Efficient plant biomass degradation by thermophilic fungus Myceliophthora heterothallica

Abstract

Rapid and efficient enzymatic degradation of plant biomass into fermentable sugars is a major challenge for the sustainable production of biochemicals and biofuels. Enzymes that are more thermostable (up to 70°C) use shorter reaction times for the complete saccharification of plant polysaccharides compared to hydrolytic enzymes of mesophilic fungi such as Trichoderma and Aspergillus species. The genus Myceliophthora contains four thermophilic fungi producing industrially relevant thermostable enzymes. Within this genus, isolates belonging to M. heterothallica were recently separated from the well-described species M. thermophila. We evaluate here the potential of M. heterothallica isolates to produce efficient enzyme mixtures for biomass degradation. Compared to the other thermophilic Myceliophthora species, isolates belonging to M. heterothallica and M. thermophila grew faster on pretreated spruce, wheat straw, and giant reed. According to their protein profiles and in vitro assays after growth on wheat straw, (hemi-)cellulolytic activities differed strongly between M. thermophila and M. heterothallica isolates. Compared to M. thermophila, M. heterothallica isolates were better in releasing sugars from mildly pretreated wheat straw (with 5% HCl) with a high content of xylan. The high levels of residual xylobiose revealed that enzyme mixtures of Myceliophthora species lack sufficient β-xylosidase activity. Sexual crossing of two M. heterothallica showed that progenies had a large genetic and physiological diversity. In the future, this will allow further improvement of the plant biomass-degrading enzyme mixtures of M. heterothallica.

Fig 1

Parsimonious consensus tree of ITS1 region of Myceliophthora species (based on reference 16). The numbers next to the branches indicate the percentage of replicate trees, in which the associated taxa clustered together in the bootstrap test (1,000 replicates).

Fig 2

Growth of thermophilic isolates of Myceliophthora on five industrial substrates at 40°C. Spruce, wheat straw, and giant reed (Arundo donax) were pretreated with 20% HCl or with a steam treatment (indicated between brackets). The pictures of M. thermophila and M. heterothallica were taken after 2 days, M. fergusii after 4 days, and M. hinnulea after 11 days.

Fig 3

Cellulase, xyloglucanase, and xylanase activities of Aspergillus niger (An), Trichoderma reesei (Tr), Myceliophthora thermophila ATCC 42464 (Mt), and Myceliophthora heterothallica CBS 202.75 and CBS 663.74 after growth on wheat straw pretreated with 20% HCl. The activities are measured by the amount of reduced sugar released and are given in nmol or μmol reduced sugar per min per mg of total protein. The averages and standard deviations represent two independent cultivations and at least four technical replicates.

Fig 4

Enzyme activities of M. thermophila ATCC 42464 and M. heterothallica CBS 202.75 and CBS 663.74 during 4 days growth on wheat straw pretreated with 20% HCl (A to F) or 5% HCl (G to L). The enzymatic activities are measured against p-nitrophenol (PNP)-linked substrates and given in nmol of PNP per min per mg of total protein. The averages and standard deviations represent two independent cultivations and six technical replicates.

Fig 5

Saccharification of wheat straw pretreated with 20% HCl (A to F) or 5% HCl (G to L) during 4 days at 45°C using an enzyme mixture of M. thermophila ATCC 42464 and M. heterothallica CBS 202.75 and CBS 663.74. The sugar concentrations are given in mg per liter of filtrate. The black lines are representing the sugar concentrations in wheat straw without an enzyme mixture. The averages and standard deviations represent two independent cultivations and six technical replicates.

Fig 6

Hierarchical clustering (UPGMA) of AFLP banding patterns of Myceliophthora progenies and their parents M. heterothallica CBS 203.75 and CBS 663.74. Similarity of the banding patterns is given in percentage.

Fig 7

Saccharification of mildly pretreated wheat straw (5% HCl) with an enzyme mixture of M. heterothallica CBS 203.75 and CBS 663.74 and their two progeny “FP 711.02” and “FP 711.14” during 4 days at 45°C. The sugar concentrations are given in mg per liter of filtrate. The black lines are representing the sugar concentrations in wheat straw without an enzyme mixture. The averages and standard deviations represent two independent cultures.