Analysis of broth rheology with cell morphology inCephalosporium fermentation

Summary Rheological properties inCephalosporium acremonium fermentation were quantitatively analysed. We obtained the correlation for consistency index (parameter of power law model) which could be estimated from the cell concentrations corresponding to morphological types. The correlated equation predicted the broth viscosity well.     

Theoretical analysis of the effect of cell recycling on recombinant cell fermentation processes.

A cell recycle system is studied for two-stage continuous fermentation. Cell recycle around the second stage provides higher cell concentrations than processes without recycle and a longer residence time of the cell, which is necessary for inducible products, especially in recombinant cell fermentation. Residence time distribution of the cell in the fermentor is important for the optimization of inducible products. The residence time distributions are studied for the cases with and without significant cell growth in the second stage. With cell growth in the second stage, three cases are considered. These are the cases of (1) zero residence time for two daughter cells after the cell division, (2) zero residence time of one daughter cell after the cell division and inherited residence time for the other daughter cell from the mother cell after the cell division, and (3) two daughter cells having the residence time of the mother cell after the cell division.     

Recycle in fermentation processes

Until recently, the recycle of the solid (microbial), liquid, or gaseous phases in microbiological processes has only been practiced rarely, with the notable exception of activated sludge processes for wastewater treatment, where recycling of a large fraction of the microbial phase is essential for process stability and performance. During the last decade, the economic impact of a number of politically motivated changes with respect to energy and feedstock costs and availability, and legislation directed towards markedly higher levels of environmental protection have encouraged the evaluation and subsequent development of recycle technology in the fermentation industry. Many of the developments have occurred in isolation and some have failed to result in either an improvement in process economics or any reduction in the quantity of pollutants discharged. This article seeks to review the present diversity of approaches to recycle technology in fermentation processes in order to provide a sensible basis for future developments.     

Effectors of amino acid transport processes in animal cell membranes

Various effectors, which act upon ion gradients, protein synthesis, membrane components or cellular functional groups, have been employed to provide insights into the nature of amino acid-membrane transport processes in animal cells. Such effectors, for example, include ions, hormones, metabolites and various organic reagents and their judicious use has allowed the following list of conclusions. Sodium ion has been found to stimulate amino acid transport in a wide variety of cell systems, although depending on the tissue and/or substrate, this ion may have no effect on such transport, or even inhibit it. Amino acid transport can be stimulated in some cell systems by other ions such as K+, Li+, H+ or Cl-. Both H+ and K+ have been found to be inhibitory in other systems. Amino acid transport is dependent in many cell systems upon an inwardly directed Na+ gradient and is stimulated by a membrane potential (negative cell interior). In some cell systems an inwardly directed Cl- and H+ gradient or an outwardly directed K+ gradient can energize transport. Structurally dissimilar effectors such as ouabain, Clostridium enterotoxin, aspirin and amiloride inhibit amino acid transport presumably through dissipation of the Na+ gradient. Inhibition by certain sugars or metabolic intermediates of the tricarboxylic acid cycle may compete with the substrate for the energy of the Na+ gradient or interact with the substrate at the carrier level either allosterically or at a common site. Stimulation of transport by other sugars or intermediates may result from their catabolism to furnish energy for transport. Insulin and glucagon stimulate transport of amino acids in a variety of cell systems by a mechanism which involves protein synthesis. Microtubules may be involved in the regulation of transport by insulin or glucagon. Some reports also suggest that insulin has a direct effect on membranes. In addition, a number of growth hormones and factors have stimulatory effects on amino acid transport which are also mediated by protein synthesis. Steroid hormones have been noted to enhance or diminish transport of amino acids depending on the nature of the hormone. These agents appear to function at the level of protein synthesis. While stimulation may involve increased carrier synthesis, inhibition probably involves synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system.(ABSTRACT TRUNCATED AT 400 WORDS)     

A direct computer control concept for mammalian cell fermentation processes

In the last 10 years, new assignments and the special demands of mammalian cells to the culture conditions caused the develoepment of complex small scale fermentation setups. The use of continuous fermentation and cell retention devices requires appropriate process control systems. An arrangement for control and data-acquisition of complex laboratory-scale bioreactors is presented. The fundamental idea was the usage of a standard personal computer, which is connected to pumps, valves and sensors via ADA-transformation. The possibility of free programming allowed the development of user-oriented software, especially designed for the far-reaching requirements of a university laboratory in the field of animal cell culture. Control of aeration, pumps, data-acquisition and data-storage are combined within one program, which allows the automation of standard operations like measurement of k_La- or OTR-values. Pump control algorithms for all common fermentation strategies (batch, fed batch, chemostat, perfusion) are included and can be selected any time during cultivation. Oxygen partial pressure and pH are controlled via direct digital control (ddc), providing simple adaption of control parameters and set points to current fermentation conditions.     

