Synthesis and Thermodynamic and Biophysical Properties of Tricyclo-DNA

The DNA analogue tricyclo-DNA, built from conformationally rigid nucleoside analogues that were linked via tertiary phosphodiester functions, can efficiently be synthesized from the corresponding phosphoramidites by conventional solid-phase cyanoethyl phosphoramidite chemistry. 5'-End phosphorylated tricyclo-DNA sequences are chemically stable in aqueous, pH-neutral media at temperatures from 0 to 90 C. Tricyclo-DNA sequences resist enzymatic hydrolysis by the 3'-exonuclease snake venom phosphodiesterase. Homobasic adenine- and thymine-containing tricyclo-DNA octa- and nonamers are extraordinarily stable A-T base-pairing systems, not only in their own series but also with complementary DNA and RNA. Base mismatch formation is strongly destabilized. As in bicyclo-DNA, the tricyclo-DNA purine sequences preferentially accept a complementary strand on the Hoogsteen face of the base. A thermodynamic analysis reveals entropic benefits in the case of hetero-backbone duplex formation (tricyclo-DNA/DNA duplexes) and both an enthalpic and entropic benefit for duplex formation in the pure tricyclo-DNA series compared to natural DNA. Stability of tricyclo-DNA duplex formation depends more strongly on monovalent salt concentration compared to natural DNA. Homopyrimidine DNA sequences containing tricyclothymidine residues form triplexes with complementary double-stranded DNA. Triple-helix stability depends on the sequence composition and can be higher when compared to that of natural DNA. The use of one tricyclothymidine residue in the center of the self-complementary dodecamer duplex (d(CGCGAAT t CGCG), t = tricyclothymidine) strongly stabilizes its monomolecular hairpin loop structure relative to that of the corresponding pure DNA dodecamer ( T m = +20 C), indicating (tetra)loop-stabilizing properties of this rigid nucleoside analogue.

Production of Ethanol from Hardwood

Diminishing crude oil and natural gas supplies, along with concern about greenhouse gas are major driving forces in the search for efficient renewable energy sources. The conversion of lignocellulosic biomass to energy and useful chemicals is a component of the solution. Ethanol is most commonly produced by enzymatic hydrolysis of complex carbohydrates to simple sugars followed by fermentation using yeast. C6Hl0O5 + H2O −Enxymes→ C6H12O6 −Yeast→ 2CH3CH2OH + 2C02 In the U.S. corn is the primary starting raw material for commercial ethanol production. However, there is insufficient corn available to meet the future demand for ethanol as a gasoline additive. Consequently a variety of processes are being developed for producing ethanol from biomass; among which is the NREL process for the production of ethanol from white hardwood. The objective of the thesis reported here was to perform a technical economic analysis of the hardwood to ethanol process. In this analysis a Greenfield plant was compared to co-locating the ethanol plant adjacent to a Kraft pulp mill. The advantage of the latter case is that facilities can be shared jointly for ethanol production and for the production of pulp. Preliminary process designs were performed for three cases; a base case size of 2205 dry tons/day of hardwood (52 million gallons of ethanol per year) as well as the two cases of half and double this size. The thermal efficiency of the NREL process was estimated to be approximately 36%; that is about 36% of the thermal energy in the wood is retained in the product ethanol and by-product electrical energy. The discounted cash flow rate of return on investment and the net present value methods of evaluating process alternatives were used to evaluate the economic feasibility of the NREL process. The minimum acceptable discounted cash flow rate of return after taxes was assumed to be 10%. In all of the process alternatives investigated, the dominant cost factors are the capital recovery charges and the cost of wood. The Greenfield NREL process is not economically viable with the cost of producing ethanol varying from $2.58 to $2.08/gallon for the half capacity and double capacity cases respectively. The co-location cases appear more promising due to reductions in capital costs. The most profitable co-location case resulted in a discounted cash flow rate of return improving from 8.5% for the half capacity case to 20.3% for the double capacity case. Due to economy of scale, the investments become more and more profitable as the size of the plant increases. This concept is limited by the amount of wood that can be delivered to the plant on a sustainable basis as well as the demand for ethanol within a reasonable distance of the plant.

Proceedings of the 13th Annual Biochemical Engineering Symposium

The symposium reported here was the thirteenth of a series devoted to talks by students on their biochemical engineering research. The first, third, fifth, ninth, and twelfth were at Kansas State University, the second and fourth were at the University of Nebraska–Lincoln, the sixth was in Kansas City and was hosted by Iowa State University, the seventh and tenth were at Iowa State, and the eighth and eleventh were at the University of Missouri–Columbia and Colorado State University, respectively. All symposia have been followed by proceedings edited by faculty of the host institution. Because final publication usually takes place elsewhere, papers here are brief, and often cover research in progress. ContentSequential Utilization of Mixed Sugars by Clostridium acetobutylicum, B. Hong, N. H. Choi, and L. T. Fan, Kansas State University The Effects of Dilution Rate on the Kinetics. of Anaerobic Acidogenesis, C. J. Huang, Colorado State University Ethanol Production by Zymomonas mobilis in Anaerobic Glucose-Limited Culture: A Yield Study, Mehmet D. Oner, Kansas State University Hydrolysis of Cellulosics by Enterobacteria, Michael R. Sierks, Iowa State University The Cellulase System of Chaetomium cellulolyticum, Nikhil Mehta, Colorado State University DNA Measurement as a Tool for Estimating Biomass Concentration in the Presence of Interfering Solids, Bamidele 0. Solomon, Kansas State University The Effect of Cellulose Crystallinity on Enzymatic Hydrolysis, Maria S. Bertran, Colorado State University High Performance Liquid Chromatography of Di- and Trisaccharides, Michael M. Meagher, Iowa State University Dynamics of Bubble Size .Distributions in Air-Lift Fermentors, c. H. Lee and Snehal A. Patel, Kansas State University A Thermal Coagulation Study of Alfalfa Leaf Proteins by Differential Scanning Calorimeter, Khalif Ahmed and Bruce Dale, Colorado State University Thermodynamic Efficiency of Photoautotrophic Growth, Hyeon Y. Lee, Kansas State University

Proceedings of the 14th Annual Biochemical Engineering Symposium

The Annual Biochemical Engineering Symposium series is devoted to presentations by students on their research topics. The fourteenth event, held in 1984, was organized at the University of Missouri–Columbia. It was attended by the biochemical engineering faculty and the students from Colorado State University, Iowa State University, Kansas State University, University of Missouri–Columbia, University of Missouri–Rolla and Washington University, St. Louis. Contents"Estimation of Product Formation Kinetics and Microbial Yield Parameters for Anaerobic Organic Acid and Solvent Production," M.D. Oner, Kansas State University "Characterization of Soy Protein Texturization in a Complex Bioreactor," J.L. Ibave, Colorado State University "Acid and Solvent Fermentations Using Mixed Cultures," D. Stevens, University of Missouri–Columbia "Preliminary Process Design for Ethanol from Sweet Sorghum Ensilage Feedstock," Keith D. Lange, Colorado State University "Lamella Settlers in Ethanol Fermentation," Yong Jayanata, University of Missouri–Columbia "Bubble Size Distribution in the Down Flow Section of an Air-Lift Column," Snehal A. Patel and C.H. Lee, Kansas State University "The Sensitivity of Plant Cells to Shear Stress," Morris Z. Resenberg and Eric H. Dunlap, Washington University, St. Louis "Estimation of Growth Yield Parameters Associated with Microbial Growth," Hyeon Y. Lee, Kansas State University "Capillary Gas Chromatography of Trimethylsilylated Trisaccharides," Etienne J.M. Selosse, Iowa State University "Subsite Mapping of an Endo-Xylanase Labeled Xylooligo-saccharides," Bernard Y. Tao, Iowa State University "Cellulase Enzyme Recycle," Kate M.V. Baptie, Colorado State University "Non-Homogeneous Poisson Renewal Reward Process for Modelling Enzymatic Hydrolysis of Cellulose," M.M. Gharpuray and L.T. Fan, Kansas State University

Proceedings of the Eighteenth Annual Biochemical Engineering Symposium

The eighteenth annual biochemical engineering symposium was held during April 22–23, 1988 at the YMCA of the Rockies conference center in Estes Park, Colorado, under the sponsorship of the University of Colorado. Previous symposia in this series have been hosted by Kansas State University (1st, 3rd, 5th, 9th, 12th, 16th), University of Nebraska-Lincoln (2nd, 4th), Iowa State University (6th, 7th, l0th, 13th, 17th), University of Missouri–Columbia (8th, 14th), and Colorado State University (11th, 15th). Next year's symposium is scheduled to be held at the University of Missouri-Columbia. The symposia are devoted to talks by students about their ongoing research. Because final publication usually takes place elsewhere, the papers included in the proceedings are brief, and often cover work in progress. ContentsApplications of mass spectrometers in biochemical engineeringJohn P. McDonald, Ayush Gupta, and Lourdes Taladriz, Kansas State University Enzymatic hydrolysis of corn gluten proteinsJulie Hardwick; Iowa State University Improved Acetone-Butanol Fermentation AnalysisZ. Buday; Colorado State University On-Line State Identification for Batch FermentationD. A. Gee and W. F. Ramirez; University of Colorado Role of Spargers in Air-Lift ReactorsPeter U. Sohn and Rakesh K. Bajpai; University of Missouri–Columbia The Interaction of Microcarriers and Turbulence within an Airlift FermenterG. Travis Jones; Kansas State University Oxygen Diffusion in the Inter-Fiber Gel/Cell Matrix of NMR-Compatible Hollow Fiber Bio-ReactorsS. L. Hanson, B. E. Dale, and R. J. Gillies; Colorado State University Characterization of Ca-alginate Gel Beads FormationHorngtwu Su, Rakesh K. Bajpai, and George W. Preckshot; University of Missouri–Columbia Metabolic Effects of Chloramphenicol Resistance in the Recombinant Host/Vector System: E. coli RRl [pBR329]William E. Bentley, Dana C. Andersen, Dhinakar S. Kompala, and Robert H. Davis; University of Colorado Genetic Engineering of Beta-Galactosidase to Aid in Fermentation Product Recovery by Polyelectrolyte PrecipitationD. E. Parker, C. E. Glatz, J. Zhao, C. F. Ford, S. M. Gendel, and M. A. Rougvie; Iowa State University Biodegradation of Organic Compounds in SoilLourdes Taladriz, L. E. Erickson, and L. T. Fan; Kansas State University Effect of Dilution, pH and Nutrient Composition on the Biodegradation of Metalworking FluidsAyush Gupta, L. E. Erickson, and L. T. Fan; Kansas State University Dissolved Hydrogen Correlation with Redox Potential in Acetone-Butanol FermentationXiangdong Zhou; Colorado State University Modeling of Ensiling Fermentation of Sweet SorghumA. K. Hilaly; Colorado State University

Proceedings of the 22nd Annual Biochemical Engineering Symposium

This is the twenty-second of a series of symposia devoted to talks and posters by students about their biochemical engineering research. The first, third, fifth, ninth, twelfth, sixteenth, and twenti~th were hosted by Kansas State University, the second and fourth by the University of Nebraska- Lincoln, the sixth, seventh, tenth, thirteenth, seventeenth, and twenty-second by Iowa State University, the eighth, fourteenth, and nineteenth by the University of Missouri-Columbia, the eleventh, fifteenth, and twenty-first by Colorado State University, and the eighteenth by the University of Colorado. Next year's symposium will be at the University of Oklahoma. Symposium proceedings are edited and issued by faculty of the host institution. Because final publication usually takes place in refereed journals, articles included here are brief and often cover work in progress. ContentsC. A. Baldwin, J.P. McDonald, and L. E. Erickson, Kansas State University. Effect of Hydrocarbon Phase on Kinetic and Transport Limitations for Bioremediation of Microporous Soil J. C. Wang, S. K. Banerji, and Rakesh Bajpai, University of Missouri-Columbia. Migration of PCP in Soil-Columns in Presence of a Second Organic Phase Cheng-Hsien Hsu and Roger G. Harrison, University of Oklahoma. Bacterial Leaching of Zinc and Copper from Mining Wastes James A. Searles, Paul Todd, and Dhinakar S. Kompala, University of Colorado. Suspension Culture of Chinese Hamster Ovary Cells Utilizing Inclined Sedimentation Ron Beyerinck and Eric H. Dunlop, Colorado State University. The Effect of Feed Zone Turbulence as Measured by Laser Doppler Velocimetry on Baker's Yeast Metabolism in a Chemostat Paul Li-Hong Yeh, GraceY. Sun, Gary A. Weisman, and Rakesh Bajpai, University of Missouri-Columbia. Effect of Medium Constituents upon Membrane Composition of Insect Cells R. Shane Gold, M. M. Meagher, R. Hutkins, and T. Conway, University of Nebraska-Lincoin. Ethanol Tolerance and Carbohydrate Metabolism in Lactobacilli John Sargantanis and M. N. Karim, Colorado State University. Application of Kalman Filter and Adaptive Control in Solid Substrate Fermentation D. Vrana, M. Meagher, and R. Hutkins, University of Nebraska-Lincoln. Product Recovery Optimization in the ABE Fermentation Kalyan R. Tadikonda and Robert H. Davis, University of Colorado. Cell Separations Using Targeted Monoclonal Antibodies Against Surface Proteins Meng H. Heng and Charles E. Glatz, Iowa State University. Charged Fusion for Selective Recovery of B-Galactosidase from Cell Extract Using Hollow Fiber Ion-Exchange Membrane Adsorption Hsiu-Mei Chen, Peter J. Reilly, and Clark Ford, Iowa State University. Site-Directed Mutagenesis to Enhance Thermostability of Glucoamylase from Aspergillus: A Rational Approach P. Tuitemwong, L. E. Erickson, and D. Y. C. Fung, Kansas State University. Applications of Enzymatic Hydrolysis and Fermentation on the Reduction of Flatulent Sugars in the Rapid Hydration Hydrothermal Cooked Soy Milk Sanjeev Redkar and Robert H. Davis, University of Colorado. Crossflow Microfiltration of Yeast Suspensions Linda Henk and James C. Linden, Colorado State University, and Irving C. Anderson, Iowa State University. Evaluation of Sorghum Ensilage as an Ethanol Feedstock Marc Lipovitch and James C. Linden, Colorado State University. Stability and Biomass Feedstock Pretreatability for Simultaneous Saccharification and Fermentation Ali Demirci, Anthony L. Pometto Ill, and Kenneth E. Johnson, Iowa State University. Application of Biofilm Reactors in Lactic Acid Fermentation Michael K. Dowd, Peter I. Reilly, and WalterS. Trahanovsky, Iowa State University. Low Molecular-Weight Organic Composition of Ethanol Stillage from Corn Craig E. Forney, Meng H. Heng, John R. Luther, Mark Q. Niederauer, and Charles E. Glatz, Iowa State University. Enhancement of Protein Separation Using Genetic Engineering J. F. Shimp, J. C. Tracy, E. Lee, L. C. Davis, and L. E. Erickson, Kansas State University. Modeling Contaminant Transport, Biodegradation and Uptake by Plants in the Rhizosphere Xiaoqing Yang, L. E. Erickson, and L. T. Fan, Kansas State University. Modeling of Dispersive-Convective Characteristics in Bioremediation of Contaminated Soil Jan Johansson and Rakesh Bajpai, University of Missouri-Columbia. Fouling of Membranes J. M. Wang, S. K. Banerji, and R. K. Bajpai, University of Missouri-Columbia. Migration of Sodium-Pentachorophenol (Na-PCP) in Unsaturated and Saturated Soil-Columns J. Sweeney and M. Meagher, University of Nebraska-Lincoln. The Purification of Alpha-D-Glucuronidase from Trichoderma reesei

