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Specificity of small ions, the Hofmeister ranking, is long-known and has many applications including medicine. Yet it evades consistent theoretical description. Here we study the effect of Hofmeister anions on gramicidin A channels in lipid membranes. Counterintuitively, we find that conductance of this perfectly cation-selective channel increases about two-fold in the H2 PO4

Interactions of lipid membranes and macromolecules in general depend on the pH of buffered solutions. However, pH buffers modify molecular interactions in more than one way [1,2]. Here we conduct in vitro experiments with mammalian and bacterial cells to determine how common buffers such as 3-(N-morpholino)propanesulfonic acid (MOPS) affect cell viability. We find that primary rat lung microvascular cells do not survive in concentrations greater than 125mM for MOPS compared to 40mM for KCl, and 50mM for PEG400. In contrast, E. coli cells survived in concentrations up to 0.75 M for KCl and 1M MOPS. This is ascribed to a protective effect from the bacterial cell wall. Results are interpreted by comparison with previous measurements of model membrane interactions in buffer solutions.[1] Koerner et al., Biophys. J. 101, 2011.[2] Peiro-Salvador et al. Biochemistry, 48, 2009.[3] van Haaren et al., Am. J. Physiol. Heart Circ. Physiol., 289, 2005.

Gramicidin A (gA) is a cation selective ion channel that has been used in many biophysical studies of lipid bilayers, in particular for investigations of lipid-protein interactions [1, 2] and membrane electrostatics [3]. In addition, it was found that ionic interactions with neutral lipid membranes also affect the kinetics of gA channels [4]. Here we report measurements of gA ion-channels for a series of sodium and potassium salts that show an anion-dependence of gA conductance. We find that gA conductance varies significantly with the anion type with ClO4 and SCN producing distinctly larger conductance values than Cl, F, and H2PO4. These results can provide new insights into ion-lipid membrane interactions and ion channel functions in general. [1] Andersen et al., Annu. Rev. Biophys. Biomol. Struct., 1996, [2] Lunbaek et al., PNAS 2010, [3] Rostovtseva et al. Biophys. J. 1998, [4] Rostovtseva et al. Biophys. J. 2008.

Lithium is a naturally occurring alkali metal which is very easily ionized to Li+, especially in aqueous solutions. Lithium is found mainly in mineral and seawater and in trace amounts in the human body. Lithium ions are being used in various fields ranging from energy storage to the treatment of mental illnesses including bipolar disorder and schizophrenia. The reason why lithium is such a peculiar ion is not exactly known except that its mode of interactions with charged surfaces depends on its hydration properties (i.e. interaction with nearby water molecules). We report two kinds of experimental measurements of lithium interactions with charged lipid membranes. One set of experiments involves X-ray scattering of multilamellar lipid vesicles in solution for which we measure how lithium salts modify van der Waals and electrostatic forces between neighboring membranes. The other set of measurements compare the effect of added lithium on the activity of gramicidin channels in neutral and negatively charged lipid bilayers. We find that in both x-ray and channel measurements, lithium ions modify lipid interactions in a manner that differs from other monovalent posititive ions.

Specific ionic effects, as captured in the Hofmeister series, have been observed in many biological phenomena including protein folding and aggregation and lipid bilayer interactions. Previously we have shown that the Hofmeister effect is present in the activity of gramicidin A channels. In particular, measurements of channel open lifetime and conductance in potassium salts clearly show the existence of two distinct ionic classes which could be identified as kosmotropic and chaotropic. To further investigate this behavior, we have measured the zeta potential of diphytanoyl phosphatidylcholine (DPhPC) liposomes in salt solutions. We observe that anions alter the surface charge of the liposomes depending on the classification of the anion as kosmotropic or chaotropic. Chaotropic anions (SCN-, ClO4-) decrease the surface charge of the liposomes while kosmotropic anions (Cl-, H2PO4-, SO42-) have the opposite effect. These results correlate with our previous studies of cation conductance through gramicidin A channels adding new insight into ionic interactions at the lipid-water interface.

