Over the last years, varieties of technologies for biofilm analysis were developed and established. They work on different principles and deliver information about biofilms on different information levels. In this work, chip-calorimetry was applied as an analytical tool that measures heat produced from biofilms. Any change of metabolism in biofilms is reflected by a changed heat flow. The heat, which is the integral of the heat flow vs. time, is quantitatively related to the growth stoichiometry of the biofilm, as described by the Hess’ Law. The heat flow is related to the growth kinetics with the reaction heat as proportionality factor. The results from the calorimetric measurement thus, deliver general information about growth stoichiometry and kinetics. The other interpretation of calorimetric results bases on the assumed proportionality between heat flow and oxygen consumption rate (- 460 kJ/mol ). This ratio is called oxycaloric equivalent. Because in case of aerobic growth the majority of oxygen is consumed in catabolic processes during the electron transport phosphorylation, calorimetry is assumed to provide information about the catabolic side of the metabolism. The newly developed chip-calorimeter applied in this work is much more suitable for biofilm studies compared to conventional microcalorimeters due to the flow-through design of the calorimetric chamber. The measurement of undisturbed growing biofilms and the comparison with conventional biofilm analysis tools (i.e. plate counts, confocal laser scanning microscopy (CLSM), and the determination of intermediates’ concentrations (e.g. ATP)) demonstrate the proper functionality of the calorimetric method and the related cultivation procedure by delivering measurement results in the range of literature values. However, when the biofilms were challenged with antimicrobial agents i.e. antibiotics, bacteriophage, and predatory bacteria, the calorimetric results surprisingly deviated from the reference analyses. By combining the results of the calorimetric and reference analyses, additional information about the antimicrobial effects on biofilms can be acquired. Combination of heat measurement and plate counts, which is one of the most conventional approaches, demonstrated that antimicrobials (especially the bactericidal acting kanamycin) could cause the loss of culturability while the cells were still metabolically active. The measurement of ATP content resulted in values out of the typical range, which indicated that antimicrobial treatments disturbed the cellular ATP regulation and the ATP concentration was no longer linearly correlated to the cell number. ATP measurements are therefore not suitable for antimicrobial susceptibility testing. The comparison of heat profiles with the biovolume determined by quantification of microscopic images shows an elevated cell specific heat production rate after the introduction of some antimicrobials (antibiotics and bacteriophage). In case of antibiotics, this can be explained as a consequence of the bacterial defense mechanisms. Most of the described defense mechanisms against antibiotics need biological energy and therefore drive the electron transport phosphorylation (ETP). In case of biofilm treatments with bacteriophage, the trigger of increasing ETP might be the synthesis of phage proteins, hull material, and genetic information molecules. In aerobic conditions, oxygen is used as terminal electron acceptor. Elevated ETP leads therefore to an increase in oxygen consumption, which correlates to the heat production using oxycaloric equivalent as a factor. These correlations explain the increase of cell specific heat productions as biofilms were challenged by antibiotics and bacteriophage. However, also a decrease of specific heat production was observed (in case of predatory bacteria). Here, the predatory bacteria activity caused various damages in host cells, including the interruption of ETP. With these experiments, chip-calorimetry was demonstrated as a promising complementary tool in biofilm research, which provides deeper insights about metabolic activity and alterations. It benefits from the noninvasive handling and the online, real-time measurement that allow the method to be applied for monitoring purposes. Furthermore, its miniaturized dimension allows easy integration in more complex analytic systems and also reduces experiment costs with minimal media/chemical consumption. This thesis also demonstrates the potential development of chip-calorimetry to be more suitable for routine analyses. The use of superparamagnetic beads as matrix to grow biofilms allows regulated transfer of biofilm samples into and from the measurement chamber. This was an initial step towards automation and higher-throughput analysis. One further outcome of the thesis is based on the highly interesting fact about the elevated heat production rate of the host cells induced by the phage infection observed in the chip- calorimetric experiments. The volume specific detection limit of the chip-calorimeter is lower compared to a commercial microcalorimeter. Thus, the infection effect of phages was additionally measured in microcalorimeter to get better quantitative information about the thermal effect of the infection. The results showed that the immediate heat increase after the addition of phage into the solution of the host cells appeared to be quantitatively related to the infection factor, MOI (Multiplicity of Infection). Unfortunately, microcalorimetric measurements in closed ampoules are often subjected to the oxygen limitation. Thus, this problem of microcalorimetric measurement has been addressed. The combination of experimental results and mathematical modeling showed that the rate of metabolism in the static ampoules is defined by the diffusion rate of oxygen into media. This factor has to be considered while designing biological experiments in closed calorimetric measuring chambers and interpreting the calorimetric results for their biological meaning. Some possible solutions to overcome the oxygen bioavailability problem are e.g. to design the experiments with low biomass, or by using media with elevated density to float the biomass at the interface to air and thus to reduce the diffusion path.

