A green and easy-to-assemble electrochemical biosensor based on thylakoid membranes for photosynthetic herbicides detection.

In this study, we report on an easy-to-assemble amperometric electrochemical biosensor incorporating thylakoid membranes for the detection of photosynthetic herbicides. These molecules interfere with the light-induced photosynthetic electron transport occurring at the level of the photosystems within the thylakoid membranes, thus reducing the current of the associated bioelectrode. Thylakoid membranes isolated from pea plants were adsorbed directly on a bare carbon paper working electrode and placed in the measurement cell in the absence of any electrochemical mediator, obtaining a fully environmental-friendly biodevice capable of photocurrent densities up to 14 µA/cm2. Three photosynthetic herbicides inhibiting Photosystem II and belonging to different chemical classes, namely diuron, terbuthylazine and metribuzin, were detected by measuring the electrode photocurrent, which decreased reproducibly in a concentration-dependent manner in a range between 10-7 - 5 × 10-5 M of each herbicide. The limit of detection for the three herbicides was between 4-6 × 10-7 M. Storage stability tests revealed for the biosensor a half-life longer than 15 days at 4 °C and full stability up to 4 months at -80 °C. This study provides a simple, environmental-friendly and cost-effective procedure for the fabrication of a mediatorless carbon paper-based electrochemical biosensor characterized by high photocurrents, long storage stability, reproducible detections and good sensitivity.

Effect of Thermal Stabilization on PAN-Derived Electrospun Carbon Nanofibers for CO2 Capture.

Carbon capture is amongst the key emerging technologies for the mitigation of greenhouse gases (GHG) pollution. Several materials as adsorbents for CO2 and other gases are being developed, which often involve using complex and expensive fabrication techniques. In this work, we suggest a sound, easy and cheap route for the production of nitrogen-doped carbon materials for CO2 capture by pyrolysis of electrospun poly(acrylonitrile) (PAN) fibers. PAN fibers are generally processed following specific heat treatments involving up to three steps (to get complete graphitization), one of these being stabilization, during which PAN fibers are oxidized and stretched in the 200-300 °C temperature range. The effect of stabilization temperature on the chemical structure of the carbon nanofibers is investigated herein to ascertain the possible implication of incomplete conversion/condensation of nitrile groups to form pyridine moieties on the CO2 adsorption capacity. The materials were tested in the pure CO2 atmosphere at 20 °C achieving 18.3% of maximum weight increase (equivalent to an uptake of 4.16 mmol g-1), proving the effectiveness of a high stabilization temperature as route for the improvement of CO2 uptake.

Channeling Anabolic Side Products toward the Production of Nonessential Metabolites: Stable Malate Production in Synechocystis sp. PCC6803.

Powered by (sun)light to oxidize water, cyanobacteria can directly convert atmospheric CO2 into valuable carbon-based compounds and meanwhile release O2 to the atmosphere. As such, cyanobacteria are promising candidates to be developed as microbial cell factories for the production of chemicals. Nevertheless, similar to other microbial cell factories, engineered cyanobacteria may suffer from production instability. The alignment of product formation with microbial fitness is a valid strategy to tackle this issue. We have described previously the "FRUITS" algorithm for the identification of metabolites suitable to be coupled to growth (i.e., side products in anabolic reactions) in the model cyanobacterium Synechocystis. sp PCC6803. However, the list of candidate metabolites identified using this algorithm can be somewhat limiting, due to the inherent structure of metabolic networks. Here, we aim at broadening the spectrum of candidate compounds beyond the ones predicted by FRUITS, through the conversion of a growth-coupled metabolite to downstream metabolites via thermodynamically favored conversions. We showcase the feasibility of this approach for malate production using fumarate as the growth-coupled substrate in Synechocystis mutants. A final titer of ∼1.2 mM was achieved for malate during photoautotrophic batch cultivations. Under prolonged continuous cultivation, the most efficient malate-producing strain can maintain its productivity for at least 45 generations, sharply contrasting with other producing Synechocystis strains engineered with classical approaches. Our study also opens a new possibility for extending the stable production concept to derivatives of growth-coupled metabolites, increasing the list of suitable target compounds.