In order to understand the cutaneous water loss in the desert-adapted and venomous lizard Heloderma suspectum, the microscopic structure and lipid composition of epidermal molts have been examined using microscopic, spectroscopic and chemical analysis techniques. The molt is formed by a variably thick, superficial beta-layer, an extensive mesos-region and few alpha-cells in its lowermost layers. The beta-layer contains most corneous beta proteins while the mesos-region is much richer in lipids. The proteins in the mesos-region are more unstructured than those located in the beta-layer. Most interestingly, among other lipids, high contents of cholesteryl-β-glucoside and cholesteryl sulfate were detected, molecules absent or present in traces in other species of squamates. These cholesterol derivatives may be involved in the stabilization and compaction of the mesos-region, but present a limited permeability to water movements. The modest resistance to cutaneous water-loss of this species is compensated by adopting other physiological strategies to limit thermal damage and water transpiration as previous eco-physiological studies have indicated. The increase of steroid derivatives may also be implicated in the heat shock response, influencing the relative behavior in this desert-adapted lizard.

Two novel protocols for the chemical valorization of polyhydroxybutyrate (PHB) were developed, aiming at the production of two bio-based molecules: methyl 3-hydroxybutyrate (MHB) and methyl 3-methoxybutyrate (MMB). Optimized reaction conditions were applied to pure PHB and PHB inclusions inside bacterial cells as starting materials. MHB was synthesized through a single-step catalytic methanolysis, while MMB was synthesized through a three-step process: thermolytic distillation to give crotonic acid (CA), esterification to give methyl crotonate (MC), and oxa-Michael addition of MeOH. The obtained MHB and MMB were tested as solvents for the recovery of PHB itself both from freeze-dried single strain cultures (SSC) and mixed microbial cultures (MMC) with low to medium contents of PHB (22-57 wt %). High PHB recovery was achieved: up to 96 ± 1% through MHB and up to 98 ± 1% through MMB. Extraction from MMC slurry (with a PHB content of 39% on dry weight) was also performed, recovering 77 ± 2% using MHB and 92 ± 2% using MMB. High purities and excellent molecular weights and polydispersity indexes of extracted PHB were obtained with both MHB and MMB. Solubility in water, octanol/water partition coefficients (log Kow), and aerobic ready biodegradability of both solvents were also evaluated.

A novel thermolytic distillation process was developed to depolymerize polyhydroxybutyrate (PHB) for the selective production of crotonic acid. The conditions adopted (170 °C, 150 mbar) were applied to pure PHB and PHB-enriched bacteria containing 60 and 30% of PHB, giving a recovery of crotonic acid of 92, 78 and 58%, respectively. The high efficiency of the developed process poses the basis for a drop-in production of bio-based crotonic acid, whose versatility as a platform chemical has been investigated through a photochemical approach. The photocatalytic addition (promoted by tetrabutylammonium decatungstate – TBADT) of aliphatic and aromatic aldehydes to crotonic acid took place under solar-simulated light irradiation. TBADT triggered the in situ formation of valuable acyl radicals from the corresponding aldehydes, thus inducing the desired hydroacylation via radical conjugate addition. Notably, the functionalization took place in a satisfactory yield quite independently of the adopted sample of crotonic acid (whether commercial or bio-based).

A novel hyphenated Pyrolysis-Anaerobic Digestion prototype (Py-AD) was tested in order to evaluate the potential of hybrid thermochemical biological process to produce methane from woody biomass. An auger intermediate pyrolyzer was directly coupled to two biological reactors optimized for the digestion of residual condensable compounds and gas produced by pyrolysis of softwood. The Py-AD was monitored for 16 months and a detailed chemical analysis of the main fractions, gas (pyrobiogas), biochar, aqueous phase and pyrolytic lignin was performed under regime conditions. The results from Py-AD and those from experiments with bench-scale pyrolysis and fermentation reactors analysis provided information on the overall performance of the Py-AD and mass and energy balance based on chemical oxygen demand. Py-AD allowed to obtain, with acceptable volumetric productivity, a pyrobiogas with a composition approaching that of biogas (47 %v/v CH4 and 45 %v/v CO2). Pyrobiogas yield was about half of the theoretical value calculated from gas and liquid fractions. A preliminary technical evaluation of the process confirmed the feasibility of Py-AD and its value to produce carbon negative fuels with simple equipment and low waste generation. Important key constraints of the process were also evidenced in the study.

