D-Galacturonic acid reduction by S. cerevisiae for L-galactonate production from extracted sugar beet press pulp hydrolysate.

Pectin-rich residues are considered as promising feedstocks for sustainable production of platform chemicals. Enzymatic hydrolysis of extracted sugar beet press pulp (SBPP) releases the main constituent of pectin, D-galacturonic acid (D-GalA). Using engineered Saccharomyces cerevisiae, D-GalA is then reduced to L-galactonate (L-GalOA) with sorbitol as co-substrate. The current work addresses the combination of enzymatic hydrolysis of pectin in SBPP with a consecutive optimized biotransformation of the released D-GalA to L-GalOA in simple batch processes in stirred-tank bioreactors. Process conditions were first identified with synthetic media, where a product concentration of 9.9 g L-1 L-GalOA was obtained with a product selectivity of 99% (L-GalOA D-GalA-1) at pH 5 with 4% (w/v) sorbitol within 48 h. A very similar batch process performance with a product selectivity of 97% was achieved with potassium citrate buffered SBPP hydrolysate, demonstrating for the first time direct production of L-GalOA from hydrolyzed biomass using engineered S. cerevisiae. Combining the hydrolysis process of extracted SBPP and the biotransformation process with engineered S. cerevisiae paves the way towards repurposing pectin-rich residues as substrates for value-added chemicals. KEY POINTS: • Efficient bioreduction of D-GalA with S. cerevisiae in stirred-tank reactors • Batch production of L-GalOA by engineered S. cerevisiae with high selectivity • Direct L-GalOA production from hydrolyzed sugar beet press pulp Bioreduction of D-galacturonic acid to L-galactonate with recombinant Saccharomyces cerevisiae enables for the first time the valorization of hydrolysates from extracted sugar beet press pulp for the sustainable production of value-added chemicals.

Production of lipids with Microchloropsis salina in open thin-layer cascade photobioreactors.

Microalgal biomass is considered as the most promising feedstock for sustainable production of liquid fuels. Lipid production with Microchloropsis salina was studied in open thin-layer cascade (TLC) photobioreactors with a surface area of 8 m2 applying a physically simulated Mediterranean summer climate. High lipid concentrations of up to 6.6 g L-1 with 46% (w w-1) total lipids in dry cell mass were achieved in two-phase batch processes applying a nitrogen limitation. The two-phase batch process was transferred into a continuously operated reactor cascade of two TLC photobioreactors. Microchloropsis salina cells were produced continuously in the first photobioreactor, whereas continuous lipid production was enabled in the second, nitrogen-limited TLC photobioreactor resulting in continuous production of 3.0 g L-1 lipids with a high overall lipid space-time-yield of 0.2 g L-1 d-1. The control of alkalinity to about 10 mM resulted in high CO2 conversion efficiencies of 84-87%.

Influence of different packing methods on the hydrodynamic stability of chromatography columns.

It is well known that packing non-uniformity may cause peak asymmetry and limit the performance of packed-bed chromatographic columns. However, understanding of the reasons leading to packing non-uniformity is still limited. Therefore, the effect of different column packing methods, i.e. dynamic axial compression (DAC), flow packing, and combinations of both on the hydrodynamic packing heterogeneity and stability of packings composed of polymer-based compressible porous resins with a mean diameter of 90µm was investigated experimentally as well as in-silico. Deterministic Euler-Lagrange modeling of a small chromatographic column with a diameter of 9.6mm and a bed height of 30mm was applied by coupling Computational Fluid Dynamics (CFD) and the Discrete Element Method (DEM). Interparticle micromechanics as well as the fluid-particle and particle-wall interactions were taken into account. Experiments and simulations revealed substantial non-uniformity of compression force transmission and axial packing density distribution during both dynamic axial compression and flow packing which was related to wall support and interparticle friction. By combining both packing methods sequentially (dynamic axial compression followed by flow packing or vice versa), the compression forces were more homogeneous resulting in improved packing procedures. Repeated alternating application of flow packing and DAC (the so-called hybrid packing method) resulted in the most homogeneous packing density distribution and the highest packing stability which was kept nearly constant during long-term operation with cyclic hydrodynamic load. The hydrodynamic stability of the chromatographic column was evaluated by calculating the integral porosity deviation and packing induced flow velocity dispersion. The hybrid packing method gave the best results for both parameters.

