Heavy-Duty Engines-Conformity Testing based on PEMS - Lessons Learned from the European Pilot Program

The European emissions legislation requires to check the conformity of heavy-duty engines with the applicable emissions certification standards during the normal life of those engines: these are the “In Service Conformity” (ISC) requirements. It was considered impractical and expensive to adopt an in-service conformity (ISC) checking scheme for heavy-duty vehicles, which require removal of engines from vehicles to test pollutant emissions against legislative limits. Therefore, it has been proposed to develop a protocol for in-service conformity checking of heavy-duty vehicles based on the use of Portable Emission Measurement Systems (PEMS). The European Commission through DG ENTR in co-operation with DG JRC launched in January 2004 a co-operative research programme to study the feasibility of PEMS in view of their application in Europe for In-Service Conformity of heavy-duty engines. The technical and experimental activities were started in August 2004 to study the feasibility of PEMS systems and to study their potential application for on-road measurements on heavy-duty vehicles.The main objectives of the above project had been defined as follows: -To assess and validate the application and performance of portable instrumentation relative to each other, and in comparison with alternative options for ISC testing; -To define a test protocol for the use of portable instrumentation within the ISC of heavy-duty vehicles; -To assess on-road data evaluation methods such as the US ‘Not To Exceed’ (NTE) approach and possibly to develop a simplified ones; -To address the need of the European industry, authorities and test houses to go through a learning process with on-vehicle emissions testing. The main objective of the present document is to report on: a. The evaluation of the test protocol, i.e. to judge whether the mandatory data and its quality were appropriate for the final evaluation (S b. The analysis conducted to evaluate the potential of the different data evaluation (Pass/Fail) methods for ISC and in particular their ability to use on-road PEMS emissions data. The candidate methods were categorized into two families: -The "control-area / data reduction methods" (Chapter 4) that use only a part of the data, depending whether the operation points considered are part of a control area and belong to a sequence of consecutive points within this control area. The US-NTE (Not To Exceed) method - already established as an official tool in the United States - falls into this category but variations of the methods could be envisaged (with another control area for instance). -The "averaging window methods" (Chapter 4.3) that use all the operation data. The main objective of task b. was to answer the following question: “Once the data has been collected correctly, what is the most appropriate method to analyze the test data measured with PEMS and to judge whether the engine is in conformity with the applicable emissions limits?”JRC.F.9-Sustainable Transport (Ispra

Investigations on the rapid transbilayer movement of phospholipids in biogenic membranes

