Biochemical characterization of hydrochars and pyrochars derived from sewage sludge and their potential as n and p slow release fertilizers and as soil amendment in agriculture.

Biochemical characterization of hydrochars and pyrochars derived from sewage sludge and their potential as N and P slow release fertilizers and as soil amendment in agriculture Marina Paneque Carmona Tesis Doctoral Universidad de Sevilla 2018 Biochemical characterization of hydrochars and pyrochars derived from sewage sludge and their potential as N and P slow release fertilizers and as soil amendment in agriculture Memoria que presenta Dña. Marina Concepción Paneque Carmona para optar al título de Doctora por la Universidad de Sevilla. Programa de Doctorado RECURSOS NATURALES Y MEDIO AMBIENTE Dpto. de Cristalografía, Mineralogía y Química Agrícola El presente trabajo se ha realizado en el marco de los proyectos PCGL201237041, CGL-2015-64811-P y CGL2016-76498-R, pertenecientes al Plan Nacional de Investigación y Desarrollo. Estos proyectos han sido financiados por el Ministerio de Economía, Industria y Competitividad y el Fondo Europeo de Desarrollo Regional (FEDER). Asimismo, se agradece al Ministerio de Educación, Cultura y Deporte la financiacion de la beca para la Formación de Profesorado Universitario de Marina Paneque Carmona (FPU 13/05831). Por último, la EU-COST Action TD1107 financió la estancia de tres meses de Marina Paneque Carmona en la Universita degli Studi di Palermo. AGRADECIMIENTOS En estos últimos años he recorrido un intenso camino, del que he aprendido casi tanto a nivel personal como profesional. Las personas que me han acompañado, de una u otra forma, han contribuido a que este trabajo sea posible y hoy quiero expresarles mi agradecimiento. En primer lugar a mis directores Heike Knicker y José María de la Rosa por su apoyo constante, desde el principio hasta el final. Gracias por vuestra paciencia, por compartir toda vuestra experiencia conmigo y por poner todos los medios posibles a mi alcance. He disfrutado trabajando con vosotros. También quiero agradecer a mi tutora, Elena Fernández Boy, profesora del Dpto. de Cristalografía, Mineralogía y Química Agrícola de la Universidad de Sevilla, por su amabilidad y eficacia a la hora de resolver cualquier duda o gestión. A Carlos Aragón, de la Fundación Centro de las Nuevas Tecnologías del Agua. Siempre dispuesto a ayudar, sin su colaboración este trabajo no hubiera sido posible. I truly appreciate the collaboration of Jürgen Kern, from the Leibniz Institute for Agricultural Engineering and Bioeconomy, in this work. He provided not only his expertise but also some necessary equipment. I want to express my gratitude to Tony Patti, from the Monash University, for giving me the opportunity to work in his research group and taking care of providing all the necessary facilities. Thanks also to Karen Little, Azita and Biplob for their assistance in the lab. Lastly, I really appreciate the kindness of the entire group, especially the nice moments with Temma and Karen. A Marta y María, por acogerme en el grupo y enseñarme todo lo que saben. Me hicisteis sentir una más desde el principio (y además nos hemos reído mucho). A Palomita, la última incorporación, que me aguanta con paciencia y está siempre dispuesta a ayudar y a tomar un té. Al resto del grupo MOSS. A Quico, porque es la base del grupo. Su experiencia y forma de ser es clave para que las cosas sean como son. A José Antonio González por su colaboración en el último capítulo de esta tesis y por estar siempre disponible a resolver mis dudas. A Alba y Desi por la eficacia y precisión con la que trabajan, que hace que pueda confiar en los datos. Aunque la tesis doctoral la he desarrollado en el IRNAS, la formación docente la he realizado en el Dpto. de Cristalografía, Mineralogía y Química Agrícola de la Universidad de Sevilla, del que ahora también me siento parte. Quiero agradecer a la Directora del Dpto., Rosario Vaz, y a mi tutora, Elena Fernández, su buena disposición y gestión a la hora de incluirme en las funciones docentes del Departamento. Además, la amabilidad y apoyo del resto de profesores ha ayudado a que mi tiempo en la ETSIA sea mucho más agradable y fructífero. En especial quiero agradecer el apoyo de las profesoras Carmen Ortega y Mª Carmen Florido, que me acogieron y guiaron desde el primer día. Muchas gracias por vuestro tiempo y dedicación. También quiero mostrar mi agradecimiento al IRNAS y su personal de administración, por poner a mi disposición los medios técnicos y humanos necesarios para desarrollar este trabajo. En especial quiero agradecer al Servicio de Análisis (Chona, Chari, Piluca...) el buen trabajo que realiza, que mezclado con el buen humor que se respira ha hecho que los finales de cada experimento sean un poco más livianos. No me puedo olvidar de mis compis (ahora amigos) del IRNAS, culpables de divertidos almuerzos, viernes de tapas, y findes de “uno al mes es menester”. MMar, Josan, Pablo, Kike, Marta, María, Desi, Alba, Palomita, Marta Gil…gracias por amenizarme la vida  A Paloma, por contagiarme su buen humor desde el principio, y su apoyo en esta etapa final. Por su amistad. Los “biólogos” (Rosendo, Edu, Josan, Pablo, Currito...) también son responsables de divertidos fines de semana, tan necesarios como dormir durante esta etapa. A María José, por apoyarme siempre, con buen humor, y no cansarse nunca de decir “fighting!”. Y a las amigas de toda la vida, MMar y Elena. Menos mal que no estáis en este “mundillo” y podemos hablar de otras cosas. Y por último, lo más importante. Mamá y Papá, os estaré eternamente agradecida por la educación y el apoyo que me habéis dado. Gracias por dejarme elegir y quererme siempre, en lo bueno y en lo malo. Por vosotros soy como soy. Enano, te has convertido en un personita estupenda, y además de ser un gran compañero de viajes haces cosas superútiles ¡como editar esta tesis! Gracias por dedicarme parte de tus “vacas” y…a los tres, gracias por los cuidados intensivos. A mis padres. RESUMEN La cantidad de fertilizantes minerales que actualmente necesita el sector agrícola para mantener su producción no es sostenible. Al mismo tiempo la sociedad actual genera una gran cantidad de residuos orgánicos que representan un problema ambiental a escala global y que deben ser gestionados. En este sentido, el reciclaje se contempla como una de las opciones más sostenibles desde el punto de vista medioambiental y económicamente viables. Se ha mostrado en diversos estudios que el uso de lodos de depuradora en agricultura no solo disminuye la cantidad de este residuo es acumulado en vertederos, sino que además se trata de una fuente de nitrógeno (N) y fósforo (P) que podría contribuir de manera significativa a reducir el uso de fertilizantes inorgánicos. Sin embargo, los nutrientes que contienen los lodos de depuradora pueden ser lixiviados después de que estos hayan sido añadidos al suelo, incluso si estos han sido sometidos a un tratamiento de estabilización previo. Teniendo en cuenta el problema anterior, investigaciones recientes sobre los procesos de carbonización de residuos orgánicos ricos en N indican que carbonización hidrotermal y la pirólisis (denominadas respectivamente en la presente Tesis Doctoral HTC y Py) podrían producir una enmienda orgánica más estable que aumente el potencial de secuestro de C en el suelo y liberar el N y el P de forma más lenta. Considerando que la estructura química de un producto es clave en su funcionalidad, esta Tesis Doctoral se ha centrado en primer lugar en la caracterización de lodos de depurada tratados mediante pirolisis (Py) y carbonización hidrotermal (HTC). Posteriormente se ha evaluado el potencial estos productos como fertilizantes de liberación lenta de N y P. La primera