In Situ Imaging for Monitoring Biomass Content and Pellet Size in Submerged Filamentous Stirred-Tank Cultivation.

Filamentous microorganisms play a central role in industrial biotechnology, where pellet morphology affects mass transfer, rheology, and process performance. However, time-resolved monitoring of filamentous biomass and pellet size dynamics in stirred-tank reactors remains challenging due to offline sampling limitations. We present a noninvasive in situ imaging approach to observe biomass-related light scattering and projected pellet size. A custom-built camera and ring-light setup with MATLAB-based image acquisition and processing was used to quantify image luminosity as a proxy for biomass content and to determine projected object area (POA) and the number of detected objects per image above a POA threshold of 0.5 mm2. To characterize systematic biases, polyoxymethylene spheres of 1-4 mm diameter served as a model system. Luminosity scaled with object concentration, whereas object detection and apparent size estimation were increasingly affected by object size and concentration. In a bench-scale cultivation of malic acid-producing Aspergillus oryzae, image luminosity followed biomass content, while in situ POA analysis revealed increasing projected pellet size coinciding with Zn2+ limitation. These observations differed from ex situ sampling, indicating that image-based in situ monitoring provides time-resolved information on biomass formation and pellet size dynamics that may not be captured by offline sampling alone.