Proceedings of the Seventeenth Annual Biochemical Engineering Symposium

This is the seventeenth of a series of symposia devoted to talks by students about their biochemical engineering research. The first, third, fifth, ninth, twelfth, and sixteenth 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, tenth, thirteenth, and seventeenth were at Iowa State University, the eighth and fourteenth were at the University of Missouri–Columbia, and the eleventh and fifteenth were at Colorado State University. Next year's symposium will be at the University of Colorado. Symposium proceedings are edited by faculty of the host institution. Because final publication usually takes place elsewhere, papers here are brief, and often cover work in progress. ContentsThe Effect of Polymer Dosage Conditions on the Properties of ProteinPolyelectrolyte Precipitates, K. H. Clark and C. E. Glatz, Iowa State University An Immobilized Enzyme Reactor/Separator for the Hydrolysis of Casein by Subtilisin Carlsberg, A. J. Bream, R. A. Yoshisato, and G. R. Carmichael, University of Iowa Cell Density Measurements in Hollow Fiber Bioreactors, Thomas Blute, Colorado State University The Hydrodynamics in an Air-Lift Reactor, Peter Sohn, George Y. Preckshot, and Rakesh K. Bajpai, University of Missouri–Columbia Local Liquid Velocity Measurements in a Split Cylinder Airlift Column, G. Travis Jones, Kansas State University Fluidized Bed Solid Substrate Trichoderma reesei Fermentation, S. Adisasmito, H. N. Karim, and R. P. Tengerdy, Colorado State University The Effect of 2,4-D Concentration on the Growth of Streptanthus tortuosis Cells in Shake Flask and Air-Lift Permenter Culture, I. C. Kong, R. D. Sjolund, and R. A. Yoshisato, University of Iowa Protein Engineering of Aspergillus niger Glucoamylase, Michael R. Sierks, Iowa State University Structured Kinetic Modeling of Hybidoma Growth and Monoclonal Antibody Production in Suspension Cultures, Brian C. Batt and Dhinakar S. Kampala, University of Colorado Modelling and Control of a Zymomonas mobilis Fermentation, John F. Kramer, M. N. Karim, and J. Linden, Colorado State University Modeling of Brettanomyces clausenii Fermentation on Mixtures of Glucose and Cellobiose, Max T. Bynum and Dhinakar S. Kampala, University of Colorado, Karel Grohmann and Charles E. Yyman, Solar Energy Research Institute Master Equation Modeling and Monte Carlo Simulation of Predator-Prey Interactions, R. 0. Fox, Y. Y. Huang, and L. T. Fan, Kansas State University Kinetics and Equilibria of Condensation Reactions Between Two Different Monosaccharides Catalyzed by Aspergillus niger Glucoamylase, Sabine Pestlin, Iowa State University Biodegradation of Metalworking Fluids, S. M. Lee, Ayush Gupta, L. E. Erickson, and L. T. Fan, Kansas State University Redox Potential, Toxicity and Oscillations in Solvent Fermentations, Kim Joong, Rakesh Bajpai, and Eugene L. Iannotti, University of Missouri–Columbia Using Structured Kinetic Models for Analyzing Instability in Recombinant Bacterial Cultures, William E. Bentley and Dhinakar S. Kompala, University of Colorado

Proceedings of the Twentieth Annual Biochemical Engineering Symposium

The 20th Annual Biochemical Engineering Symposium was held at Kansas State University on April 21,1990. The objectives of the symposium were to provide: (i) a forum for informal discussion of biochemical engineering research being conducted at the participating institutions and (ii) an opportunity for students to present and publish their work. Twenty-eight papers presented at the symposium are included in this proceedings. Some of the papers describe the progress of ongoing projects, and others contain the results of completed projects. Only brief summaries are given of the papers that will be published in full elsewhere. The program of the symposium and a list of the participants are included in the proceedings. ContentsCell Separations and Recycle Using an Inclined Settler, Ching-Yuan Lee, Robert H. Davis and Robert A. Sclafani Micromixing and Metabolism in Bioreactors: Characterization of a 14 L Fermenter, K.S. Wenger and E.H. Dunlop Production, Purification, and Hydrolysis Kinetics of Wild-Type and Mutant Glucoamylases from Aspergillus Awamori, Ufuk Bakir, Paul D. Oates, Hsiu-Mei Chen and Peter J. Reilly Dynamic Modeling of the Immune System, Barry Vant-Hull and Dhinakar S. Kompala Dynamic Modeling of Active Transport Across a Biological Cell: A Stochastic Approach, B.C. Shen, S.T. Chou, Y.Y. Chiu and L.T. Fan Electrokinetic Isolation of Bacterial Vesicles and Ribosomes, Debra T.L. Hawker, Robert H. Davis, Paul W. Todd, and Robert Lawson Application of Dynamic Programming for Fermentative Ethanol Production by Zymomonas mobilis, Sheyla L. Rivera and M. Nazmul Karim Biodegradation of PCP by Pseudomonas cepacia, R. Rayavarapu, S.K. Banerji, and R.K. Bajpai Modeling the Bioremediation of Contaminated Soil Aggregates: a Phenomenological Approach, S. Dhawan, L.E. Erickson and L.T. Fan Biospecific Adsorption of Glucoamylase-I from Aspergillus niger on Raw Starch, Bipin K. Dalmia and Zivko L. Nikolov Overexpression in Recombinant Mammalian Cells: Effect on Growth Rate and Genetic Instability, Jeffrey A. Kern and Dhinakar S. Kompala Structured Mathematical Modeling of Xylose Fermentation, A.K. Hilaly, M.N. Karim, I. C. Linden and S. Lastick A New Culture Medium for Carbon-limited Growth of Bacillus thuringiensis, W. -M. Liu and R.K. Bajpai Determination of Sugars and Sugar Alcohols by High Performance Ion Chromatography, T. J. Paskach, H.-P. Lieker, P.J. Reilly, and K. Thielecke Characterization of Poly-Asp Tailed B-Galactosidase, M.Q. Niederauer, C.E. Glatz, l.A. Suominen, C.F. Ford, and M.A. Rougvie Computation of Conformations and Energies of cr-Glucosyl Disaccharides, Jing Zepg, Michael K. Dowd, and Peter J. Reilly Pentachlorophenol Interactions with Soil, Shein-Ming Wei, Shankha K. Banerji, and Rakesh K. Bajpai Oxygen Transfer to Viscous Liquid Media in Three-Phase Fluidized Beds of Floating Bubble Freakers, Y. Kang, L.T. Fan, B.T. Min and S.D. Kim Studies on the Invitro Development of Chick Embryo, A. Venkatraman and T. Panda The Evolution of a Silicone Based Phase-Separated Gravity-Independent Bioreactor, Peter E. Villeneuve and Eric H. Dunlop Biodegradation of Diethyl Phthalate, Guorong Zhang, Kenneth F. Reardon and Vincent G. Murphy Microcosm Treatability of Soil Contaminated with Petroleum Hydrocarbons, P. Tuitemwong, S. Dhawan, B.M. Sly, L.E. Erickson and J.R. Schlup

