A general robot control system for automatic chemical analysis

Advances in instrumentation have led to increasing interest in automatic chemical analyses. Unfortunately, the potential flexibility of robot systems can often not be exploited because of complex programming languages and poor user-interface. These factors generally limit the use of laboratory robots to a few repetitive tasks. This paper describes the development of a general robot control system for automatic chemical analysis. The program is written in Turbo Pascal and Visual Basic and runs in the Microsoft Windows environment. The analytical workspace is designed by selection of an object's icon followed by its desired orientation. Positioning of this object is then performed using a ‘drag and drop’ procedure. The robotic procedure is set-up by selection of items of apparatus and their associated actions. The performance of the system is demonstrated by the determination for caffeine in tablet formulations using a continuous dilution and calibration scheme.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

Automated imunoanalysis systems for monitoring mammalian cell cultivation processes

Two different automated immunoanalysis systems are presented. Both are based on the principles of flow-injection analysis and were developed to provide reliable, rapid monitoring of relevant proteins in animal cell cultivation processes. One system uses a turbidimetric analysis, and the other employs a heterogeneous chemistry with immobilized immunocomponents. For both systems, the analysis time is in the range of a few minutes, and a complete analysis cycle, including triplicate analyses and various washing steps, is in the range of 20–30 minutes. Samples from cultivation processes can be analyzed directly without dilution. Quantitation of proteins such as rt-PA or monoclonal antibodies can be performed over an analyte concentration range of 1–1000 mg/L. Both systems were compared to conventional ELISA assays on microtiter plates. The turbidimetric analysis system also included a biosensor for simultaneous glucose determination.     

Fluctuations in continuous mammalian cell bioreactors with retention.

Continuous flow bioreactors with cell retention have been increasingly used for the cultivation of mammalian cells. The potential advantages of such bioreactors are high cell concentrations and volumetric productivities. In many reported cases, these systems have shown fluctuations in cell concentrations of various frequency and magnitude. To analyze the dynamics of the fluctuations, a model-based approach is followed. Simulations showed that large fluctuations in biomass resulted in response to fluctuations in the retention ratio when the system is operated at high dilution rate and high cell retention. The dependence of cell concentration fluctuations on variations in dilution rate and retention ratio was established by a cross-correlation statistical analysis on available experimental data. The slower dynamics and the fluctuation propensity of retention systems suggest that continuous culture without retention is more convenient for kinetic studies. In all likelihood, continuous culture with retention can be stabilized by controlling both the retention ratio and the dilution rate.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

Mixing by impeller agitation in continuous flow systems containing polysaccharide solutions

System response data for step changes in input tracer concentration have been obtained for two different impeller agitated continuous flow mixing systems containing aqueous polysaccharide solutions. The vessel volumes were 1.6 and 10.9 liters. Polysaccharide concentration, dilution rate, and impeller speed were varied according to a plan devised using dimensional analysis and assuming that bulk motion is the predominant mass transport mechanism in the system. The data show that this is not true and that serious errors may occur if scale-up calculations are based on assuming that bulk motion predominates. Under the operating conditions used, perfect mixing was not observed.     

Dynamic behavior in response to pH shift during anaerobic acidogenesis with a chemostat culture

To clarify the dynamic behavior of the anaerobic acid reactor in response to pH changes, a continuous cultivation was performed. By stepwise shifting the culture pH in the acid reactor from 6.0 to 8.0, the main products were changed from butyric acid to acetic and propionic acids. This phenomenon was reproducible, reversible and was not affected by the dilution rate. It was considered that the dominant microbial populations changed in the acid reactor due to the pH shift.     

Dynamic effects related to steady-state multiplicity in continuous Saccharomyces cerevisiae cultivations

The behavioral differences between chemostat and productostat cultivation of aerobic glucose-limited Saccharomyces cerevisiae were investigated. Three types of experiments were conducted: a chemostat, where the dilution rate was shifted up or down in stepwise manner; and a productostat, with either stepwise changed or a rampwise increased ethanol setpoint, i.e., an accelero-productostat. The transient responses from chemostat and productostat experiments were interpreted using a simple metabolic flux model. In a productostat it was possible to obtain oxido-reductive steady states at dilution rates far below Dcrit due to a strong repression of the respiratory system. However, these steady states could not be obtained in a chemostat, since a dilution rate shift-down from an oxido-reductive steady state led to a derepression of the respiratory system. It can therefore be concluded that the range of dilution rates where steady-state multiplicity can be obtained differs depending on the operation mode and that this dilution rate multiplicity range may appear larger in a productostat than in a chemostat. A more narrow multiplicity range, however, was obtained when the productostat was operated as an accelero-productostat.     