In Bakterien werden Phospholipide auf der cytoplasmatischen Seite der Plasmamembran synthetisiert. Damit ein gleichmäßiges Wachstum und somit die Stabilität biogener Membranen, d.h. Membranen, an bzw. in denen Lipidsynthese stattfindet, gewährleistet ist, muss zumindestens die Hälfte neu synthetisierter Lipide auf die entgegengesetzte Membranhälfte gelangen. Aus früheren Untersuchungen ist bereits bekannt, dass dieser transversale Phospholipidaustausch, auch als Flip-Flop bezeichnet, sehr schnell, kopfgruppenunabhängig und möglicherweise proteinabhängig ist. Dennoch sind die genauen Mechanismen dieser Prozesse noch weitgehend unverstanden. Um die oben erwähnten grundlegenden Phospholipidtransportprozesse zwischen beiden Membranhälften genauer untersuchen zu können, wandten wir einen neuartigen, sogenannten stopped-flow BSA back-extraction Assay an. Mit Hilfe dieses Assays, waren wir in der Lage, die transversale Bewegung und die Verteilung von kurzkettigen, fluoreszenzmarkierten Phospholipidanaloga über beide Membranhälften in ex vivo-Membranen zu charakterisieren. Der stopped-flow BSA back-extraction Assay basiert auf der Technik der stopped-flow-Spektroskopie und der Tatsache, dass BSA in der Lage ist, kurzkettige, fluoreszenzmarkierte Lipidanaloga aus der äußeren Leaflet von (biologischen) Membranen zu extrahieren. Wir entschieden uns für invertierte Membranvesikel der Plasmamembran (IIMV) vom E.coli Wildtypstamm MG1655 als Untersuchungsobjekt, einerseits, weil diese Vesikel nur eine Membran besitzen und zum Anderen, weil IIMV sich sehr gut als Modell für den Flip-Flop von Phospholipiden nutzen lassen. Wir beobachteten, dass kurzkettige, fluoreszenzmarkierte Analoga der beiden am häufigsten in E.coli vorkommenden Phospholipide, Phosphatidylethanolamin (PE) und Phosphatidylglycerol (PG), sehr schnell, d.h. mit Halbwertzeiten von weniger als drei Minuten, über die Membran von IIMV verteilten. Weiterhin verhielten sich kurzkettige, fluoreszenzmarkierte Analoga von den E.coli-fremden Phospholipiden, Phosphatidylcholin (PC) und Phosphatidylserin (PS), ähnlich wie die Analoga von PE und PG. Überraschenderweise, fanden wir heraus, dass alle oben genannten Phospholipidanaloga im Gleichgewichtszustand nicht gleichmässig über beide Membranhälften verteilt waren. Inwiefern Proteine an dieser transversalen Bewegung der Phospholipidanaloga beteiligt sind, sollten Messungen des Flip-Flop von Analoga an unbehandelten und mit Proteinase K inkubierten Vesikeln zeigen, die aus einem Detergenzextrakt von IIMV rekonstituiert wurden. Zunächst konnten wir zeigen, dass die schnelle Bewegung der Phospholipidanaloga über die Membran von rekonstituierten, nicht mit Proteinase K behandelten Vesikeln (Proteoliposomen) erhalten blieb. Nach Inkubation mit Proteinase K wurde jedoch der Flip-Flop von PE- und PG-Analoga vollständig inhibiert. Untersuchungen an rekonstituierten Serien von Proteoliposomen mit ansteigendem bakteriellen Proteingehalt zeigten, dass in Proteoliposomen ohne bakterielle Proteine kein Flip-Flop stattfand und somit nur 50% der fluoreszenten Analoga extrahiert wurden. In Proteoliposomen, die bakterielle Proteine enthielten, stieg das Ausmass der Extrahierbarkeit der untersuchten Analoga mit steigendem Proteingehalt. Diese Daten zeigten sehr deutlich, dass die transversale Bewegung von Phospholipiden über die innere Membran von E.coli durch Proteine vermittelt wird. Schlussfolgernd aus unseren Analysen konnten wir zeigen, dass die transversale Bewegung von Phospholipidanaloga über die Membran von IIMV sehr schnell, proteinabhängig, bidirektional und kopfgruppenunbhängig ist. Zur Identifizierung der molekularen Grundlagen der proteinvermittelten, schnellen Transversalbewegung von Phospholipiden über IIMV-Membranen, nutzen wir Ionenaustauschchromatografie. Zur unserer Überraschung mussten wir feststellen, dass in keiner der rekonstituierten Fraktionen eine nennenswerte Anreicherung der Flippaseaktivität auftrat. Möglicherweise sind mehrere Proteine, mit unterschiedlichen Nettoladungen, oder aber auch Untereinheiten, die sich nicht durch Anionenaustauscher trennen liessen, am Flip-Flop von Phospholipiden beteiligt. Weitergehende Analysen mit anderen Proteinfraktionierungsmethoden sind notwendig, um den oder die Flippasekomplex(e) zu identifizieren.In the plasma membrane of bacteria, phospholipids are synthesized on the cytoplasmic leaflet of the plasma membrane. To ensure balanced growth and thus, stability of biogenic membranes, half of the newly synthesized lipids must move to the opposing leaflet. It is known that this phospholipid transmembrane movement (flip-flop) is rapid, head-group independent and possibly protein mediated. However, the exact mechanism of this process remains elusive. To investigate these fundamental transbilayer phospholipid transport processes in biogenic membranes, a novel stopped-flow BSA back-exchange assay was utilized to characterize the transmembrane movement and transbilayer distribution of fluorescent labeled, short-chain phospholipid analogues in ex vivo membranes. This approach is based on stopped-flow fluorescence spectroscopy, and the fact that BSA is able to extract fluorescent labeled, short-chain phospholipid analogues from the outer leaflet of (bio)membranes. We chose isolated inverted inner membrane vesicles (IIMV) derived from E.coli wild type MG1655, both for their simple membrane organization and for their suitability as a simple model organism for phospholipid flip-flop. We observed that fluorescent-labeled, short-chain analogues of the major phospholipids in E.coli, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), rapidly redistributed across the IIMV bilayer with half-times of less than three minutes. Furthermore, fluorescent, short-chain phospholipid analogues of phosphatidylcholine (PC) and phosphatidylserine (PS), which are not naturally occurring phospholipids in E.coli membranes, behaved similar to the PE and PG analogues. To analyze the relevance of proteins for the transmembrane movement of fluorescent analogues, we measured flip-flop of phospholipid analogues in reconstituted and/or untreated and proteinase K treated vesicles generated from protein detergent extracts of IIMV. The amount of extractable fluorescent phospholipids analogues correlated with the amount of protein reconstituted into the proteoliposomes, strongly indicating, that protein concentrations below 100 µg/ml were not sufficient to equip every vesicle with proteins that facilitate the transmembrane movement of the fluorescent analogues. We found that the rapid transbilayer movement of phospholipid analogues across the membrane was maintained in untreated reconstituted vesicles. However, the flip-flop of fluorescent PG and PE analogues was eliminated in proteinase K treated vesicles. In conclusion, our analysis showed that the transmembrane movement of the phospholipid analogues across the membrane of IIMV was protein-mediated, very rapid, bi-directional and head-group independent. To identify the molecular basis of the protein-mediated, rapid transmembrane movement of phospholipids across IIMV membranes, we used ion exchange chromatography (IEC) to separate the IIMV proteins. To our surprise, we did not observe an enhanced flip-flop activity in any of the fractions, indicating that at least two proteins with possibly opposite net charges or several subunits, which were not separable by AEC, are involved. Further analysis using different protein separation techniques will be necessary to identify the putative flippase complex

Transbilayer movement of phospholipids at the main phase transition of lipid membranes: implications for rapid flip-flop in biological membranes.

