Producción de un biofertilizante / bioestimulante mediante un proceso biológico / enzimático a partir de subproductos orgánicos: valorización agronómica y ambiental de lodos de depuradora y plumas de matadero.

UNIVERSIDAD DE SEVILLA FACULTAD DE FARMACIA PRODUCCIÓN DE UN BIOFERTILIZANTE / BIOESTIMULANTE MEDIANTE UN PROCESO BIOLÓGICO / ENZIMÁTICO A PARTIR DE SUBPRODUCTOS ORGÁNICOS: VALORIZACIÓN AGRONÓMICA Y AMBIENTAL DE LODOS DE DEPURADORA Y PLUMAS DE MATADERO TESIS DOCTORAL Bruno Rodríguez Morgado Sevilla, 2018 DEPARTAMENTO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR FACULTAD DE FARMACIA Producción de un biofertilizante / bioestimulante mediante un proceso biológico / enzimático a partir de subproductos orgánicos: Valorización agronómica y ambiental de lodos de depuradora y plumas de matadero Memoria de Tesis Doctoral presentada por: Bruno Rodríguez Morgado Para optar al grado de Doctor en Bioquímica. Tesis realizada bajo la dirección de: Dr. Juan Parrado Rubio Dpto. Bioquímica y Biología Molecular, Universidad de Sevilla Dr. Manuel Tejada Moral Dpto. Cristalografía, Mineralogía y Química Agrícola, Universidad de Sevilla DEPARTAMENTO DE CRISTALOGRAFÍA, MINERALOGÍA Y QUÍMICA AGRÍCOLA FACULTAD DE QUÍMICA Producción de un biofertilizante / bioestimulante mediante un proceso biológico / enzimático a partir de subproductos orgánicos: Valorización agronómica y ambiental de lodos de depuradora y plumas de matadero Memoria de Tesis Doctoral presentada por: Bruno Rodríguez Morgado Para optar al grado de Doctor en Bioquímica. Tesis realizada en el programa de doctorado: Recursos Naturales y Medioambiente (R.D. 99/2011) DEPARTAMENTO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR FACULTAD DE FARMACIA La Tesis Doctoral titulada “Producción de un biofertilizante / bioestimulante mediante un proceso biológico / enzimático a partir de subproductos orgánicos: Valorización agronómica y ambiental de lodos de depuradora y plumas de matadero”, realizada por el Licenciado en Bioquímica D. BRUNO RODRÍGUEZ MORGADO, para optar al grado de Doctor en Bioquímica por la Universidad de Sevilla, se presenta bajo la modalidad de “Compendio de Publicaciones” Los Directores de la Tesis Fdo.: Juan Parrado Rubio Fdo.: Manuel Tejada Moral El Doctorando Fdo.: Bruno Rodríguez Morgado Universidad de Sevilla Departamento de Bioquímica y Biología Molecular Facultad de Farmacia C/Profesor García González, 2 41.012 Sevilla (España) Tlf. 954556755 Fax: 954556752 Dr. Juan Parrado Rubio, Catedrático del Departamento de Bioquímica y Biología Molecular de la Facultad de Farmacia, de la Universidad de Sevilla, y Dr. Manuel Tejada Moral, Profesor Titular del Departamento de Cristalografía, Mineralogía y Química Agrícola de la Facultad de Química, de la Universidad de Sevilla, Certifican que: D. Bruno Rodríguez Morgado, Licenciado en Bioquímica por la Facultad de Biología, de la Universidad de Sevilla, ha realizado en Departamento de Bioquímica y Biología Molecular de la Facultad de Farmacia de la Universidad de Sevilla, bajo nuestra dirección, el trabajo de investigación “Producción de un biofertilizante / bioestimulante mediante un proceso biológico / enzimático a partir de subproductos orgánicos: Valorización agronómica y ambiental de lodos de depuradora y plumas de matadero”, y que reúne las condiciones necesarias para ser presentada para optar al grado de Doctor por la Universidad de Sevilla. Fdo.: Dr. Juan Parrado Rubio Fdo.: Dr. Manuel Tejada Moral Índice i Página RESUMEN ......................................................................................... 1 CAPÍTULO 1 .................................................................................... 9 Introducción .................................................................................. 11 1.1. LODOS DE DEPURADORA........................................................ 13 1.1.1. Depuración de aguas residuales .......................................... 13 1.1.2. Estaciones Depuradoras de Aguas Residuales ....................... 13 1.1.3. Línea de aguas ................................................................. 14 1.1.4. Línea de lodos .................................................................. 16 1.1.5. Sistema de lodos activos .................................................... 19 1.1.6. Gestión de los lodos de depuradora ..................................... 20 1.1.7. Tratamientos convencionales de los lodos de depuradora ....... 22 1.2. PLUMAS DE POLLO ................................................................. 26 1.2.1. Origen y funciones ............................................................ 26 1.2.2. Composición ..................................................................... 26 1.2.3. Estructura y tipología de las plumas .................................... 27 1.2.4. Problemática de las plumas ................................................ 28 1.3. PRODUCCIÓN DE BIOFERTILIZANTES Y BIOESTIMULANTES ORGÁNICOS .................................................... 29 1.3.1. Tecnología enzimática........................................................ 30 1.3.1.1. Las enzimas ................................................................. 30 1.3.1.2. Hidrólisis enzimática ..................................................... 31 1.3.1.3. Hidrólisis de proteínas ................................................... 32 1.3.1.4. Proteasas .................................................................... 35 1.3.1.5. Propiedades de los hidrolizados enzimáticos: Biofertilizantes / Bioestimulantes ................................................ 36 1.3.2. Tecnología fermentativa ..................................................... 36 Índice ii 1.3.2.1. Microorganismos .......................................................... 38 1.3.2.2. Género Bacillus y Bacillus licheniformis ........................... 39 1.3.2.3. Fermentaciones ............................................................ 40 1.3.2.4. Producción de proteasas ................................................ 45 Objetivos ...................................................................................... 47 Artículos ....................................................................................... 51 - Proteomic analysis of enzyme production by Bacillus licheniformis using different feather wastes as the sole fermentation media. .......... 53 - Hydrolytic enzymes production by Bacillus licheniformis growth on fermentation media formulated with sewage sludge. .................... 61 - Obtaining biostimulant products for land application from the sewage sludge of small populations. ............................................... 75 - Obtaining edaphic biostimulants/biofertilizers from sewage sludge using fermentative processes. Short-time effects on soil biochemical properties. ........................................................... 81 Resultados y Discusión ................................................................... 89 4.1. Proteomic analysis of enzyme production by Bacillus licheniformis using different feather wastes as the sole fermentation media. ..................................................................... 91 4.2. Hydrolytic enzymes production by Bacillus licheniformis growth on fermentation media formulated with sewage sludge. ......... 97 4.3. Obtaining biostimulant products for land application from the sewage sludge of small populations. ....................................... 100 4.4. Obtaining edaphic biostimulants/biofertilizers from sewage sludge using fermentative processes. Short-time effects on soil biochemical properties. ............................................................... 104 Conclusiones ............................................................................... 107 Bibliografía .................................................................................. 111 CAPÍTULO 2 ................................................................................ 131 Introducción ................................................................................ 133 1.1. FERTILIDAD EDÁFICA .......................................................... 135 1.2. BIOESTIMULANTES .............................................................. 136 Índice iii 1.3. BIOESTIMULACIÓN MICROBIANA EN SUELOS ......................... 137 1.4. TIPOS DE BIOESTIMULANTES ............................................... 137 1.4.1. Bioestimulantes inorgánicos ............................................. 137 1.4.2. Bioestimulantes orgánicos ................................................ 138 1.5. BIOESTIMULANTES ORGÁNICOS ........................................... 138 1.5.1. Bioestimulantes orgánicos no hidrolizados .......................... 138 1.5.2. Bioestimulantes orgánicos hidrolizados .............................. 139 1.6. LODOS DE DEPURADORA Y AGRICULTURA .............................. 140 Objetivos .................................................................................... 143 Artículos ..................................................................................... 147 - Obtaining edaphic biostimulants / biofertilizers from different sewage sludges. Effects on soil biological properties. ...................... 149 - Effects of foliar fertilization of a biostimulant obtained from chicken feathers on maize yield. .................................................. 159 Resultados y Discusión ................................................................. 165 4.1. Obtaining edaphic biostimulants / biofertilizers from different sewage sludges. Effects on soil biological properties. ......... 167 4.2. Effects of foliar fertilization of a biostimulant obtained from chicken feathers on maize yield ............................................ 170 Conclusiones ............................................................................... 175 Bibliografía .................................................................................. 179 CAPÍTULO 3 ................................................................................ 193 Introducción .................................................................................. 195 1.1. CONTAMINACIÓN. SUELOS CONTAMINADOS .......................... 197 1.2. TIPOS y CAUSAS DE CONTAMINACIÓN EN SUELOS ................. 198 1.2.1. Tipos de contaminantes ................................................... 198 1.2.2. Origen de las contaminaciones .......................................... 201 1.3. DINÁMICA DE LOS CONTAMINANTES EN EL SUELO .................. 202 Índice iv 1.4. BIORREMEDIACIÓN DE SUELOS CONTAMINADOS .................... 205 1.5. BIODEGRADACIÓN MICROBIANA DE XENOBIÓTICOS ............... 206 1.6. BIOESTIMULACIÓN MICROBIANA EN SUELOS ......................... 208 1.7. TIPOS DE BIOESTIMULANTES PARA BIORREMEDIACIÓN DE SUELOS CONTAMINADOS ............................................................ 209 1.8. LODOS DE DEPURADORA Y BIORREMEDIACIÓN ...................... 209 Objetivos ...................................................................................... 211 Artículos ....................................................................................... 215 - Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers ........................ 217 - Degradation of chlorpyrifos using different biostimulants / biofertilizers: Effects on soil biochemical properties and microbial community .................................................................. 225 - Behaviour of oxyfluorfen in soils amended with edaphic biostimulants / biofertilizers obtained from sewage sludge and chicken feathers. Effects on soil biological properties ................ 233 Resultados y Discusión ................................................................... 243 4.1. Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers ........................ 245 4.2. Degradation of chlorpyrifos using different biostimulants / biofertilizers: Effects on soil biochemical properties and microbial community .................................................................. 248 4.3. Behaviour of oxyfluorfen in soils amended with edaphic biostimulants / biofertilizers obtained from sewage sludge and chicken feathers. Effects on soil biological properties ...................... 253 Conclusiones ................................................................................. 259 Bibliografía ................................................................................... 263 CONCLUSIONES FINALES ............................................................ 277 RESUMEN Resumen 3 En la sociedad industrializada actual existen infinidad de sub-productos de origen orgánico que tradicionalmente han sido considerados directamente como residuos, o como mucho, se consideran materias de bajo valor añadido. En muchos casos estos sub-productos ocasionan graves problemas medioambientales debido a la falta de una legislación adecuada que obligue a la correcta gestión de estos materiales o a la ausencia de alternativas ecológicamente amigables. Dos de estos sub-productos de origen orgánico constituyen las materias de partida de este trabajo. Por una parte están los lodos de depuradora, que si bien no pueden ser considerados como sub-productos de ninguna industria específica, dados el elevado volumen que se genera de ellos y su origen totalmente antropogénico, pueden ser considerados como un sub-producto de la actividad humana. Dependiendo del tipo de tratamiento que se haya realizado en las aguas residuales de partida, es posible obtener lodos con propiedades y características distintas, lo que aumenta la variabilidad de lodos existentes. Por otra parte están las plumas de ave, originadas en la industria avícola y caracterizadas por estar formadas mayoritariamente por la proteína queratina y grasas en mayor o menor medida. En ambos casos, su habitual gestión consiste en su eliminación por diversos medios, como puede ser la incineración o el almacenamiento en vertederos. Una alternativa a los procesos de eliminación cada vez más instaurada es el reaprovechamiento como materias primas para otros procesos, aunque el valor de los productos obtenidos es relativamente bajo (ya sea económica o cualitativamente). Estos procesos de revalorización son en el caso de los lodos de depuradora el compostaje y la metanización, y en el caso de las plumas la producción de aminoácidos de uso agrícola y en menor medida el compostaje. 1. INTRODUCCIÓN Capítulo 1 Introducción 13 1.1. LODOS DE DEPURADORA 1.1.1. Depuración de aguas residuales La contaminación de las aguas constituye uno de los problemas medioambientales más importantes debido, por una parte, a que los seres humanos y las actividades que estos realizan necesitan de un aporte de agua con una calidad mínima para su correcto desarrollo, y por otro lado, esta contaminación de las aguas afecta a los diversos ecosistemas vinculados a la masa acuífera afectada, deteriorándolos e incluso destruyéndolos. Como respuesta por parte de los gobiernos, se han promulgado diversas leyes, decretos, planes, etc. tanto a nivel nacional como europeo con el objetivo de tratar las aguas residuales generadas por la actividad humana para poder proteger los sistemas ecológicos donde se viertan estas aguas, prevenir peligros para la salud humana y/u obtener un efluente que pueda ser reutilizado (Martín y col., 2006). Una de las primeras acciones tomadas ha sido la construcción y puesta en marcha de numerosas Estaciones Depuradoras de Aguas Residuales (EDAR) por todo el territorio a fin de tratar esas aguas residuales y obtener un efluente que pueda ser vertido al medio ambiente sin riesgos ni para éste ni para el ser humano (Tchobanoglous y Burton, 1995). 1.1.2. Estaciones Depuradoras de Aguas Residuales La entrada de una EDAR está constituida por un flujo de aguas residuales, que pueden ser urbanas o industriales, adaptándose las instalaciones a sus distintas características, mientras que los efluentes son una corriente de agua “depurada” que puede ser vertida al medio o reutilizada y unos sólidos denominados comúnmente lodos o fangos de depuradora. Estos lodos son un subproducto de desecho de las EDAR que deben ser convenientemente procesados para evitar daños a la salud pública o al medio ambiente. Por todo ello, dentro de Capítulo 1 Introducción 14 una EDAR se distinguen una línea de aguas destinada al tratamiento de ésta, y por el otro, una línea de lodos donde se separarán estos de la masa de agua depurada y se tratarán convenientemente (Figura 1). Figura 1: Esquema de una Estación Depuradora de Aguas Residuales (EDAR) convencional. 1.1.3. Línea de aguas La línea de aguas de una EDAR consta de cuatro pasos o procesos básicos destinados a su depuración (Tchobanoglous y Burton, 1995): Pretratamientos: Consiste en el acondicionamiento del agua y la retirada de los sólidos en suspensión más groseros mediante rejas de desbaste, tamices, desarenadores, desengrasadores, etc. Dependiendo del tipo de influente y del destino final de las aguas residuales, estos pretratamientos pueden constituir el único proceso que sufran dichas aguas. Capítulo 1 Introducción 15 Tratamientos primarios: Principalmente consisten en procesos donde se va reducir o eliminar completamente los sólidos en suspensión de las aguas residuales que hayan superado los pretratamientos previos. Algunos de estos tratamientos primarios son la sedimentación o decantación primaria, la flotación o la neutralización. Tratamientos secundarios: Mediante estos tratamientos se busca reducir o eliminar la contaminación orgánica en solución así como aquellos sólidos en suspensión no decantables. Estos tratamientos pueden ser de naturaleza físico-química o bien de tipo biológico. En los tratamientos biológicos se emplean microorganismos que consumen la materia orgánica para dar nuevos microorganismos al tiempo que se favorece la sedimentación de la materia inorgánica. Esto se debe a que esos nuevos microrganismos son más densos que el agua y precipitan, pudiéndose separar entonces del agua. Según las condiciones de oxigenación del proceso se distinguen los tratamientos aerobios (presencia de oxígeno) de los anaerobios (ausencia de oxígeno). Independientemente del si el tratamiento es aerobio o anaerobio, las distintas tecnologías aplicables pueden agruparse en tecnologías intensivas (o convencionales) que requieren de un aporte externo de energía, como son los sistemas de lodos activos, los digestores anaerobios, los reactores secuenciales o de membranas; y tecnologías extensivas (o no convencionales) que requieren de grandes superficies de trabajo como los sistemas de lagunaje, filtros verdes, filtros de turba, etc. También existen tecnologías intermedias como son los lechos bacterianos o los contactores biológicos rotativos. El uso de unas tecnologías u otras dependerá de la disponibilidad de energía de bajo coste y de superficie donde instalarlas. Capítulo 1 Introducción 16 Tratamientos terciarios: Mediante estos tratamientos se busca obtener un efluente de gran calidad con vistas a un fin en particular. Son sistemas muy caros donde se van a retirar los sólidos en suspensión que no se hayan retirado con anterioridad, adsorber diversos contaminantes, eliminar iones perjudiciales, desinfectar las aguas u obtener una corriente de agua relativamente exenta de contaminantes mediante sistemas de membranas. 1.1.4. Línea de lodos Los lodos de depuradora presentan unas características diferentes dependiendo de las aguas residuales de origen y del tratamiento al que se sometan estas en la EDAR. En una estación depuradora convencional con tratamiento biológico se generan dos tipos de lodos:  Lodos primarios: proceden del decantador primario y lo conforman los sólidos precipitados previamente al tratamiento biológico. En su mayor parte se componen de restos fecales así como de otros desechos arrastrados por las aguas.  Lodos secundarios o biológicos: proceden del decantador secundario y se componen fundamentalmente de la biomasa microbiana generada en el tratamiento biológico de las aguas residuales. Parte de estos lodos se recirculan en el sistema de “fangos activos” y el exceso constituye el efluente sólido del sistema (Figura 2). Capítulo 1 Introducción 17 Figura 2: Lodos secundarios generados por una EDAR. Independientemente del origen de los lodos, todos se caracterizan por unos elevados contenidos de agua, materia orgánica putrescible y microorganismos patógenos. Debido a ello, los objetivos de las EDAR en cuanto al tratamiento de los lodos generados se centran en la reducción del contenido de agua, para reducir costes de transporte y mejorar la estabilidad del producto; y estabilizar la materia orgánica para evitar su putrefacción (Cieślik y col., 2015). Los distintos procesos destinados a conseguir estos objetivos son:  Espesamiento: se reduce el volumen de los lodos al eliminar agua de los mismos por medio de sistema de sedimentación gravitacional, flotación o centrifugación. Esta reducción del volumen es vital para reducir el volumen de lodos a tratar en los siguientes procesos, incidiendo directamente en el coste de inversión y operación de los mismos.  