Identification of lipophilic bioproduct portfolio from bioreactor samples of extreme halophilic archaea with HPLC-MS/MS

Extreme halophilic archaea are a yet unexploited source of natural carotenoids. At elevated salinities, however, material corrosivity issues occur and the performance of analytical methods is strongly affected. The goal of this study was to develop a method for identification and downstream processing of potentially valuable bioproducts produced by archaea. To circumvent extreme salinities during analysis, a direct sample preparation method was established to selectively extract both the polar and the nonpolar lipid contents of extreme halophiles with hexane, acetone and the mixture of MeOH/MTBE/water, respectively. Halogenated solvents, as used in conventional extraction methods, were omitted because of environmental considerations and potential process scale-up. The HPLC-MS/MS method using atmospheric pressure chemical ionization was developed and tuned with three commercially available C-40 carotenoid standards, covering the wide polarity range of natural carotenoids, containing different number of OH-groups. The chromatographic separation was achieved on a C-30 RP-HPLC column with a MeOH/MTBE/water gradient. Polar lipids, the geometric isomers of the C-50 carotenoid bacterioruberin, and vitamin MK-8 were the most valuable products found in bioreactor samples. In contrast to literature on shake flask cultivations, no anhydrous analogues of bacterioruberin, as by-products of the carotenoid biosynthesis, were detected in bioreactor samples. This study demonstrates the importance of sample preparation and the applicability of HPLC-MS/MS methods on real samples from extreme halophilic strains. Furthermore, from a biotechnological point-of-view, this study would like to reveal the relevance of using controlled and defined bioreactor cultivations instead of shake flask cultures in the early stage of potential bioproduct profiling

A reference dataset for verifying numerical electrophysiological heart models

<p>Abstract</p> <p>Background</p> <p>The evaluation, verification and comparison of different numerical heart models are difficult without a commonly available database that could be utilized as a reference. Our aim was to compile an exemplary dataset.</p> <p>Methods</p> <p>The following methods were employed: Magnetic Resonance Imaging (MRI) of heart and torso, Body Surface Potential Maps (BSPM) and MagnetoCardioGraphy (MCG) maps. The latter were recorded simultaneously from the same individuals a few hours after the MRI sessions.</p> <p>Results</p> <p>A training dataset is made publicly available; datasets for blind testing will remain undisclosed.</p> <p>Conclusions</p> <p>While the MRI data may provide a common input that can be applied to different numerical heart models, the verification and comparison of different models can be performed by comparing the measured biosignals with forward calculated signals from the models.</p

Wide-field CO isotopologue emission and the CO-to-H2_2 factor across the nearby spiral galaxy M101

Carbon monoxide (CO) emission is the most widely used tracer of the bulk molecular gas in the interstellar medium (ISM) in extragalactic studies. The CO-to-H$_2$ conversion factor, $\alpha_{\rm CO}$, links the observed CO emission to the total molecular gas mass. However, no single prescription perfectly describes the variation of $\alpha_{\rm CO}$ across all environments across galaxies as a function of metallicity, molecular gas opacity, line excitation, and other factors. Using resolved spectral line observations of CO and its isotopologues, we can constrain the molecular gas conditions and link them to a variation in the conversion factor. We present new IRAM 30-m 1mm and 3mm line observations of $^{12}$CO, $^{13}$CO, and C$^{18}$O} across the nearby galaxy M101. Based on the CO isotopologue line ratios, we find that selective nucleosynthesis and opacity changes are the main drivers of the variation in the line emission across the galaxy. Furthermore, we estimated $\alpha_{\rm CO(1-0)}$ using different approaches, including (i) the dust mass surface density derived from far-IR emission as an independent tracer of the total gas surface density and (ii) LTE-based measurements using the optically thin $^{13}$CO(1-0) intensity. We find an average value of $\alpha_{\rm CO}=4.4{\pm}0.9\rm\,M_\odot\,pc^{-2}(K\,km\,s^{-1})^{-1}$ across the galaxy, with a decrease by a factor of 10 toward the 2 kpc central region. In contrast, we find LTE-based values are lower by a factor of 2-3 across the disk relative to the dust-based result. Accounting for $\alpha_{\rm CO}$ variations, we found significantly reduced molecular gas depletion time by a factor 10 in the galaxy's center. In conclusion, our result suggests implications for commonly derived scaling relations, such as an underestimation of the slope of the Kennicutt Schmidt law, if $\alpha_{\rm CO}$ variations are not accounted for.Comment: Accepted for publication in A&A, 25 pages, 15 figure

