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In the framework of the “New Vehicle and System Technologies” technical programme funded by the German Federal Ministry of Economic Affairs and Energy, methyl-ether based fuels (DME/OME1) were investigated in the “XME Diesel” project in order to study their potential of being sustainable fuel replacements for diesel engine applications in passenger cars and commercial vehicles. In studies, which covered research activities from fundamental spray investigations over single- and multi-cylinder testing up to vehicle demonstrators, the new fuels were studied with regard to emission formation, efficiency and compatibility to conventional diesel engine technology. Here, promising findings regarding emission reduction (soot-free combustion and reduction of nitrogen oxides) and increased efficiency could be obtained. For the first time, a heavy-duty OME1 based full engine was developed in which the emission-reducing potential of methyl ethers could be demonstrated.

BackgroundThe physiological characterization of microorganisms provides valuable information for bioprocess development. Chemostat cultivations are a powerful tool for this purpose, as they allow defined changes to one single parameter at a time, which is most commonly the growth rate. The subsequent establishment of a steady state then permits constant variables enabling the acquisition of reproducible data sets for comparing microbial performance under different conditions. We performed physiological characterizations of a 3-hydroxypropionic acid (3-HP) producing Saccharomyces cerevisiae strain in a miniaturized and parallelized chemostat cultivation system. The physiological conditions under investigation were various growth rates controlled by different nutrient limitations (C, N, P). Based on the cultivation parameters obtained subsequent fed-batch cultivations were designed.ResultsWe report technical advancements of a small-scale chemostat cultivation system and its applicability for reliable strain screening under different physiological conditions, i.e. varying dilution rates and different substrate limitations (C, N, P). Exploring the performance of an engineered 3-HP producing S. cerevisiae strain under carbon-limiting conditions revealed the highest 3-HP yields per substrate and biomass of 16.6 %C-mol and 0.43ggCDW-1, respectively, at the lowest set dilution rate of 0.04h-1. 3-HP production was further optimized by applying N- and P-limiting conditions, which resulted in a further increase in 3-HP yields revealing values of 21.1 %C-mol and 0.50ggCDW-1 under phosphate-limiting conditions. The corresponding parameters favoring an increased 3-HP production, i.e. dilution rate as well as C- and P-limiting conditions, were transferred from the small-scale chemostat cultivation system to 1-L bench-top fermentersoperating in fed-batch conditions, revealing 3-HP yields of 15.9 %C-mol and 0.45ggCDW-1 under C-limiting, as well as 25.6 %C-mol and 0.50ggCDW-1 under phosphate-limiting conditions.ConclusionsSmall-scale chemostat cultures are well suited for the physiological characterization of microorganisms, particularly for investigating the effect of changing cultivation parameters on microbial performance. In our study, optimal conditions for 3-HP production comprised (i) a low dilution rate of 0.04h-1 under carbon-limiting conditions and (ii) the use of phosphate-limiting conditions. Similar 3-HP yields were achieved in chemostat and fed-batch cultures under both C- and P-limiting conditions proving the growth rate as robust parameter for process transfer and thus the small-scale chemostat system as powerful tool for process optimization.

The upcoming Stage V emissions regulation for Non-Road Heavy Duty Diesel Engines will force OEMs to adopt Diesel Particulate Filters, adding a further degree of complexity to the aftertreatment system, which in several cases already includes specific devices for NOx reduction. Since complex aftertreatment systems can rise packaging problems as well as reliability issues, the authors evaluated the performance of a Stage V compliant engine layout which can avoid dedicated aftertreatment devices for NOx. This engine features a low pressure exhaust gas recirculation system, a two-stage turbocharger and a 3000 bar injection pressure-capable fuel injection system. This paper focus on the experimental assessment of the performance of the fuel injection system, demonstrating how increasing the rail pressure from 2000 to 3000 bar can dramatically improve the NOx-Soot and NOx-Particulate Number trade-off, together with the engine efficiency, without adversely affecting the emission of nanoparticles. In particular, extremely high injection pressures in conjunction with the use of after injection as a soot reduction technique, was found to be capable of achieving a 50% smoke reduction with a more than acceptable engine efficiency degradation.

Bioconversions in industrial processes are currently dominated by single-strain approaches. With the growing complexity of tasks to be carried out, microbial consortia become increasingly advantageous and eventually may outperform single-strain fermentations. Consortium approaches benefit from the combined metabolic capabilities of highly specialized strains and species, and the inherent division of labor reduces the metabolic burden for each strain while increasing product yields and reaction specificities. However, consortium-based designs still suffer from a lack of available tools to control the behavior and performance of the individual subpopulations and of the entire consortium. Here, we propose to implement novel control elements for microbial consortia based on artificial cell-cell communication via fungal mating pheromones. Coupling to the desired output is mediated by pheromone-responsive gene expression, thereby creating pheromone-dependent communication channels between different subpopulations of the consortia. We highlight the benefits of artificial communication to specifically target individual subpopulations of microbial consortia and to control e.g. their metabolic profile or proliferation rate in a predefined and customized manner. Due to the steadily increasing knowledge of sexual cycles of industrially relevant fungi, a growing number of strains and species can be integrated into pheromone-controlled sensor-actor systems, exploiting their unique metabolic properties for microbial consortia approaches.

Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2'-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244-1250. © 2015 Wiley Periodicals, Inc.

Externally illuminated photobioreactors (PBRs) are widely used in studies on the use of phototrophic microorganisms as sources of bioenergy and other photobiotechnology research. In this work, straightforward simulation techniques were used to describe effects of varying fluid flow conditions in a continuous hydrogen-producing PBR on the rate of photofermentative hydrogen production (rH2 ) by Rhodobacter sphaeroides DSM 158. A ZEMAX optical ray tracing simulation was performed to quantify the illumination intensity reaching the interior of the cylindrical PBR vessel. 24.2% of the emitted energy was lost through optical effects, or did not reach the PBR surface. In a dense culture of continuously producing bacteria during chemostatic cultivation, the illumination intensity became completely attenuated within the first centimeter of the PBR radius as described by an empirical three-parametric model implemented in Mathcad. The bacterial movement in chemostatic steady-state conditions was influenced by varying the fluid Reynolds number. The "Computational Fluid Dynamics" and "Particle Tracing" tools of COMSOL Multiphysics were used to visualize the fluid flow pattern and cellular trajectories through well-illuminated zones near the PBR periphery and dark zones in the center of the PBR. A moderate turbulence (Reynolds number = 12,600) and fluctuating illumination of 1.5 Hz were found to yield the highest continuous rH2 by R. sphaeroides DSM 158 (170.5 mL L(-1) h(-1) ) in this study.

In this study, microalgae were cultivated in the form of suspension cultures and a new structurally organized immobilization technique called “Green Bioprinting.” This technique allows the cocultivation of microorganisms in close vicinity to, but without direct contact with microalgae, to improve the oxygen supply of different cell types by photosynthetic oxygen evolution. However, more research on the optimum culture conditions for immobilized microalgae is necessary. Therefore, Chlamydomonas reinhardtii 11.32b and Chlorella sorokiniana UTEX1230 were suspended in culture medium or embedded in hydrogels by the 3D-bioprinting process followed by cultivation under different temperatures (26°C, 30°C, or 37°C) and modes of illumination (continuous illumination or a 14/10 h light/dark cycle). The viability was monitored by either flow cytometry (suspension cultures) analysis of DiBAC4(3)-stained cells or fluorescence image analysis (hydrogel-embedded cultures). Suspended microalgae subjected to continuous illumination exhibited an increased number of membrane-depolarized cells compared to those cultivated at a 14/10 h light/dark cycle. Hydrogel immobilization resulted in a facilitated viability and stable growth rates between 0.4 and 0.7 d−1 for both microalgae strains. Concluding, the 3D-bioprinting immobilization represents a technique to cultivate microalgae at a high viability and growth rate even under nonoptimal temperature conditions.

Embedding of mammalian cells into hydrogel scaffolds of predesigned architecture by rapid prototyping technologies has been intensively investigated with focus on tissue engineering and organ printing. The study demonstrates that such methods can be extended to cells originating from the plant kingdom. By using 3D plotting, microalgae of the species Chlamydomonas reinhardtii were embedded in 3D alginate-based scaffolds. The algae survived the plotting process and were able to grow within the hydrogel matrix. Under illumination, the cell number increased as indicated by microscopic analyses and determination of the chlorophyll content which increased 16-fold within 12 days of cultivation. Photosynthetic activity was evidenced by measurement of oxygen release: within the first 24 h, an oxygen production rate of 0.05 mg L−1 h−1 was detected which rapidly increased during further cultivation (0.25 mg L−1 h−1 between 24 and 48 h). Furthermore, multichannel plotting was applied to combine human cells and microalgae within one scaffold in a spatially organized manner and hence, to establish a patterned coculture system in which the algae are cultivated in close vicinity to human cells. This might encourage the development of new therapeutic concepts based on the delivery of oxygen or secondary metabolites as therapeutic agents by microalgae.

Worldwide, combustion engines will remain as major power unit for vehicle propulsion in long-term. Consequently, immediate measures are claimed to reduce the current CO2 production from combustion engines which are accomplished by three approaches: (1) an increase of the thermal efficiency, (2) the application of fuels with low carbon content and (3) the production of fuels from renewable feedstocks. The first aspect clearly emphasizes compression ignition (CI) engines, the second aspect draws the attention to hydrogen and single C-bonded fuels and the third aspect has initiated sensitive discussions about renewable resources which lead to the commitments of first/ second/ third generation biofuels. In this context, the use of Ethers as neat or blended fuels for combustion engines has been discussed for more than 20 years. Among these, the simplest compound, Dimethylether CH3-O-CH3 (DME), has an exceptional position as a neat fuel for compression ignition (CI) engines due to its excellent ignition and combustion properties which have been well investigated published by many authors.

