The miscanthus biomass was sterilized by autoclaving, which constituted a hydrothermal pre-treatment resulting in the extraction of soluble products into the water phase of the autoclaved samples. From this pre-treated material, several fractions were separated and incorporated into different agar Petri dishes.
The growth of P. chrysosporium on solid media containing different fractions of miscanthus was followed by non-destructive microscopic observation. Two cultures were used as reference cultures: culture A which was devoid of any added miscanthus and culture B that incorporated the whole non-fractionated miscanthus. Culture A consisted of medium A containing only agar and the superimposed nitrocellulose membrane. The experiment was terminated for all the cultures when the fastest-growing culture started to reach the maximal image size (24 mm wide).
The experimental apparatus and data processing methods allowed for successive observation of P. chrysosporium growth on the different miscanthus biomass fractions incorporated in agar. An example of the image produced from a 52 h growth of P. chrysosporium on agar medium without any fraction of miscanthus is shown in Fig. 2. The left image is composed of 25 single overlapping images (tiles). The tiles were separated and reassembled into one image. Images with good black/white contrast were obtained, allowing for accurate quantification of the surface occupied by the fungus. Growth measurements (occupied area, colony diameter, germination rate, the number of objects) were determined from the processed images.
Figure 2. Example of acquired and processed images of a P. chrysosporium colony. The image acquired after 52 hours of growth on agar medium is on the left. The processed image after image treatment operations (as described in the text) is on the right. Red scale bar = 2000 µm.
The spores were visualized during the early stages of growth (Figs. 3 & 4). The number of spores decreased over time as the spores germinated, and the germ tubes grew into hyphae. The germination rate was determined between 0 and 23 hours. The mycelium developed on the surface through tip extension and branching, leading to an interconnected mycelial network and the formation of a colony that grew radially over time (Figs. 3 & 4 red circles).
Figure 3. Germination and growth of P. chrysosporium on agar (defined as negative control – substrate A). Monitoring was performed at 50X magnification. Images show the growth at time 0, 23, 26, 29, 46.5, 49.5, and 52 hours. A scale-shift of the image is indicated by the change in the color of the drawn squares from red to blue. The sides of the red square measure 9000 µm. The length of the sides of the blue squares was 11000 µm, and the image size is 12288-pixel x 12288-pixel. The red circles show the position of a single spore over time. At 23 hours, the spore was swollen. At 26 hours, a germ tube was formed. A microcolony was observed at 29 hours, where branching started. The final colony can be seen at 52 hours. The red scale bars measure 2000 µm.
The fungi growth on the nitrocellulose membrane placed on agar devoid of any miscanthus fraction (culture A) was relatively slow (Fig. 3). The final size of the colony reached 3.9 × 107 µm2. The fastest growth (Fig. 4) was observed when non-fractionated miscanthus was incorporated into the agar (culture B). The size of the colony at 52 h was 3.2 × 108 µm2, eight times greater than culture A.
Figure 4. Germination and growth of P. chrysosporium on the non-fractionated substrate (culture B). The monitoring was performed at 50X magnification. Images show the growth at time 0, 23, 26,29, 46.5, 49.5 and 52 hours. The color change of the square drawn around the colony from red to purple indicates a change in the scale of the image. The sides of the red square measure 9000 µm. The sides of the purple squares are 25000 µm long, and the image size is 24576-pixel x 24576-pixel. The red circles show the position of a single spore over time. At 23 hours, the spore was swollen, and a germ tube was formed. At 26 hours, a microcolony was obtained where branching start to take place. At 29 hours, the amplification of branching continued, and the tips extended, forming bridges with other microcolonies. One colony can be seen at 46.5 hours. The red scale bars measure 2000 µm.
