Ian W. Marison

• Dublin City University

This article reports an in-depth characterization and optimization of a fermentation process with the aim of increasing the production of high cell densities as a prerequisite for high productivity of lipids. The freshwater microalgae Chlorella protothecoides was grown heterotrophically in a bioreactor on aerated and stirred cultivation medium with...

Microcapsules for high cell density culture of mammalian cells have found an increasing interest, however, the poor stability of the microcapsules and the lack of characterisation methods led to few quantitative results. Alginate-poly-L-lysine (PLL) microcapsules have been studied in detail in order to form a basis for comparison of capsules made f...

The purpose of this study was to introduce dielectric spectroscopy and biocalorimetry as monitoring methods to follow immobilised Chinese Hamster Ovary (CHO) cell culture development. The theory behind both monitoring techniques is explained and perfusion cultures are performed in a Reaction Calorimeter (eRC1 from Mettler Toledo) as an application...

Turbidity is an important water-quality indicator as it not only indicates the presence of suspended solids but also shows the possible presence of algae, microorganisms, organic matter, and microparticulates. Generally, turbidity is removed through two very important steps in a typical water treatment process: coagulation and flocculation. At lab...

Objectives: Colorectal cancer (CRC) is a life-threatening disease that can develop as a consequence of a sustained chronic inflammatory pathology of the colon. Although not devoid of side effects, the anti-inflammatory drug celecoxib (CLX) has been shown to exert protective effects in CRC therapy. The purpose of this study was to develop and chara...

Significant improvements in the productivity and quality of therapeutic proteins produced in Chinese hamster ovary (CHO) cells have been reported since their establishment as host cells for biopharmaceutical production. Initial advances in the field focused on engineering strategies to manipulate genes associated with proliferation, apoptosis, and...

Abstract The purpose of this study was to develop a novel multipaticulate drug delivery technology suitable for the delivery of pre-solubilized celecoxib to the gastrointestinal tract and more specifically to the colon. The solubility of celecoxib in a range of oils, surfactants and co-solvents was evaluated. Celecoxib was solubilized in mixtures o...

Flocculation is an important step in water treatment as it is responsible for the separation of suspended solids and colloids. The currently used flocculants have certain limitations with respect to environmental impact and disposal as well as potentially being harmful to human health, which has encouraged the study of natural flocculants originati...

With increasing pressure from regulatory authorities on industry to develop processes embracing process analytical technology (PAT) initiatives, there is a growing demand to establish reliable tools and systems capable of meeting this need. With regard to monitoring and control of bioprocesses, this need translates to a search for robust instrument...

Understanding the growth characteristics of microorganisms is an essential step in bioprocessing, not only because product formation may be growth-associated but also because they might influence cell physiology and thereby product quality. The specific growth rate, a key variable of many bioprocesses, cannot be measured directly and relies on the...

This study proposed a methodology to evaluate the potential of mid-infrared (MIR) spectroscopy as a process analytical technology (PAT) tool for in situ (in-line) monitoring of cell culture media constituents, paving the way for on-line bioprocess monitoring and control of mammalian cell cultures. The methodology included a limit of detection (LOD)...

For over a half a century now, microencapsulation has played a very important role in many industries and in the recent decades, this versatile technology has been applied to numerous biotechnology and medical processes. However, successful application in these areas requires a methodology which has the capability to produce mono-dispersed, homogen...

Year 2001 was the dawn of a new era for the pharmaceutical industry. Over the past decades, since the Thalidomide scandal, regulatory actions ensured that quality of drugs, active pharmaceutical ingredients and biological products released to the market was not longer an issue. However, difficulties still remain with the development and continuous...

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calor...

The molecular complex "Heat shock protein 90" has become a novel target for anticancer drugs in recent times on account of its ability to perform as a chaperone toward proteins involved in cancer progression. The geldanamycin binds to this complex with high affinity and prevents it from performing correctly, which results in tumor destruction. The...

