Analysis of microbial relations in vivo using Raman Microscopy (AMIRA)
Final Report Abstract
The project “Analysis of microbial relations in vivo using Raman microscopy (AMIRA)” analyzed ammonia oxidizing bacteria (AOB), which are important players in the nitrogen cycle. We examined living AOB in planktonic cultures exposed to ongoing and past oxygen-deprivation, as well as associated in biofilm communities of up to three AOB species up into the maturation phase after four weeks. Cytochrome-c resonant Raman microscopy (CRRM) of individual, living bacteria cells provides a fast, non-invasive method for assessing the stress response and culture vitality of AOB, due to intense spectral changes caused by the oxidation status of the cytochromes-c. The technique allows to differentiate between bacteria cells with ongoing electron transport, thus metabolically active, from cells with increased electron storage, a known reaction to oxygen or nutrient deprivation, and requires only a few hundred cells instead of several milliliters of culture suspension per measurement, thus enabling closely timed monitoring even of low volume or slow growing cultures with very low cell counts that prohibit traditional, more invasive methods. Raman spectroscopy has many wide-spread applications based on the (dis)similarity of the respective Raman spectra, yet none of the distance measures found in literature are designed specifically for Raman spectra. We tested the performance of five of the most common distance measures that can be adapted to be used with Raman spectra on a set of over 50.000 resonant Raman spectra of individual bacterial cells with well-defined signal-to-noise ratio, finding that dependent on the quality of the spectra to be assessed, either the correlation function by Pearson, found to be least sensitive to low quality spectra, or the Euclidean distance measure (Minkowski p=2 metric), which provides the largest cluster separation when faced with high-quality spectra, are the best choices, while closely related distance measures to both – the Euclidean distance and the correlation function by Pearson – performed significantly worse to the point of not being usable. Biological processes for wastewater treatment involve the concerted action of several groups of bacteria; however, the speed of the entire process is limited by the speed of the slowest bacteria that is the ammonium-oxidizing bacteria (AOB). For the first time we measured the kinetic parameters for oxygen consumption, maximum oxygen uptake rate and affinity constant, of two AOB species ubiquitous in wastewater treatment plants, namely Nitrosomonas europeae and Nitrosomonas eutropha. These parameters are required for the mathematical modeling of wastewater treatment plants in order to optimize aeration and reduce costs. Fast and reliable detection ofAOB is crucial in assessing the performance and troubleshooting malfunctions in wastewater treatment plants for nitrogen removal. We developed two new probes for in situ detection of closely related ammonia-oxidizing bacteria, namely Nitrosomonas europeae and Nitrosomonas eutropha, using fluorescence microscopy that find practical application in routine analysis of AOB in biological systems. The new probes can be used to investigate how AOB diversity and 3-D structure of the microbial aggregates are related to resilience of biological systems against perturbation in the environment. AOB are fastidious growing bacteria, which explains the lack of studies investigating biofilm formation by pure cultures necessary to understand microbial interactions of the different species inhabiting the same microbial niche. We cultivated and analyzed mono, dual-, and triple species AOB biofilms for a period of four weeks and observed a synergistic interaction between the two closely related species Nitrosomonas europeae and Nitrosomonas eutropha, despite competing for the same substrate, namely oxygen and ammonium.
Publications
- „Small Sample Stress: Probing Oxygen-Deprived Ammonia-Oxidizing Bacteria with Raman Spectroscopy In Vivo” in Microorganisms 2020, 8, 432
Ann-Kathrin Kniggendorf, Regina Nogueira, Somayeh Nasiri Bahmanabad, Andreas Pommerening-Röser, Bernhard Wilhelm Roth
(See online at https://doi.org/10.3390/microorganisms8030432) - “Assessing Culture Vitality with Raman Spectral Imaging”. European Conference on Biomedical Optics | 2021
A.-K. Kniggendorf, R. Nogueira, B. W. Roth
(See online at https://doi.org/10.1117/12.2615817) - “Signal-to-Noise Sensitivity of Distance Measures in Hierarchical Cluster Analysis for Raman Spectral Imaging”. European Conference on Biomedical Optics | 2021
Ann-Kathrin Kniggendorf, Regina Nogueira, Bernhard Roth
(See online at https://doi.org/10.1117/12.2615730) - „Oxygen Stress Response of Nitrifying Bacteria monitored with Raman Spectroscopy In Vivo” CLEO® Laser Science to Photonics Applications | 2021
Ann-Kathrin Kniggendorf, Regina Nogueira, Bernhard Roth
(See online at https://doi.org/10.1364/CLEO_AT.2021.AM4P.4)
