@phdthesis{Keltsch2025,
  author    = {Keltsch, Nils G.},
  title     = {Novel insights into the bacterial world: Analysis, occurrence and transformation of quorum sensing molecules},
  doi       = {10.82549/opus4-2536},
  url       = {https://nbn-resolving.org/urn:nbn:de:hbz:kob7-25366},
  institution = {Institut f{\"u}r Integrierte Naturwissenschaften, Abt. Chemie},
  school    = {Universit{\"a}t Koblenz, Universit{\"a}tsbibliothek},
  pages     = {XII, 259},
  year      = {2025},
  abstract  = {Bacterial communication via chemical messengers, also called quorum sensing (QS), plays a major role in bioluminescence, pathogenicity and biofilm formation of bacteria. For humans, biofilm formation is undesired in areas as shipping, healthcare and water treatment because it hinders technical processes and there may be health risks for humans. To prevent the formation of unwanted biofilms (so-called biofouling), for example in shipping industry, different antifouling agents such as copper oxide are used. In water treatment, disinfectants such as sodium hypochlorite are used to reduce the occurrence of pathogens as well as build-up biofilms to ensure microbiologically safe water. Common antifouling agents in the marine industry are usually based on their toxicity and their release into the environment. These agents could be dangerous for the ecosystem and sustainable alternatives are needed. For this purpose, nanomaterials were developed which, according to the researcher's hypothesis, influence the QS-system of Gram-negative bacteria and thus prevent biofouling. The hypothesis claims that the nanomaterials mimic the defence strategy of marine organisms with vanadium-bromoperoxidases (V-BrPO) and produce via a catalytic process reactive halogen species such as hypobromous acid. Reactive halogen species also play a major role in disinfection with e.g. sodium hypochlorite. The disinfecting effect is based on oxidation processes with biological material of the microorganism. But, also an influence on the QS system of Gram-negative bacteria was described, which has not yet been investigated in detail. In this thesis, the hypothesized influence of the developed nanomaterials and that of the disinfectant free active chlorine (FAC) on the QS-system of Gram-negative bacteria are investigated. The aim of this thesis was to expand the knowledge about QS molecules (QSM) with regard to degradation processes induced by reactive halogen species and how to analyse QSMs and their transformation products (TP). This allows better assessment at the level of bacterial communication of processes such as antifouling and disinfection. In order to study the communication of Gram-negative bacteria, liquid chromatography - tandem mass spectrometry (LC-ESI-MS/MS) was used to develop a sensitive and selective method for the quantification of QSMs N-acyl-homoserine lactones (AHL), which provide information about QS. Due to the high diversity of AHLs and their low concentrations in the environment a solid-phase extraction was developed and optimized for 34 AHLs. The validated method can measure AHLs in the low ng/L concentration range in several matrices such as surface water, treated wastewater or bacterial supernatants. AHLs were detected in river water (in total 5 AHLs) and treated wastewater (in total 3 AHLs), which shows that the developed method is satisfactory for the detection of environmental concentrations. Furthermore, an unanticipated variety of AHLs was detected in the bacterial supernatants of Pseudomonas aeruginosa (in total 8 AHLs), Phaeobacter gallaeciensis (in total 6 AHLs), and Methylobacterium mesophilicum (in total 15 AHLs). To our knowledge these AHLs have not been described in the literature for these bacterial cultures so far. Quantification of AHLs was performed using a standard addition method and concentrations up to 7.3±1.0 μg/L (3-Oxo-C12-AHL in the bacterial supernatant of P. aeruginosa) were determined. Batch experiments were performed with cerium dioxide nanocrystals (NC) and V-BrPO to investigate the antifouling effects and the proposed biomimetic action of the NCs. Additionally, free active bromine (FAB) was used to elucidate the influence of reactive halogen species on QSMs and the mechanism of both catalysts. The batch experiments were performed for three QSMs and the formation of brominated, hydrolysed and oxidized TPs was identified with high resolution mass spectrometry (HRMS). For N-β-ketocaproyl-homoserine lactone (3-Oxo-C6-AHL), N-cis-tetradec-9Z-enoyl-homoserine lactone (C14:1-AHL) and 2-heptyl-4-quinolone (HHQ) a fast degradation and moiety-specific transformations were observed. In total, 17 TPs were identified at different levels of confidence. In