To identify optimal hydrogen production conditions using growing cultures of Rhodobacter sphaeroides DSM 158 the effects of varying the reactor’s volumetric power input (0.01–1.4 kW m −3 ) and irradiation intensity (5–2500 W m −2 ) were investigated in batch and continuous production modes. Irradiation intensity had a greater effect on hydrogen production than volumetric power input. Hydrogen production and photofermentative biomass formation were maximized by irradiation at 2250 W m −2 with a volumetric power input of 0.55 kW m −3 . The bacterial dry weight (2.64 g L −1 ) and rate of hydrogen production (195 mL L −1 h −1 ) achieved under these conditions were greater than any that have previously been reported for batch-mode hydrogen production by R. sphaeroides . Continuous mode experiments ( D = 0.1 h −1 ) yielded a bacterial dry weight, hydrogen production rate, productivity and hydrogen yield of 2.35 ± 0.18 g L −1 , 165 ± 6.2 mL L −1 h −1 , 3.96 L L −1 d −1 and 36.6%, respectively.

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.

Microbiological photosynthesis is a promising tool for producing hydrogen in an ecologically friendly and economically efficient way. Certain microorganisms (e.g. algae and bacteria) can produce hydrogen using hydrogenase and/or nitrogenase enzymes. However, their natural capacity to produce hydrogen is relatively low. Thus, there is a need to optimize their core photosynthetic processes as well as their cultivation, for more efficient hydrogen production. This review aims to provide a holistic overview of the recent technological and research developments relating to photobiological hydrogen production and downstream processing. First we cover photobiological hydrogen synthesis within cells and the enzymes that catalyze the hydrogen production. This is followed by strategies for enhancing bacterial hydrogen production by genetic engineering, technological development, and innovation in bioreactor design. The remaining sections focus on hydrogen as a product, that is, quantification via (in-process) gas analysis, recent developments in gas separation technology. Finally, a discussion of the sociological (market) barriers to future hydrogen usage is provided as well as an overview of methods for life cycle assessment that can be used to calculate the environmental consequences of hydrogen production.

Diesel engines remain the main powertrain concept in view of efficiency and robustness. On the one hand the emissions towards the future EU CO2 limit of 95 g/km by 2021 further demands the reduction of the fuel consumption. On the other hand the new WLTP test cycle and RDE emissions tests demand to reduce the engine-out raw emissions. So far, the main development path of the fuel injection system is focusing on increasing the rail pressure. The increase of the no. of injection events is another approach that aims for phasing the combustion in the working process of the Diesel engine efficiently. This approach is limited by the needle opening speed and capability of closed-coupled injection events. Such features were so far restricted to Piezo injectors. Recently the solenoid injector was enhanced to close this gap and it was firstly tested on an hydraulic test bench and a 4 cyl. passenger car Diesel engine. The hydraulic and engine performance is compared against the solenoid injector G4S and the Piezo injector G4P. With increasing the injection rate steepness and reducing the hydraulic intervals, a triple pilot injection strategy is applied to control the ignition delay and rate of combustion. The steep injection rate reduces the soot emissions without compromising the combustion noise. On the other hand the short interval and fast needle actuation reduces the injection and combustion duration. Thanks to this improved hydraulic performance the BSFC could be improved by 1.7% and soot emissions reduced by 36% at engine part load conditions (2000 rpm, BMEP=6 bar).

Some Lactobacillus delbrueckii ssp. bulgaricus strains are able to synthesize exopolysaccharides (EPS) and are therefore highly important for the dairy industry as starter cultures. The aim of this study was to investigate the nutritional requirements for growth and EPS production of Lactobacillus delbrueckii ssp. bulgaricus DSM 20081. A medium was developed from a semi-defined medium (SDM) in which glucose was replaced by lactose and different combinations of supplements (nucleobases, vitamins, salts, sodium formate and orotic acid) were added. Constant pH batch fermentation with the modified medium resulted in an EPS yield of approximately 210 mg glucose equivalents per liter medium. This was a 10-fold increase over flask cultivation of this strain in SDM. Although not affecting cell growth, the mixture of salts enhanced the EPS synthesis. Whereas EPS production was approximately 12 mg/g dry biomass without salt supplementation, a significantly higher yield (approximately 20 mg/g dry biomass) was observed after adding the salt mixture. In continuous fermentation, a maximal EPS concentration was obtained at a dilution rate of 0.3 1/h (80 mg EPS/L), which corresponded to a specific EPS production of 49 mg/g dry biomass. © 2011, The Society for Biotechnology, Japan. All rights reserved.

Hydrophobins are small surface-active proteins that have considerable potential for use in applications ranging from medical and technical coatings, separation technologies, biosensors, and personal care. Their wider use would be facilitated by the availability of recombinant tailor-made hydrophobins. We successfully expressed the class II hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris under the control of the constitutive GAP (glyceraldehyde 3-phosphate dehydrogenase) promoter. Avoiding the use of the AOX1 (alcohol oxidase 1) promoter prevents the costs and risks associated with the storage and delivery of methanol used as an inducer. Efficient secretion of hydrophobin was achieved using either the alpha-factor prepro-peptide or the native secretion signal of HFB1. The secreted hydrophobins have been isolated with a purity of up to 70% using in situ foam separation during the cultivation process. Coating experiments and surface pressure measurements demonstrated the activity of the hydrophobins. An immunodot assay showed the accessibility of carboxyterminally fused tags of the hydrophobin, which is necessary for potential applications using functionalized hydrophobins. The presented data show that Pichia pastoris is a suitable system for production of constitutively expressed and secreted active hydrophobin, allowing for in situ pre-purification using foam separation.

Callus cultures of Salvia tomentosa Mill. were induced and analyzed for their capacity to produce oleanolic and ursolic acids. The obtained callus lines showed high variability in their biosyntheti...

In a previous report, we showed that cell cultures of Harpagophytum procumbens, a South African plant with high medicinal value, accumulate high amounts of anti-inflammatory phenylethanoid glycosides during cultivation in shake-flasks. The aim of the present study was to transfer the phenylethanoid biosynthetic process to a 3-L stirred tank reactor and a 1-L glass-column bioreactor (operated with pulsed aeration). We found that, with stepwise increases in aeration, the stirred tank reactor yielded similar productivities of verbascoside (the major phenylethanoid glycoside in the cells) to those reported for shake-flask cultures (55.68 vs. 54.78 mg verbascoside/L/day, respectively). Transfer in the pulse-aerated column reactor resulted in 165.42 mg verbascoside/L/day, one of the highest yields reported to date. Further, to evaluate the physiological status of the suspended cells in the bioreactors cultures, we examined their hormone levels and compared them to those of cells in shake-flask cultures. While indole-3-acetic acid levels did not differ significantly between the bioreactor and shake-flask cultures, there were considerable differences in their levels of abscisic, jasmonic, and salicylic acids. These results are discussed with respect to relative stress levels in the different cultivation systems.

Calli, shoot-clumps and regenerated plants were initiated from young fruits of Pancratium maritimum L. Their genetic stability was monitored by flow cytometry before chemical studies. Apolar metabolites (alkaloids extracted at pH > 7, free fatty acids and fatty alcohols, sterols etc.) were qualitatively and quantitatively analyzed by GC-MS. The results clearly demonstrated that alkaloid synthesis in P. maritimum is closely related with tissue differentiation. The highest amounts of alkaloids and presence of homolycorine and tazettine type compounds (end products of the biosynthetic pathway of the Amaryllidaceae alkaloids) were found in highly differentiated tissues. Galanthamine accumulated in the leaves of plantlets. The amount of hordenine, a protoalkaloid, is related with the ability of tissues to synthesize alkaloids. Saturated fatty acids were found in considerably higher levels in undifferentiated callus cultures and partially differentiated shoot-clumps than in regenerated plants. Mono- and dienoic fatty acids were found at higher levels in non-photosynthesizing tissues - calli, and in vitro and intact bulbs, while α-linolenic acid (trienoic acid) was found in higher amounts in the photosynthesizing leaves of shoot-clumps and regenerated plants than in bulbs and calli. Fatty alcohols were found mainly in leaves, while sterols tended to accumulate in photosynthesizing and undifferentiated tissues.

A cell suspension culture of Devil’s claw (Harpagophytum procumbens), a South African plant with high medicinal value, cultivated under submerged conditions showed stable growth and accumulated high amounts of biomass (18.2 g l−1). Flow cytometry analyses of the suspension’s cell cycle kinetics showed that proportions of cells in G0/G1 and S phases varied insignificantly (between 69–76% and 9–13%, respectively) during the cultivation, while the proportion of G2/M-phase cells increased until day 8 of cultivation, when the exponential phase of cell growth ended. Metabolite production in the culture was studied through simultaneous determination of three bioactive phenylethanoid glycosides (verbascoside, β-OH-verbascoside and leucosceptoside A) by high performance liquid chromatography. It was found that suspended Devil’s claw cells accumulated mainly verbascoside (517.3 mg l−1), followed by leucosceptoside A (107.1 mg l−1) and β-OH-verbascoside (80.3 mg l−1). In addition, several fatty acids and β-sitosterol were identified in the cell suspension by gas chromatographic-mass spectrometry analysis. Comparison of the results with previously acquired data for Harpagophytum procumbens transformed roots indicate that cell suspensions cultures are more promising as potential commercial sources of metabolites such as phenylethanoid glycosides.