High-Light versus Low-Light: Effects on Paired Photosystem II Supercomplex Structural Rearrangement in Pea Plants.

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.

How paired PSII-LHCII supercomplexes mediate the stacking of plant thylakoid membranes unveiled by structural mass-spectrometry.

Grana are a characteristic feature of higher plants' thylakoid membranes, consisting of stacks of appressed membranes enriched in Photosystem II (PSII) and associated light-harvesting complex II (LHCII) proteins, together forming the PSII-LHCII supercomplex. Grana stacks undergo light-dependent structural changes, mainly by reorganizing the supramolecular structure of PSII-LHCII supercomplexes. LHCII is vital for grana formation, in which also PSII-LHCII supercomplexes are involved. By combining top-down and crosslinking mass spectrometry we uncover the spatial organization of paired PSII-LHCII supercomplexes within thylakoid membranes. The resulting model highlights a basic molecular mechanism whereby plants maintain grana stacking at changing light conditions. This mechanism relies on interactions between stroma-exposed N-terminal loops of LHCII trimers and Lhcb4 subunits facing each other in adjacent membranes. The combination of light-dependent LHCII N-terminal trimming and extensive N-terminal α-acetylation likely affects interactions between pairs of PSII-LHCII supercomplexes across the stromal gap, ultimately mediating membrane folding in grana stacks.

Structural and functional differentiation of the light-harvesting protein Lhcb4 during land plant diversification.

About 475 million years ago, plants originated from an ancestral green alga and evolved first as non-vascular and later as vascular plants, becoming the primary producers of biomass on lands. During that time, the light-harvesting complex II (LHCII), responsible for sunlight absorption and excitation energy transfer to the photosystem II (PSII) core, underwent extensive differentiation. Lhcb4 is an ancestral LHCII that, in flowering plants, differentiated into up to three isoforms, Lhcb4.1, Lhcb4.2 and Lhcb4.3. The pivotal position of Lhcb4 in the PSII-LHCII supercomplex (PSII-LHCIIsc) allows functioning as linker for either S- or M-trimers of LHCII to the PSII core. The increased accumulation of Lhcb4.3 observed in PSII-LHCIIsc of plants acclimated to moderate and high light intensities induced us to investigate, whether this isoform has a preferential localization in a specific PSII-LHCIIsc conformation that might explain its light-dependent accumulation. In this work, by combining an improved method for separation of different forms of PSII-LHCIIsc from thylakoids of Pisum sativum L. grown at increasing irradiances with quantitative proteomics, we assessed that Lhcb4.3 is abundant in PSII-LHCIIsc of type C2 S2 , and, interestingly, similar results were found for the PsbR subunit. Phylogenetic comparative analysis on different taxa of the Viridiplantae lineage and structural modeling further pointed out to an effect of the evolution of different Lhcb4 isoforms on the light-dependent modulation of the PSII-LHCIIsc organization. This information provides new insight on the properties of the Lhcb4 and its isoforms and their role on the structure, function and regulation of PSII.

Hydrated Salt/Graphite/Polyelectrolyte Organic-Inorganic Hybrids for Efficient Thermochemical Storage.

Hydrated salt thermochemical energy storage (TES) is a promising technology for high density energy storage, in principle opening the way for applications in seasonal storage. However, severe limitations are affecting large scale applications, related to their poor thermal and mechanical stability on hydration/dehydration cycling. In this paper, we report the preparation and characterization of composite materials manufactured with a wet impregnation method using strontium bromide hexahydrate (SBH) as a thermochemical storage material, combined with expanded natural graphite (G). In addition to these fully inorganic formulations, an organic polyelectrolyte (PDAC, polydiallyldimethylammonium chloride) was exploited in the structure, with the aim to stabilize the salt, while contributing to the sorption/desorption process. Different formulations were prepared with varying PDAC concentration to study its contribution to material morphology, by electron microscopy and X-ray diffraction, as well as water sorption/desorption properties, by thermogravimetry and differential calorimetry. Furthermore, the SBH/G/PDAC powder mixture was pressed to form tabs that were analyzed in a climatic chamber, which is evidence for an active role of PDAC in the improvement of water sorption, coupled with a significant enhancement of mechanical resistance upon hydration/dehydration cycling. Therefore, the addition of the polyelectrolyte is proposed as an innovative approach in the fabrication of efficient and durable TES devices.