Kinetics of fermentation processes

Kinetic studies on fermentation processes were made and a general equation of production rate was newly presented applying the kinetic theory on mierobial cell growth which was reported previously by the authors.^l,2 Equations for product concentration in fermentation time courses were derived by developing mathematically the general equation of production rate, and characteristic properties of fermentation processes were clarified. Some examples of fermentations were analyzed kinetically using the new kinetic theory. The calculated values of product, and cell concentrations were in good agreement with the observed values.     

Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus

Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u(t) of 1.01-2.71 ms(-1)) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production. The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers. Pellets of approximately 1200 microm initial diameter were reduced to a final stable size of approximately 900 microm when the agitation intensity was >/=600 rpm (u(t)>/=2.03 ms(-1)). A stable pellet diameter of approximately 2500 microm could be attained in less intensely agitated cultures. These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air. Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere. This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin. Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function. The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers. Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.     

Interactions of sodium transport, cell volume, and calcium in frog urinary bladder

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

Importance of bacteriophages in fermentation processes

Any bacterial strain can be infected by virulent phages or harbour one or more prophages. Therefore, bacteria-phage interactions are to be regarded as fundamental properties of bacteria. In current industrial fermentation processes phages can be advantageously employed for the identification of bacterial production strains (phage typing). In some cases phages are involved in the production of enzymes and special substances. The fundamental importance of phages in any technical fermentation process, however, is based on the peculiarities of their obligately parasitic life cycle. The propagation of phages in fermentation processes can cause complete (or at least partial) lysis of the production strains and, consequently, serious disturbances in the production process and considerable economic losses. The phage problem in the fermentation industry has not yet been completely solved. For the protection of technical processes against virulent phages five measures are discussed: phage-protected sterile fermentation, employment of alternative cultures, employment of phage-resistant mutants, employment of phage inhibitors, and employment of immobilized bacterial cells. The problem of the protection of bacterial production strains from prophage induction is more difficult and practically unsolved. Two possibilities to minimize the process risk due to temperate phages, the elimination of inducing factors during the fermentation process, and the selection of production strains which are difficult to induce, are discussed.     

A correlative evaluation of morphology and rheology of<Emphasis Type="Italic">Aspergillus terreus</Emphasis> during lovastatin fermentation

Lovastatin, a secondary metabolite, was produced by fermentation process usingAspergillus terreus in an internal loop airlift reactor. It is a highly aerobic fermentation process. Biomass concentration and cell morphology were evaluated and observed to contribute significantly to the high viscosity and pseudoplastic non-Newtonian behavior of the broth. Typical morphological changes over 10 days in the fermentation broth were studied. The viscosity increased from the start of the fermentation with an increasing cell mass content, reached to a maximum of 60 N/m²·s at 160 h and then declined after the branching of the hyphae with the formation of arthrospores. Rheological parameters like consistency index and fluidity index were evaluated. The consistency index was observed to increase from 9.8 to 66.85 N/m², while fluidity index decreased from 0.69 to 0.48 s⁻¹ during 10 days of lovastatin production. A correlation between growth and consistency index of the broth has been evaluated.     

On the controllability of continuous fermentation processes

Simulation may be used as a powerful tool for accelerating bioprocess design. This paper demonstrates the use of simulations in exploring the nature and impact of the interactions that exist in a typical bioprocess for the recovery of an intracellular protein. The study shows that an integrated approach to design must be adopted in order to achieve acceptable process designs. Data from a fed-batch fermentation, with verified models for cell harvesting, cell disruption and cell debris removal have been integrated to demonstrate the consequence of process design and operating decisions on the resulting process performance. The trade-offs between product recovery and the extent of cell debris removal for a range of operating conditions have been represented through a series of windows of operation which show how process conditions must be altered in order for given process performance levels to be realised. The capacity to account for process performance including the impact of interactions is seen as a pre-requisite for rigorous bioprocess sequence design and optimisation.     

Sliding-mode control of fermentation processes

Continuous fermentation processes described by two nonlinear differential equations with uncertain parameters are considered. Sliding mode control design for these processes is proposed. The control design is carried out with direct use of nonlinear model, expert knowledge and on-line measurement of output variable only. Chattering phenomena are avoided by realizing the sliding mode with respect to the control input derivative. The excellent performance of presented control is proved through simulation investigations.     

Knowledge-based control of fermentation processes

A decade has passed since the first applications of a knowledge-based approach to the control of bioprocesses were reported. During this period, both the development and application of intelligent control in biotechnology have undergone remarkable evolution in terms of concepts, objectives, and tools. Stimulated by rapid progress in the field of real-time expert systems, knowledge-based methodology for the control of fermentation processes has now reached a more mature phase. A growing interest among the biotechnology community and intensive, realistic, and fruitful research being undertaken both in universities and in industry suggest that large-scale application of knowledge-based systems for the control of bioprocesses is inevitable. This article provides a concise summary of the main achievements in this new area and discusses recent trends, porblems, and perspectives.     

