Feedback identification of continuous microbial growth systems

The fundamental problem of dynamic modeling of continuous culture systems for process control and optimization is addressed. Forcing a system to bifurcation via feedback control is a very promising method for model discrimination and identification. Dynamic information is obtained by using this technique, the dynamic behavior of the chemostat as predicted by unstructured models, the model with delay, and a structured model has been analyzed. The method exposes significant differences in the nonlinear dynamic structure of the various models and can be implemented to discriminate between various possible models for a continuous culture system.     

The renaissance of continuous culture in the post-genomics age

The development of continuous culture techniques 60 years ago and the subsequent formulation of theory and the diversification of experimental systems revolutionised microbiology and heralded a unique period of innovative research. Then, progressively, molecular biology and thence genomics and related high-information-density omics technologies took centre stage and microbial growth physiology in general faded from educational programmes and research funding priorities alike. However, there has been a gathering appreciation over the past decade that if the claims of systems biology are going to be realised, they will have to be based on rigorously controlled and reproducible microbial and cell growth platforms. This revival of continuous culture will be long lasting because its recognition as the growth system of choice is firmly established. The purpose of this review, therefore, is to remind microbiologists, particularly those new to continuous culture approaches, of the legacy of what I call the first age of continuous culture, and to explore a selection of researches that are using these techniques in this post-genomics age. The review looks at the impact of continuous culture across a comprehensive range of microbiological research and development. The ability to establish (quasi-) steady state conditions is a frequently stated advantage of continuous cultures thereby allowing environmental parameters to be manipulated without causing concomitant changes in the specific growth rate. However, the use of continuous cultures also enables the critical study of specified transition states and chemical, physical or biological perturbations. Such dynamic analyses enhance our understanding of microbial ecology and microbial pathology for example, and offer a wider scope for innovative drug discovery; they also can inform the optimization of batch and fed-batch operations that are characterized by sequential transitions states.     

In vitro culture of Plasmodium berghei using a new suspension system

Attempts were made to develop techniques for the continuous in vitro culture of Plasmodium berghei. The candle jar method (Trager &; Jensen, 1976) proved to be insufficient for the culture of this rodent malaria parasite. Parasitaemia decreased rapidly after the first schizogonic cycle in culture. A simple suspension technique was developed using a newly designed culture apparatus which can be placed in the laminar-flow. All manipulations necessary for the refreshment of medium and cells can be made with almost no disturbance of the culture conditions. With this system it was possible to culture P. berghei repeatedly for more than a week, completing at least four schizogonic cycles with considerable mcrozoite invasion and a 2–6-fold multiplication. Infection rates of up to 6.0% were achieved and cultures were maintained for 9 days. Several specific properties of P. berghei and the differences between the candle jar method and the new suspension method are discussed to explain the results obtained in both systems.     

Multiplying steady-state culture in multi-reactor system

Cultivation of microorganisms in batch experiments is fast and economical but the conditions therein change constantly, rendering quantitative data interpretation difficult. By using chemostat with controlled environmental conditions the physiological state of microorganisms is fixed; however, the unavoidable stabilization phase makes continuous methods resource consuming. Material can be spared by using micro scale devices, which however have limited analysis and process control capabilities. Described herein are a method and a system combining the high throughput of batch with the controlled environment of continuous cultivations. Microorganisms were prepared in one bioreactor followed by culture distribution into a network of bioreactors and continuation of independent steady state experiments therein. Accelerostat cultivation with statistical analysis of growth parameters demonstrated non-compromised physiological state following distribution, thus the method effectively multiplied steady state culture of microorganisms. The theoretical efficiency of the system was evaluated in inhibitory compound analysis using repeated chemostat to chemostat transfers.     