Betalains are water-soluble plant pigments that are widely used as food colorants, and have a wide range of desirable biological activities, including antioxidant, anti-inflammatory, hepatoprotective, anti-cancer properties. They can be produced from various plants, notably beetroot, but betalain products obtained in this way also have some undesirable properties and are difficult to standardize. A potentially attractive alternative is to use hairy root cultures. In the study reported here, we found that betalain extracts obtained from hairy root cultures of the red beetroot B. vulgaris cv. Detroit Dark Red also had higher antioxidant activity than extracts obtained from mature beetroots: six-fold higher 2,2-dyphenyl-1-picrylhydrazyl radical scavenging ability (90.7% inhibition, EC(50) = 0.11 mg, vs 14.2% inhibition, EC(50) = 0.70 mg) and 3.28-fold higher oxygen radical absorbance capacity (4,100 microM TE/g dry extract, vs 1,250 microM TE/g dry extract). The high antioxidant activity of the hairy root extracts was associated with increased concentrations (more than 20-fold) of total phenolic concomitant compounds, which may have synergistic effects with betalains. The presence of 4-hydroxybenzoic acid, caffeic acid, catechin hydrate, and epicatechin were detected in both types of extract, but at different concentrations. Rutin was only present at high concentration (1.096 mg.g(-1) dry extract) in betalain extracts from the hairy root cultures, whereas chlorogenic acid was only detected at measurable concentrations in extracts from intact plants.

One of the challenges of intracorporeal ureterolithotripsy is undesired stone migration. Stone-trapping devices have been designed to prevent this quite common phenomenon. These devices have to be effective in terms of ureteral obstruction and safe in terms of resistance to the action of commonly used lithotriptors. This work was conducted to evaluate the efficacy and safety of the recently approved Accordion® stone-trapping device in vitro. In a rigid, submerged ureteral model with two different diameters (8 and 10 mm), artificial stones were positioned in direct contact with the engaged Accordion® device. A defined number of pneumatic pulses of the LithoClast® master at different performance levels was applied and the migration distance of the stone was measured after each single pulse. As a control, the same series was repeated without the stone-trapping device. Secondly, the Accordion device was exposed to a previously defined number of pneumatic or Ho:YAG-laser pulses, in direct contact with the lithotripsy probe, up to a total activation time of 2 min. At different time points, the device was controlled for damage and functionality. The mean stone migration distance without the Accordion® device was between 39.2 and 52.8 mm and between 37.8 and 75.4 mm in the 8 and 10 mm tubes, respectively. In comparison, the stone or fragment travelling distance with the device was in the 0–2 mm range. This difference was highly significant. Both pneumatic and laser lithotriptor did not affect the functionality of the Accordion® device. The Ho:YAG laser causes small perforations of the film without affecting the devices’ stability. The Accordion device appears to be highly efficient and safe in vitro. Clinical trials will have to assess its value in endourological practice. Randomised comparative trials comparing different stone-trapping devices are needed.

Hansenula polymorpha RB11 pC10-FMD (P(FMD)- GFP) (FMD promoter gfp gene) was simultaneously cultivated in the Respiration Activity Monitoring System (RAMOS) and in the microtiter plate cultivation system "BioLector" under phosphate limitation. The light scatter signal of the BioLector, for the determination of the biomass concentration in the wells, shows a significant decrease with the onset of the phosphate limitation until a stationary level is reached. At lower initial phosphate concentration this effect is more pronounced and longer time is required until the stationary level of the scattered light is achieved. The oxygen transfer rate signal of the RAMOS and the light scatter signal of the BioLector correlate with respect to the points of time where the maxima and the stationary levels of the courses are reached. In order to understand the effect causing this light scatter behavior, the forward and side scatter properties were investigated off line by flow cytometry. The decay in the light scatter of the BioLector seems to correlate with the formation of two subpopulations of different scatter intensities detected by a flow cytometer. With ongoing cultivation the fraction of cells possessing higher light scattering properties decreases until only a population of lower light scattering properties exists. The rate of transition of the yeast from one subpopulation to the other appears to be correlated with the rate of decrease in the BioLector light scatter signal. The formation of the subpopulations may be caused by an increased asymmetry in the cell cycle due to phosphate limitation.

Der unwillkurliche Urinverlust ist fur die Betroffenen gleichbedeutend mit einem Verlust an Lebensqualitat. Die Dunkelziffer der nicht diagnostizierten und behandelten Falle ist sicherlich sehr hoch, da der Gang zum Haus- bzw. Facharzt von vielen Patientinnen verzogert bzw. vollstandig vermieden wird. Eine vollstandige sowie einfuhlsamen Anamnese, eine grundliche korperliche Befunderhebung, nichtinvasive diagnostische Masnahmen (Miktionsprotokoll, Inkontinenzfragebogen) sowie apparative Untersuchungsmethoden (Urodynamik, Zystoskopie, Radiographie, Ultraschall, MRT) liefern die notigen Informationen zur Diagnosefindung. Die Differenzierung verschiedener Harninkontinenzformen sowie Miktionsstorungen stellt aufgrund der zunehmenden Haufigkeit und Komplexitat der Pathophysiologie der Harninkontinenz eine besondere Herausforderung dar, ist jedoch unerlasslich, um ein individuelles therapeutisches Konzept entwickeln zu konnen.