addition, the same TPs were observed in the batch experiments with FAB as in the batch experiments with NC and V-BrPO. This shows on the one hand that the used cerium dioxide NCs led to a biomimetic transformation behaviour for QSMs of Gram-negative bacteria as known for the enzymes V-BrPO. And on the other hand, the results of the FAB experiments indicate that the catalytic processes take place via the formation of reactive halogen species. Furthermore, the transformation pathways for the QS groups unsaturated AHLs and alkyl quinolones have not yet been described in the literature and the knowledge about their degradation could be expanded with this study. Reactive halogen species also play a major role in disinfection processes with FAC. Additionally, FAC is one of the most potent inhibitors of QS compared to other disinfectants. However, a profound knowledge of degradation mechanisms of QSMs is lacking as well as knowledge on a possible impact on QS-controlled processes such as pathogenicity or biofilm formation. Therefore, the degradation behaviour of six representative QSMs of Gram-negative bacteria with FAC was investigated. A complete primary degradation was observed for para-coumaroyl AHL (pC-AHL), C14:1-AHL, HHQ and 3-Oxo-C14-AHL. In our study the primary kinetics and transformation pathways were studied in detail for pC-AHL, C14:1-AHL and HHQ. The reaction order varied between 1.19 (±0.07) (pC-AHL) to 1.62 (±0.13) (HHQ) at pH 7.0, indicating that different reactive species (e.g. hypochlorous acid and dichlorine monoxide) are involved in the transformation. In batch experiments with varying pH, the first-order rate constants show different trends for the investigated QSMs. For C14:1-AHL and HHQ, the first-order rate constants decreased from pH 6.0 to pH 8.5, which was mainly caused by the decreasing concentration of the reactive species hypochlorous acid and dichlorine monoxide in this pH range. In contrast, a maximum of the first order rate constant was observed for pC-AHL at pH 8.5 ranging from pH 6.0 to 10. In addition to the concentrations of reactive species, the phenol/phenolate ratio strongly influenced the first-order rate constants for pC-AHL. In total, 29 TPs (pH = 7.0) were identified by LC-ESI-HRMS and the related transformation pathways were proposed. The impact of FAC on QS can be expanded to the QSM groups unsaturated AHLs, quinolones and coumaroyl AHLs. Furthermore, the observed reaction mechanisms can be transferred to structurally similar QSMs such as 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO) to further understand QS-controlled processes during chlorination. Finally, an interdisciplinary approach with the cooperation partners (microbiology and synthetic chemistry) of the University of Mainz was carried out for a profound understanding of the antifouling effect of cerium dioxide NCs in bacterial cultures. The developed methods and the results of the degradation experiments obtained in the previous studies were used to elucidate the processes also in a more complex biological system. Gram-negative specific inhibition of the biofilm formation was observed for synthesised and characterised cerium dioxide NCs for five different bacterial cultures. Due to the different QSMs used by the bacterial species, an influence on the AHL-system of the Gram-negative bacteria was suspected. In bioassays (Agrobacterium tumefaciens), a reduced QS activity was determined for the experiments with cerium dioxide NCs and measurements of the bromide concentration indicated the consumption of bromide. However, the detected brominated TPs from the previous studies could not be observed with LC-ESI-HRMS and LC-ESI-MS/MS in liquid-liquid extracts of the bacterial supernatant. Further biological degradation processes are here suspected. However, using non-target approaches, the catalytical bromination of HQNO was observed, which can be associated with QS. The bromination was verified by an additional batch experiment with HQNO and cerium dioxide NCs. The repression of the Pseudomonas quinolone signal (PQS) production and biofilm formation in P. aeruginosa through the formed brominated HQNO on surfaces with coating (cerium dioxide NCs) indicates the non-toxic nature of this strategy. The results of this thesis provide a new insight into the occurrence of QSMs and their transformation in relation to halogenation. In addition, the mechanistic investigations on cerium dioxide NCs show the possible relevance of these transformation processes. This new knowledge can be an important basis for the development of new sustainable antifouling agents and possibly for a better understanding and optimisation of disinfection processes.},
  language  = {en}
}