Kinematic analysis of the super-extended HI disk of the nearby spiral galaxy M 83

Funding: CE, FB, AB, IB, JdB and JP acknowledge funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No.726384/Empire). TGW acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 694343). JMDK gratefully acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme via the ERC Starting Grant MUSTANG (grant agreement number 714907). SCOG acknowledges funding from the European Research Council via the ERC Synergy Grant “ECOGAL – Understanding our Galactic ecosystem: From the disk of the Milky Way to the formation sites of stars and planets” (project ID 855130). WJGdB received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 882793 ‘MeerGas’).We present new HI observations of the nearby massive spiral galaxy M83, taken with the VLA at 21″ angular resolution (≈500 pc) of an extended (1.5 deg2) 10-point mosaic combined with GBT single dish data. We study the super-extended HI disk of M83 (∼50 kpc in radius), in particular disc kinematics, rotation and the turbulent nature of the atomic interstellar medium. We define distinct regions in the outer disk (rgal > central optical disk), including ring, southern area, and southern and northern arm. We examine HI gas surface density, velocity dispersion and non-circular motions in the outskirts, which we compare to the inner optical disk. We find an increase of velocity dispersion (σv) towards the pronounced HI ring, indicative of more turbulent HI gas. Additionally, we report over a large galactocentric radius range (until rgal ∼ 50 kpc) that σv is slightly larger than thermal (i.e. > 8 km s-1). We find that a higher star formation rate (as traced by FUV emission) is not always necessarily associated with a higher HI velocity dispersion, suggesting that radial transport could be a dominant driver for the enhanced velocity dispersion. We further find a possible branch that connects the extended HI disk to the dwarf irregular galaxy UGCA365, that deviates from the general direction of the northern arm. Lastly, we compare mass flow rate profiles (based on 2D and 3D tilted ring models) and find evidence for outflowing gas at rgal ∼ 2 kpc, inflowing gas at rgal ~ 5.5 kpc and outflowing gas at rgal ~ 14 kpc. We caution that mass flow rates are highly sensitive to the assumed kinematic disk parameters, in particular, to the inclination.Publisher PDFPeer reviewe

Bio-suspensions in the ultrasonic h-shape filter

Zsfassung in dt. SpracheDer h-Separator, ein spezieller Ultraschallfilter, wurde für die Separation von Saccharomyces cerevisiae getestet. Die Separationseffizienz des Filters, also die Fähigkeit suspendierte Hefezellen zurückzuhalten, wurde gemessen. Der mögliche Einfluss des Separationsprozesses auf die Hefezellen wurde durch die Messung der Viabilität der Zellen mit Methylen Blau sowie der Proteinkonzentration des Überstandes (Messung der optische Dichte für UV-Licht) zur Detektion möglicher Zerstörung von Zellenmembranen und Messung des Zellwachstums vor und nach der Separation untersucht. Der Filter wurde mit 3W true electrical power input bei einer Frequenz von 2.1MHz betrieben. Für Suspensionen von gezüchteten Hefezellen in Malt Extract Broth konnte eine hohe Separationseffizienz von 89+/-6% gefunden werden. Für Presshefe, die in phosphatgepufferter Salzlösung (PBS, 0.9% NaCl), PBS 2x (1.8% NaCl) und Leitungswasser suspendiert war, konnten nicht so hohe Separationseffizienzen erreicht werden. Die Viabilität der Zellen wurde in keiner der Suspensionen durch Separation beeinträchtigt. In zwei Fällen kam es jedoch bei der Separation von gezüchteter Hefe in Malt Extract Broth zu Turbulenzen, die zu einer signifikanten Verminderung der Viabilität der beschallten Zellen führten. Die gemittelte Zellkonzentration der Retentat-Proben von gezüchteten Hefezellen/Malt Extract Broth-Suspensionen war 18 Stunden nach der Beschallung um 20% höher als gleich nach der Beschallung (99% Signifikanz, 2-seitiger t-test). Bei Kontrollgruppen und den Filtrat-Proben konnte keine signifikante Steigerung festgestellt werden.Für Hefezellen die in PBS suspendiert waren, wurde eine signifikante Steigerung der optischen Dichte für UV-Licht gemessen welche auf ein Auslaufen der Zellen zurückzuführen ist. Für die Suspensionen in PBS 2x und Leitungswasser konnte kein signifikanter Unterschied zwischen der optischen Dichte für UV-Licht der Kontrollgruppen und der beschallten Zellen gefunden werden.The ultrasonic h-shape filter was used for the separation of suspensions of the yeast Saccharomyces cerevisiae. The separation efficiency of the filter, i.e. the ability to retain suspended yeast cells, was assessed. To examine the influence of the separation process on the cells, cell viability by methylene blue counts, possible cell rupture assessed by the protein concentration of the supernatant and growth were investigated before and after sonication. The filter was operated at a frequency of 2.1MHz at a power input of 3W true electrical power input. The filter was shown to work well for cultured yeast suspended in Malt Extract Broth, for which a separation efficiency of 89+/-6% was found. Wet yeast suspended in phosphate buffered saline (PBS, 0.9% NaCl), PBS 2x (1.8% NaCl) and H2O tap was not retained as efficiently. For all the suspensions cell viability was not affected by sonication, remaining at high levels. However, in two cases when cultured cells suspended in Malt Extract Broth were driven through the resonator, a significant decrease of cell viability of the sonicated cells was measured. Cell concentration of retentate samples of cultured yeast/Malt Extract Broth-suspensions was 20% higher for counts 18 hours after sonication than that of counts immediately after sonication (99% significance, 2-sided t-test). For control groups and the filtrate samples no such increase was detected. Cell leakage caused by the separation process was detected by a significant increase in UV O.D.for cells suspended in PBS. For PBS 2x- and H2O tap-suspensions no significant difference in UV O.D. between control and sonicated groups, respectively, was found.4

