Hydrocracking of used cooking oil for biofuels production

Hydrocracking of used cooking oil is studied as a potential process for biofuels production. In this work several parameters are considered for evaluating the effectiveness of this technology, including hydrocracking temperature, liquid hourly space velocity (LHSV) and days on stream (DOS). Conversion and total biofuels production is favored by increasing temperature and decreasing LHSV. However moderate reaction temperatures and LHSVs are more attractive for diesel production, whereas higher temperatures and smaller LHSVs are more suitable for gasoline production. Furthermore heteroatom (S, N and O) removal increases as hydrocracking temperature increases, with de-oxygenation being particularly favorable. Saturation, however, is not favored with temperature indicating the necessity of a pre-treatment step prior to hydrocracking to enable saturation of the double bonds and heteroatom removal. Finally the impact of extended operation (catalyst life) on product yields and qualities indicates that all reactions are affected yet at different rates.     

Homogeneous,heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: A review

In the last few years, biodiesel has emerged as one of the most potential renewable energy to replace current petrol-derived diesel. It is a renewable, biodegradable and non-toxic fuel which can be easily produced through transesterification reaction. However, current commercial usage of refined vegetable oils for biodiesel production is impractical and uneconomical due to high feedstock cost and priority as food resources. Low-grade oil, typically waste cooking oil can be a better alternative; however, the high free fatty acids (FFA) content in waste cooking oil has become the main drawback for this potential feedstock. Therefore, this review paper is aimed to give an overview on the current status of biodiesel production and the potential of waste cooking oil as an alternative feedstock. Advantages and limitations of using homogeneous, heterogeneous and enzymatic transesterification on oil with high FFA (mostly waste cooking oil) are discussed in detail. It was found that using heterogeneous acid catalyst and enzyme are the best option to produce biodiesel from oil with high FFA as compared to the current commercial homogeneous base-catalyzed process. However, these heterogeneous acid and enzyme catalyze system still suffers from serious mass transfer limitation problems and therefore are not favorable for industrial application. Nevertheless, towards the end of this review paper, a few latest technological developments that have the potential to overcome the mass transfer limitation problem such as oscillatory flow reactor (OFR), ultrasonication, microwave reactor and co-solvent are reviewed. With proper research focus and development, waste cooking oil can indeed become the next ideal feedstock for biodiesel.     

Preparation of cross-linked lipase-coated micro-crystals for biodiesel production from waste cooking oil

A dual modification procedure composed of cross-linking and protein coating with K₂SO₄ was employed to modify Geotrichum sp. lipase for catalyzing biodiesel production from waste cooking oil. Compared to single modification of protein coating with K₂SO₄, the dual modification of cross-linking and lipase coating improved catalytic properties in terms of thermostable stability, organic solvent tolerance, pH stability and operational stability in biodiesel production process, although biodiesel yield and initial reaction rate for CLPCMCs were not improved. After five successive batch reactions, CLPCMCs could still maintain 80% of relative biodiesel yield. CLPCMCs retained 64% of relative biodiesel yield after incubation in a pH range of 4-6 for 4 h, and 85% of relative biodiesel yield after incubation in a range of 45-50 °C for 4 h. CLPCMCs still maintained 83% of relative biodiesel yield after both treated in polar organic solvent and non-polar organic solvent for 4 h.     

How to increase the recovery rate for waste cooking oil-to-biofuel conversion: A comparison of recycling modes in China and Japan

Improvement of recovery rates is crucial for the supply chain management of waste cooking oil-to-biofuel conversion. This paper constructs a dynamic game model between biofuel enterprises and recyclers to compare the recycling modes and recycling rate decisions of waste cooking oil-to-biofuel conversion in China and Japan. Results demonstrate that: (1) the recovery rate of Chinese mode with recyclers as price leader is not necessarily lower than that of Japanese mode theoretically, indicating that the collection-storage-transportation integrated mode in several cities led by Chinese government may not be the optimal choice. (2) The subsidies for recyclers have insignificant impact on recovery rate, while the subsidies for biofuel enterprises can significantly improve the recovery rate. (3) Decreasing difficulty coefficient and expanding the market size can greatly improve the recovery rate. Therefore, reforming the beneficiary of subsidies, improving the recycling infrastructure, implementing strict technical and administrative control and creative sales policy of biofuel finished products may benefit the recovery rate in China.     

