On-line infrared spectroscopy for bioprocess monitoring

One of the major aims of bioprocess engineering is the real-time monitoring of important process variables. This is the basis of precise process control and is essential for high productivity as well as the exact documentation of the overall production process. Infrared spectroscopy is a powerful analytical technique to analyze a wide variety of organic compounds. Thus, infrared sensors are ideal instruments for bioprocess monitoring. The sensors are non-invasive, have no time delay due to sensor response times, and have no influence on the bioprocess itself. No sampling is necessary, and several components can be analyzed simultaneously. In general, the direct monitoring of substrates, products, metabolites, as well as the biomass itself is possible. In this review article, insights are provided into the different applications of infrared spectroscopy for bioprocess monitoring and the complex data interpretation. Different analytical techniques are presented as well as example applications in different areas.     

A new electric method for non-invasive continuous monitoring of stroke volume and ventricular volume-time curves

ABSTRACT: BACKGROUND: In this paper a new non-invasive, operator-free, continuous ventricular stroke volume monitoring device (Hemodynamic Cardiac Profiler, HCP) is presented, that measures the average stroke volume (SV) for each period of 20 seconds, as well as ventricular volume-time curves for each cardiac cycle, using a new electric method (Ventricular Field Recognition) with six independent electrode pairs distributed over the frontal thoracic skin. In contrast to existing non-invasive electric methods, our method does not use the algorithms of impedance or bioreactance cardiography. Instead, our method is based on specific 2D spatial patterns on the thoracic skin, representing the distribution, over the thorax, of changes in the applied current field caused by cardiac volume changes during the cardiac cycle. Since total heart volume variation during the cardiac cycle is a poor indicator for ventricular stroke volume, our HCP separates atrial filling effects from ventricular filling effects, and retrieves the volume changes of only the ventricles. METHODS: In-vitro experiments on a post-mortem human heart have been performed to measure the effects of increasing the blood volume inside the ventricles in isolation, leaving the atrial volume invariant (which can not be done in-vivo). These effects have been measured as a specific 2D pattern of voltage changes on the thoracic skin. Furthermore, a working prototype of the HCP has been developed that uses these in-vitro results in an algorithm to decompose voltage changes, that were measured in-vivo by the HCP on the thoracic skin of a human volunteer, into an atrial component and a ventricular component, in almost real-time (with a delay of maximally 39 seconds). The HCP prototype has been tested in-vivo on 7 human volunteers, using G-suit inflation and deflation to provoke stroke volume changes, and LVot Doppler as a reference technique. RESULTS: The in-vitro measurements showed that ventricular filling caused a pattern over the thorax quite distinct from that of atrial filling. The in-vivo tests of the HCP with LVot Doppler resulted in a Pearson's correlation of R = 0.892, and Bland-Altman plotting of SV yielded a mean bias of -1.6 ml and 2SD = 14.8 ml. CONCLUSIONS: The results indicate that the HCP was able to track the changes in ventricular stroke volume reliably. Furthermore, the HCP produced ventricular volume-time curves that were consistent with the literature, and may be a diagnostic tool as well.     

Potential of on-line CIMS for bioprocess monitoring

Chemical-ionization mass spectrometry (CIMS) using flow reactors is an emerging method for on-line monitoring of trace concentrations of organic compounds in the gas phase. In this study, a flow-reactor CIMS instrument, employing the H(3)O(+) cation as the ionizing reagent, was used to simultaneously monitor several volatile metabolic products as they are released into the headspace during bacterial growth in a bioreactor. Production of acetaldehyde, ethanol, acetone, butanol, acetoin, diacetyl, and isoprene by Bacillus subtilis is reported. Ion signal intensities were related to solution-phase concentrations using empirical calibrations and, in the case of isoprene, were compared with simultaneous gas chromatography measurements. Identification of volatile and semivolatile metabolites is discussed. Flow-reactor CIMS techniques should be useful for bioprocess monitoring applications because of their ability to sensitively and simultaneously monitor many volatile metabolites on-line.     

Real-time on-line flow cytometry for bioprocess monitoring

As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000.In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R² ≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli).Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.     