Estabilización: se disminuye el contenido en materia orgánica, evitando la podredumbre de los lodos así como malos olores y la presencia de organismos Capítulo 1 Introducción 18 patógenos. Existen dos tipos de sistemas de estabilización, los biológicos y los químicos. En la digestión biológica los lodos sirven como sustrato para el crecimiento de otros microorganismos, pudiendo ser esta digestión aeróbica, donde en presencia de oxígeno la materia orgánica se oxida hasta un fango digerido conjuntamente con CO2 y agua; o bien anaeróbica en ausencia de oxígeno, donde se genera también un fango digerido junto con CO2 y metano. En la estabilización química se emplea un reactivo químico, normalmente cal para alterar las características químicas como el pH del lodo e impedir su putrefacción.  Acondicionamiento: es una etapa previa al deshidratado de los lodos para reducir la afinidad del agua por los sólidos presentes en estos. Se puede realizar mediante la adicción de reactivos químicos o mediante tratamiento térmico, de forma que los lodos pierden parte del agua intersticial al formarse agregados mayores.  Deshidratación: consistente en la práctica eliminación del contenido en agua de los lodos. Este proceso suele constituir la etapa final del tratamiento de los lodos en muchas depuradoras. Mediante la deshidratación se reducen los costes de transporte de los lodos al disminuir su peso y volumen, se reducen los problemas de malos olores y putrefacción y constituye un paso previo necesario en el caso de que el destino final de los lodos sea la incineración. Se puede realizar mediante sistemas de filtrado o centrifugado.  Destino final: el destino final de los lodos de depuradora dependerá del valor que se les dé a los mismos, pudiendo almacenarse como un residuo sin valor en vertederos (Figura 3) o utilizarse como un recurso o materia prima. Capítulo 1 Introducción 19 Figura 3: Lodos de depuradora depositados en un vertedero. 1.1.5. Sistema de lodos activos El sistema de lodos o fangos activos es el sistema biológico más ampliamente usado en la mayoría de las depuradoras de aguas residuales. En este proceso, una masa microbiana metabólicamente activa actúa sobre un sustrato orgánico, estabilizándolo por vía aerobia (Hreiz y col., 2015). Este proceso se divide en dos etapas, una de aireación, donde se metaboliza y degrada la materia orgánica por acción de los microorganismos y una segunda de sedimentación donde se separan los lodos del agua depurada. En la etapa de aireación se mezclan dentro de un reactor la materia orgánica presente en las aguas residuales con los microorganismos que la van a digerir en presencia de oxígeno suficiente para que el proceso se dé a la máxima velocidad posible. El resultado final de esta etapa es una mezcla de agua y biomasa microbiana que se ha generado tras consumir la materia orgánica disuelta en las aguas residuales. Entre los microorganismos que Capítulo 1 Introducción 26 1.2. PLUMAS DE POLLO 1.2.1. Origen y funciones Las plumas son un órgano epitelial exclusivo de la clase de las aves. Las funciones principales de estos elementos son el mantenimiento de la temperatura corporal, el vuelo, protección frente a los elementos externos, amortiguación mecánica, nado, buceo, reconocimiento, advertencia, etc. Como consecuencia de la aparición de estos órganos, las aves han sido capaces de adaptarse y colonizar nuevos nichos ecológicos a los que anteriormente no tenían acceso, como son el propio cielo, islas remotas, etc. (Stettenheim, 1972; Widelitz y col., 2007) 1.2.2. Composición Las plumas están constituidas aproximadamente en un 90% por una única proteína, la β-queratina. Esta proteína fibrosa presenta una estructura típica en lámina β antiparalela muy rica en enlaces disulfuro, lo que le otorga una gran estabilidad frente a la degradación (Figura 5). Aunque todas las queratinas son similares, su composición aminoacídica y estructura difiere ligeramente entre los distintos grupos de animales (Stettenheim, 1972). Figura 5: Estructura de lámina β antiparalela típica de la queratina. Capítulo 1 Introducción 27 1.2.3. Estructura y tipología de las plumas En general, las partes de una pluma son las que se muestran en la figura 6 y son: un eje o mástil, formado por el cálamo, cañón o plumilla, la hipopluma y el raquis; y un estandarte o vexilo, compuesto por ramificaciones que parten del raquis llamadas barbas, las cuales se subdividen en barbillas, de las que parten numerosas espinas o ganchillos que unen unas barbillas con otras y estas a su vez a las barbas contiguas, formando una estructura cohesionada y semirrígida (López-Albors y col., 1999). Figura 6: Partes de la pluma. Las plumas presentan diversas formas dependiendo de las funciones que vayan a desempeñar y de su posición en el ave. Los tipos de plumas que aparecen en un ave son: plumón, cuya función es el mantenimiento de la temperatura corporal; semipluma, intermedia entre plumón y pluma de contorno; plumón polvoriento, que acumula polvo para el acicalamiento del ave; plumas de contorno, determinan la morfología del plumaje; plumas de vuelo, encargadas de permitir y dirigir el vuelo, dividiéndose en remeras o rémiges (contorno exterior del ala), timoneras o rectrices (cola) y coberteras o tectrices; cerdas, con función sensorial y filoplumas, también con función sensorial (López-Albors y col., 1999) (Figura 7). Capítulo 1 Introducción 28 Figura 7: Diagrama comparativo de los distintos tipos de plumas 1.2.4. Problemática de las plumas La carne de pollo es la segunda carne más consumida en el mundo después de la carne de cerdo, a lo que se suma un incremento de su consumo, sobre todo en países asiáticos. Durante el procesado de los pollos por las industrias avícolas se genera este subproducto, que supone entre un 5 y un 10% del peso corporal del ave, generando una producción mundial de plumas que ronda los 9 millones de toneladas (Reddy, 2015; Callegaro y col., 2018). Aunque inicialmente se consideraban las plumas como un residuo, actualmente se encuentran recogidas dentro de la Categoría 3 de subproductos animales. Los materiales enmarcados en dicha categoría pueden ser utilizados como materia prima para procesos como la producción de alimentos para animales distintos de los de partida, compostados o ensilados, lo cual abre su posible uso como biofertilizantes o bioestimulantes una vez procesados correctamente (Reglamento CE nº 1069/2009). Capítulo 1 Introducción 29 1.3. PRODUCCIÓN DE BIOFERTILIZANTES Y BIOESTIMULANTES ORGÁNICOS Desde hace tiempo se vienen empleando de forma convencional como materias primas para la elaboración de fertilizantes y bioestimulantes orgánicos diversos sustratos de variada índole. La selección de una materia prima u otra depende en muchos casos de la composición química, la facilidad de procesado y sobre todo del precio y disponibilidad en el momento de la elección. Aunque la decisión final se toma teniendo en cuenta toda esta información y con un periodo de validez de varios años, en muchos casos, las condiciones iniciales cambian debido a agentes externos como cambios geopolíticos, crisis financieras, catástrofes ambientales, etc. o simplemente, el periodo inicial de validez a espirado y debe realizarse un estudio y valoración nuevos. A lo largo del tiempo, muchas de las materias primas que inicialmente se empleaban en la producción de fertilizantes o bioestimulantes han incrementado su precio, disminuido su disponibilidad o reducido sus usos y aplicaciones por motivos relacionados con la legislación vigente. Esto ha obligado a la búsqueda de nuevas materias primas de bajo coste y alta disponibilidad, como son los subproductos de la industria agro-alimentaria. Atendiendo únicamente a la composición química, muchos sustratos orgánicos, como los subproductos de la industria agro-alimentaria, resultan adecuados para aplicarlos como fertilizantes o bioestimulantes. Sin embargo, estos subproductos no pueden ser utilizados tal cual debido a una serie de problemas inherentes a su estructura, propiedades fisicoquímicas, etc. Entre estos parámetros limitantes a tener en cuenta destacan algunos como la solubilidad o distribución de pesos moleculares. Debido a ello, el uso agronómico o ambiental directo de estos sustratos orgánicos resulta inviable técnica y económicamente. Capítulo 1 Introducción 30 Para solventar estos problemas, los subproductos deben ser procesados de forma que se soslayen las limitaciones de los mismos. Tradicionalmente, se han empleado tratamientos de carácter físico o químico con el fin de extraer y/o modificar los componentes de interés que forman parte de esos sustratos (Prendergast, 1974). En muchos casos, estos tratamientos conllevan el empleo de condiciones extremas, como altas temperaturas, pH extremos y el uso de compuestos químicos como solventes orgánicos, agentes cáusticos, etc. Aunque estos métodos consiguen obtener el producto final requerido, en general, también se producen efectos indeseados, como la aparición de modificaciones perjudiciales en aminoácidos, residuos tóxicos, alto coste energético, etc. (Fox, 1982). Una alternativa a los tratamientos fisicoquímicos son los tratamientos biológicos o bioquímicos, como son los procesos de hidrólisis enzimática o los procesos fermentativos. 1.3.1. Tecnología enzimática 1.3.1.1. Las enzimas Las enzimas son catalizadores biológicos que, al igual que los catalizadores químicos, su función es aumentar la velocidad a la que tienen lugar la reacción que catalizan sin el consumo de ellos mismos. Las enzimas tienen la particularidad, en general, de ser bastante específicas con respecto al sustrato sobre el que actúan, la reacción que catalizan y el producto que se genera (Palmer y Bonner, 2011). Aunque las enzimas se suelen denominar de forma común teniendo en cuenta el sustrato sobre el que actúan, la reacción que catalizan, o ambos, existe una nomenclatura sistemática creada por la Enzyme Commission (EC). Esta nomenclatura consiste en 4 números separados por puntos, donde el primer dígito indica una de las seis clases principales de reacciones catalizadas (Tabla 1). El segundo dígito describe la subclase de reacción en particular. El tercer dígito es común a aquellas enzimas que catalizan reacciones similares Capítulo 1 Introducción 31 pero no exactamente iguales, mientras que el cuarto dígito define el sustrato específico (Palmer y Bonner, 2011). PRIMER DÍGITO CLASE REACCION CATALIZADA 1 Oxidorreductasas Reacciones de oxidación/reducción 2 Transferasas Transferencia de un átomo o grupo entre dos moléculas 3 Hidrolasas Reacciones de hidrólisis 4 Liasas Ruptura de una molécula (no por hidrólisis) 5 Isomerasas Reacciones de isomerización 6 Ligasas Unión de dos moléculas Tabla 1. Principales clases de reacciones catalizadas por enzimas. 1.3.1.2. Hidrólisis enzimática La hidrólisis enzimática es un tipo de reacción catalizada por una hidrolasa, donde una molécula de agua reacciona con un sustrato, de forma que se generan dos productos, uno que contiene el catión hidrógeno y el otro al anión hidroxilo, como se muestra en la ecuación 1: Eq. 1: 𝐴 − 𝐵 + 𝐻2𝑂 → 𝐴 − 𝑂𝐻 + 𝐵 − 𝐻 Este tipo de reacciones se da habitualmente en los procesos de digestión de las macromoléculas biológicas a fin de obtener los monómeros que las componen, como es el caso de las proteínas en péptidos y aminoácidos, los polisacáridos en oligosacáridos y monosacáridos, etc. Capítulo 1 Introducción 32 1.3.1.3. Hidrólisis de proteínas Las proteínas son biopolímeros compuestos por cadenas lineales de aminoácidos unidos entre sí por enlaces peptídicos y que adquiere diversas estructuras tridimensionales determinadas por la propia secuencia de aminoácidos (Berg y col., 2007). Las proteínas poseen una amplia variedad de funciones, aunque se pueden agrupar en 5 tipos principales (Tabla 2): FUNCIÓN DESCRIPCIÓN Estructurales Proporcionan estructura y soporte a las células y organismos Enzimas Catalizan las diversas reacciones químicas que tienen lugar en la célula u organismo Transporte Transportan átomos, moléculas, etc. de una parte a otra de la célula u organismo. Mensajeros Transmiten señales entre células, tejidos u órganos para coordinar diversos procesos Anticuerpos Se unen a partículas extrañas al organismo a fin de identificarlas y protegerlo. Tabla 2: Funciones de las proteínas. El nitrógeno es un elemento esencial para el desarrollo de cualquier célula y organismo debido a que forma parte de los aminoácidos y estos conforman las distintas proteínas existentes. Por ello, es necesario un aporte continuo de este elemento a lo largo de todo el desarrollo de cualquier ser vivo. Este aporte se puede dar en forma de sales de nitrógeno como nitratos, amonio, urea, etc. con los que aquellos organismos capaces de ello forman sus propios aminoácidos, o bien, pueden asimilar directamente los aminoácidos ya formados por otros organismos. Dado que en general las células no pueden incorporar directamente proteínas de gran tamaño para poder asimilar sus aminoácidos, es necesario un paso previo de hidrólisis o ruptura de esas proteínas en fragmentos de menor tamaño, los cuales reciben diferentes nombres atendiendo al peso molecular que poseen (Tabla 3) Capítulo 1 Introducción 33 PESO MOLECULAR (Daltons) DENOMINACIÓN >10.000 Proteínas 7.000-10.000 Peptonas 200-7.000 Péptidos <200 Aminoácidos Tabla 3. Distribución de pesos moleculares de las distintas especies proteicas Una de las formas de reducir el tamaño de las proteínas para hacerlas fácilmente disponibles para las células es la hidrólisis enzimática, que mediante la acción de enzimas hidrolíticas, hidrolasas o más específicamente proteasas o proteinasas, “rompen” los enlaces que unen los aminoácidos y liberan fragmentos proteicos de menor tamaño que la proteína inicial (Adler-Nissen, 1977; 1986). La unión entre los distintos aminoácidos que forman las proteínas se llama enlace peptídico, el cual es un tipo de enlace covalente. Este enlace se da entre el grupo carboxilo (- COOH) de un aminoácido y el grupo amino (-NH2) del siguiente (Figura 7). Este enlace posee una serie de características particulares, como el hecho de que actúa como un enlace doble, se suelen disponer en posición trans en vez de cis, o que presenta un carácter polar. Figura 7: Formación del enlace peptídico. Capítulo 1 Introducción 34 La hidrólisis del enlace peptídico se da a través de varias reacciones simultaneas como son la ruptura propiamente dicha del enlace, transferencia de protones, etc. (Guadix y col., 2000; Benítez y col., 2008) (Figura 8). Figura 8: Hidrólisis del enlace peptídico. Cuando esta reacción de hidrólisis está catalizada por una proteasa, el proceso tiene lugar en tres reacciones consecutivas (Adlesr-Nissen, 1986; Benítez y col., 2008): 1) Formación del complejo enzima-sustrato (proteína). 2) Ruptura del enlace peptídico y liberación de péptidos y/o aminoácidos. 3) Separación del péptido restante de la enzima tras el ataque nucleofílico de una molécula de agua. Los grupos –COOH y -NH2 de las proteínas presentan normalmente unos valores de pK comprendidos entre 3,1-3,6 y 7,5-7,8 respectivamente. (Steinhardt y Beychok, 1964; Rupley, 1967). De esta forma, a pH ácidos, los grupos carboxilo estarán parcialmente disociados y los amino protonados. Si la reacción de hidrólisis se da a estos pH, el pH irá aumentando rápidamente. Si, por el contrario, se trabaja a pH alcalino, los grupos carboxilo estarán totalmente disociados y los grupos amino parcialmente protonados, por lo que el valor de pH disminuirá constantemente. Capítulo 1 Introducción 35 1.3.1.4. Proteasas Las proteasas son enzimas esenciales para todos los seres vivos (Gupta y col., 2002a) ya que al catalizar la hidrólisis de los enlaces peptídicos, intervienen en los procesos de reciclaje de proteínas, ya sean endógenas o exógenas (Kumar y Takagi, 1999). Este tipo de enzimas constituye más del 50% del mercado mundial de enzimas con usos industriales, como son la producción de detergentes, alimentos, productos farmacéuticos, gestión de residuos, etc. (Rao y col., 1998; Gupta y col., 2002b). Las proteasas se clasifican atendiendo a una serie de parámetros (Tabla 4): PARÁMETRO TIPO DESCRIPCION Acción catalítica Endoproteasas Hidrolizan enlaces peptídicos dentro de la cadena polipeptídica, liberando péptidos Exoproteasas Hidrolizan el enlace peptídico que une el último aminoácido de la cadena, liberando aminoácidos libres pH óptimo Ácidas pH óptimo entre 8 y 12 Neutras pH óptimo cercano a 7 Alcalinas pH óptimo entre 2 y 6 Elemento catalítico en el sitio activo Aminoácidos en el sitio activo Uno o varios aminoácidos realizan las diversas reacciones de hidrólisis, los más habituales son serina (serín proteasas), cisteína (cisteín proteasas) y aspartato. Metalo-proteasas (iones metálicos en el sitio activo) Utilizan un ion metálico como el zinc como agente catalítico. Origen Animal Vegetal Bacteriana Fúngica Figura 4: Clasificación de las proteasas. Capítulo 1 Introducción 42 facilidad de producción, lo cual no ocurre en el caso de los animales o las plantas. La segunda gran ventaja radica en el uso de las nuevas tecnologías de modificación del ADN, que han permitido producir enzimas de otros orígenes en microorganismos, de forma que se obtiene un producto con las características del organismo original pero producido en masa. Además, la optimización de los procesos de producción implica otros procesos como la inducción en el momento oportuno la enzima requerida, la eliminación de represores, o la posibilidad de introducir un mayor número de copias del gen en cuestión, haciendo al microorganismo superproductor de esa enzima. INDUSTRIA USO ENZIMA FUENTE Panificación Reducción de la viscosidad de la masa, aceleración de la fermentación, aumento del volumen del pan, mejora de la suavidad de la miga y mantenimiento de la frescura. Amilasa Fúngica Mejora de la textura de la masa, reducción del tiempo de mezcla, aumento del volumen. Proteasa Fúngica / bacteriana Cervecera Maceración Amilasa Fúngica / bacteriana Turbidez Proteasa Fúngica / bacteriana Mejora de la filtración fina. β-Glucanasa Fúngica / bacteriana Cereales Alimentos para bebés, alimentos para el desayuno Amilasa Fúngica Café Fermentación de café en grano Pectinasa Fúngica Preparación de concentrados de café Pectinasa, hemicelulasa Fúngica Confitería Producción de caramelos de núcleo blando Invertasa, pectinasa Fúngica / bacteriana Sirope de maíz Producción de glucosa a partir de jarabe de maíz Amiloglucosi dasa Fúngica Fabricación de jarabes de fructosa Glucosa isomerasa Bacteriana Lechera Estabilización de la leche evaporada. Proteasa Fúngica Producción de concentrados de leche entera, helados y postres congelados. Lactasa Levaduras Zumos de frutas Clarificación Pectinasa Fúngica Eliminación de oxígeno Glucosa oxidasa Fúngica Lavandería Detergentes Proteasa, lipasa Bacteriana Capítulo 1 Introducción 43 Cuero Pelado, descarnado Proteasa Fúngica / bacteriana Cárnica Texturización Proteasa Fúngica Papelera Eliminación de ceras Lipasa Fúngica Farmacéutica Ayudas digestivas Amilasa, proteasa Fúngica Anticoagulantes Estreptoquina sa Bacteriana Análisis clínicos Numerosas Fúngica / bacteriana Biotransformación Numerosas Fúngica / bacteriana Hidrolizados proteicos Fabricación Proteasa Fúngica / bacteriana Bebidas sin alcohol Estabilización Glucosa oxidasa, catalasa Fúngica Textil Desencolado de telas Amilasa Bacteriana Vegetales Preparación de purés y sopas Pectinasa, amilasa, celulasa Fúngica Tabla 5: Principales enzimas microbianas con usos industriales. Metabolitos microbianos Durante las distintas fases de crecimiento de un cultivo microbiano se generan una serie de compuestos, que pueden ser anabolitos, si proceden de los procesos de biosíntesis, o catabolitos si proceden de los procesos de degradación. Estos productos se llaman metabolitos primarios. Aunque estos productos son generados de forma natural en cantidades suficientes para el desarrollo del organismo, la aplicación industrial de dichos procesos requiere de la optimización de las condiciones de cultivo y la mejora del propio microorganismo, aplicando para ello los avances más novedosos en genómica, proteómica y metabolómica. Todos estos avances permiten la “sobre-producción” del metabolito en cuestión. A diferencia de los metabolitos primarios, que se producen en general en todas las fases del crecimiento y son más o menos comunes a todos los organismos, los metabolitos secundarios son unos compuestos generados principalmente en la fase estacionaria del cultivo y sólo se producen en ciertas especies microbianas, mientras que en otras no. Estos Capítulo 1 Introducción 44 metabolitos secundarios proceden de otros primarios por transformación de los mismos, no siendo indispensables para el organismo productor y realizando su efecto normalmente en otro organismo distinto. Entre estos metabolitos secundarios destacan muchos antibióticos, inhibidores, moduladores, etc. (Tabla 6). Metabolito Secundario Uso comercial Penicilina, cefalosporina, estreptomicina Antibióticos Bleomicina, mitomicina Anticancerígenos Lovastatina Anticolesterolémico Ciclosporina A Inmunosupresor Avermectinas Antiparasitarios Tabla 6: Principales enzimas microbianas con usos industriales. Productos recombinantes Muchas proteínas, como factores de crecimiento, hormonas, etc. sólo se pueden obtener de forma adecuada usando cultivos de células animales (humanas), sin embargo, esto acarrea un gran número de problemas derivados del propio cultivo de este tipo de células, ya que son muy sensibles a cambios del medio, contaminaciones, etc. Gracias a los avances en recombinación de ADN es posible expresar de forma heteróloga estas proteínas en microorganismos, lo cual permite obtener dichas proteínas a partir de microorganismos, con las ventajas de cultivo de estos últimos. Procesos de modificación Además de sintetizar nuevos metabolitos a partir de los nutrientes del medio, los microorganismos son capaces de transformar ciertos compuestos en otros mediante reacciones de oxidación, deshidrogenación, hidroxilación deshidratación, descarboxilación, aminación, desaminación, etc. La ventaja principal del empleo de microorganismos para realizar estas reacciones frente a la transformación química clásica es la especificidad quiral, generándose uno de los enantiómeros en concreto y no una mezcla de ambos. La principal desventaja de Capítulo 1 Introducción 45 los procesos fermentativos de transformación es que previamente a dicha reacción, es necesario cultivar en cantidad suficiente los microorganismos, aunque una vez producidos, se pueden inmovilizar y reutilizar en numerosas ocasiones. 