Anodic microbial community analysis of microbial fuel cells based on enriched inoculum from freshwater sediment.

The characterization of anodic microbial communities is of great importance in the study of microbial fuel cells (MFCs). These kinds of devices mainly require a high abundance of anode respiring bacteria (ARB) in the anode chamber for optimal performance. This study evaluated the effect of different enrichments of environmental freshwater sediment samples used as inocula on microbial community structures in MFCs. Two enrichment media were compared: ferric citrate (FeC) enrichment, with the purpose of increasing the ARB percentage, and general enrichment (Gen). The microbial community dynamics were evaluated by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and real time polymerase chain reaction (qPCR). The enrichment effect was visible on the microbial community composition both during precultures and in anode MFCs. Both enrichment approaches affected microbial communities. Shannon diversity as well as ß-Proteobacteria and γ-Proteobacteria percentages decreased during the enrichment steps, especially for FeC (p < 0.01). Our data suggest that FeC enrichment excessively reduced the diversity of the anode community, rather than promoting the proliferation of ARB, causing a condition that did not produce advantages in terms of system performance.

Aromatic molecular junctions between graphene sheets: a molecular dynamics screening for enhanced thermal conductance.

The proper design and synthesis of molecular junctions for the purpose of establishing percolative networks of conductive nanoparticles represent an opportunity to develop more efficient thermally-conductive nanocomposites, with several potential applications in heat management. In this work, theoretical classical molecular dynamics simulations were conducted to design and evaluate thermal conductance of various molecules serving as thermal bridges between graphene nanosheets. A wide range of molecular junctions was studied, with a focus on the chemical structures that are viable to synthesize at laboratory scale. Thermal conductances were correlated with the length and mechanical stiffness of the chemical junctions. The simulated tensile deformation of the molecular junction revealed that the mechanical response is very sensitive to small differences in the chemical structure. The analysis of the vibrational density of states provided insights into the interfacial vibrational properties. A knowledge-driven design of the molecular junction structures is proposed, aiming at controlling interfacial thermal transport in nanomaterials. This approach may allow for the design of more efficient heat management in nanodevices, including flexible heat spreaders, bulk heat exchangers and heat storage devices.

Response of the thylakoid proteome of Synechocystis sp. PCC 6803 to photohinibitory intensities of orange-red light.

Photoautotrophic growth of Synechocystis sp. PCC 6803 in a flat-panel photobioreactor, run in turbidostat mode under increasing intensities of orange-red light (636 nm), showed a maximal growth rate (0.12 h-1) at 300 µmolphotons m-2 s-1, whereas first signs of photoinhibition were detected above 800 µmolphotons m-2 s-1. To investigate the dynamic modulation of the thylakoid proteome in response to photoinhibitory light intensities, quantitative proteomics analyses by SWATH mass spectrometry were performed by comparing thylakoid membranes extracted from Synechocystis grown under low-intensity illumination (i.e. 50 µmolphotons m-2 s-1) with samples isolated from cells subjected to photoinhibitory light regimes (800, 950 and 1460 µmolphotons m-2 s-1). We identified and quantified 126 proteins with altered abundance in all three photoinhibitory illumination regimes. These data reveal the strategies by which Synechocystis responds to photoinibitory growth irradiances of orange-red light. The accumulation of core proteins of Photosystem II and reduction of oxygen-evolving-complex subunits in photoinhibited cells revealed a different turnover and repair rates of the integral and extrinsic Photosystem II subunits with variation of light intensity. Furthermore, Synechocystis displayed a differentiated response to photoinhibitory regimes also regarding Photosystem I: the amount of PsaD, PsaE, PsaJ and PsaM subunits decreased, while there was an increased abundance of the PsaA, PsaB, Psak2 and PsaL proteins. Photoinhibition with 636 nm light also elicited an increased capacity for cyclic electron transport, a lowering of the amount of phycobilisomes and an increase of the orange carotenoid protein content, all presumably as a photoprotective mechanism against the generation of reactive oxygen species.

