Halophyte biorefinery for polyhydroxyalkanoates production from Ulva sp. Hydrolysate with Haloferax mediterranei in pneumatically agitated bioreactors and ultrasound harvesting.

The present study tested the outdoor cultivation of Haloferax mediterranei for PHA production from green macroalgae Ulva sp. in pneumatically agitated bioreactors and applied ultrasonic separation for enhanced settling of archaeal cells. Scaled-up cultivation (40 L) yielded maximum biomass productivity of 50.1 ± 0.11 mg·L-1·h-1 with a PHA productivity of 27 ± 0.01 mg·L-1·h-1 and conversion yield of 0.107 g PHA per gram UlvaDW. The maximum mass fraction of PHA achieved in biomass was calculated to be 56% w/w. Ultrasonic harvesting of Hfx. mediterranei cells approached 30% removal at energy inputs around 7.8 kWh·m-3, and indicated no significant aggregation enhancement by Ca2+ addition. Molecular weight analysis showed an increase in Polydispersity Index (PDI) when the corresponding air velocities were increased suggesting that the polymer was more homogeneous at lower mixing velocities. The current study demonstrated scalable processes for PHA production using Ulva sp. feedstock providing new technologies for halophilic biorefinery.

Polyhydroxyalkanoates and biochar from green macroalgal Ulva sp. biomass subcritical hydrolysates: Process optimization and a priori economic and greenhouse emissions break-even analysis.

Although macroalgae biomass is an emerging sustainable feedstock for biorefineries, the optimum process parameters for their hydrolysis and fermentation are still not known. In the present study, the simultaneous production of polyhydroxyalkanoates (PHA) and biochar from green macroalgae Ulva sp. is examined, applying subcritical water hydrolysis and Haloferax mediterranei fermentation. First, the effects of temperature, treatment time, salinity, and solid load on the biomass and PHA productivity were optimized following the Taguchi method. Hydrolysis at 170 °C, 20 min residence time, 38 g L-1 salinity with a seaweed solid load of 5% led to the maximum PHA yield of 0.104 g g-1Ulva and a biochar yield of 0.194 ± 1.23 g g-1Ulva. Second, the effect of different initial culture densities on the biomass and PHA productivity was studied. An initial culture density of 50 g L-1 led to the maximum volumetric PHA productivity of 0.024 ± 0.002 g L-1 h-1 with a maximum PHA content of 49.38 ± 0.3% w/w Sensitivity analysis shows that within 90% confidence, the annual PHA production from Ulva sp. is 148.14 g PHA m-2 year-1 with an annual biochar production of 42.6 g m-2 year-1. Priori economic and greenhouse gas break-even analyses of the process were done to estimate annual revenues and allowable greenhouse gas emissions. The study illustrates that PHA production from seaweed hydrolysate using extreme halophiles coupled to biochar production could become a benign and promising step in a marine biorefinery.

Effect of Maternal Diet and Milk Lipid Composition on the Infant Gut and Maternal Milk Microbiomes.

Inter-subject variability in human milk microbiome is well known; however, its origins and possible relationship to the mother's diet are still debated. We investigated associations between maternal nutrition, milk fatty acids composition and microbiomes in mother-infant dyads. Breast milk and infant fecal samples were collected across three time points (one week, one month and three months postpartum) from 22 mother-infant pairs. Food frequency questionnaires for the months of pregnancy and three months postpartum were collected. Milk fatty acids were analyzed by GC-MS and the microbiome in breast milk and infant feces was determined by 16S rRNA sequencing. Statistical interactions were computed using Spearman's method and corrected for multiple comparisons. We found significant negative correlation between Streptococcus relative abundance in maternal milk and intake of unsaturated fatty acids and folic acid at one month postpartum. At three months postpartum, vitamin B-12 consumption was significantly associated with a single operational taxonomic unit belonging to Streptococcus. Comparison between milk microbiome and lipid composition showed, one-month postpartum, significant negative correlation between Streptococcus relative abundance and the abundance of oleic acid. Additional correlations were detected between Staphylococcus hominis and two medium-chain saturated fatty acids. Our results reinforce the hypothesis that maternal nutrition may affect milk microbiome.

Efficient separation of conjugated polymers using a water soluble glycoprotein matrix: from fluorescence materials to light emitting devices.

Optically active bio-composite blends of conjugated polymers or oligomers are fabricated by complexing them with bovine submaxilliary mucin (BSM) protein. The BSM matrix is exploited to host hydrophobic extended conjugated π-systems and to prevent undesirable aggregation and render such materials water soluble. This method allows tuning the emission color of solutions and films from the basic colors to the technologically challenging white emission. Furthermore, electrically driven light emitting biological devices are prepared and operated.

Surface-induced conformational changes in doped bovine serum albumin self-assembled monolayers.