fase de este trabajo consistió en la pirolización y HTC de dos lodos de depuradora diferentes. La HTC se realizó a 200 y 260 ºC durante 0.5 y 3 horas mientras que la pirólisis se desarrolló a 600 ºC durante 1 hora. El producto que resulta de una HTC se denomina comúnmente por la comunidad científica “hydrochar” mientras que el que se obtiene tras una pirólisis se llama “pyrochar”. En conjunto, los hydrochars y pyrochars son productos carbonizados (pirolizados) denominados “chars”. Para estudiar la composición y propiedades de los lodos y chars así como los cambios que tienen lugar durante los tratamientos térmicos se llevó a cabo una caracterización detallada de las muestras en la que se usaron tanto técnicas analíticas rutinarias como otras más avanzadas, entre las que se incluye la espectroscopia de resonancia magnética nuclear de 13C y 15N en estado sólido. Los resultados de esta Tesis Doctoral mostraron una reducción en la presencia de grupos carboxilos y O/N alquilos así como un ligero incremento de los grupos aromáticos tras la HTC mientras que la pirólisis transformó la mayor parte de la materia orgánica en estructuras aromáticas. Además, la HTC preservó parte del N inorgánico (Ni) y estabilizó otra parte en compuestos heterocíclicos aromáticos con substituciones de N. A estas formas heterocíclicas nitrogenadas se las ha denominado “Black Nitrogen” (BN) como sugieren los trabajos científicos previos. En los chars procedentes de la pirólisis, en cambio, todo el N se encontraba en forma de BN. Estos resultados sugirieron que los hydrochars pueden tener propiedades como fertilizante de N a corto y medio plazo mientras que los pyrochars pueden aportar dosis de N más bajas durante un mayor periodo de tiempo y, además, incrementar el potencial de secuestro carbono del suelo. Para evaluar el impacto potencial de los chars producidos a partir de lodos de depuradora en el crecimiento de los cultivos se realizó un experimento de invernadero en el cual los hydrochars y pyrochars se añadieron como enmienda a un suelo en el que Lolium perenne creció durante 80 días. Los hydrochars incrementaron la producción de biomasa total a pesar de que redujeron la tasa de germinación. Los pyrochars, sin embargo, no afectaron ni a la germinación ni a la producción de biomasa de esta planta. El impacto de los hydrochas en el crecimiento de Lolium en este experimento se atribuyó a la presencia de Ni y de formas de N orgánicas fácilmente degradables. Estos resultados están en concordancia con las predicciones realizadas previamente, basadas en los datos de caracterización. El potencial de los lodos carbonizados como fertilizantes de liberación lenta de P se estudió evaluando los cambios del contenido de P en el lodo durante la pirólisis y la HTC. Este trabajo mostró que, a pesar de que ambos tratamiento produjeron un enriquecimiento en P, la cantidad de P disponible disminuyó. Este resultado se debe probablemente a un incremento de los complejos metal/P y a posibles limitaciones físicas. Sin embargo, es necesario realizar una investigación más profunda para corroborar esta hipótesis. Por otro lado, el experimento de invernadero mencionado en el párrafo anterior también se utilizó para evaluar tanto el comportamiento del P de los lodos tratados como su impacto en el suelo donde se aplicaron. Este estudio mostró que los pyrochars, y en menor medida, los hydrochars producidos a 200ºC fueron capaces de incrementar la cantidad de P disponible del suelo, por lo que serían útiles como fertilizantes de P. Además, una parte del P total que originalmente estaba no disponible en los chars pasó a la fracción disponible durante su incubación en el suelo. Este resultado apunta que estos materiales poseen cualidades como fertilizantes de liberación lenta de P. 4 HYDROTHERMAL CARBONIZATION AND PYROLYSIS OF SEWAGE SLUDGES: EFFECTS ON LOLIUM PERENNE GERMINATION AND GROWTH ............................... 48 4.1 INTRODUCTION .................................................................................................... 48 4.2 MATERIALS AND METHODS ................................................................................ 51 4.2.1 Characteristics of the sample material .................................................. 51 4.2.2 Greenhouse incubation experiment ...................................................... 54 4.2.3 Statistical analysis ........................................................................................ 55 4.3 RESULTS .................................................................................................................. 56 4.3.1 Germination and survival rates ................................................................ 56 4.3.2 Biomass production .................................................................................... 57 4.3.3 Root-to-shoot ratios .................................................................................... 59 4.3.4 Relationship between plant response and chars properties ............. 60 4.4 DISCUSSION .......................................................................................................... 61 4.4.1 Germination and survival rates ................................................................ 61 4.4.2 Biomass production and chars properties ............................................. 62 4.4.3 Root-to-shoot ratios and char properties ............................................... 63 4.4.4 Fertilization potential of thermally treated SS ........................................ 64 4.5 CONCLUSIONS ..................................................................................................... 64 4.6 REFERENCES .......................................................................................................... 65 5 HYDROCHAR AND PYROCHAR DERIVED FROM SEWAGE SLUDGE CAN INCREASE THE AVAILABLE PHOSPHORUS IN AMENDED SOILS. A GREENHOUSE STUDY. ...... 69 5.1 INTRODUCTION .................................................................................................... 69 5.2 MATERIALS AND METHODS ................................................................................ 71 5.2.1 Sewage sludge collection, treatment and characterization ............ 71 5.2.2 Greenhouse incubation experiment ...................................................... 72 5.2.3 Statistical analysis ........................................................................................ 73 5.3 RESULTS .................................................................................................................. 73 5.3.1 Characterization of non-treated SS and the produced chars .......... 73 5.3.2 Greenhouse experiment ........................................................................... 77 5.4 Summary and general discussion .................................................................... 82 5.5 CONCLUSIONS ..................................................................................................... 83 5.6 REFERENCES .......................................................................................................... 83 6 DEGRADABILITY OF ORGANIC C AND N OF A 13C AND 15N ENRICHED HYDROCHAR AND A PYROCHAR DERIVE FROM SEWAGE SLUDGE IN A SOIL AND THE AVAILABILITY OF THEIR N FOR PLANT GROWTH ...................................... 86 6.1 INTRODUCTION ..................................................................................................... 86 6.2 MATERIALS AND METHODS ................................................................................. 87 6.2.1 Production of 13C and 15N enriched SS ................................................... 87 6.2.2 Production of 13C and 15N enriched chars ............................................. 