Microbial production of homogeneously layered cellulose pellicles in a membrane bioreactor.

Microbial cellulose (MC) is being investigated for various applications in the field of biomedical engineering. Gluconacetobacter xylinus is able to produce pure cellulose in the form of a hydrogel ("pellicle"). The pellicle consists of a defined tridimensional structure that is sensitive to mechanical stress during the process of formation. The bacteria, however, are obligate aerobic and need to be supplied with oxygen. These two objectives are often conflicting. A lab-scale membrane bioreactor prototype was developed which is able to efficiently produce a MC pellicle with a homogeneous layered structure. A hydrophilic microfiltration polyethersulfone membrane separates the bacteria from the cultivation medium. This setup allows the free convective exchange of the cultivation medium, while providing mechanical support for the continuous formation of the MC layer. Thickness of the MC layer was measured online by a laser triangulation sensor. One hundred and twenty five gram cellulose dry weight/m(2) membrane surface were produced within a process time of 330 h. Membrane bioreactors may be used to produce homogenous MC layers in a variety of shapes suitable for biomedical applications.

Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD).

A novel milliliter-scale bioreactor equipped with a gas-inducing impeller was developed with oxygen transfer coefficients as high as in laboratory and industrial stirred-tank bioreactors. The bioreactor reaches oxygen transfer coefficients of >0.4 s(-1). Oxygen transfer coefficients of >0.2 s(-1) can be maintained over a range of 8- to 12-mL reaction volume. A reaction block with integrated heat exchangers was developed for 48-mL-scale bioreactors. The block can be closed with a single gas cover spreading sterile process gas from a central inlet into the headspace of all bioreactors. The gas cover simultaneously acts as a sterile barrier, making the reaction block a stand-alone device that represents an alternative to 48 parallel-operated shake flasks on a much smaller footprint. Process control software was developed to control a liquid-handling system for automated sampling, titration of pH, substrate feeding, and a microtiter plate reader for automated atline pH and atline optical density analytics. The liquid-handling parameters for titration agent, feeding solution, and cell samples were optimized to increase data quality. A simple proportional pH-control algorithm and intermittent titration of pH enabled Escherichia coli growth to a dry cell weight of 20.5 g L(-1) in fed-batch cultivation with air aeration. Growth of E. coli at the milliliter scale (10 mL) was shown to be equivalent to laboratory scale (3 L) with regard to growth rate, mu, and biomass yield, Y(XS).

Integrated L-phenylalanine separation in an E. coli fed-batch process: from laboratory to pilot scale.

Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation. An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient. Using this system with two membrane contactors, mass-transfer coefficients of up to 288 x 10(-7) cm s(-1) for the aqueous/organic and 77 x 10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach. Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase. Reactive extraction enabled a 98% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99% after L-Phe precipitation. A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.

Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation.

Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1). Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.

Evaluation of parallel operated small-scale bubble columns for microbial process development using Staphylococcus carnosus.

Shake flasks and pH-controlled small-scale bubble columns were compared with respect to their usefulness as a basic tool for process development for human calcitonin precursor fusion-protein production with Staphylococcus carnosus. Parallel control of the pH (and making use of the base addition data) is necessary to study the effects of medium composition, to identify pH-optima and to develop a medium, which minimizes the acid excretion of S. carnosus. This medium with glycerol as energy source and yeast extract as carbon and nitrogen source resulted in cell dry weight concentration in shake flasks of 5 g l(-1), which were thus improved by a factor of 10. Cell dry weight concentrations of up to 12.5 g l(-1) were measured in the batch process with pH-controlled small-scale bubble columns due to their higher oxygen transfer capability. In contrast to shake flasks it was demonstrated, that the batch process performance of recombinant S. carnosus secreting the human calcitonin precursor fusion-protein was identical within the estimation error in pH-controlled small-scale bubble columns compared to the stirred-tank reactor.

Parallel substrate feeding and pH-control in shaking-flasks.

