Esterification of Free Fatty Acids with Glycerol within the Biodiesel Production Framework

Companies in the field of the collection and treatment of waste cooking oils (WCO) for subsequent biodiesel production usually have to cope with high acidity oils, which cannot be directly transformed into fatty acid methyl esters due to soap production. Since glycerine is the main byproduct of biodiesel production, these high acidity oils could be esterified with the glycerine surplus to transform the free fatty acids (FFA) into triglycerides before performing the transesterification. In this work, commercial glycerol was esterified with commercial fatty acids and commercial fatty acid/lampante olive oil mixtures over tin (II) chloride. In the first set of experiments, the esterification of linoleic acid with glycerol excess from 20 to 80% molar over the stoichiometric was performed. From 20% glycerol excess, there was no improvement in FFA reduction. Using 20% glycerol excess, the performance of a biochar obtained from heavy metal-contaminated plant roots was compared to that of SnCl2. Then, the effect of the initial FFA content was assessed using different oleic acid/lampante olive oil mixtures. The results illustrated that glycerolysis was impeded at initial FFA contents lower than 10%. Finally, the glycerolysis of a WCO with 9.94% FFA was assayed, without success

Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine

[EN] The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrationsSIThis work was supported by grant BIO2000-272 and a fellowship (to A. de la Fuente) from the Spanish Ministry of Science and Technology (Madrid, Spain). We thank Rosario Pérez-Redondo for her help with RNA experiments

Metal Accumulation by Jatropha curcas L. Adult Plants Grown on Heavy Metal-Contaminated Soil

Jatropha curcas has the ability to phytoextract high amounts of heavy metals during its first months just after seeding. Notwithstanding, there is scarce information about metal uptake by adult J. curcas plants. To shed light on this issue, 4-year-old J. curcas L. plants were planted in a soil mixture of peat moss and mining soil (high metals content), and the biomass growth and metal absorption during 90 days were compared with those of plants growing in peat moss. The main metal found in the mining soil was Fe (31985 mg kg-1) along with high amounts of As (23717 mg kg-1). After the 90-day phytoremediation, the plant removed 29% of Fe and 44% of As from the soil mixture. Results revealed that J. curcas L. translocated high amounts of metals to its aerial parts, so that translocation factors were much higher than 1. Because of the high translocation and bioaccumulation factors obtained, J. curcas L. can be regarded as a hyperaccumulator plant. Despite the great capacity of J. curcas L. to phytoremediate heavy-metal-contaminated soils, the main drawback is the subsequent handling of the metal-contaminated biomass, although some potential applications have been recently highlighted for this biomass.University of Seville (VIPPIT-2019-I.5

Human papillomavirus genotype distribution in Madrid and correlation with cytological data

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide. Infection with certain human papillomavirus (HPV) genotypes is the most important risk factor associated with cervical cancer. This study analysed the distribution of type-specific HPV infection among women with normal and abnormal cytology, to assess the potential benefit of prophylaxis with anti-HPV vaccines. METHODS: Cervical samples of 2,461 women (median age 34 years; range 15-75) from the centre of Spain were tested for HPV DNA. These included 1,656 samples with normal cytology (NC), 336 with atypical squamous cells of undetermined significance (ASCUS), 387 low-grade squamous intraepithelial lesions (LSILs), and 82 high-grade squamous intraepithelial lesions (HSILs). HPV detection and genotyping were performed by PCR using 5'-biotinylated MY09/11 consensus primers, and reverse dot blot hybridisation. RESULTS: HPV infection was detected in 1,062 women (43.2%). Out of these, 334 (31%) samples had normal cytology and 728 (69%) showed some cytological abnormality: 284 (27%) ASCUS, 365 (34%) LSILs, and 79 (8%) HSILs. The most common genotype found was HPV 16 (28%) with the following distribution: 21% in NC samples, 31% in ASCUS, 26% in LSILs, and 51% in HSILs. HPV 53 was the second most frequent (16%): 16% in NC, 16% in ASCUS, 19% in LSILs, and 5% in HSILs. The third genotype was HPV 31 (12%): 10% in NC, 11% in ASCUS, 14% in LSILs, and 11% in HSILs. Co-infections were found in 366 samples (34%). In 25%, 36%, 45% and 20% of samples with NC, ASCUS, LSIL and HSIL, respectively, more than one genotype was found. CONCLUSIONS: HPV 16 was the most frequent genotype in our area, followed by HPV 53 and 31, with a low prevalence of HPV 18 even in HSILs. The frequency of genotypes 16, 52 and 58 increased significantly from ASCUS to HSILs. Although a vaccine against HPV 16 and 18 could theoretically prevent approximately 50% of HSILs, genotypes not covered by the vaccine are frequent in our population. Knowledge of the epidemiological distribution is necessary to predict the effect of vaccines on incidence of infection and evaluate cross-protection from current vaccines against infection with other types

