Extraction of primary and secondary metabolites.

Several new methods besides the usual organic solvent extraction have been developed over the last few years for the extraction of primary and secondary metabolites. These are: alcohol extraction with various biocompatible solvents, recovery of carboxylic acids and antibiotics with reactive extraction, dissociation extraction, aqueous two-phase extraction, and supercritical and near critical fluid extraction. Extraction and re-extraction processes are integrated into a single step by emulsion liquid membrane and solid supported liquid membrane extractions. These extraction processes are discussed and compared in this review, along with extraction with reversed micelles, and reactive extraction with the formation of a third phase at the organic-aqueous interface.

Investigations of the production of cephalosporin C by Acremonium chrysogenum.

A review is given on the morphology of Acremonium chrysogenum and the biosynthesis of cephalosporin C based on the published references. Investigations are presented on the comparison of cultivation media carried out by means of shake flask cultures. The process performance of a standard cultivation in well controlled bioreactor is presented and compared with other cultivations, which were executed with the same strain and bioreactor, but with various carbon-, nitrogen- and sulphur-sources keeping the concentrations of the key components at definite levels. Also the influence of dilution and enrichment of the medium on the process performance is explored. Mathematical models for the growth of Acremonium chrysogenum and production of cephalosporin C are reviewed and their application for control of industrial processes with complex cultivation media are discussed.

Modeling of the controlled expression of a harmful protein by a three-plasmid harboring system.

A genetically structured mathematical model was developed and used to evaluate the influence of molecular parameters involved in the expression of a harmful recombinant protein (SPA::EcoRI). The system consists of the controlled expression of the endonuclease EcoRI cloned in the plasmid pMTC48. The control is exerted by the lambda CI repressor expressed from the plasmid pRK248cIts. The deleterious effect of the activity of the enzyme EcoRI on the host DNA is prevented by the action of the EcoRI methylase that is expressed constitutively from a third plasmid, pEcoR4. The model includes molecular mechanisms involved in the regulation of the expression of these genes and is used to determine cultural conditions that maximize the production of the recombinant protein.

In-situ-fluorescence-probes: a useful tool for non-invasive bioprocess monitoring.

Optical sensors appear to be very promising for different applications in modern biotechnology. They offer the possibility to interface all the well known optical analysis techniques to bioprocesses via fiber optical cables. Thus, high sophisticated and sensitive optical analysis techniques can be coupled to a bioprocess via these light signal transporting fibers. A wide variety of sensor types for application in biotechnology has been described. Normally these sensors are non-invasive and the response times are nearly instantaneous. In particular, the use of glass fiber technology makes these sensors small, robust and reduces their costs.

Progress in monitoring, modeling and control of bioprocesses during the last 20 years.

The paper gives a review on the recent development of bioprocess engineering. It includes monitoring of product formation processes by flow injection analysis, various types of chromatographic and spectroscopic methods as well as by biosensors. The evaluation of mycelial morphology and physiology by digital image analysis is discussed also. It deals with advanced control of indirectly evaluated process variables by means of state estimation/observer, with the use of structured and hybrid models, expert systems and pattern recognition for process optimization and gives a short report on the state of the art of metabolic flux analysis and metabolic engineering.

Development of bioreaction engineering.

In addition to summarizing the early investigations in bioreaction engineering, the present short review covers the development of the field in the last 50 years. A brief overview of the progress of the fundamentals is presented in the first part of this article and the key issues of bioreaction engineering are advanced in its second part.

Recovery of proteins and microorganisms from cultivation media by foam flotation.

Foaming is often present in aerated bioreactors. It is undesired, because it removes the cells and the cultivation medium from the reactor and blocks the sterile filter. However, it can be used for the recovery of proteins and microorganisms from the cultivation medium. The present review deals with the characterization of model protein foams and foams of various cultivation media. The suppression of foaming by antifoam agents and their effect on the oxygen transfer rate, microbial cell growth and product formation are discussed. The influence of process variables on the recovery of proteins by flotation without and with surfactants and mathematical models for protein flotation are presented. The effect of cultivation conditions, flotation equipment and operational parameters on foam flotation of microorganisms is reviewed. Floatable and non-floatable microorganisms are characterized by their surface envelope properties. A mathematical model for cell recovery by flotation is presented. Possible application areas of cell recovery by flotation are discussed.