Geological, geochemical and biogeochemical data on boundary zones of the Atlantic Ocean

Results of studies in two biogeochemically active zones of the Atlantic Ocean (the Benguela upwelling waters and the region influenced by the Congo River run-off) are reported in the book. A multidisciplinary approach included studies of the major elements of the ocean ecosystem: sea water, plankton, suspended matter, bottom sediments, interstitial waters, aerosols, as well as a wide complex of oceanographic studies carried out under a common program. Such an approach, as well as a use of new methodical solutions led to obtaining principally new information on different aspects of oceanology.

Agricultural use of organic wastes as source of nitrogen and phosphorus: risks and opportunities

El nitrógeno (N) y el fósforo (P) son nutrientes esenciales en la producción de cultivos. El desarrollo de los fertilizantes de síntesis durante el siglo XX permitió una intensificación de la agricultura y un aumento de las producciones pero a su vez el gran input de nutrientes ha resultado en algunos casos en sistemas poco eficientes incrementando las pérdidas de estos nutrientes al medio ambiente. En el caso del P, este problema se agrava debido a la escasez de reservas de roca fosfórica necesaria para la fabricación de fertilizantes fosfatados. La utilización de residuos orgánicos en agricultura como fuente de N y P es una buena opción de manejo que permite valorizar la gran cantidad de residuos que se generan. Sin embargo, es importante conocer los procesos que se producen en el suelo tras la aplicación de los mismos, ya que influyen en la disponibilidad de nutrientes que pueden ser utilizados por el cultivo así como en las pérdidas de nutrientes de los agrosistemas que pueden ocasionar problemas de contaminación. Aunque la dinámica del N en el suelo ha sido más estudiada que la del P, los problemas importantes de contaminación por nitratos en zonas vulnerables hacen necesaria la evaluación de aquellas prácticas de manejo que pudieran agravar esta situación, y en el caso de los residuos orgánicos, la evaluación de la respuesta agronómica y medioambiental de la aplicación de materiales con un alto contenido en N (como los residuos procedentes de la industria vinícola y alcoholera). En cuanto al P, debido a la mayor complejidad de su ciclo y de las reacciones que ocurren en el suelo, hay un mayor desconocimiento de los factores que influyen en su dinámica en los sistemas suelo-planta, lo que supone nuevas oportunidades de estudio en la evaluación del uso agrícola de los residuos orgánicos. Teniendo en cuenta los conocimientos previos sobre cada nutriente así como las necesidades específicas en el estudio de los mismos, en esta Tesis se han evaluado: (1) el efecto de la aplicación de residuos procedentes de la industria vinícola y alcoholera en la dinámica del N desde el punto de vista agronómico y medioambiental en una zona vulnerable a la contaminación por nitratos; y (2) los factores que influyen en la disponibilidad de P en el suelo tras la aplicación de residuos orgánicos. Para ello se han llevado a cabo incubaciones de laboratorio así como ensayos de campo que permitieran evaluar la dinámica de estos nutrientes en condiciones reales. Las incubaciones de suelo en condiciones controladas de humedad y temperatura para determinar el N mineralizado se utilizan habitualmente para estimar la disponibilidad de N para el cultivo así como el riesgo medioambiental. Por ello se llevó a cabo una incubación en laboratorio para conocer la velocidad de mineralización de N de un compost obtenido a partir de residuos de la industria vinícola y alcoholera, ampliamente distribuida en Castilla-La Mancha, región con problemas importantes de contaminación de acuíferos por nitratos. Se probaron tres dosis crecientes de compost correspondientes a 230, 460 y 690 kg de N total por hectárea que se mezclaron con un suelo franco arcillo arenoso de la zona. La evolución del N mineral en el suelo a lo largo del tiempo se ajustó a un modelo de regresión no lineal, obteniendo valores bajos de N potencialmente mineralizable y bajas contantes de mineralización, lo que indica que se trata de un material resistente a la mineralización y con una lenta liberación de N en el suelo, mineralizándose tan solo 1.61, 1.33 y 1.21% del N total aplicado con cada dosis creciente de compost (para un periodo de seis meses). Por otra parte, la mineralización de N tras la aplicación de este material también se evaluó en condiciones de campo, mediante la elaboración de un balance de N durante dos ciclos de cultivo (2011 y 2012) de melón bajo riego por goteo, cultivo y manejo agrícola muy característicos de la zona de estudio. Las constantes de mineralización obtenidas en el laboratorio se ajustaron a las temperaturas reales en campo para predecir el N mineralizado en campo durante el ciclo de cultivo del melón, sin embargo este modelo generalmente sobreestimaba el N mineralizado observado en campo, por la influencia de otros factores no tenidos en cuenta para obtener esta predicción, como el N acumulado en el suelo, el efecto de la planta o las fluctuaciones de temperatura y humedad. Tanto el ajuste de los datos del laboratorio al modelo de mineralización como las predicciones del mismo fueron mejores cuando se consideraba el efecto de la mezcla suelo-compost que cuando se aislaba el N mineralizado del compost, mostrando la importancia del efecto del suelo en la mineralización del N procedente de residuos orgánicos. Dado que esta zona de estudio ha sido declarada vulnerable a la contaminación por nitratos y cuenta con diferentes unidades hidrológicas protegidas, en el mismo ensayo de campo con melón bajo riego por goteo se evaluó el riesgo de contaminación por nitratos tras la aplicación de diferentes dosis de compost bajo dos regímenes de riego, riego ajustado a las necesidades del cultivo (90 ó 100% de la evapotranspiración