1.3.2.4. Producción de proteasas Dentro de toda la amplia variedad de enzimas hidrolíticas que el género Bacillus puede excretar y en particular B. licheniformis, destacan sobre las demás las proteasas, enzimas implicadas en los procesos de reciclaje celular y de adquisición de nutrientes del medio, de ahí que su producción sea constitutiva (Gupta y col., 2002a). Esta enzima resulta, además, de vital importancia en los procesos de digestión de la materia orgánica al constituir las proteínas un importante componente estructural de la misma, por lo que existe un creciente interés en su producción a partir de organismos sobre-productores como B. licheniformis. Es conocido que la producción de estas enzimas extracelulares está unida al paso de la fase de estacionaria a la de latencia, es decir, a la esporulación, proceso regulado por la existencia de fuentes de carbono y nitrógeno en el medio (Gupta y col., 2002a). Sin embargo, ambos procesos son independientes entre sí, aunque parece ser que el desencadenante en los dos casos es la falta de nutrientes (Khan, 2000). Una de las limitaciones a la hora de producir comercialmente proteasas a partir de microorganismos es el elevado coste de los medios de cultivo (Romero y col., 2007), constituyendo hasta el 40% del coste total de la enzima comercial (Genesse, 1997), por lo que la mayoría de los estudios actuales se centran en la reducción de estos costes mediante la búsqueda de nuevas materias primas más económicas conjuntamente con la optimización de los parámetros de producción (Reddy y col., 2007). 2. OBJETIVOS Capítulo 1 Objetivos 49 Como consecuencia de la necesidad de crear nuevos productos con capacidad bioestimulante tanto agronómicos como ambientales, así como de buscar alternativas para revalorizar subproductos que actualmente se encuentran infrautilizados o considerados directamente como residuos, se plantean los siguientes objetivos del presente capítulo: 1. Caracterizar físico-químicamente dos subproductos de la industria agroalimentaria, como son los lodos de depuradora y las plumas de pollo. 2. Desarrollar una metodología para obtener nuevos biofertilizantes / bioestimulantes a partir de los subproductos anteriormente mencionados. 3. Producir y caracterizar nuevos productos biofertilizantes / bioestimulantes agronómicos y ambientales obtenidos a partir de lodos de depuradora y plumas de pollo. 3. ARTICULOS 6 J. Parrado et al. / Enzyme and Microbial Technology 57 (2014) 1–7 Table 3 Protein identification of extracellular proteins in B. licheniformis growing in different feathers, TG-Feather and CEBAS-Feather, by MASCOT (Matrixscience, UK) searching engine over NCBI database. Mass (Da) Score Queries matched emPAIaProtein name TG-Feather 31,266 441 14 0.49 KerA [Bacillus licheniformis] Subtilisin precursor [Bacillus licheniformis] 66,111 270 7 0.16 Glycoside hydrolase family 18 protein [Bacillus licheniformis] 64,048 193 4 0.10 Gamma-glutamyltranspeptidase (Ggt) [Bacillus licheniformis] 14,050 166 8 0.91 Hypothetical protein BL00275 [Bacillus licheniformis] MU-Feather 31,266 640 18 1.02 KerA [Bacillus licheniformis] Subtilisin precursor [Bacillus licheniformis] 435 468 16 0.44 Chain A, Chitosanase [Bacillus sp.] 79,565 419 9 0.22 Glycoside hydrolase family protein [Bacillus licheniformis] 66,111 273 5 0.10 Glycoside hydrolase family 18 protein [Bacillus licheniformis] 64,048 221 3 0.10 Gamma-glutamyltranspeptidase (Ggt) [Bacillus licheniformis] 3201 218 5 0.21 Flagellin [Bacillus licheniformis] 85,573 184 5 0.08 Extracellular serine protease [Bacillus licheniformis] 14,050 173 9 0.24 Hypothetical protein BL00275 [Bacillus licheniformis] 73,549 109 2 0.04 Putative acylaminoacyl-peptidase YuxL [Bacillus licheniformis] LB 33,201 910 72 7.87 Flagellin [Bacillus licheniformis] 50,616 348 11 0.37 Chitosanase [Bacillus sp.] 33,888 232 7 0.45 Intracellular serine protease[Bacillus licheniformis] 64,048 222 6 0.22 Gamma-glutamyltranspeptidase (Ggt) [Bacillus licheniformis] 22,530 187 11 0.51 Superoxide dismutase [Bacillus licheniformis] 17,909 157 4 0.19 Metalloregulation DNA-binding stress protein [Bacillus licheniformis] 31,266 123 5 0.11 KerA [Bacillus licheniformis] Subtilisin precursor [Bacillus licheniformis] 24,831 122 4 0.29 Metal-dependent hydrolase [Bacillus licheniformis] 14,050 166 8 0.91 Hypothetical protein BL00275 [Bacillus licheniformis] 19,722 116 3 0.17 Chitosanase [Bacillus sp.] 66,111 81 2 0.10 Glycoside hydrolase family 18 protein [Bacillus licheniformis] aExponentially modified protein abundance index. The second most important protein produced quantitatively was a chitosanase. Chitosanases (EC 3.2.1.132) are endo-hydrolytic enzymes acting on internal glycosidic bonds within the biopolymer chains thereby releasing low molecular weight oligomers. There was no chitosan in the chemical composition of the feathers, thus the secretion of this enzyme could be due to a dual and unspecific activity of lipase. This dual activity has been attributed to the similarity of the active sites of both enzymes chitosan and lipase [34,35]. It is known that lipases of various origins depolymerize chitosans [36,37,38]. The nonspecific activity is justified by the simplicity of the enzymatic hydrolytic mechanism and, once again, the similarity of the active sites of both enzymes [39,40]. Data also show that B. licheniformis expresses some of the exoenzyme genes and secretes the corresponding hydrolyzing enzymes, such as glycoside hydrolase family proteins, when a carbon source is present in the medium. Therefore, a proteomic analysis of the fermentation broth suggests that the majority of the proteins identified tend to be exocellular enzymes. However, there were also proteins that were secreted regardless of the growth conditions, such as flagellin (Hag). Furthermore, most of the proteases were secreted in response to all conditions, although not to the same extent. KerA was the main protease secreted by B. licheniformis in both by-products, but the MU-Feather induced the generation of other proteases such as extracellular serine protease and putative acylaminoacylpeptidase. The presence of a greater diversity of proteases leads to greater diversity in the breaking of peptide bonds, which could be the reason why the hydrolysis products found in the fermentation broth of the MU-Feather medium are smaller in size (see Fig. 1). The growth of B. licheniformis on an enriched medium like LB as a standard control, showed a wide variety of secreted extracellular proteins and cell wall-associated proteins (Table 3). Few of these proteins associated with flagellum, flagellin and Hag [41,42] were secreted at baseline levels. However, the secretion of certain proteases, such as KerA and Ggt, were secreted in all types of culture media. The present work, carried out on different kind of feathers, clearly demonstrates the influence of the chemical composition of the medium on the excretion protein profiles of the microorganism. 4. Conclusions We have established the feasibility of using organic waste from the poultry industry as an efficient fermentation medium to produce hydrolytic enzymes as proteases and lipases. To conclude, our study shows the influence of the chemical composition of feathers on protein secretion by B. licheniformis. 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Journal of Biotech Research [ISSN: 1944-3285] 2018; 9: 14-26 14 Hydrolytic enzymes production by Bacillus licheniformis growth on fermentation media formulated with sewage sludge Alberto Renato Inca-Torres1, 2, Anabell Del Rocío Urbina-Salazar1, 2, Gonzalo Falcón- García1, Pilar Carbonero-Aguilar1, Bruno Rodríguez Morgado1, Juan Parrado1, Juan Bautista1, * 1Department of Biochemistry and Molecular Biology, University of Seville, C/ Professor García González nº2, 41012-Seville, Spain. 2Polytechnical High School of Chimborazo, Faculty of Science, Panamericana Sur km 1 1/2, EC060155- Riobamba, Ecuador. Received: January 23, 2018; accepted: March 27, 2018. In this work, the production of hydrolytic enzymes by Bacillus licheniformis grown in media formulated with sewage sludge as the main carbon and nitrogen source with induction by keratins has been studied. The three main types of enzymes of industrial interest produced were proteases, lipases and cellulases. The addition of an inductor, keratin in our case, improves the production of these enzymes to reach a productivity of 16.89 mU/mL· day, 0.25 mU/mL·day, and 0.51 mU/mL·day for proteases, lipases and cellulases, respectively. The secretion of proteins and enzymes into the fermentation media was studied by electrophoretic and proteomic methods, which revealed the presence of proteases, lipases, and cellulases in the fermentation media, among other excreted proteins. Our results show that the growth of B. licheniformis in fermentation media formulated with sewage sludge as the main carbon and nitrogen sources, supplemented with keratin from feather meal as an inductor could be used for the industrial production of these enzymes, particularly of proteases. Keywords: Hydrolytic enzymes; Bacillus licheniformis; sewage sludge; proteases; lipases; cellulases. Abbreviations: GRAS: generally regarded as safe; CDDGs: corn dried distillers grains with solubles; TCA: trichloroacetic acid; DNS: dinitrosalicylic acid; EA: enzymatic activity; p-NPG: p-nitrophenyl-β-glucopyranoside; MUB: Modified Universal Buffer; CBB: Coomassie Brilliant Blue. *Corresponding author: Juan Bautista, Department of Biochemistry and Molecular Biology, University of Seville, C/ Professor García Gonzalez nº2, 41012-Seville, Spain. Phone: +34 954556854. E-mail: jdbau[email protected]. Introduction Industrial application of enzymes began in the mid-sixteen centuries, although its stable implementation was not achieved until the midtwenty centuries, between 1950-1960 [1]. The main industrial applications of enzymes are in the production of food, textiles and detergents, representing approximately 90% of the market volume. Other applications, such as organic synthesis, medical and pharmaceutical applications, agronomic uses, etc., are much less used [2]. However, recently, the application of enzymes in agricultural and environmental applications has experienced a significant increase, mainly due to the development of new production methods using cheap culture media derived from agricultural products and urban residues [3]. Most industrial enzymes are bacterial enzymes, although fungal enzymes have lately become increasingly important, both quantitatively and qualitatively. Bacterial enzymes produced by the genus Bacillus are of special interest for both research and industrial applications due to the Journal of Biotech Research [ISSN: 1944-3285] 2018; 9: 14-26 15 high rate of growth of these bacteria and their ability to secrete a high volume of enzymes into culture medium (20-25 g/L) [4]. In addition, these microorganisms are “generally regarded as safe” (GRAS). The Bacillus strains considered of industrial interest are the species B. licheniformis and B. subtilis. One of the major bottlenecks for the industrial application of enzymes is the high production cost, while the cultivation medium may represent 30-40% of total production costs [5]. This has produced great interest in the development of efficient and cheap cultivation media. Among the media developed to address this issue, those derived from agroindustrial byproducts (whey, brewers grains, rice bran, defatted sunflower flour, and corn dried distillers grains with solubles (CDDGs)) and urban wastes (water and sewage sludge) are two types of promising substrates [6]. In addition to the economic benefit associated with the use of agroindustrial byproducts and urban waste, another important advantage is the resulting contribution to environment maintenance [7, 8]. In industrialized countries, the high rates of production of household and industrial liquid waste results in a high polluting effect [9, 10]. The treatment of these wastes in depuration plants generates two main products: purified water and sewage sludge. While the purification of water has received great attention, much less attention has been devoted to the management of sewage sludge, which is a dangerous source of environmental pollution if not treated properly. However, it has recently been shown that sewage sludge, when adequately processed, may serve as a useful substrate in the treatment and regeneration of soil and/or as an inexpensive cultivation media for the production of microorganisms and enzymes [11]. Sewage sludge is one of the most abundant and inexpensive substrates available for the culture of microorganisms and has practically zero cost. Sewage sludge may be processed to modify and/or adjust its physical, chemical, and biological properties to yield acceptable material for use as fertilizer and/or culture medium for the production of substances of high added value [12, 13]. Currently, sewage sludge is transformed mainly into bio-fertilizer, while only a small portion is used for the production of high value-added products such as enzymes. The goal of this work was to study the use of sludge from water treatment plants as a cultivation medium for the production of hydrolytic enzymes (proteases, cellulases, lipases, acid phosphatase, and β-glucosidase) using Bacillus licheniformis. Materials and methods Microorganisms Bacillus licheniformis strain 21415 provided by the American Type Culture Collection (ATCC) (Manassas, VA, USA) was used for the production of enzymes in this study. The bacteria were stored frozen at -80˚C and refreshed in LB medium (10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl, adjusted to pH 7) 24 h before inoculation. Briefly, after thawing at 4˚C overnight, 2 mL of bacteria was inoculated into a flask containing 30 mL of LB medium at pH 7 and 37˚C, and then grown until the transmittance at 620 nm reached 20%. Substrates: Sludge samples, medium composition, and fermentation Sludge samples were provided by the experimental treatment plant of Carrión de los Céspedes (Sevilla, Spain) of the CENTA Foundation. Thickened sludge with a humidity of 90±1.5% was used both alone and in the preparation of cultivation medium (table 1). Culture medium was sterilized at 121˚C for 30 min prior to inoculation to eliminate pre-existing sludge bacteria. Six hundred mL sterile media was inoculated with 30 mL inoculums and grown over 14 days at 37˚C with constant agitation (150 rpm). During growth, 10 mL samples were taken each day and stored at -80˚C until use for measurement of biomass and enzymatic activities, including protease, cellulase, lipase, acid phosphatase, and βglucosidase. Journal of Biotech Research [ISSN: 1944-3285] 2018; 9: 14-26 16 Table 1. Compositions of culture media formulations. Sample preparation Samples were thawed at 4˚C and either used directly for biomass determination or centrifuged at 12,000 × g for 20 min to obtain the supernatant used for enzyme activity assays. (1) Determination of biomass. Biomass was determined indirectly by measuring the protein content of samples at different growth times using the Bradford method [14]. Biomass concentration was determined in μg/mL by interpolation from a standard curve plotting biomass (μg/mL) against protein concentration (μg/mL) (Figure 1). (2) Enzymatic assays The supernatant obtained after centrifugation was used for determination of enzyme activities. Protease activity assay The total extracellular protease activity was determined following the method described by Beynon and Bond [15]. Briefly, 250 μL of the sample was mixed with 250 μL of a reaction mixture containing 0.1 g azocasein and 0.2 mL of ethanol dissolved in 4.8 mL of 0.1 M phosphate buffer at pH 7. Samples were then incubated for 10 min at 40˚C. The reaction was then terminated by adding 2.5 mL of 5% (w/v) TCA solution. The reaction mixture was centrifuged at 10,000 × g for 2 min. The absorbance of the supernatant at 440 nm was measured. One unit of proteolytic activity was defined as the amount of enzyme required to produce an increase in optical density of 0.001. Cellulase activity assay The total cellulase activity was determined by the method described by Galindo [16], modified for application to cellulolytic enzymes. Briefly, 250 μL of a 2% soluble microcrystalline cellulose (Avicel PH-101, Sigma- Aldrich, Barcelona, Spain) solution in 0.1 M acetate buffer (pH 5) was incubated with 250 μL of the sample for 2 hours at 37˚C. The reaction was stopped by adding 1 mL of DNS reagent, and the mixture was heated to 95˚C for 10 min. The reaction mixture was then cooled to room temperature and the absorbance at 575 nm was measured. A standard curve was obtained using glucose for used in the calculation of cellulase activity. One unit of cellulase activity was defined as the amount of enzyme required to liberate 1 mg/mL of reduced sugars into the test solution. Lipase activity assay The total extracellular lipase activity was determined using a modified version of the method described by Kilcawley et al. [17]. Briefly, 1.75 mL of buffer (0.1 M sodium phosphate pH 7, 0.15 M NaCl, and 0.5% (v/v) Triton-X) was mixed with 0.25 mL of sample that had been previously centrifuged for 30 min at 12,000 × g, along with 20 μL of 50 mM pnitrophenol laurate in acetonitrile. This mixture was then incubated for 30 min at 37˚C. Afterward, the mixture was cooled in an ice bath for 5 min, then centrifuged for 1 min at 9,000 × g. The supernatant absorbance at 400 nm was measured. Activity was quantified using the molar extinction coefficient of p-nitrophenol (14,800 /M·cm) at 400 nm. The activity units were defined as one unit corresponding to the release of 1 nmol of p-nitrophenol/min·mg protein under the test conditions. β-glucosidase activity assay The β-glucosidase activity was determined using the colorimetric method described by Eivazi and Tabatabai [18] with slight modifications. Into 250 μL of the sample, 2 mL of modified universal buffer (MUB, pH 6) and 0.5 mL of p-nitrophenyl- β-glucopyranoside 25 mM (7.53 mg/mL) were added. The mixture was then incubated at 37˚C for 30 to 60 min. The reaction was stopped by placing the mixture in an ice bath. Subsequently, 2 mL of 0.5 M NaOH was added and the solution absorbance at 400 nm was measured. The Journal of Biotech Research [ISSN: 1944-3285] 2018; 9: 14-26 17 Figure 1. Standard curve of biomass versus protein concentration for Bacillus licheniformis grown in a defined medium. enzymatic activity (EA) was expressed in μmol/g of sample·min and was calculated by the following formula: EA = C × V / T × G where C is the concentration of liberated pnitrophenyl (mM); V is the sample volume (mL); T is the incubation time (min); and G is the sample weight (g). One β-glucosidase unit of activity was defined as the amount of βglucosidase needed to release 1 nmol pnitrophenol from p-nitrophenyl-β- glucopyranoside in 1 min under the specified conditions. Acid phosphatase activity assay (APHA) Acid phosphatase activity was determined by the method established by Tabatabai and Bremmer [19] with slight modifications. The procedure was initiated by creating a mixture containing 250 μL of sample, 2 mL of MUB, pH 6, and 0.5 mL of p-nitrophenyl-phosphate 25 mM (9.28 mg/mL). This mixture was incubated at 37˚C for 30-60 min. Afterward, the samples were cooled in an ice bath to stop the reaction. Then, 2 mL of 0.5M NaOH was added to the sample and the solution absorbance at 400 nm was measured. The EA was expressed in μmol/g of sample·min and was calculated by the following formula: EA = C × V / T × G where, C is the concentration of liberated pnitrophenyl (mM); V is the sample volume (mL); T is the incubation time (min); and G is the sample weight (g). One phosphatase unit of activity was defined as to the amount of acid phosphatase needed to release 1 nmol pnitrophenol from p-nitrophenyl-phosphate in 1 min under the specified conditions. Spectrophotometric detection of the artificial molecule p-nitrophenol manifesting yellow as a product of acid phosphatase activity was used to quantify the enzyme activity in these samples. Protein assay Protein concentration was quantified by the method described by Bradford, using bovine serum albumin as the protein standard [14]. SDS-PAGE protein profiling The extracellular protein profile was investigated by SDS-PAGE analysis. After acid-acetone Journal of Biotech Research [ISSN: 1944-3285] 2018; 9: 14-26 18 precipitation of the supernatant obtained after fermentation broth centrifugation at 12,000 x g, the pellet was solubilized in sample preparation buffer (1 mM Tris–HCl, pH 6.8) containing 5% (w/v) SDS and 7% (v/v) β-mercaptoethanol. The samples were kept at room temperature for 2 h and then centrifuged at 12,000 × g for 15 min. The supernatant was collected and run on 12% SDS-PAGE gel in triplicate, with each run containing 20 μL of supernatant (approximately 50 μg of protein) and 5 μL of loading buffer (0.5 M Tris-HCl, pH 6.8, 50% (v/v) glycerol, 10% (w/v) SDS, and 5% (v/v) bromophenol blue). The samples were boiled for 5 min, centrifuged at 10,000 × g for 10 min, and cooled before being loaded on the gel. Electrophoresis was carried out at constant intensity (25 mA) using a Tris– glycine buffer system containing 0.1% SDS until the bromophenol front ran off of the gel. After completion of electrophoresis, the gels were stained with Coomassie Brilliant Blue (CBB) G- 250 (Bio-Rad, Hercules, CA, USA) and/or silver staining solution (GE Healthcare, Barcelona, Spain) and digitized on a Gel Doc™ XR + Imaging system (Bio-Rad, Hercules, CA, USA). Zymogram analysis Proteolytic activity was detected via electrophoresis on 12% polyacrylamide gels prepared with 1% SDS and 0.1% (w/v) gelatin. After completion of electrophoresis, the gels were incubated in 0.05 M Tris–HCl buffer (pH 8.0) containing 2% (v/v) Triton X-100 at 35°C for 1 h, and then again in the same buffer without detergent at 35°C for 3 h. After CBB staining and subsequent destaining, proteolytic activity was determined from the presence of non-stained zones and bands on the stained background composed of nondigested gelatin [20]. Proteomics A proteomics study was carried out according to the procedure described in Parrado et al. [21]. Briefly, to remove contaminants such as cells and debris in suspension, the samples were centrifuged at 12,000 × g for 20 min and the supernatants were recovered. Then, 10 μL of the supernatant was treated with 40 μL of methanol and vortex mixed for approximately 1 min. 10 μL of chloroform was added and the mixture was shaken again for 1 min before 30 μL of Milli-Q water was added and mixed well. The mixture was centrifuged at 14,000 × g for 5 min and the supernatant was removed. 