Application of systems biology for bioprocess development

Random mutagenesis or genetic modification of an organism without consideration of its consequences to the entire system might cause unwanted changes in cellular metabolism. Systems metabolic engineering thus aims to develop strains by performing metabolic engineering within a systems biology framework, in which entire cellular networks are optimized and fermentation and downstream processes are considered at early stages. Thus, regulatory, metabolic and other cellular networks are engineered in an integrated manner. Here, we review the applications of systems biology for the development of strains and bioprocesses by means of several successful examples and, furthermore, discuss future prospects.     

A systems approach to plant bioprocess optimization

A dynamic model for plant cell metabolism was used as a basis for a rational analysis of plant production potential in in vitro cultures. The model was calibrated with data from 3-L bioreactor cultures. A dynamic sensitivity analysis framework was developed to analyse the response curves of secondary metabolite production to metabolic and medium perturbations. Simulation results suggest that a straightforward engineering of cell metabolism or medium composition might only have a limited effect on productivity. To circumvent the problem of the dynamic allocation of resources between growth and production pathways, the sensitivity analysis framework was used to assess the effect of stabilizing intracellular nutrient concentrations. Simulations showed that a stabilization of intracellular glucose and nitrogen reserves could lead to a 116% increase in the specific production of secondary metabolites compared with standard culture protocol. This culture strategy was implemented experimentally using a perfusion bioreactor. To stabilize intracellular concentrations, adaptive medium feeding was performed using model mass balances and estimations. This allowed for a completely automated culture, with controlled conditions and pre-defined decision making algorithm. The proposed culture strategy leads to a 73% increase in specific production and a 129% increase in total production, as compared with a standard batch culture protocol. The sensitivity analysis on a mathematical model of plant metabolism thus allowed producing new insights on the links between intracellular nutritional management and cell productivity. The experimental implementation was also a significant improvement on current plant bioprocess strategies.     

Near-infrared spectroscopy for bioprocess monitoring and control.

This article describes the calibration of a spectroscopic scanning instrument for the measurement of selected contaminants in a complex biological process stream. Its use is for the monitoring of a process in which contaminants are to be removed selectively by flocculation from yeast cell homogenate. The main contaminants are cell debris, protein, and RNA. A low-cost instrument has been developed for sensitivity in the region of the NIR spectrum (from 1900 to 2500 nm) where preliminary work found NIR signatures from cell debris, protein, and RNA. Calibration models have been derived using a multivariate method for concentrations of these contaminants, such as would be found after the flocculation process. Two strategies were compared for calibrating the NIR instrument. In one case, samples were prepared by adding materials representative of the contaminants to clarified yeast homogenate so the contaminant levels were well known but outside the range of interest. In the other case, where samples were like those from the process stream after flocculation and floc removal, there was uncertainty of analysis of contaminant level, but the calibration was in the range of interest. Calibration using process stream samples gave results close to those derived from traditional assays. When the calibration models were used to predict the contaminant concentrations in previously unseen samples, the correlation coefficients between measurements and predictions were above 90% in all cases but one. The prediction errors were similar to the errors in the traditional assays.     

The use of continuous monitoring and computer-assisted image analysis for the histochemical quantification of hexokinase activity

The effect of endogenous 6-phosphogluconate dehydrogenase (6PGDH) on the histochemical quantification of low-Km hexokinase activity in rat submandibular salivary glands has been investigated using a Seescan Solitaire Plus image analysis system and a modified black and white Newvicon TV camera. Absorbance readings of neutral density filters were close to their known absorbance values. A significant correlation was found between the absorbance of Nitro BT reduction products within sections, with and without the use of a 588 nm interference filter. Furthermore, absorbance readings obtained from 8 microns-thick sections were 1.92 and 2.22 times greater than values obtained from 4 microns-thick sections using 'white' and 588 nm light respectively. The level of background illumination was not critical for absorbance measurements provided it was below the level that saturated the Newvicon camera and was not changed between background and image capture. The greatest variations in absorbance readings on tissue sections were associated with changes in zoom and objective combinations. Our studies indicate that this relatively low cost image analysis system can give reproducible absorbance readings from various structures defined in digitized, captured images of tissue sections. Results from continuous monitoring studies indicated that the high levels of 6PGDH activity in excretory ducts caused a 1.67-fold overestimation of hexokinase activity as assessed by absorbance readings performed throughout a 22-minute reaction period. By contrast, overestimation of hexokinase activity in salivary gland acini only became apparent after 8 min incubation with a 1.4-fold overestimation being seen at 22 min. This difference appears to reflect the relatively low hexokinase and 6PGDH activities present in acini compared with excretory ducts.     