88 6.2.3 Characterization analyses ......................................................................... 88 6.2.4 Greenhouse experiment ............................................................................ 89 6.2.5 Statistical analysis ........................................................................................ 91 6.3 RESULTS AND DISCUSSION .................................................................................. 91 6.3.1 Characterization of the amendments .................................................... 91 6.3.2 Greenhouse incubation experiment ....................................................... 93 6.4 CONCLUSIONS ................................................................................................... 101 6.5 REFERENCES ........................................................................................................ 101 7 CONCLUSIONS ............................................................................................................ 103 1. INTRODUCTION 1 1 INTRODUCTION 1.1 Chemical fertilizers The amount of nitrogen (N) and phosphorus (P) in farmlands greatly determine crop yields. Traditionally, agriculture relied on natural levels of soil nutrients and on the addition of locally available organic matter (Cordell et al., 2009). Chemical N fertilizers were first used in the 19th century. However, it was not until the beginning of the 20th century, with the development of the Haber-Bosch process, that its production and application at high-scale was feasible (Razon et al. 2018). During the same period, inorganic high-grade P fertilizers, derived from mined phosphate rocks, appeared on the market (Cordell et al., 2009). The extensive application of these fertilizers, along with the introduction of new crop varieties during the so called “Green Revolution” hugely raised crop yields. However, they have been and are still overused which can result in the eutrophication of water bodies, threatening delicate ecosystems, and in an increase of greenhouse gas emission. Thus, N fertilizers have potential impacts on global climate change as well as ozone depletion (Razon et. 2018). Rosmarin et al. (2004) estimated that close to 25% of the 1 billion tons of P mined since 1950 have ended up in water bodies or has been buried in landfills. Nowadays, the use of these products is more environmentally conscious and more stringent regulated (European Nitrate Directive, Directive 91/676/EEC), which has reduced N surpluses substantially. In addition, the demand for P in most developed countries has stabilized or is decreasing after its use during the first decades of the Green Revolution since it increased the P background levels of many agricultural soils (Cordell et al., 2009). However, intensive agriculture still depends on huge amounts of chemical fertilizers. At present, the annual P need in agriculture of the European Union (EU) is estimated to be 3.85 million tons per year (Toth et al., 2014) whereas the global N fertilization is projected to reach 236x106 MT per year in 2050. (Tilman et al., 2001). Nowadays, fertilizer production is oil-dependent and consumes 1.2% of the annually produced world’s total energy, of which 94% is used for the synthesis of ammonia (Razon et al., 2018; IFA, 2009). Considering that world oil-reserves are depleting (Kerschner et al., 2013) and that the remaining phosphate rocks may be exhausted in the next 50-100 years (Cordell et al., 2009), alternative N and P sources are urgently needed. INTRODUCTION 2 1.2 Slow-release fertilizers The release rate of P and N from conventional mineral fertilizers is higher than the respective plant uptake. In addition, P fertilizers can be transformed into nonavailable forms before the crops use them. Consequently, their nutrient use efficiency is low and ranges between 30-35% for N and between 18-20% for P (Guo et al., 2018). This means not only a waste of energy and materials used for their production but also an important waste of money for farmers. In addition, the nonused fertilizers can either leach or volatilize, which jeopardize the environment. Therefore, there is a great interest and need in developing fertilizers with high nutrient-use efficiency-rates. Various formulations where the nutrients are slowly released or release in a controlled manner have already been proposed (Trenkel, 1997; Rose et al., 2016; Guo et al., 2018). According to Trenkel (1997), slowor controlled-release fertilizers are those containing a plant nutrient in a form, which either (a) delays its availability for plant uptake and use after application, or (b) is available to the plant significantly longer than a reference “rapidly available nutrient fertilizer”. According to Rose et al. (2016) most of the present slow-release N fertilizers consist of: - polymer coatings that delay fertilizer release; - higher-stability chemical fertilizers that take more time to solubilize; - granules coated or mixed with specific compounds that inhibit N transformation pathways, such as urease or nitrification inhibitors. However, the addition of N in the form of N-rich soil organic amendments is also an option. In this case, the N release rate will depend on the degradation rate of the organic N (Norg) into available plant-forms. The development of slow-release P fertilizers barely exists (Landel et al., 2003). Nevertheless, the traditional use of local organic matter (OM) such as manure and human excreta as P source meets with the approach of the N-rich soil organic amendments previously mentioned. 1.3 Organic waste Human economic and social activities are associated with the generation of huge amounts of waste, which poses not only environmental problems, but has also a negative impact on human health. Therefore, the EU has created a substantial INTRODUCTION 3 body of regulations to address this problem and turn Europe into a recycling society. The EU policies which regulate the biodegradable wastes are:  The Sewage Sludge Directive (86/278/EEC), which encourages the application of sewage sludge (SS) in agriculture and regulates its use to prevent harmful effects on soil, vegetation, animals and humans. It was initiated more than 30 years ago and currently, the European Commission is considering its revision.  The Landfill Directive (1999/31/EC), which obliges the member states to reduce the amount of biodegradable municipal waste that they landfill.  The Waste Framework Directive (2008/98/EC), which prioritizes the prevention of waste production, followed by the preparation of waste for reuse and recycling, being waste disposal the least preferable option. The Report from the European commission on the implementation of EU waste legislation for the period 2010-2012 (last one published) shows some positive data: - During this period, approximately 45% of the produced sludge was recycled for agriculture. - Most member states reported a year-by-year reduction of landfilling of biodegradable waste. - The 2020-target of recycling 50% of household and similar waste was already reached or in the way to be reached for at least half of the Member States. However, despite these promising data, the achievements of the different EU countries vary remarkable and some member states still need to work hard to efficiently implement the above-mentioned directives. These directives are in line with the EU Action Plan for the Circular Economy, which supports the transition to a sustainable development based on the principles of a low carbon, resource efficient and competitive economy. This legal framework has promoted the transformation of organic wastes into added-value products usable in agriculture. INTRODUCTION 4 1.4 Production of sewage sludge In the EU, one major source of organic waste is sewage sludge (SS), since the implementation of the Urban