30 μL of methanol was added to the pellet and the mixture was vortexed again for 1 min. Samples were centrifuged at 16,000 × g for 5 min. The supernatant was discarded, and the pellets were dried by lyophilization. The pellet proteins were resuspended in Milli-Q water. A total of 30 μg of resuspended protein was added into 30 μL of 6 M urea, 200 mM ammonium bicarbonate, 10 mM DTT and allowed to remain at room temperature for 30 min for protein reduction. 10 μL of 100 mM iodoacetamide was added to promote protein alkylation, and the samples were kept at room temperature in the dark for 30 min. Samples were then dialyzed against Milli-Q water until the urea concentration was below 0.1 M and subsequently treated with trypsin (Promega, Madison, WI, USA) in a protein-to-enzyme ratio of 50:1. Digestion was carried out at 37°C overnight. The mixture was then acidified with TFA and concentrated using a Speed C18/18 column (Applied Separations, Allentown, PA, USA). Peptides were eluted in 400 μL of 70% acetonitrile containing 0.1% TFA. After the samples were completely dried, they were resuspended in 15 μL of 5% acetonitrile containing 0.1% formic acid for use in tandem liquid chromatography–mass spectrometry (LC– MS) analysis. LC–MS analysis was performed in a Surveyor HPLC system in tandem with a Finnigan LTQ mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA). A total of 5 μL of sample was injected into a C18 PepMap100- Precolumn Cartridge (Dionex, Netherlands) for pre-concentration and washing, then resolved in a Biobasic C18 75 µm × 10 cm column (ThermoFisher Scientific, Waltham, MA, USA). Peptides were eluted at a nominal post-split flow rate of 250 L/min using a 120-min gradient of 5% acetonitrile with 0.1% formic acid to 40% acetonitrile with 0.1% formic acid. The LTQ mass spectrometer was run in positive-ion mode using the nanospray source. The spray voltage was set Journal of Biotech Research [ISSN: 1944-3285] 2018; 9: 14-26 19 Figure 2. Biomass and protease production by B. licheniformis grown in media formulations M1, M2, and M3. at 2 kV, and the capillary temperature was set to 170°C. The samples were scanned in the range of 400–1500 m/z using the full scan mode, and data-dependent MS/MS analysis with collisioninduced dissociation (CID) was performed on the top five ions with dynamic exclusion. The data was converted to SEQUEST format (DTA) and compared against the NCBI database (version 11/10/2007) using an in-house MASCOT (Matrixscience, London, UK) search engine with taxonomy restrictions set to Firmicutes and with carboximethylated cysteine as a fixed modification. Statistical analysis Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). All experiments were completed in triplicate, and the mean of the three data sets is presented for each experiment. A level of significance of α < 0.05 was used to calculate significant differences. One-way ANOVA analysis revealed that the results of the triplicate analyses for each experiment did not differ significantly. Results and discussion In an attempt to find an abundant and cheap cultivation medium for the production of hydrolytic enzymes using Bacillus licheniformis, we present in this work the results obtained from the use of sewage sludge as the main fermentation source in the culture of B. licheniformis. As observed in figures 2 to 4, measurement of the protein production indicated that the microorganism grew well, although the biomass production was not high (2.76 μg/mL). Biomass estimation was carried out by protein determination using the Bradford method. As figure 1 shows, a linear relationship (R2 = 0.995) between protein concentration and Journal of Biotech Research [ISSN: 1944-3285] 2018; 9: 14-26 20 Figure 3. Biomass and lipase production by B. licheniformis grown in media formulations M1, M2, and M3. dry biomass was observed, from which biomass concentration can be estimated by interpolation. Figures 2 to 4 also show hydrolase production by B. licheniformis grown in media M1, M2, and M3, specifically, proteases, lipases, and cellulases. Among these hydrolases, proteases are of the highest industrial interest. Proteases (EC 3.4.21- 24 and 99) are industrially useful hydrolytic enzymes that cleave peptide bonds between amino acid residues and are the dominant enzyme in the worldwide market. Two-thirds of the proteases produced annually are used in the detergent industry. Microbial proteases, especially from Bacillus species, have traditionally comprised the predominant portion of industrial enzymes on the market. The major application of microbial proteases is in the formulation of various detergents, which constitutes a significant share of worldwide enzyme sales [22]. Many bacteria in the genus Bacillus excrete large amounts of enzymes into culture medium. Alkaline serine protease, one of the most important enzymes industrially, is excreted into culture medium by strains of B. licheniformis or B. pumilus [23]. These alkaline proteases (i.e., subtilisin) operate in a high alkaline pH range of 8.5-10 and are thus optimal for use in the formulation of detergents. In addition to their use in the detergents industry [24], these alkaline proteases have several other industrial applications, including in the production of foods, pharmaceuticals, leathers, and diagnostic reagents [25]. Proteases are also used for the bioconversion of chitinous materials as a waste-treatment alternative for the disposal of shellfish wastes [26, 27]. As shown in figure 2, B. licheniformis produces extracellular proteases during exponential growth and at the start of the stationary growth phase [28], after which point the production decreased. The analysis of the results obtained in medium M2 (without the addition of feather meal) and M3 (with added feather meal) show that the production of proteases by B. licheniformis is induced by the Ecological Engineering 50 (2013) 31– 36 Contents lists available at SciVerse ScienceDirect Ecological Engineering j ourna l ho me page: www.elsevier.com/locate/ecoleng Obtaining biostimulant products for land application from the sewage sludge of small populations Manuel Tejadaa, Ana M. García-Martínezb,∗, Bruno Rodríguez-Morgadoc, Modesto Carballod, Daniel García-Antrasc, Carlos Aragónb, Juan Parradoc aDpto. Cristalografía, Mineralogía y Química Agrícola, E.T.S.I.A., Universidad de Sevilla, Crta. Utrera, km 1, 41013 Sevilla, Spain bFundación “Centro de las Nuevas Tecnologías del Agua”, Autovía Sevilla-Huelva (A-49), km 28, 41820 Carrión de los Céspedes (Sevilla), Spain cDpto. Bioquímica y Biología Molecular, Facultad de Farmacia Universidad de Sevilla, C/Profesor García González, 2, 41012 Sevilla, Spain dCITIUS, Servicio Biología, Universidad de Sevilla, Avda. Reina Mercedes 4, 41012 Sevilla, Spain a r t i c l e i n f o Article history: Received 17 February 2012 Received in revised form 5 July 2012 Accepted 15 July 2012 Available online 9 August 2012 Keywords: Biofertiliser Enzymatic hydrolysis Protease Sewage sludge Small wastewater treatment plants Soil enzymatic activities a b s t r a c t Large amounts of sewage sludge are generated in wastewater treatment plants during the water purification process. In small populations – defined as urban settlements with less than 2000 population equivalent – sewage sludge is normally produced in anaerobic systems (e.g. septic tanks, Imhoff tanks or anaerobic ponds) or in extended aeration systems. According to the European Directives 86/278/EEC and 2006/12/EC, sewage sludge must be treated before its final fate. Although there are various alternatives, land application of sewage sludge after its composting has been widely promoted because of its high nutrient content. However, the employment of composted sludge as a soil amendment has some limitations, including social rejection. In this paper, a hydrolytic process for obtaining an agricultural biofertiliser from sewage sludge has been assessed. The enzymatic hydrolysis of sludge produced two by-products: an insoluble paste and a nutrient-enriched liquid that constituted the fertilising product. This biofertiliser had higher contents of organic matter, proteins, potassium (K) and sulphur (S) compared to the fresh sludge. Protein content was characterised by a low molecular size, thus increasing the bioavailability of nitrogen (N). Further, high levels of enzymatic activity were detected in soil after the addition of the obtained biofertiliser. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The treatment and management of residual solid waste generated during sewage treatment remains as a very important issue since the sludge concentrates all the pollutants that are removed from wastewater (Aragon et al., 2009). In order to regulate the management of these wastes, the European Union has promulgated different Directives: Directive 2006/12/EC on Waste; Council Directive 86/278/EEC on the protection of the environment, and in particular of the soil, when sewage sludge (SS) is used in agriculture. The main goals of the above-mentioned Directives have been to improve the management of SS, and optimise its agricultural application (promoting sludge recycling), and to protect the Abbreviations: SS, sewage sludge; BF, biofertiliser; IP, insoluble paste; WWTP, wastewater treatment plant; DHA, dehydrogenase activity; PHA, phosphatase activity; GA, ß-glucosidase activity; INT, 2-p-iodo-3-nitrophenyl-5-phenyl tetrazolium chloride; INTF, iodonitrotetrazaolium formazan. ∗Corresponding author. E-mail address: [email protected] (A.M. García-Martínez). environment and, especially, the quality of the soil. The current policy of the European Commission is to provide the adequate quality of sludge to encourage its beneficial use on land and, consequently, consolidate this long-term sustainable solution to sludge disposal (Schowanek et al., 2004; Carbonell et al., 2009). Furthermore, land application is the least expensive method of SS management. Soils are often subject to severe degradation processes, which are linked to an organic matter loss, which affects adversely the soil’s fertility and increases the risks of erosion and desertification, especially in agricultural ecosystems (Fernández et al., 2007). The usual way to recover the soil’s quality and its physical, chemical and biological functions is the use of SS as a resource of organic matter (Navas et al., 1998; Wong et al., 1998; Kizilkaya and Bayrakli, 2005; Pascual et al., 2007; Fernández et al., 2007). Thus, SS can be employed as an organic amendment for both agricultural purposes and the restoration of degraded areas. SS can be used as fertiliser because of its high N and P content and low C/N ratio (Boyd et al., 1980; Iakimenko et al., 1996). However, the direct use of these wastes has major drawbacks that may cause problems derived from the presence of heavy metals, pathogenic microorganisms, bad odours or phytotoxicity. The heavy metal content 0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ecoleng.2012.07.006 32 M. Tejada et al. / Ecological Engineering 50 (2013) 31– 36 in SS may prevent its use as biofertiliser. Therefore, an adequate stabilisation and disinfection treatment is required before application of SS on land. Composting is the main biological approach to stabilise the SS. Composts represent an important resource to maintain and restore soil fertility and are of great value nowadays, particularly in those countries where the organic matter content of the soil is low (Castaldi et al., 2008; Tejada et al., 2009). However, the composting of SS has several problems including the long time of production, the low control of the process, the low value of the final products, the presence of large size solids that avoid some kind of agronomic applications and the slow assimilation by soil microorganisms and plants (Ranalli et al., 2001).In recent years, there has been an increasing use of organic biofertilisers obtained from the hydrolysis of different organic materials (Romero et al., 2007; Parrado et al., 2008; García-Martínez et al., 2010). These products generally comprise peptides, amino acids, polysaccharides, humic substances, phytohormones, etc., that are directly absorbed by plants, thus spending a smaller amount of energy in the absorption process. This fact has a positive effect not only on growth but also in the quality and production of fruit or grain harvested. Thus, the aim of these products is not only to provide nutrition, but also to encourage and stimulate plant metabolism, stress reduction, etc. (Parrado et al., 1991, 2006, 2007, 2008). In fact, the development of new fertilisers and soil-stimulating products has become a focus of interest. Similarly, the application of these biofertilisers to the soil not only leads to an increased content of organic matter and macro- and micro-nutrients, but also a significant activation of the soil’s microbial community. In this study, a biofertiliser from the SS generated in a small wastewater treatment plant (WWTP) (serving less than 2000 population equivalent) has been obtained by applying an enzymatic hydrolysis process and, additionally, its quality and its effects on soils’ biochemical properties has been assessed. 2. Materials and methods 2.1. SS sampling SS was collected from the waste sludge line of an extended aeration unit located at the Experimental Plant at the Foundation Centre for New Water Technologies (CENTA) manages in Carrión de los Céspedes (Seville, Spain) (Fahd et al., 2007). The pilot unit was treating around 30 m3/d of urban wastewater, which meant an F/M rate of 0.1 kg BOD5kg MLVSS−1d−1. The sludge age was 20 days and the excess sludge production was estimated to be 0.35 kgVSS kg BOD5−1. This sludge was taken once at the beginning of the trial and was physically and chemically characterised as described in Section 2.3. 2.2. Enzymatic hydrolysis process The SS was hydrolysed according to the pH-stat method (Adler-Nissen, 1977), using an endoprotease obtained by liquid fermentation of Bacillus lichiniformis ATCC 21415 as the hydrolytic agent. This process took place in a bioreactor operating under controlled temperature and pH, agitation and NaOH consumption (Parrado et al., 2008). The sludge hydrolysis was carried out under the following conditions: (a) substrate: 15–20% of SS; (b) solvent: water; (c) catalytic agent: subtilisin produced by the fermentation of Bacillus lichiniformis ATCC 21415, 0.15% (v/v) (Romero et al., 2007); (d) Enzyme concentration: 1 ml l−1of substrate; (e) temperature: 55–60◦C; (f) pH: 8.5, controlled by the addition of 10 M NaOH; (g) time: 120 min. Finally, the hydrolysed product was centrifuged obtaining two by-products: the hydrolysed liquid, which constituted the biofertiliser (BF) and an insoluble paste (IP). Both products were characterised as described in Section 2.3. 2.3. Analytical characterization of SS and by-products obtained by enzymatic hydrolysis 2.3.1. Physico-chemical characterisation For SS, as well as for the IP and the BF, the determination of organic matter was performed using the dry combustion method (MAPA, 1986). Organic matter content was determined by combustion at 550◦C for 3 h and estimated by difference after obtaining the ash percentage. Total nitrogen was determined by the Kjeldahl method (AOAC, 1990). The protein content was determined by multiplying the total nitrogen content in a protein by the conversion factor 6.25, which is applicable to most proteins. For SS and IP, macro- and micro-nutrients were determined after digestion with nitric and perchloric acid. The nutrients were measured by atomic emission spectrometry and ICP plasma-AES (Fisons-ARL 3410). For BF, the measurements of macro- and micro-nutrients were made directly on the hydrolysed product using the same equipment above mentioned. 2.3.2. Molecular weight analysis by size-exclusion chromatography For SS and BF, the molecular weight distribution of proteins was determined by size-exclusion chromatography using an ÄKTA- purifier (GE Healthcare), according to the procedure described by Bautista et al. (1996) employing a Superdex PeptideTM 10/300GL column (optimum separation range 0.1–7 kDa). Samples were centrifuged at 13,300 × g for 15 min at 4◦C to remove insoluble particles, and the supernatant was filtered through a 0.2 ␮m filter and loaded into a 0.1 ml loop connected to an ÄKTA purifier system. The column was equilibrated and eluted with Na2HPO450 mM (pH 7.5) in isocratic mode, at a flow-rate of 0.5 ml min−1. Protein standard mixture (cytochrome C, 12,500 Da; aprotinin, 6512 Da; vitamin B12, 1255 Da; cytidine, 246 Da; glycine 75 Da) was used to cover the range 100–7000 Da. 2.4. Experimental design for the soil stimulation assessment 2.4.1. Soil characterisation In order to assess the potential use of SS and BF as an agricultural biostimulant, an experimental soil classified as Plagic Antrosol was employed (FAO, 1989). The soil was collected from the University of Seville’s experimental research field, from the 0–25 cm surface layer. Soil characteristics determined are shown in Table 1. Soil pH was monitored in distilled water with a glass electrode (soil:H2O ratio 1:10, MAPA, 1986). Soil texture was determined by the Robinson’s pipette method (SSEW, 1982) and quantification and the dominant clay types were determined by X-ray diffraction. Total carbonates were measured by quantifying the CO2produced after HCl addition by standard methods (MAPA, 1986). Soil organic matter was determined by the method of Yeomans and Bremner (1988). Total N was determined by the Kjeldhal method (MAPA, 1986). After nitric and perchloric acid digestion, total Fe, Cu, Mn, Zn, Cd, Pb, Ni and Cr concentrations were determined by atomic absorption spectrometry, according to MAPA methods (MAPA, 1986). 2.4.2. Biostimulation experiment design Soil microcosms were employed to investigate the effects of SS and BF on soil microbial activity. Those microcosms consisted of 300 g of dried and 2 mm-sieved soil mixed and packed into a plastic container. Soil was pre-incubated at 25◦C for 7 days at 30–40% of their water-holding capacity according to Moreno et al. (2003) M. Tejada et al. / Ecological Engineering 50 (2013) 31– 36 33 Table 1 Physico-chemical characteristics of the experimental soil expressed as mean ± standard error. Data are the mean of four samples. pH (H2O) 8.6 ± 0.2 CO32−(g kg−1) 203 ± 12 Fine sand (g kg−1) 142 ± 35 Coarse sand (g kg−1) 387 ± 26 Silt (g kg−1) 242 ± 19 Clay (g kg−1) 229 ± 10 Clay types Smectite: 66% Kaolinite: 20% Illite: 14% Organic matter (g kg−1) 4.1 ± 0.8 Total N (g kg−1) 0.4 ± 0.1 Fe (mg kg−1) 35.8 ± 3.7 Cu (mg kg−1) 9.7 ± 1.3 Mn (mg kg−1) 11.3 ± 2.1 Zn (mg kg−1) 8.1 ± 1.5 Cd (mg kg−1) 6.5 ± 1.2 Pb (mg kg−1) 0.36 ± 0.11 Ni (mg kg−1) 2.9 ± 0.7 Cr (mg kg−1) 5.3 ± 0.6 prior to the experiment. After this pre-incubation period, soil samples were mixed with SS and BF at 0.2%, 0.5% and 1% (w/w) and then adjusted to a water-holding capacity of 60%. A non-amended soil was used as a control. Soil microcosms were incubated in darkness at 25◦C in an incubation chamber for 0, 1, 5, 7, 14 and 30 days. For each product and each incubation time, three soil sub-samples were taken (10 g of soil per sample). The incubation conditions were the following: (a) control: unamended soil; (b) SS 0.2: amended soil with SS at 0.2% (w/w); (c) SS 0.5: amended soil with SS at 0.5% (w/w); (d) SS 1: amended soil with SS at 1% (w/w); (e) BF 0.2: amended soil with BF at 0.2% (w/w); (f) BF 0.5: amended soil with BF at 0.5% (w/w); (g) BF 1: amended soil with SS at 1% (w/w). 