Real-time analyte monitoring of a fungal fermentation, at pilot scale, using in situ mid-infrared spectroscopy

Robust in situ biochemical monitoring is essential for the development of substrate feed control to optimize fermentation processes. The scale up of the fermentation for the fungus Glarea lozoyensis can benefit from such technology to improve the yield of the pharmaceutically important pneumocandin of interest and control the levels of unwanted analogues. A new in situ probe, using a diamond attenuated total reflection element, was evaluated at pilot scale for the quantitative measurement of fermentation analytes using Fourier transform mid-IR spectrometry. The new technology was shown to be stable, unaffected by reactor operation conditions of agitation, airflow, and backpressure, but sensitive to temperature control. Both glucose and phosphate were simultaneously monitored during a seed fermentation at 280 L pilot scale using complex medium with detection to 0.1 g/L for both analytes. Fructose, glutamate, and proline were monitored at 75 L scale using production media with detection limits of 0.1, 0.5, and 0.5 g/L respectively. Partial least squares calibration/prediction models were created for analytes of interest using off-line reference measurements and specific spectral regions. Good fits were obtained between off-line measurements and those predicted by in situ mid-IR. Standard errors of prediction (SEP) for glucose (range 18-0.1 g/L) and phosphate (range 11-7.5 g/L) were 0.16 and 1.8 g/L respectively with mean percentage errors (MPEs) around 2.5%. SEP values for the production process: fructose (range 20-0.1 g/L), glutamate (8-0.5 g/L), and proline (12-0.5 g/L) were 0.44, 0.6, and 0.5 g/L respectively with MPEs of 2.2, 5.3, and 10.1%. The technology effectively demonstrates quantitative multicomponent analysis of fermentation processes using in situ monitoring.     

Real-time monitoring of yeast cell division by dielectric spectroscopy.

To assay cell cycle progression in synchronized culture of yeast we have applied dielectric spectroscopy to its real-time monitoring. The dielectric monitoring is based on the electromagnetic induction method, regarded as a nonelectrode method, which has resolved the problems encountered in measurements with metal electrodes, namely electrode polarization and bubble formation on electrodes. In the synchronized culture with temperature-sensitive cell division cycle mutants, the permittivity of the culture broth showed cyclic changes at frequencies below 300 kHz. The increase and decrease in the cyclic changes of the relative permittivity correspond to the increase in cell length and bud size and to the septum formation between mother and daughter cells, respectively.     

Real-time monitoring of epithelial cell-cell and cell-substrate interactions by infrared surface plasmon spectroscopy

The development of novel technologies capable of monitoring the dynamics of cell-cell and cell-substrate interactions in real time and a label-free manner is vital for gaining deeper insights into these most fundamental cellular processes. However, the label-free technologies available today provide only limited information on these processes. Here, we report a new (to our knowledge) infrared surface plasmon resonance (SPR)-based methodology that can resolve distinct phases of cell-cell and cell-substrate adhesion of polarized Madin Darby canine kidney epithelial cells. Due to the extended penetration depth of the infrared SP wave, the dynamics of cell adhesion can be detected with high accuracy and high temporal resolution. Analysis of the temporal variation of the SPR reflectivity spectrum revealed the existence of multiple phases in epithelial cell adhesion: initial contact of the cells with the substrate (cell deposition), cell spreading, formation of intercellular contacts, and subsequent generation of cell clusters. The final formation of a continuous cell monolayer could also be sensed. The SPR measurements were validated by optical microscopy imaging. However, in contrast to the SPR method, the optical analyses were laborious and less quantitative, and hence provided only limited information on the dynamics and phases of cell adhesion.     

Rapid measurement of phytate in raw soymilk by mid-infrared spectroscopy

The phytate content in soymilk is known to affect tofu curdling. A rapid measurement of phytate from a water extract of soybean (raw soymilk) in an early stage of tofu processing was investigated using mid-infrared spectroscopy (IR) with an ATR accessory. IR absorption of phytate was observed from 1200 cm-1 to 900 cm-1, and saccharide and protein in the extract also had IR absorption in the same region. In order to separate phytate from other components, the phytate was precipitated completely by the addition of calcium under alkaline condition (pH 11.5). The precipitate was dissolved in citrate buffer (pH 6.0) and then used for IR measurement. The absorbance at 1070 cm-1 correlated well with the phytate content of the soymilk. The measurement of phytate in raw soymilk can be done rapidly by FT-IR measurement with an ATR accessory and gives reproducible values, which can be used for the measurement of phytate content in various soybeans for tofu making.     

Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy

An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µ_s′ with CDW was observed in the range of 5–69 g L⁻¹ (R² = 0.98). The growth rates calculated based on µ_s′ were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µ_s′ as a function of the absorption coefficient µ_a allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.     

Real-time in situ monitoring of freely suspended and immobilized cell cultures based on mid-infrared spectroscopic measurements.

Glucose and lactate profiles in Chinese hamster ovary cell cultures were accurately monitored in real time and in situ during three bioreactor batch cultures lasting 11,15, and 15 days performed within a 60-day period. Monitoring was accomplished using in situ-collected mid-infrared spectra analyzed with a priori one-time established partial least-squares regression models. The robustness of the technique was demonstrated by application of these models without modification after 2.3 years. Neither recalibration nor instrument maintenance was required during the 2.3-year period, except for the daily filling of liquid nitrogen for detector cooling during operation. The lactate calibration model yielded accurate absolute concentration estimations during each of the batch cultures with standard errors of estimate from 1 to 3 mM. The a priori-established glucose calibration model yielded concentration estimations with an off-set, which was constant throughout a culture. Adjustment of the off-set before inoculation resulted in accurate concentration estimations with Standard errors of estimate of approximately 1 mM for each of the bioreactor cultures. Sensitivity in detecting differences of 0.5 mM and selectivity against variation of one metabolite while the other was kept constant was demonstrated during standard additions of either glucose or lactate. The sensor system proved to be reliable, simple, accurate, sterile, and capable of long-term automatic operation and is considered to be mature enough to be routinely applied for in situ (on-line) cell culture monitoring.     

Developments in mid-infrared FT-IR spectroscopy of selected carbohydrates

FT-IR spectroscopy has become a powerful research tool for elucidating the structure, physical properties and interactions of carbohydrates. It provides a new interpretive and experimental framework for the study of complicated systems of natural polymers. This paper gives an overview over new infrared applications in the study of carbohydrates, both small compounds and macromolecules. These include a wide range of studies of carbohydrates in different physical states, from the crystalline solid state to aqueous solution, and special techniques, which expand the experimental framework to the in-muro studies of plant materials, and quantitative determination.     

Real-time update of calibration model for better monitoring of batch processes using spectroscopy

In order to reduce the large calibration matrix usually required for calibrating multiwavelength optical sensors, a simple algorithm based on the addition in process of new standards is proposed. A small calibration model, based on 14 standards, is periodically updated by spectra collected on-line during fermentation operation. Concentrations related to these spectra are reconciled into best-estimated values, by considering carbon and oxygen balances. Using this method, fructose, acetate, and gluconacetan were monitored during batch fermentations of Gluconacetobacter xylinus 12281 using mid-infrared spectroscopy. It is shown that this algorithm compensates for noncalibrated events such as production or consumption of by-products. The standard error of prediction (SEP) values were 0.99, 0.10, and 0.90 g/L for fructose, acetate, and gluconacetan, respectively. By contrast, without an updating of the calibration model, the SEP values were 2.46, 0.92, and 1.04 g/L for fructose, acetate, and gluconacetan, respectively. Using only 14 standards, it was therefore possible to approach the performance of an 88-standard-based calibration model having SEP values of 1.11, 0.37, and 0.79 g/L for fructose, acetate, and gluconacetan, respectively. Therefore, the proposed algorithm is a valuable approach to reduce the calibration time of multiwavelength optical sensors.     

On-line monitoring of nine different batch cultures of E. coli by mid-infrared spectroscopy, using a single spectra library for calibration

A single spectra library was used to monitor on-line, by mid-infrared spectroscopy, nine different batch cultures of Escherichia coli performed with various medium compositions, including chemically complex formulations. Whereas the classic chemometrics approach would have required the preparation and measurement of hundreds of standards, only six spectra were included in the library. These included the molar absorbance of the four main metabolites (i.e. glucose, glycerol, ammonium and acetate), and the remaining two were drift spectra found by factor analysis. The accuracy of the prediction was not altered by a change of the carbon source, the ammonium concentration or even the addition of chemically undefined compounds, such as yeast extract and peptone. The standard errors of prediction averaged over the nine experiments were 8.0, 12.3, 5.9 and 5.6 mM for glucose, glycerol, ammonium and acetate, respectively. Inclusion of two drift spectra in the library provided an estimation of how noisy an experiment was. This also allowed detection of batch cultures that require further investigation, namely runs which were subject to large signal drift or during which an unexpected compound was produced, without having to carry out time-consuming off-line analyses.     

Terpenoid biotransformations by Mucor species

Terpenoids are natural products of great interest due to their widespread use in agrochemicals, drugs, fragrances, flavouring and pigments. Biocatalysts are increasingly being used in the search for new derivatives with improved properties especially to obtain structurally novel leads for new drugs which are difficult to obtain using conventional organic chemical methods. This review, covering up to the end of 2012, reports on the application of Mucor species as catalysts in terpenoid biotransformation to obtain new drug targets, enhance pharmacological activity or decrease the unwanted effects of starting material.     

Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region

Fourier transform spectroscopy in the mid-infrared (400–5,000 cm⁻¹) (FT-IR) is being recognized as a powerful tool for analyzing chemical composition of food, with special concern to molecular architecture of food proteins. Unlike other spectroscopic techniques, it provides high-quality spectra with very small amount of protein, in various environments irrespective of the molecular mass. The fraction of peptide bonds in α-helical, β-pleated sheet, turns and aperiodic conformations can be accurately estimated by analysis of the amide I band (1,600–1,700 cm⁻¹) in the mid-IR region. In addition, FT-IR measurement of secondary structure highlights the mechanism of protein aggregation and stability, making this technique of strategic importance in the food proteomic field. Examples of applications of FT-IR spectroscopy in the study of structural features of food proteins critical of nutritional and technological performance are discussed.     

Validation of chemometric models for the determination of deoxynivalenol on maize by mid-infrared spectroscopy

Validation methods for chemometric models are presented, which are a necessity for the evaluation of model performance and prediction ability. Reference methods with known performance can be employed for comparison studies. Other validation methods include test set and cross validation, where some samples are set aside for testing purposes. The choice of the testing method mainly depends on the size of the original dataset. Test set validation is suitable for large datasets (>50), whereas cross validation is the best method for medium to small datasets (<50). In this study the K-nearest neighbour algorithm (KNN) was used as a reference method for the classification of contaminated and blank corn samples. A Partial least squares (PLS) regression model was evaluated using full cross validation. Mid-Infrared spectra were collected using the attenuated total reflection (ATR) technique and the fingerprint range (800–1800 cm⁻¹) of 21 maize samples that were contaminated with 300 – 2600 μg/kg deoxynivalenol (DON) was investigated. Separation efficiency after principal component analysis/cluster analysis (PCA/CA) classification was 100%. Cross validation of the PLS model revealed a correlation coefficient of r=0.9926 with a root mean square error of calibration (RMSEC) of 95.01. Validation results gave an r=0.8111 and a root mean square error of cross validation (RMSECV) of 494.5 was calculated. No outliers were reported. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Organic and inorganic carbon in paddy soil as evaluated by mid-infrared photoacoustic spectroscopy

Paddy soils are classified as wetlands which play a vital role in climatic change and food production. Soil carbon (C), especially soil organic C (SOC), in paddy soils has been received considerable attention as of recent. However, considerably less attention has been given to soil inorganic carbon (SIC) in paddy soils and the relationship between SOC and SIC at interface between soil and the atmosphere. The objective of this research was to investigate the utility of applying Fourier transform mid-infrared photoacoustic spectroscopy (FTIR-PAS) to explore SOC and SIC present near the surface (0-10 μm) of paddy soils. The FTIR-PAS spectra revealed an unique absorption region in the wavenumber range of 1,350-1,500 cm(-1) that was dominated by C-O (carbonate) and C-H bending vibrations (organic materials), and these vibrations were used to represented SIC and SOC, respectively. A circular distribution between SIC and SOC on the surface of paddy soils was determined using principal component analysis (PCA), and the distribution showed no significant relationship with the age of paddy soil. However, SIC and SOC were negatively correlated, and higher SIC content was observed near the soil surface. This relationship suggests that SIC in soil surface plays important roles in the soil C dynamics.     