Hydraulic Performance of the Denitrification Bioreactor

Denitrification bioreactors, also known as woodchip bioreactors, are a new strategy for improving drainage water quality before these flows arrive at local streams, rivers, and lakes. A bioreactor is an excavated, woodchip-filled pit that is capable of supporting native microbes that convert nitrate in the drainage water to nitrogen gas. The idea of these edgeof-field treatment systems is still relatively new, meaning it is important for investigations to be made into how to design these “pits” and to determine how drainage water moves through the woodchips. Because the bioreactor at the ISU Northeast Research Farm (NERF) is one of the best monitored bioreactor sites in the state, it provided an ideal location to not only monitor bioreactor nitrate-reduction performance, but also to investigate design hydraulics.

Nitrification rates in the NW Mediterranean Sea

During spring, ammonium oxidation and nitrite oxidation rates were measured in the NW basin of the Mediterranean Sea, from mesotrophic sites (Ligurian Sea and Gulf of Lions) to oligotrophic sites (Balearic Islands). Nitrification rates (average values for 37 measurements) ranged from 72 to 144 nmol of N oxidised/l/d, except in the Rhône River plume area where the rates increased to 264-504 nmol/l/d because of the riverine inputs of nitrogen. Maximal rates were located around the peak of nitrite within the nitracline at about 40 to 60 m and just above the phosphacline. At 1 station, relatively high values of nitrification (50 to 130 nmol/l/d) were also measured deep in the water column (240 m). Day-to-day variations were measured demonstrating the response within a few hours to hydrological stress (wind-induced mixing of the water column) and showing the role of hydrological characteristics on the distribution of nitrification rates. Because of the homogenous temperature (13°C) in the Mediterranean Sea, the spatial (geographical and vertical) fluctuations of nitrifying rates were linked to the presence of substrate due to mineralisation processes and/or Rhône River inputs. We estimate the contribution of nitrate produced by nitrification to the N demand of phytoplankton to range from 16% at mesotrophic to 61% at oligotrophic stations.

CPI, d13C, Corg and diploptene from ODP Hole 169-1034B varved sediments

Saanich Inlet has been a highly productive fjord since the last glaciation. During ODP Leg 169S, nearly 70 m of Holocene sediments were recovered from Hole 1034 at the center of the inlet. The younger sediments are laminated, anaerobic, and rich in organic material (1-2.5 wt.% Corg), whereas the older sediments below 70 mbsf are non-laminated, aerobic, with glacio-marine characteristics and have a significantly lower organic matter content. This difference is also reflected in the changes of interstitial fluids, and in biomarker compositions and their carbon isotope signals. The bacterially-derived hopanoid 17alpha(H),21beta(H)-hop-22(29)-ene (diploptene) occurs in Saanich Inlet sediments throughout the Holocene but is not present in Pleistocene glacio-marine sediments. Its concentration increases after ~6000 years BP up to present time to about 70 µg/g Corg, whereas terrigenous biomarkers such as the n-alkane C31 are low throughout the Holocene (<51 µg/g Corg) and even slightly decrease to 36 µg/g Corg at the most recent time. The increasing concentrations of diploptene in sediments younger than ~6000 years BP separate a recent period of higher primary productivity, stronger anoxic bottom waters, and higher bacterial activity from an older period with lesser activity, heretofore undifferentiated. Carbon isotopic compositions of diploptene in the Holocene are between ~31.5 and ~39.6 per mil PDB after ~6000 years BP. These differences in the carbon isotopic record of diploptene probably reflect changes in microbial community structure of bacteria living at the oxic-anoxic interface of the overlying water column. The heavier isotope values are consistent with the activity of nitrifying bacteria and the lighter isotope values with that of aerobic methanotrophic bacteria. Therefore, intermediate delta13C values probably represent mixtures between the populations. In contrast, carbon isotopic compositions of n-C31 are roughly constant at ~31.4 ± 1.1 per mil PDB throughout the Holocene, indicating a uniform input from cuticular waxes of higher plants. Prior to ~6000 years BP, diploptene enriched in 13C of up to -26.3 per mil PDB is indicative of cyanobacteria living in the photic zone and suggests a period of lower primary productivity, more oxygenated bottom waters, and hence lower bacterial activity during the earliest Holocene.