Waste Water Treatment Directive 91/271/EEC (CEC, 1991) forced EU countries to improve their wastewater collecting and treatment systems, leading to a steadily increase of its annual production. Despite some member states (i.e. Austria, France, Germany, Sweden) have stabilized or even slightly decreased their SS production over the last years, the EU generates more than 10 million tons of SS (dry solid) every year (Table 1.1). Table 1.1. Total quantities of sludge production (Thousand tons) in EU countries. Source: Eurostat, 2018 (personal compilation) 1991 2006 2010 2015 Belgium 127.5 176.3 157.2 (2012) Bulgaria 38 49.8 57.4 Czech Republic 166.9 203.4 196.3 210.2 Denmark 141 141 (2010) Germany 2,956 2,099.9 3,656.60 1,820.60 Estonia 27.6 27.9 34.1 (2013) Ireland 77.7 90 58.4 Greece 48.2(1990) 126 151.5 (2009) 116.1 (2014) Spain 270 1,065 1,205.10 1082.7 (2012) France 865(1992) 1059.8 (2004) 966.4 961.5 (2014) Croatia 57.6 25.5 Italy 1056.4 (2005) 1,102.70 1102.7 (2010) Cyprus 8.3 (2005) 7.1 6.7 Latvia 23.9 21.4 22.8 (2013) Lithuania 51.3 42.9 Luxembourg 15.2 9.7 9.2 Hungary 237.6 184.3 171.8 Malta 1.2 8.4 Netherlands 320 (1990) 372.7 551 551.2 (2014) Austria 254.6 262.8 239 (2014) Poland 501.3 895.1 951.5 Portugal 8 189.1 (2007) 344.3 (2009) 338.8 (2012) Romania 225.6 376.1 411.2 Slovenia 19.5 30.1 29.1 Slovakia 54.78 54.8 56.2 Finland 162 148.8 142.7 141.2 (2012) Sweden 217 (1990) 207.1 203.5 197.5 United Kingdom 1,072 1,809 1,419.10 1136.7 (2012) Norway 45.5 Switzerland 210 210 (2009) 194.5 (2013) Albania 91.5 Serbia 303 (2009) 14.3 Turkey 1181.9 (2008) 850 (2014) Bosnia and Herzegovina 0.8 0.7 1.3 (2013) Total EU 6130.6 10,159.6 14,071.40 11,233.40 INTRODUCTION 5 Sludge derives from the process of treatment of wastewater, which consists basically in the following steps (Werther and Ogada, 1999):  Firstly, the wastewater passes through racks, screens and grid chambers, where coarse and heavy solid materials are separated.  Secondly, readily settable solids and floating materials are removed in sedimentation tanks. The separated material forms the primary sludge.  A biological treatment phase follows. The activated sludge system is the most common intensive biological treatment of wastewater. During this process the microorganisms convert the colloidal and dissolved OM into various gases which are released into the atmosphere, or incorporated into cell tissues, which subsequently precipitates. The precipitated sludge removed at this stage is called secondary sludge. Some Wastewater Treatment Plants (WWTPs) perform advance biological treatments which also remove N and P dissolved in the wastewater. This occurs in modified activated sludge systems with anaerobic-aerobic or anaerobicdenitrifying spatial-temporal unit processes. Nitrogen elimination is usually achieved by firstly oxidizing the ammonia to nitrate (nitrification phase) and thereafter converting the nitrate into volatile N (Werther and Ogada, 1999). In contrast, the phosphorous compounds are incorporated into the microorganisms which, thereafter, will be removed through sedimentation (Gebremarian et al., 2011). These intensive biological treatments achieve a wastewater treatment with reduced space and during a short period of time. In extensive systems microorganisms degrade the colloidal and dissolved OM with low efficiency. They can consist in artificial ponds or in more sophisticated infiltration-percolation or wetland systems. The extensive systems require more space than the intensive one, but are cheaper, use less energy and the operation and maintenance is lower (Rodíguez-Roda et al, 2008). As such, these technologies are often used for small urban agglomerations. The waste water treatments tend to concentrate heavy metals, poorly biodegradable trace organic compounds and potentially pathogenic organisms into the SS. However, the heavy metal concentrations in SS have steadily declined in the EU since the mid 80s due to better control of use and discharge of dangerous substances, voluntary agreements and improved industrial practices. In INTRODUCTION 6 contrast, nanoparticles, pharmaceuticals and personal care products are now new emerging contaminants which must not be ignored (Fijalkowski et al., 2017). Use of SS in agriculture In addition to heavy metals and organic compounds, SS contains also valuable OM and is rich in nutrients such as N and P. Thus, the application of this waste on agricultural soils as a fertilizer or as organic soil improver has been promoted by the EU (Sewage Sludge Directive (86/278/EEC). During 2016, in the EU, the average total N and P contents of sludge recycled for agriculture were 3.6 and 2.8 %, respectively. For Spain those values were 4.5 and 3.6 % (EC, 2008b). The N plant availability in SS ranges between 15-85% compared to the availability of N in inorganic fertilizers and depends on how the sludge was stabilized before its addition to the soil. In contrast, the plant availability of P from SS is less influenced by the treatment process and can account to 50% in most sludge products (EC, 2008b). Despite the Sludge Directive allows the application of untreated sludge on soil under specific conditions, it is rarely applied and in some member states it is even prohibited (EC, 2008a). The most common stabilization process consists in a mesophilic anaerobic digestion, or aerobic digestion. After this, it is treated with polymers and dewatered using filter presses, vacuum filters or centrifuges. Other treatment processes include long-term storage, conditioning with lime, thermal drying and composting (EC, 2008b). The latter, however, consumes space and increases greenhouse gas emission by releasing CO2 and volatile N. Alternatively, thermal treatment such as pyrolysis or hydrothermal carbonization (HTC) may be used for efficient hygienization with concomitant stabilizing organic C and N within a relative short process time. Of course, during thermal treatment greenhouse gases are also released, but this emission may be compensated by recycling the produced thermal energy for other energy requiring purposes. Former solid-state 15N nuclear magnetic resonance (NMR) spectroscopic studies indicated that N in N-rich chars can be bioavailable (De la Rosa and Knicker, 2011) but is less bioavailable than inorganic N (López-Martín et al., 2017). Thus, if the biochemical resistant of charred organic residues is not as high as thought but l, these materials can still be degraded, carbonized SS may be applicable as slowrelease fertilizers. Thus, a major goal of the present work was to test if this approach is feasible and to elucidate if amendment of charred SS can add to the reduction of the need of mineral P and N. INTRODUCTION 7 1.5 Hydrothermal carbonization and dry pyrolysis These technologies consist in the thermochemical carbonization of biomass at elevated temperatures in the absence or restricted amounts of oxygen. During these processes, liquid and gaseous (by-) products are formed due to the degradation of biomacromolecules, whereas the solid residue considerably differs from the original biomass. During pyrolysis, the biomass is carbonized in a largely inert atmosphere such as N2 and CO2 at temperatures between 300 and 700 °C (Lehmann and Joseph, 2015). The resulting product is named pyrochar and can be considered as biochar if it accomplishes with certain characteristics, described below. Hydrothermal carbonization (HTC) carbonizes the biomass in the presence of water at 180-250 ˚C during 0.5-12 h under autogenous pressure and the solid product is called hydrochar (Libra et al., 2011). The term ‘wet pyrolysis’ also refers to HTC whereas ‘dry pyrolysis’ is used for pyrolysis. Pyrolysis can destroy hazardous organic compounds, present in the feedstock but may also produce them