The spore germination percentage and the calculated spore germination rate for the two cultures (Table 2) showed a large difference in their initial growth rates. Culture B, containing the substrate, grew much more rapidly. The percentage of spore germination was significantly greater in culture B, which was reflected in the spore germination rate. The only apparent inconsistency in these observations was in culture A, wherein the germination rate was greater between 23 and 26 hours. This observation must be interpreted by considering that the majority of the spore population germinated during this time in culture A.
Table 2
The percentage of germinated spores during the first 29 hours. All objects observed at time 0 h were considered to be spores. The average size of these objects was 560µm2.
| | Spore germination percentage | Spore germination rate (spore/h) |
| Time (h) | 23 | 26 | 29 | 23 | 26 | 29 |
Substrate | A | 17 | 64 | 89 | 5 | 110 | 60 |
B | 78 | 90 | 94 | 23 | 28 | 11 |
C | 66 | 90 | 96 | 19 | 51 | 13 |
D | 64 | 93 | 97 | 18 | 65 | 7 |
E | 14 | 72 | 81 | 4 | 126 | 18 |
The deposition of the inoculum droplet containing spores left a marked area on the nitrocellulose membrane, corresponding to the maximum area where germination would first occur. Even after complete absorption of the droplet into the paper, this mark persisted. Colonization of the membrane surface in all experiments occurred first within this marked area. It was noted that the spores near the edge of the mark showed a tendency to first grow inwards rather than outside of the marked area.
Comparative extent of P. chrysosporium growth on substrate fractions
Growth as measured by spore germination rate
Observing the percentage spores’ germination, culture A and culture E demonstrated very similar results (Table 2). The growth rate measured in this way was very similar for cultures B, C, and D. The final spore germination percentage was very high for all cultures (81–87%), demonstrating good spore viability. Culture B gave a slight but significantly higher growth rate as measured by this method (Table 2).
Figure 5. The area occupied by P. chrysosporium mycelia after 52 hours. The blue and orange bars indicate the data of duplicate cultures for each substrate. The corresponding images visually represent the growth and morphology of the colonies at each time point. The upper row of images corresponds to the data represented by the blue bars, and the lower images correspond to the data represented by the orange bars. Each side of the purple square is 25000 µm. The mycelium in culture B covered 3.0 ± 0.2 × 108µm2, 8-fold higher than the negative control (culture A). At the same time point, culture C was 78% of culture B; culture D was 42% of culture B, and culture E was 26% of culture B. The use of non-fractionated miscanthus (Fig. 1) resulted in the fastest growth of the organism followed by the soluble fraction (culture C), the unwashed solid fraction (culture D), and the washed solid fraction (culture E). If the growth area on agar alone (culture A) is subtracted from all growth data, the sum of the growth measured on cultures C and D provided a value close to that of culture B. Thus, the covered area on culture C at 52 hours represented 64% of the covered area of culture B; the soluble fraction is largely responsible for the extent of mycelial growth.
Growth rates as measured by particle coalescence
At time zero, all objects were considered to be spores. Coalescence was defined as the rate at which the number of objects decreased. The objects consisting of spores and microcolonies (germ tubes and mycelia) were counted at each time point. The variation in the number of objects demonstrated a rapid growth measurement in the early stages of colony development.
The coalescence dynamics were followed for the first 29 h of incubation, after which time it was no longer possible to follow the number of individual objects. The decline in the number of objects accelerated after 26 hours, even with the washed solid fraction (Fig. 6 III). The change in the number of objects correlated well with the average size of counted objects (Table 4). At 29 hours, the percentage of objects remaining from the initial spores was 85, 30, 18, 60, and 80% on cultures A, B, C, D, and E, respectively. Thus, the fastest growth was observed in culture C, and cultures A and E demonstrated very similar growth rates.
Growth rates as measured by radial extension
Once complete coalescence had occurred, the mycelial mat could be considered a single colony. The radial extension of the colony was the internal distance between the circumferences of each colony.
