Toward a Noninvasive, Label-Free Screening Method for Determining Spore Inoculum Quality of Penicillium chrysogenum Using Raman Spectroscopy.

We report on a label-free, noninvasive method for determination of spore inoculum quality of Penicillium chrysogenum prior to cultivation/germination. Raman microspectroscopy providing direct, molecule-specific information was used to extract information on the viability state of spores sampled directly from the spore inoculum. Based on the recorded Raman spectra, a supervised classification method was established for classification between living and dead spores and thus determining spore inoculum quality for optimized process control. A fast and simple sample preparation method consisting of one single dilution step was employed to eliminate interfering signals from the matrix and to achieve isolation of single spores on the sample carrier (CaF2). Aiming to avoid any influence of the killing procedure in the Raman spectrum of the spore, spores were considered naturally dead after more than one year of storage time. Fluorescence staining was used as reference method. A partial least squares discriminant analysis classifier was trained with Raman spectra of 258 living and dead spores (178 spectra for calibration, 80 spectra for validation). The classifier showed good performance when being applied to a 1 µL droplet taken from a 1:1 mixture of living and dead spores. Of 135 recorded spectra, 51% were assigned to living spores while 49% were identified as dead spores by the classifier. The results obtained in this work are a fundamental step towards developing an automated, label-free, and noninvasive screening method for assessing spore inoculum quality.

Comparison of Fiber Optic and Conduit Attenuated Total Reflection (ATR) Fourier Transform Infrared (FT-IR) Setup for In-Line Fermentation Monitoring.

The performance of a fiber optic and an optical conduit in-line attenuated total reflection mid-infrared (IR) probe during in situ monitoring of Penicillium chrysogenum fermentation were compared. The fiber optic probe was connected to a sealed, portable, Fourier transform infrared (FT-IR) process spectrometer via a plug-and-play interface. The optical conduit, on the other hand, was connected to a FT-IR process spectrometer via a knuckled probe with mirrors that had to be adjusted prior to each fermentation, which were purged with dry air. Penicillin V (PenV) and its precursor phenoxyacetic acid (POX) concentrations were determined by online high-performance liquid chromatography and the obtained concentrations were used as reference to build partial least squares regression models. Cross-validated root-mean-square errors of prediction were found to be 0.2 g L-1 (POX) and 0.19 g L-1 (PenV) for the fiber optic setup and 0.17 g L-1 (both POX and PenV) for the conduit setup. Higher noise-levels and spectrum-to-spectrum variations of the fiber optic setup lead to higher noise of estimated (i.e., unknown) POX and PenV concentrations than was found for the conduit setup. It seems that trade-off has to be made between ease of handling (fiber optic setup) and measurement accuracy (optical conduit setup) when choosing one of these systems for bioprocess monitoring.

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 f(p)) 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 f(p) 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.

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 C40 carotenoid standards, covering the wide polarity range of natural carotenoids, containing different number of OH-groups. The chromatographic separation was achieved on a C30 RP-HPLC column with a MeOH/MTBE/water gradient. Polar lipids, the geometric isomers of the C50 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.

Multi-analyte quantification in bioprocesses by Fourier-transform-infrared spectroscopy by 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.

Determination of carbohydrates present in Saccharomyces cerevisiae using mid-infrared spectroscopy and partial least squares regression.

A fast and simple method to control variations in carbohydrate composition of Saccharomyces cerevisiae, baker's yeast, during fermentation was developed using mid-infrared (mid-IR) spectroscopy. The method allows for precise and accurate determinations with minimal or no sample preparation and reagent consumption based on mid-IR spectra and partial least squares (PLS) regression. The PLS models were developed employing the results from reference analysis of the yeast cells. The reference analyses quantify the amount of trehalose, glucose, glycogen, and mannan in S. cerevisiae. The selection and optimization of pretreatment steps of samples such as the disruption of the yeast cells and the hydrolysis of mannan and glycogen to obtain monosaccharides were carried out. Trehalose, glucose, and mannose were determined using high-performance liquid chromatography coupled with a refractive index detector and total carbohydrates were measured using the phenol-sulfuric method. Linear concentration range, accuracy, precision, LOD and LOQ were examined to check the reliability of the chromatographic method for each analyte.

Ultrasonic manipulation of yeast cells in suspension for absorption spectroscopy with an immersible mid-infrared fiberoptic probe.

Recent advances in combining ultrasonic particle manipulation with attenuated total reflection infrared spectroscopy of yeast suspensions are presented. Infrared spectroscopy provides highly specific molecular information about the sample. It has not been applicable to in-line monitoring of cells during fermentation, however, because positioning cells in the micron-thin measurement region of the attenuated total reflection probe was not possible. Ultrasonic radiation forces exerted on suspended particles by an ultrasonic standing wave can result in the buildup of agglomerates in the nodal planes, hence enabling the manipulation of suspended cells on the microscopic scale. When a chamber setup and a prototype in-line applicable probe were used, successful control over the position of the yeast cells relative to the attenuated total reflection sensor surface could be proven. Both rate of increase and maximum mid-infrared absorption of yeast-specific bands during application of a pushing frequency (chamber setup: 1.863 MHz, in-line probe: 1.990 MHz) were found to correlate with yeast cell concentration.

Combining light microscopy, dielectric spectroscopy, MALDI intact cell mass spectrometry, FTIR spectromicroscopy and multivariate data mining for morphological and physiological bioprocess characterization of filamentous organisms.

Along with productivity and physiology, morphological growth behavior is the key parameter in bioprocess design for filamentous fungi. Lacking tools for fast, reliable and efficient analysis however, fungal morphology is still commonly tackled by empirical trial-and-error techniques during strain selection and process development procedures. Bridging the gap, this work presents a comprehensive analytical approach for morphological analysis combining automated high-throughput microscopy, multi-frequency dielectric spectroscopy, MALDI intact cell mass spectrometry and FTIR spectromicroscopy. Industrial fed-batch production processes were investigated in fully instrumented, automated bioreactors using the model system Penicillium chrysogenum. Physiological process characterization was based on the determination of specific conversion rates as scale-independent parameters. Conventional light microscopic morphological analysis was based on holistic determination of time series for more than 30 morphological parameters and their frequency distributions over the respective parameter range by automated high-throughput light microscopy. Characteristic protein patterns enriched in specific morphological and physiological states were further obtained by MALDI intact cell mass spectrometry. Spatial resolution of molecular biomass composition was facilitated by FTIR spectromicroscopy. Real-time in situ monitoring of morphological process behavior was achieved by linking multi-frequency dielectric spectroscopy with above outlined off-line methods. Data integration of complementing orthogonal techniques for morphological and physiological analysis together with multivariate modeling of interdependencies between morphology, physiology and process parameters facilitated complete bioprocess characterization. The suggested approach will thus help understanding morphological and physiological behavior and, in turn, allow to control and optimize those complex processes.