Application of balancing methods in modeling the penicillin fermentation

This paper shows the application of elementary balancing methods in combination with simple kinetic equations in the formulation of an unstructured model for the fed-batch process for the production of penicillin. The rate of substrate uptake is modeled with a Monod-type relationship. The specific penicillin production rate is assumed to be a function of growth rate. Hydrolysis of penicillin to penicilloic acid is assumed to be first order in penicillin. In simulations with the present model it is shown that the model, although assuming a strict relationship between specific growth rate and penicillin productivity, allows for the commonly observed lag phase in the penicillin concentration curve and the apparent separation between growth and production phase (idiophase-trophophase concept). Furthermore it is shown that the feed rate profile during fermentation is of vital importance in the realization of a high production rate throughout the duration of the fermentation. It is emphasized that the method of modeling presented may also prove rewarding for an analysis of fermentation processes other than the penicillin fermentation.     

Application of mass and energy balance regularities in fermentation

Material and energy balances for fermentation processes are developed based on the facts that the heat of reaction per electron transferred to oxygen for a wide variety of organic molecules, the number of available electrons per carbon atom in biomass, and the weight fraction carbon in biomass are relatively constant. Mass–energy balance equations are developed which relate the biomass energetic yield coefficient to sets of variables which may be determined experimentally. Organic substrate consumption, biomass production, oxygen consumption, carbon dioxide production, heat evolution, and nitrogen consumption are considered as measured variables. Application of the balances using direct and indirect methods of yield coefficient estimation is illustrated using experimental results from the literature. Product formation is included in the balance equations and the effect of product formation on biomass yield estimates is examined. Application of mass–energy balances in the optimal operation of continuous single-cell protein production facilities is examined, and the variation of optimal operating conditions with changes in yield are illustrated for methanol as organic substrate.     

Dynamic flux balance modeling of microbial co-cultures for efficient batch fermentation of glucose and xylose mixtures

Sequential uptake of pentose and hexose sugars that compose lignocellulosic biomass limits the ability of pure microbial cultures to efficiently produce value-added bioproducts. In this work, we used dynamic flux balance modeling to examine the capability of mixed cultures of substrate-selective microbes to improve the utilization of glucose/xylose mixtures and to convert these mixed substrates into products. Co-culture simulations of Escherichia coli strains ALS1008 and ZSC113, engineered for glucose and xylose only uptake respectively, indicated that improvements in batch substrate consumption observed in previous experimental studies resulted primarily from an increase in ZSC113 xylose uptake relative to wild-type E. coli. The E. coli strain ZSC113 engineered for the elimination of glucose uptake was computationally co-cultured with wild-type Saccharomyces cerevisiae, which can only metabolize glucose, to determine if the co-culture was capable of enhanced ethanol production compared to pure cultures of wild-type E. coli and the S. cerevisiae strain RWB218 engineered for combined glucose and xylose uptake. Under the simplifying assumption that both microbes grow optimally under common environmental conditions, optimization of the strain inoculum and the aerobic to anaerobic switching time produced an almost twofold increase in ethanol productivity over the pure cultures. To examine the effect of reduced strain growth rates at non-optimal pH and temperature values, a break even analysis was performed to determine possible reductions in individual strain substrate uptake rates that resulted in the same predicted ethanol productivity as the best pure culture.     

Application of dynamic flux balance analysis to an industrial Escherichia coli fermentation

We have developed a reactor-scale model of Escherichia coli metabolism and growth in a 1000L process for the production of a recombinant therapeutic protein. The model consists of two distinct parts: (1) a dynamic, process specific portion that describes the time evolution of 37 process variables of relevance and (2) a flux balance based, 123-reaction metabolic model of E. coli metabolism. This model combines several previously reported modeling approaches including a growth rate-dependent biomass composition, maximum growth rate objective function, and dynamic flux balancing. In addition, we introduce concentration-dependent boundary conditions of transport fluxes, dynamic maintenance demands, and a state-dependent cellular objective. This formulation was able to describe specific runs with high-fidelity over process conditions including rich media, simultaneous acetate and glucose consumption, glucose minimal media, and phosphate depleted media. Furthermore, the model accurately describes the effect of process perturbations—such as glucose overbatching and insufficient aeration—on growth, metabolism, and titer.     

Energy balance in solid state fermentation processes

It was applied a macroscopic energy balance to a solid state fermentation process and an electron balance in order to estimate the temperature and the heat evolved in the process. There were employed several equations that describe the development of the system and offer the possibility to design or control such fermentations.     

A macroscopic model describing yield and maintenance relationships in aerobic fermentation processes

The present paper presents a generalized treatment of the principles of elemental and enthalpy balances which are applied to aerobic fermentation processes. It is shown that strict relations do exist between the various yield factors of biomass or product on substrate, oxygen, carbon dioxide, and between the various maintenance coefficients. These relations are confirmed from the existing body of literature data on yield and maintenance coefficients. Another consequences of the application of elemental balances is the existence of limits for the maximum biomass yield on substrate and oxygen, which depend on the degree of reduction of the substrates with different degree of reduction. It appears from this model that substrates with a high degree of reduction are C limited and substrates with a low degree of reduction are energy limited. Finally the effects of temperature on yield and maintenance coefficients are analyzed from the existing body of literature data. It can be concluded that the maintenance coefficients follow an Arrhenius type of relationship and that yield is temperature independent. The literature data seem to indicate that a degree of reduction of about 4 is optimal for the carbon and energy needs for biomass formation.     

