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The composition of plant biomass varies depending on the feedstock and pre-treatment conditions and influences its processing in biorefineries. In order to ensure optimal process conditions, the quantitative proportion of the main polymeric components of the pre-treated biomass has to be determined. Current standard procedures for biomass compositional analysis are complex, the measurements are afflicted with errors and therefore often not comparable. Hence, new powerful analytical methods are urgently required to characterize biomass. In this contribution, Differential Scanning Calorimetry (DSC) was applied in combination with multivariate data analysis (MVA) to detect the cellulose content of the plant biomass pretreated by Liquid Hot Water (LHW) and Organosolv processes under various conditions. Unlike conventional techniques, the developed analytic method enables the accurate quantification of monosaccharide content of the plant biomass without any previous sample preparation. It is easy to handle and avoids errors in sample preparation.
For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 µL can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.
Sieker, Tim ; Ulber, Roland ; Dimitrova, Darina ; Bart, Hans-Jörg ; Neuner, Andreas ; Heinzle, Elmar ; Tippkötter, Nils
In Anbetracht des zu erwartenden Rückgangs der Verfügbarkeit fossiler Rohstoffe müssen nicht nur für den Energiesektor, sondern auch für die Herstellung industrieller Produkte alternative Rohstoffe gefunden werden. Ein Beispiel für einen nicht in Nahrungsmittelkonkurrenz stehenden nachwachsenden Rohstoff ist grüne Biomasse wie Gras und Klee. Diese lassen sich in Deutschland auf großen Flächen anbauen und enthalten eine Vielzahl potenzieller Substrate für Fermentationen.
Durch die Kombination von Oligonukleotid-Liganden (Aptameren) hoher Bindungsaffinitäten
mit hochselektiv abtrennbaren magnetisierbaren Mikropartikeln
wird eine einstufige Separation von Zielmolekülen aus mikrobiologischen
Produktionsansätzen möglich. Die Aptamere werden hierfür reversibel
auf den Partikeloberflächen gebunden und für die spezifische Isolierung von
Bioprodukten eingesetzt. Die Abtrennung der beladenen Partikel erfolgt
durch einen neuen Rotor-Stator-Separator mit Hochgradient-Magnetfeld.
Kappler-Tanudyaya, Nathalie ; Schmitt, Heike ; Tippkötter, Nils ; Meyer, Lina ; Lenzen, Sigurd ; Ulber, Roland
Mannoheptulose is a seven-carbon sugar. It is an inhibitor of glucose-induced insulin secretion due to its ability to selectively inhibit the enzyme glucokinase. An improved procedure for mannoheptulose isolation from avocados is described in this study (based upon the original method by La Forge). The study focuses on the combination of biotransformation and downstream processing (preparative chromatography) as an efficient method to produce a pure extract of mannoheptulose. The experiments were divided into two major phases. In the first phase, several methods and parameters were compared to optimize the mannoheptulose extraction with respect to efficiency and purity. In the second phase, a mass balance of mannoheptulose over the whole extraction process was undertaken to estimate the yield and efficiency of the total extraction process. The combination of biotransformation and preparative chromatography allowed the production of a pure mannoheptulose extract. In a biological test, the sugar inhibited the glucokinase enzyme activity efficiently.
Due to the interfering effects of acetic acid in many fermentation processes, a gas-diffusion technique was developed for the online determination of acetic acid. The measurements were accomplished with a flow diffusion analysis (FDA) unit from the TRACE Analytics GmbH, Braunschweig, Germany. The diffusion analysis is based on the UV-absorbance of acetic acid at 205 nm. The measurement was achieved by the separation of an acceptor and a carrier stream (acidified fermentation broth) using a gas permeable polytetrafluoroethylene (PTFE) membrane, whereby broth constituents that would otherwise disturb the UV-measurement of acetic acid, are held back efficiently. Merely, the fermentation by-products, e.g. formic acid, is capable of diffusing through the membrane. While formic acid can disturb the measurement, carbon dioxide does not absorb at 205 nm. The method operates with time-dependent sample enrichment. During the analysis, a small volume of the acceptor stream is stopped for a defined time interval in the acceptor chamber. During this period, the gaseous acetic acid diffuses through the membrane and is enriched in the acceptor chamber. Subsequently after the enrichment, the acceptor stream flows through a UV-detector. The intensity of the signal is proportional to the acetic acid concentration. Online measurements in bioreactors via a sterile filtration probe have been accomplished. A linear calibration in the range of 0.5–5.0 g/L acetic acid with a relative standard deviation of <5 % was obtained. A sampling rate of 8 samples per hour was possible. The system was applied for the determination of acetic acid in E. coli fermentation broth. The instrument is easy to clean, very user-friendly and does not require any toxic or expensive reagents.