Geochemical analysis of sediments and interstitial water in sediment cores from the North-West African continental margin and from the Central Pacific

A. Continental slope sediments off Spanish-Sahara and Senegal contain up to 4% organic carbon and up to 0.4% total nitrogen. The highest concentrations were found in sediments from water depths between 1000 and 2000 m. The regional and vertical distribution of organic matter differs significantly. Off Spanish-Sahara the organic matter content of sediment deposited during glacial times (Wuerm, Late Riss) is high whereas sediments deposited during interglacial times (Recent, Eem) are low in organic matter. Opposite distribution was found in sediments off Senegal. The sediments contain 30 to 130 ppm of fixed nitrogen. In most sediments this corresponds to 2-8 % of the total nitrogen. Only in sediments deposited during interglacial times off Spanish-Sahara up to 20 % of the total nitrogen is contained as inorganically bound nitrogen. Positive correlations of the fixed nitrogen concentrations to the amounts of clay, alumina, and potassium suggest that it is primarily fixed to illites. The amino acid nitrogen and hexosamine nitrogen account for 17 to 26 % and 1.3 to 2.4 %, respectively of the total nitrogen content of the sediments. The concentrations vary between 200 and 850 ppm amino acid nitrogen and 20 to 70 ppm hexosamine nitrogen, both parallel the fluctiations of organic matter in the sediment. Fulvic acids, humic acids, and the total organic matter of the sediments may be clearly differentiated from one another and their amino acid and hexosamine contents and their amino acid composition: a) Fulvic acids contain only half as much amino acids as humic acids b) The molar amino acid/hexosamine ratios of the fulvic acids are half those of the humic acids and the total organic matter of the sediment c) The amino acid spectra of fulvic acids are characterized by an enrichment of aspartic acid, alanine, and methionine sulfoxide and a depletion of glycine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, and arginine compared to the spectra of the humic acids and those of the total organic matter fraction of the sediment. d) The amino acid spectra of the humic acids and those of the total organic matter fraction of the sediments are about the same with the exception that arginine is clearly enriched in the total organic matter. In general, as indicated by the amino compounds humic acids resemble closer the total organic matter composition than the low molecular fulvic acids do. This supports the general idea that during the course of diagenesis in reducing sediments organic matter stabilizes from a fulvic-like structure to humic-like structure and finally to kerogen. The decomposition rates of single aminio acids differ significantly from one another. Generally amino acids which are preferentially contained in humic acids and the total organic matter fraction show a smaller loss with time than those preferably well documented in case of the basic amino acids lysine and arginine which- although thermally unstable- are the most stable amino acids in the sediments. A favoured incorporation of these compounds into high molecular substances as well as into clay minerals may explain their relatively high "stability" in the sediment. The nitrogen loss from the sediments due to the activity of sulphate-reducing bacteria amounts to 20-40 % of the total organic nitrogen now present. At least 40 % of the organic nitrogen which is liberated by sulphate-reducing bacteria can be explained ny decomposition of amino acids alone. B. Deep-sea sediments from the Central Pacific The deep-seas sediments contain 1 to 2 orders of magnitude less organic matter than the continental slope sediments off NW Africa, i.e. 0.04 to 0.3 % organic carbon. The fixed nitrogen content of the deep-sea sediments ranges from 60 to 270 ppm or from 20 to 45 % of the total nitrogen content. While ammonia is the prevailing inorganic nitrogen compound in anoxic pore waters, nitrate predominates in the oxic environment of the deep-sea sediments. Near the sediment/water interface interstital nitrate concentrations of around 30 µg-at. N/l were recorded. These generally increase with sediment depth by 10 to 15 µg-at. NO3- N/l. This suggests the presence of free oxygen and the activity of nitrifying bacteria in the interstitial waters. The ammonia content of the interstitial water of the oxic deep-sea sediments ranges from 2 to 60 µg-at. N/l and thus is several orders of magnitude less than in anoxic sediments. In contrast to recorded nitrate gradients towards the sediments/water interface, there are no ammonia concentration gradients. However, ammonia concentrations appear to be characteristic for certain regional areas. It is suggested that this regional differentiation is caused by ion exchange reactions involving potassium and ammonium ions rather than by different decomposition rates of organic matter. C. C/N ratios All estimated C/N ratios of surface sediments vary between 3 and 9 in the deep-sea and the continental margin, respectively. Whereas the C/N ratios generally increase with depth in the sediment cores off NW Africa they decrease in the deep-sea cores. The lowest values of around 1.3 were found in the deeper sections of the deep-sea cores, the highest of around 10 in the sediments off NW Africa. The wide range of the C/N ratios as well as their opposite behaviour with increasing sediment depth in both the deep-sea and continental margin sediment cores, can be attributed mainly to the combination of the following three factors: 1. Inorganic and organic substances bound within the latticed of clay minerals tend to decrease the C/N ratios. 2. Organic matter not protected by absorption on the clay minerals tends to increase C/N ratios 3. Diagenetic alteration of organic matter by micro-organisms tends to increase C/N ratios through preferential loss of nitrogen The diagenetic changes of the microbially decomposable organic matter results in both oxic and anoxic environments in a preferential loss of nitrogen and hence in higher C/N ratios of the organic fraction. This holds true for most of the continental margin sediments off NW Africa which contain relatively high amounts of organic matter so that factors 2 and 3 predominate there. The relative low C/N ratios of the sediments deposited during interglacial times off Spanish-Sahara, which are low in organic carbon, show the increasing influence of factor 1 - the nitrogen-rich organic substances bound to clay minerals. In the deep-sea sediments from the Central Pacific this factor completely predominates so that the C/N rations of the sediments approach that of the substance absorbed to clay minerals with decreasing organic matter content. In the deeper core sections the unprotected organic matter has been completely destroyed so that the C/N ratios of the total sediments eventually fall into the same range as those of the pure clay mineral fraction.

