolytic activity in 72 h +Fe compared to 48 h +Fe cultures is most likely caused by degradation of the proteases.

Figure 4

Positive correlation of CGQ-mediated growth monitoring and classical biomass measurement of A. fumigatus AfS77 during 14–72 h liquid shake culturing under +Fe and −Fe conditions. Data were taken from the experiments described in Figure 2 and Table 1. (a) Time course graphs of mean final backscatter (final BS) and mean dry-weight (DW) ± standard deviation of 3 runs each. (b) Final BS plotted vs. DW with colored dots representing endpoints of individual cultures grown for the times indicated. Pearson correlation coefficient and corresponding p-values are displayed in the upper left side of the panels. Regression lines and corresponding equations based on linear models fitted to the scatter plots are shown.

Figure 5

Pellet morphology differs in +Fe and −Fe conditions. (a) A. fumigatus AfS77 pellets were removed for microscopic analysis when base-line-corrected BS reached values of approximately 1000 mAU, after growth times of 17 h and 24 h for +Fe and −Fe conditions, respectively. (b) Violin plots showing the distribution of pellet cross-sectional area in +Fe and −Fe cultures at BS~1000 mAU. Box plots showing medians (0.72 mm² and 0.30 mm² for +Fe and −Fe, respectively), interquartile ranges, and spikes extending to the upper- and lower-adjacent values in each group are overlaid. Outliers are not shown.

Figure 6

CGQ-mediated growth monitoring of A. fumigatus AfS77 cultured for 24 h at different days (experiments 1–3) under +Fe and −Fe conditions. Each experiment included biological triplicates. Details of the graph are as in the caption of Figure 2.

Figure 7

CGQ-mediated growth monitoring of A. fumigatus AfS77 cultured in biological triplicates for 22 h with 20 mM, 10 mM, or 5 mM glutamine (Gln). Details of the graph are as in the caption of Figure 2.

Figure 8

CGQ-mediated growth monitoring of A. fumigatus AfS77 (WT) compared to ΔhapX and ΔsrbA mutant strains cultured in biological triplicates for 24 h under +Fe and −Fe conditions. Details of the graph are as in the caption of Figure 2.

Figure 9

Colony morphology of A. fumigatus AfS77 compared to ∆hapX and ∆srbA cultured for 48 h at 37 °C under +Fe and −Fe conditions. For inoculation of fungal strains, suspensions containing 10⁴ spores were dotted onto minimal media solidified with 1.5% agar.

Figure 10

CGQ-mediated growth monitoring of A. fumigatus AfS77 compared to A1160p+, AfS35, and Af293 cultured for 24 h and 48 h under +Fe and −Fe conditions. Details of the graph are as in the caption of Figure 2.

Figure 11

Colony morphology of A. fumigatus AfS77 compared to A1160p+, AfS35, and Af293 cultured for 48 h at 37 °C under +Fe and −Fe conditions. For inoculation of fungal strains, suspensions containing 10⁴ spores were dotted onto minimal media solidified with 1.5% agar.