Ein viel versprechender erneuerbarer Rohstoff für die Produktion von Chemikalien und Treibstoffen ist Lignocellulose aus pflanzlicher Biomasse. Die darin enthaltenen Zucker können mittels enzymatischer Hydrolyse freigesetzt und fermentativ zu Ethanol umgesetzt werden. Ein interessanter Ansatz ist dabei die simultane Verzuckerung und Fermentation. Hefen und Enzyme haben mit 30 °C bzw. 50 °C zwar unterschiedliche Temperaturoptima, es konnte aber gezeigt werden, dass auch bei den niedrigeren Temperaturen eine Umsetzung der Cellulose zu Glucose erfolgt, wenn auch langsamer als bei optimalen Bedingungen. Außerdem konnte in Vorversuchen gezeigt werden, dass Ethanol in den zu erwartenden Konzentrationen keinen Einfluss auf die enzymatische Umsetzung hat.
Sieker, Tim ; Neuner, Andreas ; Dimitrova, Darina ; Tippkötter, Nils ; Bart, Hans-Jörg ; Heinzle, Elmar ; Ulber, Roland
Grassilage stellt einen nachwachsenden Rohstoff mit großem Potenzial dar. Neben Cellulose und Hemicellulose enthält sie auch organische Säuren, insbesondere Milchsäure. In einem Bioraffinerie-Projekt wird die Milchsäure aus der Silage isoliert und mit gentechnisch optimierten Stämmen zu L-Lysin weiterverarbeitet. Die Lignocellulose wird hydrolysiert und zu Ethanol fermentiert. Ein besonderes Augenmerk liegt auf der Integration der unterschiedlichen Prozesse sowie der einzelnen Prozessschritte zu einem Gesamtprozess, der sämtliche Inhaltsstoffe der Silage verwertet.
Paracoccus denitrificans for the effluent recycling during continuous denitrification of liquid food
(2010)
Tippkötter, Nils ; Roikaew, Wipa ; Ulber, Roland ; Hoffmann, Alexander ; Denzler, Hans-Jörg ; Buchholz, Heinrich
Nitrate is an undesirable component of several foods. A typical case of contamination with high nitrate contents is whey concentrate, containing nitrate in concentrations up to 25 l. The microbiological removal of nitrate by Paracoccus denitrificans under formation of harmless nitrogen in combination with a cell retention reactor is described here. Focus lies on the resource-conserving design of a microbal denitrification process. Two methods are compared. The application of polyvinyl alcohol-immobilized cells, which can be applied several times in whey feed, is compared with the implementation of a two step denitrification system. First, the whey concentrate's nitrate is removed by ion exchange and subsequently the eluent regenerated by microorganisms under their retention by crossflow filtration. Nitrite and nitrate concentrations were determined by reflectometric color measurement with a commercially available Reflectoquant® device. Correction factors for these media had to be determined. During the pilot development, bioreactors from 4 to 250 mg·L-1 and crossflow units with membrane areas from 0.02 to 0.80 m2 were examined. Based on the results of the pilot plants, a scaling for the exemplary process of denitrifying 1,000 tons per day is discussed.
Lignocellulosic biorefinery: Process integration of hydrolysis and fermentation (SSF process)
(2011)
The aim of the present work is the process integration and the optimization of the enzymatic hydrolysis of wood and the following fermentation of the products to ethanol. The substrate is a fiber fraction obtained by organosolv pre-treatment of beech wood. For the ethanol production, a co-fermentation by two different yeasts (Saccharomyces cerevisiae and Pachysolen tannophilus) was carried out to convert glucose as well as xylose. Two approaches has been followed: 1. A two step process, in which the hydrolysis of the fiber fraction and the fermentation to product are separated from each other. 2. A process, in which the hydrolysis and the fermentation are carried out in one single process step as simultaneous saccharification and fermentation (SSF). Following the first approach, a yield of about 0.15 g ethanol per gram substrate can be reached. Based on the SSF, one process step can be saved, and additionally, the gained yield can be raised up to 0.3 g ethanol per gram substrate.