Advances in bioprocess monitoring by mid-infrared spectroscopy

Zusammenfassung in deutscher SpracheIm Rahmen dieser Dissertation wurde ein neues Messverfahren zur selektiven Aufnahme von Spektren von biologischen Zellen in Suspension im mittleren Infrarot (MIR) entwickelt. MIR Messungen mittels abgeschwächter Totalreflexion (ATR) wurden dazu mit Ultraschall-Partikel-Manipulation kombiniert. Ein Ultraschall-Aufsatz wurde entworfen, ein Prototyp gefertigt und mit einer speziell angefertigten ATR Sonde kombiniert. Mit dieser ultraschallgestützten ATR Sonde konnten Spektren von Saccharomyces cerevisiae in Suspension erfolgreich off-line aufgenommen werden. Mit den gewonnen Erkenntnissen wurde ein weiterer Prototyp entworfen und gefertigt, der zum Einsatz in situ in einem Bioreaktor geeignet war. Dieser wurde mit einem kommerziellen Prozessspektrometer kombiniert welches mit einer faser-optischen in-line ATR Sonde ausgestattet war. Mit diesem Aufbau konnten, nach bestem Wissen der Autorin, zum ersten Mal Spektren von Zellen in einem gerührten, begasten Bioreaktor aufgenommen werden. Die Änderung der Polysaccharidzusammensetzung von S. cerevisiae konnte off-line quantifiziert werden. Durch eine gezielt herbeigeführte Stickstoff-Limitation akkummulierten die Zellen Trehalose und Glykogen. MIR Spektren von gewaschenen, getrockneten Zell-Proben wurden mit einem Infrarotmikroskop aufgenommen, diese konnten mittels Partial Least Squares Regression (PLS-R) auf Referenzwerte aus nasschemischen Verfahren und High Pressure Liquid Chromatography kalibriert werden (root-mean-square Kreuzvalidierungsfehler: 0.33 % Trockengewicht (%DW) für Trehalose, 0.55 %DW für Glykogen und 1.17 %DW für Mannan). Mit der neu-entwickelten ultraschallgestützten faser-optischen ATR Sonde und Hauptkomponentenanalyse konnten ähnliche Änderungen in den Spektren der Zellen in der Fermentationsbrühe in-line verfolgt werden. Durch die simultane Änderung der Biomassekonzentration und des Polysaccharidgehalts der Zellen war eine quantitative Bestimmung der Analyten aus in-line Spektren nicht möglich. Darüber hinaus wurde die Eignung von Stand-der-Technik-MIR-Instrumentierung für die in-line Quantifizierung von im Medium gelösten Stoffen untersucht. Am Beispiel von Penicillium chrysogenum Fermentationen wurden PLS-R und multivariate curve resolution - alternating least squares (MCR-ALS) für die quantitative Bestimmung von Phenoxyessigsäure und Penicillin V verwendet. Die beiden Methoden ergaben vergleichbare Kalibrationsmodelle mit Detektionsschwellen im niedrigen g L-1 Bereich. Für MCR-ALS Modelle benötigt man mehr Wissen über den Prozess als für PLS-R, die Selektivität der MCR-ALS Modelle kann jedoch durch den Vergleich der errechneten 'pure component' Spektren mit gemessen Referenzspektren leichter überprüft werden. Im gleichen biologischen System wurden eine faser-optische und eine Sonde mit einem optischen Conduit simultan verwendet um Spektren in-line aufzunehmen. Die PLS-R Modelle für die beiden oben erwähnten Analyten waren von vergleichbarer Qualität, die Conduit-Sonde wies jedoch eine höhere Stabilität (Spektrum-zu-Spektrum-Änderungen) und ein besseres Signal-Rausch-Verhältnis auf.In this thesis two techniques, attenuated total reflection (ATR) mid-infrared (mid-IR) spectroscopy and ultrasonic particle manipulation, were successfully combined for the acquisition of mid-IR spectra of cells in suspension. A prototype ultrasound accessory was designed, realized and combined with a custom fiber optic ATR probe. The working principle was successfully