Studies on fermentation processes in silage

Zusammenfassung Die Menge vergärbarer Kohlenhydrate in Klee und Luzerne ist meistens zu gering, um eine zureichende Milchsäuregärung zu gewährleisten. Nur ein geringer Teil der gesamten Kohlenhydrate des Grünfutters wird zu Milchsäure vergoren. Durch Hydrolyse kann ein Teil der nicht direkt vergärbaren Kohlenhydrate für die Milchsäurebakterien zugänglich werden. Eine solche Hydrolyse scheint bei der Grünfuttergärung vor sich zu gehen, besonders bei einem Material mit relativ hohem Trockensubstanzgehalt, wahrscheinlich weil bei den hohenph-Werten, die in solchem Grünfutter vorliegen, die Pflanzen- und Bakterien-Carbohydrasen aktiv sind und weil die Carbohydrasen produzierenden Bakterien am Anfang der Gärung gut wachsen.Zusatz von verschiedenen Zuckerarten, wie Glucose, Maltose, Saccharose und auch Dextrin, begünstigen die Milchsäuregärung. Verschiedene Stärkearten, in fester Form fein verteilt zugesetzt, werden von den Pflanzen- und Bakterienamylasen nicht oder nur unbedeutend angegriffen und können deshalb durch die Milchsäurebakterien nicht vergoren werden. Malzamylasen vermögen dagegen solche Stärkearten zu hydrolysieren und sind im Grünfutter hoch aktiv. In allen Versuchen begünstigt deshalb Zusatz von Malz oder Malz + Stärke die Milchsäuregärung in hohem Grade. Wahrscheinlich vermögen die Amylasen und die übrigen Carbohydrasen des Malzes auch einen Teil der Polysaccharide des Pflanzenmaterials zu hydrolysieren.     

Metabolic regulation of fermentation processes

To compete in nature against other forms of life, microorganisms possess regulatory mechanisms which control production of their metabolites, thus, protecting against overproduction and excretion of these primary and secondary metabolites into the environment. To effect such an economical form of life, they possess regulatory mechanisms which control production of these metabolites and protect against overproduction and excretion into the environment of excess concentrations. In the field of industrial fermentation, the opposite concept prevails. Fermentation microbiologists search for a rare overproducing strain in nature, then further deregulate the microorganism so that it overproduces huge quantities of a desired commercially important product such as a metabolite or an enzyme. Deregulation is brought about by nutritional as well as classical and molecular genetic manipulations to bypass and/or remove negative regulatory mechanisms and to enhance positive regulatory mechanisms. These mechanisms include induction, nutritional regulation by sources of carbon, nitrogen and phosphorus, and feedback control. The controls and their modification by biotechnologists are the subjects of this review.     

Studies on fermentation processes in silage

Zusammenfassung Die Beimischung von mäßigen Mengen von Mehl verschiedener Getreidearten in Silage fördert die Milchsäuregärung nicht in nennenswertem Grad, da rohe Stärke nur in unbedeutendem Umfang von den Amylasen (hauptsächlich -Amylasen) des Getreidemehls oder des Grünfutters in für Milchsäurebakterien vergärbaren Zucker hydrolysiert werden kann. Durch -Amylase, welches Enzym in Malz in großen Mengen vorkommt, wird dagegen die rohe Stärke derart angegriffen, daß dadurch in der Silage ein fortgesetzter Abbau zu Milchsäure durch die sich dort befindenden Milchsäurebakterien erfolgt.Durch Beimischung verhältnismäßig geringer Mengen enzymreichen Malzmehles ist es demnach unter praktischen Bedingungen möglich, einen großen Teil der Stärke, welche dem Futter mit den verschiedenen Getreidemehlen zugeführt wurde, den Milchsäurebakterien zugänglich zu machen. Die starke amylolytische Enzymaktivität bei Mischungen von Malz-und Getreidemehl ist ein additiver Effekt von der -Amylase des Malzes und der -Amylase des Malzes, des Getreides und des Grünfutters.     

Studies on fermentation processes in silage

Zusammenfassung Kohlenhydrat-Zusatzmittel dienen bei der natürlichen Gärfutterbereitung einerseits als Nährstoffe für die Mikroorganismen, andererseits beeinflussen sie die Feuchtigkeit des Gärfutters durch ihr Absorptionvermögen.Bei der Milchsäuregärung kann Zucker vollständig ausgenützt werden, Stärke und Cellulose dagenen nicht, oder nur unbedeutend. Zusatz von Gerstenmalz hat wegen seines höheren Zuckerinhaltes und wegen seiner diastasewirkung einen wesentlich besseren Effekt als Gerstegrütze.Malz, Grütze und Cellulose besitzen ein hohes Absorptionsvermögen und können dadurcheine günstige Wirkung bei der Gärfutterbereitung ausüben.

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These studies are still in progress. We are indebted to the Swedish Foundation: Fonden för främjande av forskinings- och försöksverksamheten på jordbrukets område, for generous financial support.