Durch den exzessiven Gebrauch von Antibiotika haben sich in den letzten Jahren zunehmend Resistenzen herausgebildet. Eine potentielle Alternative zu konventionellen Antibiotika sind prädatorische Bakterien. Das Bakterium Bdellovibrio bacteriovorus hat einen zweiphasigen Lebenszyklus bestehend aus einer Angriffsphase, in der es andere gram-negative Bakterien jagt, und einer Wachstumsphase, in der es das Zytoplasma eines Wirtes für die eigene Reproduktion nutzt. Für einen künftigen Einsatz von B. bacteriovorus als Antibiotikum müssen die Prozesse des Lebenszyklus verstanden werden. Das Proteom von B. bacteriovorus wurde bisher jedoch nur sehr wenig untersucht. Daher wurden in dieser Arbeit mithilfe der Massenspektrometrie Proteine von verschiedenen Zeitpunkten des Lebenszyklus von B. bacteriovorus relativ quantifiziert. Es konnten zahlreiche Proteine identifiziert werden, die zu spezifischen Zeitpunkten des Lebenszyklus hoch- oder herabreguliert werden. Die größten Unterschiede im Proteinmuster konnten zwischen der Angriffs- und der Wachstumsphase beobachtet werden. In der Angriffsphase sind einige Proteine herabreguliert, die mit der Proteinexpression im Zusammenhang stehen. Weiterhin wurde bestätigt, dass sich junge und gealterte Zellen der Angriffsphase deutlich voneinander unterscheiden, womit die Angriffsphase eigentlich aus zwei Phasen besteht. Auf Grundlage der Ergebnisse und eines Vergleiches mit Transkriptionsdaten wurde die Vermutung aufgestellt, dass B. bacteriovorus Proteine, welche spezifisch für die Angriffsphase sind, bereits während der Wachstumsphase synthetisiert. Im Zusammenhang mit der Forschung an B. bacteriovorus konnten auch neue Impulse bezüglich der MeCAT-basierten massenspektrometrischen Proteinquantifizierung angestoßen werden. In dieser Arbeit wurde unter anderem ein MeCAT-Reagenz mit Acrylamidfunktionalität entwickelt, welches erfolgreich als interner Standard für die Laserablation-ICP-MS von Polyacrylamidgelen verwendet werden kann. / Due to the excessive use of antibiotics, antibiotic resistance has increased over the last years. A potential alternative to conventional antibiotics are predatory bacteria. The predatory bacterium Bdellovibrio bacteriovorus has a biphasic life cycle consisting of an attack phase in which it hunts other gram-negative bacteria, and a growth phase in which it uses the cytoplasm of a prey cell as a substrate for its own reproduction. For future application of B. bacteriovorus as an antibiotic, it is necessary to understand the processes that occur during the life cycle. However, almost no information has been obtained regarding the proteome of B. bacteriovorus yet. Using mass spectrometry and an isotopic labelling strategy, proteins from different time points in the life cycle of B. bacteriovorus were quantified relatively to each other in this work. Numerous proteins were identified that are up- or down-regulated at specific time points in the life cycle. The largest differences in protein pattern existed between the attack phase and the growth phase, whereas only minor differences occurred within the growth phase. For instance, several proteins that appear to be down-regulated during the attack phase are related to protein expression. Furthermore, it was confirmed that there is a significant difference between young and aged cells of the attack phase. Therefore, the attack phase actually consists of two phases. Based on the results and on a comparison with transcription data, it was suggested that attack phase specific proteins of B. bacteriovorus are already synthesized during the growth phase. In connection with the research on B. bacteriovorus, new impulses regarding the MeCAT based protein quantification with mass spectrometry could be initiated. In this work, a MeCAT reagent with acrylamide functionality was developed, which can be used successfully as an internal standard for laser ablation ICP-MS of polyacrylamide gels.