Thylakoid proteome modulation in pea plants grown at different irradiances: quantitative proteomic profiling in a non-model organism aided by transcriptomic data integration.

Plant thylakoid membranes contain hundreds of proteins that closely interact to cope with ever-changing environmental conditions. We investigated how Pisum sativum L. (pea) grown at different irradiances optimizes light-use efficiency through the differential accumulation of thylakoid proteins. Thylakoid membranes from plants grown under low (LL), moderate (ML) and high (HL) light intensity were characterized by combining chlorophyll fluorescence measurements with quantitative label-free proteomic analysis. Protein sequences retrieved from available transcriptomic data considerably improved thylakoid proteome profiling, increasing the quantifiable proteins from 63 to 194. The experimental approach used also demonstrates that this integrative omics strategy is powerful for unravelling protein isoforms and functions that are still unknown in non-model organisms. We found that the different growth irradiances affect the electron transport kinetics but not the relative abundance of photosystems (PS) I and II. Two acclimation strategies were evident. The behaviour of plants acclimated to LL was compared at higher irradiances: (i) in ML, plants turn on photoprotective responses mostly modulating the PSII light-harvesting capacity, either accumulating Lhcb4.3 or favouring the xanthophyll cycle; (ii) in HL, plants reduce the pool of light-harvesting complex II and enhance the PSII repair cycle. When growing at ML and HL, plants accumulate ATP synthase, boosting both cyclic and linear electron transport by finely tuning the ΔpH across the membrane and optimizing protein trafficking by adjusting the thylakoid architecture. Our results provide a quantitative snapshot of how plants coordinate light harvesting, electron transport and protein synthesis by adjusting the thylakoid membrane proteome in a light-dependent manner.

Analysis of the light intensity dependence of the growth of Synechocystis and of the light distribution in a photobioreactor energized by 635 nm light.

Synechocystis gathered momentum in modelling studies and biotechnological applications owing to multiple factors like fast growth, ability to fix carbon dioxide into valuable products, and the relative ease of genetic manipulation. Synechocystis physiology and metabolism, and consequently, the productivity of Synechocystis-based photobioreactors (PBRs), are heavily light modulated. Here, we set up a turbidostat-controlled lab-scale cultivation system in order to study the influence of varying orange-red light intensities on Synechocystis growth characteristics and photosynthetic activity. Synechocystis growth and photosynthetic activity were found to raise as supplied light intensity increased up to 500 µmol photons m-2 s-1 and to enter the photoinhibition state only at 800 µmol photons m-2 s-1. Interestingly, reverting the light to a non-photo-inhibiting intensity unveiled Synechocystis to be able to promptly recover. Furthermore, our characterization displayed a clear correlation between variations in growth rate and cell size, extending a phenomenon previously observed in other cyanobacteria. Further, we applied a modelling approach to simulate the effects produced by varying the incident light intensity on its local distribution within the PBR vessel. Our model simulations suggested that the photosynthetic activity of Synechocystis could be enhanced by finely regulating the intensity of the light incident on the PBR in order to prevent cells from experiencing light-induced stress and induce their exploitation of areas of different local light intensity formed in the vessel. In the latter case, the heterogeneous distribution of the local light intensity would allow Synechocystis for an optimized usage of light.

Controlling the melt dripping of polyester fabrics by tuning the ionic strength of polyhedral oligomeric silsesquioxane and sodium montmorillonite coatings assembled through Layer by Layer.