Evidence for considerable stabilization of doped bovine serum albumin (BSA) molecules upon adsorption on gold surfaces is provided. This is compared to the surface-induced conformational changes of the bare BSA and its corresponding monolayer. The BSA unfolding phenomenon is correlated with dehydration, which in turn enables improved monolayer coverage. The stabilization mechanism is found to be partially controllable via nanodoping of the BSA molecules, upon which the dehydration process is suppressed and molecular rigidity can be varied. Our experimental data and calculations further point to the intermixing of structural characteristics and inherent molecular properties in studies of biological monolayers.

Controlled electroluminescence from films composed of mixed bio-composites and nanotubes.

Good things come in threes: A new type of light emitting bio-composites allowing for the nanometric separation of the active components is demonstrated. A protein with large host-guest capacities is used for the encapsulation of a water-soluble composite dye in a nano-sized shell, which efficiently reduces Förster resonance energy transfer and related mechanisms. Blending of this bio-composite with multi-walled nanotubes increases the charge injection efficiency, in the electro-luminescent device.

Doped biomolecules in miniaturized electric junctions.

Control over molecular scale electrical properties within nano junctions is demonstrated, utilizing site-directed C(60) targeting into protein macromolecules as a doping means. The protein molecules, self-assembled in a miniaturized transistor device, yield robust and reproducible operation. Their device signal is dominated by an active center that inverts affinity upon guest incorporation and thus controls the properties of the entire macromolecule. We show how the leading routes of electron transport can be drawn, spatially and energetically, on the molecular level and, in particular, how the dopant effect is dictated by its "strategic" binding site. Our findings propose the extension of microelectronic methodologies to the nanometer scale and further present a promising platform for ex situ studies of biochemical processes.

Bio-inspired synthesis of chiral silver nanoparticles in mucin glycoprotein--the natural choice.

A method of synthesizing stable chiral Ag nanoparticles inside a natural mucin glycoprotein is demonstrated. The reaction is carried out without the help of an external reducing agent, by utilizing the reducing properties of the host mucin. A chiral spectrum is detected in the visible range, indicating the formation of a new type of chiral Ag nanoparticles-containing biomaterial.

Enhanced bioavailability of polyaromatic hydrocarbons in the form of mucin complexes.

Increasing exposure of biological systems to large amounts of polycyclic aromatic hydrocarbons is of great public concern. Organisms have an array of biological defense mechanisms, and it is believed that mucosal gel (which covers the respiratory system, the gastrointestinal tract, etc.) provides an effective chemical shield against a range of toxic materials. However, in this work, we demonstrate, for the first time, that, upon complexation of polyaromatic hydrocarbons with mucins, enhanced bioavailability and, therefore, toxicity are obtained. This work was aimed to demonstrate how complexation of various highly hydrophobic polycyclic aromatic hydrocarbons with representative mucin glycoprotein could lead to the formation of previously undescribed materials, which exhibit increased toxicity versus pristine polycyclic aromatic hydrocarbons. In the present work, we show that a representative mucin glycoprotein, bovine submaxillary mucin, has impressive and unprecedented capabilities of binding and solubilizing water-insoluble materials in physiological solution. The complexes formed between the mucin and a series of polycyclic aromatic hydrocarbons were comprehensively characterized, and their toxicity was evaluated by both in vivo and in vitro assays. In addition, the bioavailability and membrane-penetration capabilities were tested using an internalization assay. Our results provide, for the first time, evidence of an unknown route by which hydrophobic materials may achieve higher bioavailability, penetrating some of the biological defense systems, in the form of water-soluble complexes with mucosal proteins.

Resolving the Mystery of the Elusive Peak: Negative Differential Resistance in Redox Proteins.

Vertical molecular transistors are used to explain the nonconformal electron transfer results obtained for redox proteins. The transport characteristics of a negative differential resistance peak as appears in the transport data of azurin and its nonredox derivative are explored. A correlation between the peak and its redox center is demonstrated.

Mucin complexes of nanomaterials: first biochemical encounter.

In recent years, the exposure of biological systems to various nanomaterials has become an issue of great public concern. Although living organisms have arrays of biological defense mechanisms against exposure to exogenous compounds, the biochemical mechanisms allowing various nanomaterials to enter the body are not well understood. A unique example of a typical mucosal glycoprotein capable of binding and solubilizing nanomaterials in physiological solution is provided, suggesting a possible route for entry into biological systems.

Organic reactions promoted by mucin glycoproteins.

Mucins are heavily glycosylated proteins that exhibit a broad range of adhesive interactions with various hydrophobic organic materials. However, there has been no clear evidence that mucins are capable of promoting chemical reactions. Here we provide the first demonstration that bovine submaxillary mucin and porcine gastric mucin accelerate the rate of fatty acid p-nitrophenol ester hydrolysis up to 337 times and a carbon-carbon bond-forming Diels-Alder reaction between N-propylmaleimide and anthracene up to 200 times relative to the rates of the reference processes, which were performed in water and chloroform, respectively. The discovered property of mucins to accelerate organic reactions provides a new and unique example of natural nonenzymatic proteins capable of promoting reactions of hydrophobic materials in aqueous solution. A better understanding of the interactions of mucins with hydrophobic molecules is needed for the development of superior drug-delivery systems and implantable devices.

