In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time-efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non-invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed-batch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor.

中文翻译：

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微生物反应器辅助培养工作流程，用于以分批和补料分批模式对产蛋白黑曲霉进行高效的表型分析

近年来，许多真菌基因组已公开可用。结合新的基因编辑工具，这可以加速菌株的构建，使丝状真菌对生产有价值的产品更加有趣。然而，除了它们非凡的生产和分泌能力外，真菌最常表现出具有挑战性的形态，需要对其进行筛选以获得最佳操作窗口。因此，将遗传多样性与各种环境参数相结合会产生很大的参数空间，从而对高效的表型技术提出了强烈的需求。微生物反应器系统已经为细菌有机体建立了良好的环境，可通过并行化和小型化提高培养产量，并通过无创在线监测增强对过程的洞察力。然而，文献中关于丝状真菌微量滴定板培养的报道很少，在线监测的报道更少。此外，在微尺度分批条件下进行筛选，当大规模进行补料分批过程时，甚至可能导致对优化参数的错误识别。因此，在本研究中，一种新颖的工作流程开发了黑曲霉，可以分批和分批补料模式进行多达 48 个平行的微生物反应器培养。该工作流程已针对实验室规模的生物反应器培养进行了验证，以证明可扩展性。通过优化的培养方案，测试了三种不同的微量补料分批策略，以确定细胞内模型产物 GFP 的最佳蛋白质生产条件。随后，最佳进料策略再次在实验室规模的生物反应器中得到验证。