The effect of lithium chloride on the biooxidation of aqueous methanol/acetone mixtures

Lithium chloride, more specifically the lithium cation, has been implicated in interference in biological systems. In the case of Escherichia coli, interference involves the Na+(Li+)/H+ antiporter transport system. The study reported here concerns the effects of LiCl on a mixed enrichment culture that is able to biodegrade both methanol and acetone under aerobic conditions. The results obtained using unsteady state continuous flow culture techniques demonstrate a significant disruptive effect of LiCl on culture performance. In addition, a reduction in the substrate-based biomass yield coefficient, which is a clear advantage as far as biotreatment process performance is concerned, also occurs. The ultimate fate of the LiCl was not determined.     

Marginal modeling of multilevel binary data with time-varying covariates

We propose and compare two approaches for regression analysis of multilevel binary data when clusters are not necessarily nested: a GEE method that relies on a working independence assumption coupled with a three-step method for obtaining empirical standard errors, and a likelihood-based method implemented using Bayesian computational techniques. Implications of time-varying endogenous covariates are addressed. The methods are illustrated using data from the Breast Cancer Surveillance Consortium to estimate mammography accuracy from a repeatedly screened population.     

The automation of culturing and harvesting of human chromosome specimens

This paper describes the development of a machine for the automated culturing and harvesting of human chromosome specimens. The machine is capable of handling different preparation methods simultaneously, such as standard blood cell, blast cell and bone marrow cultures, and can be programmed to perform the prophase synchronization techniques. It is composed of a culture tray, centrifuge, mixer, input and output station and a head assembly capable of transporting samples between the various stations and which is equipped with an aspirator needle to suck off the supernatant and fluid dispensers for the dosing of the various chemicals. A microprocessor system controls all hardware functions and schedules the manipulations of all samples thereby preventing, that more than one sample requires servicing at the same time. The samples (up to a maximum of 255) are processed by the machine in small batches of maximal 16, which can be activated at any time according to one of 16 culture procedures. The cytogeneticist can modify these procedures using a simple interpretive language specifying both the types of manipulations, such as centrifugation, addition of chemicals, sucking of supernatant and the minimal and maximal variation in time, which is allowed between two sequential manipulations. Besides the hardware setup and software organization of the machine, the first preliminary results and future prospects for the machine are presented.     

Multilayer adaptive control of continuous bioprocesses using optimising control technique. Case study: Bakers' yeast culture

High costs associated with many fermentation processes in an increasingly competitive industry make any prompt application of modern control techniques to industrial bioprocesses very desirable. However, this is often hampered by the lack of adequate mathematical models, on the one hand, and by the absence of continuous, on-line measurement of the most relevant process variables, on the other hand. This paper addresses these problems and offers a new strategy to control continuous bioprocesses using a hierarchical structure such that neither structured process models nor continuous measurement of all relevant variables have to be available. The control system consists of two layers. The lower layer represents a dynamic adaptive follow-up control of a continuously measured output — in our case dissolved oxygen concentration. This variable is supposed to be strongly correlated with the key output variable — in our case cellular concentration which is not continuously available for measurement. The higher layer is then designed to maintain a desired profile of the process key output using a set-point optimising control technique. The Integrated System Optimisation and Parameter Estimation method used operates on an appropriately chosen steady-state performance criterion. A prerequisite for successful application of the proposed approach is an approximate steady-state model, describing the relationship between the measured output and the process key output variable. Furthermore, occasional in situ, off-line or laboratory measurement values of the key output variable are needed. Promising simulation results of the biomass concentration control, by manipulating the air flow-rate in the continuous bakers' yeast culture are presented.     

Fast on-line polarity correlation algorithms for muscle fibre conduction velocity measurement

Polarity cross-correlation is a useful technique for the measurement of muscle fibre conduction velocity using surface electromyography. Owing to the nature and volume of computation involved in the correlation function, standard techniques for its estimation by a microprocessor are too slow for an on-line application. In this paper two algorithms suitable for on-line estimation of polarity function are presented. Some useful features of the correlation function, as well as careful programming and careful choice of instructions, made it possible to use a standard microprocessor to achieve higher sampling rates than those reported recently.     