by recondensation of volatiles (Sharma et al., 2004). In contrast to organic compounds, metals are not removed and will be enriched in the final char. The chemical reactions involved in both processes include hydrolysis, dehydration, decarboxylation, aromatization and recondensation (Lehmann and Joseph, 2015; Funke and Ziegler, 2010). However, the reactions mechanisms are scarcely understood. Aside from realizing that the chemistry of the substrates defines the properties of the product, it is known that the different reaction media during the dry-pyrolysis and HTC play a defining role. During pyrolysis, the temperature was reported to be the main factor determining the dominating reactions. In contrast, the HTC starts breaking the biomacromolecules into fragments through hydrolysis, which may allow the initiation of completely different chemical pathways than in pyrolysis. Thus, despite pyrochars and hydrochars have some similarities they also differ considerably both in their chemistry and characteristics (Libra et al., 2011). To which extend those differences affect the behavior as soil amendment still has to be explored and will be a part of the present research. The HTC usually achieves a higher solid yield than pyrolysis. In addition, the nature of the feedstock, process temperature and reaction time are the main factors influencing the composition and properties of hydrochars and pyrochars (Lehmann and Joseph, 2015; Libra et al., 2011). Generally, pyrolysis is supposed to INTRODUCTION 14 Guo H., White J.C., Wang Z., Xing B. (2018) Nano-enabled fertilizers to control the release and use efficiency of nutrients. Current Opinion in Environmental Science & Health, In press, https://doi.org/10.1016/j.coesh.2018.07.009 Hammes K., Smernik R.J., Skjemstad J.O., Schmidt M.W.I., (2008) Characterisation and evaluation of reference materials for black carbon analysis using elemental composition, colour, BET surface area and 13C NMR spectroscopy. Applied Geochemistry. 23, 2113–2122. Haider G., Steffens D., Moser G., Christoph Müller C., Kammann C.I. (2017) Biochar reduced nitrate leaching and improved soil moisture content without yield improvements in a four-year field study. Agriculture, Ecosystems & Environment 237, 80-94. Jeffery S., Abalos D., Prodana M., Bastos A.C., van Groenigen J.W., Hungate B.A., Verheijen F. (2017) Biochar boosts tropical but not temperate crop yields. Environ. Res. Lett. 12, 053001 Jeffery S., Verheijen F.G.A., van der Velde M., Bastos A.C. 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INTRODUCTION 15 López-Martín M., Nowak K.M., Milter A., Knicker H. (2017) Incorporation of N from burnt and unburnt 15N grass residues into the peptidic fraction of fire affected and unaffected soils. J Soils Sediments 17, 1554–1564. Lian L.Y., Roberts G., 2011. ‘Introduction’, in: Lian L.Y. and Roberts G, Protein NMR Spectroscopy: Practical Techniques and applications, John Wiley and Sons Ltd, United Kingdom. 1-4. Libra J., Ro K., Kammann C., Funke A., Berge N., Neubauer Y., Titirici M., Fuhner C., Bens O., Kern J., Emmerich K. (2011) Hydrothermal carbonization of biomass residuals: a comparative review of the chemistry, processes and applications of wet and dry pyrolysis, Biofuels 2, 89–124. Michener R., Lajtha K. (2007) Stable Isotopes in Ecology and Environmental Science, second ed., Blackwell Publishing, Oxford. Razon L.F. (2018) Reactive nitrogen: A perspective on its global impact and prospects for its sustainable production. Sustainable Production and Consumption 15, 35-48. 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(2001) Forecasting agriculturally driven global environmental change. Science, 292 281-284. INTRODUCTION 16 Tóth G., Guicharnaud R.A., Tóth B. Hermann T. (2014) Phosphorus levels in croplands of the European Union with implications for P fertilizer use. European Journal of Agronomy 55, 42-52. Trenkel M.E. (1997) Improving Fertilizer Use Efficiency: Controlled-Release and Stabilized Fertilizers in Agriculture. The International Fertilizer Industry Association, Paris, France. Werther J., Ogada T. (1999) Sewage sludge combustion, Prog. Energy Combust. Sci. 25, 55–116. Wilson M.A. (1987) NMR Techniques and Applications in Geochemistry and Soil Chemistry. Pergamon Press, Oxford. 2. RATIONAL AND OBJECTIVES 17 2 RATIONAL AND OBJECTIVES There is an increasing urge to reduce the use of mineral fertilizers. At the same time, modern society produces a huge amount of organic waste, rich in N and P, which needs to be treated or preferentially recycled as value-added product. Although SS has been identified as having the potential to satisfy both needs, nutrient leaching has been observed after its application to soil both directly and after stabilization. In addition, hygienization as well as destruction of potential organic pollutants have to be performed prior to its application to soils. Therefore, the present PhD proposes pyrolysis and hydrothermal carbonization (HTC) as a mean to convert SS into a hygienized product which after its application to soil degrades only slowly but still provides its N and P for plant growth. As such, it may have the potential to act as a slow-release fertilizer. This would reduce the amount of organic waste for disposal on the one hand and the dependence on chemical fertilizers as well as nutrient leaching on the other hand. It is well known that the chemical structure of a product defines its function. However, presently the knowledge about the chemical reactions that occur during the thermal treatment of SS is still scarce. Therefore the main objectives of the present work were: - to bring some light on the thermally induced alteration by an in-detail characterization of the chemical composition and some physical properties of the treated SS produced under different process conditions. - to elucidate if the thermally altered products allow plant growth and how the latter is affected by the different N and P forms of the chars. - To obtain some insights about the stability of the organic N forms of the charred SS (Black Nitrogen) in soils after their amendment and to elucidate their potential for C sequestration. The actual knowledge on the chemistry of N-rich charred residues as well as on its behavior after its addition to soil bases the initial hypotheses of this work: i) During the pyrolyzation and HTC of SS at least part of its N will be integrated into the aromatic network as heterocyclic N which will be mobilized only slowly for plant growth after its amendment to soil. ii) Although the dynamic and chemistry of P is not comparable to that of the N, we expect a similar behavior for both during the pyrolysis and HTC of at least the organic P fraction. 