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 µs ' with CDW was observed in the range of 5-69 g L-1 (R2 = 0.98). The growth rates calculated based on µs ' were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µs ' as a function of the absorption coefficient µa 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.

Depletion-Induced Flocculation of Concentrated Emulsions Probed by Photon Density Wave Spectroscopy.

Stable, creaming-free oil in water emulsions with high volume fractions of oil (ϕ = 0.05-0.40, density matched to water) and polysorbate 80 as an emulsifier were characterized without dilution by Photon Density Wave spectroscopy measuring light absorption and scattering behavior, the latter serving as the basis for droplet size distribution analysis. The emulsion with ϕ = 0.10 was used to investigate flocculation processes induced by xanthan as a semi-flexible linear nonabsorbing polymer. Different time regimes in the development of the reduced scattering coefficient µs' could be identified. First, a rapid, temperature-dependent change in µs' during the depletion process was observed. Second, the further decrease of µs' follows a power law in analogy to a spinodal demixing behavior, as described by the Cahn-Hilliard theory.

Limitations of turbidity process probes and formazine as their calibration standard.

Turbidity measurements are frequently implemented for the monitoring of heterogeneous chemical, physical, or biotechnological processes. However, for quantitative measurements, turbidity probes need calibration, as is requested and regulated by the ISO 7027:1999. Accordingly, a formazine suspension has to be produced. Despite this regulatory demand, no scientific publication on the stability and reproducibility of this polymerization process is available. In addition, no characterization of the optical properties of this calibration material with other optical methods had been achieved so far. Thus, in this contribution, process conditions such as temperature and concentration have been systematically investigated by turbidity probe measurements and Photon Density Wave (PDW) spectroscopy, revealing an influence on the temporal formazine formation onset. In contrast, different reaction temperatures do not lead to different scattering properties for the final formazine suspensions, but give an access to the activation energy for this condensation reaction. Based on PDW spectroscopy data, the synthesis of formazine is reproducible. However, very strong influences of the ambient conditions on the measurements of the turbidity probe have been observed, limiting its applicability. The restrictions of the turbidity probe with respect to scatterer concentration are examined on the basis of formazine and polystyrene suspensions. Compared to PDW spectroscopy data, signal saturation is observed at already low reduced scattering coefficients.

Process analytical approaches for the coil-to-globule transition of poly(N-isopropylacrylamide) in a concentrated aqueous suspension.

The coil-to-globule transition of poly(N-isopropylacrylamide) (PNIPAM) microgel particles suspended in water has been investigated in situ as a function of heating and cooling rate with four optical process analytical technologies (PAT), sensitive to structural changes of the polymer. Photon Density Wave (PDW) spectroscopy, Focused Beam Reflectance Measurements (FBRM), turbidity measurements, and Particle Vision Microscope (PVM) measurements are found to be powerful tools for the monitoring of the temperature-dependent transition of such thermo-responsive polymers. These in-line technologies allow for monitoring of either the reduced scattering coefficient and the absorption coefficient, the chord length distribution, the reflected intensities, or the relative backscatter index via in-process imaging, respectively. Varying heating and cooling rates result in rate-dependent lower critical solution temperatures (LCST), with different impact of cooling and heating. Particularly, the data obtained by PDW spectroscopy can be used to estimate the thermodynamic transition temperature of PNIPAM for infinitesimal heating or cooling rates. In addition, an inverse hysteresis and a reversible building of micrometer-sized agglomerates are observed for the PNIPAM transition process.

Optical monitoring of chemical processes in turbid biogenic liquid dispersions by Photon Density Wave spectroscopy.

In turbid biogenic liquid material, like blood or milk, quantitative optical analysis is often strongly hindered by multiple light scattering resulting from cells, particles, or droplets. Here, optical attenuation is caused by losses due to absorption as well as scattering of light. Fiber-based Photon Density Wave (PDW) spectroscopy is a very promising method for the precise measurement of the optical properties of such materials. They are expressed as absorption and reduced scattering coefficients (µ a and µ s', respectively) and are linked to the chemical composition and physical properties of the sample. As a process analytical technology, PDW spectroscopy can sense chemical and/or physical processes within such turbid biogenic liquids, providing new scientific insight and process understanding. Here, for the first time, several bioprocesses are analyzed by PDW spectroscopy and the resulting optical coefficients are discussed with respect to established mechanistic models of the chosen processes. As model systems, enzymatic casein coagulation in milk, temperature-induced starch hydrolysis in beer mash, and oxy- as well as deoxygenation of human donor blood were investigated by PDW spectroscopy. The findings indicate that also for very complex biomaterials (i.e., not well-defined model materials like monodisperse polymer dispersions), obtained optical coefficients allow for the assessment of a structure/process relationship and thus for a new analytical access to biogenic liquid material. This is of special relevance as PDW spectroscopy data are obtained without any dilution or calibration, as often found in conventional spectroscopic approaches.

Industrial applications of photon density wave spectroscopy for in-line particle sizing [Invited].

Optical spectroscopy in highly turbid liquid material is often restricted by simultaneous occurrence of absorption and scattering of light. Photon Density Wave (PDW) spectroscopy is one of the very few, yet widely unknown, technologies for the independent quantification of these two optical processes. Here, a concise overview about modern PDW spectroscopy is given, including all necessary equations concerning the optical description of the investigated material, dependent light scattering, particle sizing, and PDW spectroscopy itself. Additionally, it is shown how the ambiguity in particle sizing, arising from Mie theory, can be correctly solved. Due to its high temporal resolution, its applicability to highest particle concentrations, and its purely fiber-optical probe, PDW spectroscopy possesses all fundamental characteristics for optical in-line process analysis. Several application examples from the chemical industry are presented.