Sieker, Tim ; Neuner, Andreas ; Dimitrova, Darina ; Tippkötter, Nils ; Muffler, Kai ; Bart, Hans-Jörg ; Heinzle, Elmar ; Ulber, Roland
Grass silage provides a great potential as renewable feedstock. Two fractions of the grass silage, a press juice and the fiber fraction, were evaluated for their possible use for bioethanol production. Direct production of ethanol from press juice is not possible due to high concentrations of organic acids. For the fiber fraction, alkaline peroxide or enzymatic pretreatment was used, which removes the phenolic acids in the cell wall. In this study, we demonstrate the possibility to integrate the enzymatic pretreatment with a simultaneous saccharification and fermentation to achieve ethanol production from grass silage in a one-process step. Achieved yields were about 53 g ethanol per kg silage with the alkaline peroxide pretreatment and 91 g/kg with the enzymatic pretreatment at concentrations of 8.5 and 14.6 g/L, respectively. Furthermore, it was shown that additional supplementation of the fermentation medium with vitamins, trace elements and nutrient salts is not necessary when the press juice is directly used in the fermentation step.
Biotechnological downstream processing is usually an elaborate procedure, requiring a multitude of unit operations to isolate the target component. Besides the disadvantageous space-time yield, the risks of cross-contaminations and product loss grow fast with the complexity of the isolation procedure. A significant reduction of unit operations can be achieved by application of magnetic particles, especially if these are functionalized with affinity ligands. As magnetic susceptible materials are highly uncommon in biotechnological processes, target binding and selective separation of such particles from fermentation or reactions broths can be done in a single step. Since the magnetizable particles can be produced from iron salts and low priced polymers, a single-use implementation of these systems is highly conceivable. In this article, the principles of magnetizable particles, their synthesis and functionalization are explained. Furthermore, applications in the area of reaction engineering, microfluidics and downstream processing are discussed focusing on established single-use technologies and development potential.
Thiel, Alexander ; Tippkötter, Nils ; Suck, Kirstin ; Sohling, Ulrich ; Ruf, Friedrich ; Ulber, Roland
Commercial materials with polyvinylpolypyrrolidone and polymeric amberlites (XAD7HP, XAD16) are commonly used for the adsorptive downstream processing of polyphenols from renewable resources. In this study, beta-zeolite-based adsorbent systems were examined, and their properties were compared to organic resins. Batch adsorption experiments were conducted with synthetic solutions of major polyphenols. Adsorption isotherms and desorption characteristics of individual adsorbent were determined based on these results. Maximum adsorption capacities were calculated using the Langmuir model. For example, the zeolites had capacities up to 203.2 mg/g for ferulic acid. To extend these results to a complex system, additional experiments were performed on rapeseed meal and wheat seed extracts as representative renewable resources. HPLC analysis showed that with 7.5% w/v, which is regarded as the optimum amount of zeolites, zeolites A and B could bind 100% of the major polyphenols as well as release polyphenols at high yields. Additionally, regeneration experiments were performed with isopropyl alcohol at 99°C to evaluate how zeolites regenerate under mild conditions. The results showed only a negligible loss of adsorption capacity and no loss of desorption capacity. In summary, it was concluded that beta-zeolites were promising adsorbents for developing new processes to isolate polyphenols from renewable resources.
Al-Kaidy, Huschyar ; Duwe, Anna ; Huster, Manuel ; Muffler, Kai ; Schlegel, Christin ; Sieker, Tim ; Stadtmüller, Ralf ; Tippkötter, Nils ; Ulber, Roland
Biotechnologie und die mit ihr verbundenen technischen Prozesse prägen seit Jahrtausenden die Entwicklung der Menschheit. Ausgehend von empirischen Verfahren, insbesondere zur Herstellung von Lebensmitteln und täglichen Gebrauchsgütern, haben sich diese Disziplinen zu einem der innovativsten Zukunftsfelder entwickelt. Durch das immer detailliertere Verständnis zellulärer Vorgänge können mittlerweile Produktionsstämme gezielt optimiert werden. Im Zusammenspiel mit moderner Prozesstechnik können so eine Vielzahl von Bulk- und Feinchemikalien sowie Pharmazeutika effizient hergestellt werden. In diesem Artikel werden exemplarisch einige der aktuellen Trends vorgestellt.
Adsorptive Vorbehandlung von Rohglycerin für die 1,3-Propandiol Fermentation mit Clostridium diolis
(2014)
Wiesen, Sebastian ; Tippkötter, Nils ; Muffler, Kai ; Suck, Kirstin ; Sohling, Ulrich ; Ruf, Nils ; Ulber, Roland
Bei der Gewinnung von Fettsäuren aus Pflanzenölen, z. B. zur Herstellung von Biopolymeren, oder bei der Biodiesel- und Seifenproduktion, fällt Glycerin als Nebenprodukt an. Bei der Biokonversion dieses Rohstoffes zu 1,3-Propandiol wird der Produktionsorganismus Clostridium diolis durch Verunreinigungen im Rohglycerin gehemmt. Als inhibierende Substanzen konnten freie Fettsäuren identifiziert werden. Mithilfe eines adsorptiven Aufarbeitungsverfahrens ist es gelungen, die Fettsäuren zu entfernen und die Konversionseffizienz zu 1,3-Propandiol zu erhöhen.
