Future diesel engine legislation Tier 4 / Stage V and EU6d demand further improvements to reduce CO₂ while keeping the already low NOₓ emissions levels. For US trucks a more strict limit of 0.2 ...g/bhp-hr NOₓ emissions need to be achieved. In this trade-off, system costs and complexity of the after-treatment are defining the constraint in which the common rail fuel injection system layout has to be defined. The increase of rail pressure was in the past the major step to control the soot emissions in view of low engine-out NOₓ emissions by applying massive EGR. With the on-going development of NOₓ-aftertreatment by Selective Catalytic Reduction (SCR), conversion efficiencies of up to 97% allow to reduce the EGR usage and rail pressure usage. In that context, the steepness of injection rate, the nozzle flow rate and the injection pressure are remaining parameters to control the NOₓ emissions. A shallow injection rate in combination with larger nozzle flow rates is beneficial to reduce the NOₓ emissions thanks to a reduced premixing of fuel with air. To study this effect, the latest solenoid injector with improved magnetic actuation is utilized. The influence of the steepness of injection rate is studied on a 6 cylinder heavy-duty Diesel engine on 3 representative part- load points of the WLTC cycle. Depending on the engine-out NOₓ emissions requirements, two scenarios are considered. In case of a strong NOₓ aftertreatment, the shallow injection rate steepness is beneficial for the fuel consumption. In case of less NOₓ aftertreatment, high EGR rates are required and soot emissions can be controlled through the steepness of injection rate.
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.
►Metabolites in
Pancratium maritimum.
Hansenula polymorpha RB11 pC10-FMD (PFMD- 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. Biotechnol. Bioeng. 2009; 104: 554-561
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.
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.
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.
Phenylethanoids are a group of natural water-soluble compounds with high biological value, which could potentially be commercially produced by hairy root cultures. Thus, we have examined the capacity ...of transformed root cultures of Devil's claw (Harpagophytum procumbens) to accumulate four phenylethanoid glycosides -beta-OH-verbascoside, verbascoside, leucosceptoside A, and martynoside--in shake-flasks and a 3-L stirred tank reactor. Verbascoside was found to be the major phenylethanoid, and its maximal contents were the same (1.12 mg/g dry weight) in both kinds of culture. However, peak leucosceptoside A contents were 1.6-times higher in bioreactor cultures than in shake-flask cultures. Flow cytometry analysis revealed that G0 + G1-phase cells predominated throughout the growth of the cultures, which was in accordance with the very high proportion of quiescent cells in the transformed roots. The results provide the first demonstration of the potential utility of Devil's claw hairy roots as biofactories for producing high-value phenylethanoid glycosides.
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.
Three often cited systems for the extraction of plant nuclei for flow cytometric measurement (CyStain PI, Partec GmbH, Munster, 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.
