6 results on '"Hippler, Michael"'
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2. On-line analysis of bacterial metabolism by modern spectroscopic laser techniques
- Author
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Metcalfe, George Derek and Hippler, Michael
- Abstract
In order to best determine how bioprocesses develop and how to run bioreactors most efficiently, innovative new analytical techniques are required to supplement, or even supersede, conventional methods, many of which are invasive, require sampling and/or do not provide on-line data analysis. Spectroscopic laser techniques are powerful analytical tools that are capable of real-time, non-invasive monitoring of multiple variables simultaneously. Furthermore, spectroscopy offers high selectivity and sensitivity, including the ability to distinguish different isotopomers and isotopologues, which enables isotopic labelling studies to provide greater mechanistic insights into metabolic pathways. This thesis describes the development of several spectroscopic techniques and their applications in studying different metabolic modes of Escherichia coli batch cultures. On-line analysis is achieved in the gas-phase using cavity-enhanced Raman spectroscopy (CERS), White cell FTIR spectroscopy and photoacoustic detection in a differential Helmholtz resonator (DHR) as well as in the liquid-phase using Raman spectroscopy. The spectral analysis and quantitation of over twenty parameters is discussed, including growth substrates such as glucose and ammonia, metabolites such as acetate, ethanol and formate, headspace gases such as H2, O2 and CO2, and other process variables measured in situ such as the pH and optical density (OD). The first bacterial study conducted is a revisitation of the classical E. coli experiment of glucose-lactose diauxie. A new approach for studying mixed sugar metabolism is presented using both the CERS and DHR techniques to distinguish 13CO2 produced from 13C-glucose metabolism from the subsequent production of 12CO2 from unlabelled lactose. Next, gas-phase FTIR and liquid-phase Raman are developed for batch culture analysis and applied to monitoring mixed-acid fermentation. Finally, two further isotopic labelling studies are conducted by using CERS alongside FTIR and liquid-phase Raman analysis. Nitrate and nitrite reduction by E. coli to the major and minor end-products of ammonium and nitrous oxide is studied, respectively. 15N-labelling is used to give mechanistic insights through interpretation of the different 14N/15N-isotopomer products. The final study focuses on the fermentative pathways of E. coli in the absence and presence of O2. Using 13C- and D-labelled formate, evidence is found that the formate hydrogenlyase (FHL) complex can be assembled and functional under micro-aerobic conditions, which could remove some barriers to biotechnological applications such as biohydrogen generation.
- Published
- 2022
3. Vibrational spectroscopic applications of Fourier transform infrared and Raman spectroscopy in biochemistry and microbiology
- Author
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Alrasheedi, Muneera and Hippler, Michael
- Abstract
Vibrational spectroscopy has a number of applications due to its high sensitivity and selectivity, including the distinction of isotopomers, making it a powerful analytical technique. Unlike typical chromatographic methods that require sampling, vibrational spectroscopic methods give in situ data acquisition in a closed system, with high time resolution. These advantages make vibrational spectroscopy suitable for monitoring rapidly developing biological processes. A number of applications of both infrared and Raman spectroscopies are presented in this thesis, including the monitoring of metabolites produced and consumed by microbiological cultures and volatiles released during fruit ripening. A home-built 2.0 m pathlength multiple-pass absorption White cell was constructed, characterised and employed for long-path Fourier Transform Infrared (FTIR) Spectroscopy. CO2, acetaldehyde and ethanol production were monitored by FTIR spectroscopy during both Escherichia coli growth and banana ripening. Other volatiles, such as ethyl acetate and the ripening hormone ethylene, were also monitored during fruit ripening. Cavity Enhanced Raman Spectroscopy (CERS) was used to monitor the consumption of IR inactive O2 by bacterial cultures and bananas transitioning from aerobic respiration to fermentation. H2 production was also monitored using CERS for anaerobic cultures of E. coli. Alongside FTIR and CERS, TMAO reduction by E. coli cultures was monitored using in situ optical density measurements and liquid phase Raman spectroscopy to monitor TMAO, glucose, acetate, formate and monobasic and dibasic phosphate. Phosphate anion concentration could be used to calculate the pH of the growth medium using a modified Henderson Hasselbalch equation. Deuterated formate (formate-d) could be distinguished from unlabelled formate allowing the monitoring of exogenous formate metabolism and the link to H2 production in the presence of TMAO. FTIR and CERS are proven to be cost-effective, highly specific analytical methods in biochemistry and bioscience, complementing and in some cases superseding existing conventional techniques. They also offer new possibilities for mechanistic insights in biochemistry and display a significant promise for measuring isotopically labelled bioassays.