CB2 cannabinoid receptor activation promotes colon cancer progression via AKT/GSK3β signaling pathway

The pharmacological activation of the cannabinoid receptor type 2, CB2, has been shown to elicit anti-tumoral mechanisms in different cancer types. However, little is known about its endogenous role in tumor pathophysiology, and different studies have attributed pro-tumorigenic properties to this receptor. In a previous work, we showed that CB2 expression is a poor prognostic factor in colon cancer patients. Here we report that activation of CB2 with low doses of specific agonists induce cell proliferation and favor the acquisition of aggressive molecular features in colon cancer cells. We show that sub-micromolar concentrations of CB2-specific agonists, JWH-133 and HU-308, promote an increase in cell proliferation rate through the activation of AKT/PKB pathway in colon cancer in vitro and in vivo. AKT activation promotes GSK3β inhibition and thus, a more aggressive cell phenotype with the subsequent elevation of SNAIL levels, E-cadherin degradation and β-catenin delocalization from cell membrane. Taken together, our data show that CB2 activation with sub-micromolar doses of agonists, which could be more similar to endogenous levels of cannabinoids, promote colon cancer progression, implicating that CB2 could have a pro-tumorigenic endogenous role in colon cancerThis work was supported by grants from Fondo de Investigaciones Sanitarias (ISCIII-PI10/00879 to JMG; Plan Nacional de I+D+I 2008-2011, FEDER funds co-financed), Red Temática de Investigación Cooperativa en Cáncer (ISCIII-RETIC RD12/0036/0041; Plan Estatal de I+D+I 2013-2016, FEDER funds cofinanced). JMG and PM were supported by ISCIII CP08/00217 and JR14/0018 contracts, respectively. EMM was recipient of ISCIII PFIS PhD studentship (FI11/00696) (Plan Nacional de I+D+I 2008-2011, FEDER funds co-financed); AMR was recipient of PhD contract from Department of Medical Oncology of H.U. Puerta de Hierro; VC was recipient of attending physician contract in Medical Oncology Department from H.U. Puerta de Hierro; MP was supported by Universidad Autónoma de Madrid (UAM) with Full Professor contrac

Characterization of a two-gene operon epeRA involved in multidrug resistance in Streptomyces clavuligerus

[EN] Two genes, epeR and epeA, are located downstream of argH in the Streptomyces clavuligerus genome. EpeR belongs to the TetR family of transcriptional regulators. It is homologous to PqrA of Streptomyces coelicolor (74.3% identity) and to NfxB of Pseudomonas aeruginosa (30.9% identity). EpeA encodes a protein with 14 transmembrane spanning domains (TMS) of the major facilitator superfamily. It shares 68.9% identity to PqrB of S. coelicolor and 46.5% identity to LfrA, conferring resistance to fluoroquinolones in Mycobacterium smegmatis. Disruption of epeR results in a S. clavuligerus epeR::aph mutant which shows increased resistance to ethidium bromide and proflavine (16- and 32-fold higher than the wild type). Taking into consideration the sensitivity to drugs of different transformants carrying functional copies of either epeR or epeA, it might be concluded that both genes appear to be co-transcribed, with epeR encoding a regulatory protein which controls the expression of epeASIThis work was supported by CICYT grant BIO2003-3274 and GEN2003-20245. We thank Matthew Smith (University of Nottingham) and María Alvarez (University of León, Spain) for critical reading of the manuscript

CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus

[EN] The putative regulatory CcaR protein, which is encoded in the β-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter regionSIThis work was supported by the Spanish Ministry of Science and Technology (FD97-1419-CO2-O2). I. Santamarta received a fellowship from the University of León. We are grateful to A. de la Fuente, F. J. Enguita, and C. de Torre for their interest and helpful discussions, to A. Jiménez for revising the manuscript, and to M. Mediavilla for technical assistance

Determination of the Composition of Bio-Oils from the Pyrolysis of Orange Waste and Orange Pruning and Use of Biochars for the Removal of Sulphur from Waste Cooking Oils

Waste generated in the agri-food sector is a potential source of biomass and other products of high added value. In this work, the pyrolysis of orange waste and orange pruning was carried out to produce adsorbent biochars and characterise the bio-oils aiming for high-added-value compounds. Pyrolysis was carried out in a vertical tubular furnace on the laboratory scale modifying the temperature (400–600 °C), the heating ramp (5–20 °C·min−1) to reach the previous temperature and the inert gas flow rate (30–300 mL Ar·min−1) throughout the furnace. The most suitable conditions for obtaining biochar were found to be 400 °C, 5 °C·min−1, and 150 mL Ar·min−1 for orange waste, and 400 °C, 10 °C·min−1, and 150 mL Ar·min−1 for orange pruning. Thermogravimetric analysis showed higher thermal stability for orange pruning due to its higher lignin content (20% vs. 5% wt. on a wet basis). The bio-oil composition was determined by GC-MS. Toluene and 5-hydroxymethylfurfural were the main compounds found in orange waste bio-oils, while orange pruning bio-oils were composed mainly of 4-hydroxy-4-methyl-2-pentanone. Finally, the removal of the sulphur content from waste cooking oil was assayed with the biochars from both orange waste and orange pruning, whose BET surface areas were previously determined. Despite their low specific surface areas (≤1 m2·g−1 for orange waste biochars and up to 24.3 m2·g−1 for orange pruning biochars), these biochars achieved a reduction of the initial sulphur content of the waste cooking oil between 66.4% and 78.8%.European Union under the LIFE 13 BIOSEVILLE Programme ENV/ES/1113 (analysis, materials and salaries)European Regional Development Fund (ERDF) through the CARBOENERGY project (materials and salaries) granted by the FEDER INNTERCONECT

Energetic Valorisation of Olive Biomass: Olive-Tree Pruning, Olive Stones and Pomaces

Olive oil industry is one of the most important industries in the world. Currently, the land devoted to olive-tree cultivation around the world is ca. 11 106 ha, which produces more than 20 106 t olives per year. Most of these olives are destined to the production of olive oils. The main by-products of the olive oil industry are olive-pruning debris, olive stones and di erent pomaces. In cultures with traditional and intensive typologies, one single ha of olive grove annually generates more than 5 t of these by-products. The disposal of these by-products in the field can led to environmental problems. Notwithstanding, these by-products (biomasses) have a huge potential as source of energy. The objective of this paper is to comprehensively review the latest advances focused on energy production from olive-pruning debris, olive stones and pomaces, including processes such as combustion, gasification and pyrolysis, and the production of biofuels such as bioethanol and biodiesel. Future research e orts required for biofuel production are also discussed. The future of the olive oil industry must move towards a greater interrelation between olive oil production, conservation of the environment and energy generation

A Microplate-Based Bioluminescence Assay of Mitochondrial Calcium Uptake

Producción CientíficaMitochondrial Ca2+ homeostasis is crucial for regulating vital functions such as respiration or apoptosis. Targeted aequorins are excellent probes to measure subcellular Ca2+. Ca2+ concentration in mitochondria ([Ca2+]M) is low at rest (about 10−7 M) and can increase to the micromolar or even approach the millimolar range, upon cell activation. Here we describe a new quantitative luminescent protocol to directly measure mitochondrial Ca2+ uptake, optimized for high throughput. The sensitivity of the method allows detection of changes in either the capacity or the affinity of mitochondrial Ca2+ transport.Ministerio de Economía, Industria y Competitividad (Project BFU2014-534698P)Instituto de Salud Carlos III (RD16/0011/0003