Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.     

The use of Aspergillus niger cultures for biotransformation of terpenoids

Aspergillus niger is a well-known fungus that has been used for many different biotransformations of organic compounds. The terpenoids include a large variety of natural hydrocarbons and their derivatives, mostly obtained from plant essential oils, but some obtained from animals or fungi. They may be acyclic or have one or more rings of various sizes, and they show a variety of biological activities that include antibacterial, antifungal, antiparasitic, antiviral, and anticancer activities. Terpenoids are classified as monoterpenoids (C₁₀), sesquiterpenoids (C₁₅), diterpenoids (C₂₀), triterpenoids (C₃₀), and others. This review summarizes experimental processes that use cultures of various A. niger strains to carry out stereoselective biochemical reactions in terpenoids, including related epoxides, lactones, N-phenylcarbamates, and saponins, to produce metabolites that may be useful as flavors and fragrances or as new experimental drug candidates. Cultures of A. niger that add hydroxyl, carbonyl, and other groups at specific positions or reduce double bonds have resulted in the production of valuable new compounds.     

Production of glucosyl-transferring enzyme by Aspergillus niger in batch cultures

Aspergillus niger produced an extracellular glucosyltransferase with a high transglucosylating activity. Among carbon sources tested, maltose gave the highest enzyme production: 0.20-0.26 units/ml.     

On-line state estimation of biomass based on acid production in Zymomonas mobilis cultures

The concentrations of biomass, substrate and product are very important state variables of almost every bioprocess and generally unable to be measured directly in?situ due to the lack of reliable sensors. In this paper, an adaptive observer of the biomass concentration is proposed for an anaerobic fermentation process where only the measurement of the acid product is available on-line. The observer was tested to be effective by several experiments under various operating conditions. In this experimental system, an auto-sampling device was connected between the bioreactor for the fermentation of Zymomonas mobilis and a HPLC so that the concentrations of glucose and ethanol could be directly measured through such implementation.     

Biomass estimation of Aspergillus niger growing on real and model supports in solid state fermentation

Summary Direct hydrolysis of Aspergillus niger mycelium growth on amberlite IRA-900 or sugar cane bagasse on solid state fermentation followed by the analysis of soluble protein by the dye binding method was carried out. Hydrolysis with phosphoric acid 0.25M during 7 min allowed maxima protein extraction available to be measured. Color interference of medium components was not observed, allowing the use of this method for biomass estimation when amberlite IRA-900 or sugar cane bagasse are used as support in solid state fermentation processes.     

Hybrid modeling approach to on-line estimation of yeast biomass concentration in industrial bioreactor

The industrial fed-batch yeast cultivation process has been divided into four different metabolic phases (adaptation, carbon limited, oxygen limited and maturation) by a neuro-fuzzy classification model that consists of 4 applied linguistic rules on 2 state variables: oxygen uptake rate and liquid volume. The membership functions have been automatically adapted by this fuzzy perceptron, i.e., by a supervised learning algorithm initialized by prior operator's knowledge. Process compartmentalization has made easier and more realistic a subsequent state estimation of the biomass concentration with separate artificial neural networks combined with balance equations. Static networks with local recurrent memory structures were used, and the inputs were standard cultivation state variables: respiratory quotient, molasses feed rate, ethanol concentration, etc. This hybrid approach is generally applicable to state estimation or prediction when different sources of process information and knowledge have to be integrated.     

Correlation of Aspergillus niger broth rheological properties with biomass concentration and the shape of mycelial aggregates

Aspergillus niger was grown in a 7-L chemostat at biomass levels of 7 to 9 gL(-1); dilution rates of 0.03, 0.05, 0.075, and 0.009 h(-1); and dissolved oxygen tensions of 7%, 12%, and 40% of air saturation. Broth rheological measurements were made on-line, while off-line image analysis was used to measure mycelial morphology, including characterization of mycelial aggregates (clumps). Under all conditions, more than 87% of the hyphase were in clumps, the shape of which determined the rheological characteristics of the broth. In particular, the power law consistency index could be correlated with the biomass concentration and the roughness factor of the clumps, which describes their hairiness. A decrease in specific growth rate decreased roughness, possibly due to changes in the amount of clump breakup. However, decreases of roughness with increasing dissolved oxygen tension might rather imply some effect on hyphal-hyphal interactions within the clumps. (c) 1993 John Wiley & Sons, Inc.     

Microbial biomass product from apple pomace in batch and fed batch cultures

Summary Apple pomace, a solid waste from ap-ple processing industries, was used as the raw ma-terial for production of a protein enriched pro-duct. The experiments were conducted on a small scale in Erlenmeyer flasks or 4-1 fermentors in batch and fed-batch processes usingSaccharo-mycopsis lipolytica and Trichoderma reesei in sin-gle and mixed cultures. The results obtained indi-cate the technical feasibility of the process for ob-taining products containing 13% to 15% protein, on dry basis, which is adequate for cattle feed-ing.     

Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures

The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.     

Yeast concentration estimation and prediction with static and dynamic neural network models in batch cultures

The second fermentation is one of the most important steps in Champagne production. For this purpose, yeasts are grown on a wine based medium to adapt their metabolism to ethanol. Several models built with various static and dynamic neural network configurations were investigated. The main objective was to achieve real-time estimation and prediction of yeast concentration during growth. The model selected, based on recurrent neural networks, was first order with respect to the yeast concentration and to the volume of CO₂ released. Temperature and pH were included as model parameters as well. Yeast concentration during growth could thus be estimated with an error lower than 3% (±1.7×10⁶ yeasts/ml). From the measurement of initial yeast population and temperature, it was possible to predict the final yeast concentration (after 21 hours of growth) from the beginning of the growth, with about ±3×10⁶ yeasts/ml accuracy. So a predictive control strategy of this process could be investigated.     