Low-grade oils and fats: effect of several impurities on biodiesel production over sulfonic acid heterogeneous catalysts

Different lipidic wastes and low-grade oils and fats have been characterized and evaluated as feedstocks for the acid-catalyzed production of FAME. The characterization of these materials has revealed significant contents of free fatty acids, Na, K, Ca, Mg, P, unsaponifiable matter and humidity. Arenesulfonic acid-functionalized SBA-15 silica catalyst has provided yields to FAME close to 80% in the simultaneous esterification-transesterification of the different feedstocks, regardless of their nature and properties, using methanol under the following reaction conditions: 160 °C, 2 h, methanol to oil molar ratio of 30, 8 wt.% catalyst loading, and 2000 rpm stirring rate. Nevertheless, reutilization of the catalyst is compromised by high levels of impurities, especially because of deactivation by strong interaction of unsaponifiable matter with the catalytic sites. The conditioning of these materials by aqueous washing in the presence of cationic-exchange resin Amberlyst-15, followed by a drying step, resulted in a lower deactivation of the catalyst.     

A high-oleic-acid and low-palmitic-acid soybean: agronomic performance and evaluation as a feedstock for biodiesel

Phenotypic characterization of soybean event 335-13, which possesses oil with an increased oleic acid content (> 85%) and reduced palmitic acid content (< 5%), was conducted across multiple environments during 2004 and 2005. Under these conditions, the stability of the novel fatty acid profile of the oil was not influenced by environment. Importantly, the novel soybean event 335-13 was not compromised in yield in both irrigated and non-irrigated production schemes. Moreover, seed characteristics, including total oil and protein, as well as amino acid profile, were not altered as a result of the large shift in the fatty acid profile. The novel oil trait was inherited in a simple Mendelian fashion. The event 335-13 was also evaluated as a feedstock for biodiesel. Extruded oil from event 335-13 produced a biodiesel with improved cold flow and enhanced oxidative stability, two critical fuel parameters that can limit the utility of this renewable transportation fuel.     

Isolation of 2-methyl branched unsaturated very long fatty acids from marine sponge Halichondria panicea and identification of them by GC-MS and NMR

In order to study the biomarker fatty acids of symbionts in the marine sponge Halichondria panicea, purification and structural identification of two new 2-methyl branched monoenoic very long fatty acids (2-Me-24:1 n-7 and 2-Me-26:1 n-9) were performed for the first time. These acids amounted to 7.1% of total sponge FAs, but our attempts to determine their structures by one-step GC-MS analysis were unsuccessful because of low yields of the correspondent N-acyl pyrrolidide derivatives. Silver-ion thin-layer chromatography isolated enriched fractions of monoenoic fatty acids extracted from the sponge. Further purification of unknown fatty acid methyl esters was carried out by reversed-phase high-performance liquid chromatography. Determination of the chain length, degree and position of unsaturations was achieved by gas chromatography-mass spectrometry on methyl esters and dimethyldisulfide adducts. Structures, position of methyl substitution, and double bonds cis isomery were confirmed by 1H nuclear magnetic resonance.     

SESAME: A least-squares approach to the evaluation of protein structures computed from NMR data

Summary A method is proposed for defining a probability distribution on an ensemble of protein conformations from a 2D NOE spectrum, while at the same time back-calculating the experimental spectrum from the ensemble. This enables one to assess the relative quality and significance of the conformations, and to test the consistency of the ensemble as a whole with the experimental spectrum. The method eliminates the need to integrate the cross-peak intensities and is surprisingly insensitive to random noise in the spectrum. In this communication, these advantages are demonstrated by applying the method to simulated data, for which the correct result is already known.Abbreviations NOE nuclear Overhauser effect - BPTI basic pancreatic trypsin inhibitor - rmsd root-mean-square deviation     

A computer-based protocol for semiautomated assignments and 3D structure determination of proteins

Summary A strategy is presented for the semiautomated assignment and 3D structure determination of proteins from heteronuclear multidimensional Nuclear Magnetic Resonance (NMR) data. This approach involves the computer-based assignment of the NMR signals, identification of distance restraints from nuclear Overhauser effects, and generation of 3D structures by using the NMR-derived restraints. The protocol is described in detail and illustrated on a resonance assignment and structure determination of the FK506 binding protein (FKBP, 107 amino acids) complexed to the immunosuppressant, ascomycin. The 3D structures produced from this automated protocol attained backbone and heavy atom rmsd of 1.17 and 1.69 Å, respectively. Although more highly resolved structures of the complex have been obtained by standard interpretation of NMR data (Meadows et al. (1993) Biochemistry, 32, 754–765), the structures generated with this automated protocol required minimal manual intervention during the spectral assignment and 3D structure calculations stages. Thus, the protocol may yield an approximate order of magnitude reduction in the time required for the generation of 3D structures of proteins from NMR data.     

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons

The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.     

Sampling and efficiency of metric matrix distance geometry: A novel partial metrization algorithm

Summary In this paper, we present a reassessment of the sampling properties of the metric matrix distance geometry algorithm, which is in wide-spread use in the determination of three-dimensional structures from nuclear magnetic resonance (NMR) data. To this end, we compare the conformational space sampled by structures generated with a variety of metric matrix distance geometry protocols. As test systems we use an unconstrained polypeptide, and a small protein (rabbit neutrophil defensin peptide 5) for which only few tertiary distances had been derived from the NMR data, allowing several possible folds of the polypeptide chain. A process called metrization in the preparation of a trial distance matrix has a very large effect on the sampling properties of the algorithm. It is shown that, depending on the metrization protocol used, metric matrix distance geometry can have very good sampling properties'indeed, both for the unconstrained model system and the NMR-structure case. We show that the sampling properties are to a great degree determined by the way in which the first few distances are chosen within their bounds. Further, we present a new protocol (partial metrization) that is computationally more efficient but has the same excellent sampling properties. This novel protocol has been implemented in an expanded new release of the program X-PLOR with distance geometry capabilities.     

Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry

The Aconitum alkaloids aconitine, mesaconitine, and hypaconitine are the main toxic components in a commonly used traditional Chinese herbal medicine Fu Zi. To provide guidelines for the safe use of this medicine, metabolic changes in Wistar rats caused by these compounds were investigated by means of integrated analysis of two metabonomic approaches: ¹H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF–MS). Rats were given a single dose of aconitine, mesaconitine, hypaconitine, or vehicle. The largest metabolic changes were observed 6 h after treatment. Every group receiving a dose had higher urine concentrations of glucose, acetate, dimethylglycine, succinate, and alanine and had lower concentrations of creatinine, citrate, 2-oxoglutarate, N-acetylated metabolites, and trimethylamine-N-oxide (TMAO) than did the control group. These results may reflect the perturbation of renal tubular function within the first 24 h after treatment. The results also revealed a larger perturbation of metabolic profiles in the aconitine group than in the mesaconitine and hypaconitine groups, illustrating how these alkaloids exhibit different toxicities. An analysis of plasma samples collected 7 days postdose showed that there were higher levels of lactate, alanine, and lipids along with lower levels of glucose, β-hydroxybutyrate, and creatine in the plasma of the aconitine and mesaconitine groups than there were in the control and hypaconitine groups. The GC/TOF–MS data from the plasma samples showed that the number of metabolites, with significant changes or with a tendency to change, in the aconitine and mesaconitine groups were dissimilar, suggesting a possible difference in the acute toxicity mechanisms of these alkaloids.     

NMR- and GC/MS-based metabolomics of sulfur mustard exposed individuals: a pilot study

Sulfur mustard (SM) is a potent alkylating agent and its effects on cells and tissues are varied and complex. Due to limitations in the diagnostics of sulfur mustard exposed individuals (SMEIs) by noninvasive approaches, there is a great necessity to develop novel techniques and biomarkers for this condition. We present here the first nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC/MS) metabolic profiling of serum from and healthy controls to identify novel biomarkers in blood serum for better diagnostics. Of note, SMEIs were exposed to SM 30 years ago and that differences between two groups could still be found. Pathways in which differences between SMEIs and healthy controls are observed are related to lipid metabolism, ketogenesis, tricarboxylic acid (TCA) cycle and amino acid metabolism.     

Blood group A glycolipid (Ax) with globo-series structure which is specific for blood group A1 erythrocytes: one of the chemical bases for A1 and A2 distinction

A new blood group A-active glycolipid fraction, termed Ax, showing a chromatographic mobility between Aa and Ab was found in blood group A1 erythrocytes but not in A2 erythrocytes. Ax was identified by its conversion to "globo H" by alpha-N-acetylgalactosaminidase and by 1H-NMR spectroscopy as GalNAc alpha l----3[Fuc alpha l----2]Gal beta l----3GalNAc beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer. Globo-H (Fuc alpha l----2Gal beta l----3GalNac beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer) was found in blood group A, and O but not in A1 erythrocytes. Thus, one of the A1-specific determinants must be an A determinant carried by globo-series structure.     

Assembling NMR structures for the intracellular loops of the human thromboxane A2 receptor: implication of the G protein-coupling pocket

It has been reported that the multiple intracellular loops (iLPs) of the thromboxane A₂ receptor (TP) are involved in the receptor G protein coupling. In this study, a high-resolution 2D NMR technique was used to determine the 3D structures of the first, second, and third iLPs of the TP using synthetic peptides constrained into the loop structures. 2D ¹H NMR spectra, TOCSY and NOESY were obtained for the two peptides from proton NMR experiments. The NMR data was processed and assigned through the Felix 2000 program. Standard methods were used to acquire sequence-specific assignments. Structure calculations were processed through DGII and NMR refinement programs within the Insight II program. We were able to calculate and use the NOE constraints to obtain the superimposed structure of 10 structures for each iLP peptide. The NMR-determined structures of the iLP peptides were used to refine a homology model of the TP. A 3D G-protein-binding cavity, formed by the three intracellular loops, was predicted by the docking of the C-terminal domain of the Gαq. Based on the structural model and the previous mutagenesis studies, the residues, R130, R60, C223, F138, L360, V361, E358 and Y359, which are important for interaction with the G protein, were further highlighted. These results reveal the possibly important molecular mechanisms in TP signaling and provide structural information to characterize other prostanoid receptor signalings.     

A monolithic lipase reactor for biodiesel production by transesterification of triacylglycerides into fatty acid methyl esters

An enzymatic reactor with lipase immobilized on a monolithic polymer support has been prepared and used to catalyze the transesterification of triacylglycerides into the fatty acid methyl esters commonly used for biodiesel. A design of experiments procedure was used to optimize the monolithic reactor with variables including control of the surface polarity of the monolith via variations in the length of the hydrocarbon chain in alkyl methacrylate monomer, time of grafting of 1-vinyl-4,4-dimethylazlactone used to activate the monolith, and time used for the immobilization of porcine lipase. Optimal conditions involved the use of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monolith, grafted first with vinylazlactone, then treated with lipase for 2 h to carry out the immobilization of the enzyme. Best conditions for the transesterification of glyceryl tributyrate included a temperature of 37°C and a 10 min residence time of the substrate in the bioreactor. The reactor did not lose its activity even after pumping through it a solution of substrate equaling 1,000 reactor volumes. This enzymatic reactor was also used for the transesterification of triacylglycerides from soybean oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.     

Resolution of heterogeneous charged antibody aggregates via multimodal chromatography: A comparison to conventional approaches

Clearance of aggregates during protein purification is increasingly paramount as protein aggregates represent one of the major impurities in biopharmaceutical products. Aggregates, especially dimer species, represent a significant challenge for purification processing since aggregate separation coupled with high purity protein recovery can be difficult to accomplish. Biochemical characterization of the aggregate species from the hydrophobic interaction and cation exchange chromatography elution peaks revealed two different charged populations, i.e. heterogeneous charged aggregates, which led to further challenges for chromatographic removal. This paper compares multimodal versus conventional cation exchange or hydrophobic chromatography methodologies to remove heterogeneous aggregates. A full, mixed level factorial design of experiment strategy together with high throughput experimentation was employed to rapidly evaluate chromatographic parameters such as pH, conductivity, and loading. A variety of operating conditions were identified for the multimodal chromatography step, which lead to effective removal of two different charged populations of aggregate species. This multimodal chromatography step was incorporated into a monoclonal antibody purification process and successfully implemented at commercial manufacturing scale. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:636–645, 2014     

Low frequency-low voltage alternating electric current-induced anoxic granulation in biofilm-electrode reactor: A study of granule properties

We characterized anoxic nitrate granules produced by an alternating current biofilm electrode reactor operating at low voltage-low frequency under optimum conditions. Hydrodynamic results revealed that the settling velocity for the granules ranged from 0.12 to 2.85 cm/s, while that of settling velocities ranged from 0.07 to 3.42 cm/s. Granule diameter varied, with a mean mass of 3.65 ± 1.29 mm and corresponding dry mass ranging from 0.52 to 5.64 mg. Roundness ratio of the sampled granules was determined to be 0.78 ± 0.11. Integrity coefficient obtained from a shear strength test was 87.05 ± 2.07% after 2 min and 74.1 ± 4.14% after 5 min. An adhesion test revealed hydrophilic properties of bacteria. The Most probable number (MPN) value was 2.0 × 10⁶ for HDB and 2.0 × 10³ for ADB. An apoptosis assay by flow cytometry confirmed that the majority of cells (87.7%) were viable and non-apoptotic (Annexin V-PI) and dehydrogenase activity was 15.05 ± 1.76 μg TF/mg biomass cm⁻² d. Comparison of seed and granules by ¹H NMR spectra showed different signals in the range of 0.279–1, 1–1.5, and 1.5–7.5 ppm. Therefore, the biofilm in ACBER can be easily granulated and used to generate dense and fast-settling sludge granules.     

INCA 2.0: A tool for integrated,dynamic modeling of NMR- and MS-based isotopomer measurements and rigorous metabolic flux analysis

Metabolic flux analysis (MFA) combines experimental measurements and computational modeling to determine biochemical reaction rates in live biological systems. Advancements in analytical instrumentation, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), have facilitated chemical separation and quantification of isotopically enriched metabolites. However, no software packages have been previously described that can integrate isotopomer measurements from both MS and NMR analytical platforms and have the flexibility to estimate metabolic fluxes from either isotopic steady-state or dynamic labeling experiments. By applying physiologically relevant cardiac and hepatic metabolic models to assess NMR isotopomer measurements, we herein test and validate new modeling capabilities of our enhanced flux analysis software tool, INCA 2.0. We demonstrate that INCA 2.0 can simulate and regress steady-state ¹³C NMR datasets from perfused hearts with an accuracy comparable to other established flux assessment tools. Furthermore, by simulating the infusion of three different ¹³C acetate tracers, we show that MFA based on dynamic ¹³C NMR measurements can more precisely resolve cardiac fluxes compared to isotopically steady-state flux analysis. Finally, we show that estimation of hepatic fluxes using combined ¹³C NMR and MS datasets improves the precision of estimated fluxes by up to 50%. Overall, our results illustrate how the recently added NMR data modeling capabilities of INCA 2.0 can enable entirely new experimental designs that lead to improved flux resolution and can be applied to a wide range of biological systems and measurement time courses.