Comparative analysis of ethanolic fermentation in two continuous flocculation bioreactors and effect of flocculation additive

Two types of bioreactor using a flocculating strain of Saccharomyces cerevisiae and continuous ethanolic fermentation as model were compared in terms of start-up evolution, overall performance and power costs. Also, the effect of adding to the medium a polymer — Magna Floc LT25 — that increases floc porosity was studied. The main difference between the reactors lies on the system that is used to recycle the flocculated cells — one presents an external loop with mechanically forced recycling and the other has an airlift configuration with an internal loop. During start-up of both bioreactors, no significant differences between the fermentation kinetics were established, either with or without Magna Floc. In the airlift bioreactor no positive effect of the dilution rate on substrate uptake was observed. Concerning ethanol productivity, both systems behave in a similar way. The best ethanol productivity, 12.9 kg/kg/h, was obtained for the airlift system. This value is 7 times higher than in conventional systems and justifies the interest devoted to flocculation bioreactors. The results also indicate that the activity of the cells that are kept inside the airlift bioreactor is higher and compensates its lower cell retention capacity at higher dilution rates. The addition of Magna Floc to the medium causes a reduction on the ethanol yield on glucose for the external loop system, but allows for an increase in the maximal dilution rate for total glucose consumption. Such a behavior is not observed for the airlift system. The analysis of the power cost associated with the operation of the two bioreactors indicates that the differences between them are only relevant at laboratory and pilot scales. However, from an industrial scale point of view the airlift bioreactor is advantageous because no mechanical parts are involved in recycling.     

Neutral zone and range of motion in the spine are greater with stepwise loading than with a continuous loading protocol. An in vitro porcine investigation

Biomechanical testing of the spine has traditionally been performed to help understand the normal function of the spine as well as to evaluate the effects of injury and surgical procedures on spinal behaviour. The overall objective of this investigation was to compare traditional stepwise loading with the recently introduced continuous loading protocol, determining the effect of loading protocol on the mechanical behaviour of the spine. For all tests, a custom spine testing machine was used to apply pure moments of flexion extension, axial rotation, and lateral bending to a maximum of 2 Nm, using six porcine cervical spine specimens (C2-C4). Motions of C2 with respect to C4 were measured with an optoelectronic camera system. Motion parameters calculated were range of motion (ROM), neutral zone (NZ), and the ratio of NZ and ROM. The continuous loading protocol had smaller values for all motion parameters in each loading direction (p<0.05). ROM for the continuous test ranged between 88% and 93% of that of stepwise for the three loading directions. The continuous protocol NZ was 56-75% of that of the stepwise test. The findings of the study demonstrate that the two loading protocols provide differing spinal behaviours.     

Induction of interleukin-5 responsiveness in resting B cells by engagement of the antigen receptor and perception of a second polyclonal activation signal.

Experiments were performed to examine the nature of agents which could induce IL-5 responsiveness in small, resting splenic B lymphocytes. First, IL-5 increased plaque forming cell responses to the TI-1 antigen TNP-LPS. A second set of experiments using anti-IgM + LPS which allowed limiting dilution analysis showed induction of IL-5 responsiveness in about 20% of the resting B cell population. In the same system, IL-4 increased the percentage of proliferating cells by about 40%. A third system using the TI-2 analog conjugate anti-IgD-dextran (anti-delta-dextran) also rendered small, resting B cells responsive to IL-5. An additional system employing anti-IgM plus dextran sulfate, which also allowed limiting dilution analysis, induced IL-5 responsiveness in at least 10% of resting B cells. The features common to all four systems inducing B cell IL-5 responsiveness are at least twofold. Each system directly accesses the B cell antigen receptor and causes crosslinking. Second, each system also provides an additional polyclonal activating moiety, some of which may be similar to those in thymus independent antigens. These results suggest that some resting B cells may become IL-5 receptive after perception of at least two kinds of signals one of which perturbs sIg and the second being nonspecific and polyclonally activating.     