A simple stoichiometric model is proposed linking the biomass yield to the enthalpy and Gibbs energy changes in chemo-heterotrophic, mixotrophic, and photo-autotrophic microbial growth. A comparison with calorimetric experiments on the algae Chlorella vulgaris and Chlorella sorokiniana confirmed the trends but revealed large calorimetric measuremen...

Control of bioprocesses requires reliable and robust on- or in-line monitoring tools providing real-time information on process dynamics. Heat generation related to metabolic activity of living systems is currently gaining importance in bioprocess industry due to its non-invasive and essentially instantaneous characteristics. This study deals with...

Our spinning disk atomization (SDA) can, relative to other existing techniques, produce micron-sized particles of very narrow size distribution. The aim of this work is to present this technology for the production of alginate microspheres. We have atomized and gelled aqueous alginate solutions into very narrowly dispersed microspheres with sizes r...

Reliable control of the specific growth rate (μ) in fed-batch fermentations depends on the availability of accurate online estimations of the controlled variable. Due to difficulties in measuring biomass, μ is typically estimated using reference models relating measurements of substrate consumption or oxygen uptake rate to biomass growth. However,...

The chemical modification and immobilisation of lipase B from Candida antarctica (CalB) onto three different types of mesoporous silicate (MPS) were undertaken. Soluble CalB was modified by two bifunctional reagents, ethylene glycol bis(succinimidyl succinate) (EGNHS) and glutaraldehyde, and by the monofunctional citraconic anhydride. Both chemical...

In recent years ever-increasing amounts of pharmaceuticals are being detected in the aquatic environment and in some cases, they have even been discovered in drinking water. Their presence is attributed mainly to the inability of sewage treatment plants to adequately remove these compounds from the sewage influent. The aim of this study was to inve...

Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore, is a key sequence for the step-up from Quality by Analysis to Quality by Design. A novel and robust Process Control Tool designed to achieve close control of the specific growth rate in fed-batch...

The control of production costs in the food industry must be very strict as a result of the relatively low added value of food products. Since a wide variety of enzymes and/or cells are employed in the food industry for starch processing, cheese making, food preservation, lipid hydrolysis and other applications, immobilization of the cells and/or e...

Real-time data reconciliation of concentration estimates of process analytes and biomass in microbial fermentations is investigated. A Fourier-transform mid-infrared spectrometer predicting the concentrations of process metabolites is used in parallel with a dielectric spectrometer predicting the biomass concentration during a batch fermentation of...

The press-cake (PC) of oleaginous species (Brassica napus, Moringa oleifera, Glycine max) has been investigated as a biosorbent for metals removal from contaminated water. Sorption isotherms for copper (Cu) have been established for different pH values and fitted with Langmuir isotherms. The maximum sorption capacity was observed at pH 5 (qmax = 11...

This work evaluates three techniques of calibrating capacitance (dielectric) spectrometers used for on-line monitoring of biomass: modeling of cell properties using the theoretical Cole-Cole equation, linear regression of dual-frequency capacitance measurements on biomass concentration, and multivariate (PLS) modeling of scanning dielectric spectra...

This paper describes the development of a new method to obtain aqueous-core microcapsules from organic-core capsules. The direct production of microcapsules, using tripropionin as organic material, followed by the hydrolysis of the core by a lipase was investigated. The enzymatic study showed that the enzyme obeyed a Michaelis-Menten mechanism and...

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 acet...

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...

Due to residual oil retained within it, rapeseed press-cake has been shown to be effective for the removal of atrazine from water through an absorption mechanism. However, it is difficult to put this into practice due to the hygroscopic nature of the press-cake resulting in considerable swelling, together with the formation of a thick paste which h...

Oil-bodies are minute plant organelles (0.5-2.0microm diameter) consisting of an oil core surrounded by a phospholipid monolayer/proteinaceous membrane. Oil-bodies have been isolated from rapeseed seeds and demonstrated to constitute a novel type of micro-capsule suitable for the extraction of hydrophobic organic compounds from aqueous environments...