Impact of elevated levels of CO2 on animal mediated ecosystem function: The modification of sediment nutrient fluxes by burrowing urchins

A mesocosm experiment was conducted to quantify the relationships between the presence and body size of two burrowing heart urchins (Brissopsis lyrifera and Echinocardium cordatum) and rates of sediment nutrient flux. Furthermore, the impact of seawater acidification on these relationships was determined during this 40-day exposure experiment. Using carbon dioxide (CO2) gas, seawater was acidified to pHNBS 7.6, 7.2 or 6.8. Control treatments were maintained in natural seawater (pH = 8.0). Under normocapnic conditions, burrowing urchins were seen to reduce the sediment uptake of nitrite or nitrate whilst enhancing the release of silicate and phosphate. In acidified (hypercapnic) treatments, the biological control of biogeochemical cycles by urchins was significantly affected, probably through the combined impacts of high CO2 on nitrifying bacteria, benthic algae and urchin behaviour. This study highlights the importance of considering biological interactions when predicting the consequences of seawater acidification on ecosystem function.

La reutilización de efluentes depurados en España: retrospectiva, desarrollo del marco normativo, estudio de las tecnologías de regeneración frente a los biorreactores de membrana y sus costes en función del uso

La reutilización de efluentes depurados siempre ha sido una opción en lugares con déficit coyuntural o estructural de recursos hídricos, se haya o no procedido a la regulación y planificación de esta práctica. La necesidad se crea a partir de las demandas de una zona, normalmente riego agrícola, que ven un mejor desarrollo de su actividad por contar con este recurso. España es el país de la UE que más caudal reutiliza, y está dentro de los diez primeros a nivel mundial. La regulación de esta práctica por el RD 1620/2007, ayudó a incorporar la reutilización de efluentes depurados a la planificación hidrológica como parte de los programas de medidas, con objeto de mitigar presiones, como son las extracciones de agua superficial y subterránea, o mejoras medioambientales evitando un vertido. El objeto de este trabajo es conocer la situación de la reutilización de efluentes depurados en España, los diferentes escenarios y planteamientos de esta actividad, el desarrollo del marco normativo y su aplicabilidad, junto a los tratamientos que permiten alcanzar los límites de calidad establecidos en la normativa vigente, en función de los distintos usos. Además, se aporta un análisis de costes de las distintas unidades de tratamiento y tipologías de líneas de regeneración, tanto de las utilizadas después de un tratamiento secundario como de otras opciones de depuración, como son los biorreactores de membrana (MBRs). Para el desarrollo de estos objetivos, en primer lugar, se aborda el conocimiento de la situación de la reutilización en España a través de una base de datos diseñada para cubrir todos los aspectos de esta actividad: datos de la estación depuradora de aguas residuales (EDAR), de la estación regeneradora (ERA), caudales depurados, reutilizados, volúmenes utilizados y ubicación de los distintos usos, tipos de líneas de tratamiento, calidades del agua reutilizada, etc. Las principales fuentes de información son las Confederaciones Hidrográficas (CCHH) a través de las concesiones de uso del agua depurada, las entidades de saneamiento y depuración de las distintas comunidades autónomas (CCAA), ayuntamientos, Planes Hidrológicos de Cuenca (PHC) y visitas a las zonas más emblemáticas. Además, se revisan planes y programas con el fin de realizar una retrospectiva de cómo se ha ido consolidando y desarrollando esta práctica en las distintas zonas de la geografía española. Se han inventariado 322 sistemas de reutilización y 216 tratamientos de regeneración siendo el más extendido la filtración mediante filtro arena seguido de una desinfección mediante hipoclorito, aunque este tratamiento se ha ido sustituyendo por un físico-químico con decantación lamelar, filtro de arena y radiación ultravioleta, tratamiento de regeneración convencional (TRC), y otros tratamientos que pueden incluir membranas, tratamientos de regeneración avanzados (TRA), con dosificación de hipoclorito como desinfección residual, para adaptarse al actual marco normativo. El uso más extendido es el agrícola con el 70% del caudal total reutilizado, estimado en 408 hm3, aunque la capacidad de los tratamientos de regeneración esperada para 2015, tras el Plan Nacional de Reutilización de Aguas (PNRA), es tres veces superior. Respecto al desarrollo normativo, en las zonas donde la reutilización ha sido pionera, las administraciones competentes han ido desarrollando diferentes recomendaciones de calidad y manejo de este tipo de agua. El uso agrícola, y en zonas turísticas, el riego de campos de golf, fueron los dos primeros usos que tuvieron algún tipo de recomendación incluso reglamentación. Esta situación inicial, sin una normativa a nivel estatal ni recomendaciones europeas, creó cierta incertidumbre en el avance de la reutilización tanto a nivel de concesiones como de planificación. En la actualidad sigue sin existir una normativa internacional para la reutilización y regeneración de efluentes depurados. Las recomendaciones de referencia a nivel mundial, y en concreto para el uso agrícola, son las de la OMS (Organización Mundial de la Salud) publicadas 1989, con sus posteriores revisiones y ampliaciones (OMS, 2006). Esta norma combina tratamientos básicos de depuración y unas buenas prácticas basadas en diferentes niveles de protección para evitar problemas sanitarios. Otra normativa que ha sido referencia en el desarrollo del marco normativo en países donde se realiza esta práctica, son las recomendaciones dadas por la Agencia Medioambiente Estadunidense (USEPA, 2012) o las publicadas por el Estado de California (Título 22, 2001). Estas normas establecen unos indicadores y valores máximos dónde el tratamiento de regeneración es el responsable de la calidad final en función del uso. Durante 2015, la ISO trabajaba en un documento para el uso urbano donde se muestra tanto los posibles parámetros que habría que controlar como la manera de actuar para evitar posibles riesgos. Por otro lado, la Comisión Europea (CE) viene impulsando desde el 2014 la reutilización de aguas depuradas dentro del marco de la Estrategia Común de Implantación de la Directiva Marco del Agua, y fundamentalmente a través del grupo de trabajo de “Programas de medidas”. Para el desarrollo de esta iniciativa se está planteando sacar para 2016 una guía de recomendaciones que podría venir a completar el marco normativo de los distintos Estados Miembros (EM). El Real Decreto 1620/2007, donde se establece el marco jurídico de la reutilización de efluentes depurados, tiende más a la filosofía implantada por la USEPA, aunque la UE parece más partidaria de una gestión del riesgo, donde se establecen unos niveles de tolerancia y unos puntos de control en función de las condiciones socioeconómicas de los distintos Estados, sin entrar a concretar indicadores, valores máximos o tratamientos. Sin embargo, en la normativa estadounidense se indican una serie de tratamientos de regeneración, mientras que, en la española, se hacen recomendaciones a este respecto en una Guía sin validez legal. Por tanto, queda sin regular los procesos para alcanzar estos estándares de calidad, pudiendo ser éstos no apropiados para esta práctica. Es el caso de la desinfección donde el uso de hipoclorito puede generar subproductos indeseables. En la Guía de recomendaciones para la aplicación del RD, publicada por el Ministerio de Agricultura y Medioambiente (MAGRAMA) en 2010, se aclaran cuestiones frecuentes sobre la aplicación del RD, prescripciones técnicas básicas para los sistemas de reutilización, y buenas prácticas en función del uso. Aun así, el RD sigue teniendo deficiencias en su aplicación siendo necesaria una revisión de la misma, como en las frecuencias de muestreo incluso la omisión de algunos parámetros como huevos de nematodos que se ha demostrado ser