A novel microplate‐based screening strategy to assess the cellulolytic potential of Trichoderma strains

Abstract

Bioconversion of lignocellulosic biomass to fuel requires a hydrolysis step to obtain fermentable sugars, generally accomplished by fungal enzymes. An assorted library of cellulolytic microbial strains should facilitate the development of optimal enzyme cocktails specific for locally available feedstocks. Only a limited number of strains can be simultaneously assayed in screening based on large volume cultivation methods, as in shake flasks. This study describes a miniaturization strategy aimed at allowing parallel assessment of large numbers of fungal strains. Trichoderma strains were cultivated stationary on microcrystalline cellulose using flat bottom 24-well plates containing an agarized medium. Supernatants obtained by a rapid centrifugation step of the whole culture plates were evaluated for extracellular total cellulase activity, measured as filter paper activity, using a microplate-based assay. The results obtained were consistent with those observed in shake-flask experiments and more than 300 Trichoderma strains were accordingly characterized for cellulase production. Five strains, displaying on shake-flasks at least 80% of the activity shown by the hyper-cellulolytic mutant Trichoderma Rut-C30, were correctly recognized by the screening on 24-well plates, demonstrating the feasibility of this approach. Cellulase activity distribution for the entire Trichoderma collection is also reported. One strain (T. harzianum Ba8/86) displayed the closest profile to the reference strain Rut-C30 in time course experiments. The method is scalable and addresses a major bottleneck in screening programs, allowing small-scale parallel cultivation and rapid supernatant extraction. It can also be easily integrated with high-throughput enzyme assays and could be suitable for automation.

Overexpression of an exotic thermotolerant β-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw

“…Three biological replicates were done for each transformant. To examine β-glucosidase activity and total cellulase activity, a time course trial was conducted similar to that of Cianchetta et al [38]. Specifically, the flasks were incubated at 30°C for a total of 144 hours.…”

Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains

“…There is, therefore, a need to correlate qualitative results from screening with quantitative results obtained for cellulase production during cultivation in a fermentation process. A microplate-based screening method to assess the cellulolytic potential of Trichoderma strains has recently been reported [14] and the results were compared with those obtained using submerged shake flask cultivations. Since the natural habitats of these filamentous fungi are solid media, the SSF procedure offers considerable advantages over submerged fermentation (SmF).…”

A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains

“…Recently, Cianchetta et al described a screening strategy consistent with cultivations in batch shake flasks for the identification of hypercellulolytic T. reesei strains [21]. However in their shake flask protocol, the strains were cultivated with excess cellulose so the results cannot be extrapolated to the industrial carbon-limited protocol.…”