The transbilayer movement of fluorescent phospholipid analogs in liposomes was studied at the lipid phase transition of phospholipid membranes. Two NBD-labeled analogs were used, one bearing the fluorescent moiety at a short fatty acid chain in the sn-2 position (C(6)-NBD-PC) and one headgroup-labeled analog having two long fatty acyl chains (N-NBD-PE). The transbilayer redistribution of the analogs was assessed by a dithionite-based assay. We observed a drastic increase of the transbilayer movement of both analogs at the lipid phase transition of DPPC (T(c) = 41 degrees C) and DMPC (T(c) = 23 degrees C). The flip-flop of analogs was fast at the T(c) of DPPC with a half-time (t(1/2)) of ~6-10 min and even faster at the T(c) of DMPC with t(1/2) on the order of <2 min, as shown for C(6)-NBD-PC. Suppressing the phase transition by the addition of cholesterol, the rapid transbilayer movement was abolished. Molecular packing defects at the phase transition are assumed to be responsible for the rapid transbilayer movement. The relevance of those defects for understanding of the activity of flippases is discussed

Engaging Stakeholders to Solve Complex Environmental Problems Using the Example of Micropollutants

Current and future challenges such as the climate crisis, demographic change and achieving the objectives of the Water Framework Directive require holistic and precautionary approaches within the framework of national and supranational strategies. Specific measures and projects resulting from these strategic activities are required to successfully meet the challenges. In 2016, the German Environment Agency (UBA) and the Federal Ministry for the Environment (BMUV) commissioned the process for the development of the Federal Government’s micropollutants strategy, which was later named the Trace Substance Strategy. The essential core instrument herein was a multi-stakeholder dialogue aimed at giving sufficient consideration to the different interests of the various stakeholders. The goal was to develop a balanced mix of measures and to initiate implementations in order to reduce emissions of micropollutants as effectively and efficiently as possible, at the source, in their application and in the downstream areas. The various measures were tested in a pilot phase, and the activities were evaluated before being transferred into the subsequent consolidation phase. This article describes the outcomes of the stakeholder dialogue as an instrument. This is complemented by the results of a stakeholder evaluation of the process itself and the results achieved. Important outcomes of the stakeholder dialogue are a Committee for the Identification of Relevant Micropollutants and the use of roundtables as an important instrument in which the manufacturers and the users of the substances can make an important contribution to reducing emissions. To address the opportunities and necessities of additional wastewater treatment, an “orientation framework” for municipal wastewater treatment plants was also established. Additionally, the German Centre for Micropollutants (SZB) was founded to continuously organize, support and accompany the various outcomes that became relevant pillars of the German government’s Trace Substance Strategy. The evaluation has shown that new approaches and new instruments have been created within the framework of the stakeholder dialogue, which enable flexible and short-term options for action and allow for the involvement of stakeholders in a manner appropriate to the polluter-pays principle. Specific emission reductions could not be expected within the time frame of the dialogue. However, stakeholders agreed that the strategic process chosen is preferable to purely regulatory steps due to the holistic approach involving all stakeholders concerned in this complex matter. It is expected that in the future, if implemented consistently, the approach could achieve a lasting and sustainable impact on a broad scale

Development of an Official Test Method for On-Board PM Measurements from Heavy-Duty Diesel Engines in the European Union

Portable Emissions Measurement Systems (PEMS) are becoming part of the emissions control regulations, as evidenced by the latest requirements introduced in the United States regulations for on-highway and non-road machinery. The European Union is currently following the same route to check the in-use behaviour of heavy-duty diesel vehicles. The current research programmes tend to demonstrate that both the instrumentation and the test methods are mature for gaseous emissions. For PM emissions, the development of portable PM instruments and their test protocols remain a complex challenge, as simultaneous progress take place in the engine after-treatment technologies and the official homologation procedures (the PMP programme in particular). The present paper discusses the current research strategy proposed in the EU for the development of an on-board PM test protocol. Case studies from the EU-PEMS project are presented. For the equivalency tests between portable and laboratory system, results are presented for the partial flow dilution associated with the filter mass based principle. Portable commercial PM instruments are evaluated under controlled laboratory conditions (i.e. on reference test cycles) against reference laboratory instruments using a Euro III heavy-duty engine running on different fuels, including bio-fuel. Other support instruments, measuring real-time soot or the total PM composition, are also used. Under these test conditions, the study has demonstrated the equivalency between the tested portable technology and its laboratory counterpart. Another part of the work is to evaluate the applicability of the methodology for on-board testing: results and lessons learned from on board measurements conducted on diesel city buses on urban test routes are presented and discussed showing very good test-to-test repeatability. Issues, such as filter handling and conditioning on board, need particular attention for the adaptation of the test method to the future generations of vehicles.JRC.H.4-Transport and air qualit