Comparing the physiochemical parameters of three celluloses reveals new insights into substrate suitability for fungal enzyme production

Abstract

Background: The industrial applications of cellulases are mostly limited by the costs associated with their production. Optimized production pathways are therefore desirable. Based on their enzyme inducing capacity, celluloses are commonly used in fermentation media. However, the influence of their physiochemical characteristics on the production process is not well understood. In this study, we examined how physical, structural and chemical properties of celluloses influence cellulase and hemicellulase production in an industrially-optimized and a non-engineered filamentous fungus: Trichoderma reesei RUT-C30 and Neurospora crassa. The performance was evaluated by quantifying gene induction, protein secretion and enzymatic activities.

Results: Among the three investigated substrates, the powdered cellulose was found to be the most impure, and the residual hemicellulosic content was efficiently perceived by the fungi. It was furthermore found to be the least crystalline substrate and consequently was the most readily digested cellulose in vitro. In vivo however, only RUT-C30 was able to take full advantage of these factors. When comparing carbon catabolite repressed and de-repressed strains of T. reesei and N. crassa, we found that cre1/cre-1 is at least partially responsible for this observation, but that the different wiring of the molecular signaling networks is also relevant.

Conclusions: Our findings indicate that crystallinity and hemicellulose content are major determinants of performance. Moreover, the genetic background between WT and modified strains greatly affects the ability to utilize the cellulosic substrate. By highlighting key factors to consider when choosing the optimal cellulosic product for enzyme production, this study has relevance for the optimization of a critical step in the biotechnological (hemi-) cellulase production process.

Keywords: Cellulase production; Cellulose crystallinity; Microcrystalline cellulose; Neurospora crassa; Powdered cellulose; RUT-C30; Trichoderma reesei.

Fig. 1

Scanning electron micrographs of cellulose substrates obtained at 8.0 kV accelerating voltage and ×100 magnification (Jeol JSM-IT100). Images show a representative picture for each substrate out of several technical replicates

Fig. 2

Solid state ¹³C NMR spectra of cellulose samples. Depicted are normalized spectra between 50 and 110 ppm showing the assignment of peaks to the carbons in a glucopyranose repeat unit. Shown is a single but representative spectrum for each cellulose

Fig. 3

Enzymatic digestion of the cellulose substrates in vitro. All celluloses were digested by a N. crassa-derived cellulase cocktail (filtered culture supernatant after 5 days growth on Avicel) for a total of 24 h. a Shown are representative HPAEC-PAD chromatograms of the reaction supernatants after 8 h as well as the quantification results for monosaccharides at an initial time point (1 h; inset in a ). The peaks of d-glucose (d-Glc) and higher cellodextrins (not quantified) are indicated. Note: in this run (CarboPac^® PA200 column), the other monosaccharides will also migrate at the same speed as d-Glc, but the amounts are substantially lower. The quantifications represent means of triplicate reactions. The error bars represent standard deviations. Letters indicate data groups that are significantly different (one-way ANOVA, p-values < 0.05 were considered significant). b–m Representative scanning electron micrographs out of technical triplicates for each cellulose substrate obtained at 8.0 kV accelerating voltage and × 30–× 2000 magnification (Jeol JSM-IT100)

Fig. 4

Gene expression induction of selected genes used as proxies for the fungal cellulolytic and hemicellulolytic response. Sucrose pre-grown N. crassa cultures were exposed to the celluloses or no carbon source for 4 h before RNA was harvested. Gene induction was measured by RT-qPCR. Shown is the mean fold-induction over the no carbon (No C) starvation condition derived from biological and technical triplicates. Error bars denote standard deviation. Letters indicate data groups that are significantly different (one-way ANOVA, p-values < 0.05 were considered significant)

Fig. 5

Cellulase and hemicellulase production by N. crassa and T. reesei RUT-C30 on the cellulose substrates. Performance was measured by analysis of culture supernatant aliquots taken after 3 and 6 days, respectively. Secreted protein was measured by Bradford assay, endo-glucanase activity by Azo-CMC assay and endo-xylanase activity by Azo-Xylanase assay as described in Methods. Values are the mean of biological triplicates. Error bars show standard deviation. Letters indicate data groups that are significantly different (separately for both fungi and sample days; one-way ANOVA, p-values < 0.05 were considered significant)

Fig. 6

Total protein secreted by N. crassa (WT and Δcre-1) and T. reesei (QM6a and RUT-C30) on the cellulose substrates. Performance was measured by analysis of culture supernatant aliquots taken after 2 and 3 days, respectively. Protein concentration was measured by Bradford assay as described in Methods. Values are the mean of biological triplicates. Error bars show standard deviation. Letters indicate data groups that are significantly different (separately for each strain; one-way ANOVA, p-values < 0.05 were considered significant)

Fig. 7

Cellulase and hemicellulase production by N. crassa (WT and Δcre-1) and T. reesei (QM6a and RUT-C30) on the cellulose substrates. Performance was measured by analysis of culture supernatant aliquots taken after 2 and 3 days, respectively. Endo-glucanase activity was measured by Azo-CMC assay and endo-xylanase activity by Azo-Xylanase assay as described in Methods. Values are the mean of biological triplicates. Error bars show standard deviation. Letters indicate data groups that are significantly different (separately for each strain; one-way ANOVA, p-values < 0.05 were considered significant). ND indicates that no values could be determined