del cultivo (ETc)) o riego excedentario (120% ETc). A lo largo del ciclo de cultivo se estimó semanalmente el drenaje mediante la realización de un balance hídrico, así como se tomaron muestras de la solución de suelo y se determinó su concentración de nitratos. Para evaluar el riesgo de contaminación de las aguas subterráneas asociado con estas prácticas, se utilizaron algunos índices medioambientales para determinar la variación en la calidad del agua potable (Índice de Impacto (II)) y en la concentración de nitratos del acuífero (Índice de Impacto Ambiental (EII)). Para combinar parámetros medioambientales con parámetros de producción, se calculó la eficiencia de manejo. Se observó que la aplicación de compost bajo un régimen de riego ajustado no aumentaba el riesgo de contaminación de las aguas subterráneas incluso con la aplicación de la dosis más alta. Sin embargo, la aplicación de grandes cantidades de compost combinada con un riego excedentario supuso un incremento en el N lixiviado a lo largo del ciclo de cultivo, mientras que no se obtuvieron mayores producciones con respecto al riego ajustado. La aplicación de residuos de la industria vinícola y alcoholera como fuente de P fue evaluada en suelos calizos caracterizados por una alta capacidad de retención de P, lo cual en algunos casos limita la disponibilidad de este nutriente. Para ello se llevó a cabo otro ensayo de incubación con dos suelos de diferente textura, con diferente contenido de carbonato cálcico, hierro y con dos niveles de P disponible; a los que se aplicaron diferentes materiales procedentes de estas industrias (con y sin compostaje previo) aportando diferentes cantidades de P. A lo largo del tiempo se analizó el P disponible del suelo (P Olsen) así como el pH y el carbono orgánico disuelto. Al final de la incubación, con el fin de estudiar los cambios producidos por los diferentes residuos en el estado del P del suelo se llevó a cabo un fraccionamiento del P inorgánico del suelo, el cual se separó en P soluble y débilmente enlazado (NaOH-NaCl-P), P soluble en reductores u ocluido en los óxidos de Fe (CBD-P) y P poco soluble precipitado como Ca-P (HCl-P); y se determinó la capacidad de retención de P así como el grado de saturación de este elemento en el suelo. En este ensayo se observó que, dada la naturaleza caliza de los suelos, la influencia de la cantidad de P aplicado con los residuos en el P disponible sólo se producía al comienzo del periodo de incubación, mientras que al final del ensayo el incremento en el P disponible del suelo se igualaba independientemente del P aplicado con cada residuo, aumentando el P retenido en la fracción menos soluble con el aumento del P aplicado. Por el contrario, la aplicación de materiales orgánicos menos estabilizados y con un menor contenido en P, produjo un aumento en las formas de P más lábiles debido a una disolución del P retenido en la fracción menos lábil, lo cual demostró la influencia de la materia orgánica en los procesos que controlan el P disponible en el suelo. La aplicación de residuos aumentó el grado de saturación de P de los suelos, sin embargo los valores obtenidos no superaron los límites establecidos que indican un riesgo de contaminación de las aguas. La influencia de la aplicación de residuos orgánicos en las formas de P inorgánico y orgánico del suelo se estudió además en un suelo ácido de textura areno francosa tras la aplicación en campo a largo plazo de estiércol vacuno y de compost obtenido a partir de biorresiduos, así como la aplicación combinada de compost y un fertilizante mineral (superfosfato tripe), en una rotación de cultivos. En muestras de suelo recogidas 14 años después del establecimiento del experimento en campo, se determinó el P soluble y disponible, la capacidad de adsorción de P, el grado de saturación de P así como diferentes actividades enzimáticas (actividad deshidrogenasa, fosfatasa ácida y fosfatasa alcalina). Las diferentes formas de P orgánico en el suelo se estudiaron mediante una técnica de adición de enzimas con diferentes substratos específicos a extractos de suelo de NaOH-EDTA, midiendo el P hidrolizado durante un periodo de incubación por colorimetría. Las enzimas utilizadas fueron la fosfatasa ácida, la nucleasa y la fitasa las cuales permitieron identificar monoésteres hidrolizables (monoester-like P), diésteres (DNA-like P) e inositol hexaquifosfato (Ins6P-like P). La aplicación a largo plazo de residuos orgánicos aumentó el P disponible del suelo proporcionalmente al P aplicado con cada tipo de fertilización, suponiendo un mayor riesgo de pérdidas de P dado el alto grado de saturación de este suelo. La aplicación de residuos orgánicos aumentó el P orgánico del suelo resistente a la hidrólisis enzimática, sin embargo no influyó en las diferentes formas de P hidrolizable por las enzimas en comparación con las observadas en el suelo sin enmendar. Además, las diferentes formas de P orgánico aplicadas con los residuos orgánicos no se correspondieron con las analizadas en el suelo lo cual demostró que éstas son el resultado de diferentes procesos en el suelo mediados por las plantas, los microorganismos u otros procesos abióticos. En este estudio se encontró una correlación entre el Ins6P-like P y la actividad microbiana (actividad deshidrogenasa) del suelo, lo cual refuerza esta afirmación. Por último, la aplicación de residuos orgánicos como fuente de N y P en la agricultura se evaluó agronómicamente en un escenario real. Se estableció un experimento de campo para evaluar el compost procedente de residuos de bodegas y alcoholeras en el mismo cultivo de melón utilizado en el estudio de la mineralización y lixiviación de N. En este experimento se estudió la aplicación de tres dosis de compost: 1, 2 y 3 kg de compost por metro lineal de plantación correspondientes a 7, 13 y 20 t de compost por hectárea respectivamente; y se estudió el efecto sobre el crecimiento de las plantas, la