Integrated processing of biotechnology products.

Integrated bioprocessing in which a potentially inhibitory product is continuously removed from the fermentation broth as it is produced, has important advantages in improving yield and conversion relative to conventional processes. This review discusses integrated processing for ethanol, butanol, organic acids, antibiotics, and other products. A variety of recovery operations can be used to isolate the product, as discussed. Use of some of the available options is compared.

On-line and off-line monitoring of the production of cephalosporin C by Acremonium chrysogenum.

Process monitoring of cephalosporin C formation by Acremonium chrysogenum in laboratory investigations is considered. The goal of these investigations is the identification of bottlenecks in the biosynthesis and the improvement of the process performance. Based on reports of other research groups and own experience the key parameters were selected, which influence the process performance. They are: dissolved oxygen and pH values. In addition the concentrations of biomass, DNA, glucose and reducing sugars (glucose, maltose, maltotriose and oligosaccharides), methionine, other nitrogen sources (ammonium ion, other amino acids), organic acids, phosphate, sulfate, dissolved organic carbon, proteins, product and precursors in the cell free cultivation medium are monitored. In addition the intracellular concentrations of RNA, DNA, proteins, amino acids as well as the activities of the enzymes of the biosynthesis of cephalosporin C are determined. The influence of these parameters on the biosynthesis is discussed.

Flow-injection sandwich ELISA for bioprocess monitoring.

A fully automated flow-injection immunoassay based on sandwich enzyme-linked immunosorbent assay (ELISA) is described for the model system: protein G-sepharose, rabbit IgG and horseradish peroxidase (HRP)-labelled protein A. After injecting rabbit IgG and HRP-labelled protein A into a cartridge containing protein G-sepharose sequentially, a mixture of hydrogen peroxide and the redox indicator, 2.2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) is passed through the cartridge. The HRP-labelled protein A bound in the cartridge is directly proportional to the concentration of rabbit IgG. The colour variation of ABTS caused during the reaction between HRP and H202 in the cartridge is detected photometrically. The whole assay procedure is controlled and evaluated by a computer. Rabbit IgG and HRP-labelled protein A are also detected by a fluorometer, which is introduced into the flow system. In the flow-injection sandwich ELISA, the slope of the calibration curve is 0.4491 in the range of 0 and 300 microg ml(-1) rabbit IgG, while it is 0.1274 in the heterogeneous immunoassay. So the flow-injection sandwich ELISA system is found to be more sensitive than a heterogeneous immunoassay for the monitoring of the model protein.

Influence of the process parameters on the morphology and enzyme production of Aspergilli.

Several papers have been published dealing with various fungi to determine their morphology, enzyme production or process performance. However, no publication considered all of these aspects simultaneously. In the case of the production of xylanase by Aspergillus awamori the interrelationship of various key parameters are investigated. The influence of the reactor type (shake flasks, stirred tank and airlift tower loop reactor), the medium composition (semisynthetic and complex medium with wheat bran of different sizes, respectively as well as different concentrations of phosphate), and the specific power input (stirrer speed) on the growth, morphology, physiology, and productivity of the fungus are investigated. The results reveal a complex interrelationship which explains why the published results are contradictory. Without considering all of the relevant parameters, it is not possible to make general conclusions.

The inhibitory mechanisms of glucose and carbon dioxide on the biosyntheses of penicillins and cephalosporins.

The inhibitory mechanism of glucose and CO2 on the biosyntheses of penicillins and cephalosporins is discussed in the present paper. 6-aminopenicillanic acid (6-APA) is considered to be an intermediate product, and the reaction between 6-APA and glucose may play an important role in the yield and rate of biosyntheses of beta-lactam antibiotics. According to this hypothesis the experimental phenomena taking place in biosynthesis of penicillin and cephalosporin, such as the inhibition by glucose and carbon dioxide and the reduction of the yield, can be satisfactorily explained. The stability of 6-aminopenicillanic acid (6-APA) in bicarbonate solution, the reaction of 6-APA with sugars, the determination of the concentration of the 6-APA-sugar compound and the effect of these reactions on the biosynthesis of penicillin G are investigated to present evidences for this hypothesis.