A recombinant avidin-producing Mut+ Pichia pastoris strain was used as a model organism to study the influence of the methanol feeding strategy on the specific product productivity (q(p)) and protein glycosylation. Fed-batch cultivations performed at various specific growth rates (micro) and residual methanol concentrations showed that the specific...

A general overview of methods that help maintain the on-line reliability and robustness ofspectroscopic monitoring instruments used in bioprocess monitoring is presented. Trulypredictive spectroscopic monitoring techniques, capable of providing continuous, liveinformation about process analytes, biomass and bioproducts are highly desirable as they...

An aqueous–organic two-layer partitioning and continuous process was designed to make efficient use of growing cells for the biodesulfurization of diesel oils. This biphasic system was shown to have a significant advantage over batch and fed-batch processes in the maintenance of the biodesulfurization activity for an extended period of time. Specif...

The advantages of mixed feeds of sorbitol and methanol for the production of recombinant proteins with Pichia pastoris were analyzed quantitatively. The influence of the methanol-sorbitol ratio in the feed medium was investigated on growth stoichiometry and recombinant protein productivity with a P. pastoris Mut(+) strain secreting avidin by perfor...

Oilseed press-cake (PC) is proposed as a novel material for the removal of hydrophobic organic pollutants (HOPs) from water. Sorption of the pesticides carbaryl, atrazine and parathion, with log K(ow) being, respectively, 1.59, 2.55 and 3.83, was demonstrated using cold-pressed rapeseed (Brassica napus), moringa (Moringa oleifera) and soybean (Glyc...

A study of alginate lyase was carried out to determine if this enzyme could be used to remove alginate present in the core of alginate/poly-L-lysine (AG/PLL) microcapsules in order to maximize cell growth and colonization. A complete kinetic study was undertaken, which indicated an optimal activity of the enzyme at pH 7-8, 50 degrees C, in the pres...

In the methylotrophic yeast Pichia pastoris, alcohol oxidase (AOX) is a key enzyme involved in the dissimilation of methanol. Heterologous proteins are usually expressed under the control of the AOX1 promoter, which drives the expression of alcohol oxidase 1 in the wild-type strain. This study investigates the regulation of the alcohol oxidase enzy...

The main limitation in the use of spin-filters during perfusion cultures of animal cells was revealed to be filter fouling. This phenomenon involves cell-sieve interactions as well as cell attachment to, and growth on, the filter surface. The cell attachment effect has been analysed in the present study during long-term perfusion simulations with C...

In order to significantly reduce the time involved in mid-infrared spectroscopy calibrations, a novel approach based on a library of pure component spectra was developed and tested with an aerobic Saccharomyces cerevisiae fermentation. Instead of the 30-50 standards that would have been required to build a chemometric model, only five solutions wer...

Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-L: -Lysine microcapsules, containing recombinant CHO cells, have been investigated:...

Transient continuous cultures constitute a means to speed up strain characterization, by avoiding the need for many time-consuming steady-state experiments. In this study, mixed substrate growth on glycerol and methanol of a Pichia pastoris strain expressing and secreting recombinant avidin was characterized quantitatively by performing a nutrient...

Mid-infrared FTIR spectroscopy is an efficient tool for the monitoring of bioprocesses, since it is fast and able to detect many compounds simultaneously. However, complex and time-consuming calibration procedures are still required, and have inhibited the spreading of these instruments. A simple and quick method to calibrate a FTIR instrument was...

Due to its very high affinity to biotin, avidin is one of the most widely exploited proteins in modern biotechnological and biomedical applications. Since biotin is an essential vitamin for the growth of many microorganisms, we examined the effect of biotin deficiency on growth for a recombinant Pichia pastoris strain expressing and secreting a rec...

