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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.
Lately there has been an increasing concern about uranium toxicity in some districts of Punjab State located in the North Western part of India after the publication of a report (Blaurock-Busch et al. 2010) which showed that the concentration of uranium in hair and urine of children suffering from physical deformities, neurological and mental disorder from Malwa region (Fig. 1) of Punjab State was manifold higher than the reference ranges. A train which connects the affected region with the nearby city of Bikaner which has a Cancer Hospital has been nicknamed as Cancer Express due to the frenzy generated on account of uranium related toxicity.
The invention relates to a method for production of single-stranded macronucleotides by amplifying and ligating an extended monomeric single-stranded target nucleic acid sequence (targetss) into a repetitive cluster of double-stranded target nucleic acid sequences (targetds), and subsequently cloning the construct into a vector (aptagene vector). The aptagene vector is transformed into host cells for replication of the aptagene and isolated in order to optain single-stranded target sequences (targetss). The invention also relates to single-stranded nucleic acids, produced by a method of the invention.
We present an effective and simple multiscale method for equilibrating Kremer Grest model polymer melts of varying stiffness. In our approach, we progressively equilibrate the melt structure above the tube scale, inside the tube and finally at the monomeric scale. We make use of models designed to be computationally effective at each scale. Density fluctuations in the melt structure above the tube scale are minimized through a Monte Carlo simulated annealing of a lattice polymer model. Subsequently the melt structure below the tube scale is equilibrated via the Rouse dynamics of a force-capped Kremer-Grest model that allows chains to partially interpenetrate. Finally the Kremer-Grest force field is introduced to freeze the topological state and enforce correct monomer packing. We generate 15 melts of 500 chains of 10.000 beads for varying chain stiffness as well as a number of melts with 1.000 chains of 15.000 monomers. To validate the equilibration process we study the time evolution of bulk, collective, and single-chain observables at the monomeric, mesoscopic, and macroscopic length scales. Extension of the present method to longer, branched, or polydisperse chains, and/or larger system sizes is straightforward.
The Kremer-Grest (KG) bead-spring model is a near standard in Molecular Dynamic simulations of generic polymer properties. It owes its popularity to its computational efficiency, rather than its ability to represent specific polymer species and conditions. Here we investigate how to adapt the model to match the universal properties of a wide range of chemical polymers species. For this purpose we vary a single parameter originally introduced by Faller and Müller-Plathe, the chain stiffness. Examples include polystyrene, polyethylene, polypropylene, cis-polyisoprene, polydimethylsiloxane, polyethyleneoxide and styrene-butadiene rubber. We do this by matching the number of Kuhn segments per chain and the number of Kuhn segments per cubic Kuhn volume for the polymer species and for the Kremer-Grest model. We also derive mapping relations for converting KG model units back to physical units, in particular we obtain the entanglement time for the KG model as function of stiffness allowing for a time mapping. To test these relations, we generate large equilibrated well entangled polymer melts, and measure the entanglement moduli using a static primitive-path analysis of the entangled melt structure as well as by simulations of step-strain deformation of the model melts. The obtained moduli for our model polymer melts are in good agreement with the experimentally expected moduli.
The metabolic activity of Chinese hamster ovary (CHO) cells was observed using a light-addressable potentiometric sensor (LAPS). The dependency toward different glucose concentrations (17–200 mM) follows a Michaelis–Menten kinetics trajectory with Kₘ = 32.8 mM, and the obtained Kₘ value in this experiment was compared with that found in literature. In addition, the pH shift induced by glucose metabolism of tumor cells transfected with the HPV-16 genome (C3 cells) was successfully observed. These results indicate the possibility to determine the tumor cells metabolism with a LAPS-based measurement device.
A new microfluidic assembly method for semiconductor-based biosensors using 3D-printing technologies was proposed for a rapid and cost-efficient design of new sensor systems. The microfluidic unit is designed and printed by a 3D-printer in just a few hours and assembled on a light-addressable potentiometric sensor (LAPS) chip using a photo resin. The cell growth curves obtained from culturing cells within microfluidics-based LAPS systems were compared with cell growth curves in cell culture flasks to examine biocompatibility of the 3D-printed chips. Furthermore, an optimal cell culturing within microfluidics-based LAPS chips was achieved by adjusting the fetal calf serum concentrations of the cell culture medium, an important factor for the cell proliferation.
In the present work an optical sensor in combination with a spectrally resolved detection device for in-line particle-size-monitoring for quality control in beer production is presented. The principle relies on the size and wavelength dependent backscatter of growing particles in fluids. Measured interference structures of backscattered light are compared with calculated theoretical values, based on Mie-Theory, and fitted with a linear least square method to obtain particle size distributions. For this purpose, a broadband light source in combination with a process-CCD-spectrometer (charge ? coupled device spectrometer) and process adapted fiber optics are used. The goal is the development of an easy and flexible measurement device for in-line-monitoring of particle size. The presented device can be directly installed in product fill tubes or vessels, follows CIP- (cleaning in place) and removes the need of sample taking. A proof of concept and preliminary results, measuring protein precipitation, are presented.
BACKGROUND
Currently, several techniques exist for the downstream processing of protein, phytic acid and sinapic acid from rapeseed and rapeseed meal, but no technique has been developed to separate all of the components in one process. In this work, two new downstream processing strategies focusing on recovering sinapic acid, phytic acid and protein from rapeseed meal were established.
