Bernhard Lendl

On-Line Fermentation Monitoring by Mid-infrared Spectroscopy

A new method for on-line monitoring of fermentations using mid-infrared (MIR) spectroscopy has been developed. The method has been used to predict the concentrations of glucose and ethanol during a bakers yeast fermentations. A completely automated flow system was employed as an interface between the bioprocess under study and the Fourier transform infrared (FT-IR) spectrometer, which was equipped with a flow cell housing a diamond attenuated total reflection (ATR) element. By using the automated flow system, experimental problems related to adherence of CO2 bubbles to the ATR surface, as well as formation of biofilms on the ATR surface, could be efficiently eliminated. Gas bubbles were removed during sampling, and by using rinsing steps any biofilm could be removed from the ATR surface. In this way, constant measuring conditions could be guaranteed throughout prolonged fermentation times (∼8 h). As a reference method, high-performance liquid chromatography (HPLC) with refractive index detection was used. The recorded data from different fermentations were modeled by partial least-squares (PLS) regression comparing two different strategies for the calibration. On the one hand, calibration sets were constructed from spectra recorded from either synthetic standards or from samples drawn during fermentation. On the other hand, spectra from fermentation samples and synthetic standards were combined to form a calibration set. Differences in the kinetics of the studied fermentation processes used for calibration and prediction, as well as the precision of the HPLC reference method, were identified as the main chemometric sources of error. The optimal PLS regression method was obtained using the mixed calibration set of samples from fermentations and synthetic standards. The root mean square errors of prediction in this case were 0.267 and 0.336 g/L for glucose and ethanol concentration, respectively.

Direct, reagent-free determination of free fatty acid content in olive oil and olives by Fourier transform Raman spectrometry

Abstract Fourier transform (FT) Raman spectrometry in combination with partial least squares (PLS) regression was used for direct, reagent-free determination of free fatty acid (FFA) content in olive oils and olives. Oils were directly investigated in a simple flow cell. Milled olives were measured in a dedicated sample cup, which was rotated eccentrically to the horizontal laser beam during spectrum acquisition in order to compensate sample heterogeneity. Both external and internal (leave-one-out) validation were used to assess the predictive ability of the PLS calibration models for FFA content (in terms of oleic acid) in oil and olives in the range 0.20–6.14 and 0.15–3.79%, respectively. The root mean square error of prediction (RMSEP) was 0.29% for oil and 0.28% for olives. The predicted FFA contents were used to classify oils and olives in different categories according to the European Union regulations. Ninety percent of the oil samples and 80% of the olives were correctly classified. These results demonstrate that the proposed procedures can be used for screening of good quality olives before processing, as well as, for the on-line control of the produced oil.

Application of Mid-Infrared Transmission Spectrometry to the Direct Determination of Glucose in Whole Blood

The capability of FT-IR transmission spectrometry was examined for the direct determination of glucose in whole blood without any sample preparation. For these investigations, the whole blood samples were automatically aspirated by a syringe pump into the transmission cell. Infrared spectra were recorded in the 1500–900 cm−1 range. Despite the high water background absorption and the complex blood matrix, significant spectral changes due to different glucose concentrations were observed. Chemometric (partial least-squares) models were applied for the determination of glucose. A standard error of calibration of 13.8 mg/dL was obtained by using a partial least-squares calibration model containing five ranks. The residues for an independent test set were less than 15 mg/dL.

High-performance liquid chromatography with real-time Fourier-transform infrared detection for the determination of carbohydrates, alcohols and organic acids in wines

Abstract The coupling of high-performance liquid chromatography (HPLC) with Fourier-transform infrared spectroscopy (FTIR) is presented as a new and versatile tool for the direct determination of the main components of wine, which are glucose, fructose, glycerol, ethanol, acetic, citric, lactic, malic, succinic and tartaric acid. An ion-exchange resin based column (counterion: H+) was employed as the stationary phase and 0.005 M sulfuric acid as the mobile phase. FTIR detection in the spectral region from 1600 to 900 cm−1 was performed in a 25-μm flow cell without elimination of the solvent. Characteristic FTIR spectra were obtained subsequent to the injection and separation of 2 mg/ml of each component. The average standard deviation of the investigated compounds was found to be 66 μg/ml in standard solutions containing 1–10 mg/ml of the analyte. The method was furthermore applied to real wine samples taken from a round robin test. An average deviation of 0.16 mg/ml from the external reference data was obtained.