- Published
- 2021
4. Monitoring trace gases in the biological sciences and petrochemistry by photoacoustic and Raman spectroscopy
- Author
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Alahmari, saeed and Hippler, Michael
- Subjects
540 - Abstract
Photoacoustic spectroscopy (PAS) and Cavity-Enhanced Raman Spectroscopy (CERS) can detect headspace gases above a microbiological culture in a closed system. PAS and CERS have a detection limit of few ppm per volume. These techniques were used to investigate the aerobic respiration of Escherichia coli (E. coli). Both techniques are able to monitor O2 and CO2 and its isotopomers with excellent sensitivity and time resolution to characterise bacterial growth and metabolism. CO2 can be detected using CERS and a differential Helmholtz Resonator (DHR) because it has Raman and IR active vibrations. However, homonuclear diatomic molecules, such as O2, have only symmetric stretching vibrations that are Raman active but not IR active. In PAS, O2 can be detected by exciting a formally forbidden electronic absorption band in the red, the b 1Σg+ (ν = 0) ← X 3Σg- (ν = 0) band (the "A band") near 760 nm. Identification of different growth phases and changes in the aerobic metabolic activity of E. coli was possible by taking simultaneous measurements of O2 consumption and CO2 production using CERS and DHR in PAS, including optical density (OD) measurements. We demonstrate how 13C isotopic labelling of sugars combined with spectroscopic detection allows the study of bacterial mixed sugar metabolism, to establish whether sugars are sequentially or simultaneously metabolised. For E. coli, we have characterised the shift from glucose to lactose metabolism without a classic diauxic lag phase. DHR and CERS are shown to be cost-effective and highly selective analytical tools in the biosciences and in biotechnology, complementing and superseding existing, conventional techniques. They also provide new capabilities for mechanistic investigations in biochemistry and show a great deal of promise for use in stable isotope bioassays. Finally, PAS in a differential Helmholtz resonator has been employed with near-IR detection of CO2 and H2S in natural gas, in static and flow cell measurements. The set-up has also been used for simultaneous in situ monitoring of O2, CO2 and H2S in the cysteine metabolism of microbes (E.coli), and for the analysis of CO2 and H2S impurities in natural gas.
- Published
- 2019
5. New applications of gas phase vibrational spectroscopies in biochemistry and microbiology
- Author
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Smith, Thomas W. and Hippler, Michael F. A.