Tippkötter, Nils ; Wollny, Steffen ; Suck, Kirstin ; Sohling, Ulrich ; Ruf, Friedrich ; Ulber, Roland
A major part of edible oil is subjected to bleaching procedures, primarily with minerals applied as adsorbers. Their recycling is currently done either by regaining the oil via organic solvent extraction or by using the spent bleaching earth (SBE) as additive for animal feed, etc. As a new method, the reutilization of the by-product SBE for the microbiologic formation of acetone, butanol, and ethanol (ABE) is presented as proof-of-concept. The SBE was taken from a palm oil cleaning process. The recycling concept is based on the application of lipolytic clostridia strains. Due to considerably long fermentation times, co-fermentation with Candida rugosa and enzymatic hydrolyses of the bound oil with a subsequent clostridia fermentation are shown as alternative routes. Anaerobic fermentations under comparison of different clostridia strains were performed with glycerol media, enzymatically hydrolyzed palm oil and SBE. Solutes, side product compositions and productivities were quantified via HPLC. A successful production of ABE solutes from SBE has been done with a yield of 0.15 g butanol per gram of bound glycerol. Thus, the biotechnological recycling of the waste stream is possible in principle. Inhibition of the substrate suspension has been observed. A chromatographic ion-exchange of substrates increased the biomass concentration.
High gradient magnetic separation (HGMS) has been established since the early 1970s. A more recent application of these systems is the use in bioprocesses. To integrate the HGMS in a fermentation process, it is necessary to optimize the separation matrix with regard to the magnetic separation characteristics and permeability of the non-magnetizable components of the fermentation broth. As part of the work presented here, a combined fluidic and magnetic force finite element model simulation was created using the software COMSOL Multiphysics and compared with separation experiments. Finally, as optimal lattice orientation of the separation matrix, a transversal rhombohedral arrangement was defined. The high suitability of the new filter matrix has been verified by separation experiments.
Thiel, Alexander ; Muffler, Kai ; Tippkötter, Nils ; Suck, Kirstin ; Sohling, Ulrich ; Ruf, Friedrich ; Ulber, Roland
Aufarbeitung von Polyphenolen aus Weizenmittels Zeolithen am Beispiel der Ferulasa¨ ureAlexander Thiel1, Kai Muffler1, Nils Tippko¨ tter1, Kirstin Suck2, Ulrich Sohling2, Friedrich Ruf3und Roland Ulber1,*DOI: 10.1002/cite.201400031Bei der Ferulasa¨ure handelt es sich um einen Wertstoff, der aus Weizen gewonnen und in der Lebensmittel- und Pharma-industrie eingesetzt werden kann. Der Einsatz von Weizen als nachwachsende Rohstoffquelle ist allerdings nur dann wirt-schaftlich durchfu¨hrbar, wenn eine Prozessintegration in die bestehenden industriellen Verfahren gewa¨hrleistet oder einedirekte Konkurrenz zur Mehl- und Sta¨rkeindustrie vermieden werden kann. In diesem Artikel wird ein Verfahren aufge-zeigt, welches hohe Ausbeuten ermo¨glicht und eine Konkurrenz zu bestehenden Verwertungspfaden vermeidet.
Simultane Atline-Quantifizierung von Magnetpartikeln und Mikroorganismen bei einer HGMS-Filtration
(2015)
Schumann, Christiane ; Rogin, Sabine ; Schneider, Horst ; Tippkötter, Nils ; Oster, Jürgen ; Kampeis, Percy
Es wird eine neue Atline-Messmethode vorgestellt, mit der während einer Hochgradienten-Magnetseparation (HGMS)-Filtration eine simultane Quantifizierung von Magnetpartikeln und Mikroorganismen im Filtrat vorgenommen werden kann. Dabei gelingt die Quantifizierung signifikant besser als mit bisher verwendeten Messmethoden. Mit dieser Methode ist es möglich, die Trennleistung einer HGMS-Filtration zu bestimmen und einen Filterdurchbruch durch Konzentrationsanstiege im Bereich einiger µg L−1 von Magnetpartikeln im Filtrat frühzeitig zu detektieren, ohne dass nennenswerte Partikelmengen verloren gehen.