Tutorial published on: http://www.chemometrics.se/File/VirtualCalibrations.pdf

Atrazine, one of the most widely used pesticides, is a relatively hydrophobic organic pollutant (HOP) that contaminates ground and surface waters. Bioremediation of this pesticide by the bacterium Pseudomonas sp. strain ADP could successfully be performed in a pure aqueous system, with a specific growth rate of 0.0575 h−1 utilizing 100 mg/L of atra...

Pichia pastoris is increasingly used as a host for the expression of heterologous proteins both in academic research and at industrial scale. It is therefore important to have a good understanding of the regulation of its expression system. Usually, heterologous proteins are expressed under the control of the AOX1 promoter, which drives the express...

The stereospecific hydrolysis of cis-epoxysuccinate (CES) to L-(+)-tartrate, catalysed by the action of CES hydrolase activity in whole Nocardia tartaricans bacterial cells, has been chosen as a model biotransformation system. The performance of cells was studied by immobilization in polyelectrolyte complex (sodium alginate–cellulose sulfate–poly(m...

This paper attempts to review in how far thermodynamic analysis can be used to understand and predict the performance of microorganisms with respect to growth and bio-product synthesis. In the first part, a simple thermodynamic model of microbial growth is developed which explains the relationship between the driving force for growth in terms of Gi...

A novel reactive perstraction system has been developed based on liquid-core capsules, involving an enzyme-catalyzed reaction coupled with simultaneous in situ product recovery. Lipase-catalyzed reactions, hydrolysis of triprionin and nitrophenyl laurate, were selected to test the system and demonstrate the feasibility of immobilization of enzymes...

New bacteria were enriched and isolated from oil-contaminated soils and desulphurisation activity was compared with reported bacterial strains. Of the six different bacterial strains, a newly isolated Rhodococcus globerulus DAQ3 showed the highest activity and stability for desulphurisation of dibenzothiophene (DBT) in a two-phase system. The overa...

A new approach combining electrostatic and covalent bonds was established for the formation of resistant capsules with long-term stability under physiological conditions. Three kinds of interactions were generated in the same membrane: (1) electrostatic bonds between alginate and poly-L-lysine (PLL), (2) covalent bonds (amides) between propylene-gl...

The activity of penicillin acylase has been studied in aqueous and organic solvents, as free enzyme as well as immobilized within the membrane of liquid-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely phenyl acetic acid (PAA). In order to overcome this inhibition a range...

Hairy roots obtained by transformation via Agrobacterium rhizogenes provide an artificial plant material devoid of aerial parts with high growth on hormone-free media. Fundamental knowledge of hairy root physiology is essential to develop and control its culture. In contrast to shake-flask cultures, a bioreactor set-up combined with on-line data lo...

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions cont...

A novel perstraction system using liquid-core microcapsules for pesticide and herbicide removal from aqueous environments is proposed. The microcapsules contain an oil, dibutyl sebacate, surrounded by a hydrogel membrane. The extraction efficiency of the capsules was demonstrated with atrazine, methylparathion, ethylparathion, and 2,4-dichloro-phen...

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 relat...

In order to extend the shelf life of refrigerated dough, a new method based on thermo-gelling immobilization is proposed. This slows fermn. at low temps. (<10 DegC) while enabling it at ambient temp. The principle of immobilization is to create a phys. barrier between the substrate and the enzymes or the yeast in order to reduce the prodn. of CO2....

A novel chemistry has been developed for the production of capsules composed of a hydrophobic liquid core surrounded by a cross-linked polyacrylamide/alginate membrane. These liquid-core capsules may be used in capsular perstraction for the removal of inhibitory products from bioprocesses and bioconversions. They have the advantage of having a high...

Applying a vibration on a laminar jet for controlled break-up into monodisperse microcapsules is one among different extrusion technologies for encapsulation of animal and plant cells, microbes, enzymes and liquids [1]. The vibration technology is based on the principle that a laminar liquid jet breaks up into equally sized droplets by a superimpos...