An intermittent feeding system for shaking-flasks was developed to close the gap between batch operated shaking-flasks and fed-batch operated as well as pH-controlled stirred tank reactors. A precise syringe pump was connected via a substrate distribution system to individual 2/2-way miniature valves, one for each of up to 16 shaking-flask. The shaking-flasks were equipped with pH-probes. A process computer controls the intermittent feeding of substrates by tracking predefined individual feeding profiles as well as the base (or acid) addition for individual pH-control of the shaking-flasks. Higher concentrations of aerobic cells with higher cellular activities were achieved in fed-batch operated and pH-controlled shaking-flasks as compared to the conventional batch operation. Physiological effects of an intermittent feeding were studied in a stirred tank reactor with a recombinant E. coli strain, which expressed the GDP-mannose-pyrophosphorylase enzyme under the control of the lac-promoter.

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract).

Experimental design for fermentation media development: statistical design or global random search?

The diversity of combinatorial interactions of medium components with the metabolism of cells as well as the large number of medium constituents necessary for cellular growth and production do not permit satisfactory detailed modelling. For this reason, experimental search procedures in simultaneous shaking flask experiments are used to optimise fermentation media. As an alternative to the methods of statistical experimental design employed in this field for many decades, the use of stochastic search procedures has been evaluated recently, since these require neither the unimodality of the response surface nor limitations in the number of medium components under consideration. Genetic algorithms were selected due to their basic capability for efficient exploration of large variable spaces. Using a genetic algorithm, it has been experimentally verified, with the aid of process examples, that process improvements can be achieved both for microbial and enzymatic conversions and for cell cultures despite the large number of medium components under simultaneous consideration (about 10 or more). In exploring a new variable space, process improvements of more than 100% were generally achieved. For initial reaction conditions previously 'optimised' via standard procedures it has been possible in most cases to achieve a further improvement of 20-40% of the target quantity. Although the genetic algorithm can be very efficient for exploration of large variable spaces, it is improbable that a 'global optimum' can be precisely identified because of the relatively small number of shaking flask experiments usually performed. As a consequence, a combination of highly directed random searches to explore the n-dimensional variable space with the genetic algorithm and subsequent application of classical statistical experimental design is recommended for media development.

Automated sampling device for monitoring intracellular metabolite dynamics.

An automated sampling device coupled to a stirred tank reactor was developed for monitoring intracellular metabolite dynamics. Sample flasks fixed in transport magazines were moved by a step engine in a way that each sample flask was filled within 220 ms, resulting in a sampling rate of 4.5 s-1. Rapid inactivation of the metabolism was achieved by spraying the samples into 60% methanol at -50 degrees C. After centrifugation of the quenched cells at -20 degrees C the metabolites were extracted with perchloric acid and analyzed biochemically or with HPLC. The automated sampling device was applied for investigation of the intracellular metabolite dynamics of glycolysis in Escherichia coli after rapid glucose addition to a glucose-limited steady-state culture. For the first time oscillations of intracellular metabolite concentrations like glucose-6-phosphate, phosphoenolpyruvate, glyceraldehyde 3-phosphate, dihydroxyacetonphosphate, 3-phosphoglycerate, and pyruvate were quantified on a subseconds to seconds scale in E. coli. As an example, the kinetics of the decomposition of fructose 1, 6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetonphosphate were investigated by use of a well-known mechanistic kinetic model and the measured in vivo metabolite dynamics.

Sampling tube device for monitoring intracellular metabolite dynamics.

Continuous sampling of microorganisms from a controlled bioreactor with rapid inactivation of metabolism and extraction of metabolites using precooled -40 degrees C perchloric acid solution (35%) was achieved with a sampling tube, thus fixing fast dynamic reactions at a certain position in the tube. After sampling was stopped (200 s) the tube was frozen at -80 degrees C and divided into identical parts and the extracted metabolites were analyzed enzymatically. A high resolution in time was achieved due to the axial dispersion of the metabolites in the sampling tube: The events of 1 s in the cells of the reactor were represented by 15 parts of the sampling tube. Axial dispersion was determined quantitatively with tracer measurements. The performance of the sampling tube device was evaluated with dynamic investigations on glucose-metabolism of Zymomonas mobilis. The dynamics of intracellular glucose 6-phosphate, glyceraldehyde 3-phosphate, and 3-phosphoglycerate concentrations were monitored after adding a glucose pulse to a glucose-limited steady-state culture.

Experimental design for the identification of macrokinetic models and model discrimination.

