Towards a Miniaturized Culture Screening for Cellulolytic Fungi and Their Agricultural Lignocellulosic Degradation

Abstract

The substantial use of fungal enzymes to degrade lignocellulosic plant biomass has widely been attributed to the extensive requirement of powerful enzyme-producing fungal strains. In this study, a two-step screening procedure for finding cellulolytic fungi, involving a miniaturized culture method with shake-flask fermentation, was proposed and demonstrated. We isolated 297 fungal strains from several cellulose-containing samples found in two different locations in Thailand. By using this screening strategy, we then selected 9 fungal strains based on their potential for cellulase production. Through sequence-based identification of these fungal isolates, 4 species in 4 genera were identified: Aspergillus terreus (3 strains: AG466, AG438 and AG499), Penicillium oxalicum (4 strains: AG452, AG496, AG498 and AG559), Talaromyces siamensis (1 strain: AG548) and Trichoderma afroharzianum (1 strain: AG500). After examining their lignocellulose degradation capacity, our data showed that P. oxalicum AG452 exhibited the highest glucose yield after saccharification of pretreated sugarcane trash, cassava pulp and coffee silverskin. In addition, Ta. siamensis AG548 produced the highest glucose yield after hydrolysis of pretreated sugarcane bagasse. Our study demonstrated that the proposed two-step screening strategy can be further applied for discovering potential cellulolytic fungi isolated from various environmental samples. Meanwhile, the fungal strains isolated in this study will prove useful in the bioconversion of agricultural lignocellulosic residues into valuable biotechnological products.

Keywords: Lignocellulosic biomass; cellulase; enzymatic saccharification; fungal degradation; hemicellulase; microplate-based screening.

Fig. 1. Schematic of the screening workflow for cellulolytic fungi.

Two proposed steps: (1) preliminary screening using a microplate-based cultivation step; (2) secondary screening using shake-flask cultivation.

Fig. 2. Scatter plot representing CMCase activity using a small-scale assay and the amount of secreted protein of different fungal strains based on a microplate-based screening assay.

A. aculeatus BCC199 (reference strain; black border dot), selected strains (black dot), non-selected strains (gray dot) are shown.

Fig. 3. Scatter plot representing total protein content and cellulase activity against filter paper (FPase; A) and CMC (CMCase; B) as substrates of crude enzymes from selected fungal strains in a 250-ml shake flask.

A. aculeatus BCC199 (reference strain; black border dot), selected strains (black dot), nonselected strains (gray dot) are shown.

Fig. 4

Enzymatic hydrolysis of pretreated sugarcane trash and bagasse, cassava pulp and coffee silverskin using secreted enzymes from newly isolated fungal strains.