Research Centre for Industrial Crops, Agriculture Research Council, Bologna, Italy.
Biotechnol Bioeng. 2010 Oct 15;107(3):461-8. doi: 10.1002/bit.22816.
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.
木质纤维素生物质转化为燃料需要水解步骤以获得可发酵糖，通常通过真菌酶完成。各种纤维素分解微生物菌株库应有助于开发针对当地可用饲料的最佳酶混合物。由于大容量培养方法（如摇瓶）的限制，只有有限数量的菌株可以同时在基于筛选的方法中进行检测。本研究描述了一种小型化策略，旨在允许同时评估大量真菌菌株。使用含有琼脂培养基的平底 24 孔板，在微晶体纤维素上静止培养木霉菌株。通过整个培养板的快速离心步骤获得上清液，并使用基于微孔板的测定法评估细胞外总纤维素酶活性，以滤纸活性为衡量标准。所得结果与摇瓶实验观察到的结果一致，因此对超过 300 株木霉菌株进行了纤维素酶生产特性的描述。在摇瓶实验中，有 5 株菌株的活性至少为高纤维素酶突变体 Trichoderma Rut-C30 的 80%，通过 24 孔板筛选正确识别，证明了这种方法的可行性。还报告了整个木霉收集的纤维素酶活性分布。一株菌株（T. harzianum Ba8/86）在时间过程实验中与参考菌株 Rut-C30 的接近程度最高。该方法可扩展，解决了筛选计划中的主要瓶颈，允许小规模平行培养和快速上清液提取。它还可以轻松与高通量酶测定法集成，并且适合自动化。