Novel oral microscope gives mechanistic insights into colloidal drivers of friction in oral biofilms.

Texture and mouthfeel are central to the sensory enjoyment of food and beverages. Yet our incomplete understanding of how food boluses are transformed in the mouth limits our texture prediction ability. As well as thin film tribology, the interaction of food colloids with the oral tissue and salivary biofilms plays a key role in texture perception via mechanoreceptors in the papillae. In this study we describe the development of an oral microscope capable of quantitative characterization of the inactions of food colloids with papillae and their concurrent saliva biofilm. We also highlight how the oral microscope revealed key microstructural drivers of several topical phenomena (oral residue formation, coalescence in-mouth, grittiness of protein aggregates and finally microstructural origin of polyphenol astringency) in the domain of texture creation. The coupling of a fluorescent food grade dye with image analysis enabled specific and quantitative determination of the microstructural changes in mouth. Emulsions either underwent no aggregation, small aggregation, or extensive aggregation depending on whether their surface charge facilitated complexation with the saliva biofilm. Quite surprisingly cationic gelatin emulsions that were already aggregated with saliva in mouth underwent coalescence if subsequently exposed to tea polyphenols (EGCG). Large protein aggregates were found to aggregate with the saliva coated papillae, increasing their size tenfold and possibly explaining why there are perceived as gritty. An exciting observation was the oral microstructural changes that occurred upon exposure to tea polyphenols (EGCG). Filiform papillae shrunk, and the saliva biofilm was seen to precipitate/collapse, exposing a very rough tissue surface. These tentative early steps are the first in vivo microstructural insights into the different food oral transformations that are drivers of key texture sensation.

Inline monitoring of high cell density cultivation of Scenedesmus rubescens in a mesh ultra-thin layer photobioreactor by photon density wave spectroscopy.

OBJECTIVE: Due to multiple light scattering that occurs inside and between cells, quantitative optical spectroscopy in turbid biological suspensions is still a major challenge. This includes also optical inline determination of biomass in bioprocessing. Photon Density Wave (PDW) spectroscopy, a technique based on multiple light scattering, enables the independent and absolute determination of optical key parameters of concentrated cell suspensions, which allow to determine biomass during cultivation. RESULTS: A unique reactor type, called "mesh ultra-thin layer photobioreactor" was used to create a highly concentrated algal suspension. PDW spectroscopy measurements were carried out continuously in the reactor without any need of sampling or sample preparation, over 3 weeks, and with 10-min time resolution. Conventional dry matter content and coulter counter measurements have been employed as established offline reference analysis. The PBR allowed peak cell dry weight (CDW) of 33.4 g L-1. It is shown that the reduced scattering coefficient determined by PDW spectroscopy is strongly correlated with the biomass concentration in suspension and is thus suitable for process understanding. The reactor in combination with the fiber-optical measurement approach will lead to a better process management.

Process Characterization of Polyvinyl Acetate Emulsions Applying Inline Photon Density Wave Spectroscopy at High Solid Contents.

The high solids semicontinuous emulsion polymerization of polyvinyl acetate using poly (vinyl alcohol-co-vinyl acetate) as protective colloid is investigated by optical spectroscopy. The suitability of Photon Density Wave (PDW) spectroscopy as inline Process Analytical Technology (PAT) for emulsion polymerization processes at high solid contents (>40% (w/w)) is studied and evaluated. Inline data on absorption and scattering in the dispersion is obtained in real-time. The radical polymerization of vinyl acetate to polyvinyl acetate using ascorbic acid and sodium persulfate as redox initiator system and poly (vinyl alcohol-co-vinyl acetate) as protective colloid is investigated. Starved-feed radical emulsion polymerization yielded particle sizes in the nanometer size regime. PDW spectroscopy is used to monitor the progress of polymerization by studying the absorption and scattering properties during the synthesis of dispersions with increasing monomer amount and correspondingly decreasing feed rate of protective colloid. Results are compared to particle sizes determined with offline dynamic light scattering (DLS) and static light scattering (SLS) during the synthesis.

Fiber optic plasmonic sensors: Providing sensitive biosensor platforms with minimal lab equipment.

A simple, convenient, and inexpensive method to fabricate optical fiber based biosensors which utilize periodic hole arrays in gold films for signal transduction is reported. The process of hole array formation mainly relies on self-assembly of hydrogel microgels in combination with chemical gold film deposition and subsequent transfer of the perforated film onto an optical fiber tip. In the fabrication process solely chemical wet lab techniques are used, avoiding cost-intensive instrumentation or clean room facilities. The presented method for preparing fiber optic plasmonic sensors provides high throughput and is perfectly suited for commercialization using batch processing. The transfer of the perforated gold film onto an optical fiber tip does not affect the sensitivity of the biosensor ((420 ±â€¯83) nm/refractive index unit (RIU)), which is comparable to sensitivities of sensor platforms based on periodic hole arrays in gold films prepared by significantly more complex methods. Furthermore, real-time and in-line immunoassay studies with a specially designed 3D printed flow cell are presented exploiting the presented optical fiber based biosensors.

In-Line Monitoring of Polyhydroxyalkanoate (PHA) Production during High-Cell-Density Plant Oil Cultivations Using Photon Density Wave Spectroscopy.

Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient µs' and the absorption coefficient µa at 638 nm. A correlation of µs' with the cell dry weight (CDW) and µa with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse feed cultivations. The correlation was used to predict CDW, RCDW, and PHB formation in a high-cell-density fed-batch cultivation with a productivity of 1.65 gPHB·L-1·h-1 and a final biomass of 106 g·L-1 containing 73 wt% PHB. The new method applied in this study allows in-line monitoring of CDW, RCDW, and PHA formation.