2.4.3. Biochemical soil assays: enzymatic activity determinations Dehydrogenase activity (DHA) was determined by the modified method of García et al. (1993) based on the measurement of iodonitrophenylformazan formed by the reduction of 2-p-iodo- 3-nitrophenyl-5-phenyl tetrazolium chloride (INT) (García et al., 1993). In this procedure, 0.5 g of soil was exposed to 2 ml of 0.2% INT and 1 ml of 20 mM sodium succinate in distilled water for 30 min at room temperature in darkness. The iodonitrotetrazaolium formazan (INTF) formed was extracted with 5 ml of ethylacetate by shaking vigorously for 20 min. INTF was measured spectrophotometrically at 490 nm using a spectrophotometer (GeneQuant 1300, GE Healthcare Bio-Sciences AB, USA). Controls were prepared without substrate.Phosphatase activity (PHA) was measured using p-nitrophenyl phosphate disodium as substrate (Tabatabai and Bremner, 1969). Enzymatic activity was measured using 0.025 M p-nitrophenyl phosphate. 0.5 g of soil was incubated at 37◦C in malate buffer MUB (pH 6.5) for 30 min. 0.5 M CaCl2and 0.5 M NaOH were added to stop the reaction and to extract the product, i.e. pnitrophenol. The concentration of p-nitrophenol was determined spectrophotometrically at 410 nm. Controls were prepared without substrate. ␤-Glucosidase activity (GA) was determined using pnitrophenyl-b-d-glucopyranoside as substrate (Masciandaro et al., 1994). Enzymatic activity was measured using 0.025 M p-nitrophenyl-b-d-glucopyranoside. 0.5 M NaOH was added to stop the reaction and extract the product, i.e. p-nitrophenol. The product concentrations were determined spectrophotometrically at 410 nm. Controls were prepared without substrate Table 2 Average composition of SS, IP and BF. Different letters following the number indicate a significant difference at p < 0.05. SS IP BF pH (1/10) 7.9a ± 0.2 8.2a ± 0.3 7.8a ± 0.2 Organic matter (g kg−1) 392a ± 13 327a ± 15 555b ± 19 Total N (g kg−1) 27.0b ± 1.4 15.8a ± 1.1 29.3b ± 1.6 Proteins (g kg−1) 168.7b ± 9.1 102.7a ± 7.1 190.4b ± 10.4 2.5. Statistical analysis Analysis of variance (ANOVA) was performed using the Statgraphics v. 5.0 software package. Means were separated by the LSD test, considering a significance level of p < 0.05 throughout the study. For the ANOVA, triplicate data were used for each product and every day of incubation. 3. Results and discussion 3.1. Chemical properties of the hydrolysis products In this study, the enzymatic hydrolysis of SS generated two byproducts: IP and BF. Table 2 shows the average composition of both by-products, showing that these materials are highly bioavailable. As observed in Table 2, soluble organic matter content in the BF raised significantly (p < 0.05) up to 555 ± 1.6 g kg−1, which represents an increase of 41.6% compared to the original SS. On the other hand, the protein content was increased in BF by 12.8%, although, the statistical analysis indicated that this increase was not significant. Moreover, there was a notable difference in both K and S content (Table 3), elements that have a great significance on the physical properties of soil since they constitute the basis of plant nutrition. To this respect, K and S contents were 96.5% and 28.4% lower, respectively, in SS than in BF. Regarding the remaining nutrients analysed, their concentration decreased in BF compared with the SS content (being increased in IP due to the precipitation process) (Table 3). The decrease in P content in BF of around 89.5% is remarkable (p < 0.05; Table 3). The chemical properties of the BF had a great effect on the use of BF as biofertiliser and soil stimulant. An increase in the organic matter content and in fundamental elements for plant nutrition, such as K, has a major influence in converting BF to a more advantageous product for agronomic use than the starting raw material, SS, whose characteristics depend upon the quality of sewage and type of treatment processes followed (Singh and Agrawal, 2008). SS may substitute for fertiliser but the usual way for using this residue from wastewater treatment is the composting process. The application of SS (Albiach et al., 2001) Table 3 Elemental composition of SS, IP and BF. SS IP BF P (g kg−1) 20.9a†± 1.6 21.5a ± 1.3 2.2b ± 0.4 K (g kg−1) 2.9a†± 0.3 194.2c ± 12.1 83.2b ± 15.6 S (g kg−1) 6.3a†± 1.0 6.7a ± 1.1 8.8b ± 0.9 Ca (g kg−1) 85.7a†± 3.3 100.5a ± 4.7 26.5b ± 1.9 Mg (g kg−1) 6.3a†± 1.6 7.8b ± 1.4 5.4a ± 0.8 B (g kg−1) 70.8a†± 2.6 82.1a ± 3.9 50.1b ± 2.1 Fe (g kg−1) 27.8a†± 1.6 20.1a ± 2.3 0.6b ± 0.1 Cu (mg kg−1) 300a†± 10.9 327a ± 11.8 33.8b ± 1.5 Mn (mg kg−1) 196.1ab†± 14.8 237.3b ± 19.6 116.1a ± 12.3 Zn (mg kg−1) 736a†± 16 778a ± 22 87.6b ± 2.3 Cd (mg kg−1) 1.4a†± 0.3 1.5a ± 0.6 0.15b ± 0.05 Pb (mg kg−1) 51.2a†± 1.9 56.7a ± 1.6 ND Ni (mg kg−1) 36a†± 8 31.9a ± 5.5 4.3b ± 0.9 Hg (mg kg−1) 1.6a†± 0.3 2.2a ± 0.4 0.04b ± 0.002 †Different letters following the number indicate a significant difference at p < 0.05. 34 M. Tejada et al. / Ecological Engineering 50 (2013) 31– 36 or compost (Tejada and González, 2003) to soil is a current environmental and agricultural practice for maintaining soil organic matter, reclaiming degraded soils and supplying plant nutrients. However, SS compost use has drawbacks such as the high amount of time required to generate compost, odour generation from composting operations (Sikora, 1998) and lack of process control which avoid SS compost bioavailability. Moreover, the low bioavailability of nutrients and the presence of toxic metals restrict its uses (Ranalli et al., 2001). In fact, the major limitation of soil application of SS compost is the total heavy metal content and their availability to the soil–plant system (Amir et al., 2005). To this regard, when comparing heavy metal contents in the product to that of the original SS, it is observed that their concentration significantly decreased (p < 0.05) in the fertilising soluble product and most of them were retained in the IP (Table 3). The reduction in Fe content was 97.8%, 88.7% for Cu, 40.8% for Mn, 88.1% for Zn, 89.3% for Cd, 88.1% for Ni and 97.5% for Hg. Pb concentrations in BF were not detectable. As mentioned above, the proposed hydrolytic process occurs at an alkaline pH (pH 8.5), and in those circumstances the heavy metals precipitate and are concentrated in the IP. In the environment, heavy metals have been identified as the most dangerous pollutants, so attention is increasingly being given to the potential health hazard presented by heavy metals in the environment. Heavy metals are not only biodegradable and toxic at some concentrations, but also they tend to accumulate along the food chain where man is the last link (Dudka and Miller, 1999). Bioavailability of heavy metals and their transfer to plants, groundwater, animals and, finally, to humans depends on the SS quality (Katsoyiannis and Samara, 2007). During composting, heavy metal bioavailability is dependent on several physico-chemical properties of the medium besides total metal content such as decomposition of organic matter, humic substances content and pH (Amir et al., 2005), which may complicate the composting process and the removal of heavy metal content. However, the low heavy metal content found in the BF product has important environmental implications, since the final use of BF as fertiliser will not cause dangerous damage to the ecosystem. Thus, the enzymatic hydrolytic process could constitute in an effective treatment to remove heavy metals from SS. Nevertheless, heavy metals are accumulated in the IP and, therefore, this by-product requires an adequate treatment. Another aspect of the enzymatic hydrolysis process on SS is the protein content, which was increased in BF compared to the raw material, although without statistical significance. However, the analysis by size-exclusion chromatography of soluble protein components of SS and BF showed differences in protein solubility (Fig. 1). The distribution of the chromatogram obtained for the SS indicates that the proteins were highly insoluble. During the hydrolytic process, the protein component was broken down, and so the peptide and free amino acid content was increased until the end of the process (120 min), displaying different chromatographic signals throughout the process. As a final product, BF showed a soluble protein component characterised mainly by small molecular peptides, and even free amino acids (<10 kDa). The decrease in the molecular size indicates that N is in a form readily bioavailable to soil-living organisms, such as plants (Higgings and Payne, 1982) and microorganisms (Vasileva-Tonkova et al., 2007), since peptides are an ideal vehicle to transport N in soils, better than free amino acids (Higgings and Payne, 1982). Our results show several benefits of the molecular size of the proteins on the biostimulation process. Proteins must be hydrolysed to peptides and amino acids by extracellular proteases to be absorbed by plants and microorganism, due to the lack of carrier proteins that transfer proteins intact through cell membranes (Jan et al., 2009). Recent studies have suggested that plants are able to absorb N easily from soil in the form of amino acids, although the significance of this in the regulation of 0 10 20 30 40 50 Abs 214nm (mAU) -200 0 200 400 600 800 1000 1200 1400 BF Intermediate BF (1h) EV (ml) SS Fig. 1. Proteins’ molecular weight distribution in time of SS and the hydrolysed product (BF) determined by size-exclusion chromatography (ÄKTA-purifier, GE Healthcare, employing a Superdex Peptide 10/300GL column. productivity of plants is unknown (Jones et al., 2009). On the other hand, amino acids and small peptides can be absorbed easily by soil microbial populations through a series of membrane transporters (Jones and Hodge, 1999; Walker and Altaman, 2005). This suggests that the breakdown of proteins in soil is the bottleneck step in the N incorporation for soil microorganisms. 3.2. Biochemical soil assays Fig. 2 shows the evolution of enzymatic activities (dehydrogenase, phosphatase and ␤-glucosidase) in soils amended with SS and BF in different dosages. The application of both SS and BF products to the soil caused a stimulatory effect on DHA. Land application of both products serves as a good source of microbial nutrient and the organic constituents providing beneficial soil-conditioning properties (Logan and Harrison, 1995; Singh and Agrawal, 2008). This stimulating effect was dose-dependent and changed with the type of organic material. Thus, for SS-amended soil, DHA was progressively increased until the end of the incubation period. However, in BF-amended soil, the stimulant effect was extremely marked from the first day of incubation. Moreover, as it is shown, after 5 days the values of DHA measured in BF-amended soils increased significantly compared to the control soil and those amended with SS. At that time, the increase observed was not dose-dependent and was about 35 mmol INTF g−1soil−1min−1in BF-amended soils. Over the experiment, DHA was higher in BF-amended soils compared to the SS-amended treatments. After 14 days, treatments with BF showed how DHA increased significantly up to almost 21-, 13.5- and 11- fold in BF 0.2, BF 0.5 and BF 1, respectively, when compared to SS-amended soil. Regarding the dosage effect, this appeared along the incubation period: after 14 days, the BF 1 treatment showed a 1.7- and 2-fold higher DHA than in BF 0.5 and BF 0.2 treatments, respectively. At the end of the experiment, the dose effect remained and the DHA levels were increased significantly in BF 1 treatment compared with BF 0.5 and BF 0.2 treatments. The results obtained for DHA, an intracellular enzyme involved in microbial respiratory metabolism (Masciandaro et al., 2001), demonstrate that the biostimulant capacity of SS and BF are different, being significantly higher in BF, which suggests that it is a consequence of the lower molecular size of the protein content in BF. The higher values of this enzyme in BF-amended soils suggest that the bioavailability of the organic material is significantly higher and this causes an increase in the microbial activity. The enzymatic hydrolysis process solves the problem of the N-cycle in soil: the breakdown of proteins into peptides and free amino acids (Jones et al., 2009). In fact, the M. Tejada et al. / Ecological Engineering 50 (2013) 31– 36 35 Fig. 2. Evolution of enzymatic activities (dehydrogenase, phosphatase and ␤- glucosidase) in soils amended with SS and BF. Data are expressed as mean values ± standard error. Columns (mean ± S.E.) followed by the same letter(s) are not significantly different (p < 0.05). INTF, 2-p-iodo-3-nitrophenyl; PNP, p-nitrophenol. constituent proteins of the organic matter tend to be quite resistant to microbial attack due to physico-chemical protection provided by soil colloids (Schulten and Schnitzer, 1998) and the range of mineralisation depends on the ecosystem (Joanisse et al., 2008). This demonstrates that the proteins are not as accessible as peptides and free amino acids. In addition, the application of SS and BF to the soil caused a stimulation of PHA. However, this stimulation was different to that detected for DHA. In fact, the stimulating effect was higher in soils amended with SS than with BF. Once again, the application rate greatly influenced the stimulation. At the end of the incubation period and when compared with treatment BF 1, PHA was up to 60.2% higher in the SS1 microcosm. Compared with the control treatment, the PHA showed a minor stimulation for the soils amended with BF compared with SS. To understand these results, it should be noted that microorganisms produce this enzyme due to the demand for P (García et al., 1994). In SS, there is a high content of P (20.9 g kg−1), while in BF, the P content drastically decreases (2.2 g kg−1). However, most of the P from SS was part of the insoluble paste. In BF, the existing P is in soluble form, and although its concentration is lower, it is accessible and easily absorbed. Regarding GA activity, an increase was observed after the application of both organic amendments to the soil. This stimulation was similar to that obtained for DHA. At the end of the incubation period and compared to the SS1 treatment, GA was significantly higher (41%) in the BF1 treatment. Moreover, a dose-dependent effect was observed during the incubation time. 4. Conclusions The hydrolysis of SS generated in WWTPs is an alternative process to composting for land application purposes. In this study, a new enzymatic process has been designed for obtaining a new fertilising product (BF) from SS. This product is mainly characterised by its low content of heavy metals and low molecular size of proteins. The hydrolysed product obtained presents a high stability and bioavailability thus enhancing the enzymatic activities of the soil’s microbial community. BF is mainly constituted by peptides and free amino acids that have a potential biostimulatory capacity, higher than SS which is mainly composed by large proteins. Thus, the results of enzymatic activities in soil suggest that this new product has a high potential for stimulating soil microorganisms, and therefore could be used for fertilisation and soil bioremediation.Unlike other processes such as composting and chemical acid hydrolysis, the enzymatic hydrolysis has important advantages in terms of both product quality and technological level. Firstly, the process is technologically controllable (type of enzyme used in the hydrolysis, reaction time, etc.), leading to a uniform product and the possible design of different types of hydrolysates. Secondly, all peptides and amino acids are conserved and there are no nutritional losses, so the quality of BF formed principally of peptides is superior to that of a mixture of free amino acids. This characteristic is very important, since free amino acids are generally unstable and hydrophobic amino acids (tryptophan, valine, isoleucine, leucine, etc.) cannot be administered via liquid due to their insolubility. In addition, the process is biological and respectful to the environment, being non-pollutant (neutral pH, no utilisation of chemical products, etc.). Acknowledgments This work was supported by the Ministry of Science and Technology (Spain), Plan Nacional I+D CTM2007-60210/TECNO and CTM2011-29930-C03-01 References Adler-Nissen, J., 1977. Enzymatic hydrolysis of food protein. 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Short-time effects on soil biochemical properties Bruno Rodríguez-Morgado a , Pablo Caballero a , Patricia Paneque b , Isidoro Gómez b , Juan Parrado a and Manuel Tejada b a Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain; b Grupo de Investigación Edafología Ambiental, Departamento de Cristalografía, Mineralogía y Química Agrícola, E.T.S.I.A. Universidad de Sevilla, Sevilla, Spain ABSTRACT In this manuscript, we study the manufacture and effect on soils of different edaphic biostimulants/ biofertilizers (BS) obtained from sewage sludge using Bacillus licheniformis as biological tool. These BS consist of different combinations of organic matter, bacteria and enzymes that were subjected to several treatments. These BS were applied in soil in order to observe their influence on the biochemical properties (enzymatic activities and ergosterol content). Dehydrogenase, urease, βglucosidase, phosphatase activities and ergosterol content were measured at different incubation days. Only dehydrogenase activity and ergosterol content were significantly stimulated after the application of BS1 and BS4. Rest of the extracellular activities were not stimulated probably because B. licheniformis practically has digested all organic substrates during fermentation process. ARTICLE HISTORY Received 27 July 2017 Accepted 4 October 2017 KEYWORDS Sewage sludge; Bacillus licheniformis; fermentation process; edaphic biostimulant/biofertilizer; soil biochemical properties 1. Introduction Currently, the continuous and progressive growth in the world population as well as the awareness of the need to treat wastewater has led to an increase in the volume of sewage sludge produced [1,2]. Also, all this amount of sewage sludge requires new methods for its use and disposal [2]. Due to high content of organic matter as well as essential nutrients, application of sewage sludge to agricultural soils is of great interest in order to improve physical, chemical and biological properties of such soils and to provide essential nutrients to plants [3–6]. However, as they could often contain considerable amounts of heavy metals, organic contaminants and pathogens (e.g. helminth eggs, protozoan cysts, Escherichia coli, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, halogenated organic compounds, linear alkylbenzene sulfonates, bis (2-ethylhexyl) phthalate, nonylphenol ethoxylates, polychlorinated dibenzodioxines and dibenzofuranes) and may pose health risks, use of sewage sludges is not feasible [7–12]. Therefore, sewage sludge composting for its posterior use is a good solution. In addition to eliminating potentially harmful organisms, this technique helps stabilize organic matter, converting sewage sludge into a stabilized, nutrient-rich compost suitable for agricultural applications [13,14]. It is well known that composting sewage sludge entail several problems. The high moisture content and low C/N ratio are responsible for significant losses of N in the form of ammonia during composting process, thus decrease the quality of the compost obtained [15,16]. In addition, long processing time, low level of control over the process and the presence of large solids that hinder some agronomic applications must be taken into account [17]. This has led several researchers to seek alternatives for composting in order to increase the use of sewage sludge. In previous works, Rodríguez-Morgado et al. [17] and Tejada et al. [18] obtained an edaphic biostimulant/biofertilizer (BS) from sewage sludge using proteolytic enzymes. That BS was characterized by a low concentration of heavy metals and a high concentration of low molecular-weight peptides (<300 Daltons), which can be assimilated directly by soil microorganisms, stimulating them significantly. These authors demonstrated that microbial stimulation is essential for the degradation of xenobiotic substances in soil such as pesticides and hydrocarbons [19–22]. The use of Bacillus licheniformis as a tool to obtain BS has been described. Furthermore, this bacteria is able to produce and secrete numerous hydrolytic enzymes that enable the microorganisms to degrade many different © 2017 Informa UK Limited, trading as Taylor & Francis Group CONTACT Manuel Tejada [email protected] Grupo de Investigación Edafología Ambiental, Departamento de Cristalografía, Mineralogía y Química Agrícola, E.T.S.I.