acumulación de N y P en la planta, así como la producción y calidad del cultivo. La aplicación del compost produjo un ligero incremento en la biomasa vegetal acompañado por una mejora significativa de la producción con respecto a las parcelas no enmendadas, obteniéndose la máxima producción con la aplicación de 2 kg de compost por metro lineal. Aunque los efectos potenciales del N y P fueron parcialmente enmascarados por otras entradas de estos nutrientes en el sistema (alta concentración de nitratos en el agua de riego y ácido fosfórico suministrado por fertirrigación), se observó una mayor acumulación de P uno de los años de estudio que resultó en un aumento en el número de frutos en las parcelas enmendadas. Además, la mayor acumulación de N y P disponible en el suelo al final del ciclo de cultivo indicó el potencial uso de estos materiales como fuente de estos nutrientes. ABSTRACT Nitrogen (N) and phosphorus (P) are essential nutrients in crop production. The development of synthetic fertilizers during the 20th century allowed an intensification of the agriculture increasing crop yields but in turn the great input of nutrients has resulted in some cases in inefficient systems with higher losses to the environment. Regarding P, the scarcity of phosphate rock reserves necessary for the production of phosphate fertilizers aggravates this problem. The use of organic wastes in agriculture as a source of N and P is a good option of management that allows to value the large amount of wastes generated. However, it is important to understand the processes occurring in the soil after application of these materials, as they affect the availability of nutrients that can be used by the crop and the nutrient losses from agricultural systems that can cause problems of contamination. Although soil N dynamic has been more studied than P, the important concern of nitrate pollution in Nitrate Vulnerable Zones requires the evaluation of those management practices that could aggravate this situation, and in the case of organic wastes, the evaluation of the agronomic and environmental response after application of materials with a high N content (such as wastes from winery and distillery industries). On the other hand, due to the complexity of soil P cycle and the reactions that occur in soil, there is less knowledge about the factors that can influence its dynamics in the soil-plant system, which means new opportunities of study regarding the evaluation of the agricultural use of organic wastes. Taking into account the previous knowledge of each nutrient and the specific needs of study, in this Thesis we have evaluated: (1) the effect of the application of wastes from the winery and distillery industries on N dynamics from the agronomic and environmental viewpoint in a vulnerable zone; and (2) the factors that influence P availability in soils after the application of organic wastes. With this purposes, incubations were carried out in laboratory conditions as well as field trials that allow to assess the dynamic of these nutrients in real conditions. Soil incubations under controlled moisture and temperature conditions to determine N mineralization are commonly used to estimate N availability for crops together with the environmental risk. Therefore, a laboratory incubation was conducted in order to determine the N mineralization rate of a compost made from wastes generated in the winery and distillery industries, widely distributed in Castilla-La Mancha, a region with significant problems of aquifers contamination by nitrates. Three increasing doses of compost corresponding to 230, 460 and 690 kg of total N per hectare were mixed with a sandy clay loam soil collected in this area. The evolution of mineral N in soil over time was adjusted to a nonlinear regression model, obtaining low values of potentially mineralizable N and low constants of mineralization, indicating that it is a material resistant to mineralization with a slow release of N, with only 1.61, 1.33 and 1.21% of total N applied being mineralized with each increasing dose of compost (for a period of six months). Furthermore, N mineralization after the application of this material was also evaluated in field conditions by carrying out a N balance during two growing seasons (2011 and 2012) of a melon crop under drip irrigation, a crop and management very characteristic of the area of study. The mineralization constants obtained in the laboratory were adjusted to the actual temperatures observed in the field to predict N mineralized during each growing season, however, this model generally overestimated the N mineralization observed in the field, because of the influence of other factors not taken into account for this prediction, as N accumulated in soil, the plant effect or the fluctuations of temperature and moisture. The fitting of the laboratory data to the model as well as the predictions of N mineralized in the field were better when considering N mineralized from the soil-compost mixture rather than when N mineralized from compost was isolated, underlining the important role of the soil on N mineralization from organic wastes. Since the area of study was declared vulnerable to nitrate pollution and is situated between different protected hydrological units, the risk of nitrate pollution after application of different doses compost was evaluated in the same field trial with melon under two irrigation regimes, irrigation adjusted to the crop needs (90 or 100% of the crop evapotranspiration (ETc)) or excedentary irrigation (120% ETc). Drainage was estimated weekly throughout the growing season by conducting a water balance, samples of the soil solution were taken and the concentration of nitrates was determined. To assess the risk of groundwater contamination associated with these practices, some environmental indices were used to determine the variation in the quality of drinking water (Impact Index (II)) and