18 iii) Once applied to land, these chars will be attacked and degraded by microorganisms, leading to a slow mobilization of its N and P. iv) Relative to untreated SS, the chars will show a higher biochemical resistance and have the potential to add to the C sequestration potential of soils. As a consequence some of the organic N will also stay in the soil and may be usable for plant growth at a later time. In order to achieve those objectives the following goals were define: 1. The processing conditions (pyrolysis/HTC, temperature, residence time, etc) influence the composition and properties of the final product, which in turn determine its potential use. Consequently, the first goal of this work was a detailed characterization of the composition and chemical changes occurring during HTC and pyrolysis of SS. Different temperatures and residence times were used. This characterization was not limited to the organic C fraction but included also the N-forms. (Chapter 3) 2. Despite char properties can indicate its potential as soil amendment and nutrient source, its use for plant growth has to be tested. Therefore, the second task of this work was to evaluate the impact of the hydrochars and pyrochars on the germination and growth of Lolium perenne under the controlled conditions of a greenhouse incubation study. (Chapter 4) 3. The potential of pyrolyzed and HTC-treated SS as slow-release P fertilizer depends on the speciation of this element in the chars as well as on the potential changes of this speciation once the chars are applied to the soil. Therefore, we studied the alteration of the P contained in the SS during the HTC and pyrolysis processes as well as the possible changes in the P availability of the chars after their soil addition. (Chapter 5) 4. The biochemical resistance of SS-derived hydrochars and pyrochars determines their degradation rate and thus their potential for C sequestration. However they are also related to their potential as slow release N fertilizers. Therefore, 13C and 15N enriched SS was produced in the laboratory and transformed into hydroand pyrochar, which were amended to soil on which Lolium perenne grew during a 10-month greenhouse incubation experiment. The recalcitrance of the organic C and N in the chars was monitored by determining their stable isotopic ratios in the soil and plants as a function of incubation time. (Chapter 6) 3. Hydrothermal carbonization and pyrolysis of sewage sludges: what happen to carbon and nitrogen? 19 3 HYDROTHERMAL CARBONIZATION AND PYROLYSIS OF SEWAGE SLUDGES: WHAT HAPPEN TO CARBON AND NITROGEN? Abstract Hygienization by thermochemical carbonization may be one option to enable the use of sewage sludge (SS) as soil amendment and nitrogen (N) fertilizer. To evaluate this possibility, SS derived from different water purification processes of a rural waste water treatment plant were hydrothermal carbonized (HTC) at 200 and 260 °C for 0.5 and 3.0 h, and pyrolyzed at 600 °C for 1h. During HTC, temperature rather than residence time affected the chemical alteration. Solid-state 13C and 15N nuclear magnetic resonance (NMR) spectroscopy showed considerably lower aromaticity in the hydrochars than in the pyrochars. Whereas the aromatic network of the hydrochars is dominated by polyfurans and N-heterocyclic aromatic units that of the pyrochars is mainly composed of arene structures. The highest total and inorganic N contents were obtained via HTC at 200 °C for 30 min, thus this material may be applied if both immediate and slow N release is needed. Keywords Slow-release N fertilization, hygienization, pyrochar, hydrochar, solid-state NMR spectroscopy 3.1 INTRODUCTION In the European Union (EU), the major N and P sources in agriculture are synthetic fertilizers although inputs from animal manure remain important, especially in regions of high livestock density. The use of synthetic fertilizers has dramatically increased food production worldwide, but the unintended costs to the environment and human health due to surplus and inefficient application have also been substantial. Thus, alternatives are needed. One of those represents the valorization of organic waste, the amount of which increased exponentially during the last decades. Its estimated annual increase is around 25 million tons (WRAP, 2008). A major source of organic waste is sewage sludge (SS). Containing high amounts of N, with a median of 3.3 % (Sommers et al., 1977), P and OM, this material offers CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 20 an excellent feedstock for the production of soil amendments that can reduce the need of synthetic P and N fertilizer (Kern et al., 2008; Theobald et al., 2016). At the same time it may increase the C-sequestration potential of cropped soils. In addition, sludge recycling as fertilizer or organic amendment helps to reduce the amount of organic waste by returning it into the bio-cycle while at the same time soil physical and chemical properties are restored (Goss et al., 2013; Werther et al., 1999). Indeed, the Sewage Sludge Directive (Council Directive 1986/278/EEC) and the working document on sludge (European Commission, 2000) describe the use of SS on soils as beneficial but also seek to encourage a safe use of this material in agriculture and to regulate its application to prevent harmful effects on soil, vegetation, animals and humans (Kelessidis and Stasinakis, 2012). Thus, in order to enable the use of SS as soil application, it first has to be decontaminated by removing organic pollutants and pathogen bacteria. Different treatments can be used for the latter: thermal drying, anaerobic digestion, conditioning with lime, etc. In general, the process of removing pathogen bacteria of SS is called hygienization. Heavy metals content also need to be taken into account. In cases their concentrations are above the allowed thresholds, strategies have to be developed to avoid secondary contamination due to SS application. The thermochemical carbonization of SS at elevated temperatures in the absence of oxygen has recently been considered to achieve sludge stabilization and hygienization. In the presence of water, this kind of carbonization is called hydrothermal carbonization (HTC), whereas in the absence of water it is known as pyrolysis. During pyrolysis, temperatures between 300-700°C are used (Lehmann and Joseph, 2015). The solid product is considered as biochar, according to the European Biochar Foundation, if it derives from a feedstock approved for this organization, its organic carbon content is above 50% and its atomic H/C ratio is < 0.7 (European Biochar Foundation, 2012). Considering that these thresholds are rarely reached with carbonized SS, in the following we refer to pyrolyzed SS as pyrochar. Hydrochars represent the solid residue after heating biomass together with water at 180-250˚C for 1-12 h under autogenous pressure (Libra et al., 2011). Due to the high humidity of SS, the application of HTC avoids the necessity of an additional drying step. Both types of carbonization include reactions such as dehydration, decarboxylation, aromatization and recondensation (Funke et al., 2010; Libra et al., 2011). However, the different process conditions lead to significant differences of both the chemistry and physical characteristics of the solid products (Libra et al., 2012). Accordingly, hydrochars contain a high amount of furan-type structures. On the other hand, pyrochars produced at high temperatures are composed of an arene-rich core (Falco et al., 2011; Baccile et al., 2009; Baccile et al., 2011). CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 21 Despite alterations of the organic C fraction during the pyrolysis and HTC of biomass have already been focus of many investigations (Lehmann and Joseph, 2015; Lu and Berge, 2014; Reza et al., 2014a, 2014b) the knowledge about changes of organic N is still scarce. Previous studies have shown that after incomplete combustion of N-rich plant residues and casein at 350 °C for several minutes a considerable part of their organic N was incorporated into heterocyclic aromatic structures, the so called pyrogenic organic nitrogen (Knicker et al., 2008; Knicker, 2010). Although this nitrogen is less bio-available than mineral fertilizer it was mobilized and used by plants and microbes for the neo-formation of biomass and amides (De la Rosa and Knicker, 2011). Thus, N-rich carbonized OMs may be prime candidates for the development of slow N-release fertilizers, since the mobilization of their N is expected to be sufficiently low to avoid fast N losses due to leaching or nitrification shortly after fertilizer application. A deeper knowledge of the N transformations during the thermochemical carbonization of SS is necessary in order to evaluate its potential to fulfill this function. Wei et al. (Wei et