Gene expression enhancement due to plasmid maintenance.

Analysis of the chromosomic beta-galactosidase activity in strains of Escherichia coli with and without plasmids indicated that plasmid maintenance enhances gene expression. Cyclic AMP (cAMP) determinations confirmed that the gene enhancement observed in strains carrying plasmids was due to a small increase in the intracellular concentration of cAMP. Also, cells carrying plasmids displayed higher specific glucose uptake rates than did cells without plasmids. The increases in the expression of beta-galactosidase and the glucose uptake rate suggest a cAMP-mediated release of the glucose effect due to plasmid maintenance. Our results suggested that this effect is independent of the host and type and number of plasmids.

Segregated mathematical model for the fed-batch cultivation of a high-producing strain of Penicillium chrysogenum.

A new segregated mathematical model for the penicillin fed-batch process is presented and applied to the growth of the pellet-forming, industrially used high-producing strain Penicillium chrysogenum S2. The model comprises two kinds of biomass (growing and producing, nongrowing and still producing), cell lysis, and complex medium as an important substrate for primary growth. In accordance with our experimental observation, product formation is not inhibited by glucose, but related to the growth rate. Maintenance metabolism is cell age-dependent. The model was verified with two sets of experimental data including exhaust gas measurements while keeping the estimated parameters almost constant. The presented model derived from that of Bajpai and Reuss (1980, 1981) does not describe our data properly. The particular influence of the pellet structure on the model is discussed. The necessity of new model assumptions (e.g., different kinds of biomass, non-constant maintenance coefficient) is explained by cell damage by lysis and shear forces, and the succeeding repair processes.

Transient behaviour of baker's yeast during enforced periodical variation of dissolved oxygen concentration.

The aim of the investigation was to find out the influence of the variation of the dissolved oxygen concentration in the microenvironment of yeast cells on their physiological behaviour in small laboratory reactors and estimate their behaviour in large industrial reactors. Since the morphology of the laboratory and industrial yeasts differed considerably, their transient behaviour was investigated and compared. For this purpose, the strain Saccharomyces cerevisiae H620 and an industrial strain were cultivated on synthetic as well as on complex medium in batch operation during periodical variation of the dissolved oxygen concentration, monitoring the most important key parameters. Also the yeast was cultivated in batch as well as in continuous operation, the cell-containing culture medium was recirculated through a nonaerated loop at different recirculation rates (residence times of the cells in the loop), and the key operation variables were monitored. It was found that the transient behaviour of laboratory and industrial yeasts differed slightly. Since cells growing in batch culture are more sensitive to dissolved oxygen concentration variation than cells growing in continuous culture, the transient behavior of cells cultivated in batch operation varied from those in continuous operation. If the anaerobic phase was longer than 1 min, ethanol was produced. However, it was consumed during the aerobiosis again, provided that phase was considerably longer than the anaerobic phase. This means that the yeast cultivation was not influenced by the periodic operation of the dissolved oxygen. Judging from the measurements in large stirred tank and airlift tower loop reactors, in general, the cells would spend more time in the aerobic than in the anaerobic flow region, and they would spend less than 1 min in the anaerobic flow region. Therefore, no considerable effect of the periodically varied dissolved oxygen concentration on the cell cultivation can be expected in large-scale reactors. In the stirred tank-loop-system at high pumping rates/high frequencies of periodically varied dissolved oxygen concentration, unexpectedly, the formation of ethanol was observed, which might be caused by stress imposed on the cells.

Analysis of plasmid-DNA and cell protein of recombinant Escherichia coli using capillary gel electrophoresis.

Plasmid DNA prepared from cultivation samples of recombinant Escherichia coli was analyzed by capillary gel electrophoresis. We used this method to control the genetic stability during a fed-batch culture. The plasmid DNA and a standard DNA mixture with molecular weights in the size range of 3000-22,000 base pairs (bp) were analyzed in capillaries that were filled with solutions of non-cross-linked polyacrylamide. With this method the plasmid DNA from recombinant E. coli cultivation samples was analyzed within 30 min. Separation parameters such as gel concentration, capillary length and current were optimized for that purpose. The method was modified to allow the analysis of proteins. Crude cell lysate samples could be screened for the protein pattern within 15 min in sodium dodecyl sulfate-polyacrylamide filled capillaries. The method was also used to verify product purity after the down-stream process.

