Dirk Weuster-Botz

Succinic acid from renewable resources as a C4 building-block chemical : a review of the catalytic possibilities in aqueous media

Aqueous hydrogenation of bio-based succinic acid has been reported for the production of value added chemicals, e.g. 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, 2-pyrrolidone or N-methyl-2-pyrrolidone. A variety of heterogeneous metallic catalysts, active under quite severe conditions have previously been studied, whereas research into organometallic complexes is thus far limited to solvent reactions or to aqueous reactions producing succinic acid.

Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid.

The production of bio-based succinic acid is receiving great attention, and several predominantly prokaryotic organisms have been evaluated for this purpose. In this study we report on the suitability of the highly acid- and osmotolerant yeast Saccharomyces cerevisiae as a succinic acid production host. We implemented a metabolic engineering strategy for the oxidative production of succinic acid in yeast by deletion of the genes SDH1, SDH2, IDH1 and IDP1. The engineered strains harbor a TCA cycle that is completely interrupted after the intermediates isocitrate and succinate. The strains show no serious growth constraints on glucose. In glucose-grown shake flask cultures, the quadruple deletion strain Δsdh1Δsdh2Δidh1Δidp1 produces succinic acid at a titer of 3.62 g L(-1) (factor 4.8 compared to wild-type) at a yield of 0.11 mol (mol glucose)(-1). Succinic acid is not accumulated intracellularly. This makes the yeast S. cerevisiae a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale.

Recovery of succinic acid from fermentation broth

Succinic acid is of high interest as bio-feedstock for the chemical industry. It is a precursor for a variety of many other chemicals, e.g. 1,4-butandiol, tetrahydrofuran, biodegradable polymers and fumaric acid. Besides optimized production strains and fermentation processes it is indispensable to develop cost-saving and energy-effective downstream processes to compete with the current petrochemical production process. Various methods such as precipitation, sorption and ion exchange, electrodialysis, and liquid–liquid extraction have been investigated for the recovery of succinic acid from fermentation broth and are reviewed critically here.

Modifying the product pattern of Clostridium acetobutylicum Physiological effects of disrupting the acetate and acetone formation pathways

Clostridial acetone–butanol–ethanol (ABE) fermentation is a natural source for microbial n-butanol production and regained much interest in academia and industry in the past years. Due to the difficult genetic accessibility of Clostridium acetobutylicum and other solventogenic clostridia, successful metabolic engineering approaches are still rare. In this study, a set of five knock-out mutants with defects in the central fermentative metabolism were generated using the ClosTron technology, including the construction of targeted double knock-out mutants of C. acetobtuylicum ATCC 824. While disruption of the acetate biosynthetic pathway had no significant impact on the metabolite distribution, mutants with defects in the acetone pathway, including both acetoacetate decarboxylase (Adc)-negative and acetoacetyl-CoA:acyl-CoA transferase (CtfAB)-negative mutants, exhibited high amounts of acetate in the fermentation broth. Distinct butyrate increase and decrease patterns during the course of fermentations provided experimental evidence that butyrate, but not acetate, is re-assimilated via an Adc/CtfAB-independent pathway in C. acetobutylicum. Interestingly, combining the adc and ctfA mutations with a knock-out of the phosphotransacetylase (Pta)-encoding gene, acetate production was drastically reduced, resulting in an increased flux towards butyrate. Except for the Pta-negative single mutant, all mutants exhibited a significantly reduced solvent production.

Miniature bioreactors for automated high-throughput bioprocess design (HTBD): reproducibility of parallel fed-batch cultivations with Escherichia coli

To verify the reproducibility of cultivations of Escherichia coli in novel millilitre‐scale bioreactors, fully automated fed‐batch cultivation was performed in seven parallel‐operated ml‐scale bioreactors with an initial volume of 10 ml/reactor. The process was automatically controlled by a liquid‐handling system responsible for glucose feeding, titration and sampling. Atline analysis (carried out externally of the reaction vessel with a short time delay) comprised automated pH and attenuance measurements. The partial pressure of oxygen (pO2) was measured online by a novel fluorimetric sensor block measuring the fluorescence lifetime of fluorophors immobilized inside the millilitre‐scale bioreactors. Within a process time of 14.6 h, the parallel cultivation yielded a dry cell weight of 36.9±0.9 g·l−1. Atline pH measurements were characterized by an S.D. of <1.1% throughout the process. Computational‐fluid‐dynamics simulation of single‐phase flow yields a mean power input of 21.9 W·l−1 at an impeller speed of 2800 rev./min corresponding to a power number (NP) of 3.7.