inexistentes tras un tratamiento de regeneración convencional. En este sentido, existe una tendencia a nivel mundial a reutilizar las aguas con fines de abastecimiento, incluir indicadores de presencia de virus o protozoos, o incluir ciertas tecnologías como las membranas u oxidaciones avanzadas para afrontar temas como los contaminantes emergentes. Otro de los objetivos de este trabajo es el estudio de tipologías de tratamiento en función de los usos establecidos en el RD 1620/2007 y sus costes asociados, siendo base de lo establecido a este respecto en la Guía y PNRA anteriormente indicados. Las tipologías de tratamiento propuestas se dividen en líneas con capacidad de desalar y las que no cuentan con una unidad de desalación de aguas salobres de ósmosis inversa o electrodiálisis reversible. Se realiza esta división al tener actuaciones en zonas costeras donde el agua de mar entra en los colectores, adquiriendo el agua residual un contenido en sales que es limitante en algunos usos. Para desarrollar este objetivo se han estudiado las unidades de tratamiento más implantadas en ERAs españolas en cuanto a fiabilidad para conseguir determinada calidad y coste, tanto de implantación como de explotación. El TRC, tiene un coste de implantación de 28 a 48 €.m-3.d y de explotación de 0,06 a 0,09 €. m-3, mientras que, si se precisara desalar, este coste se multiplica por diez en la implantación y por cinco en la explotación. En caso de los usos que requieren de TRA, como los domiciliarios o algunos industriales, los costes serían de 185 a 398 €.m-3.d en implantación y de 0,14 a 0,20 €.m-3 en explotación. En la selección de tecnologías de regeneración, la capacidad del tratamiento en relación al coste es un indicador fundamental. Este trabajo aporta curvas de tendencia coste-capacidad que sirven de herramienta de selección frente a otros tratamientos de regeneración de reciente implantación como son los MBR, u otros como la desalación de agua de mar o los trasvases entre cuencas dentro de la planificación hidrológica. En España, el aumento de las necesidades de agua de alta calidad en zonas con recursos escasos, aumento de zonas sensibles como puntos de captación para potables, zonas de baño o zonas de producción piscícola, y en ocasiones, el escaso terreno disponible para la implantación de nuevas plantas depuradoras (EDARs), han convertido a los MBRs, en una opción dentro del marco de la reutilización de aguas depuradas. En este trabajo, se estudia esta tecnología frente a los TRC y TRA, aportando igualmente curvas de tendencia coste-capacidad, e identificando cuando esta opción tecnológica puede ser más competitiva frente a los otros tratamientos de regeneración. Un MBR es un tratamiento de depuración de fangos activos donde el decantador secundario es sustituido por un sistema de membranas de UF o MF. La calidad del efluente, por tanto, es la misma que el de una EDAR seguida de un TRA. Los MBRs aseguran una calidad del efluente para todos los usos establecidos en el RD, incluso dan un efluente que permite ser directamente tratado por las unidades de desalación de OI o EDR. La implantación de esta tecnología en España ha tenido un crecimiento exponencial, pasando de 13 instalaciones de menos de 5.000 m3. d-1 en el 2006, a más de 55 instalaciones en operación o construcción a finales del 2014, seis de ellas con capacidades por encima de los 15.000 m3. d-1. Los sistemas de filtración en los MBR son los que marcan la operación y diseño de este tipo de instalaciones. El sistema más implantado en España es de membrana de fibra hueca (MFH), sobre todo para instalaciones de gran capacidad, destacando Zenon que cuenta con el 57% de la capacidad total instalada. La segunda casa comercial con mayor número de plantas es Kubota, con membranas de configuración placa plana (MPP), que cuenta con el 30 % de la capacidad total instalada. Existen otras casas comerciales implantadas en MBR españoles como son Toray, Huber, Koch o Microdym. En este documento se realiza la descripción de los sistemas de filtración de todas estas casas comerciales, aportando información de sus características, parámetros de diseño y operación más relevantes. El estudio de 14 MBRs ha posibilitado realizar otro de los objetivos de este trabajo, la estimación de los costes de explotación e implantación de este tipo de sistemas frente a otras alternativas de tratamiento de regeneración. En este estudio han participado activamente ACA y ESAMUR, entidades públicas de saneamiento y depuración de Cataluña y Murcia respectivamente, que cuentan con una amplia experiencia en la explotación de este tipo de sistemas. Este documento expone los problemas de operación encontrados y sus posibles soluciones, tanto en la explotación como en los futuros diseños de este tipo de plantas. El trabajo concluye que los MBRs son una opción más para la reutilización de efluentes depurados, siendo ventajosos en costes, tanto de implantación como de explotación, respecto a EDARs seguidas de TRA en capacidades por encima de los 10.000 m3.d-1. ABSTRACT The reuse of treated effluent has always been an option in places where a situational or structural water deficit exists, whether regulatory and/or planning efforts are completed or not. The need arises from the demand of a sector, commonly agricultural irrigation, which benefits of this new resource. Within the EU, Spain is ahead in the annual volume of reclaimed water, and is among the top ten countries at a global scale. The regulation of this practice through the Royal Decree 1620/2007 has helped to incorporate the water reuse to the hydrological plans as a part of the programme of measures to mitigate pressures such as surface or ground water extraction, or environmental improvements preventing discharges. The object of this study is to gain an overview of the state of the water reuse in Spain, the different scenarios and approaches to this activity, the development of the legal framework and its enforceability, together with the treatments that achieve the quality levels according to the current law, broken down by applications. Additionally, a cost analysis of technologies and regeneration treatment lines for water reclamation is performed, whereas the regeneration treatment is located after a wastewater treatment or other options such as membrane bioreactors (MBR). To develop the abovementioned objectives, the state of water reuse in Spain is studied by means of a database designed to encompass all aspects of the activity: data from the wastewater treatment plants (WWTP), from the water reclamation plants (WRP), the use of reclaimed water, treated water and reclaimed water annual volumes and qualities, facilities and applications, geographic references, technologies, regeneration treatment lines, etc. The main data providers are the River Basin authorities, through the concession or authorization for water reuse, (sanitary and wastewater treatment managers from the territorial governments, local governments, Hydrological Plans of the River Basins and field visits to the main water reuse systems. Additionally, a review of different plans and programmes on wastewater treatment or water reuse is done, aiming to put the development and consolidation process of this activity in the different regions of Spain in perspective. An inventory of 322 reuse systems and 216 regeneration treatments has been gathered on the database, where the most extended regeneration treatment line was sand filtration followed by hypochlorite disinfection, even though recently it is being replaced by physical–chemical treatment with a lamella settling system, depth sand filtration, and a disinfection with ultraviolet radiation and hypochlorite as residual disinfectant, named conventional regeneration treatment (CRT), and another treatment that may include a membrane process, named advanced regeneration treatment (ART), to adapt to legal requirements. Agricultural use is the most extended, accumulating 70% of the reclaimed demand, estimated at 408 hm3, even though the expected total capacity of WRPs for 2015, after the implementation of the National Water Reuse Plan (NWRP) is three times