The automation of culturing and harvesting of human chromosome specimens

Summary This paper describes the development of a machine for the automated culturing and harvesting of human chromosome specimens. The machine is capable of handling different preparation methods simultaneously, such as standard blood cell, blast cell and bone marrow cultures, and can be programmed to perform the prophase synchronization techniques.It is composed of a culture tray, centrifuge, mixer, input and output station and a head assembly capable of transporting samples between the various stations and which is equipped with an aspirator needle to suck off the supernatant and fluid dispensers for the dosing of the various chemicals. A microprocessor system controls all hardware functions and schedules the manipulations of all samples there-by preventing, that more than one sample requires servicing at the same time.The samples (up to a maximum of 255) are processed by the machine in small batches of maximal 16, which can be activated at any time according to one of 16 culture procedures. The cytogeneticist can modify these procedures using a simple interpretive language specifying both the types of manipulations, such as centrifugation, addition of chemicals, sucking of supernatant and the minimal and maximal variation in time, which is allowed between two sequential manipulations.Besides the hardware setup and software organization of the machine, the first preliminary results and future prospects for the machine are presented.In honour of Prof. P. van Duijn     

A versatile data acquisition system for physiological modelling of laboratory fermentation processes

Summary A versatile data acquisition system for physiological modelling of laboratory fermentation processes is given. This system meets the stringent qualifications of modern modelling techniques in terms of signal to noise ratios and sampling rates using readily available equipment. The data are portable to any data processing system for modelling and analysing the culture characteristics. Two completely equipped fermentation systems are sampled with a sampling period of six seconds. The data acquisition system is used to acquire data of a set of continuous fermenters showing sustained oscillations. Oscillation data was used to show the performance of the data acquisition system. With the high sampling rate signal to noise ratios of 30 dB or higher are achieved. It was possible to determine periods of oscillatory behaviour and delay times. The system has shown to be versatile in alaboratory set up. Addition or subtraction of sensors or complete fermentation systems can be done without major alterations to the system.     

Conditions for a continuous production of transient microbial products in a two-stage fermentation system

Summary The optimal residence time in the inducing reactor of a continuous two-stage system has been studied in order to maximize the yield in such a process. The attention has been focused on the case in which the product (or one of its forms) appears in the culture only transiently after the induction. Whereas in some cases the two-stage system is able to improve the yield and to allow a continuous product concentration in the outgoing stream, in others, and depending on how the product is accumulated in the induced culture, no optimal residence time can be found, and a higher productivity will be achieved by using the one-stage continuous strategy or a batch fermentation.     

Physiology of,an enzyme production by,plant cell cultures

Red kidney bean (Phascolus vulgaris) cells, derived from roof, callus, were grown in suspension culture in shake flasks and in laboratory fermentors using batch and continuous batch culture techniques. The medium contained casein hydrolysate, sucrose, inorganic salts, vitamins, and growth hormones. In continuous batch culture yields of up to 171 g wet weight, (8.5 g dry weight) per liter were obtained in 7 days. Organic nitrogen was used preferentially. Growth on nitrate was considerably slower than on organic nitrogen sources. Indole acetic and naphthalene acetic acids were not essential for good growth of the cells whereas kinetin and 2, 4-D were. The optimum pH for growth was about p11 4.5. The presence of amylase and peroxidase was detected in culture filtrates. Amylase activity was low in either the presence or the absence of starch in the medium. Peroxidase production could be related directly with growth of the culture. Maximum peroxidase yield, as measured by the guaiacol method and expressed as horse radish peroxidase, was 1.25 × 10^?8 M.     

Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system

We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols.     

A continuous-flow method of organ culture

Summary A method of perfusion organ culture is described in which explants cultured at the airmedium interface are bathed by a continuous flow of nutrient medium. Morphological studies on the fetal rat lung indicate that explant development in this system is comparable to that obtained using standard organ-culture dishes. Medium supply is easily manipulated and continuous sampling of the effluent stream is possible without disturbing the immediate explant environment. The basic design facilitates secretory-response studies on cultured organ explants as demonstrated by a study of glucose-stimulated insulin release by the neonatal rat pancreas. This work was supported by U. S. Public Health Service Training Grant No. GM 00114.     