Potentials and limitations of miniaturized calorimeters for bioprocess monitoring

In theory, heat production rates are very well suited for analysing and controlling bioprocesses on different scales from a few nanolitres up to many cubic metres. Any bioconversion is accompanied by a production (exothermic) or consumption (endothermic) of heat. The heat is tightly connected with the stoichiometry of the bioprocess via the law of Hess, and its rate is connected to the kinetics of the process. Heat signals provide real-time information of bioprocesses. The combination of heat measurements with respirometry is theoretically suited for the quantification of the coupling between catabolic and anabolic reactions. Heat measurements have also practical advantages. Unlike most other biochemical sensors, thermal transducers can be mounted in a protected way that prevents fouling, thereby minimizing response drifts. Finally, calorimetry works in optically opaque solutions and does not require labelling or reactants. It is surprising to see that despite all these advantages, calorimetry has rarely been applied to monitor and control bioprocesses with intact cells in the laboratory, industrial bioreactors or ecosystems. This review article analyses the reasons for this omission, discusses the additional information calorimetry can provide in comparison with respirometry and presents miniaturization as a potential way to overcome some inherent weaknesses of conventional calorimetry. It will be discussed for which sample types and scientific question miniaturized calorimeter can be advantageously applied. A few examples from different fields of microbiological and biotechnological research will illustrate the potentials and limitations of chip calorimetry. Finally, the future of chip calorimetry is addressed in an outlook.     

Biohydrogen production and bioprocess enhancement: A review

This paper provides an overview of the recent advances and trends in research in the biological production of hydrogen (biohydrogen). Hydrogen from both fossil and renewable biomass resources is a sustainable source of energy that is not limited and of different applications. The most commonly used techniques of biohydrogen production, including direct biophotolysis, indirect biophotolysis, photo-fermentation and dark-fermentation, conventional or “modern” techniques are examined in this review. The main limitations inherent to biochemical reactions for hydrogen production and design are the constraints in reactor configuration which influence biohydrogen production, and these have been identified. Thereafter, physical pretreatments, modifications in the design of reactors, and biochemical and genetic manipulation techniques that are being developed to enhance the overall rates and yields of biohydrogen generation are revisited.     

An artificial intelligence tool for bioprocess monitoring: application to continuous production of gluconic acid by immobilized Aspergillus niger

Experimental data on continuous fermentation of sucrose and glucose solution at low pH to gluconic acid by Asprgillus niger immobilized on cellulose fabric show complex dynamic behaviour including a decline in yield. The data have been analyzed using an artificial intelligence based symbolic regression technique to provide a mathematical model for predicting values of conversion 5, 10 and 15 h ahead values of conversion. These predictions can be used during continuous operations to monitor the bioprocess and adjust the residence time of fermentation to get complete and more efficient conversion of sucrose or glucose to gluconic acid.     

A review on sample entropy applications for the non-invasive analysis of atrial fibrillation electrocardiograms

The application of non-linear metrics to physiological signals is a valuable tool because “hidden information” related to underlying mechanisms can be obtained. In this respect, approximate entropy (ApEn) is the most popular non-linear regularity index that has been applied to physiological time series. However, ApEn presents some shortcomings, such as bias, relative inconsistency and dependence on the sample length. A modification of ApEn, named sample entropy (SampEn), was introduced to overcome these deficiencies. Recently, in the context of electrocardiography, SampEn has been applied to study non-invasively atrial fibrillation (AF), which is the most common arrhythmia encountered in clinical practice with unknown mechanisms provoking its onset and termination. Useful clinical information, that could help for a better understanding of AF mechanisms, has been obtained through the application of SampEn to electrocardiographic (ECG) recordings. This work reviews its application in the context of non-invasive analysis of AF. During this arrhythmia, atrial and ventricular components can be regarded as unsynchronized activities, whereby, the application of SampEn to the analysis of each component will be described separately. In first place, clinical challenges in which SampEn has been successfully applied to estimate AF organization from the atrial activity pattern are presented. The AF organization study can provide information on the number of active reentries, which can help to improve AF treatment and to take the appropriate decisions on its management. Next, the heart rate variability study via SampEn, to characterize ventricular response and predict AF onset, is described. Through the aforementioned applications it is remarked throughout this review that SampEn can be considered as a very promising and useful tool towards the non-invasive understanding of AF.     