RESULTS
The sinapic acid content was enhanced by a factor of 4.5 with one method and 5.1 with the other. The isolation of sinapic acid was accomplished using a zeolite-based adsorbent with high adsorptive and optimal desorption characteristics. Phytic acid was isolated using the anion-exchange resin Purolite A200®. In addition, the processes resulted in two separated protein fractions. The ratios of globulin and albumin ratio to the total protein were 59.2% and 40.1%, respectively. The steps were then combined in two different ways: (a) a ‘sequential process’ using the zeolite and A200 in batch processes; and (b) a ‘parallel process’ using only A200 in a chromatographic system to separate all of the compounds.
CONCLUSIONS
It can be concluded that isolation of all three components was possible in both processes. These could enhance the added value of current processes using rapeseed meal as a protein source. © 2015 Society of Chemical Industry
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.
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.
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.
The development of a cost-effective hydrolysis for crude cellulose is an essential part of biorefinery developments. To establish such high solid hydrolysis, a new solid state reactor with static mixing is used. However, concentrations >10% (w/w) cause a rate and yield reduction of enzymatic hydrolysis. By optimizing the synergetic activity of cellulolytic enzymes at solid concentrations of 9%, 17% and 23% (w/w) of crude Organosolv cellulose, glucose concentrations of 57, 113 and 152 g L⁻¹ are reached. However, the glucose yield decreases from 0.81 to 0.72gg⁻¹ at 17% (w/w). Optimal conditions for hydrolysis scale-up under minimal enzyme addition are identified. As result, at 23% (w/w) crude cellulose the glucose yield increases from 0.29 to 0.49gg⁻¹. As proof of its applicability, biobutanol, succinic and itaconic acid are produced with the crude hydrolysate. The potential of the substrate is proven e.g. by a high butanol yield of 0.33gg⁻¹.
In comparison to crude oil, biorefinery raw materials are challenging in concerns of transport and storage. The plant raw materials are more voluminous, so that shredding and compacting usually are necessary before transport. These mechanical processes can have a negative influence on the subsequent biotechnological processing and shelf life of the raw materials. Various approaches and their effects on renewable raw materials are shown. In addition, aspects of decentralized pretreatment steps are discussed. Another important aspect of pretreatment is the varying composition of the raw materials depending on the growth conditions. This problem can be solved with advanced on-site spectrometric analysis of the material.
Paracoccus denitrificans for the effluent recycling during continuous denitrification of liquid food
(2010)
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.
In traditional microbial biobutanol production, the solvent must be recovered during fermentation process for a sufficient space-time yield. Thermal separation is not feasible due to the boiling point of n-butanol. As an integrated and selective solid-liquid separation alternative, solvent impregnated resins (SIRs) were applied. Two polymeric resins were evaluated and an extractant screening was conducted. Vacuum application with vapor collection in fixed-bed column as bioreactor bypass was successfully implemented as butanol desorption step. In course of further increasing process economics, fermentation with renewable lignocellulosic substrates was conducted using Clostridium acetobutylicum. Utilization of SIR was shown to be a potential strategy for solvent removal from fermentation broth, while application of a bypass column allows for product removal and recovery at once.
An immunochromatographic lateral flow dipstick assay for the fast detection of microcystin-LR was developed. Colloid gold particles with diameters of 40 nm were used as red-colored antibody labels for the visual detection of the antigen. The new dipstick sensor is capable of detecting down to 5 µg·l−1 (ppb; total inversion of the color signal) or 1 ppb (observation of color grading) of microcystin-LR. The course of the labeling reaction was observed via spectrometric wave shifts caused by the change of particle size during the binding of antibodies. Different stabilizing reagents showed that especially bovine serum albumin (BSA) and casein increase the assays sensitivity and the conjugate stability. Performance of the dipsticks was quantified by pattern processing of capture zone CCD images. Storage stability of dipsticks and conjugate suspensions over 115 days under different conditions were monitored. The ready-to-use dipsticks were successfully tested with microcystin-LR-spiked samples of outdoor drinking- and salt water and applied to the tissue of microcystin-fed mussels.
Magnetisierbare Partikel als Träger von Katalysatoren können durch Anlegen eines magnetisches Feldes einfach und schnell abgetrennt werden. Die Wiedergewinnung von wertvollen Enzymen unter geringem Energie- und Materialeinsatz der magnetischen Abtrennung eröffnet einen Wettbewerbsvorteil für Produktionsprozesse. Die Abtrennung von magnetisierbaren Partikeln vom Überstand wird üblicherweise entweder durch Anlegen eines äußeren Magnetfelds und der resultierenden Ablagerung der Partikel an den Reaktorwänden oder durch Hochgradientenmagnetseparation (HGMS)durchgeführt. Beide Verfahren resultieren meist in der Bildung eines Filterkuchens aus Magnetpartikeln und den Feststoffen des Reaktionsmediums. Das magnetische horizontale Wirbelbett ermöglicht simultan eine kontinuierliche Reaktionsführung und die Rückhaltung der Partikel im Durchfluss. Die Partikelsuspension fließt durch einen Rohrreaktor, der in einem Magnetfeld mit wechselnden Feldgradienten eingebracht ist. Die Änderung des Magnetfeldgradienten erfolgt entgegen der Strömungsrichtung der Reaktionslösung. Durch alternierende Feldmaxima an den beiden Seiten des Reaktors werden die magnetisierbaren Partikel zu dessen Wänden gezogen. Bei Umkehrung des Feldes wandern die Partikel an die gegenüberliegende Reaktorwand. Durch Wahl einer geeigneten Wechselfrequenz kann eine kontinuierliche Durchmischung und Rückhaltung der Mikropartikel im durchströmten Rohr erreicht werden. Somit können Immobilisierungsreaktionen und Biotransformationen mit den Partikelsystemen im Durchfluss durchgeführt werden.