Small-scale cultivation systems with real-time-monitoring of suspension parameters are important for high-throughput bioprocess development. This manuscript describes the design and characterization of a new photobioreactor (PBR) approach using 3D-printing and organic light emitting diodes (OLEDs) in the design step. The structurally complex miniaturized PBR periphery was manufactured from polyamide using the selective laser sintering technology. The MicrOLED-PBR - the first Flat-Panel-Airlift photobioreactor (FPA-PBR) with a working volume below 20 mL - was equipped and validated with non-invasive optical sensors for cell- (microalgal dry weight concentration and chlorophyll fluorescence) and suspension parameters (pH, dO 2 and dCO 2 ) allowing multiparametric high-resolution physiological studies of microalgae growth at low photon flux densities. The OLED modules used in the MicrOLED-PBR were characterized with respect to their spectral photosynthetically active radiation efficiency (35.31%), maximum photon flux density (83 μmol m − 2 s − 1 ) and resulting photon flux density profiles across the layer thickness of the FPA-cultivation chamber (10 mm) according to Lambert-Beers law (150 μmol m − 2 s − 1 for dual-plane external illumination). The hydrodynamic properties of the FPA-cultivation chamber, i.e. its volumetric oxygen transfer coefficient k L a (1.5–57 h − 1 ), superficial gas velocity (0.8–42 m h − 1 ), mixing time (1.5–34.5 s) and gas hold-up (0.016–0.2) were comparable to those for lab- and production-scale FPA-PBRs at volumetric aeration rates of 0.5–5.0 L h − 1 . The application of the MicrOLED-PBR was demonstrated for optimizing the CO 2 conditions during batch-mode growth of Chlamydomonas reinhardtii 11-32b. By analyzing the suspension dynamics in real-time limitations of dissolved carbon dioxide were identified at a CO 2 amount of 0.1 vol% whereas 2.0 vol% CO 2 was identified as optimum conditions for growing C. reinhardtii 11-32b in the MicrOLED-PBR.

Externally illuminated photobioreactors (PBRs) are widely used in studies on the use of phototrophic microorganisms as sources of bioenergy and other photobiotechnology research. In this work, straightforward simulation techniques were used to describe effects of varying fluid flow conditions in a continuous hydrogen-producing PBR on the rate of photofermentative hydrogen production (rH2 ) by Rhodobacter sphaeroides DSM 158. A ZEMAX optical ray tracing simulation was performed to quantify the illumination intensity reaching the interior of the cylindrical PBR vessel. 24.2% of the emitted energy was lost through optical effects, or did not reach the PBR surface. In a dense culture of continuously producing bacteria during chemostatic cultivation, the illumination intensity became completely attenuated within the first centimeter of the PBR radius as described by an empirical three-parametric model implemented in Mathcad. The bacterial movement in chemostatic steady-state conditions was influenced by varying the fluid Reynolds number. The "Computational Fluid Dynamics" and "Particle Tracing" tools of COMSOL Multiphysics were used to visualize the fluid flow pattern and cellular trajectories through well-illuminated zones near the PBR periphery and dark zones in the center of the PBR. A moderate turbulence (Reynolds number = 12,600) and fluctuating illumination of 1.5 Hz were found to yield the highest continuous rH2 by R. sphaeroides DSM 158 (170.5 mL L(-1) h(-1) ) in this study.

A laboratory-scale microbubble dispersion (MBD) generator was shown to improve oxygen transfer to aerobic microorganisms when connected to a conventional air sparger. However, this process setup had not been tested with fungi, where the morphology of microorganisms causes a very viscous broth rheology that impedes the oxygen mass transfer. Trichoderma reesei RUT C30 was chosen as a model fungus as it shows both the challenging rheology and a cellulase enzyme complex product that is shear-sensitive. Conventional and MBD sparged fermentations where carried out to compare the effect of aeration methods on oxygen transfer and product formation. MBD sparged fermentations showed fivefold higher kLa values and nearly twofold higher cell mass productivities. The cellulase enzyme activities were similar for both the MBD and the conventional sparged fermenters.