higher. According to the development of the water reuse legal framework, there were pioneer areas where competent authorities developed different quality and use recommendations for this new resource. Agricultural use and golf course irrigation in touristic areas were the first two uses with recommendations and even legislation. The initial lack of common legislation for water reuse at a national or European level created some doubts which affected the implementation of water reuse, both from a planning and a licensing point of view. Currently there is still a lack of common international legislation regarding water reuse, technologies and applications. Regarding agricultural use, the model recommendations at a global scale are those set by the World Health Organization published in 1989, and subsequent reviews and extensions about risk prevention (WHO, 2006). These documents combine wastewater treatments with basic regeneration treatments reinforced by good practices based on different levels of protection to avoid deleterious health effects. Another relevant legal reference for this practices has been the Environmental Protection Agency of the US (USEPA, 2012), or those published by the State of California (Title 22, 2001). These establish indicator targets and maximum thresholds where regeneration treatment lines are responsible for the final quality according to the different uses. During 2015, the ISO has worked on a document aimed at urban use, where the possible parameters to be monitored together with risk prevention have been studied. On the other hand, the European Commission has been promoting the reuse of treated effluents within the Common Implementation Strategy of the Water Framework Directive, mainly through the work of the Programme of Measures Working Group. Within this context, the publication of a recommendation guide during 2016 is intended, as a useful tool to fill in the legal gaps of different Member States on the matter. The Royal Decree 1620/2007, where the water reuse regulation is set, resembles the principles of the USEPA more closely, even though the EU shows a tendency to prioritize risk assessment by establishing tolerance levels and thresholds according to socioeconomic conditions of the different countries, without going into details of indicators, maximum thresholds or treatments. In contrast, in the US law, regeneration treatments are indicated, while in the Spanish legislation, the only recommendations to this respect are compiled in a non-compulsory guide. Therefore, there is no regulation on the different treatment lines used to achieve the required quality standards, giving room for inappropriate practices in this respect. This is the case of disinfection, where the use of hypochlorite may produce harmful byproducts. In the recommendation Guide for the application of the Royal Decree (RD), published by the Ministry of Agriculture and Environment (MAGRAMA) in 2010, clarifications of typical issues that may arise from the application of the RD are given, as well as basic technical parameters to consider in reuse setups, or good practices according to final use. Even so, the RD still presents difficulties in its application and requires a review on issues such as the sampling frequency of current quality parameters or even the omission of nematode eggs indicator, which have been shown to be absent after CRT. In this regard, there is a global tendency to employ water reuse for drinking water, including indicators for the presence of viruses and protozoans, or to include certain technologies such as membranes or advanced oxidation processes to tackle problems like emerging pollutants. Another of the objectives of this study is to provide different regeneration treatment lines to meet the quality requirements established in the RD 1620/2007 broken down by applications, and to estimate establishment and operational costs. This proposal has been based on what is established in the above mentioned Guide and NWRP. The proposed treatment typologies are divided in treatment trains with desalination, like reverse osmosis or reversible electrodialisis, and those that lack this treatment for brackish water. This separation is done due to coastal facilities, where sea water may permeate the collecting pipes, rising salt contents in the wastewater, hence limiting certain uses. To develop this objective a study of the most common treatment units set up in Spanish WRPs is conducted in terms of treatment train reliability to obtain an acceptable relationship between the required quality and the capital and operational costs. The CRT has an establishment cost of 28 to 48 €.m-3.d and an operation cost of 0.06 to 0.09 €.m-3, while, if desalination was required, these costs would increase tenfold for implementation and fivefold for operation. In the cases of uses that require ART, such as residential or certain industrial uses, the costs would be of 185 to 398 €.m-3.d for implementation and of 0.14 to 0.20 €.m-3 for operation. When selecting regeneration treatment lines, the relation between treatment capacity and cost is a paramount indicator. This project provides cost-capacity models for regeneration treatment trains. These may serve as a tool when selecting between different options to fulfill water demands with MBR facilities, or others such as sea water desalination plants or inter-basin water transfer into a water planning framework. In Spain, the requirement for high quality water in areas with low resource availability, the increasing number of sensitive zones, such as drinking water extraction, recreational bathing areas, fish protected areas and the lack of available land to set up new WWTPs, have turned MBRs into a suitable option for water reuse. In this work this technology is analyzed in contrast to CRT and ART, providing cost-capacity models, and identifying when and where this treatment option may outcompete other regeneration treatments. An MBR is an activated sludge treatment where the secondary settling is substituted by a membrane system of UF or MF. The quality of the effluent is, therefore, comparable to that of a WWTP followed by an ART. MBRs ensure a sufficient quality level for the requirements of the different uses established in the RD, even producing an effluent that can be directly treated in OI or EDR processes. The implementation of this technology in Spain has grown exponentially, growing from 13 facilities with less than 5000 m3.d-1 in 2006 to above 55 facilities operating by the end of 2014, 6 of them with capacities over 15000 m3.d-1. The membrane filtration systems for MBR are the ones that set the pace of operation and design of this type of facilities. The most widespread system in Spain is the hollow fiber membrane configuration, especially on high flow capacities, being Zenon commercial technology, which mounts up to 57% of the total installed capacity, the main contributor. The next commercial technology according to plant number is Kubota, which uses flat sheet membrane configuration, which mounts up to 30% of the total installed capacity. Other commercial technologies exist within the Spanish MBR context, such as Toray, Huber, Koch or Microdym. In this document an analysis of all of these membrane filtration systems is done, providing information about their characteristics and relevant design and operation parameters. The study of 14 full scale running MBRs has enabled to pursue another of the objectives of this work: the estimation of the implementation and operation costs of this type of systems in contrast to other regeneration alternatives. Active participation of ACA and ESAMUR, public wastewater treatment and reuse entities of Cataluña and Murcia respectively, has helped attaining this objective. A number of typical operative problems and their possible solutions are discussed, both for operation and plant design purposes. The conclusion of this study is that MBRs are another option to consider for water reuse, being advantageous in terms of both implementation and operational costs, when compared with WWTPs followed by ART, when considering flow capacities above 10000 m3.d-1.