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, Spain ENVIRONMENTAL TECHNOLOGY 2019, VOL. 40, NO. 3, 399–406 https://doi.org/10.1080/09593330.2017.1393016 substrates and grow in a wide range of nutrient sources [23]. These new products, which also act as biostimulants of soil microbial community, could be very useful in organic-xenobiotics-contaminated soils’bioremediation. The objective of this paper was to study the influence of different BSs obtained by fermentation process from a sewage sludge on the biochemical characteristics of a Mediterranean agricultural soil. 2. Material and methods 2.1. Sampling of sewage sludge and fermentation process The sewage sludge was supplied by the Experimental Water Treatment Plant of the Center for New Water Technologies (CENTA) located in Carrión de los Céspedes (Seville, Spain). The sludge had a maturity of 4 months and its chemical characteristics are described in Table 1. The methodology used in determination of pH, macro and micronutrients and molecular mass distribution was the same, as described in Rodriguez-Morgado et al. [17]. In order to obtain the different experimental BS, the sewage sludge was subjected to a fermentation process. For this purpose, sewage sludge was autoclaved, inoculated with B. licheniformis ATCC21415 (2% w/w) and incubated under agitation for 7 days at 37°C, thus obtaining what was termed as BS1 (Figure 1). Once this BS was obtained, the following treatments were performed to obtain the rest of experimental BS: (1) Fermented sludge was centrifuged, obtaining a liquid fraction that was discarded and a solid fraction, consisting mainly of microbial biomass generated during the process. This new product was called BS2 (Figure 2). (2) Fermented sludge was centrifuged, obtaining a liquid fraction that was subjected to heat treatment in order to eliminate undesirable enzymes and a solid fraction. Both fractions were mixed again to obtain BS3 (Figure 3). Table 1. Chemical characteristics and protein molecular-weight distribution (mean ± standard error, n= 3) of sewage sludge and biostimulants obtained by fermentation processes. SS BS1 BS2 BS3 BS4 Dry matter (%) 5.1a a ± 0.4 5.3a ± 0.3 16.7b ± 1.1 5.2a ± 0.2 5.3a ± 0.3 pH 6.6a a ± 0.2 8.1b ± 0.1 8.3b ± 0.2 8.0b ± 0.2 8.2b ± 0.2 Organic matter (g kg −1 ) 472a a ± 14 470a ± 16 475a ± 18 470a ± 11 473a ± 15 N(gkg −1 ) 30.5a a ± 7.9 30.3a ± 7.0 21.6a ± 5.1 31.2a ± 7.1 30.0a ± 6.9 P(gkg −1 ) 12.2a a ± 1.4 12.0a ± 1.6 17.1a ± 1.9 12.1a ± 1.9 12.2a ± 1.5 K(gkg −1 ) 6.2a a ± 1.6 6.4a ± 1.2 6.2a ± 1.7 6.1a ± 1.6 6.4a ± 1.3 S(gkg −1 ) 16.6a a ± 1.4 16.6a ± 1.2 13.9a ± 1.1 16.9a ± 1.4 16.1a ± 1.2 Ca (g kg −1 ) 44.8a ± 4.1 43.5a ± 3.6 45.1a ± 2.7 45.1a ± 4.2 43.8a ± 3.2 Mg (g kg −1 ) 7.1a a ± 1.7 7.3a ± 1.4 8.1a ± 1.6 7.0a ± 1.6 7.3a ± 1.3 Fe (g kg −1 ) 18.7a a ± 1.5 18.9a ± 1.3 20.8a ± 1.8 19.0a ± 1.2 18.5a ± 1.3 Si (g kg −1 ) 25.5a a ± 2.6 25.9a ± 2.4 21.3a ± 1.7 25.9a ± 2.6 25.3a ± 2.1 Cu (mg kg −1 ) 341a a ± 14 344a ± 17 401a ± 15 344a ± 18 347a ± 12 Mn (mg kg −1 ) 166a a ± 4 160a ± 5 162a ± 5 165a ± 4 164a ± 4 Zn (mg kg −1 ) 864a a ± 15 874a ± 14 887a ± 22 860a ± 14 862a ± 17 Protein molecular-weight distribution (Da) >10,000 98.5b a ± 1.1 43.3a ± 1.4 ND 42.2a ± 2.1 43.9a ± 2.9 10,000–5000 0.0a a ± 0.0 14.7b ± 1.1 ND 15.1b ± 1.3 14.1b ± 1.6 5000–1000 1.5a a ± 0.8 11.5b ± 1.8 ND 12.6b ± 1.4 10.5b ± 1.4 1000–300 0.0a a ± 0.0 1.7b ± 0.3 ND 1.3b ± 0.2 1.2b ± 0.2 <300 0.0a a ± 0.0 26.7b ± 2.1 ND 28.8b ± 1.9 30.3b ± 2.2 Notes: SS: sewage sludge; BS1: biostimulant/biofertilizer 1; BS2: biostimulant/biofertilizer 2; BS3: biostimulant/biofertilizer 3; BS4: biostimulant/biofertilizer 4. ND: Not determined because the compound is insoluble. a Files followed by the same letter(s) are not significantly different according to the Tukey test (p< .05). Figure 1. Process for obtaining the edaphic biostimulant/biofertilizer 1. 400 B. RODRÍGUEZ-MORGADO ET AL. (3) To obtain BS4, fermented sludge was heat treated in order to destroy both enzymes and microorganisms (bacteria). BS4 is composed of organic matter only, with no biological or biochemical activity (Figure 4). Chemical composition of these new biostimulants is shown in Table 1. As with sewage sludge, methodology used to determine all chemical parameters analysed is detailed in Rodríguez-Morgado et al. [17]. 2.2. Biostimulation experiment design Soil used in this experiment is a Calcaric Regosol [24]. Soil samples were collected at a depth of 0–25 cm. Main soil characteristics have already been reported [19] and are summarized in Table 2. Soil microcosms were used to investigate the effects of four BS on soil biochemical properties. The microcosms consisted of 300 g grams of dry soil, sieved to 2 mm and placed in dark glass vessels. Soil was amended with the four BSs obtained at a rate of 1% of organic matter due to the fact that the organic content of each BS was different. In this respect, soil samples were mixed with 60 g of BS1 (S + BS1 treatment), 38.6 g of BS2 (S + BS2 treatment), 60 g of BS3 (S + BS3 treatment) and 71.4 g of BS4 (S + BS4 treatment). An unamended soil was used as control (S). All experimental BSs were liquid, having been solubilized Figure 2. Process for obtaining the edaphic biostimulant/biofertilizer 2. Figure 3. Process for obtaining the edaphic biostimulant/biofertilizer 3. ENVIRONMENTAL TECHNOLOGY 401 Capítulo 1 Resultados y Discusión 91 4.1. Proteomic analysis of enzyme production by Bacillus licheniformis using different feather wastes as the sole fermentation media Dado que la optimización de los procesos de producción resulta clave en la biotecnología en general y en los procesos fermentativos en particular, en este trabajo se han evaluado las plumas de pollo como medio de cultivo y de producción de enzimas para el organismo B. licheniformis a través de la manipulación de parámetros nutricionales y físicoquímicos, estudiándose además el secretoma mediante técnicas proteómicas. Caracterización química de las plumas Las plumas están compuestas principalmente por queratina, una proteína fibrilar insoluble, rica en azufre y relativamente resistente a la degradación. Además de queratina, en las plumas existen otros componentes secundarios como son pequeñas cantidades de lípidos y carbohidratos. En este estudio se han empleado dos tipos de plumas, la pluma TG, con un contenido en proteínas de más del 90% y un 2% de lípidos, y la pluma MU, con un contenido en proteínas en torno al 65% y un contenido en lípidos del 30%. Estas diferencias en el contenido en lípidos pueden ser determinantes a la hora de determinar las diferencias de crecimiento y producción extracelular observadas (Kim y col., 2001; Fakhfakh-Zouari y col., 2010; Kornillowicz-Kowalska y Bohacz, 2011). Proceso de fermentación Bacillus licheniformis es capaz de solubilizar en condiciones aeróbicas de ambos tipos de plumas a las concentraciones estudiadas sin el aporte de fuentes externas de carbono, nitrógeno u otros elementos. Esta solubilización es inversamente proporcional a la concentración de plumas, de forma que a los 5 días de incubación y a las concentraciones más Capítulo 1 Resultados y Discusión 92 bajas (0,2 y 1%) la solubilización es total, no apreciándose restos sólidos de pluma. A la concentración más alta estudiada (5%), esta solubilización no fue completa, existiendo unos remanentes insolubles del 57 y 35% en las muestras MU y TG respectivamente. Figura 10: Cultivo de B. licheniformis en bioreactor de laboratorio. Estas diferencias en las tasas de solubilización pueden ser debidas a una reducción en la excreción de enzimas o bien a un crecimiento menor de la bacteria. Con el fin de determinar si el caso se corresponde con la última hipótesis, se ha estudiado la actividad microbiana de los cultivos mediante la determinación de la actividad deshidrogenasa, enzima intracelular implicada en los procesos de fosforilación oxidativa e indicador de la actividad microbiana (Bolton y col., 1985). Cuando B. licheniformis se cultiva en un medio rico (medio LB), se observa un rápido incremento de esta actividad a las 18 horas de incubación gracias a la alta disponibilidad de los nutrientes presentes. Sin embargo, cuando se cultiva en los medios compuestos por Capítulo 1 Resultados y Discusión 93 plumas, este máximo de actividad se retrasa en las muestras TG y Mu (1%) a las 42 y 90 horas respectivamente. El retraso observado entre ambos tipos de plumas puede ser debido a la presencia de grasas que impiden la colonización superficial de la pluma por B. licheniformis. La actividad deshidrogenasa no está vinculada directamente con la solubilización de la queratina, lo que sugiere que la excreción de enzimas no está relacionada con el nivel de crecimiento microbiano, pero si puede estar inducida por las condiciones de fermentación. De forma contraria a la solubilización de la pluma, la actividad microbiana está directamente relacionada con la concentración y tipo de pluma empleado, observándose los valores más altos en las muestras MU a concentraciones del 5%. Cuando se estudió el perfil de pesos moleculares de las proteínas solubles mediante cromatografía de exclusión molecular, se observan diferencias entre las plumas MU y TG (1%). La muestra MU muestra un contenido mayor en la fracción de bajo peso molecular probablemente debido a la expresión diferencial de enzimas hidrolíticas observadas entre ambos tipos de plumas. Producción de proteasas Bacillus licheniformis es un microorganismo ampliamente empleado en la producción de enzimas de uso industrial principalmente proteasas (Pillai y col., 2011; Zaghloul y col., 2011), así como se ha descrito el uso de plumas como sustrato para la producción de proteasas por diversos bacilos (Wang y col., 2005; Fakhfakh-Zouari y col., 2010; Daroit y col., 2011). La producción de proteasas por B. licheniformis cultivado en un medio queratinoso, al igual que la solubilización del sustrato, está inversamente relacionada con la concentración de pluma, independientemente de la pluma utilizada. A concentraciones relativamente altas (>1%), la producción de proteasas se encuentra afectada negativamente. Capítulo 1 Resultados y Discusión 94 La excreción de proteasas por parte del genero Bacillus es una respuesta al agotamiento de nutrientes en el medio (Van Putten y col., 1996; Calik y col., 2002; Romero Ramírez y col., 2007), lo que explica la producción de esta enzima a bajas concentraciones de sustrato. Además, la presencia de un sustrato con una alta relación carbono / nitrógeno parece producir una mayor inducción de estas enzimas. Producción de lipasas Varias especies de Bacillus, entre ellas B. licheniformis, están reconocidas como productoras de lipasas, unas enzimas de uso industrial capaces de hidrolizar los triacil glicéridos (Nthangeni y col., 2001; Chen y col., 2004). Las plumas de pollo constituyen un sustrato adecuado para la producción de estas enzimas, estando su excreción directamente relacionada con la concentración de sustrato. De esta forma, los mayores valores de actividad lipasa se han encontrado en las muestras al 5%. Aunque ambos tipos de pluma inducen esta enzima, las muestras TG presentan unos valores sensiblemente inferiores a los encontrados en las muestras MU, probablemente debido al mayor contenido en lípidos de esta última. Comparando la producción de proteasas y lipasas, la pluma MU es la que produce una mayor inducción de ambas enzimas a causa del mayor contenido en fuentes de carbono de esta pluma (Kapoor y Gupta, 2002; Gupta y Rathi, 2004). De esta forma queda corroborado que la composición del medio afecta al secretoma de B. licheniformis. Análisis proteómico Para analizar la producción de proteínas extracelulares de Bacillus licheniformis cultivado en medios elaborados con plumas de pollo se han utilizado las concentraciones intermedias de sustrato (1%) así como la muestra crecida en medio rico (medio LB). En cualquiera de los medios utilizados B. licheniformis ha sido capaz de crecer y de excretar Capítulo 1 Resultados y Discusión 95 diversas proteínas (Sinchaikul y col., 2002; Voigt y col., 2004; Harwood y Cranenburgh, 2007; Van Dyk y col., 2010). Muchas especies del género Bacillus son capaces de producir diversas hidrolasas en respuesta a la presencia de ciertos inductores en el medio, como son los propios sustratos macromoleculares a hidrolizar (Chu y col., 2000; Sinchaikul y col., 2002). Esta inducción en la excreción de enzimas al medio extracelular tiene como objetivo la captación de nutrientes y energía. La pluma TG induce principalmente la producción de una proteasa, la queratinasa (Ker A), probablemente debido al hecho de que esta pluma se compone en más de un 90% de proteínas (queratinas mayoritariamente). Esta enzima posee numerosos usos tanto industriales como agronómicos. Junto con la queratinasa, aunque en menor medida, se excreta la enzima Ggt o gamma-glutamil transpeptidasa (EC 2.3.2.2), que cataliza la transferencia de restos glutamilo desde diversos compuestos que lo contienen hasta una amplia variedad de aminoácidos y dipéptidos (Suzuki y col., 2003; Wu y col., 2006). Ambas enzimas se asocian para incrementar el espectro de acción de la queratinasa así como su potencial catalítico (Tiwary y Gupta, 2010a; b). Además de estas dos enzimas, B. licheniformis también excreta otras proteínas, como son glicosidasa a fin de obtener los azúcares existentes en forma de polisacáridos en las plumas TG, etc. La pluma MU es capaz de inducir una mayor variedad de proteínas probablemente debido a que su composición es más compleja que la pluma TG. Aun así, la proteína más inducida en ambos casos es la queratinasa asociada a la gamma-glutamil transpeptidasa. La segunda proteína más producida por Bacillus licheniformis es una quitosanasa (EC 3.2.1.132), que cataliza la hidrólisis del quitosano. Dado que no existe quitosano en la composición de la pluma MU, este enzima puede aparecer debido a una actividad lipasa secundaria e inespecífica. Esta actividad es posible debido a la similitud entre los sitios activos de las Capítulo 1 Resultados y Discusión 96 quitosanasas y las lipasas (Muzzarelli y col., 1995; Struszczyk y col., 2009). Se conocen varios casos de lipasas capaces de hidrolizar quitosano (Qin y col., 2002; Sashiwa y col., 2002; Muzzarelli y col., 2004), probablemente debido a la simplicidad y similitud de los mecanismos hidrolíticos implicados (Fu y col., 2003; Roy y col., 2003). De forma similar al medio formulado con pluma TG, la pluma MU también induce la excreción de otras hidrolasas como las glicosidasas. Aunque tanto en las muestras TG como MU se produce la inducción de la enzima Ker A, el segundo tipo de pluma también induce la producción de otras proteasas con mecanismos de acción y especificidades distintas, lo que podría explicar el hecho de que el tamaño molecular de los péptidos en la muestra MU es menor. Cuando B. licheniformis se crece en un medio rico (medio LB), se observa una amplia variedad de proteínas extracelulares y otras asociadas a la pared bacteriana, todas ellas producidas de forma basal (Antelmann y col., 2005; Voigt y col., 2005). También se detectó la presencia de Ker A y Ggt, lo que indica que estas proteínas se excretan de forma constitutiva. Capítulo 1 Resultados y Discusión 97 4.2. Hydrolytic enzymes production by Bacillus licheniformis growth on fermentation media formulated with sewage sludge Con el objetivo de encontrar un medio de cultivo económico y abundante para la producción de enzimas hidrolíticas por parte de B. licheniformis, hemos usado lodos de depuradora. En este medio, este organismo es capaz de crecer, aunque sin una gran producción de biomasa. Sin embargo, si se produce una importante producción de enzimas hidrolíticas extracelulares, como son proteasas, lipasas o celulasas, todas ellas de gran interés comercial e industrial. La producción de enzimas extracelulares por microrganismos suele estar altamente influenciada por la composición del medio, por parámetros como la relación carbono / nitrógeno, presencia de fuentes de nutrientes fácilmente disponibles o iones metálicos (Kole y col., 1988; Ferrero y col., 1996; Varela y col., 1996). A esto se le unen otros factores como la oxigenación, la densidad del inóculo, pH, temperatura etc. (Nehete y col., 1985; Hameed y col., 1999). Las proteasas son probablemente las enzimas más comercializadas del mundo, empleándose más de dos tercios de la producción en la industria de los detergentes (Gupta y col., 2002a). Este uso se debe principalmente a la existencia de las proteasas alcalinas, muchas de las cuales son excretadas por especies del género Bacillus (Ward, 1983). Estas proteasas alcalinas trabajan a pH entre 8,5 y 10, el cual es óptimo para muchas formulaciones de detergentes (Maurer, 2004). Otros usos de estas proteasas son la producción de alimentos, la industria farmacéutica, las curtidurías, etc. (Gupta y col., 2002b). Bacillus licheniformis produce proteasas extracelulares durante la fase exponencial y al comienzo de la fase estacionaria (Kole y col., 1988), disminuyendo a continuación. Para aumentar los rendimientos de producción de proteasas, se ha estudiado la adicción de plumas Capítulo 1 Resultados y Discusión 98 al medio de cultivo, produciéndose una inducción en la producción de estas hidrolasas (Ferrero y col., 1996; Varela y col., 1996, Parrado y col., 2014). Las lipasas, enzimas que catalizan la hidrolisis de los triacil gliceroles, tienen un especial interés en industrias como la de los detergentes, alimentaria, química o farmacéutica (Patel y col., 2016). Bacillus licheniformis, organismo ampliamente reconocido como productor de estas enzimas, es capaz de producir lipasas cuando se cultiva en medios de fermentación formulados con lodos de depuradora. Además, la adición de plumas produce una inducción aún mayor. Esta inducción puede deberse al relativamente alto contenido en grasas de las plumas. No obstante, se observa como la producción en los medios sin suplementar empieza al inicio del cultivo, mientras que en el medio con plumas esta producción se retrasa hasta 4 días. También se ha estudiado la producción de enzimas capaces de degradar los materiales celulósicos, como son las celulasas, ya que estas enzimas tienen un gran uso en procesos de biorrefinería como los de obtención de biocarburantes. B. licheniformis, reconocido también como productor de estas enzimas, es capaz de excretar celulasas cuando se cultiva en un medio con lodos de depuradora, comenzando esta producción al inicio del cultivo y alcanzando su valor máximo tras 5 días. Cuando se añade pluma de pollo al medio, también se produce una importante inducción de esta enzima, aunque se desconoce el motivo. Aunque se determinaron otras actividades enzimáticas, como la actividad βglucosidasa o la actividad fosfatasa ácida, no se detectaron indicios de estas. Las únicas actividades enzimáticas detectadas fueron proteasas, lipasas y celulasas, cuya producción fue inducida por la presencia de plumas de pollo en el medio de cultivo. Esto puede ser debido a que B. licheniformis excreta estas enzimas en respuesta a bajas concentraciones de fuentes de carbono o nitrógeno. Capítulo 1 Resultados y Discusión 99 Cuando se estudiaron las proteínas extracelulares presentes en los distintos medios de cultivo, se observó un incremento en el número de proteínas detectadas por SDS-PAGE como consecuencia del crecimiento de Bacillus licheniformis, detectándose por medio de un análisis zimográfico tres proteasas distintas en las muestras M2 y M3. Tras el análisis proteómico de las muestras, se observa como B. licheniformis excreta al medio diversas enzimas a fin de obtener los nutrientes necesarios para su supervivencia (Sinchaikul y col., 2002; Voigt y col., 2004; Harwood y Cranenburgh, 2007; Van Dyk y col., 2010). Entre estas enzimas se encuentran numerosas hidrolasas de macromoléculas, como proteasas, lipasas o polisacaridasas. La inducción de unas enzimas u otras depende de la presencia de compuestos inductores en el medio, como xilano, lípidos, etc. (Chu y col., 2000; Sinchaikul y col., 2002). Cuando se añaden plumas de pollo a un medio de cultivo, estas actúan como fuente de nitrógeno así como de inductor en la secreción de enzimas hidrolíticas como proteasas, lipasas, etc. El análisis proteómico de las proteínas excretadas por B. licheniformis en el medio de cultivo M3 muestra que el 80% de estas eran hidrolasas, siendo predominante la queratinasa Ker A (proteasa). Esta enzima es típicamente producida por este organismo cuando se cultiva en medios con fuentes de nitrógeno insolubles como la queratina. Otra proteína identificada es la Ggt o gamma-glutamil transpeptidasa (EC 2.3.2.2), enzima que cataliza la transferencia de residuos glutamilo (Suzuki y col., 2003; Wu y col., 2006) y que se asocia a Ker A, aumentando el espectro de acción de la proteasa y mejorando su actividad catalítica. Este fenómeno tiene importantes aplicaciones industriales (Tiwary y Gupta, 2010a; b). Capítulo 1 Resultados y Discusión 106 deshidrogenasa. Sin embargo, a diferencia de la actividad enzimática, el valor máximo de ergosterol se detectó a los 12 días de incubación, probablemente debido a que el crecimiento de los hongos es más lento que el de las bacterias (Starke y col., 2016), digiriendo los cuerpos celulares de estás tras haber consumido los nutrientes aportados por los bioestimulantes (Miltner y col., 2009). 5. CONCLUSIONES Capítulo 1 Conclusiones 109 Tras haber desarrollado nuevos procesos para obtener biofertilizantes / bioestimulantes a partir de lodos de depuradora y plumas de pollo aplicando dos tecnologías distintas y después de caracterizar los productos obtenidos, se exponen a continuación las conclusiones más relevantes: 1. Es posible obtener productos biofertilizantes / bioestimulantes mediante el uso de tecnología enzimática o tecnología fermentativa a partir de subproductos agroindustriales como son los lodos de depuradora o las plumas de pollo. 