Fed-batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60-L working volume airlift tower loop reactor.

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60-L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony-forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl-thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria-Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity.

Cephalosporin C production by a highly productive Cephalosporium acremonium strain in an airlift tower loop reactor with static mixers.

The production of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC) and deacetylcephalosporin C (DAC), with a highly productive strain of Cephalosporin acremonium, was investigated in an 80-1 airlift tower loop reactor with four static mixer modules (Type SMV, Sulzer) (ATLRM) on a complex medium containing 50 g l-1 peanut flour (PF). The most important key parameters such as glucose concentration and cell mass concentration were monitored during a fed-batch cultivation process. The concentrations of products CPC, PEN N, DAOC an DAC were determined on line by HPLC. The influences of four motionless mixers on the dissolved oxygen concentration (DOC), oxygen transfer rate, the cell growth and the CPC production, as well as the reactor performance, were evaluated. The results were compared with the performance of an airlift tower loop reactor (ATLR) without static mixers as well as with a stirred tank reactor (STR). A comparison of cultivations in the ATLRM and ATLR with 50 g l-1 PF indicates that the obtained maximal CPC concentration and the (CPC + DAC + DAOC) concentration were 7% and 22% higher in the ATLRM (4.96 and 7.46 g l-1) than in the ATLR (4.63 and 6.13 g l-1) respectively. The maximal CPC volumetric productivity in the ATLRM (55.1 mg l-1 h-1) was also considerably higher than that in the ATLR (48.5 mg l-1 h-1). The specific power input was reduced from 2.36 to 1.5 kW m-3, the specific productivity pertaining to the power input was improved from 1.96 to 3.31 g W-1. On the other hand, cultivation in the ATLRM had a lower maximum CPC concentration and volumetric productivity than those in STR (7.2 g l-1 and 71.2 mg l-1 h-1) with the same medium due to the lower shear stress levels and the lower specific power input (1.5 vs. 3.0 kW m-3); but the specific power imput-based yield coefficient was in the ATLRM (3.31 g W-1) higher than in the STR (2.40 g W-1). By increasing the amount of PF, it was possible to enhance the CPC concentration and volumetric productivity in the STR. However, the performance of the ATLRM was limited to using a medium containing maximal 50 g l-1 PF because of the high viscosity of the medium, the limited energy input and thus the limited oxygen supply.

Production of fusion protein SpA::EcoRI in batch culture in a 60-L airlift tower loop reactor.

Escherichia coli JM103 carrying the expression plasmid pMTC48, repressor plasmid pRK248, and protection plasmid pEcoR4 was grown in a 60-L working volume airlift tower loop reactor on M9 minimal medium. Production of fusion protein SpA::EcoRI was induced by a temperature shift from 30 to 38 (optimum), 40, or 42 degrees C. The following parameters were monitored: cell mass concentration (X), total cell counts (TCC), number of colony-forming units (CFU), concentrations of glucose, acetate, ethanol, pyruvate, lactate, succinate, amino acids, and ammonia, and soluble and total protein content, as well as product concentration (enzyme activity of the fusion protein), dissolved oxygen concentration, oxygen utilization rate (OUR), CO2 production rate (CPR), respiration quotient (RQ), and volumetric mass-transfer coefficients (kLa). Product formation by temperature shift was only observed if LB concentrate was added to the culture at the same time the aeration rate was increased to avoid oxygen-transfer limitation. No product accumulation was observed with glucose and ammonia supplementation. During gene expression, X and TCC increased, CFU decreased, acetate increased, and the primary metabolite (ethanol, pyruvate, lactate) concentrations as well as OUR and CPR passed a maximum while RQ changed only slightly. These facts indicate that, during gene expression, the metabolic activity of the cell passes a maximum, and after that it decreases. With increasing aeration rate, the volumetric productivity increased, but the specific productivity with respect to the cell concentration decreased.