the nitrates concentration in the groundwater (Environmental Impact Index (EII)). To combine environmental parameters together with yield parameters, the Management Efficiency was calculated. It was observed that the application of compost under irrigation adjusted to the plant needs did not represent a higher risk of groundwater contamination even with the application of the highest doses. However, the application of large amounts of compost combined with an irrigation surplus represented an increase of N leaching during the growing season compared with the unamended plots, while no additional yield with respect to the adjusted irrigation strategy is obtained. The application of wastes derived from the winery and distillery industry as source of P was evaluated in calcareous soils characterized by a high P retention capacity, which in some cases limits the availability of this nutrient. Another incubation experiment was carried out using two soils with different texture, different calcium carbonate and iron contents and two levels of available P; to which different materials from these industries (with and without composting) were applied providing different amounts of P. Soil available P (Olsen P), pH and dissolved organic carbon were analyzed along time. At the end of the incubation, in order to study the changes in soil P status caused by the different residues, a fractionation of soil inorganic P was carried out, which was separated into soluble and weakly bound P (NaOH-NaCl- P), reductant soluble P or occluded in Fe oxides (CBD-P) and P precipitated as poorly soluble Ca-P (HCl-P); and the P retention capacity and degree of P saturation were determined as well. Given the calcareous nature of the soils, the influence of the amount of P applied with the organic wastes in soil available P only occurred at the beginning of the incubation period, while at the end of the trial the increase in soil available P equalled independently of the amount of P applied with each residue, increasing the P retained in the least soluble fraction when increasing P applied. Conversely, the application of less stabilized materials with a lower content of P resulted in an increase in the most labile P forms due to dissolution of P retained in the less labile fraction, demonstrating the influence of organic matter addition on soil P processes that control P availability in soil. As expected, the application of organic wastes increased the degree of P saturation in the soils, however the values obtained did not exceed the limits considered to pose a risk of water pollution. The influence of the application of organic wastes on inorganic and organic soil P forms was also studied in an acid loamy sand soil after long-term field application of cattle manure and biowaste compost and the combined application of compost and mineral fertilizer (triple superphosphate) in a crop rotation. Soil samples were collected 14 years after the establishment of the field experiment, and analyzed for soluble and available P, P sorption capacity, degree of P saturation and enzymatic activities (dehydrogenase, acid phosphatase and alkaline phosphatase). The different forms of organic P in soil were determined by using an enzyme addition technique, based on adding enzymes with different substrate specificities to NaOH-EDTA soil extracts, measuring the hydrolyzed P colorimetrically after an incubation period. The enzymes used were acid phosphatase, nuclease and phytase which allowed to identify hydrolyzable monoesters (monoester-like P) diesters (DNA-like P) and inositol hexakisphosphate (Ins6P-like P). The long-term application of organic wastes increased soil available P proportionally to the P applied with each type of fertilizer, assuming a higher risk of P losses given the high degree of P saturation of this soil. The application of organic wastes increased soil organic P resistant to enzymatic hydrolysis, but no influence was observed regarding the different forms of enzyme hydrolyzable organic P compared to those observed in the non-amended soil. Furthermore, the different forms of organic P applied with the organic wastes did not correspond to those analyzed in the soil which showed that these forms in soil are a result of multifaceted P turnover processes in soil affected by plants, microorganisms and abiotic factors. In this study, a correlation between Ins6P-like P and the microbial activity (dehydrogenase activity) of soil was found, which reinforces this claim. Finally, the application of organic wastes as a source of N and P in agriculture was evaluated agronomically in a real field scenario. A field experiment was established to evaluate the application of compost made from wine-distillery wastes in the same melon crop used in the experiments of N mineralization and leaching. In this experiment the application of three doses of compost were studied: 1 , 2 and 3 kg of compost per linear meter of plantation corresponding to 7, 13 and 20 tonnes of compost per hectare respectively; and the effect on plant growth, N and P accumulation in the plant as well as crop yield and quality was studied. The application of compost produced a slight increase in plant biomass accompanied by a significant improvement in crop yield with respect to the unamended plots, obtaining the maximum yield with the application of 2 kg of compost per linear meter. Although the potential effects of N and P were partially masked by other inputs of these nutrients in the system (high concentration of nitrates in the irrigation water and phosphoric acid supplied by fertigation), an effect of P was observed the first year of study resulting in a greater plant P accumulation and in an increase in the number of fruits in the amended plots. In addition, the higher accumulation of available N and P in the topsoil at the end of the growing season indicated the potential use of this material as source of these nutrients.