2. Los productos obtenidos por ambas tecnologías son ricos en péptidos bajo peso molecular y aminoácidos libres, lo que les confiere una elevada capacidad bioestimulante. 3. Es posible crecer ciertos microorganismos, en particular la bacteria Bacillus licheniformis, en medios de cultivo formulados con lodos de depuradora, plumas de pollo o combinaciones de ambos. 4. 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Olive farming is one of the most important in the Mediterranean, particularly in Spain, and is located predominantly in Andalusia. In recent years, there have been repeated events of herbicide contamination in soils cultivated with olive and in aquifers near these crops, which has caused some public alarm. This has led to certain herbicides, such as atrazine, simazine, diuron and terbuthylazine, before they were widely used in olive cultivation and be replaced by other less soluble herbicides. Therefore, it is increasingly common to use herbicides such as oxyfluorfen for weed control in the cultivation of olive trees. Oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)- 4-(trifluoromethyl) benzene] is a diphenyl ether herbicide with residual activity and contact, is selective and is more readily absorbed by the leaves, especially by the roots of the buds, with little translocation. However, despite that in recent years, oxyfluorfen is widely used, and there is no information about the toxic effects of this herbicide on the soil activity and microbial population. No studies have been reported using different BS obtained from sewage sludge and chicken feathers to remediate oxyfluorfen-contaminated soil. For this reason, the objective of this study was to investigate the influence of different BS in the behaviour of oxyfluorfen and its influence on soil biological properties. Material and methods Soil, organic wastes and herbicide characteristics The study was conducted from March to June 2013 near Seville City (Andalusia, Spain). The soil used in this experiment is a Calcaric Regosol (FAO 1989). Soil samples were collected from the 0–25-cm surface layer. The main soil characteristics are shown in Table 1. Soil pH was determined in distilled water with a glass electrode (soil/H 2 O ratio 1:2.5). Soil texture was determined by Robinson’s pipette method (SSEW 1982). Total N was determined by the Kjeldahl method (MAPA 1986). Soil organic matter was determined by the method of Yeomans and Bremner (1988). The herbicide used in this experiment was the oxyfluorfen. The commercial formulation Fenfen (24 % p v −1 ,240gl −1 ) was purchased from Lainco, S.A. (Spain). The rate applied to the soil was 4 l ha −1 (recommended application rate). Two edaphic BS were used: (1) BS derived from sewage sludge (SS) by enzymatic hydrolysis process and (2) BS derived from chicken feathers (CF) also obtained by enzymatic hydrolysis. Figure 1shows schematically how both protein hydrolysates were obtained. Both types of BS were chemically analysed (Table 2). Organic matter content was determined by combustion at 550 °C for 6 h. Phosphorus and sulphur were determined after combustion and analysed by inductively coupled plasma atomic emission spectrometry (ICP-AES) using a Fisons- ARL 3410 sequential multielement instrument equipped with a data acquisition and control system. The standard operational conditions of this instrument are summarized as follows: The carrier gas, coolant gas and plasma gas are argon at 80 psi of pressure, the carrier gas flow rate is 0.8 l min −1 , the coolant gas flow rate is 7.5 l min −1 , the plasma gas flow rate is 0.8 l min −1 , and the integration time is 1 s. One mini-torch consumes argon gas at a radio frequency power of 650 W. Crude fat was determined gravimetrically after extraction with hexane for 12 h in a Soxhlet extractor (Clemente et al. 1997). Total nitrogen was determined by the Kjeldahl method (AOAC 1990). The molecular mass distribution of protein in the samples was determined by size-exclusion chromatography using an ÄKTApurifier (GE Healthcare), using a Superdex Peptide TM 10/300 GL column (optimum separation range 0.1–7kDa) (Table 3). Samples were centrifuged at 12,000×gfor 30 min at 4 °C to remove insoluble molecules, and the supernatant was passed through a 0.2-μm filter and loaded into a 0.1-ml loop connected to the ÄKTApurifier system. The column was equilibrated and eluted with 0.25 M Tris–HCl buffer (pH 7.0) in isocratic mode, at a flow rate of 0.5 ml min −1 , and proteins/peptides were detected at 280 and 215 nm with a GE Healthcare UV900 module coupled to the column elution. Experimental layout The experimental layout was a randomized, complete block design with six treatments and three replicates per treatment. The plot size was 4 m×3 m. Oxyfluorfen was surface broadcast on 11 March 2013. Two days after application of oxyfluorfen to soil, both BS were also applied to the soil. Soil Table 1 Characteristics of the experimental soil (mean ± standard error) Data are the means of three samples pH (H 2 O) 7.9±0.2 Coarse sand (g kg −1 )486±49 Fine sand (g kg −1 )130±25 Silt (g kg −1 )123±29 Clay (g kg −1 )260±35 Total N (g kg −1 ) 0.93±0.08 Organic matter (g kg −1 )17±1 11028 Environ Sci Pollut Res (2014) 21:11027–11035 samples were mixed with 6.6 l of SS (5 t organic matter (OM) ha −1 ) or 10.8 l of CF (5 t OM ha −1 ), applying to the soil the same amount of organic matter with each BS. Both BS were liquid and were solubilized in water (500 l ha −1 )beforethe application. An unamended soil was used as control. The treatments are detailed as follows: 1. C, control soil, soil non-organic amended and without oxyfluorfen 2. H, soil with oxyfluorfen and non-organic amended 3. SS, soil without oxyfluorfen and amended with SS 4. CF, soil without oxyfluorfen and amended with CF 5. SS+H, soil with oxyfluorfen and amended with SS 6. CF+H, soil with oxyfluorfen and amended with CF Soil analysis For 2, 4, 7, 9, 20, 30, 60, 90 and 120 days of the application of herbicide to the soil and for each treatment, the activity levels of four soil enzymes were measured. Dehydrogenase activity was measured as the reduction of 2-p-iodo-3-nitrophenyl-5- phenyl tetrazolium chloride to iodonitrophenyl formazan (García et al. 1993). Urease activity was determined by the buffered method of Kandeler and Gerber (1988), using urea as substrate. The β-glucosidase activity was determined using pnitrophenyl-β-D-glucopyranoside as substrate (Masciandaro et al. 1994). Phosphatase activity was measured using pnitrophenyl phosphate as substrate (Tabatabai and Bremner 1969). For 2, 7, 30 and 120 days of the application of herbicide to the soil and for each treatment, phospholipids were extracted (three replicates per treatment) using a chloroform–methanol Enzymatic process -pH: 9 - Temperature : 55 °C -Time: 180 min -Proteolytic enzime: subtilisin , 0.15% (v/v) - Enzyme concentration : 1 ml l -1 of substrate - Substrate concentration : 10% Chichen feathers Centrifugation Concentration Biofertilizer /Bioestimulant Fig. 1 Enzymatic hydrolysis process used for obtaining edaphic biostimulants/ biofertilizers from sewage sludge and chicken feathers Table 2 Chemical composition of both edaphic biofertilizers/ biostimulants Biostimulants/biofertilizers Sewage sludge Chicken feathers Density (g ml −1 ) 1.18a±0.04 1.20a±0.06 Organic matter (g kg −1 ) 773b±21 463a±48 N-Kjeldahl (g kg −1 ) 34.9b±2.3 14.1a±1.6 Total carbohydrates (g kg −1 ) 42a±19 65b±11 P(gkg −1 )2.9a±0.127b±8 S(gkg −1 )5.9a±1.619b±4 Fat (g kg −1 ) 18a±3 20a±2 Data are the means of three samples. Rows (mean ± S.E.) followed by the same letter(s) are not significantly different (p>0.05) Table 3 Molecular weight distribution of both edaphic biostimulants/ biofertilizers Molecular weight (Da) Biostimulant/biofertilizer Sewage sludge Chicken feathers >10000 21.4a±2.6 23.8a±2.4 10,000–5,000 7.3a±1.5 9.1a±1.1 5,000–1,000 5.5a±2.0 25.1b±2.6 1,000–300 1.8a±0.4 8.0b±1.5 <300 64.0b±3.6 34.0a±3.1 Data are the means of three samples. Columns (mean ± S.E.) followed by the same letter(s) are not significantly different (p>0.05) Environ Sci Pollut Res (2014) 21:11027–11035 11029 extraction based on Bligh and Dyer (1959) and fractionated and quantified using the procedure described by Frostegard et al. (1993a) and Bardgett et al. (1996). Twenty-six separated fatty acid methyl esters were identified using gas chromatography and a flame ionization detector. The phospholipids were transformed by alkaline methanolysis into fatty acid methyl esters (FAMEs), which were quantified with a gas chromatograph (GC/FID, AutoSystem XL Gas Chromatograph, Varian Saturno 2000) fitted with a 50-m capillary column, using helium as the carrier gas. The injector temperature was 260 °C, the flame ionization detector temperature was 280 °C and the initial temperature was 70 °C (for 2 min); it was increased to 160 °C at 30 °C min −1 and then to 280 °C at 3°Cmin −1 . To estimate the various proportions of the main taxa in the samples according to the phospholipid fatty acids (PLFAs), the biomarkers i15:0, a15:0, i16:0, 16:1ω7c, 17:0, i17:0, cy17:0, 18:1ω9c and cy19:0 were used to represent bacterial biomass (bacPLFA) (Frostegard et al. 1993b; Bardgett et al. 1996) and 18:2ω6 (fungPLFA) was taken to indicate fungal biomass (Federle et al. 1986). The ratio of bacPLFA to fungPLFA (bacPLFA/fungPLFA) represents the ratio between bacterial and fungal biomass (Bardgett et al. 1996). The gram + -specific fatty acids i15:0, a15:0, i16:0 and i17:0 and the gram − -specific fatty acids cy17:0, 18:1ω9c and cy19:0 were taken as a measure of the ratio of the gram + and gram − bacterial biomass (gram + /gram − ). All results are given in nanomole per gram. For each treatment and each experimental day, 20 g of soil was taken. Soil samples were stored in sealed polyethylene bags at 4 °C for 15 days, prior to analysis of the enzymatic activities (Tejada et al. 2011), and at −20 °C prior to phospholipid analysis (Wu et al. 2011). Oxyfluorfen determination in soil The extraction of oxyfluorfen from soil was realized using the Anastassiades et al. (2003) method. Oxyfluorfen was extracted with a mixture of triphenyl phosphate and acetonitrile (1:25). Once the supernatant had been shaken and centrifuged, magnesium sulphate was added to it and it was stirred and centrifuged again. The supernatant was concentrated, and the dried residue was recomposed with 1 ml of cyclohexane/ethyl acetate (9:1). Oxyfluorfen was determined using a tandem mass spectrometer and electron impact, where the chromatographic conditions were as follows: carrier gas, He at 1 ml min −1 ; initial injector temperature, 70 °C for 0.50 min and 310 to 100 °C min −1 for 10 min; column, 30 m×0.25 mm ID; initial temperature of the column oven, 70 °C for 3.5 min and 180 to 35 °C min −1 and 300 °C at 10 °C min −1 for 5 min; temperature detector, Trap to 250 °C, Manifold to 60 °C and Xfer Line to 280 °C; injection volume, 5 μl. The MS/MS parameters were the following: excitation storage level (m z −1 ), 100; CID, 3.5 V; parent ion (m z −1 ), 300; quantification ion (m z −1 ), 223. The precursor ion was excited using a resonant waveform. Statistical analysis Data were submitted to two-way ANOVA with treatment and sampling time as factors followed by Tukey’s significant difference as a post hoc test, considering a significance level of p<0.05 throughout the study. The ANOVA was performed using the Statgraphics Plus 2.1 software package. For the ANOVA, triplicate data were used for each treatment and every experimental day. Results Evolution of soil biological properties Statistical analysis indicated a significant (p<0.05) stimulation of the dehydrogenase activity during the first days after the application of both BS, mainly at 7 days after the beginning of the experiment (Table 4). In this respect, and compared to the control, the soil dehydrogenase activity significantly increased by 92.1 and 88.2 % in the SS and CF treatments, respectively. After the first 7 days, the dehydrogenase activity began to decline gradually. At the end of the experimental period, all treatments studied had very similar values. The application of oxyfluorfen in soil showed a significant decrease in dehydrogenase activity (Table 4). At the end of the experimental period and compared with the control treatment, dehydrogenase activity significantly (p<0.05) decreased by 38.1 %. The application of both BS in soils with oxyfluorfen caused a minor decrease in dehydrogenase activity (Table 4). In this respect, at 7 days after applying the herbicide in the soil, and compared to that SS treatment, it decreased the dehydrogenase activity in the treatment by 66.6 % SS+H. Compared to the CF treatment, the dehydrogenase activity decreased by 57.4 % in the CF+H treatment. At 30 days after the beginning of the experiment, this decrease in soil dehydrogenase activity was progressively reduced during the experimental period, noting that in soils amended with both BS, this activity showed similar values than the amended soil without oxyfluorfen. Unlike the dehydrogenase activity, the soil urease activity was not stimulated after the application of the two BS studied (Table 4). At the end of the experimental period, the soil urease activity decreased slightly. However, the statistical analysis shows no significant differences (p>0.05) between 11030 Environ Sci Pollut Res (2014) 21:11027–11035 these values throughout the experimental period. Similar to the dehydrogenase activity, when oxyfluorfen was applied to the unamended soil, there was a significant (p<0.05) decrease of this enzyme activity throughout the experimental period. These same results were also observed when the two BS studied were applied to the soil with the herbicide. Similar to the dehydrogenase activity, the β-glucosidase activity was also stimulated in organically amended soils, mainly at 7 days after the beginning of the experiment (Table 5). Again, this stimulation (p<0.05) was significantly higher in the SS treatment than in the CF treatment (24.4 %). Theresponseoftheβ-glucosidase activity to the application of oxyfluorfen in soil was very similar to other enzyme activities studied (Table 5).Thus, it was observed an inhibition of this enzyme in the soils with oxyfluorfen. The application of both BS in the soil with herbicide also showed a similar response to the other enzymatic activities studied. In this respect, the application of BS to the soil decreased the inhibition of this enzymatic activity. At 7 days of beginning the experiment, this decrease was higher in the SS+H treatment than the CF+H treatment (9.7 %). At 30 days after the beginning of the experimental period, the decrease in soil βglucosidase activity was progressively reduced, noting that in soils amended with both BS, this activity showed similar values than the amended soil without oxyfluorfen. The soil phosphatase activity was also significantly (p<0.05) stimulated after the application of both BS to the soil (Table 5). Similar to the dehydrogenase and βglucosidase activities, the soil phosphatase activity showed a higher stimulation at 7 days after the beginning of the experiment. In this respect, and compared to the C treatment, soil phosphatase activity significantly increased by 89.7 and 82.7 % in the SS and CF treatments, respectively. This stimulation decreased as the experimental period progressed. At the end of the experimental period, all experimental treatments had similar values. Also, oxyfluorfen applying to the soil inhibited this enzymatic activity during the experimental period. Similar to the enzymes studied, when BS was applied in the soil with herbicide, the inhibition of phosphatase activity decreased. This decrease was higher in the SS+H treatment than in the CF+H treatment. The application of both BS increased the soil bacteria and fungi population (Table 6). Similar to the results of the enzymatic activities, this increase was higher in the SS treatment than in the CF treatment. Again and in the organic treatments, at 7 days of beginning the experiment, the highest population of bacteria and fungi was found. These populations were decreasing during the experimental period. At the end of the experiment, the bacteria population was similar to the one in the control treatment. The bacgram + /gram − and bacPLFA/ fungPLFA rates increased in all organic treatments, also indicating the variability in the biodiversity of these soils during the first days of the experiment (Table 4). At the end of the experimental period, these rates showed similar values to those obtained in the control treatment. When the herbicide was applied to the soil, the total bacterial population significantly (p<0.05) decreased while the fungal population decreased slightly. However, and with Table 4 Evolution of dehydrogenase and urease activities in soils amended with the edaphic biostimulants/biofertilizers and with oxyfluorfen during the experimental period Dehydrogenase activity (μgINTFg −1 h −1 ) 2 4 7 9 20 30 60 90 120 C 2.3±0.5 2.4±0.4 2.4±0.5 2.3±0.5 2.2±0.6 2.2b±0.4 2.3b±0.5 2.1b±0.4 2.1b±0.5 H 1.3a±0.2 1.4a±0.3 1.3a±0.3 1.3a±0.4 1.4a±0.2 1.3a±0.4 1.2a±0.2 1.3a±0.2 1.3a±0.3 SS 16.0±1.2 30.5e±2.4 20.2d±1.8 10.6±1.9 4.2b±1.1 3.5b±1.3 2.7b±0.8 2.2b±0.2 CF 14.7c±1.4 20.4d±1.6 17.3d±1.2 7.1±1.1 3.1b±0.6 3.3b±0.8 2.6b±0.5 2.3b±0.3 SS+H 11.9c±1.1 10.2c±1.2 11.4c±1.1 6.9±1.0 4.2b±0.9 3.6b±0.4 2.8b±0.3 2.2b±0.4 CF+H 7.8c±1.1 8.7c±1.5 5.1c±0.9 4.4b±0.8 3.4b±0.6 3.2b±0.5 2.6b±0.5 2.1b±0.4 Urease activity (μgNH 4+ g −1 h −1 ) C 2.1b±0.5 1.9b±0.3 1.9b±0.4 1.8b±0.3 1.8b±0.2 1.8b±0.4 1.9b±0.3 1.9b±0.3 1.9b±0.4 H 1.2a±0.3 1.1a±0.4 1.1a±0.2 0.9a±0.3 0.9a±0.1 1.0a±0.2 1.1a±0.4 1.1a±0.2 1.0a±0.3 SS 1.8b±0.3 1.9b±0.3 1.8b±0.2 1.7b±0.2 1.7b±0.3 1.8b±0.4 1.8b±0.3 1.7b±0.2 CF 1.8b±0.5 2.1b±0.4 2.2b±0.4 1.8b±0.4 1.8b±0.3 1.9b±0.2 1.8b±0.4 1.8b±0.4 SS+H 1.0a±0.2 1.1a±0.3 1.0a±0.2 0.9a±0.1 0.9a±0.1 1.0a±0.1 0.9a±0.2 1.0a±0.2 CF+H 1.1a±0.3 1.0a±0.2 1.1a±0.3 0.9a±0.1 0.9a±0.2 0.9a±0.1 1.0a±0.2 0.9a±0.1 Data are expressed as mean values ± standard error. Columns followed by the same letter(s) are not significantly different (p>0.05) INTF 2-p-iodo-3-nitrophenyl formazan, SS sewage sludge, CF chicken feathers Environ Sci Pollut Res (2014) 21:11027–11035 11031 respect to the control treatment, no significant (p>0.05) differences were found in bacgram + /gram − and bacPLFA/ fungPLFA rates (Table 4). The application of BS to soils with oxyfluorfen caused a minor decrease in the bacteria and fungi populations. This decrease was higher when the BS applied to the soil was SS. Table 5 Evolution of β-glucosidase and phosphatase activities in soils amended with the edaphic biostimulants/biofertilizers and with oxyfluorfen during the experimental period β-glucosidase activity (mmol PNP g −1 h −1 ) 2 4 7 9 20 30 60 90 120 C 1.4b±0.3 1.4b±0.2 1.4b±0.3 1.3b±0.1 1.2b±0.3 1.2b±0.2 1.1b±0.2 1.1b±0.1 1.0b±0.2 H 0.5a±0.1 0.5a±0.1 0.5a±0.1 0.6a±0.2 0.7a±0.1 0.6a±0.1 0.7a±0.2 0.6a±0.1 0.6a±0.1 SS 3.5c±0.4 5.3d±0.7 4.5c±0.9 3.3c±0.6 2.7b±0.4 1.9b±0.4 1.2b±0.2 1.1b±0.3 CF 2.9c±0.4 4.1c±0.9 3.3c±0.4 2.6c±0.7 2.0b±0.4 1.3b±0.3 1.0b±0.2 1.0b±0.2 SS+H 2.0b±0.3 3.1c±0.6 2.5b±0.5 2.0b±0.4 1.5b±0.2 1.2b±0.2 1.1b±0.2 1.0b±0.2 CF+H 1.6b±0.2 2.8c±0.4 1.8b±0.3 1.4b±0.3 1.3b±0.2 1.2b±0.2 1.0b±0.1 1.0b±0.1 Phosphatase activity (μmol PNP g −1 h −1 ) C 3.6b±1.3 3.6b±1.1 3.5b±1.4 3.5b±1.2 3.5b±1.4 3.4b±1.7 3.4b±1.3 3.3b±1.2 3.2b±1.4 H 2.3a±0.5 2.0a±0.9 1.8a±0.6 2.0a±0.5 1.8a±0.3 1.7a±0.2 1.8a±0.4 1.7a±0.3 1.8a±0.5 SS 19.1d±2.3 34.0e±2.9 26.2d±1.5 19.1d±1.9 8.0c±1.4 4.6b±1.2 4.2b±1.3 3.0b±1.1 CF 18.7d±2.8 28.1d±3.3 20.4d±2.1 13.5c±1.1 6.9b±1.3 4.5b±1.5 4.4b±1.4 3.2b±0.9 SS+H 13.2c±1.7 20.2d±3.1 12.1c±1.9 8.4c±1.5 6.3b±1.3 4.2b±1.1 3.8b±1.1 3.1b±1.2 CF+H 10.3c±1.3 14.8c±2.2 9.6c±1.4 6.1b±1.2 4.8b±1.6 3.7b±0.9 3.6b±0.8 3.2b±1.0 Data are expressed as mean values ± standard error. Columns followed by the same letter(s) are not significantly different (p>0.05) PNP p-nitrophenol, SS sewage sludge, CF chicken feathers Table 6 Evolution of bacterial gram + , bacterial gram − , total bacterial and fungal PLFAs (nmol g −1 ), gram + /gram − and bacteria/fungi during the experimental period bacgram + bacgram − Total bacterial PLFA Fungal PLFA bacgram + /bacgram − bacPLFA/fungPLFA C (2 days) 13.1b±2.1 1.4b±0.2 14.7±1.8 0.9a±0.1 8.2a±1.1 16.3a±1.4 C (7 days) 12.8b±1.3 1.4b±0.4 14.2±1.4 0.9a±0.1 9.1a±1.0 15.8a±1.5 C (30 days) 13.0b±1.4 1.3b±0.2 14.5±1.1 0.8a±0.1 10.0a±1.5 18.1a±1.2 C (120 days) 14.0b±1.4 1.5b±0.3 15.5±1.5 0.9a±0.1 9.3a±1.2 17.2a±1.3 H (7 days) 8.8a±0.6 0.9a±0.2 9.7±1.1 0.7a±0.1 9.8a±1.3 13.9a±1.4 H (30 days) 9.3a±0.9 0.9a±0.2 10.2±1.1 0.6a±0.2 10.3a±1.2 17.0a±1.3 H (120 days) 8.9a±0.7 1.0a±0.3 9.9±1.2 0.6a±0.1 8.9a±1.2 16.5a±1.3 SS (7 days) 43.4d±2.6 4.6c±1.1 88.0±3.4 2.1c±0.3 9.4a±1.5 41.9c±3.9 SS (30 days) 16.3b±1.5 1.6b±0.4 17.9±1.5 1.2b±0.2 10.2a±1.2 14.9a±1.7 SS (120 days) 14.4b±1.5 1.4b±0.2 15.8±1.4 1.0a±0.2 10.3a±1.5 15.8a±1.3 CF (7 days) 34.0c±2.1 3.2b±0.9 37.2±2.6 1.6b±0.2 10.6a±1.3 23.2b±2.6 CF (30 days) 15.1b±1.2 1.5b±0.3 16.6±1.3 1.0a±0.2 10.1a±1.3 16.8a±1.4 CF (120 days) 14.6b±1.3 1.5b±0.2 15.1±1.2 0.9a±0.1 9.7a±1.2 16.8a±1.5 SS+H (7 days) 30.3c±2.8 3.7bc±1.5 34.0±3.1 1.5b±0.4 8.2a±1.3 22.7b±2.3 SS+H (30 days) 15.2b±1.1 1.5b±0.2 16.1±1.2 1.1a±0.2 10.1a±1.3 14.6a±1.6 SS+H (120 days) 13.8b±1.3 1.5b±0.3 14.3±1.3 1.0a±0.2 9.2a±1.2 14.3a±1.5 CF+H (7 days) 21.1bc±1.6 2.2b±1.2 43.3±3.4 1.3b±0.3 9.6a±1.3 33.3b±3.4 CF+H (30 days) 14.0b±1.2 1.4b±0.2 15.4±1.1 1.0a±0.2 