This work deals with the Layer by Layer (LbL) assembly of hybrid organic/inorganic flame retardant coatings made of Octa-ammonium POSS (polyhedral oligomeric silsesquioxane) and sodium montmorillonite clay on polyester (PET) fabrics. The effects of ionic strength on the achieved flame retardancy properties were investigated. The coating growth asa function of different ionic strengths was evaluated by infrared spectroscopy. 0.10M NaCl was found able to promote the highest adsorption of each coating species at each deposition step strongly affecting clay stacking as evaluated by X-ray diffraction measurements. The coatings on PET assembled at high ionic strength turned out to be more homogeneous and thicker than the reference one. Thanks to the increased thickness and better surface coverage, the same coatings efficiently suppressed the melt dripping phenomenon and significantly slowed down flame spread rate in horizontal flammability tests with only 2wt% of coating mass added. Furthermore, the same performances were maintained after 1h washing at 70°C. By cone calorimetry, coated fabrics showed a strong reduction in the combustion kinetics by nearly halving the peak of heat release rate. This paper provides an important insight on the viability of tuning deposition of LbL coatings on fabrics employing industrial-like processes by simple modification of ionic strength.

Thermally and Electrically Conductive Nanopapers from Reduced Graphene Oxide: Effect of Nanoflakes Thermal Annealing on the Film Structure and Properties.

In this study, we report a novel strategy to prepare graphene nanopapers from direct vacuum filtration. Instead of the conventional method, i.e., thermal annealing nanopapers at extremely high temperatures prepared from graphene oxide (GO) or partially reduced GO, we fabricate our graphene nanopapers directly from suspensions of fully reduced graphene oxide (RGO), obtained after RGO and thermal annealing at 1700 °C in vacuum. By using this approach, we studied the effect of thermal annealing on the physical properties of the macroscopic graphene-based papers. Indeed, we demonstrated that the enhancement of the thermal and electrical properties of graphene nanopapers prepared from annealed RGO is strongly influenced by the absence of oxygen functionalities and the morphology of the nanoflakes. Hence, our methodology can be considered as a valid alternative to the classical approach.

Pea PSII-LHCII supercomplexes form pairs by making connections across the stromal gap.

In higher plant thylakoids, the heterogeneous distribution of photosynthetic protein complexes is a determinant for the formation of grana, stacks of membrane discs that are densely populated with Photosystem II (PSII) and its light harvesting complex (LHCII). PSII associates with LHCII to form the PSII-LHCII supercomplex, a crucial component for solar energy conversion. Here, we report a biochemical, structural and functional characterization of pairs of PSII-LHCII supercomplexes, which were isolated under physiologically-relevant cation concentrations. Using single-particle cryo-electron microscopy, we determined the three-dimensional structure of paired C2S2M PSII-LHCII supercomplexes at 14 Å resolution. The two supercomplexes interact on their stromal sides through a specific overlap between apposing LHCII trimers and via physical connections that span the stromal gap, one of which is likely formed by interactions between the N-terminal loops of two Lhcb4 monomeric LHCII subunits. Fast chlorophyll fluorescence induction analysis showed that paired PSII-LHCII supercomplexes are energetically coupled. Molecular dynamics simulations revealed that additional flexible physical connections may form between the apposing LHCII trimers of paired PSII-LHCII supercomplexes in appressed thylakoid membranes. Our findings provide new insights into how interactions between pairs of PSII-LHCII supercomplexes can link adjacent thylakoids to mediate the stacking of grana membranes.

The physiology of trace elements in biological methane production.