tested off-line with suspensions of Saccharomyces cerevisiae. A second prototype fit for use in a semi-industrial bioprocess environment was designed and built. It comprised the optimized ultrasound accessory and a commercial process spectrometer equipped with an in-line fiber optic ATR probe. Using this instrumental development, to the best of the author-s knowledge, mid-IR spectra of cells inside a stirred, aerated bioreactor could be successfully recorded for the first time. Changes of the carbohydrate content of S. cerevisiae undergoing nitrogen-limited growth, i.e. the accumulation of trehalose and glycogen, could be followed quantitatively off-line. Mid-IR spectra of washed, dried cells were recorded and analyzed by partial least squares regression (PLS-R) with reference values obtained by wet chemistry and liquid chromatography (root-mean-square error of cross validation: 0.33 % dry weight (%DW) for trehalose, 0.55 %DW for glycogen, and 1.17 %DW for mannan). Similar spectral changes could also be observed in-line using the novel ultrasound enhanced fiber optic ATR probe and principal components analysis of spectra of cells present in the fermentation broth. Quantification of the analytes proved to be difficult because changes in biomass and in the carbohydrate content occurred simultaneously, and this correlation could not be broken. Complimentary, the applicability of state-of-the-art mid-IR technology for quantitative analysis of solutes in-line was explored. On the example of Penicillium chrysogenum fermentations, PLS-R and multivariate curve resolution-alternating least squares (MCR-ALS) were applied for the simultaneous determination of penicillin V and phenoxyacetic acid. The obtained results show that models are of comparable quality with LODs in the low gL-1 range. MCR-ALS requires more process knowledge than PLS-R, but provides an objective assessment for model validity by comparison of the calculated pure component with the respective reference spectra. Using the same biological system, a fiber optic and a conduit ATR probe were simultaneously applied in-line. No significant difference in performance for quantification of the two analytes by PLS-R could be found. However, higher spectrum-to-spectrum stability and better signal-to-noise were found for the optical conduit set-up.17

Emulsion templating: Unexpected morphology of monodisperse macroporous polymers

Hypothesis: We synthesised monodisperse macroporous polymers via polymerisation of water-in-monomer droplet emulsions and obtained non-spherical pores with layered pore walls. We hypothesise that this morphology is caused by surfactant diffusion and phase separation during polymerisation.Experiments: We varied the surfactant mass fraction of the emulsions and polymerised the templates with a water-soluble initiator. From the resulting macroporous polymers we determined the shape of the pores and thickness of the layers via scanning election microscopy. The response of the monomer/surfactant mixture to polymerisation was studied by a ternary phase diagram that simulated polymerisation. Findings:The emergence of non-spherical pores with layered pore walls is indeed caused by surfactant diffusion and phase separation. During polymerisation the surfactant molecules diffuse either to the water/monomer interface or deeper into the continuous monomer phase. The first process results in non-spherical pores, while the second process generates layered pore walls

Is the ‘bat population’ an operational concept for statutory species conservation?