10.0a±1.2 15.4a±1.3 CF+H (120 days) 13.2b±1.1 1.3b±0.2 14.5±1.2 1.0a±0.1 10.1a±1.3 14.5a±1.1 Data are the means of three samples. Columns (mean ± S.E.) followed by the same letter(s) are not significantly different (p>0.05) PLFA phospholipid fatty acid, SS sewage sludge, CF chicken feathers 11032 Environ Sci Pollut Res (2014) 21:11027–11035 Evolution of oxyfluorfen in soil Figure 2shows the evolution of oxyfluorfen in the experimental soil. The application of both BS to soil decreased the soil oxyfluorfen concentration. However, this decrease depended on the BS type applied to the soil. Comparing both the SS+H and CF+H treatments, the values suggest a more rapid degradation of the herbicide when the SS was applied to the soil with oxyfluorfen. Discussion Our results indicated that the oxyfluorfen herbicide caused a toxic effect on soil enzymatic activity and soil diversity. These results are in agreement with those obtained by García-Orenes et al. (2010) who observed an important decrease in the biochemical properties of an agricultural soil after the application of 1.5 kg ha −1 oxyfluorfen to the soil. Sheeba Singh et al. (2011) observed the toxic effect of this herbicide on soil microorganisms, especially in different soil cyanobacterias. Also, Nadiger et al. (2013) highlight the toxic effect on microorganisms when applied oxyfluorfen in agricultural soils cropped with maize. According to Renella et al. (2005), the decrease in the biological properties of soils contaminated by xenobiotics may be a consequence of a decrease in the release of extracellular enzymes from soil microorganisms or an inhibition of these extracellular enzymes. However, there are also authors who found positive effects when they applied oxyfluorfen to the soil. Das et al. (2003) and Das and Debnath (2006) indicate that the application of this herbicide in soil caused an increase in both the population and microbial activity, because this chemical acted as a source of carbon and nutrients for the microorganisms of the soil. Also, El Hussein et al. (2012) found that the application of oxyfluorfen to soil at low concentrations causes a positive effect on microbial growth. However, these authors suggested that the effect of oxyfluorfen on soil microorganisms depends on both the pesticide concentration and the soil physicochemical characteristics (texture, organic matter, etc.). The results obtained show a high persistence of oxyfluorfen in soil. These results are in agreement with those obtained by other authors. Baruah and Mishra (1986) indicate that oxyfluorfen has a long persistence in soil (halflife of 72 to 160 days) in natural conditions. Ying and Williams (2000) found a persistence time of 119 days of this herbicide in soil. Also, Mantzos et al. (2014) found that oxyfluorfen soil dissipation is better described by first-order kinetics with half-life ranging between 45 and 52.9 days. Our results suggest that the application of both BS caused an increase in the soil microbial activity. These results are in agreement with those obtained by García-Martínez et al. (2010a,b), Parrado et al. (2008) and Tejada et al. (2010b, 2013) who indicate that the incorporation of different BS obtained from wheat condensed distiller soluble, carob germ enzymatic, rice bran extract and SS causes a soil microbial activity stimulation. Since soil microorganisms degrade the soil organic matter through the production of a wide variety of extracellular enzymes, the application of both BS explained the increase in these enzymatic activities. Dehydrogenase activity showed a significant stimulation in soils amended with both BS. Dehydrogenase activity is an intracellular enzyme, and therefore, it is present in all intact, viable microbial cells (García et al. 1993). Dehydrogenase activity suggests that its stimulation was higher in the soil amended with BS SS than in the one amended with BS CF. Possibly, this fact is a consequence of the different chemical composition of the protein hydrolysates obtained. We believe that the main reason for the increased stimulation of the dehydrogenase activity is the size of the proteins present in both hydrolysates. The BS SS has a higher content of lowmolecular-weight proteins (<300 Da) than the BS CF. These results agree with those obtained by Tejada et al. (2013). 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 247 306090120 Oxyfluorfen (mg kg-1) H SS+H CF+H a aa a a a a cc c c c b b b b b a bb b a bb a Fig. 2 Evolution of oxyfluorfen (mean ± S.E.) in soils during the experimental period. Data are the means of three samples. Columns (mean ± S.E.) followed by the same letter(s) are not significantly different (p>0.05) Environ Sci Pollut Res (2014) 21:11027–11035 11033 These authors found that after applying the BS obtained from SS to the soil, the dehydrogenase activity showed a higher increase when the BS contained a higher proportion of lowmolecular-weight proteins. The decrease in protein molecular size indicates that N is in a more readily available form for soil microorganisms. This aspect promotes an increased proliferation of soil microorganisms (Vasileva-Tonkova et al. 2007). This greater assimilation of low-molecular-weight proteins is possibly responsible for the fact that the soil urease activity exhibits no significant stimulation after the application of both protein hydrolysates. Since soil microorganisms can obtain this N without any energy expenditure, microorganisms do notneedtoexcreteanyenzymetoobtainthiseasilyavailable N. These results also agree with those obtained by García- Martínez et al. (2010a,b) and Tejada et al. (2013), who found that after the application to the soil of different protein hydrolysates, no stimulation was observed in the soil urease activity due to the fact that these chemical compounds were rich in low-molecular-weight proteins. The soil β-glucosidase activity showed similar values to those obtained for the dehydrogenase activity, highlighting the positive influence of BS applied to the soil in the stimulation of this enzymatic activity, at least during the first days of the experimental period. These results agree with those obtained by García-Martínez et al. (2010a,b), Parrado et al. (2008)and Tejada et al. (2010b,2013), who observed a significant stimulation of this enzymatic activity after the application of different organic compounds to the soil. Soil phosphatase activity was also stimulated after the application of both BS. The results indicate that this stimulation was dependent on the amount and chemical composition of the BS applied to the soil. The highest values of this enzymatic activity were found in the soil amended with the SS. These results suggest that most of the P applied by the feather usually has an organic origin, because this stimulation occurs when soil microorganisms may not find an easily available soil inorganic P and therefore need to excrete this phosphatase. With respect to the soil microbial diversity, Marschner et al. (2003) found that the degradation of organic matter requires enzymes that are produced by a limited number of microbial species and may increase the competitive ability of microorganisms. It is very probably that this is the reason that microbial biodiversity of soils amended with both BS would increase in the first days of the experimental period. Once these easily degradable organic complexes have been metabolized, the structure of the soil microbial population is reestablished again. Also, these authors note that the availability of substrate is a reason to find differences in the structure of the soil bacterial community. Our results indicate that the application of both BS significantly stimulated the soil microbial activity. Therefore, when BS was added in soils with oxyfluorfen, a less inhibition of the enzymatic activities studied occured. Furthermore, the toxic effect of the herbicide in soil was considerably reduced. Coinciding with the results obtained by Tejada et al. (2010b)to apply different BS obtained from wheat condensed distiller soluble, carob germ enzymatic and rice bran extract in a soil with the MCPA herbicide, BS stimulate soil microorganisms and therefore favour and accelerate degradation of the xenobiotic compound in the soil. This degradation was faster in the soil amended with SS than with CF, probably due to higher stimulation of soil microorganisms in the soil treated with SS. Conclusions It can be concluded that the oxyfluorfen herbicide caused a negative effect on soil enzymatic activities and microbial diversity. The application of BS decreased the toxic action of oxyfluorfen on soil biological properties. However, this effect depended on the chemical composition of the BS applied to the soil. Comparing both the BS, when SS was applied in soils with herbicide occurred a higher decrease in the inhibition of the soil biological properties, probably due to their higher content of lower molecular weight peptides, easily assimilated by soil microorganisms. Acknowledgments This work was supported by the Ministry of Science and Innovation (Spain), Plan Nacional I+D CTM2011-29930-01, CTM2011-29930-03 and by Proyecto Excelencia, Junta de Andalucía P11-RNM-7887. 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Este comportamiento se debe a que la estimulación o inhibición de estas actividades enzimáticas está directamente relacionada con la actividad microbiana reflejada en la actividad deshidrogenasa, la cual a su vez es una consecuencia de la adicción de materia orgánica biodisponible al suelo (Gan y col., 1998; Perucci y col., 2000). Cuando la actividad deshidrogenasa aumenta significa que las poblaciones microbianas crecen y/o aumentan su actividad, o justamente lo contrario cuando decae. Esto es así porque en respuesta a las necesidades nutricionales, los microorganismos excretan enzimas con las que poder captar los nutrientes necesarios para su actividad y multiplicación, reduciéndose cuando no es necesario, de ahí esa vinculación entre las actividades βglucosidasa, fosfatasa y arilsulfatasa con la actividad deshidrogenasa (García-Martínez y col., 2010a, b). Sin embargo, la actividad ureasa no fue estimulada cuando se añadieron los BS al suelo, mientras que la adicción del insecticida si produjo un efecto inhibitorio sobre esta actividad. La disminución de la actividad en presencia del contaminante se puede explicar del mismo modo que el resto de actividades enzimáticas estudiadas, es decir, una disminución de la actividad y tamaño de las poblaciones microbianas conlleva una disminución en la producción de enzimas destinadas a la captación de nutrientes. Cuando se enmendaron los suelos con los BS, no se produjo una estimulación de esta actividad a pesar de que si se produjo una estimulación de los microorganismos. Esto es debido a que estos productos ofrecen una fuente rica y fácilmente disponible de Capítulo 3 Resultados y Discusión 251 nitrógeno por lo que los microorganismos no tienen la necesidad de excretar una enzima destinada a su captación a partir de otras fuentes más complejas (García-Martínez y col., 2010a, b). Con respecto a las comunidades microbianas del suelo, se observó como el insecticida tuvo un efecto inhibitorio sobre las mismas (Rangaswamy y Venkateswarlu, 1992; Ahmed y Ahmad, 2006), al mismo tiempo que los BS tuvieron un efecto inductor similar al observado con las actividades enzimáticas determinadas anteriormente. Se comprueba de nuevo como los bioestimulantes con un mayor contenido en la fracción proteica de bajo peso molecular producen la mayor inducción en las poblaciones microbianas (Marschner y col., 2003). Cuando se estudiaron los PLFA en suelos enmendados con los BS previamente contaminados con clorpirifos, se observa como estos productos son capaces de revertir de nuevo el efecto causado por el insecticida de forma similar a la observada con las actividades enzimáticas. En todos los casos, después de que los nutrientes aportados por los bioestimulantes son consumidos, las poblaciones se restablecieron gradualmente para finalmente volver a ser similares a las observadas en la muestra control. Después de aplicar clorpirifos al suelo se observa como su concentración va disminuyendo gradualmente en todas las muestras. Sin embargo, esta desaparición se encuentra acelerada en las muestras enmendadas con los diferentes bioestimulantes. Esto se debe a que la actividad general y biodegradativa en particular de los microorganismos se encuentra estimulada, al tiempo que la biodisponibilidad del contaminante es menor que en los suelos no enmendados, lo que favorece el desarrollo de microorganismos que normalmente a esa dosis de insecticida se encontrarían inhibidos. La suma de ambos factores es la causante de esta degradación acelerada del contaminante. Al igual que con las actividades enzimáticas o la estructura de las Capítulo 3 Resultados y Discusión 252 poblaciones microbianas, el BS con mayor contenido en péptidos de bajo peso molecular es el que induce una mayor tasa de biodegradación del insecticida. Capítulo 3 Resultados y Discusión 253 4.3. Behaviour of oxyfluorfen in soils amended with edaphic biostimulants / biofertilizers obtained from sewage sludge and chicken feathers. Effects on soil biological properties El oxifluorfeno es un herbicida por contacto con actividad residual y alta persistencia que actúa inhibiendo la ruta de síntesis de la clorofila y citocromos y provocando una acumulación de especies reactivas de oxígeno (Kunert, 1984, Lee y col. 2000). Debido a su mecanismo de acción, resulta tóxico tanto para las plantas como para los microorganismos del suelo. Tras su aplicación al suelo se observa como permanece en el mismo un largo periodo de tiempo (Baruah y Mishra, 1986; Ying y Williams, 2000; Mantzos y col., 2014), aunque su concentración va disminuyendo progresivamente, señal de que probablemente está siendo degradado por organismos del suelo tolerantes al mismo. Cuando se enmendaron suelos contaminados por oxifluorfeno con los bioestimulantes estudiados, se observó un aumento en la tasa de desaparición de este compuesto. Esto puede estar debido a la bioestimulación de las poblaciones microbianas del suelo y/o al aumento de la biodisponibilidad del herbicida, que es altamente insoluble en agua, pudiendo actuar los BS como surfactantes (Mantzos y col., 2014; Tripathy y col. 2018). Las diferencias entre un bioestimulante y el otro se deben a la diferente composición de cada uno, siendo SS el que induce la mayor tasa de eliminación del oxifluorfeno. Cuando se estudió la actividad deshidrogenasa en suelos enmendados con ambos BS, se observó cómo se producía una rápida y significativa inducción de esta actividad, siendo mayor en el caso de SS que en el de CF. Este incremento en la actividad deshidrogenasa es un indicativo de que las poblaciones microbianas del suelo han aumentado en número y actividad como consecuencia de este aporte de nutrientes Capítulo 3 Resultados y Discusión 254 (Parrado y col., 2008; García-Martínez y col., 2010a, b; Tejada y col., 2010b, 2013). Además, el relativo alto contenido en péptidos de bajo peso molecular (>300 Da) y aminoácidos libres de los productos causa un efecto de bioestimulación microbiana al aportar una fuente de nitrógeno, cuya concentración en el suelo es normalmente limitante, fácilmente biodisponible (Tejada y col., 2013). Las diferencias observadas entre ambos bioestimulantes se deben a la diferente composición de cada uno y probablemente en particular al diferente contenido en el componente proteico de bajo peso molecular (Vasileva-Tonkova y col., 2007). Cuando se aplicó el herbicida al suelo se detectó una inhibición en la actividad deshidrogenasa del mismo. Esto se debe al efecto causado por el oxifluorfeno al inhibir una ruta fundamental del metabolismo. Esta inhibición se mantuvo durante todo el periodo experimental, indicativo de la persistencia de este compuesto. Por otra parte, cuando se aplicaron los bioestimulantes en suelos contaminados con oxifluorfeno se observó una reversión de la inhibición de la actividad deshidrogenasa causada por el herbicida. Los mayores valores de actividad deshidrogenada detectados se dieron a los 7 días de iniciado el ensayo, siendo superiores en los suelos tratados con SS que en los enmendados con CF. Una vez alcanzados los valores máximos de actividad, esta decayó gradualmente hasta alcanzar valores similares a los del suelo sin contaminar. El hecho de que los valores observados sean superiores para SS que para CF puede ser debido de nuevo a que el contenido en péptidos de bajo peso molecular (>300 Da) y aminoácidos libres es superior en el primero que en el segundo. Capítulo 3 Resultados y Discusión 255 Figura 21: Microcosmos a los 7 días de incubación. a) C; b) H; c) SS; d) SS+H; e) CF; f) CF+H. La reversión del efecto del contaminante sobre la actividad deshidrogenasa se debe en parte a que los bioestimulantes inducen en gran medida el crecimiento y actividad de las poblaciones microbianas del suelo, ignorando aparentemente el efecto tóxico del oxifluorfeno, y por otra parte, los BS actúan aumentando la biodisponibilidad del herbicida para los organismos degradadores y reducen su concentración aparente al Capítulo 3 Resultados y Discusión 256 actuar adsorbiéndolo gracias a su contenido en materia orgánica. Gracias a ello, los organismos tolerantes son capaces de degradar más eficientemente el contaminante, permitiendo que rápidamente otros organismos menos resistentes a este compuesto también puedan desarrollarse y continuar degradando el oxifluorfeno. De forma similar a la actividad deshidrogenasa, las actividades β-glucosidasa y fosfatasa se vieron estimuladas significativamente en los suelos enmendados con los bioestimulantes con respecto al suelo control. Del mismo modo, se inhibieron en los suelos contaminados con oxifluorfeno. Esta inducción e inhibición son consecuencia directa del aumento o disminución de las poblaciones microbianas y de su actividad, reflejada en la actividad deshidrogenasa. Cuando las poblaciones de microorganismos aumentan su tamaño o su actividad, requieren de un mayor aporte de nutrientes, principalmente a partir de compuestos ya mineralizados, o en los casos donde no haya formas de los mismos fácilmente disponibles, por mineralización de la materia orgánica (Marschner y col., 2003). De este modo, la materia orgánica aportada por los bioestimulantes actúa como inductor de la producción y secreción de estas enzimas hidrolíticas, que en primer lugar hidrolizan los compuestos orgánicos complejos en unidades estructurales menores y a continuación los mineralizan en forma de iones fácilmente translocables al interior celular. Debido a la vinculación entre las enzimas β-glucosidasa y fosfatasa con la actividad deshidrogenasa (indicativo de la actividad microbiana), estás siguen una dinámica similar. Los valores máximos para ambas enzimas se detectaron a los 7 días de iniciarse el experimento de bioestimulación, decayendo posteriormente hasta valores similares al suelo sin contaminar, mientras que la inhibición de las mismas en suelos contaminados con el herbicida se mantuvo durante todo el ensayo. Capítulo 3 Resultados y Discusión 257 Cuando se añadieron los bioestimulantes estudiados a un suelo contaminado con oxifluorfeno, de nuevo se observa una reversión del efecto tóxico del herbicida similar a la observada con la actividad deshidrogenasa. Ambas actividades enzimáticas alcanzan sus valores máximos a los 7 días, decayendo gradualmente hasta los valores del suelo control hacia el final del ensayo. En todos los suelos ensayados, SS siempre presenta unos valores supriores a los de CF. Esto puede ser debido de nuevo al mayor contenido en péptidos de bajo peso molecular y aminoácidos libres del primero con respecto al segundo. Con respecto a la actividad ureasa, enzima implicada en la mineralización de nitrógeno, ninguno de los BS estudiados indujo una estimulación a la misma. Esto puede ser debido a que el componente proteico de bajo peso molecular de los bioestimulantes es una fuente de nitrógeno altamente biodisponible, por lo que los microorganismos no necesitan gastar energía en producir enzimas para su adquisición, incorporando directamente al interior celular esos compuestos nitrogenados (García- Martínez y col., 2010a, b; Tejada y col., 2013). Por otra parte, en los suelos contaminados con oxifluorfeno si se observó una inhibición de esta actividad, debido al efecto tóxico del xenobiótico, independiente de la presencia o no de los bioestimulantes. La aplicación de ambos bioestimulantes al suelo incrementó significativamente las poblaciones de bacterias y hongos, siendo mayor el efecto causado por SS que el de CF y alcanzando los valores máximos a los 7 días de incubación. De nuevo, el efecto tóxico del oxifluorfeno causó una disminución de las poblaciones microbianas de acuerdo a lo observado con la actividad deshidrogenasa, efecto que se mantuvo hasta el final del experimento. Cuando se añadieron los BS a suelos contaminados con el herbicida se observó un incremento de las poblaciones microbianas, aunque siempre menor al observado en Capítulo 3 Resultados y Discusión 258 los suelos enmendados sin contaminar. Hacia el final del ensayo, los valores de todos los suelos enmendados fueron similares al control, lo que sugiere que las poblaciones microbianas se restablecieron de forma parecida al suelo sin contaminar ni enmendar. 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