Gallera method of chick embryo culture in vitro supports better growth compared with original New method

An avian embryo is a valuable model system for vertebrate embryology. Easy availability, accessibility to various developmental stages and amenability of organ fields makes the chick embryo one of the favored model systems. Seminal discoveries regarding organogenesis and vertebrate morphogenesis have been made using chick embryos cultured in vitro . Dennis A.T. New revolutionized chick embryo culture methodology with his development of a single glass ring explantation technique. Many modifications and/or embellishments were introduced after the New era of embryo culture. A double glass ring method for chick embryo culture introduced by Gallera and Nicolet is compared with the original New method and the EASY method in this study. In addition, a video of culture methods is presented as a valuable tool in learning about and/or teaching techniques of chick embryo culture.     

The Continuous Culture of Luminous Bacteria: a Luminostat

A method has been designed for the continuous culture of luminous bacteria. The control system for the culture uses a combination of luminescence and optical density as a light signal received by a photomultiplier. This combined signal operates pumps which exchange the growth medium. Using this method, a culture of brightly luminescing bacteria was maintained for periods up to 3 weeks.     

A Chamber Designed for Continuous, Long-term Monitoring of Legume Root Respiration

A chamber is described which allows for continuous, long-termmonitoring of legume root respiration by infrared gas analysisof CO₂ production. The chamber can accommodate either an automaticor manually operated irrigation system and is constructed fromreadily available raw materials. A series of trials throughout the entire growth period (66 d)of cowpea cv. K 2809 have demonstrated that vegetative growthand reproductive development are closely similar for plantsrooted in the respiration chamber and those grown in standardpots using conventional culture techniques. Ontogenetic changesin root respiration rate were recorded and are discussed inrelation to vegetative and reproductive periods of plant growth.     

Single-cell protein production from spent sulfite liquor utilizing cell-recycle and computer monitoring

Spent sulfite liquor (SSL), a waste product of the paper pulping industry, is produced at a rate of 1 ton (dry basis) per ton of pulp. The sugar content of SSL is about 30 g/L. To reduce the biological oxygen demand (BOD) of SSL before disposal, torula yeast (Candida utilis) is produced by a continuous culture process, the productivity of which is limited by sugar concentration and cell growth rate. To increase productivity, a recycle system has been designed and tested. Cells were sedimented continuously with a flocculating agent (bentonite) before being recycled to the fermentor. A bentonite concentration of 0.02 g/g cell was required. A computer monitoring system based on material balancing techniques was developed to monitor and control the recycle system. With this computer system, productivity was raised to 6.1 g/L h, with cell concentration up to 65 g/L in the recycle stream and 24 g/L in the fermentor. This represents a productivity increase of 150% over continuous culture with no recycle.     

Continuous Fermentation of Novobiocin

Continuous fermentation trials with Streptomyces niveus in a nine-stage fermentation system (7-liter reaction volume per stage) indicated that the cultures used gradually lost their ability to produce novobiocin when cultured over periods from 10 to 25 days. It was found that mycelial degeneration could be circumvented by operational means during continuous culture using the following technique: Two interchangeable 24-liter stages were installed at the front end of the nine-stage system and connected in parallel with the latter. Alternatingly one of these two tanks was then used as first stage of the continuous fermentation system. The holdup time in the first vessel was adjusted to limit cell growth chiefly to this stage so that most of the antibiotic production took place in subsequent stages. The first stages were switched at approximately weekly intervals. Each of the new tanks was prepared as a batch, inoculated with a high-producing cell population, and allowed to grow for 3 days before it was connected to the remaining system for continuous operation. Using this technique no evidence of culture degeneration was encountered in subsequent novobiocin production stages over a period of 33 days. In conventional runs without periodic replacement of the first stage, culture degeneration with the novobiocin fermentation occurred within a period of 10 to 25 days of continuous operation. This observation indicates that the described technique offers a solution to the problem of maintaining high steady-state titers in continuous novobiocin fermentations. Extension of this technique to other continuous fermentations where culture degeneration is a problem is indicated.