Design, simulation and application of a new micromixing device for time resolved infrared spectroscopy of chemical reactions in solution

We present a novel micromachined fast diffusion based mixing unit for the study of rapid chemical reactions in solution with stopped-flow time resolved Fourier transform infrared spectroscopy (TR-FTIR). The presented approach is based on a chip for achieving lamination of two liquid sheets of 10 microm thickness and approximately 1 mm width on top of each other and operation in the stopped-flow mode. The microstructure is made on infrared transmitting calcium fluoride discs and built up with two epoxy negative photoresist layers and one silver layer in between. Due to the highly laminar flow conditions and the short residence time in the mixer there is hardly any mixing when the two liquid streamlines pass through the mixing unit, which allows one to record a mid-IR transmission spectrum of the analytes prior to reaction. When the flow is stopped, the reactant streams are arrested in the flow-cell and rapidly mixed by diffusion due to the reduced interstream distances and the reaction can be directly followed with hardly any dead time. On the basis of two model reactions-neutralisation of acetic acid with sodium hydroxide as well as saponification of methyl monochloroacetate-the performance of the mixing device was tested revealing proper functioning of the device with a time for complete mixing of less than 100 ms. The experimental results were supported by numerical simulations using computational fluid dynamics (CFD), which allowed a reliable, quantitative analysis of concentration, pressure and flow profiles in the course of the mixing process.

Mid-infrared spectroscopy coupled to sequential injection analysis for the on-line monitoring of the acetone–butanol fermentation process

Fourier transform infrared (FTIR) spectroscopy, coupled to sequential injection analysis (SIA) was employed for the on-line monitoring of an acetone–butanol fermentation by simultaneously determining acetone, acetate, n-butanol, butyrate and glucose from the mid-IR spectra of the samples. The analysis system (SIA-FTIR) developed was completely computer controlled, requiring only minute amounts of NaOH and Na2CO3 as reagents and was capable of performing 30 analyte determinations per hour. From a batch fermentation run of 55 h duration, samples were automatically drawn and analysed at 14 different points of time. The analysis by SIA-FTIR comprised of triplicate injections, from each of which two spectra one with 4 cm −1 the other with 8 cm −1 spectral resolution were obtained. In addition, triplicate reference analysis by gas chromatography and enzymatic test kits were also done at corresponding points of time. Based on the data of the reference analysis a partial least squares (PLS) model was established and validated by a leave-one-out cross validation. Best results were obtained using the second derivatised spectra collected at 8 cm −1 resolution. The regression coefficients and standard errors of prediction (S.E.P.) were as follows: acetone, r = 0.999, S.E.P. = 0.077 g/l; acetate, r = 0.998, S.E.P. = 0.063 g/l; butyrate, r = 0.955 and S.E.P. = 0.058 g/l; n-butanol, r = 0.999, S.E.P. = 0.301 g/l and glucose, r = 0.999, S.E.P. = 0.493 g/l. The high quality of the results obtained, and the observation that the precision of the SIA-FTIR analysis was as good and often better than those of the reference methods, showed that SIA-FTIR is a powerful tool for rapid on-line bioprocess monitoring.

A rapid method for peroxide value determination in edible oils based on flow analysis with Fourier transform infrared spectroscopic detection

The development of an automated, rapid and highly precise method for determination of the peroxide value in edible oils based on a continuous flow system and Fourier transform infrared (FTIR) spectroscopic detection is described. The sample stream was mixed with a solvent mixture consisting of 25% (v/v) toluene in hexanol which contained triphenylphosphine (TPP). The hydroperoxides present in the sample reacted stoichiometrically with TPP to give triphenylphosphine oxide (TPPO) which has a characteristic and intense absorption band at 542 cm-1. A 10% (m/v) TPP solution in the solvent mixture and a 100 cm reaction coil were necessary for complete reaction. FTIR transmission spectra were recorded using a flow cell equipped with CsI windows having an optical pathlength of 100 microns. By using tert-butyl hydroperoxide spiked oil standards and evaluation of the band formed at 542 cm-1 a linear calibration graph covering the range 1-100 PV (peroxide value; mequiv O2 kg-1 oil) was obtained. The relative standard deviation was 0.23% (n = 11) and the throughput 24 samples h-1. The developed system was also applied to the determination of PV in olive, sunflower and corn oils, showing good agreement with the official reference method of the European Community which is based on titration using organic solvents. The results obtained clearly show that the developed method is superior to the standard wet chemical method, hence suggesting its application in routine analysis and quality control.