Data model for the elimination of matrix effects in enzyme-based flow-injection systems

This contribution presents a new conceptional enzyme-based flow injection analysis (FIA) system for the process and quality control of food processing and biotechnological systems. It provides the determination of different analytes in distinct process media on the base of a common experimental set-up. In contrast to known comparable systems, analysis is performed without the commonly used sample preparation and dilution steps. Instead, the adaptation to the necessary measurement range is realized by optimization of intrinsic system parameters. The central principle of the work presented is the elimination of occurring interferences by the heterogeneous matrix of the process sample. Based on a particular injection mode, the application of dehydrogenases as indicator enzymes and a specially developed data model using cognitive methods, cross sensitivities of the detector as well as disturbed reaction rates of the enzymes could be almost completely compensated. Two applications are presented, the analysis of ethanol in non-alcoholic beer and the online determination of D-/L-lactate during a lactic acid fermentation, which reveal the advantage of the developed system.     

Preparation and physicochemical characterization of dioctyl sodium sulfosuccinate (aerosol OT) microemulsion for oral drug delivery

The performance of dioctyl sodium sulfosuccinate (aerosol OT) in the development of a pharmaceutically acceptable, stable, self-emulsifying water continuous microemulsion with high dilution efficiency was assessed. A pseudoternary microemulsion system was constructed using aerosol OT/medium-chain triglycerides with oleic acid/glycerol monooleate and water. The model microemulsion was characterized with regard to its electroconductive behavior, eosin sodium absorption, interfacial tension, and droplet size measurements after dilution with water. The percolation transition law, which makes it possible to determine the percolation threshold and to identify bicontinuous structures, was applied to the system. The interfacial tension changes associated with the microemulsion formation revealed ultralow values up to 30% oil at a surfactant/cosurfactant ratio of 3∶1. Moreover, the investigated particle size and polydispersity using photon correlation spectroscopy after dilution with excess of the continuous phase proved the efficiency of the microemulsion system as a drug carrier that ensures an infinitely dilutable, homogeneous, and thermodynamically stable system.     

Production of 2,3-butanediol in a membrane bioreactor with cell recycle

Summary The production of 2,3-butanediol by Enterobacter aerogenes DSM 30053 was studied in a cell recycle system with a microfiltration module. Emphasis was put on the influence of oxygen supply, cell residence time, dilution rate, and pH. Under optimal conditions a productivity as high as 14.6 g butanediol + acetoin/l per hour was achieved with a product concentration of 54 g/l and a product yield of 88%. This productivity is three times higher than that of an ordinary continuous culture. The achievable final product concentration of a cell recycle system was limited by the accumulation of the inhibiting by-product acetic acid, which increased very rapidly at low dilution rate. To maximize product concentration a fed-batch fermentation was carried out with stepwise pH adaption at high cell density. A final product concentration of 110 g/l was obtained with a productivity of 5.4 g/l per hour and a yield of 97%.     

Effect of variable nutrient supply rates on the RNA level of a heterotrophic bacterial strain

The heterotrophic bacterial strain HIS 53 was grown in a continuous culture under chemostat conditions and at sinusoidal or stepwise variations of the dilution rate; aspartate, ammonium, and phosphate were the growth-limiting nutrients. Within a specific nutrient limitation the growth yield was constant and independent of the applied environmental conditions. Compared with the reference chemostat culture, sinusoidal variations of the dilution rate increased the cellular RNA level by 19%–53% dependent on the growth limitation; stepwise variations caused an increase of the RNA level by 28%–41%. It was hypothesized that under the variable environmental conditions in the natural habitat the physiological potential of the organism is enhanced by some such increase of the cellular RNA level. As a consequence these increased RNA levels influence the competition between heterotrophic bacteria and, as a result, also the composition of the population of heterotrophic bacteria.     

Metabolic flux analysis of hybridoma continuous culture steady state multiplicity.

Physiological state multiplicity was observed in continuous cultures of the hybridoma cell line ATCC CRL-1606 cultivated in glutamine-limited steady state chemostats. At the same dilution rate (0.04 h-1), two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations (7.36 x 10(5) and 1.36 x 10(6) cells/mL). Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the TCA cycle for energy generation. The low-efficiency steady state was achieved after innoculation by growing the cells in a nutrient rich environment, first in batch mode followed by a stepwise increase of the dilution rate to its set point at 0.04 h-1. The high-efficiency steady state was achieved by reducing the dilution rate to progressively lower values to 0.01 h-1 resulting in conditions of stricter nutrient limitation. The high energetic efficiency attained under such conditions was preserved upon increasing the chemostat dilution rate back to 0.04 h-1 with a higher nutrient consumption, resulting in approximate doubling of the steady state cell concentration. This metabolic adaptation is unlikely due to favorable genetic mutations and could be implemented for improving cell culture performance by inducing cellular metabolic shifts to more efficient flux distribution patterns.     