Encapsulation is the process by which a gaseous, liquid or solid encapsulant, is surrounded by a continuous film or coating. As a result the core of the capsule contains the encapsulant, which is prevented from contact with the surroundings by the capsule wall or membrane. Capsules are frequently classified with respect to size: macro-, micro-, and...

In this work, dielectric spectroscopy was used to monitor two CHO perfusion culture experiments (B14 and B16). The capacitance of the cell suspension was recorded every 20 minutes over an excitation frequency range of 0.2 MHz to 10.0 MHz. A phase plot of the capacitance at a low excitation frequency vs. the value at a higher frequency proved to be...

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis r...

Since the yeast Phaffia rhodozyma was first described some 35 years ago, there has been significant interest in the development of commercial processes to exploit its ability to produce carotenoids (approximately 80% astaxanthin). However, the optimal conditions for carotenoid production are not well understood. A key limitation has been the lack o...

A novel in situ product removal (ISPR) method that uses microcapsules to extract inhibitory products from the reaction suspension is introduced into fermentation technology. More specifically, L-phenylalanine (L-Phe) was transformed by Saccharomyces cerevisiae to 2-phenylethanol (PEA), which is inhibitory toward the yeast. In order to continuously...

The influence of residual ethanol on metabolism of food grade Gluconacetobacter xylinus I 2281 was investigated during controlled cultivations on 35 g/l glucose and 5 g/l ethanol. Bacterial growth was strongly reduced in the presence of ethanol, which is unusual for acetic acid bacteria. Biomass accumulated only after complete oxidation of ethanol...

This contribution analyzes the position of biochemical engineering in general and bioprocess engineering particularly in the force fields between fundamental science and applications, and between academia and industry. By using culture technology as an example, it can be shown that bioprocess engineering has moved slowly but steadily from an empiri...

In many microorganisms, flux limitations in oxidative metabolism lead to the formation of overflow metabolites even under fully aerobic conditions. This can be avoided if the specific growth rate is controlled at a low enough value. This is usually accomplished by controlling the substrate feeding profile in a fed-batch process. The present work pr...

An in-situ, mid-infrared sensor was used to monitor the major analyte concentrations involved in the cultivation of Gluconacetobacter xylinus and the production of gluconacetan, a food-grade exopolysaccharide. To predict the analyte concentrations, three different sets of standard spectra were used to develop calibration models, applying partial le...

The bioconversion of L-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae in fed-batch experiments has shown that concentrations of 2-phenylethanol of >2.9 g/L have a negative impact on the oxidative capacity of the yeast. Without tight control on ethanol production, and hence on the feed rate, ethanol rapidly accumulates in the culture m...

The inhibitory impacts of the bioconversion of L-phenylalanine (L-Phe) to 2-phenylethanol (PEA), a very important natural aroma compound, on the metabolism of Saccharomyces cerevisiae Giv 2009 were investigated in batch and chemostat cultures. The bioconversion was found to be completely growth associated and lead to a maximal final PEA concentrati...

Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinki...

The effect of the presence of metabolism-induced concentration correlations in the calibration samples on the prediction performance of partial least-squares regression (PLSR) models and mid-infrared spectra from Chinese hamster ovary cell cultures was investigated. Samples collected from batch cultures contained highly correlated metabolite concen...

Calorimetry has shown real potential at bench-scale for chemical and biochemical processes. The aim of this work was therefore to scale-up the system by adaptation of a standard commercially available 300-L pilot-scale bioreactor. To achieve this, all heat flows entering or leaving the bioreactor were identified and the necessary instrumentation im...

For many years, calorimetry has been recognized as a powerful and universal tool for monitoring chemical and biological processes. A laboratory-scale reaction calorimeter (RC1, Mettler–Toledo), initially developed for chemical reaction studies with a sensitivity of 100–150 mW/l, has been improved to enable the monitoring of very low heat production...