The development and applications of thermal biosensors for bioprocess monitoring

Enzyme thermistors are biosensors that use thermal resistors to measure the heat change caused by an enzymatic reaction. They combine the selectivity of enzymes with the sensitivity of biosensors and allow continuous analysis in a flow-injection mode. They can be used to monitor fermentation systems, biocatalysis, enzyme-catalysed synthesis and clinical and food technology. This article gives an overview of the general principles of enzyme thermistors, the sampling process and the ongoing developments in the field of bioprocess monitoring.     

Control of substrate concentration in a continuous bioprocess

This paper is the first one presenting an application of the control law based on the ratio of measured and optimal values for a real fermentation pilot plant operated in continuous mode. A controller of this type takes into account the process variables and may be designed by using classical linear methods. A simple algorithm is applied for a real CSTR to control the substrate concentration. The results obtained confirm that this approach offers the possibility to combine simplicity and effectiveness in bioprocess control.     

Lens-free shadow image based high-throughput continuous cell monitoring technique

A high-throughput continuous cell monitoring technique which does not require any labeling reagents or destruction of the specimen is demonstrated. More than 6000 human alveolar epithelial A549 cells are monitored for up to 72h simultaneously and continuously with a single digital image within a cost and space effective lens-free shadow imaging platform. In an experiment performed within a custom built incubator integrated with the lens-free shadow imaging platform, the cell nucleus division process could be successfully characterized by calculating the signal-to-noise ratios (SNRs) and the shadow diameters (SDs) of the cell shadow patterns. The versatile nature of this platform also enabled a single cell viability test followed by live cell counting. This study firstly shows that the lens-free shadow imaging technique can provide a continuous cell monitoring without any staining/labeling reagent and destruction of the specimen. This high-throughput continuous cell monitoring technique based on lens-free shadow imaging may be widely utilized as a compact, low-cost, and high-throughput cell monitoring tool in the fields of drug and food screening or cell proliferation and viability testing.     

Multi-stage continuous high cell density culture systems: A review

A multi-stage continuous high cell density culture (MSC-HCDC) system makes it possible to achieve high productivity together with high product titer of many bioproducts. For long-term continuous operation of MSC-HCDC systems, the cell retention time and hydraulic retention time must be decoupled and strains (bacteria, yeast, plant, and animal cells) must be stable. MSC-HCDC systems are suitable for low-value high-volume extracellular products such as fuel ethanol, lactic acid or volatile fatty acids, and high-value products such as monoclonal antibodies as well as intracellular products such as polyhydroxybutyric acid (PHB), microbial lipids or a number of therapeutics. Better understanding of the fermentation kinetics of a specific product and reliable high-density culture methods for the product-generating microorganisms will facilitate timely industrialization of MSC-HCDC systems for products that are currently obtained in fed-batch bioreactors.     

Chemical and biological characterisation of a sensor surface for bioprocess monitoring

This paper describes the step-wise fabrication and characterisation of a multi-layer dual polarization interferometry (DPI) based biosensor utilising Protein G (ProG) as the bio-recognition layer for the detection of a fragment antibody (Fab'). The biosensor is capable of monitoring the concentration of Fab' product within the extracellular medium of a fed-batch fermentation after leakage from Escherichia coli (E.coli). The activity, stability and functionality of each sensor layer were analysed in situ using DPI, whilst the chemical identity and homogeneity of the chemical layers were assessed ex situ using X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Two different biotin linkers were found to produce hugely differing surfaces after the capture of NeutrAvidin? (NA) and biotinylated Protein G (b-ProG). The hydrophilic (PEG)(4)-biotin linker resulted in a surface where the b-ProG layer was deposited and organised above the NA layer producing an active and stable surface, whilst the hydrophobic LC-biotin linker generated a surface where the b-ProG layer was buried within the NA layer leading to variable surfaces and poor binding of the Fab' target. The biosensor has a detection limit of 1.7 μg/ml with a dynamic range covering two orders of magnitude. The sensor can detect the onset of Fab' leakage as early as 2h following product induction, with high signal-to-noise ratios and little interference from extracellular components. Leakage of Fab' followed a biphasic profile, switching to a more rapid rate 20 h after induction, indicating accelerated product loss and the need for cultivation harvest.