The effect of iron concentration on pectin decomposition by Aspergillus niger and Aspergillus awamori]

The effect of cations Fe2+ and Fe3+ on the decomposition of apple pectin by the enzymic preparations of Asp. niger and Asp. awamori has been examined. Fe ions have delayed the process of enzymic decomposition of the pectin molecule. In the presence of Fe3+ far less amounts of monogalacturonic acid are formed. The presence of Fe ions makes the pectin molecule more stable to the effect of pectolytic enzymes.     

Elevated temperature effects on the oxidant/antioxidant balance in submerged batch cultures of the filamentous fungus Aspergillus niger B1-D

In the present study the relationship between oxidative stress and elevated culture temperature was examined in an industrially relevant fungal culture, Aspergillus niger B1-D. For the first time, both the intracellular levels of the main stressor species (superoxide radical [O(2) (.-)]) and activities of cellular defensive enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxide [GPx]) were quantified at varying temperature (25, 30, 35, 40 degrees C) to more fully characterize culture response in different growth phases. Elevated culture temperature led to increased O(2) (.-) levels in various culture phases. In the exponential phase this was due to an enhanced generation of O(2) (.-), whereas in stationary phase a decreased dismutation rate may also have contributed. CAT activities generally increased with culture temperature, whereas GPx activity changed little as temperature rose, indicating that GPx played only a minor role in destroying H(2)O(2) in this A. niger. The combination of elevated temperature (35 degrees C) and increased O(2) supply (50% enrichment) led to decreased levels of O(2) (.-) compared to the cultivation at 35 degrees C gassed with air, probably due to enhanced activity of the alternative fungal respiratory pathway. Our findings indicate that while elevated cultivation temperature does clearly induce oxidative stress events, mechanistically, it does so by a rather more complex route than previous studies indicate. Elevated temperature caused a marked disparity in the activities of SOD and CAT, very distinct from the integrated increase in activity of these enzymes in response to oxidative stress.     

Citric acid and polyols production by Aspergillus niger at high glucose concentration in solid state fermentation on inert support

Summary Aspergillus niger cultures at high initial glucose concentration (up to 400 g/1) on Amberlite as inert support were carried out. Citric acid was accumulated in the support showing high concentration (94.54 g/l) and productivity (1.35 g/l h) without inhibition related to the presence of metals (Mn²⁺, Zn²⁺, Co²⁺, Cu²⁺, and Ca²⁺) at high concentrations. Citric acid accumulation was clearly associated with both, glycerol production and to the age of the culture. Glycerol and erythritol, the major osmoregulator metabolites, were also produced (8.16 and 24.57 g/l respectively) at 400 g/l of glucose.     

A simple capillary viscosity meter for the on-line measurement and control of the biomass concentration in high-density cultures

A capillary viscosity meter was used for the on-line determination of the biomass concentration in a fermentation broth. At high cell densities the viscosity of the broth increases, which can be measured as the pressure drop over a capillary. Calibration aspects of this viscosity meter are presented, and the use of the device for the control of the biomass concentration in a membrane recycle fermentor is demonstrated.     

Production of the Phanerochaete flavido-alba laccase in Aspergillus niger for synthetic dyes decolorization and biotransformation

We investigated the expression of Phanerochaete flavido-alba laccase gene in Aspergillus niger and the physical and biochemical properties of the recombinant enzyme (rLac-LPFA) in order to test it for synthetic dye biotransformation. A. niger was able to produce high levels of active recombinant enzyme (30 mgL^?1), whose identity was further confirmed by immunodetection using Western blot analysis and N-terminal sequencing. Interestingly, rLac-LPFA exhibited an improved stability at pH (2–9) and organic solvents tested. Furthermore, the percentage of decoloration and biotransformation of synthetic textile dyes, Remazol Brilliant Blue R (RBBR) and Acid Red 299 (NY1), was higher than for the native enzyme. Its high production, simple purification, high activity, stability and ability to transform textile dyes make rLac-LPFA a good candidate for industrial applications.     

On-line estimation of biomass concentration using a neural network and information about metabolic state

This paper deals with the design of a neural network-based biomass concentration estimation system. This system is enhanced by the incorporation of information about the actual metabolism of the microorganism cultivated, which is taken from an on-line knowledge-based system. Two different design approaches have been investigated using the fed-batch cultivation of bakers yeast as the model process. In the first, metabolic state (MS) data were passed as additional input to the neural network; in the second, these data were used to select a neural network suitable for the specific MS. Two neural network types—feed-forward (Levenberg-Marquardt) and cascade correlation—were applied to this system and tested, and the performances of these neural networks were compared.     

Microbial transformations of flavanone by Aspergillus niger and Penicillium chermesinum cultures

As a result of biotransformation of flavanone (1) by the strain Aspergillus niger MB (being the UV mutant) and by the wild strain Penicillium chermesinum 113 the products of hydroxylation at C-6 (2) and C-4′ (5) were obtained. Additionally, three dihydrochalcones with hydroxyl groups at C-2′ (4), C-2′ and C-5′ (3) and C-2′ and C-4 (6) were formed.