Over the last years, varieties of technologies for biofilm analysis were developed and established. They work on different principles and deliver information about biofilms on different information levels. In this work, chip-calorimetry was applied as an analytical tool that measures heat produced from biofilms. Any change of metabolism in biofilms is reflected by a changed heat flow. The heat, which is the integral of the heat flow vs. time, is quantitatively related to the growth stoichiometry of the biofilm, as described by the Hess’ Law. The heat flow is related to the growth kinetics with the reaction heat as proportionality factor. The results from the calorimetric measurement thus, deliver general information about growth stoichiometry and kinetics. The other interpretation of calorimetric results bases on the assumed proportionality between heat flow and oxygen consumption rate (- 460 kJ/mol ). This ratio is called oxycaloric equivalent. Because in case of aerobic growth the majority of oxygen is consumed in catabolic processes during the electron transport phosphorylation, calorimetry is assumed to provide information about the catabolic side of the metabolism. The newly developed chip-calorimeter applied in this work is much more suitable for biofilm studies compared to conventional microcalorimeters due to the flow-through design of the calorimetric chamber. The measurement of undisturbed growing biofilms and the comparison with conventional biofilm analysis tools (i.e. plate counts, confocal laser scanning microscopy (CLSM), and the determination of intermediates’ concentrations (e.g. ATP)) demonstrate the proper functionality of the calorimetric method and the related cultivation procedure by delivering measurement results in the range of literature values. However, when the biofilms were challenged with antimicrobial agents i.e. antibiotics, bacteriophage, and predatory bacteria, the calorimetric results surprisingly deviated from the reference analyses. By combining the results of the calorimetric and reference analyses, additional information about the antimicrobial effects on biofilms can be acquired. Combination of heat measurement and plate counts, which is one of the most conventional approaches, demonstrated that antimicrobials (especially the bactericidal acting kanamycin) could cause the loss of culturability while the cells were still metabolically active. The measurement of ATP content resulted in values out of the typical range, which indicated that antimicrobial treatments disturbed the cellular ATP regulation and the ATP concentration was no longer linearly correlated to the cell number. ATP measurements are therefore not suitable for antimicrobial susceptibility testing. The comparison of heat profiles with the biovolume determined by quantification of microscopic images shows an elevated cell specific heat production rate after the introduction of some antimicrobials (antibiotics and bacteriophage). In case of antibiotics, this can be explained as a consequence of the bacterial defense mechanisms. Most of the described defense mechanisms against antibiotics need biological energy and therefore drive the electron transport phosphorylation (ETP). In case of biofilm treatments with bacteriophage, the trigger of increasing ETP might be the synthesis of phage proteins, hull material, and genetic information molecules. In aerobic conditions, oxygen is used as terminal electron acceptor. Elevated ETP leads therefore to an increase in oxygen consumption, which correlates to the heat production using oxycaloric equivalent as a factor. These correlations explain the increase of cell specific heat productions as biofilms were challenged by antibiotics and bacteriophage. However, also a decrease of specific heat production was observed (in case of predatory bacteria). Here, the predatory bacteria activity caused various damages in host cells, including the interruption of ETP. With these experiments, chip-calorimetry was demonstrated as a promising complementary tool in biofilm research, which provides deeper insights about metabolic activity and alterations. It benefits from the noninvasive handling and the online, real-time measurement that allow the method to be applied for monitoring purposes. Furthermore, its miniaturized dimension allows easy integration in more complex analytic systems and also reduces experiment costs with minimal media/chemical consumption. This thesis also demonstrates the potential development of chip-calorimetry to be more suitable for routine analyses. The use of superparamagnetic beads as matrix to grow biofilms allows regulated transfer of biofilm samples into and from the measurement chamber. This was an initial step towards automation and higher-throughput analysis. One further outcome of the thesis is based on the highly interesting fact about the elevated heat production rate of the host cells induced by the phage infection observed in the chip- calorimetric experiments. The volume specific detection limit of the chip-calorimeter is lower compared to a commercial microcalorimeter. Thus, the infection effect of phages was additionally measured in microcalorimeter to get better quantitative information about the thermal effect of the infection. The results showed that the immediate heat increase after the addition of phage into the solution of the host cells appeared to be quantitatively related to the infection factor, MOI (Multiplicity of Infection). Unfortunately, microcalorimetric measurements in closed ampoules are often subjected to the oxygen limitation. Thus, this problem of microcalorimetric measurement has been addressed. The combination of experimental results and mathematical modeling showed that the rate of metabolism in the static ampoules is defined by the diffusion rate of oxygen into media. This factor has to be considered while designing biological experiments in closed calorimetric measuring chambers and interpreting the calorimetric results for their biological meaning. Some possible solutions to overcome the oxygen bioavailability problem are e.g. to design the experiments with low biomass, or by using media with elevated density to float the biomass at the interface to air and thus to reduce the diffusion path.