Large-scale fabrication of 4-nm-channel vertical protein-based ambipolar transistors.

We suggest a universal method for the mass production of nanometer-sized molecular transistors. This vertical-type device was fabricated using conventional photolithography and self-assembly methods and was processed in parallel fashion. We used this transistor to investigate the transport properties of a single layer of bovine serum albumin protein. This 4-nm-channel device exhibits low operating voltages, ambipolar behavior, and high gate sensitivity. The operation mechanism of this new device is suggested, and the charge transfer through the protein layer was explored.

Biodelivery of a fullerene derivative.

Most current nanotoxicology research is focused on examining the influence of nanomaterials at the tissue and cellular levels. To explore these interactions on the molecular level, new carboxyfullerenes interact with transport proteins at the molecular level. The carboxyfullerenes exhibited an unusual mode of binding outside the calyx of beta-lactoglobulin (a typical representative of lipocalin family of barrier liquid proteins). The complexes were studied by various techniques, including mass spectrometry, UV/vis and circular dichroism spectroscopy, chromatographic methods, gel electrophoresis, and dynamic light scattering. The fullerene ligands were transferred from beta-lactoglobulin to human serum albumin (a representative of a blood transport protein), thus providing a model of how fullerene-based nanomaterials interact with biomolecules and are transported in biological systems.

Can apomyoglobin form a complex with a spherical ligand? Interactions between apomyoglobin and [C60] fullerene derivative.

The preparation and characterization of the stable equine skeletal muscle apomyoglobin and eee-isomer of tris-malonic acid [C60] fullerene complex is reported. For this new bio-nanomaterial preparation, a procedure of complexation-during-protein-refolding was used and the obtained compound sustained separation by gel-filtration and ion-exchange chromatography. The apomyoglobin-tris-malonic acid [C60] fullerene complex was characterized by UV-vis spectroscopy, steady state fluorescence, time-resolved fluorescence, and circular dichroism spectroscopies. Important information provided by this study, regarding the stability and properties of new material, may lead to a better understanding of the apomyoglobin protein binding characteristics, as well as to development of novel antioxidant and photodynamic therapeutic agents and components for bioelectronic devices.

Formation of a soluble stable complex between pristine C60-fullerene and a native blood protein.

Concern is growing about the potential impact of human exposure to carbonaceous nanomaterials (such as fullerenes) in the environment. A valid biological study of how native biomolecules interact with nanomaterials at the molecular level in physiological conditions requires the preservation of their physicochemical properties, yet most investigations rely on the use of modified fullerene conjugates or aggregates. We report the formation of a stable, water-soluble, well-defined complex between a single molecule of pristine C(60)-fullerene and a native protein, bovine serum albumin protein (BSA), with the normal three-dimensional structure of BSA preserved. The ability to produce a pristine C(60)-fullerene-BSA hybrid at a physiological pH range lays a solid foundation for studying carbonaceous materials, biodelivery systems, and transport mechanisms and for characterizing the potential effects of nanomaterials on wildlife and human health, both in vitro and in vivo.

Interaction of c(60)-fullerene and carboxyfullerene with proteins: docking and binding site alignment.

The unique properties of fullerenes have raised the interest of using them for biomedical applications. Within this framework, the interactions of fullerenes with proteins have been an exciting research target, yet little is known about how native proteins can bind fullerenes, and what is the nature of these interactions. Moreover, though some proteins have been shown to interact with fullerenes, up to date, no crystal structure of such complexes was obtained. Here we report docking studies aimed at examining the interactions of fullerene in two forms (C60 nonsubstituted fullerene and carboxyfullerene) with four proteins that are known to bind fullerene derivatives: HIV protease, fullerene-specific antibody, human serum albumin, and bovine serum albumin. Our work provides docking models with detailed binding pockets information, which closely match available experimental data. We further compare the predicted binding sites using a novel multiple binding site alignment method. A high similarity between the physicochemical properties and surface geometry was found for fullerene's binding sites of HIV protease and the human and bovine serum albumins.

Formation and characterization of stable human serum albumin-tris-malonic acid [C60]fullerene complex.

The preparation and characterization of the stable human serum albumin (HSA)-C3 isomer of tris-malonic acid [C60]fullerene complex is reported. Other than the anti-fullerene antibody, a stable protein-fullerene complex with a native protein has never been observed. This study may provide valuable answers to the growing concern regarding the effects of carbonaceous nanomaterials on human health on one hand and, on the other, may lead to the development of novel antioxidant therapeutic agents, radiopharmaceuticals, and components for bioelectronic devices.