Detection of suppressor T lymphocytes and estimation of their frequency in limiting dilution assays by generalized linear regression modeling

The estimate of the frequency of suppressor T lymphocytes in unfractionated cell populations remains challenging, mainly because these regulatory cells do not display specific immunophenotypic markers. In this paper, we describe a novel theoretical approach for quantifying the frequency of suppressor cells. This method is based on limiting dilution data modeling, and allows the simultaneous estimation of the frequencies of both proliferating and suppressor cells. We used previously published biological data, characterizing the inhibiting activity of suppressor T cell clones. Starting from these data, we propose a mathematical model describing the interaction between suppressor and proliferating T cells, and applied to a Poisson process. Limiting dilution data corresponding to this non-single-hit, suppressor two-target Poisson model were artificially generated, then modeled according to a generalized linear regression procedure. Deviation from the single-hit Poisson model was revealed by a statistical slope test, and a stepwise analysis of the regression appeared to be an efficient method that strongly argued in favor of the presence of suppressor cells. By using the frequency of proliferating T cells calculated in the first step of the regression, we demonstrated the possibility to provide a reasonable estimate of the frequency of suppressor T cells. Based on these findings, a practical decision-making procedure is given to perform standard analyses of limiting dilution data.     

Continuous-culture studies of synthesis and regulation of extracellular beta(1-3) glucanase and protease enzymes from Oerskovia xanthineolytica

This article describes the synthesis and regulation of beta(1-3)glucanase and protease enzymes from the cell lytic system of Oerskovia xanthineolytica LL-G109 in continuous culture using different concentrations of carbon source (glucose) and inducer (glucan). These two enzyme activities are the main components of a lytic system capable of lysing and disrupting whole yeast cells; it is subject to catabolite repression by glucose and is induced by yeast glucan. Peaks of beta(1-3)glucanase and protease activity are obtained at dilution rates of between 0.05 and 0.15 h(-1). The glucanase-protease ratio is very high compared to other strains. At dilution rates above 0.15 h(-1) all activities are similar to those obtained in batch culture. The lytic enzyme system appears to contain several beta(1-3)glucanase enzymes. In continuous culture both productivity and enzyme concentrations are greatly in creased when compared to batch culture, 11- and 4.4-fold, respectively.     

Development of yeast molecular display systems focused on therapeutic proteins, enzymes, and foods: functional analysis of proteins and its application to bioconversion

Molecular display systems using yeast have been developed for industrial, medical, pharmaceutical, and biological studies. Although several host cells are available to construct a molecular display system, the yeast Saccharomyces cerevisiae is a well-established and convenient organism in eukaryotes. A wide variety of prokaryotic and eukaryotic proteins have been displayed on yeast cell surfaces. In addition, functional analyses and applications to bioconversion have been performed on the cell surface, and cells are conveniently engineered by molecular display systems. In this review, we focus on the yeast molecular display system with regard to therapeutic proteins, several enzymes, and food ingredients. In addition, recent patents on molecular display using yeast cell for production of those compounds, screening technology and related techniques are introduced. Development of devices for functional analysis of created and modified proteins in the yeast display system is also described.     

Continuous glutamate production using an immobilized whole-cell system

For the purpose of saving the energy and raw materials required in glutamate fermentation, an immobilized whole-cell system was prepared and its performance in a continuous reactor system was evaluated. Corynebacterium glutamicum (a mutant strain of ATCC 13058) whole cell was immobilized in K-carrageenan matrix and the gel structure was strengthened by treatment with a hardening agent. The effective diffusivities of carrageenan gel for glucose and oxygen were found to decrease significantly with an increase in carrageenan concentration, while the gel strength showed an increasing trend. Based on the physical and chemical properties of carrageenan gel, the immobilization method was improved and the operation of the continuous reactor system was partially optimized. In an air-stirred fermentor, the continuous production of glutamate was carried out. The effect of the dilution rate on glutamate production and operational stability were investigated. The performance of the continuous whole-cell reactor system was evaluated by measuring glutamate productivity for a period of 30 days; it was found to be far superior to the performance of conventional batch reactor systems using free cells.