In the European Union (EU), all bat species are legally protected by the Habitats Directive (HD). Especially the goal of keeping or restoring a favourable conservation status for protected species and habitats is an outstanding innovation in the law of nature conservation. The Directive does not only focus on rare species close to extinction, but has the goal of viable populations for all species included in the pertinent annexes. However, most bat populations are still far away from reaching this goal, which leads to the question why the Habitats Directive may have failed to achieve its objective.We argue that the pluralism of definitions of the concept of ‘population’ is a major problem in the application of the Directive. Concerning bats, several population definitions are possible that fit biological aims as well as aims of legal protection. But without clear assignment which definition should be used in the application of nature conservation law, the pluralism of the population definition offers the possibility of arbitrary use, and with it the failure of bat conservation. In consequence, we describe different population definitions and give suggestions to avoid arbitrary delimitation. When applying species protection law, the population must be specified through ecological parameters rather than merely through statistical approaches. In case of migrating bats, we found that the objectives of the HD can only be achieved with individual-based approaches. Population-based protection is doomed to failure because of the lack of knowledge about meaningful population delimitation. Thus, strong EU-wide effort is necessary to completely implement legal bat protection

Multi-analyte quantification in bioprocesses by FTIR spectroscopy using Partial Least Squares Regression and Multivariate Curve Resolution

This paper presents the quantification of Penicillin V and phenoxyacetic acid, a precursor, inline during Pencillium chrysogenum fermentations by FTIR spectroscopy and partial least squares (PLS) regression and multivariate curve resolution – alternating least squares (MCR-ALS). First, the applicability of an attenuated total reflection FTIR fiber optic probe was assessed offline by measuring standards of the analytes of interest and investigating matrix effects of the fermentation broth. Then measurements were performed inline during four fed-batch fermentations with online HPLC for the determination of Penicillin V and phenoxyacetic acid as reference analysis. PLS and MCR-ALS models were built using these data and validated by comparison of single analyte spectra with the selectivity ratio of the PLS models and the extracted spectral traces of the MCR-ALS models, respectively. The achieved root mean square errors of cross-validation for the PLS regressions were 0.22 g L−1 for Penicillin V and 0.32 g L−1 for phenoxyacetic acid and the root mean square errors of prediction for MCR-ALS were 0.23 g L−1 for Penicillin V and 0.15 g L−1 for phenoxyacetic acid. A general work-flow for building and assessing chemometric regression models for the quantification of multiple analytes in bioprocesses by FTIR spectroscopy is given.Fil: Koch, Cosima. Vienna University of Technology. Institute of Chemical Technologies and Analytics; AustriaFil: Posch, Andreas E.. Vienna University of Technology. Institute of Chemical Engineering. Research Area Biochemical Engineering. Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses; AustriaFil: Goicoechea, Hector Casimiro. Universidad Nacional del Litoral. Laboratorio de Desarrollo Analítico y Quimiometría; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe; ArgentinaFil: Herwig, Christoph. Vienna University of Technology. Institute of Chemical Engineering. Research Area Biochemical Engineering. Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses; AustriaFil: Lendl, Bernhard. Vienna University of Technology. Institute of Chemical Technologies and Analytics; Austri

Ultrasound-enhanced attenuated total reflection mid-infrared spectroscopy in-line probe: Acquisition of cell spectra in a bioreactor

This article presents a novel method for selective acquisition of Fourier transform infrared (FT-IR) spectra of microorganisms in-line during fermentation, using Saccharomyces cerevisiae as an example. The position of the cells relative to the sensitive region of the attenuated total reflection (ATR) FT-IR probe was controlled by combing a commercially available ATR in-line probe with contact-free, gentle particle manipulation by ultrasonic standing waves. A prototype probe was successfully constructed, assembled, and tested in-line during fed-batch fermentations of S. cerevisiae. Control over the position of the cells was achieved by tuning the ultrasound frequency: 2.41 MHz was used for acquisition of spectra of the cells (pushing frequency fp) and 1.87 MHz, for retracting the cells from the ATR element, therefore allowing spectra of the medium to be acquired. Accumulation of storage carbohydrates (trehalose and glycogen) inside the cells was induced by a lack of a nitrogen source in the feed medium. These changes in biochemical composition were visible in the spectra of the cells recorded in-line during the application of fp and could be verified by reference spectra of dried cell samples recorded off-line with a FT-IR microscope. Comparison of the cell spectra with spectra of trehalose, glycogen, glucose, and mannan, i.e., the major carbohydrates present in S. cerevisiae, and principal components analysis revealed that the changes observed in the cell spectra correlated well with the bands specific for trehalose and glycogen. This proves the applicability and capability of ultrasound-enhanced in-line ATR mid-IR spectroscopy as a real-time PAT method for the in situ monitoring of cellular biochemistry during fermentation