The electric charge of pigment granules in pigment cells

Black pigment cells called melanophores change colour in response to environmental changes and have lately been studied as promising biosensors. To further elucidate the intracellular processes involved in the colour changes of these cells, and to find optimal biosensing principles, the electric charge of intracellular pigment granules, melanosomes, has been determined in vitro by electrophoresis. Melanosomes from the two extreme states in the cell colour change (aggregated and dispersed melanosomes) were measured. The charge was found to be -1.5 x 10(-16) and -1.7 x 10(-16) C, aggregated and dispersed melanosomes, respectively, without significant difference between the two conditions. This charge is of the same order of magnitude as the one of 1000 electrons. The origin of the melanosome charge, and the use of these findings in new biosensor principles, is discussed.     

Electric charge balance mechanism of extended soluble proteins

Extended proteins such as calmodulin and troponin C have two globular terminal domains linked by a central region that is exposed to water and often acts as a function-regulating element. The mechanisms that stabilize the tertiary structure of extended proteins appear to differ greatly from those of globular proteins. Identifying such differences in physical properties of amino acid sequences between extended proteins and globular proteins can provide clues useful for identification of extended proteins from complete genomes including orphan sequences. In the present study, we examined the structure and amino acid sequence of extended proteins. We found that extended proteins have a large net electric charge, high charge density, and an even balance of charge between the terminal domains, indicating that electrostatic interaction is a dominant factor in stabilization of extended proteins. Additionally, the central domain exposed to water contained many amphiphilic residues. Extended proteins can be identified from these physical properties of the tertiary structure, which can be deduced from the amino acid sequence. Analysis of physical properties of amino acid sequences can provide clues to the mechanism of protein folding. Also, structural changes in extended proteins may be caused by formation of molecular complexes. Long-range effects of electrostatic interactions also appear to play important roles in structural changes of extended proteins.     

On the estimation of the pseudo-stoichiometric matrix for macroscopic mass balance modelling of biotechnological processes

In this paper we propose a methodology to determine the structure of the pseudo-stoichiometric coefficient matrix kappa in a macroscopic mass balance based model. The first step consists in estimating the minimal number of reactions that must be taken into account to represent the main mass transfer within the bioreactor. This provides the dimension of kappa. Then we discuss the identifiability of the components of kappa and we propose a method to estimate their values. Finally we present a method to select among a set of possible macroscopic reaction networks those which are in agreement with the available measurements. These methods are illustrated with three examples: real data of the growth and biotransformation of the filamentous fungi Pycnoporus cinnabarinus, real data of an anaerobic digester involving a bacterial consortium degrading a mixture of organic substrates and a process of lipase production from olive oil by Candida rugosa.     

Elucidation of enzyme control mechanisms using macroscopic measurements in a mixed substrate fermentation system

The objective of this work was to relate macroscopically measurable on-line fermentation parameters such as dissolved oxygen, off-gas oxygen and carbon dioxide, and cell mass, to the controlled production of key intracellular enzymes under carbon limited conditions. Both batch and perturbed batch aerobic fermentations were performed using two different strains of Escherichia coli, with glucose and lactose as the sole carbon sources. The two strains differed from each other only in the lac operon region of their genome. The parent strain, E. coli 3000, was inducible for the enzyme beta-galactosidase. The other strain, E. coli 3300, was a constitutive mutant in the production of beta-galactosidase. In all experiments, off-line assays of sugars and beta-galactosidase activity were performed. It was observed that there is a clear relationship between the macroscopic on-line measurements, dissolved oxygen tension, carbon dioxide evolution rate and oxygen uptake rate, and the microscopic control phenomena of catabolite repression, catabolite inhibition, and inducer repression.     

Effect of environmental pH on the fermentation balance of Lactobacillus reuteri

The influence of environmental pH on the regulation of glucose catabolism by Lactobacillus reuteri was examined in anaerobic batch cultures. Under acidic conditions both glucose consumption and end-products formation were low. Maximum biomass was reached at pH 5·0, with a specific growth rate of µ= 0·78 h^-1. The shift in pH values from 4.3 to 6.5 reflected an increase in glucose uptake as well as in the yield ( Y _p/x) of acetate, lactate and ethanol after 12 h of incubation. Ethanol was the major metabolite produced at all pH values assayed.     

Shifts in the fermentation balance of Lactobacillus reuteri. In the presence of glycerol

Summary The influence of glycerol (100 mM) on the growth kinetic and the end-products formation by Lactobacillus reuteri growing on maltose (29 mM) was examined in aerobiosis and anaerobiosis. The presence of glycerol in aerated cultures changed the maltose consumption but not the growth rate nor the fermentation balance. In a nitrogen atmosphere, L. reuteri was not able to grow on maltose alone. In the presence of both, maltose and glycerol, L. reuteri changed its fermentation pattern, producing 58 mM 1,3-propanediol as the major end product.     

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-- >E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s- 1.