An overview of the main polyhydroxyalkanoates (PHA) recovery methods is here reported, by considering the kind of PHA-producing bacteria (single bacterial strains or mixed microbial cultures) and the chemico-physical characteristics of the extracted polymer (molecular weight and polydispersity index). Several recovery approaches are presented and categorized in two main strategies: PHA recovery with solvents (halogenated solvents, alkanes, alcohols, esters, carbonates and ketones) and PHA recovery by cellular lysis (with oxidants, acid and alkaline compounds, surfactants and enzymes). Comparative evaluations based on the recovery, purity and molecular weight of the recovered polymers as well as on the potential sustainability of the different approaches are here presented.

In order to deal with hydrothermal liquefaction wastewater (HTWW), a new anaerobic-aerobic continuous process was developed. The process, which included a sequence of Up-Flow Anaerobic Sludge Blanket (UASB) and downstream aerobic Continuously Stirred Tank Reactor (CSTR), was tested on the HTWW obtained from Waste to Fuel® demo plant developed by ENI s.p.a. [ 1 ]. Performance of the system was evaluated in term of methane yield and chemical oxygen demand (COD) abatement capability. Detailed fate of organic compounds was evaluated through different analytical techniques, highlighting main issues and potential of HTWW biological treatment. The system was fed with neat HTWW (189 gCOD L−1) for 2.5 y, with variable organic loading rate (OLR) and minimal external inputs. UASB reactors converted most of HTWW organics into volatile fatty acids (VFA) and methane with concurrent precipitation of oily like insoluble, whereas aerobic CSTR removed VFA from anaerobic effluent. Under regime conditions (ORL equal to 0.5 gCOD L−1 d−1) COD decreased from 189 to 6.6 gCOD L-1, showing 97 % COD abatement with the coupled anaerobic-aerobic treatment. Such a COD abatement was obtained by means of multiple effects, namely biomethanation, precipitation of organic matter and aerobic oxidation of fermentation products (VFA produced in anaerobic digestion) and subsequent aerobic oxidation (in downstream aerobic reactor). These effects accounted for 43, 40 and 17 % of the total COD decrease, respectively. Inhibition phenomena were the key challenge for improving methane yields and system productivities. The overall results confirmed that valorization of HTWW is a feasible task, albeit rather challenging.

In view of the potential application of pyrolysis-based biotechnologies, it is crucial to look for novel microorganisms able to convert pyrolysis-derived products, in particular bio-oil water-soluble constituent, into valuable compounds. For the first time, this paper proposed a survey on a collection of bacterial, yeast, and fungal strains with well-known industrial properties as well as new bacterial isolates in order to select microbes able to both tolerate bio-oil inhibitors and convert bio-oil into valuable products. This survey found that bio-oil aqueous phase (BOAP) obtained from intermediate pyrolysis could be metabolized as it is by fungal strains whereas several dilutions are needed to do not hamper cell viability of many tested yeast and bacterial isolates. To process BOAP into valuable products, the yeast Saccharomyces cerevisiae L13, selected as the most industrially relevant tested strain, was adopted to convert bio-oil aqueous fraction hydrolysate into ethanol without any detoxification step. The fermenting performances were much greater than those of the benchmark yeast strain and S. cerevisiae L13 proved to be a strong candidate for bioethanol production from BOAP hydrolysates. This study demonstrated that the search for microorganisms is a promising approach to the future development of pyrolysis oil-based biorefinery platforms.

