RED, PEF, and EPD: Conflicting rules for determining the carbon footprint of biofuels give unclear signals to fuel producers and customers

Biofuel producers and other commodity suppliers are increasingly affected by conflicting rules for life cycle assessment (LCA). They may get multiple requests for LCAs to be used in various contexts, which require the application of different methodological approaches that vary in scope, system boundaries, data demand, and more. This results in increased cost and competence requirements for producers, as well as confusion among other actors including their customers. Differences in methodologies might also lead to various outcomes, conclusions and conflicting guidance regarding which fuels to prioritize or develop. We have analyzed the actual differences when applying three different frameworks: the EU Renewable Energy Directive (RED), the EU framework for Product Environmental Footprints (PEF), and the framework of Environmental Product Declarations (EPD), which have different modeling requirements. We analyzed the methods from a conceptual point of view and also applied the methods to estimate the carbon footprint on a wide range of biofuel production pathways: (i) ethanol from corn, (ii) fatty acid methyl ester (FAME) from rapeseed oil, (iii) biogas from food waste, (iv) hydrogenated vegetable oil (HVO) from rapeseed oil, and (v) HVO from used cooking oil. Results obtained for a specific fuel could differ substantially depending on the framework applied and the assumptions and interpretations made when applying the different frameworks. Particularly, the results are very sensitive to the modeling of waste management when biofuel is produced from waste. Our results indicate a much higher climate impact for, e.g., biogas and HVO produced from used cooking oil when assessed with the PEF framework compared to the other frameworks. This is because PEF assigns at least part of the production of primary materials and energy to the use of recycled material and recovered energy. Developing Category Rules for biofuels for PEF and EPD ought to help clarifying remaining ambiguities

Methods for Assessing Mitochondrial Function in Diabetes

A growing body of research is investigating the potential contribution of mitochondrial function to the etiology of type 2 diabetes. Numerous in vitro, in situ, and in vivo methodologies are available to examine various aspects of mitochondrial function, each requiring an understanding of their principles, advantages, and limitations. This review provides investigators with a critical overview of the strengths, limitations and critical experimental parameters to consider when selecting and conducting studies on mitochondrial function. In vitro (isolated mitochondria) and in situ (permeabilized cells/tissue) approaches provide direct access to the mitochondria, allowing for study of mitochondrial bioenergetics and redox function under defined substrate conditions. Several experimental parameters must be tightly controlled, including assay media, temperature, oxygen concentration, and in the case of permeabilized skeletal muscle, the contractile state of the fibers. Recently developed technology now offers the opportunity to measure oxygen consumption in intact cultured cells. Magnetic resonance spectroscopy provides the most direct way of assessing mitochondrial function in vivo with interpretations based on specific modeling approaches. The continuing rapid evolution of these technologies offers new and exciting opportunities for deciphering the potential role of mitochondrial function in the etiology and treatment of diabetes

Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome c oxidase

Redox-driven proton pumps such as cytochrome c oxidase (CcO) are fundamental elements of the energy transduction machinery in biological systems. CcO is an integral membrane protein that acts as the terminal electron acceptor in respiratory chains of aerobic organisms, catalyzing the four-electron reduction of O(2) to H(2)O. This reduction also requires four protons taken from the cytosolic or negative side of the membrane, with an additional uptake of four protons that are pumped across the membrane. Therefore, the proton pump must embody a “gate,” which provides alternating access of protons to one or the other side of the membrane but never both sides simultaneously. However, the exact mechanism of proton translocation through CcO remains unknown at the molecular level. Understanding pump function requires knowledge of the nature and location of these structural changes that is often difficult to access with crystallography or NMR spectroscopy. In this paper, we demonstrate, with amide hydrogen/deuterium exchange MS, that transitions between catalytic intermediates in CcO are orchestrated with opening and closing of specific proton pathways, providing an alternating access for protons to the two sides of the membrane. An analysis of these results in the framework of the 3D structure of CcO indicate the spatial location of a gate, which controls the unidirectional proton flux through the enzyme and points to a mechanism by which CcO energetically couples electron transfer to proton translocation

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O(2). The reduction of O(2), accompanied by the uptake of substrate protons to form H(2)O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H(2)O the uptake and release reactions occur with the same rates, in D(2)O the substrate and pumped protons are taken up first (τ(D) ≅ 200 μs, “peroxy” to “ferryl” transition) followed by a significantly slower proton release to the other side of the membrane (τ(D) ≅ 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560–3,800 cm-1 range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(−), and 3,607(+) cm-1. A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D2O and H218O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism