Use of waste frying oil and coconut pulp for the production, isolation, and characterization of a new lipase from Moesziomyces aphidis.

Lipases comprise the third most commercialized group of enzymes worldwide and those of microbial origin are sought for their multiple advantages. Agro-industrial waste can be an alternative culture medium for producing lipases, reducing production costs and the improper disposal of waste frying oil (WFO). This study aimed to produce yeast lipases through submerged fermentation (SF) using domestic edible oil waste as inducer and alternative culture medium. The optimal culture conditions, most effective inducer, and purification method for a new lipase from Moesziomyces aphidis BRT57 were identified. Yeast was cultured in medium containing green coconut pulp and WFO waste for 72 h. The maximum production of lipases in SF occurred in a culture medium containing WFO and yeast extract at 48 and 72 h of incubation, with enzyme activities of 8.88 and 11.39 U mL-1, respectively. The lipase was isolated through ultrafiltration followed by size exclusion chromatography, achieving a 50.46 % recovery rate. To the best of our knowledge, this is the first study to report the production and purification of lipases from M. aphidis, demonstrating the value of frying oil as inducer and alternative medium for SF, contributing to the production of fatty acids for biodiesel from food waste.

Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography.

In recent years, the study of extracellular vesicles (EVs) in the context of various diseases has dramatically increased due to their diagnostic and therapeutic potential. Typically, EVs are isolated in vitro from the cell culture of primary cells or cell lines or from bodily fluids. However, these cell culture methods do not represent the whole complexity of an in vivo microenvironment, and bodily fluids contain a high heterogeneous population of vesicles since they originate from different tissues. This highlights the need to develop new methods to isolate EVs directly from tissue samples. In the present study, we established a protocol for isolating EVs from hepatic and adipose tissue of mice, using a combination of ultracentrifugation and iodixanol-sucrose density gradient separation. EV isolation was confirmed with EV protein marker enrichment in Western blot assays, total protein quantification, and transmission electron microscopy. Regarding the liver tissue, we additionally implemented size exclusion chromatography (SEC) to further increase the purity grade of the EVs. The successful isolation of EVs from tissue samples will allow us to uncover a more precise molecular composition and functions, as well as their role in intercellular communication in an in vivo microenvironment.

Proteínas recombinantes de interesse na área da saúde

Recombinant proteins have revolutionized modern medicine and therapeutics. Currently recombinant proteins are used to treat diseases of great worldwide impact, as they allow maintenance and improvement of the life’s quality of patients with rare genetic diseases, metabolic syndromes, and immunological diseases. Since 1982, after the production of the first therapeutic recombinant protein (PRT), recombinant insulin, all major pharmaceutical companies have tried to develop PRT and supported its growth in the market. The right choice of expression systems and purification processes become essential steps to produce high-quality recombinant proteins with high yield. The recombinant protein Lsa63, the target of this project, is a leptospiral adhesin involved in the bacteria adhesion to the host. Lsa63 is expressed on the surface of patogenic Leptospiras and is absent in saprophytic ones. That said, the use of this protein in the development of new therapies and diagnosis for leptospirosis could be interesting. During this project, Lsa63 was purified from the frozen biomass of E. coli previously produced using liquid chromatography techniques). In all purification steps Lsa63 was quantified by BCA (bicinchoninic acid) and analyzed by SDS-PAGE electrophoresis and Western Blotting. The relative purity of Lsa63 was estimated by densitometry of SDS-PAGE bands. Immobilized-metal affinity chromatography (IMAC) was chosen as the first purification step and 24.4% of Lsa63 was yield with relative purity of 54.8%, followed by size-exclusion chromatography (SEC) with recovery of 95.2% and last step ion-exchange chromatography (IEC) with 7.9% and 80.3% of relative purity. Despite having obtained 80.3% of relative purity the global yield was low (7.9%).

As proteínas recombinantes revolucionaram a medicina moderna e a terapêutica. Por isso, atualmente, as proteínas recombinantes são utilizadas para tratamento de doenças de grande impacto mundial, pois permitem manutenção e melhoria da qualidade de vida de pacientes portadores de doenças genéticas raras, síndromes metabólicas e doenças imunológicas. Desde 1982, após a produção da primeira proteína recombinante terapêutica (PRT), insulina recombinante, todas as grandes farmacêuticas começaram a desenvolver PRT e impulsionaram o crescimento destas no mercado. A escolha correta dos sistemas de expressão e processos de purificação tornam-se etapas essenciais para produção de proteínas recombinantes de alta qualidade e em alto rendimento. A proteína recombinante Lsa63, alvo deste projeto, é uma adesina de leptospira envolvida na adesão da bactéria no organismo dos hospedeiros. A Lsa63 é expressa na superfície de Leptospiras patogênicas e apresenta-se ausente em cepas saprofíticas. Dito isto, a utilização desta proteína, no desenvolvimento de novas terapias e diagnóstico para leptospirose pode ser interessante. Durante o decorrer deste projeto, a Lsa63 foi purificada a partir da biomassa congelada de E. coli utilizando técnicas de cromatografia líquida. Em todas as etapas de purificação a Lsa63 foi quantificada por BCA (ácido bicinchonínico) e analisada por eletroforese SDS-PAGE e Western Blotting. A pureza relativa da Lsa63 foi estimada por densitometria das bandas do gel de eletroforese. A cromatografia de afinidade por íons metálicos imobilizados (IMAC) foi escolhida como primeira etapa de purificação sendo que a Lsa63 foi obtida com rendimento de 24,4% e pureza relativa de 54,8%, seguida pela cromatografia de exclusão molecular com recuperação de 95,2% e a última etapa a cromatografia de troca iônica com recuperação de 52,0% e pureza relativa de 80,3%. Apesar de ter obtido uma pureza relativa de 80,3% a recuperação global incluindo todas as etapas cromatográficas foi baixa sendo 7,9%.

A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

Validation of an Analytical Method for the Simultaneous Determination of Hyaluronic Acid Concentration and Molecular Weight by Size-Exclusion Chromatography.

The hyaluronic acid (HA) global market growth can be attributed to its use in medical, cosmetic, and pharmaceutical applications; thus, it is important to have validated, analytical methods to ensure confidence and security of its use (and to save time and resources). In this work, a size-exclusion chromatography method (HPLC-SEC) was validated to determine the concentration and molecular distribution of HA simultaneously. Analytical curves were developed for concentration and molecular weight in the ranges of 100-1000 mg/L and 0.011-2.200 MDa, respectively. The HPLC-SEC method showed repeatability and reproducibility greater than 98% and limits of detection and quantification of 12 and 42 mg/L, respectively, and was successfully applied to the analysis of HA from a bacterial culture, as well as cosmetic, and pharmaceutical products.

Removal efficiency of dissolved organic matter from secondary effluent by coagulation-flocculation processes.

Wastewater reuse has been widely discussed as an essential strategy to minimize the consumption of drinking water for less noble purposes. During biological wastewater treatment, organic matter is converted into a complex matrix containing a variety of soluble organic compounds. The objective of the present study was to evaluate the removal efficiency of the residual organic load in the final effluent from wastewater treatment plant with a conventional activated sludge process by different coagulants and parameters of coagulation-flocculation process, using dissolved organic carbon (DOC) concentration, molecular weight (MW) size distribution by size exclusion chromatography (SEC) coupled to mass spectrometry (MS), and zeta potential (ZP) analyses. The results showed a DOC removal efficiency up to 45% with iron chloride, and of 38% for aluminum sulfate and 31% for PAC coagulants. ZP was also measured during the procedures and authors conclude that the ZP also does not have a determining role in these removals. SEC and MS assessment was able to detect changes on secondary effluent molecular weight distribution profile after effluent coagulation-flocculation, this technique might be a promising tool to understand the composition of effluent organic matter and be helpful to estimate and optimize the performance of wastewater effluents treatment processes.

Extracellular vesicles containing the transferrin receptor as nanocarriers of apotransferrin.

Previous work by our group has shown the pro-differentiating effects of apotransferrin (aTf) on oligodendroglial cells in vivo and in vitro. Further studies showed the remyelinating effect of aTf in animal demyelination models such as hypoxia/ischemia, where the intranasal administration of human aTf provided brain neuroprotection and reduced white matter damage, neuronal loss, and astrogliosis in different brain regions. These data led us to search for a less invasive and controlled technique to deliver aTf to the CNS. To such end, we isolated extracellular vesicles (EVs) from human and mouse plasma and different neuron and glia conditioned media and characterized them based on their quality, quantity, identity, and structural integrity by western blot, dynamic light scattering, and scanning electron microscopy. All sources yielded highly pure vesicles whose size and structures were in keeping with previous literary evidence. Given that, remarkably, EVs from all sources analyzed contained Tf receptor 1 (TfR1) in their composition, we employed two passive cargo-loading strategies which rendered successful EV loading with aTf, specifically through binding to TfR1. These results unveil EVs as potential nanovehicles of aTf to be delivered into the CNS parenchyma, and pave the way for further studies into their possible clinical application in the treatment of demyelinating diseases.

Content/Potency Assessment of Botulinum Neurotoxin Type-A by Validated Liquid Chromatography Methods and Bioassays.

Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks purity. The method was applied to assess the content and high-molecular-weight (HMW) forms of BoNTA in biopharmaceutical products, and the results were compared with those of the LD50 mouse bioassay, the T-47D cell culture assay, and the reversed-phase chromatography (RPC) method, giving mean values of 0.71% higher, 0.36% lower, and 0.87% higher, respectively. Aggregated forms showed significant effects on cytotoxicity, as well as a decrease in the bioactivity (p < 0.05). The employment of the proposed method in conjunction with the optimized analytical technologies for the analysis of the intact and altered forms of the biotechnology-derived medicines, in the correlation studies, enabled the demonstration of the capability of each one of the methods and allowed for great improvements, thereby assuring their safe and effective use.

Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3.

Enzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins from the Complement System. However, the production of this recombinant protein using traditional refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs) solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against complement C3 α-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to establish efficient HHP-based processes for refolding of bioactive proteins from IBs.

Novel method for metalloproteins determination in human breast milk by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry.

Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277 Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.

Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

Size-exclusion chromatography (HPLC-SEC) technique optimization by simplex method to estimate molecular weight distribution of agave fructans.

Agave fructans are increasingly important in food industry and nutrition sciences as a potential ingredient of functional food, thus practical analysis tools to characterize them are needed. In view of the importance of the molecular weight on the functional properties of agave fructans, this study has the purpose to optimize a method to determine their molecular weight distribution by HPLC-SEC for industrial application. The optimization was carried out using a simplex method. The optimum conditions obtained were at column temperature of 61.7°C using tri-distilled water without salt, adjusted pH of 5.4 and a flow rate of 0.36mL/min. The exclusion range is from 1 to 49 of polymerization degree (180-7966Da). This proposed method represents an accurate and fast alternative to standard methods involving multiple-detection or hydrolysis of fructans. The industrial applications of this technique might be for quality control, study of fractionation processes and determination of purity.

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus.

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.

An unexpected cell-penetrating peptide from Bothrops jararaca venom identified through a novel size exclusion chromatography screening.

Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell-penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography-MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP - interestingly, a classical bradykinin-potentiating peptide - in the peptide-rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence-labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell-cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label-free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Analysis of streptokinase by validated liquid chromatography methods and correlation with an in vitro bioassay.

Reversed-phase and size-exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed-phase method was carried out on a Jupiter C4 column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size-exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25-250 µg/mL (25.75-25 750 IU/mL) (r2 = 0.9997) and 5-80 µg/mL (515-8240 IU/mL) (r2 = 0.9996), respectively, for reversed-phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.

Role of cell wall deconstructing enzymes in the proanthocyanidin-cell wall adsorption-desorption phenomena.

The transference of proanthocyanidins from grapes to wine is quite low. This could be due, among other causes, to proanthocyanidins being bound to grape cell wall polysaccharides, which are present in high concentrations in the must. Therefore, the effective extraction of proanthocyanidins from grapes will depend on the ability to disrupt these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the behavior of proanthocyanidin-cell wall interactions when commercial maceration enzymes are present in the solution. The results showed that cell wall polysaccharides adsorbed a high amount of proanthocyanidins and only a limited quantity of proanthocyanidins could be desorbed from the cell walls after washing with a model solution. The presence of enzymes in the solution reduced the proanthocyanidin-cell wall interaction, probably through the elimination of pectins from the cell wall network.

Influence of the extraction time on macromolecular parameters of galactomannans.

This study evaluated the aqueous extraction of galactomannans from the seeds of Mimosa scabrella (GM), Stryphnodendron adstringens (GS) and Schizolobium parahybae (GG) for 1, 2, 3, 4, 6, 24 and 48 h. The efficiency of extraction processes was assessed in terms of yield, carbohydrate and protein content. The extraction process, as well as the source of the galactomananns generated molecules with differences in molar mass, viscosity and rigidity analyzed by HPSEC-MALLS/RI/VIS. The extraction time results for each species, based on minimum extraction time and HPSEC-MALLS/RI/VIS results, were 4 h (GM4h), 6 h (GS6h) and 2 h (GG2h) for GM, GS and GG, respectively. In most cases, the apparent persistence length, as determined by viscometry, indicated that aggregates remained in galactomannans after centrifugation and filtration. Results suggest an effective extraction time for each plant source of galactomannan based on its performance and its macromolecular behavior in solution.

Sample preparation for arsenic speciation in terrestrial plants--a review.

Arsenic is an element widely present in nature. Additionally, it may be found as different species in several matrices and therefore it is one of the target elements in chemical speciation. Although the number of studies in terrestrial plants is low, compared to matrices such as fish or urine, this number is raising due to the fact that this type of matrix are closely related to the human food chain. In speciation analysis, sample preparation is a critical step and several extraction procedures present drawbacks. In this review, papers dealing with extraction procedures, analytical methods, and studies of species conservation in plants cultivated in terrestrial environment are critically discussed. Analytical procedures based on extractions using water or diluted acid solutions associated with HPLC-ICP-MS are good alternatives, owing to their versatility and sensitivity, even though less expensive strategies are shown as feasible choices.

Evaluations of the TiO2/simulated solar UV degradations of XAD fractions of natural organic matter from a bog lake using size-exclusion chromatography.

This work reports on the changes in compositions of humic acids (HAs) and fulvic acids (FAs) during photocatalytic degradation. The HAs and FAs were obtained from the XAD-resin fractionation of natural-organic matter (NOM) from a bog lake (Lake Hohloh, Black Forest, Germany). Degussa P-25 titanium dioxide (TiO2) in a suspension and a solar UV simulator (batch reactor) were used in the experiments. The photocatalytic degradation of the HAs and FAs were monitored using size-exclusion chromatography (SEC) equipped with dissolved organic carbon (DOC) and ultraviolet (UV254) detection (SEC-DOC and SEC-UV254) and UV-Vis spectrophotometry. The evolutions of the photocatalytic degradations of the HA and FA fractions were selective. The photocatalytic degradation started with the degradations of high molecular weight compounds with relatively high UV254 absorbances in the HA and FA fractions to yield low molecular weight compounds showing less specific UV254 absorbances. Observance of the same tendency for the original NOM from Lake Hohloh indicates that these XAD-fractions still having complex compound mixtures. However, the larger molecular weight fractions of the FAs showed higher preferential adsorptions onto TiO2, which caused their faster degradation rates. Furthermore, FAs showed a greater reduction of the total THM formation potential (TTHMFP) and the organic halogen compounds adsorbable on activated carbon formation potential (AOXFP), in comparison with the HAs.

Efecto del uso la cobertura del suelo sobre e perfil de polidispersidad de áácidos húmicos extraídos de un andisol del departamento de caldas, Colombia / Effectof the usee and the cover of the soil on the profile of polydispersity of humic acids extracted of an andisol from the department of caldas,Colombia

Con el objeto de conocer el posible efecto del uso y la cobertura del suelo, sobre el perfil de polidispersidadde los ácidos húmicos, se analizaron los horizontes A de tres muestras de suelo de un andisol (Melanudands) del departamento de Caldas, Colombia, con diferentes condiciones de manejo y cobertura: bajo bosque de guadua, bajo café con sombrío de guamo y bajo café de libre exposición. Para ello, en la fracción inferior a 50 µm, se realizó la extracción secuencial de las sustancias húmicas con soluciones de tetraborato (Na2B4O7 0,05 M), pirofosfato (Na4P2O7 0,025 M) e hidróxido de sodio (NaOH 0,1M) y la separación y purificación de los respectivos ácidos húmicos. Para obtener los perfiles se acoplaron las técnicas de separación de cromatografía de exclusión por tamaño utilizando Sephadex G-75 y ultracentrifugación por gradiente de densidad con sacarosa y se elaboraron gráficas de distribución por tamaño (absorbancia (450 nm) vs. volumen eluido). Se encontró que los ácidos húmicos extraídos con hidróxido de sodio presentaron en todas las muestras menor polidispersidad, mayor tamaño, peso y aromaticidad evaluada por la relación E4/E6. Los resultados no mostraron, en general, efecto drástico del uso y la cobertura del suelo, sobre el perfil de polidispersidad y el coeficiente de sedimentación de los ácidos húmicos, lo cual está relacionado con el tamaño, peso y densidad molecular de los mismos. Sin embargo, se encontró que los ácidos húmicos de las muestras de suelo bajo café con sombrío de guamo y café de libre exposición son muy similares entre sí y diferentes de la muestra bajo bosque de guadua, lo que se asoció con un inicio del efecto del uso y la cobertura del suelo sobre el tamaño de los ácidos húmicos del suelo; se presume que a largo término este efecto podría acentuarse y eventualmente ser considerado como indicador de degradación del componente orgánico del suelo.

In order to know the possible effect of the use and the cover of soil, on the polydispersity profile of humic acids were analyzed the A horizon of three samples of an andisol (Melanudand) from the Department of Caldas, Colombia, that have presented different management and coverland as soils under forest of bamboo, coffee with somber of guamo and under free exposition coffee. For this in the lower fraction to 50 µm, the sequential extraction of humic substances was carried out with solutions of sodium tetraborate (Na2B4O7 0.05 M), sodium pyrophosphate (Na4P2O7 0.025 M) and sodium hydroxide (NaOH 0.1M) and the later separation and purification of the respective humic acids. To obtain the polydispersity profiles was applied the exclusion chromatography separation by size technique utilizing Sephadex G-75 and ultracentrifugation by gradient of density with sucrose and they devised graphics of distribution by size (absorbance (450 nm) vs. volume eluted). It was found that the humic acids extracted with sodium hydroxide presented smaller polydispersity, bigger size and molecular weight, as well as, degree of aromatic condensation. The results did not show drastic effect of the use and the coverland , on the polidispersity profile and the coefficient of sedimentation of the humic acids, which is related to the size, weight and molecular density of the same ones. Nevertheless, it was found that the húmic acids of the samples of soil under coffee with somber of guamo and free exposition coffee are very similar among it and different from the sample under forest of bamboo. This fact was associated with a beginning of the effect of the use and the cover of the soil on the size of the humic acids. It presumed that in the long term the effect is accentuated and eventually could be considered as indicator of degradation of the organic component of the soil.