In situ microscopy for analysis of filamentous bacteria: optics end image evalaution

Abstract

In the activated sludge process, problems of foaming and filamentous bulking can occur due to overgrowth of certain filamentous bacteria. Nowadays, these microorganisms are typically monitored by means of light microscopy combined with staining techniques. As drawbacks, these methods are susceptible to human errors, subjectivity and limited by the use of discontinuous microscopy. The present project aims the application of an in situ microscope (ISM) for continuous monitoring of filamentous bacteria, providing real-time examination, automated analysis and elimination of sampling, preparation and transport of samples. The ISM previously developed at the Hochschule Mannheim required adaptations for use within wastewater environment, specially in terms of impermeability and development of a cleaning mechanism. With a new objective lens design, the system was simplified to a single tubus and an externally activated cleaning system based on magnetism was created. A proper image processing algorithm was designed for automated recognition and measurement of filamentous objects, allowing real-time evaluation of images without any staining, phase-contrast or dilution techniques. Three main operations are performed: preprocessing and binarization; recognition of filaments using distance-maps and shape descriptors; measurement and display of total extended filament length. A 3D-printed prototype was used for experiments with respect to the new ISM’s design, providing images with resolution very close to the ones acquired with the previous microscope. The designed cleaning system has shown to be effective, removing dirt settled above the lens during tests. For evaluation of the image processing algorithm, samples from an industrial activated sludge plant were collected weekly for a period of twelve months and imaged without any prior conditioning, replicating real environment conditions. Experiments have shown that the developed algorithm correctly identifies trends of filament growth rate, which is the most important parameter for decision making. For reference images whose filaments were marked by specialists, the algorithm correctly recognized 72% of the filaments pixels, with a false positive rate of at most 14%. An average execution time of 0.7 second per image was achieved, demonstrating the algorithm suitability for real-time monitoring.