On-line FT-Raman spectroscopic monitoring of starch gelatinisation and enzyme catalysed starch hydrolysis

Abstract The hydrolysis of starch by α-amylase and amyloglucosidase and the kinetics of these technically important reactions were investigated by FT-Raman spectroscopy. During the technical starch hydrolysis process, three reactions proceed one after the other but partly in parallel: the gelatinisation, the hydrolysis of starch to dextrin (liquefaction) by α-amylase, and the hydrolysis of dextrin to glucose (saccharification) by glucoamylase. These three reactions were studied separately. Potato starch or dextrin in concentrations of 50 g/l were suspended or dissolved in water, the reaction chamber was placed in a heated water bath, and defined, varied amounts of enzymes in solution were added. The reactions were monitored on-line in a bypass loop. The greatest spectral changes were observed due to the swelling and gelatinisation of starch. The liquefaction of starch to dextrin was started with the addition of α-amylase at a temperature of 80°C and the spectral changes were monitored. In a similar way, a solution/suspension of dextrin was used to investigate the reaction of glucoamylase at 50°C. Both enzymatic reactions were performed at four different enzyme activities. All reactions showed distinctive spectral changes, which can, in principle, be evaluated for the determination of the degree of starch hydrolysis. As a Nd:YAG laser at 1064 nm was used, fluorescence excitation was not observed despite the use of crude, technical-grade enzymes. The findings demonstrate the potential use of Raman spectroscopy in monitoring and control of technical enzyme reactions.

Automated sample preparation and analysis using a sequential-injection-capillary electrophoresis (SI-CE) interface

A fully automated sequential-injection-capillary electrophoresis (SI-CE) system was developed using commercially available components as the syringe pump, the selection and injection valves and the high voltage power supply. The interface connecting the SI with the CE unit consisted of two T-pieces, where the capillary was inserted in one T-piece and a Pt electrode in the other (grounded) T-piece. By pressurising the whole system using a syringe pump, hydrodynamic injection was feasible. For characterisation, the system was applied to a mixture of adenosine and adenosine monophosphate at different concentrations. The calibration curve obtained gave a detection limit of 0.5 microg g(-1) (correlation coefficient of 0.997). The reproducibility of the injection was also assessed, resulting in a RSD value (5 injections) of 5.4%. The total time of analysis, from injection, conditioning and separation to cleaning the capillary again was 15 minutes. In another application, employing the full power of the automated SIA-CE system, myoglobin was mixed directly using the flow system with different concentrations of sodium dodecyl sulfate (SDS), a known denaturing agent. The different conformations obtained in this way were analysed with the CE system and a distinct shift in migration time and decreasing of the native peak of myoglobin (Mb) could be observed. The protein samples prepared were also analysed with off-line infrared spectroscopy (IR), confirming these results.

Towards functional group-specific detection in high-performance liquid chromatography using mid-infrared quantum cascade lasers.

A distributed feedback quantum cascade laser was applied for the first time as a powerful light source for mid-infrared (MIR) detection in liquid chromatography. Fructose and glucose in red wine were separated with an isocratic HPLC system, which was connected to a custom-made flow cell. This flow cell was constructed of two diamond windows with adjustable spacing and two hollow wave-guides for guiding the incoming and outgoing light. The HPLC column based on an ion-exchange resin with calcium(II) counter ion was run at 80 degrees C with 0.04% formic acid as the mobile phase. Under these conditions the carbohydrates could not be completely separated from the organic acids also present in wine. However, the emission of the laser at 1067 cm(-1) matches the absorption maximum of fructose and glucose, whereas the organic acids do not absorb appreciably at this wavenumber. Thus group-specific detection could be achieved. Additionally, the optical path length could be increased from 25 to 125 microm, which is very promising in gaining enhanced sensitivity compared to Fourier transform IR detection.