The recycling of polyhydroxybutyrate (PHB) via depolymerization-polymerization is here proposed and assessed for the first time. Effective depolymerization of PHB to crotonic acid through a thermolytic distillation at mild conditions (170 °C and 150 mbar), and without the need of any catalyst, gives a crotonic acid-enriched condensate (94% yield and 98% selectivity towards trans-crotonic acid) that was used without any further purification as a substrate for producing renewed PHB with a culture of Cupriavidus necator. The yield of PHB (0.6 g PHB per g of crotonic acid), final PHB content (63%), and PHB-molecular weight (1.5 MDa) confirmed the technical feasibility of this tandem approach, with a 55% overall yield. A preliminary evaluation of the energy consumption of various depolymerization approaches indicated that the thermolytic distillation consumes 20–25% less energy than previously reported methods; the energy requirement for producing crotonic acid from PHBwaste with 50% moisture has a still acceptable energy demand (4 MJ/kgcrotonic acid), comparable to the energy required for obtaining fermentable sugars (4.4 MJ/kgsugar) to be used for feeding bacteria that accumulate PHB.

Dimethicone (poly(dimethylsiloxane), PDMS) and its copolymers bearing a poly(ethylene glycol) (PEG) side chain (PEG-12 dimethicone and PEG-8 dimethicone) are utilised as ingredients in personal care products. These materials along with bis-PEG-18 methyl ether dimethyl silane were analysed by Py-GC–MS. The pyrolysates of dimethicone was dominated by cyclic dimethyl siloxanes from D3 to over D10. Besides Dn, the pyrolysates of PEG-dimethicone were featured by the presence of linear dimethyl siloxanes Ln. Hydroxylated siloxanes were also tentatively identified. Pyrograms were also characterised by PEG oligomers with different terminal groups (hydroxyl, ethyl, ethenyl). The molecular structure of pyrolysis products from the combined structural units of PDMS and PEG could not be assigned. Internal standard calibration protocols using pyrolysis products of PEG-12 dimethicone (L6, L8, L9, and triethyleneglycol monoethyl ether) exhibited satisfactory linearity (R2 > 0.992) in the 0.75−58 μg range and repeatability (RSD < 15 %). Three samples of dried sewage sludge from a municipal water treatment plant were extracted with tetrahydrofuran and the extracts analysed by Py-GC–MS. The pyrograms were characterised by intense signals due to the lipid matrix (hydrocarbons, fatty acids, steranes, sterenes, sterols). Dn and Ln were identified indicative for the presence of siloxanes at levels around 60−290 μg gdw−1, while pyrolytic markers of PEG were not revealed. The recovery of PEG-12 dimethicone (17 μg) in spiked sample was 76 %. Galaxolide, galaxolide lactone and triclosan were identified in the pyrograms. The results of this study evidenced the potential of Py-GC–MS as a screening tool for the determination of cosmetic ingredients in sewage sludge.

Polyhydroxyalkanoates (PHA) are a key constituent of excess sludge produced by Aerobic Sewage Sludge Treatment plants. The accumulation of significant amount of PHA inside aerobic microbial cells occurs when a surplus of an easily degradable carbon source (e.g., volatile fatty acids, VFA) is found in combination with other nutrients limitation. Herein, hydrothermal treatment (HT) of PHA-containing sludge at 300 and 375 °C was demonstrated to be effective in converting most (>70% w/w) of the bacterial PHA stored inside microbial cells into alkene/CO2 gas mixtures. Simultaneously, most of non-PHA biomass was converted into water-soluble compounds (50% carbon yield) that were acidogenic fermented to produce volatile fatty acids, ideal substrate to feed aerobic bacteria and produce more PHA. According to results here presented, HT of excess sludge with moderate (13%) PHA content can produce about 50 kg of alkenes per tonne of suspended solids treated, with a significant reduction of sludge mass (80% reduction of wet sludge volume) and consequent disposal cost.