al., 2015) found that ammonia (NH3) is the main product of protein-N during SS pyrolysis and that the majority of pyridine N originally present in SS tends to be converted into hydrogen cyanide (HCN) at 400-600 °C. Studying HTC as a potential tool to reduce NH3 and HCN emissions during the pyrolysis of SS-derived residues, Liu et al. (Liu et al., 2017) confirmed the conversion of nitrogen into more stable forms by the application of HTC. Since chemical composition and physical properties of a soil amendment define its function, the goal of the present research was a detailed characterization of the chemical changes during HTC and pyrolysis of two different SS. This analysis was not limited to the organic C fraction but included also the N-forms. The SS used in this study derived from a rural waste water treatment plant of a community with approximately 3000 inhabitants. They were collected from an extensive and an intensive cleaning process. It is important to bear in mind that physical and chemical properties of the SS depend on the wastewater treatment process by which it was produced and on the characteristics of the treated effluents (Zielinska et al., 2015). For example, the commonly called primary SS is formed from suspended organic material which is separated by sedimentation; whereas secondary sludge constitutes mainly residues of bacteria biomass which is involved in the digestion of the easily metabolized organic fraction of the water. Thus, primary sludge is expected to be biochemically less stable than secondary sludge (Goss et al., 2013). This may also affect the chemical alteration during thermal carbonization, which was applied for hygienization before it can be applied as soil amendment. In order to evaluate the potential of the products as slow-release N fertilizer, their chemical structures was not only analyzed by Infrared (IR-FT) and CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 28 The total N content of A_SS and T_SS are 19 and 32 g kg-1, respectively (Table 3.1). These values are within the range reported for this material (Porta et al., 2003). Compared to C, higher losses of N were observed for both hydrochars and pyrochars. However, in all the charred samples the N contents are still > 9 g kg-1, most of which occurs in organic forms (> 97 %) (Table 3.2). Therefore, the organic N sequestered in these chars is likely to affect the N cycling in soils once this material has been applied. The bioavailability of organic N in the chars is influenced by its composition and by the chemical and biological changes it undergoes after its addition to soil, but still, it will be lower than that expected for inorganic N forms (Ni). After all charring treatments, the Corg/N ratio (w/w) increased slightly, most tentatively due to deamination and degradation of amino acids and amino sugars, but also by volatilization of NH3 and nitrous oxides. However, even after inclusion the pyrochars, the C/N ratios do not exceed the value of 20, which is considerably lower than biochars from wood (De la Rosa et al., 2014). Thus, if the organic N in chars occurs in bioavailable forms, both the hydroand pyrochar can serve as an efficient additional N-source for biomass production provided sufficient bioavailable C is present, too. The contribution of Ni to total N increased after HTC from 0.5 to 2.6 % and from 0.4 to 2.2 % for A and T hydrochars, respectively. However, after pyrolysis all Ni was lost (Table 3.2). Regarding the different available inorganic nitrogen forms, for both A_SS and T_SS the NH4-N contents are considerably higher (115 and 123 mg kg-1, respectively) than their NO2-N and NO3-N contributions. This trend was also observed by Alvarenga et al. (2015), Adegbidi and Briggs (2003) and Hossain et al. (2011). Increasing NH4-N levels after HTC at 200 °C are best explained with deamination reactions. Increasing the temperature leads to further transformation and to N losses into the gaseous phase. Here, one has to bear in mind that the liquid phase which was discarded can represent a further considerable sink for inorganic N. Comparable trends, but at a much lower concentration level, are observed for NO2-N and NO3-N. The increase of NH4+ after HTC is in agreement with the results obtained by Sohn and Ho (1995), who demonstrated that after heating aqueous solutions of amino acids at 180 °C during 2 h, considerable amounts of NH3 were released. On the other hand, Jin-hong et al. (2014) reported a considerable decrease of available NH4-N and NO3-N for SS hydrochars produced at 190 and 260 °C during 1, 6, 12, 18 and 24 h. They may have achieved the lack of Ni in their samples because of intense rinsing of the HTC-chars with distilled water. Thus, post-treatment processes also need to be taken into account to evaluate the fertilizer potential of HTC chars. Here one has to bear in mind that efficient removal of Ni may counteract fertilization purpose since this easily available N form would be accessible already during the early plant growing stage. CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 29 Table 3.2. pH, Corg/N ratios, and inorganic nitrogen (Ni) contents of primary (A_SS) and secondary (T_SS) sewage sludges and their respective hydrochars produced at 200 °C (_HTC_200) and 260 °C (_HTC_260) for 0.5 and 3 hours (_0.5, _3, respectively), as well as the pyrochars produced at 600 °C for 1h (_Py__600_1). Sample pH Corg/N NH4-N NO2-N NO3-N Nia of the total N (w/w) mg kg-1 mg kg-1 mg kg-1 % A_SS 7.4 11.9 115 0.03 0.18 0.5 A_HTC_200_0.5 6.5 14.0 417 0.36 0.59 2.6 A_HTC_200_3 6.5 15.4 349 0.20 0.34 2.5 A_HTC_260_0.5 6.4 19.1 169 0.06 0.15 1.5 A_HTC_260_3 6.6 18.2 227 0.05 0.003 1.9 A_Py_600_1 9.3 15.5 0.2 0.003 -b 0.0 T_SS 7.5 7.6 123 0.04 0.57 0.4 T_HTC_200_0.5 6.7 9.3 542 0.76 1.26 2.2 T_HTC_200_3 6.2 9.7 341 0.36 0.44 1.4 T_HTC_260_0.5 6.3 11.4 239 0.09 0.47 1.2 T_HTC_260_3 6.4 11.8 256 0.04 - 1.4 T_Py_600_1 10.0 10.3 0.3 - - 0.0 a) Ni : sum of NH4-N,NO2-N, and NO3-N in %; b) - : below the detection limit In contrast to the HTC chars, the pyrochars did not accumulate Ni and consequently, their remaining N must have been sequestered in organic constituents of the char as it was suggested by Knicker et al. (2010). Thus, whereas pyrolysis turns almost the entire liberated N into gaseous compounds HTC leads to deamination and accumulation of NH4+. However, some of the N will be also lost through the liquid phase. 3.3.2 Solid-state 13C and 15N NMR spectroscopy 3.3.2.1. Sewage sludge materials The solid-state 13C NMR spectra of both A_SS and T_SS are dominated by signals in the chemical shift region of alkyl C (0-45 ppm). Both lipids and peptides can contribute to this region, which comprises 43 for A_SS and 38 % for T_SS of the total 13C signal intensity (Table 3.3). Their carboxyl C and amide C add to the signal at 174 ppm in the chemical shift region between 220 and 160 ppm (Figure 3.2). The Oalkyl C region (90-60 ppm) accounts for 17 and 23 % of the total 13C intensity for A_SS and T_SS, respectively. The latter comprises signals from C2-C5 in CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 30 carbohydrates, alcohols and ethers. The region between 110 and 90 ppm is commonly assigned to anomeric C (C1) in pentoses and hexoses but can also derive from C2 and C6 in lignin derivatives. However, lignin compounds are of minor importance in SS, which is mainly composed of proteins, carbohydrates, fats and oils (Werther and Ogada, 1999). In addition, A_SS contains slightly more aromatic C (160 to 110 ppm) than T_SS. Considering the low contribution of lignin to SS, most of this signal intensity derives from aromatic amino acids or olefinic structures, which contribute to this chemical shift region, too. Jin-hong et al. (2014) showed also a dominance of the