Objectives: A new lithotriptor for intracorporeal lithotripsy was developed combining the two most effective lithotriptors. A combination of the mechanically driven Lithoclast Master and a new ultrasonic device was constructed. Efficacy was tested in standardized model stones and in patient treatment. Material: The new lithotriptor is composed of a Lithoclast Master and an ultrasonic device (EMS, Nyon, Switzerland). The 1.0 mm Lithoclast probe is advanced off-center through the hollow 3.3 mm ultrasonic probe and protrudes about 1 mm. Five different artificial stones of defined hardness and density were used as model stones for disintegration. Time until first fragmentation and complete fragmentation (particles smaller than 2.2 mm to fit through the ultrasonic probe), percent disintegration after 1 min and time until 50% disintegration were determined for the new device as well as lithoclast and ultrasound alone. A total of 68 patients were treated by percutaneous nephrolitholapaxy (PNL) with the new device from February 1999 to August 2001. Lithotripsy was performed after fluoroscopically guided puncture of the lower calix and dilatation of the nephrostomy tract with coaxial bougies. Thirty-five patients had complete and 33 patients partial staghorn calculi. Results: First fragmentation was reached 25–200 times faster with the combination as with either mode alone. Disintegrated stone mass after 1 min was 1.5–4 times larger in combined lithotripsy and 50% disintegration time 30–50% shorter. Clinically, complete stone free rate (KUB and ultrasound) was 66% after the first PNL. Sixteen out of 68 patients had a second look PNL with an overall stone free rate of 89.7% by dismission. Stone composition was calcium-oxalate–monohydrate in 13%, Ca-ox–monohydrate/uric acid in 35%, apatite in 20% and cystine in 11%. Conclusion: In in vitro experiments and clinically the new lithotriptor provides easy handling and high effectivity in fragmentation of all stones regardless of their composition.

Objective To assess a new lithotripter for intracorporal lithotripsy, which combines the mechanically driven pneumatic LithoClast™ (Electro-Medical Systems, Nyon, Switzerland) and a new ultrasonic device (Electro-Medical Systems), for use in percutaneous nephrolitholapaxy (PNL). Patients and methods The new lithotripter consists of a LithoClast Master with 12 Hz repetition rate and a new ultrasonic device. The 1.0 mm LithoClast probe is advanced off-centre through the hollow 3.3 mm ultrasonic probe and protrudes about 1 mm. A new irrigation system with a pinch valve compressing the irrigation tube, a foot-switch for activating the ultrasound, the LithoClast and both together, and a stone bucket at the outlet tube are new features. Between February 1999 and August 2001, 68 patients were treated by PNL with the new device; 35 had complete and 33 had partial staghorn calculi. PNL was administered under fluoroscopic control and with the patient prone. Results The mean (range) duration of surgery was 61 (42–119) min. The complete stone-free rate was 66% after the first PNL; of the 68 patients, 16 received a second PNL, giving a final stone-free rate of 76% and 80%. The stone was composed of calcium oxalate monohydrate (COM) in 13%, COM with uric acid in 35%, apatite in 20% and cystine in 11%. Clinically the new lithotripter was very effective, producing smaller stone particles and thus fewer residual stone fragments after PNL than with the LithoClast or ultrasonic fragmentation alone. Conclusion The new lithotripter provides easily managed and highly effective stone fragmentation of all stones, regardless of their composition.

Three often cited systems for the extraction of plant nuclei for flow cytometric measurement (CyStain PI, Partec GmbH, Münster, Germany, the method of Arumuganathan and Earle, and LB01 buffer) failed, when applied to the hairy roots of red beet (Beta vulgaris). By combining these systems and introducing a centrifugation step, the extraction, staining, and analysis of nuclei from this tissue were performed successfully.

Mass production of value-added molecules (including native and heterologous therapeutic proteins and enzymes) by plant cell culture has been demonstrated as an efficient alternative to classical technologies [i.e. natural harvest and chemical (semi)synthesis]. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up plant cell culture-based processes (most notably to produce paclitaxel) and several commercial processes have been established so far. The choice of a suitable bioreactor design (or modification of an existing commercially available reactor) and the optimization of its internal environment have been proven as powerful tools toward successful mass production of desired molecules. This review highlights recent progress (mostly in the last 5 years) in hardware configuration and optimization of bioreactor culture conditions for suspended plant cells.

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.

The ploidy levels of the cells in different organs (leaves, petioles and roots) of red beet (Beta vulgaris L.) plants of different ages, as well as of different in vitro systems (transformed hairy roots, calli derived from leaves and rhizogenic calli), were investigated using flow cytometry. Two callus lines with red and yellow phenotypes, derived by mechanical separation of the morphologically heterogeneous rhizogenic callus, were also examined. All investigated samples experienced several cycles of endoreduplication. The older organs exhibited higher levels of polysomaty than the young ones. The highest degree of endoreduplication was found in old petiole tissue and the lowest in the red callus line (cycle values of 1.81 and 0.55, respectively). Interestingly, the callus derived from leaves did not exhibit a 2Cx peak, but was tetraploid, probably due to genetic instability, which may have been caused by prolonged cultivation under in vitro conditions. Red and yellow calli showed significantly lower polysomaty (cycle values of 0.55 and 0.59, respectively) than the primary rhizogenic callus (cycle value of 1.09). The DNA profiles of the two phenotypes differed, possibly reflecting differences in their metabolism.

More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered.