An analytical procedure based on off-line analytical pyrolysis was utilized to study the bioaccumulation of polystyrene microspheres with different size (3 μm and 45 μm) by gills and digestive glands of Mytilus galloprovincialis exposed for 4 days at different concentrations of the polymer (3 μm: 0.015–1.5 μg L−1; 45 μm: 5−500 μg L−1). The mussel tissues were treated with 10 % KOH at 90 °C with good digestion efficiency (97 ± 1 %) and extracted with toluene. After evaporation the residue was pyrolysed at 500 °C with a resistively heated platinum filament. Pyrolysis products were trapped onto a charcoal cartridge, eluted with toluene and analysed by GC–MS under selected ion monitoring. Polystyrene concentrations were determined by the internal standard method from the peak areas of styrene. The procedure provided information on the effect of particle size, exposure level and tissue type on the bioaccumulation of polystyrene particles in terms of mass concentrations. Accumulation was observed in digestive glands, not in gills. The accumulation of larger particles in digestive glands increased with increasing concentrations of polystyrene in water and peaked at 14 ± 4 μg g−1. The reduction of lysosome membrane stability was observed for both particles size at a water concentration of 10,000 particles per litre, that corresponded to a lower mass concentration for the smaller particles. This study confirmed the capability of analytical pyrolysis to provide microplastic concentrations in terms of mass concentration in complex matrices.

Volatile fatty acids (VFAs) are ideal substrates for mixed microbial cultures to synthesize polyhydroxyalkanoates. Therefore, VFA pertraction from anaerobic diluted fermentation broths to slightly alkaline aerobic medium becomes a crucial step to develop a closed loop concept in which VFAs are used for polyhydroxyalkanoate production. New liquid membranes (LMs) based on lipophilic amines and biodiesel were proposed for this purpose. The performance of each LM was tested through a VFA distribution coefficient test and pertraction kinetic experiments. The transfer of VFAs from LM to receiving solution was the main limiting step of the process, with flux rates of 0.001–0.04 cm3 cm−2 h−1 (best LM with 10 wt% of trioctylamine). The biocompatibility of trioctylamine -biodiesel LM towards anaerobic bacteria was confirmed through a 15-d acidogenic fermentation test. Trioctylamine-biodiesel LM was finally tested in a specifically designed continuous pertraction module, where VFA transfer rates of 1–3 h−1 were achieved.

A transdisciplinary approach that integrates thermochemical conversions and biological transformations for valorizing C-content and chemical energy of anaerobically digested sewage sludge and producing biopolymers (polyhydroxyalkanoates, PHA) and drop-in chemicals (crotonic acid) has been presented here. Hydrothermal carbonization has increased by 10-times the soluble COD of sewage sludge, allowing it to split 44-54% of its COD into an aqueous phase (HTCap) enriched in small fermentable organic molecules; the coupling of acidogenic fermentation and aerobic fermentation converted these substrates first into volatile fatty acids (20% of CODHTCap) and then into PHA (about 20% of CODVFA). The extraction of microbial biomass with dimethyl carbonate (DMC) allowed high quality PHA to be produced, with a high molecular weight (0.9 MDa) and a percentage of medium chain monomers (hydroxyvalerate and hydrohexanoate) close to 12%. The postextraction microbial biomass, still containing a relevant portion of "unextractable" PHA, was last treated at 300 °C to selectively convert the PHA-backbone into crotonic acid with a 42% yield on PHA content basis.