Due to the observation that initial growth took place within the marked area where the spores were deposited, no radial growth was observed during the first 29 hours. After this period, the colony diameters for cultures B and C increased linearly but accelerated for the other cultures (Fig. 6I). After 29 hours, the fastest growth was observed in culture B; cultures C and D were growing slower and at a similar rate. Culture E was growing considerably slower than cultures B-D, and finally, culture E was growing very slowly (Table 3).
Figure 6. Three types of measurements describe the growth of P. chrysosporium on different substrates. Graph (I) shows the colony growth measured by radial expansion. Graph (II) shows the colony growth by surface coverage of the membrane by the mycelium. Histograms (III) show the coalescence patterns of objects; each spore is counted as an individual particle at time zero. Graph (IV) shows the number of objects versus the total area occupied by P. chrysosporium.
Growth rates as measured by surface colonization
Image analysis allowed for the determination of the total surface occupied by the spores and mycelia at any given time. This total area consisted of the sum of all the white pixels present on the image. The inferred growth is related specifically to the superficial growth area in the marked surface.
Growth measured in terms of the occupied area was exponential during two distinct periods; 23–29 h and 29–52 h (Fig. 6 II) (Table 3). Between 23 h and 29 h, the exponential growth rate of P. chrysosporium in culture C was the highest (0.326 h− 1), followed, in order, by cultures B, D, E, and the agar control (culture A) (Table 3). The growth rates of cultures D and E were very similar.
During the second period (29–52 h), the ranking from high to low culture growth rates was B, E, C, D, and A (Fig. 6 II). The growth rates of cultures B and E were very similar, and C and D were also similar. Culture E showed a rapid increase in growth during this phase compared to all other cultures (Table 3).
Table 3
Occupation rate (µ) on different substrates. The values of the mean extension rate (v) of the P. chrysosporium colony are correlated with the growth measured by surface area occupation for the same time intervals. The coefficient of determination (R2) was calculated for each regression line.
Growth rate Culture | Occupied area | Radial expansion |
23 h to 29 h (3 points) | 29 h to 52 h (4 points) | 29 h to 52 h (4 points) |
µ1 (h− 1) exponential fit | R2 | µ2 (h− 1) exponential fit | R2 | v (µm/h) linear fit | R2 |
A | 0.195 | 0.999 | 0.101 | 0.999 | 71 | 0.813 |
B | 0.290 | 0.997 | 0.134 | 0.995 | 633 | 0.999 |
C | 0.326 | 0.999 | 0.124 | 0.996 | 528 | 0.996 |
D | 0.257 | 0.997 | 0.121 | 0.998 | 497 | 0.986 |
E | 0.248 | 0.957 | 0.133 | 0.989 | 347 | 0.970 |
Table 4
Average size of counted objects during the growth period 0–29 hours.
| | Occupied area in µm2, (no. of objects) |
| Time (h) | 0 | 23 | 26 | 29 |
Culture | A | 5.64 × 102 (715) | 1.76 × 103 (680) | 3.23 × 103 (645) | 6.35 × 103 (605) |
B | 5.20 × 102 (687) | 4.39 × 103 (627) | 1.24 × 104 (485) | 7.48 × 104 (209) |
C | 6.64 × 102 (648) | 3.65 × 103 (588) | 1.26 × 104 (472) | 1.27 × 105 (120) |
D | 5.83 × 102 (660) | 3.10 × 103 (598) | 8.12 × 103 (533) | 2.04 × 104 (424) |
E | 6.48 × 102 (645) | 1.53 × 103 (628) | 4.42 × 103 (601) | 8.11 × 103 (524) |
Legend: The area is reported in µm2; the number of individual objects counted is reported in brackets. After 29 hours, it was no longer possible to count the number of objects due to the high degree of coalescence. At the next time point, 46.5 h, there remained 26 individual objects on culture A and just a single object on each of the other cultures. The number of spores deposited on the membrane at time zero varied between 645 and 715, indicating the reproducibility of the inoculation procedure. |
Chemical analysis of solid and soluble fractions of pre-treated miscanthus by FTIR |
FTIR spectroscopy of the multiple miscanthus fractions used for P. chrysosporium growth provided rapid semi-quantitative information on the functional groups present, and therefore, on the polymers and molecules potentially available for fungal growth within the substrates. All the substrates in cultures B to E were analyzed (Table 1). The correspondence between the name of the FTIR sample and the type of the substrate from which it is originated is in Table 1 and Fig. 1. |
Table 5
Selected absorption band data which correspond to the wavelength of the spectra for the solid and soluble fractions.