Alginate-polylysine-alginate (APA) microcapsules are of particular interest for their application as implants or for bioreactor cultures. Although their formation has been widely studied, there is still a lack of quantitative data describing resistance, membrane thickness and permeability. In this study, the quantitative application of a Texture An...

Alginate beads, as well as microcapsules based on alginate, cellulose sulphate and polymethylene-co-guanidine, were produced at diameters of 0.4, 1.0 and 1.5 mm. These standard materials were tested, by independent laboratories, in regards to water activity, bead or capsule size, mechanical resistance and transport behaviour. The water activity and...

In Saccharomyces cerevisiae, the genes HXK2 and REG1 are believed to play a central role in carbon catabolite repression, acting on multiple pathways in the central metab. The present study aimed at quantifying the effect of deletion of genes HXK2 or REG1, resp., in the S. cerevisiae strain CEN.PK113-7D on the regulation of invertase and respirator...

A unique bench-scale heat flux calorimeter is described, which is capable of detecting the low heat signal of an animal cell culture (a max. of 200 mW/l is produced with a batch culture of SF9 insect cells). After the tech. modifications that enhanced the sensitivity of a com. heat flux reaction calorimeter, RC1 from Mettler-Toledo AG. Switzerland,...

The high-pressure freezing (HPF) technique was applied to the cryo-immobilization of alginate gels and the quality of the freezing analyzed on a TEM by comparison of the segregation pattern of samples of decreasing thickness. Dynamic simulations of heat transfer within an idealized slab of pure water surrounded by two walls of aluminium were perfor...

Calcium alginate beads were thermally treated at temperatures ranging from 25°C to 130°C for periods of up to 30 minutes. Important modifications to the structure of the alginate beads were shown to be a function of the temperature and period of incubation at each temperature. Modifications to the alginate beads included reduction in size, mechanic...

Spores of Bacillus subtilis ATCC 6051 and Bacillus stearothermophilus NCTC 10003 were immobilized in monodisperse alginate beads (diameter, 550 microm +/- 5%), and the capacity of the immobilized bioindicators to provide accurate and reliable F-values for sterilization processes was studied. The resistance of the beads to abrasion and heat was stro...

A math. model describing a novel sweeping gas membrane distn. (SGMD) configuration is presented and the sepn. of two volatile components from binary mixts. by this membrane process is theor. investigated. SGMD configuration is modified by using a thermostated sweeping gas in order to enhance the driving forces decrease along the module. A Stefan-Ma...

The bioconversion of L-phenylalanine (L-Phe) to 2-phenylethanol (PEA) by the yeast Saccharomyces cerevisiae is limited by the toxicity of the product. PEA extraction by a separate organic phase in the fermenter is the ideal in situ product recovery (ISPR) technique to enhance productivity. Oleic acid was chosen as organic phase for two-phase fed-ba...

Aq. solns. of formic acid were exptl. investigated in a modified sweeping gas membrane distn. (SGMD) configuration. A thermostated sweeping gas was used in order to enhance the mass transfer performance. A new tubular module was designed and built for this purpose. The effects of the relevant process parameters on the permeate flux and selectivity...

The effective biotechnol. process development depends on the system for monitoring the activity of the culture during cultivation. The feasibility of monitoring the metabolic flux during the cultivation of Bacillus thuringiensis var. galleriae (Btg) in pilot scale bioreactor was studied. All possible sources of heat generation and transfer in the s...

Animal cell (Chinese Hamster Ovary) concentration was determined on-line in a packed bed process using dielectric spectroscopy. This enabled the evaluation of the effect of temperature on specific metabolic rates during 3 months of continuous culture. The effect of low cultivation temperature on cell growth and metabolism was monitored, and the dat...

Dielectric spectroscopy was applied to two industrial high cell density culture processes and used to determine on-line the concentration of CHO cells immobilized on macroporous microcarriers in a stirred bioreactor and in a packed-bed of disk carriers. The cell concentration predicted from the spectroscopic data was in excellent agreement with off...