This study proposes FTIR spectroscopy in the far-infrared region (FarIR) as an alternative method for the characterisation of natural resins. To this purpose, standards of natural resins belonging to four different categories (sesquiterpenic, i.e. elemi, shellac; diterpenic, i.e. colophony, Venice turpentine; diterpenic with polymerised components, i.e. copal, sandarac; triterpenic, i.e. mastic and dammar) used as paint varnishes have been analysed by FarIR spectroscopy in ATR mode. Discrimination between spectral data and repeatability of measurements have been magnified and verified using principal component analysis, in order to verify the effectiveness of the method in distinguishing the four resin categories. The same samples were analysed in the MidIR range, but the spectral differences between the different categories were not evident. Moreover, the method has been tested on historical samples from the painting "La Battaglia di Cialdiran" (sixteenth century) and from a gilded leather (seventeenth century). In the first case, FarIR spectroscopy allowed confirmation of the results obtained by analytical pyrolysis. In the latter, FarIR spectroscopy proved successfully, effective in the identification of the superficial resin layer that could not be detected with the bulk chromatographic analyses.

Lipid extraction is a critical step in the development of biofuels from microalgae. The use of toxic and polluting organic solvents should be reduced and the sustainability of the extraction procedures improved in order to develop an industrial extraction procedure. This could be done by reducing solvent amounts, avoiding use of harmful solvents, or eliminating the solvent at all. Here we describe two new processes to extract hydrocarbons from dried and water-suspended samples of the microalga Botryococcus braunii. The first one is a solvent-based procedure with switchable polarity solvents (SPS), a special class of green solvents easily convertible from a non-ionic form, with a high affinity towards non-polar compounds as B. braunii hydrocarbons, into an ionic salt after the addition of CO2, useful to recover hydrocarbons. The two SPS chosen for the study, based on equimolar mixtures of 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) and an alcohol (DBU/octanol and DBU/ethanol), were tested for the extraction efficiency of lipids from freeze-dried B. braunii samples and compared with volatile organic solvents extraction. The DBU/octanol system was further evaluated for the extraction of hydrocarbons directly from algal culture samples. DBU/octanol exhibited the highest yields of extracted hydrocarbons from both freeze-dried and liquid algal samples (16 and 8.2%, respectively, against 7.8 and 5.6% with traditional organic solvents). The second procedure here proposed is the thermochemical conversion of algal biomass by using pyrolysis; this process allowed to obtain three valuable fractions, exploitable for energy purpose, fuel production, and soil carbon storage: a volatile fraction (37% on dry biomass weight), a solid fraction called biochar (38%) and, above all, a liquid fraction named bio-oil (25%), almost entirely composed by hydrocarbon-like material, thus directly usable as fuel.

Background Cyclosporin A is a powerful immunosuppressive drug with considerable impact on transplants and is able to modify extracellular matrix (ECM) composition. It has recently been demonstrated that cyclosporin A stimulates the production of the cytokine family. Cytokines such as interleukin, transforming growth factor β 1 , and bone morphogenetic protein induce the deposition of glycosaminoglycans (GAGs), proteoglycans, and collagen fibers in the connective ECM. ECM composition is very important for normal tissue development and function. In this work, we examine the effects caused by cyclosporin A on cultures of normal human palate fibroblasts in order to evaluate interleukin, transforming growth factor β II, and bone morphogenetic protein II membrane receptor induction and extracellular GAG changes such as hyaluronic acid, heparin sulfate, and chondroitin sulfate. Methods Palate fibroblasts were maintained for 24 h in serum-free 199 medium containing 5 μg/mL 3 H glucosamine hydrochloride. After this time, TGF II and BMP II receptors were determined by microarray analysis and GAG classes by the biochemical method. Results The results show that TGFβ 1 II and BMP II membrane receptors are significantly inhibited in cyclosporin A-treated cultures as compared to controls, whereas IL-1R2 membrane receptors are stimulated. The behavior of total intra- and extracellular GAGs is significantly increased in cyclosporin A-treated cultures, whereas the ratio between non-sulfated/sulfated GAGs decreases ( p ≤0.01) vis-a-vis controls. Conclusions Because they form a highly complicated macromolecular network in the ECM, which provides an indication of cell function and gene expression and modulates growth factor activities, GAG changes are related to modification of ECM functions. Our data show that cyclosporin A causes biochemical changes to ECM through alterations in cytokines and respective membrane receptor linkages.