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.

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 pastorisis presented. Making use of the P. pastorisAOX1‐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.

Reaction engineering analysis of hydrogenotrophic production of acetic acid by Acetobacterium woodii

Great interest has emerged in biological CO₂-fixing processes in the context of current climate change discussions. One example for such a process is the hydrogenotrophic production of acetic acid by anaerobic microorganisms. Acetogenic microorganisms make use of carbon dioxide in the presence of hydrogen to produce acetic acid and biomass. In order to establish a process for the hydrogenotrophic production of acetic acid, the formation of acetate by Acetobacterium woodii was studied in a batch-operated stirred-tank bioreactor at different hydrogen partial pressures (pH₂) in the gas phase. The volumetric productivity of the batch processes increased with increasing hydrogen partial pressure. A maximum of the volumetric productivity of 7.4 g(acetate) L⁻¹ day⁻¹ was measured at a pH₂ of 1,700 mbar. At this pH(2) a final acetate concentration of 44 g L⁻¹ was measured after a process time of 11 days, if the pH was controlled at pH 7.0 (average cell density of 1.1 g L⁻¹ cell dry weight). The maximum cell specific actetate productivity was 6.9 g(acetate) g(cdw)⁻¹ day⁻¹ under hydrogenotrophic conditions.

Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations

Single-use stirred tank bioreactors on a 10-mL scale operated in a magnetic-inductive bioreaction block for 48 bioreactors were equipped with individual stirrer-speed tracing, as well as individual DO- and pH-monitoring and control. A Hall-effect sensor system was integrated into the bioreaction block to measure individually the changes in magnetic field density caused by the rotating permanent magnets. A restart of the magnetic inductive drive was initiated automatically each time a Hall-effect sensor indicates one non-rotating gas-inducing stirrer. Individual DO and pH were monitored online by measuring the fluorescence decay time of two chemical sensors immobilized at the bottom of each single-use bioreactor. Parallel DO measurements were shown to be very reliable and independently from the fermentation media applied in this study for the cultivation of Escherichia coli and Saccharomyces cerevisiae. The standard deviation of parallel pH measurements was pH 0.1 at pH 7.0 at the minimum and increased to a standard deviation of pH 0.2 at pH 6.0 or at pH 8.5 with the complex medium applied for fermentations with S. cerevisiae. Parallel pH-control was thus shown to be meaningful with a tolerance band around the pH set-point of ± pH 0.2 if the set-point is pH 6.0 or lower.

Selective enhancement of autotrophic acetate production with genetically modified Acetobacterium woodii

Great interest has emerged in the recent past towards the potential of autotrophic acetogenic bacteria for the sustainable production of fuels and chemicals. This group of microorganisms possesses an ancient pathway for the fixation of carbon dioxide in the presence of hydrogen, making them highly attractive for the utilization of gas mixtures as a cheap and abundant carbon and energy source. As more and more genome sequence data of acetogens becomes available, the genetic tools are being developed concomitantly. Here, we demonstrate for the first time the genetic modification of the well-characterized acetogen Acetobacterium woodii. This microorganism selectively produces acetate under autotrophic conditions, but seems to be limited at high acetate concentrations. To increase the carbon flow through the Wood-Ljungdahl pathway and therefore increase the efficiency of CO2 fixation, genes of enzyme groups of this pathway were selectively overexpressed (the four THF-dependent enzymes for the processing of formate as well as phosphotransacetylase and acetate kinase to enhance an ATP-generation step). Acetate production with genetically modified strains was increased in a batch process under pH-controlled reaction conditions in a stirred-tank reactor with continuous sparging of H2 and CO2. Final acetate concentrations of more than 50gL(-1) acetate were thus measured with the recombinant strains at low cell concentrations of 1.5-2gL(-1) dry cell mass in less than four days under autotrophic conditions.