X-Ray Scattering Reveals Two Mechanisms of Cellulose Microfibril Degradation by Filamentous Fungi

Abstract

Mushroom-forming fungi (Agaricomycetes) employ enzymatic and nonenzymatic cellulose degradation mechanisms, the latter presumably relying on Fenton-generated radicals. The effects of the two mechanisms on the cellulose microfibrils structure remain poorly understood. We examined cellulose degradation caused by litter decomposers and wood decomposers, including brown-rot and white-rot fungi and one fungus with uncertain wood decay type, by combining small- and wide-angle X-ray scattering. We also examined the effects of commercial enzymes and Fenton-generated radicals on cellulose using the same method. We detected two main degradation or modification mechanisms. The first characterized the mechanism used by most fungi and resembled enzymatic cellulose degradation, causing simultaneous microfibril thinning and decreased crystalline cellulose. The second mechanism was detected in one brown-rot fungus and one litter decomposer and was characterized by patchy amorphogenesis of crystalline cellulose without substantial thinning of the fibers. This pattern did not resemble the effect of Fenton-generated radicals, suggesting a more complex mechanism is involved in the destruction of cellulose crystallinity by fungi. Furthermore, our results showed a mismatch between decay classifications and cellulose degradation patterns and that even within litter decomposers two degradation mechanisms were found, suggesting higher functional diversity under current ecological classifications of fungi. IMPORTANCE Cellulose degradation by fungi plays a fundamental role in terrestrial carbon cycling, but the mechanisms by which fungi cope with the crystallinity of cellulose are not fully understood. We used X-ray scattering to analyze how fungi, a commercial enzyme mix, and a Fenton reaction-generated radical alter the crystalline structure of cellulose. Our data revealed two mechanisms involved in crystalline cellulose degradation by fungi: one that results in the thinning of the cellulose fibers, resembling the enzymatic degradation of cellulose, and one that involves amorphogenesis of crystalline cellulose by yet-unknown pathways, resulting in a patchy-like degradation pattern. These results pave the way to a deeper understanding of cellulose degradation and the development of novel ways to utilize crystalline cellulose.

Keywords: Fenton chemistry; X-ray scattering; biodegradation; brown-rot fungus; cellulose; filamentous fungi; litter decomposer; white-rot fungus.

FIG 1

Full scattering profiles for the original filter paper (two replicates each of OP1 and OP2) and the control filter paper sample (two replicates each of C25-1, C25-2, C20-1, and C20-2), incubated for 40 days at 25°C and 20°C. The association of the scattering regions with the relevant structure of a cellulose fiber is shown. The red line was obtained by fitting the OP1 SAXS profile with equation 1 in the range 0.004 to 0.45 Å⁻¹. The intensity was normalized for the transmitted beam. The absolute intensity was obtained from the OP thickness, 0.430 mm. The inset highlights WAXS results and shows the main reflections of cellulose I. The numbers in the inset represent Miller indices (57).

FIG 2

Transmission (T) values for all samples. T values were obtained by averaging the transmission value of each pattern collected at the three sample-to-detector distances and divided by the average value for the original paper (OP).

FIG 3

Full scattering profiles of the original filter paper and following exposure to enzymes, the Fenton reaction, and fungal species. (Left) Full scattering profiles of the original filter paper (OP), the filter paper after 24- and 48-h exposure to commercial enzyme concentrations C1 and C2 (C1-24h, C1-48h, C2-24h, and C2-48h), and after exposure to the Fenton reaction radicals (Fe1, Fe2). (Right) Full scattering profiles of OP and after fungal degradation by selected species (see Table 1 for species abbreviations). The data are reported along with the model (equation 1). The insets for both panels highlight the WAXS region.

FIG 4

SAXS and WAXS fitting parameters and estimates of amorphous cellulose. (Top) The A and B fitting parameters of equation 1 relative to the data in Fig. 1 and 3 and the square root of the C parameter obtained at the q position of 1.63 ± 0.04 Å⁻¹. A and B were normalized for the values obtained from the OP (A₀ and C₀, respectively). (Bottom) Calculated ratios between the scattering intensity at the expected position of the amorphous peak at 1.38 Å⁻¹ and the sum of all the intensities of the peaks between 1 and 1.6 Å⁻¹, including the intensity at 1.38 Å⁻¹ (40). The intensity was obtained by deconvolution (see the “Additional description of analyses” in the supplemental material). The red line represents the average values obtained from the original samples, the autoclaved samples, and the control samples at 20°C and 25°C. The dotted lines mark the areas of relative deviation. LD, litter decomposer; WR, white rot; BR, brown rot; UWD, uncertain wood decay strategy; Fe, Fenton reaction. Species names corresponding to the acronyms are shown in Table 1. LN and HN after the acronym indicate species that were incubated in medium with low or high nitrogen content, respectively.

FIG 5

Relationship between overall fiber volume, proportional to (A/A₀)², and the total volume of crystalline domains, proportional to C/C₀. The relationship between (A/A₀)² and C/C₀ provides a schematic summary of the two types of cellulose degradation mechanisms detected in this study. Gloeophyllum sp. and Leucoagaricus leucothites cause melting of the crystallites and generation of amorphous cellulose without concomitant thinning of the fibrils. We refer to this as the patchy-like mechanism. In contrast, Coprinellus angulatus uses an enzymatic degradation mechanism during which the fibrils are degraded from their surface inwards with simultaneous, proportional loss of crystalline cellulose, which we refer to as a thinning mechanism. We assume for both these mechanisms that the number of fibers remain constant on the experimental time scale. A/A₀ versus C/C₀ was plotted in the case of enzymatic (C1-24h, C1-48h, C2-24h, C2-48h) and Fenton (Fe-12h, Fe-24h) degradation, due to a decrease in scattering (fiber) volume at a constant fiber radius, R. Colors stand for different ecologies or treatments: yellow, white-rot fungi; brown, brown-rot fungi; green, litter decomposers; pink, uncertain wood decay type; blue, enzymatic treatments; red, Fenton treatments; open circles, control sample.