Diseño y evaluación de un procedimiento on-line para la biorremediación de aguas radiactivas en centrales nucleares

Recientemente se ha demostrado la existencia de microorganismos en las piscinas de almacenamiento de combustible nuclear gastado en las centrales nucleares utilizando técnicas convencionales de cultivo en el laboratorio. Estudios posteriores han puesto de manifiesto que los microorganismos presentes eran capaces de colonizar las paredes de acero inoxidable de las piscinas formando biopelículas. Adicionalmente se ha observado la capacidad de estas biopelículas de retener radionúclidos, lo que hace pensar en la posibilidad de utilizarlas en la descontaminación de las aguas radiactivas de las piscinas. En la presente tesis se plantea conocer más profundamente la biodiversidad microbiana de las biopelículas utilizando técnicas de biología molecular como la clonación, además de desarrollar un sistema de descontaminación a escala piloto con el objetivo de valorar si el proceso podría resultar escalable a nivel industrial. Para ello se diseñaron y fabricaron dos biorreactores en acero inoxidable compatibles con las condiciones específicas de seguridad sísmica y protección frente a la radiación en la zona controlada de una central nuclear. Los biorreactores se instalaron en la Central Nuclear de Cofrentes (Valencia) en las proximidades de las piscinas de almacenamiento de combustible nuclear gastado y precediendo a las resinas de intercambio iónico, de forma que reciben el agua de las piscinas permitiendo el análisis in situ de la radiación eliminada del agua de las mismas. Se conectó una lámpara de luz ultravioleta a uno de los biorreactores para poder comparar el desarrollo de bipelículas y la retención de radiactividad en ambas condiciones. En estos biorreactores se introdujeron ovillos de acero inoxidable y de titanio que se extrajeron a diversos tiempos, hasta 635 días para los ovillos de acero inoxidable y hasta 309 días para los ovillos de titanio. Se analizaron las biopelículas desarrolladas sobre los ovillos por microscopía electrónica de barrido y por microscopía de epifluorescencia. Se extrajo el ADN de las biopelículas y, tras su clonación, se identificaron los microorganismos por técnicas independientes de cultivo. Asimismo se determinó por espectrometría gamma la capacidad de las biopelículas para retener radionúclidos. Los microorganismos radiorresistentes identificados pertenecen a los grupos filogenéticos Alpha-proteobacteria, Gamma-proteobacteria, Actinobacteria, Deinococcus-Thermus y Bacteroidetes. Las secuencias de estos microorganismos se han depositado en el GenBank con los números de acceso KR817260-KR817405. Se ha observado una distribución porcentual ligeramente diferente en relación con el tipo de biorreactor. Las biopelículas han retenido fundamentalmente radionúclidos de activación. La suma de Co-60 y Mn-54 ha llegado en ocasiones al 97%. Otros radionúclidos retenidos han sido Cr-51, Co-58, Fe-59, Zn-65 y Zr-95. Se sugiere un mecanismo del proceso de retención de radionúclidos relacionado con el tiempo de formación y desaparición de las biopelículas. Se ha valorado que el proceso escalable puede ser económicamente rentable. ABSTRACT The existence of microorganisms in spent nuclear fuel pools has been demonstrated recently in nuclear power plants by using conventional microbial techniques. Subsequent studies have revealed that those microorganisms were able to colonize the stainless steel pool walls forming biofilms. Additionally, it has been observed the ability of these biofilms to retain radionuclides, which suggests the possibility of using them for radioactive water decontamination purposes. This thesis presents deeper knowledge of microbial biofilms biodiversity by using molecular biology techniques such as cloning, and develops a decontamination system on a pilot scale, in order to assess whether the process could be scalable to an industrial level. Aiming to demonstrate this was feasible, two stainless steel bioreactors were designed and manufactured, both were compatible with seismic and radiation protection standards in the controlled zone of a nuclear plant. These bioreactors were installed in the Cofrentes Nuclear Power Plant (Valencia) next to the spent nuclear fuel pools and preceding (upstream) ion exchange resins. This configuration allowed the bioreactors to receive water directly from the pools allowing in situ analysis of radiation removal. One ultraviolet lamp was connected to one of the bioreactors to compare biofilms development and radioactivity retention in both conditions. Stainless steel and titanium balls were introduced into these bioreactors and were removed after different time periods, up to 635 days for stainless steel balls and up to 309 days for titanium. Biofilms developed on the balls were analyzed by scanning electron microscopy and epifluorescence microscopy. DNA was extracted from the biofilms, was cloned and then the microorganisms were identified by independent culture techniques. Biofilms ability to retain radionuclides was also determined by gamma spectrometry. The identified radioresistant organisms belong to the phylogenetic groups Alphaproteobacteria, Gamma-proteobacteria, Actinobacteria, Deinococcus-Thermus and Bacteroidetes. The sequences of these microorganisms have been deposited in GenBank (access numbers KR817260-KR817405). A different distribution of microorganisms was observed in relation to the type of bioreactor. Biofilms have essentially retained activation radionuclides. Sometimes the sum of Co-60 and Mn-54 reached 97%. Cr-51, Co-58, Fe-59, Zn-65 and Zr-95 have also been retained. A radionuclide retention process mechanism related to biofilms formation and disappearance time is suggested. It has been assessed that the scalable process can be economically profitable.