- Subjects
540 - Abstract
Vibrational spectroscopy has a number of strengths which make it a powerful analytical tool such as high selectivity and sensitivity as well as the ability to distinguish different isotopomers and isotopologues. Unlike conventional gas detection techniques, vibrational spectroscopy can be used to monitor the headspace of a reactor or fermenter online and in situ with no sampling requirements, high time resolution and with little involvement from the user. These strengths make it particularly suited to monitoring rapidly evolving biological processes. This thesis describes a number of new applications of both Infrared and Raman spectroscopies in monitoring the production and consumption of gases by microorganisms and biologically relevant carbon monoxide (CO) releasing molecules (CORMs). A new 8 meter path length multiple pass gas absorption cell is characterised and applied to the simultaneous detection of microbial nitrous oxide (N2O) and carbon dioxide (CO2) by Fourier Transform Infrared (FTIR) Spectroscopy. Distinct phases in both N2O and CO2 formation are observed from anaerobic Escherichia coli treated with either nitrate or nitrite. CO release from two metal-based CORMs has been measured using FTIR spectroscopy in the gas phase. [Mn(CO)3(tpa-?3N)]Br, a promising antimicrobial agent, was found to release CO photolytically even in dense bacterial cell suspensions. [Ru(CO)3Cl(glycinate)] (CORM-3) was found to rapidly loose its ability to release CO via a dithionite / sulfite triggered mechanism in phosphate buffer, defined minimal salts mediumand complex bacterial and mammalian media. The first report of photolytic CO release from CORM-3 is also reported. Cavity Enhanced Raman Spectroscopy (CERS), is introduced in its first reported application in monitoring molecular hydrogen (H2) metabolism in anaerobic cultures of E. coli. Similar yields of H2 are observed from glycerol and glucose. Using D2, hydrogen production and uptake by the bacteria have been monitored simultaneously. Both techniques show great promise as novel bioanalytical tools.
- Published
- 2017
6. Advanced laser based spectroscopic techniques for trace gas detection based on optical cavity enhancement and multipass absorption cells
- Author
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Chu, Johnny Chung Leung and Hippler, Michael
- Subjects
621.381 - Abstract
In this thesis, three advanced experiments based on laser spectroscopy are introduced for the first time which address several experimental topics for trace gas analysis. The implementation of a diode laser based gas phase Raman detector is introduced, capable of parts per million (ppm) detection limits. The spectrometer features a low power laser diode (10 mW) which is enhanced by power build up in an optical cavity. This new technique is characterised by recording spectra of N2, O2, H2, CH4 and benzene. A second advanced laser based spectroscopy technique for trace gas detection, mid infrared cavity enhanced resonant photoacoustic spectroscopy (mid-IR CERPAS) is set up and characterised. This scheme uses optical cavity power build-up, optical feedback stabilisation and resonant photoacoustics. A single-mode continuous wave quantum cascade laser is coupled to a three mirror V-shape optical cavity. Gas phase species absorbing in the mid-IR are detected using the photoacoustic (PA) technique. Mid-IR CERPAS was characterised by measuring acetylene (limit of detection 50 ppt) and nitromethane (0.8 ppb). The mid-IR CERPAS equipment was also used to detect explosives’ vapours; TNT (1.2 and 5.5 ppb), 2, 4-DNT (7 ppb), TATP (4 ppb) and explosives’ taggants such as DMNB (11 ppb). Significant interferences from ambient water in lab air are observed and are overcome. Normalized noise-equivalent absorption coefficients are determined as » 6 x 10-10 cm-1 s1/2 (1 s integration time) and 6 x 10-11 cm-1 s1/2 W (1 s integration time and 1 W laser power). Finally, a near infrared Herriott cell enhanced resonant photoacoustic spectroscopy spectrometer is set up and characterised. This scheme uses enhancement from the absorption pathlength by a multipass Herriott cell and detection of the gas phase species by resonant photoacoustics, Herriott cell enhanced resonant photoacoustics, HERPAS. A single-mode continuous wave near infrared external cavity diode laser is coupled to a Herriott cell. Absorbing gas phase species are detected using the photoacoustic (PA) technique which was characterised by measuring acetylene (150 ppb detection limit at 100 ms integration time). HERPAS was extended to measure several toxic industrial gases including hydrogen sulfide, ammonia and carbon monoxide. Normalized noise-equivalent absorption coefficients are determined for H2S as » 5.3 x 10-9 cm-1 s1/2 (1 s integration time) and 1.6 x 10-10 cm-1 s1/2 W (1 s integration time and 1 W laser power). These three novel advanced spectroscopic techniques allow the detection of IR-inactive and IR- active gas phased species with great sensitivities and selectivity and improve significantly current capabilities for trace gas phase detection.
- Published
- 2015
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