Occurrence and analysis of selected pharmaceutical compounds in soil from Spanish agricultural fields

This work describes the analysis of 15 pharmaceutical compounds, belonging to different therapeutic classes (anti-inflammatory/analgesics, lipid regulators, antiepileptics, ?-blockers and antidepressants) and with diverse physical?chemical properties, in Spanish soils with different farmland uses. The studied compounds were extracted from soil by ultrasound-assisted extraction (UAE) and determined, after derivatization, by gas chromatography with mass spectrometric detection (GC?MS). The limits of detection (LODs) ranged from 0.14 ng g?1 (naproxen) to 0.65 ng g?1 (amitriptyline). At least two compounds where detected in all samples, being ibuprofen, salicylic acid, and paracetamol, the most frequently detected compounds. The highest levels found in soil were 47 ng g?1 for allopurinol and 37 ng g?1 for salicylic acid. The influence of the type of crop and the sampling area on the levels of pharmaceuticals in soil, as well as their relationship with soil physical?chemical properties, was studied. The frequent and widespread detection of some of these compounds in agricultural soils show a diffuse contamination, although the low levels found do not pose a risk to the environment or the human health.

Spatio-temporal distribution of pyrethroids in soil in Mediterranean paddy fields

Purpose The demand of rice by the increase in population in many countries has intensified the application of pesticides and the use of poor quality water to irrigate fields. The terrestrial environment is one compartment affected by these situations, where soil is working as a reservoir, retaining organic pollutants. Therefore, it is necessary to develop methods to determine insecticides in soil and monitor susceptible areas to be contaminated, applying adequate techniques to remediate them. Materials and methods This study investigates the occurrence of ten pyrethroid insecticides (PYs) and its spatio-temporal variance in soil at two different depths collected in two periods (before plow and during rice production), in a paddy field area located in the Mediterranean coast. Pyrethroids were quantified using gas chromatography?mass spectrometry (GC?MS) after ultrasound-assisted extraction with ethyl acetate. The results obtained were assessed statistically using non-parametric methods, and significant statistical differences (p < 0.05) in pyrethroids content with soil depth and proximity to wastewater treatment plants were evaluated. Moreover, a geographic information system (GIS) was used to monitor the occurrence of PYs in paddy fields and detect risk areas. Results and discussion Pyrethroids were detected at concentrations ?57.0 ng g?1 before plow and ?62.3 ng g?1 during rice production, being resmethrin and cyfluthrin the compounds found at higher concentrations in soil. Pyrethroids were detected mainly at the top soil, and a GIS program was used to depict the obtained results, showing that effluents from wastewater treatment plants (WWTPs) were the main sources of soil contamination. No toxic effects were expected to soil organisms, but it is of concern that PYs may affect aquatic organisms, which represents the worst case scenario. Conclusions A methodology to determine pyrethroids in soil was developed to monitor a paddy field area. The use of water fromWWTPs to irrigate rice fields is one of the main pollution sources of pyrethroids. It is a matter of concern that PYs may present toxic effects on aquatic organisms, as they can be desorbed from soil. Phytoremediation may play an important role in this area, reducing the possible risk associated to PYs levels in soil.

Methyl Jasmonate Induces Lauric Acid ω-Hydroxylase Activity and Accumulation of CYP94A1 Transcripts but Does Not Affect Epoxide Hydrolase Activities in Vicia sativa Seedlings1

Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mm solution of MetJA stimulated ω-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min−1 mg−1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid ω-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.