Tissue engineering of cartilage in space

Tissue engineering of cartilage, i.e., the in vitro cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth. Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10−4–10−6 g) or Earth (1 g). This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue. Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen. Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior. The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues. These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development.

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1.

The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(II) sepulchrate trichloride] for the electrocatalytically driven omega-hydroxylation of lauric acid. Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 [mRat4A1/mRatOR]L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase. Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor. These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals.

Low-cost disposable ALT electrochemical microsensors for in-vitro hepatotoxic assessment

Liver-on-chip systems are widely seen as having the potential to replace animal testing for long-term liver toxicity assessments. However, such systems necessitate solutions, such as electrochemical microsensors, to provide information about the cells exposed to chemical compounds in a confined space. This study describes the development of microsensors for the detection of alanine-aminotransferase (ALT), an intracellular enzyme found in hepatocytes, for monitoring the viability of in-vitro hepatic cell cultures. The electrochemical sensors were developed by using screen printed electrodes functionalized by drop-casting. These technologies are intended to produce disposable and low-cost sensors that can easily be exchanged once their performance is degraded. The sensors are capable of measuring ALT in a microfluidic environment through the detection of changes in glutamate concentration. The microsensors were found to be stable for more than 60 days and were successfully tested using hepatocellular lysates to assess their capability to quantify ALT activity in a hepatic cell culture. These results open the way to their integration in liver bioreactors to assess hepatocellular toxicity in-vitro.

Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair

INTRODUCTION: Around 80% of people are affected by low back pain at least once in their life, often caused by trauma provoking intervertebral disc (IVD) herniation and/or IVD degeneration. Apart from some promising approaches for nucleus pulposus repair, so far no treatment or repair is available for the outer fibrous tissue, annulus fibrosus (AF). We aimed for sealing and repairing an AF injury in a bovine IVD organ culture model in vitro over 14 days under different loading conditions. For this purpose, a silk fleece composite from Bombyx mori silk was combined with genipin-enhanced fibrin hydrogel [1]. METHODS: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue, followed by cutting out the IVDs [2]. Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described. On the next day, injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35- 55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d. Complex loading was applied by a custom built 2 degree of freedom bioreactor [3]. After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy-proline) were determined. Finally, real-time qPCR of major IVD marker genes was performed. RESULTS: The silk seal closing the injury site could successfully withstand the forces of all three loading conditions with no misplacement over the two weeks’ culture. Nevertheless, disc height of the repaired discs did not significantly differ from the injured group. The disc phenotype could be maintained as demonstrated by biochemical analysis of gene expression, cell activity, DNA-, collagen- and GAG content. The silk itself was evaluated to be highly biocompatible for hMSC, as revealed by cytotoxicity assays. DISCUSSION & CONCLUSIONS: The silk can be considered a highly-elastic and biocompatible material for AF closure and the genipin-enhanced fibrin hydrogel has also good biomechanical properties. However, the cyto-compatibility of genipin seems rather poor and other hydrogels and/or cross-linkers should be looked into. REFERENCES: 1 C.C. Guterl et al. (2014) Characterization of Mechanics and Cytocompatibility of Fibrin Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules, Tissue Eng Part A 2 S.C. Chan, B. Gantenbein-Ritter (2012) Preparation of intact bovine tail intervertebral discs for organ culture, J Vis Exp 3 B Gantenbein et al. (2015) Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy, Curr Stem Cell Res Ther [epub ahead of print] ACKNOWLEDGEMENTS: This project is supported by the Gebert Rüf Stiftung project # GRS-028/13.

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