An experimental design method for the identification of macrokinetic models was developed applying an extended D-optimal design criterion. The D-optimal design criterion was modified to consider variable measurement variances as well as multivariate macrokinetic models. The macrokinetics of formate dehydrogenase (FDH) production with Candida boidinii were thus identified within 10 steady state experiments in a labscale continuous stirred tank reactor (10 model parameters). Closed loop control (nutristat) was applied to set-up the operating states suggested by this experimental design method. After each set of steady state experiments the quality of macrokinetic parameters was characterized statistically. For model discrimination a parameter discrimination algorithm based on entropy formulations was adapted. Again a multivariate criterion considering variable measurement variances was developed. This discrimination algorithm was applied to discriminate the macrokinetic model of FDH production with Candida boidinii out of 10 different macrokinetic approaches. An unequivocal discrimination result could be obtained calculating model specific probabilities. These were compared with commonly used sum of squares values. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 564-576, 1997.

Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities.

A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400-MHz wide-bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation-integrated cross-flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients k(L)a of more than 1.0 s(-1) are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate >15,000 mg O(2) .(L h)(-1)] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo (31)P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP-sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo (13)C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 g(cell dry weight) . L(-1), the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L-lactate, L-glutamate, succinate, and L-lysine with a time resolution of 10 min after impressing a [1-(13)C]-glucose pulse.

Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum.

To identify potential L-lysine export limitations by Corynebacterium glutamicum in the L-lysine production process, the excretion of L-lysine was studied in continuous and fed-batch operated stirred tank reactors. A structured biochemical model of the L-lysine excretion mechanism was used to determine the activity of the export carrier and to calculate a cell-specific concentration of the export carrier. For the biochemical characterization of this specific carrier concentration a standardized L-lysine efflux test was developed. Carrier activity, cell-specific carrier concentration, and the specific L-lysine export rate were identified as a function of pH value and L-lysine concentration in the reactors. Also, the correlation of these parameters to the metabolic state of C. glutamicum was determined. The pH value in the reactor governs the carrier activity (maximum at pH 6.5) and the specific carrier concentration (maximum at pH 8.0). The specific L-lysine export rate, as the product of carrier activity and specific carrier concentration, revealed a maximum at pH 7.0. Decreasing L-lysine productivities also correlated with decreasing specific carrier concentrations. The L-lysine concentration in the reactor had no effect on the specific carrier concentration but strongly inhibited the carrier activity. The specific export rate was reduced to 50% at 400 mM L-lysine compared to the specific export rate at 80 mM L-lysine. (c) 1996 John Wiley & Sons, Inc.

Reaction engineering methods to study intracellular metabolite concentrations.

The analysis of intracellular metabolite concentrations is of basic importance for metabolic engineering of microorganisms. In vivo NMR-spectroscopy as a non-invasive technique to measure intracellular metabolite concentrations and rapid sampling devices as invasive techniques are reviewed. The methods are discussed from a reaction engineering point of view. The objective is to obtain intracellular concentration data under well defined physiological conditions in balanced steady state and defined transitional states as well. Application examples are given for a membrane-cyclone-reactor configuration designed to achieve high signal sensitivity with in vivo 31P-NMR and 13C-NMR spectroscopy as well as for a sampling tube device designed for high sampling rates (2s-1). This sampling device enables the measurement of dynamic metabolite profiles at a time scale of a few seconds.

Large-scale production of a soluble human beta-1,4-galactosyltransferase using a Saccharomyces cerevisiae expression system.

We report in this communication the first large-scale heterologous expression of a glycosyltransferase in yeast. A soluble form of a human beta-1,4-galactosyltransferase (EC 2.4.1.38) was expressed using a Saccharomyces cerevisiae expression system. Fermentation technology afforded the means to increase the expression level of the beta-1,4-galactosyltransferase up to a concentration of 700 mU/liter. The enzyme was produced at a scale of 200 units. The recombinant soluble enzyme was purified 766-fold to a specific activity of approx. 2 U/mg using a purification protocol based on sequential affinity chromatography on N-acetylglucosaminyl- and alpha-lactalbumin-Sepharose, respectively. This study demonstrates that heterologous expression of a glycosyltransferase is possible on a large scale and offers an alternative to natural sources like human breast milk or bovine colostrum.