Trace element (TE) requirements of Methanothermobacter okinawensis and Methanothermobacter marburgensis were examined in silico, and using closed batch and fed-batch cultivation experiments. In silico analysis revealed genomic differences among the transport systems and enzymes related to the archaeal Wood-Ljungdahl pathway of these two methanogens. M. okinawensis responded to rising concentrations of TE by increasing specific growth rate (µ) and volumetric productivity (MER) during closed batch cultivation, and can grow and produce methane (CH4) during fed-batch cultivation. M. marburgensis showed higher µ and MER during fed-batch cultivation and was therefore prioritized for subsequent optimization of CO2-based biological CH4 production. Multiple-parameter cultivation dependency on growth and productivity of M. marburgensis was finally examined using exponential fed-batch cultivation at different medium-, TE- and sulphide dilution rates, and different gas inflow rates. MER of 476mmolL-1h-1 and µ of 0.69h-1 were eventually obtained during exponential fed-batch cultivations employing M. marburgensis.

Supernucleation and Orientation of Poly(butylene terephthalate) Crystals in Nanocomposites Containing Highly Reduced Graphene Oxide.

The ring-opening polymerization of cyclic butylene terephthalate into poly(butylene terephthalate) (pCBT) in the presence of reduced graphene oxide (RGO) is an effective method for the preparation of polymer nanocomposites. The inclusion of RGO nanoflakes dramatically affects the crystallization of pCBT, shifting crystallization peak temperature to higher temperatures and, overall, increasing the crystallization rate. This was due to a supernucleating effect caused by RGO, which is maximized by highly reduced graphene oxide. Furthermore, combined analyses by differential scanning calorimetry (DSC) experiments and wide-angle X-ray diffraction (WAXS) showed the formation of a thick α-crystalline form pCBT lamellae with a melting point of ∼250 °C, close to the equilibrium melting temperature of pCBT. WAXS also demonstrated the pair orientation of pCBT crystals with RGO nanoflakes, indicating a strong interfacial interaction between the aromatic rings of pCBT and RGO planes, especially with highly reduced graphene oxide.

Improving efficiency and stability of perovskite solar cells with photocurable fluoropolymers.

Organometal halide perovskite solar cells have demonstrated high conversion efficiency but poor long-term stability against ultraviolet irradiation and water. We show that rapid light-induced free-radical polymerization at ambient temperature produces multifunctional fluorinated photopolymer coatings that confer luminescent and easy-cleaning features on the front side of the devices, while concurrently forming a strongly hydrophobic barrier toward environmental moisture on the back contact side. The luminescent photopolymers re-emit ultraviolet light in the visible range, boosting perovskite solar cells efficiency to nearly 19% under standard illumination. Coated devices reproducibly retain their full functional performance during prolonged operation, even after a series of severe aging tests carried out for more than 6 months.

Dynamic reorganization of photosystem II supercomplexes in response to variations in light intensities.

Plants are sessile organisms and need to acclimate to ever-changing light conditions in order to survive. These changes trigger a dynamic reorganization of the membrane protein complexes in the thylakoid membranes. Photosystem II (PSII) and its light harvesting system (LHCII) are the major target of this acclimation response, and accumulating evidences indicate that the amount and composition of PSII-LHCII supercomplexes in thylakoids are dynamically adjusted in response to changes in light intensity and quality. In this study, we characterized the PSII-LHCII supercomplexes in thylakoid membranes of pea plants in response to long-term acclimation to different light intensities. We provide evidence of a reorganization of the PSII-LHCII supercomplexes showing distinct changes in their antenna moiety. Mass spectrometry analysis revealed a specific reduction of Lhcb3, Lhcb6 and M-LHCII trimers bound to the PSII cores, while the Lhcb4.3 isoform increased in response to high light intensities. The modulation of Lhcb protein content correlates with the reduction of the functional PSII antenna size. These results suggest that the Lhcb3, Lhcb4.3 and Lhcb6 antenna subunits are major players in modulation of the PSII antenna size upon long-term acclimation to increased light levels. PsbS was not detected in the isolated PSII-LHCII supercomplexes at any light condition, despite an increased accumulation in thylakoids of high light acclimated plants, suggesting that PsbS is not a constitutive component of PSII-LHCII supercomplexes.

Isolation of novel PSII-LHCII megacomplexes from pea plants characterized by a combination of proteomics and electron microscopy.

In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-D-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12-39), as (C2S2)4 and (C2S2 + C2S2M2)2 megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.