Prediction of soil carbon levels in calcareous soils of Iran by mid-infrared reflectance spectroscopy

The objective of this study was to assess the predictive performance of midDRIFTS-PLSR models in quantifying total carbon (TC), total organic carbon (TOC), total inorganic carbon (TIC), total nitrogen (TN), hot water extractable carbon and nitrogen (C_HWE, N_HWE), pH, and the clay, silt, and sand content of soils. A total of 68 soil samples were taken across an agroecological region in southwest Iran, and analyzed in the laboratory using mid-DRIFTS-PLSR. midDRIFTS-PLSR calibration models were developed, and external validation was performed for each of the soil properties via an independent algorithm. The calibration and validation models allowed for a sufficient prediction of TC, TIC, and TOC with residual prediction deviations ≥3 and R² values >0.9. The precise prediction ofcarbon fractions, such as TC, TIC, and TOC, in a rapid and inexpensive manner confirmed that midDRIFTS analysis was a rapid-throughput and cost-effective technique for monitoring soil carbon at the regional scale.     

The production of fine chemicals by biotransformations

Today, biocatalysis is a standard technology for the production of chemicals. An analysis of 134 industrial biotransformations reveals that hydrolases (44%) and redox biocatalysts (30%) are the most prominent categories. Most products are chiral (89%) and are used as fine chemicals. In the chemical industry, successful product developments involve on average a yield of 78%, a volumetric productivity of 15.5 g/(L.h) and a final product concentration of 108 g/L. By contrast, the pharmaceutical industry focuses on time-to-market. The implications of this for future research and development on biocatalysis are discussed.     

pH prediction and control in bioprocesses using mid-infrared spectroscopy

An on-line pH monitoring method based on mid-infrared spectroscopy relevant to bioprocesses is presented. This approach is non-invasive and does not require the addition of indicators or dyes, since it relies on the analysis of species of common buffers used in culture media, such as phosphate buffer. Starting with titrations of phosphoric and acetic acid solutions over almost the entire pH range (2-12), it was shown that the infrared spectra of all samples can be expressed as a linear combination of the molar absorbance of the acids and their deprotonated forms. In other words, pH had no direct influence on the molar infrared spectra themselves, but only on deprotonation equilibria. Accurate prediction (standard error of prediction for pH < 0.15 pH units) was achieved by taking into account the non-ideal behavior of the solutions, using the Debye-Hückel theory to estimate the activity coefficients. Batch cultures of E. coli were chosen as a case study to show how this approach can be applied to bioprocess monitoring. The discrepancy between the spectroscopic prediction and the conventional electrochemical probe never exceeded 0.12 pH units, and the technique was fast enough to implement a feedback controller to maintain the pH constant during cultivation.     

Real-time monitoring of polyacrylamide gel electrophoresis by schlieren optics

A modernized schlieren optics was applied to follow protein bands visually during polyacrylamide gel electrophoresis. A band containing as little as 0.3 microgram of a protein could be detected. Besides the protein bands, usually overlooked phenomena such as boundary migration of the buffer components could be visualized. As an example, electrophoretic patterns thus obtained for SDS-polyacrylamide gel electrophoresis with a discontinuous buffer system are presented. The use of a split-type colored filter was found to be useful for coloring a particular location on the gel red or blue depending on the sign of the refractive index gradient. This means of detection also seems useful in that, firstly, it makes electrophoretic mobility measurement more quantitative and, secondly, it allows localization of a protein band and sampling in the intact state.     

Real-time monitoring of ubiquitination in living cells by BRET

Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET), we measured the ubiquitination of beta-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of beta-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.     

Real-time monitoring of recombinant bacterial proteins by mass spectrometry

Genetically altered bacteria manipulated to express green fluorescent protein (GFP) were used in an investigation of real-time monitoring for recombinant protein expression in cell by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). A significant advantage to whole cell MALDI MS is its ability to analyze bacterial cultures without pretreatment other than concentration. This paper describes the simultaneous analysis of overexpressed GFP recombinant Escherichia coli JM101 by MALDI-TOF MS and standard fluorescence measurements. Cells were harvested from liquid culture media during a 12 h GFP induced expression cycle to demonstrate the feasibility of near real-time monitoring of induced protein expression. The results show that although MALDI MS is not as sensitive as fluorescence measurements, expression levels of the targeted protein can easily be determined. Data available only through MALDI MS measurements reveal the presence of both native GFP and GFP-(histidine)(6) proteins. Additionally, biochemical processes not yet fully understood are observed in the presence and absence of ribosomal protein constituents. Thus, the work presented here demonstrates the ability of MALDI MS to monitor and characterize in real time the expression of targeted and unexpected genetically recombinant proteins in active cell cultures.