Plant in vitro systems are valuable sources for the production of biological active substances. However, changed profiles of secondary metabolites, and low, variable yields possibly caused by genetic instabilities complicate their industrial implementation. DNA profiling of plant in vitro cultures may provide data for the selection of highly producing in vitro cultures. Diploid and tetraploid Datura stramonium and Hyoscyamus niger plant as well as calli, and hairy root lines derived from them were analyzed by flow cytometry. Plant in vitro cultures undergo several cycles of endoreduplication more than the explants from which they were obtained. The highest cycle values were observed in calli (e.g. 1.19 for diploid H. niger) possibly induced by the growth factors. However, hairy roots cultivated without growth factor exhibited significant degrees of endoreduplication (cycle value 0.88 for diploid H. niger). Sets of five hairy root lines from each plant and ploidy level showed consistent within-set ploidy patterns. The ploidy profiles of investigated plant in vitro and in vivo differ. For the first time we report that hairy roots of two Solanaceae species undergo endoreduplication. Theploidy profiles of in vitro cultures (hairy roots and calli) seem to be influenced by the genome size, the growth factors applied, and the type of in vitro culture. The transformation of several hairy root lines showed no differences in the ploidy patterns. © 2008 International Society for Advancement of Cytometry

Hyoscyamine biosynthesis in Datura stramonium hairy roots with different ploidy levels was investigated. For the first time, we report that hairy roots undergo endoreduplication and therefore consist mainly of cells with doupled sets of chromosomes of primary plant tissues, used for Agrobacterium transformation. The alkaloid profiles of hairy roots obtained from diploid and tetraploid plants were similar in terms of the major compounds, but they differed significantly with respect to the minor compounds (here defined as those that accounted for

For over twenty years, DME has shown itself to be a most promising fuel for diesel combustion. DME is produced by simple synthesis of such common sources as coal, natural gas, biomass, and waste feedstock. DME is a flammable, thermally-stable liquid similar to liquefied petroleum gas (LPG) and can be handled like LPG. However, the physical properties of DME such as its low viscosity, lubricity and bulk modulus have negative effects for the fuel injection system, which have both limited the achievable injection pressures to about 500 bar and DME's introduction into the market. To overcome some of these effects, a common rail fuel injection system was adapted to operate with DME and produce injection pressures of up to 1000 bar. To understand the effect of the high injection pressure, tests were carried out using 2D optically accessed nozzles. This allowed the impact of the high vapour pressure of DME on the onset of cavitation in the nozzle hole to be assessed and improve the flow characteristics.

Objectives A new device for percutaneous nephrolithotomy, combining Lithoclast (LC) and ultrasound (US) lithotripsy, was developed. Under standardized in vitro conditions, we evaluated the efficacy of the new technique using artificial stones. Combined application of pneumatic and US lithotripsy was compared with each of the two components alone. Methods Five different artificial stones of defined hardness were used. Disintegration was performed under defined pressure in a water bath. The time until the first fragmentation and until complete disintegration to fragments of 2 mm or smaller was measured for LC and US alone and for combined lithotripsy. Furthermore, the disintegrated partition after 1 minute and time until 50% disintegration of each stone was determined. Results With regard to first fragmentation and complete disintegration, LC and US combination showed superior efficacy. First fragmentation was achieved 25 to 200 times faster and complete disintegration in a range of 11 to 15 minutes. No complete disintegration was possible by LC and US alone within a time limit of 20 minutes. The disintegrated stone mass after 1 minute was 1.5 to 4 times larger in combined lithotripsy and the 50% disintegration time was 30% to 50% compared with LC or US alone. No technical defects occurred. Conclusions Combining LC and US in one device for percutaneous nephrolithotomy shows promising in vitro results in an artificial stone model. It seems to provide superior efficacy in the disintegration parameters important for clinical practice.

Transrectal multi-core biopsies of the prostate can cause substantial discomfort with the need for high dose systemic analgesics. In a prospective randomised trial we investigated the efficacy of fine-needle administered local anaesthesia for bilateral prostatic nerve block prior to transrectal ultrasound (TRUS) guided prostate biopsy.One hundred and eight men suspected of having cancer of the prostate were randomised to receive TRUS-guided bilateral prostate nerve block prior to biopsy or not, when having no history of previous prostate biopsies (groups I and II, n=68). In group III (n=40) all patients with history of previous biopsies exclusively received local anaesthesia injection. Patients routinely underwent a 10-core biopsy regimen. For repeat biopsy 12 cores were taken. The consented patients documented pain on a visual analogue pain score.In the randomised groups I and II average pain score was 1.85 with versus 3.29 without periprostatic nerve block (p0.0001). In group III the difference in pain stated for the present biopsy with local anaesthesia nerve block in comparison to the pain experienced with the previous biopsy solely under transrectal lidocaine gel was even higher (1.71 versus 4.59; p0.0001). Pain relief was independent of the number of biopsy cores sampled. Overall cancer detection rate was 46% (50/108).Bilateral local anaesthesia nerve block prior to multi-core TRUS-guided prostate biopsy significantly reduces pain independent of the number of cores taken.