Functional groups | HC | L | L | L | L-C | C-HC | C | C-HC | L |
Wavenumbers (cm− 1) | 1734 | 1605 | 1514 | 1463 | 1427 | 1376 | 1322 | 1055 | 1036 | 896 |
Samples & absorbances | FTIR-C C1 | 0.2 | 0.6 | 0.2 | 0.2 | 0.2 | 0.2 | 0.8 | 0.8 | 0.7 | 0.0 |
FTIR-Ew C2 | 0.2 | 0.6 | 0.2 | 0.2 | 0.2 | 0.2 | 0.7 | 0.7 | 0.8 | 0.0 |
FTIR-B | 0.5 | 0.5 | 0.3 | 0.3 | 0.3 | 0.3 | 1.3 | 1.3 | 1.2 | 0.1 |
FTIR-D | 0.6 | 0.5 | 0.3 | 0.3 | 0.3 | 0.3 | 1.2 | 1.2 | 1.2 | 0.2 |
FTIR-E | 0.5 | 0.5 | 0.3 | 0.3 | 0.3 | 0.3 | 1.2 | 1.2 | 1.2 | 0.1 |
Legend
Cellulose (C), hemicellulose (HC), and lignin (L). C1 represents 1.5% and C2 0.25% of the total substrate in culture B. The main differences between the fractions are highlighted in black or grey.
The main functional groups were assigned to cellulose (C), hemicellulose (HC) and lignin (L) in FTIR spectra according to the literature: a(Stewart and Morrison 1992), b(Sun et al. 2000), c(Geng et al. 2003), d(Schwanninger et al. 2004), e(Sun et al. 2005), f(Xu et al. 2006a), g(Xu et al. 2006b), h(Belmokhtar 2012), i(Xu et al. 2013), k(Ferrer et al. 2016), l(Lara-Serrano et al. 2018), m(Li et al. 2018), and n(Lavarda et al. 2019).
The FTIR spectra of substrates used in cultures B, D, and E are almost identical, suggesting few changes in the relative content of lignin, hemicellulose, and cellulose due to the pre-treatment. All these substrates contained solid fractions. Results for samples C and Ew were similar but different from samples B, D, and E, especially for the functional groups related to xylan (1734 cm− 1) and aromatic compounds (1605 cm− 1). The soluble material remaining in substrate D was similar to substrate C (Table 5), demonstrating that the liquid fraction is also present in the unwashed solids fraction.
The elemental (CHNSO) analysis of miscanthus, agar, and the nitrocellulose membrane showed the total absence of nitrogen and sulfur in miscanthus, while the two elements existed in the nitrocellulose membrane (Table 6).
Table 6
Elemental analysis of untreated miscanthus, agar, and the nitrocellulose membrane.
CHNOS composition Element of device | % C | % H | % N | % S | % O |
Miscanthus x giganteus | 45.9 ± 1.6 | 6.0 ± 0.2 | 0.0 | 0.0 | 48.1 ± 1.8 |
Nitrocellulose membrane | 26.3 ± 1.9 | 2.6 ± 0.5 | 11.0 ± 1.0 | 0.6 ± 0.1 | 59.4 ± 3.6 |
Agar | 42.7 ± 0.2 | 6.4 ± 0.1 | 0.0 | 0.9 ± 0.0 | 50.0 ± 0.3 |
Legend
The average percentage by mass of each element of total CHNSO is represented for duplicates of samples at 0% moisture.