alkyl C region for SS, with a value of 64 %. This is due to the large contribution of fat acids but also peptides typically present in domestic SS (Werther and Ogada, 1999). Calculating the alkyl C/carboxyl C ratios from the intensity distribution of the 13C NMR spectra as an index for the average chain length of the alkyl residues, values of 4.7 and 3.5 (Table 3.3) are obtained confirming that in our SS samples, lipids comprise a major fraction. This is in line with results by Jin-hong et al. (2014). The higher value of this ratio obtained for A_SS may be due to the anaerobic conditions during its production, which leads to a preferential preservation of long chain paraffinic structures. In addition, the higher N content of T_SS compared to A_SS is consistent with the higher 13C-intensity in the region of N-alkyl C spectrum. This points to a higher proportion of proteinaceous material for the former than for the latter, which contributes to decrease the alkyl C/carboxyl C ratio. Carbohydrates are commonly preferentially degraded during thermal treatments, leading to the relative enrichment of all other compound classes. Based on this, Baldock et al. (1997) introduced the ratio alkyl C/O-alkyl C as an index for the degradation degree. Calculating this ratio, a value of 2.5 was obtained for A_SS and 1.7 for T_SS (Table 3.3), confirming that the material of the anaerobic pond (A) is more humified, thus biochemically stabilized, than that derived from the “thickener pond” (T). The solid-state 15N NMR spectra of both A_SS and T_SS are dominated by a signal at -260 ppm in the chemical shift region of amide N (from -248 to -285 ppm), Figure 3.3. This signal contributes with 69 and 74 % to the total 15N intensity of the spectra of A_SS and T_SS, respectively (Table 3.5). Most of this nitrogen occurs as peptides and, to a lower amount, as amides in amino sugars. Note that some amide structures are also contributing to the shoulder between -230 and -248 ppm. The respective N-alkyl C of these compounds gives rise to signals in the chemical shift region between 60 and 45 ppm of the 13C NMR spectra. This signal contributes with 13 and 14 % to the total 13C intensity of the spectra of A_SS and E_SS, respectively (Table 3.3). Although lignin is not considered as a major component of sewage sludge, its methoxyl C would contribute to the signal of this region, too. The peak at CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 31 -346 ppm, which appears in both A_SS and T_SS 15N NMR spectra, corresponds to free aliphatic amino groups of amino acids or amino sugars, but the low signal-tonoise ratios of the spectra does not allow an unbiased differentiation of such a signal from the noise. Regarding pyrrole compounds (from -150 to -248 ppm) A_SS shows a higher contribution, with a 22 %, than T_SS with a 17 % (Table 3.5). However, in contrast to Liu et al. (2017) who applied X-ray photoelectron spectroscopy (XPS), no indications for the presence of pyridine N or nitrile N was evidenced. In studies about the nitrite fixation to humic material, their chemical shifts were found between -65 to -80 ppm and -90 to -140 ppm, respectively (Thorn et al., 2016). 3.3.2.1. Hydrochars and pyrochars After HTC, the contribution of the chemical shift region of alkyl C to the total 13C intensity increased for the A samples from 43 (A_SS) to 55 % (260 °C, 30 min) and for the T set from 38 (T_SS) up to 52 % (260 °C, 3 h) (Table 3.3). An augmentation in this region was also observed by Jin-hong et al. (2014) after HTC of SS at 190 and 260 °C for 1 h. Table 3.4, showing the recovery of C in the different C groups after HTC treatment, indicates that there was no net-synthesis or degradation of lipids, but a relative enrichment due to the loss of other compounds. Thus, lipids were not affected by HTC, which is in line with previous works of Popov et al. (2016) and Shin et al. (2012). The latter demonstrated that three kinds of fatty acids were stable under subcritical water conditions at temperatures of 300 °C or below. As expected, HTC resulted in decarboxylation which is revealed by the diminishment of the recovery of C as carboxyl C. Whereas Oand N-alkyl C were degraded, as HTC charring temperature and time increased, aromatic C was formed, most likely by dehydration and cyclization of carbohydrates and peptideous material. This is indicated by the increase of C which occurs as aryl C and Oand N-aryl C for both, the “A” and “T” hydrochars (Table 3.4). The latter suggests the formation of furans and N-heterocyclic aromatic structures. Jin-hong et al. (2014) reported comparable results for SS hydrochars. Considering that O/Naryl C constitutes a third of the aromatic C in the hydrochars, a predominantly polyfuran core with additional N-heterocyclic aromatic units instead of a benzoidal graphenic network (arene structures) has to be assumed (Baccile et al., 2009). As expected, an increase in the alkyl C/O-alkyl C ratio occurred after HTC. This increase was more affected by temperature than by time with HTC. The highest values were obtained for hydrochars produced at 260 ºC for 3 h, with 17.8 and 16.9 for “A” and “T” hydrochars, respectively (Table 3.3). CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 32 Table 3.3. Intensity distribution (%) of the solid-state 13C NMR spectra of primary (A_SS) and secondary (T_SS) sewage sludges and their respective hydrochars produced at 200 °C (_HTC_200) and 260 °C (_HTC_260) for 0.5 and 3 hours (_0.5, _3, respectively), as well as the pyrochars produced at 600 °C for 1 h (_Py__600_1). Carboxyl/ Amide C O/NAryl C Aryl C O-Alkyl C N-Alkyl/ Methoxyl C Alkyl C Alkyl C/ Carboxyl C Alkyl C/ O-Alkyl C ppm 220-160 160-140 140-110 110-90 90-60 60-45 45-0 A_SS 9.1 2.8 10.4 4.3 17.3 12.9 43.1 4.7 2.5 A_HTC_200_0.5 6.3 5.0 16.3 3.6 12.0 9.6 47.2 7.5 3.9 A_HTC_200_3 6.4 5.6 18.1 2.5 8.9 7.5 51.1 8.0 5.7 A_HTC_260_0.5 5.4 5.9 20.3 2.2 4.2 6.7 55.4 10.3 13.2 A_HTC_260_3 4.8 6.1 22.0 2.1 3.1 6.7 55.2 11.5 17.8 A_Py_600_1 6.0 10.8 68.5 4.2 3.3 1.8 5.4 0.9 1.6 T_SS 10.7 1.9 7.5 4.9 22.6 14.3 37.6 3.5 1.7 T_HTC_200_0.5 10.1 5.0 19.3 2.8 8.4 9.2 45.2 4.5 5.4 T_HTC_200_3 7.4 6.1 24.1 2.7 5.1 6.1 48.6 6.6 9.5 T_HTC_260_0.5 7.3 6.5 25.2 1.9 3.3 4.8 51.0 7.0 15.5 T_HTC_260_3 5.0 6.1 25.2 2.8 3.1 5.7 52.3 10.5 16.9 T_Py_600_1 5.8 12.1 68.6 4.9 2.8 1.3 4.5 0.8 1.6 CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 33 Figure 3.2. Solid-state 13C NMR spectra of primary (A_SS) and secondary (T_SS) sewage sludges and their respective hydrochars produced at 200 °C (_HTC_200) and 260 °C (_HTC_260) for 0.5 and 3 hours (_0.5, _3, respectively), as well as the pyrochars produced at 600 °C for 1h (_Py__600_1). CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 34 Table 3.4. C-recovery of the different C groups of primary (A_SS) and secondary (T_SS) sewage sludges as a function of charring conditions (% of C present in the original sewage sludge). Carboxyl/ amide C O/ NAryl C Aryl C O-Alkyl C N Alkyl/ Methoxyl C Alkyl C ppm 220-160 160-140 140-110 110-90 90-60 60-45 45-0 A_SS 9 3 10 4 17 13 43 A_HTC_200_0.5 5 4 14 3 10 8 40 A_HTC_200_3 5 5 15 2 7 6 42 A_HTC_260_0.5 4 5 16 2 3 5 43 A_HTC_260_3 4 5 17 2 3 5 44 A_Py_600_1 3 5 29 2 1 1 2 T_SS 11 2 8 5 23 14 38 T_HTC_200_0.5 8 4 15 2 7 7 36 T_HTC_200_3 6 5 18 2 4 5 37 T_HTC_260_0.5 5 5 18 1 2 3 35 T_HTC_260_3 3 4 17 2 2 4 35 T_Py_600_1 3 5 31 2 1 1 2 CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 35 Pyrolysis resulted in more drastic chemical changes, which are expressed in an almost complete transformation of organic matter into aromatic structures. In the spectra of A_Py_600_1 and T_Py_600_1, the aryl C region between 140 and 110 ppm accounts for 69 % of the total 13C intensity (Table 3.3) and the Oand N-aryl C region for only 11 to 12 %, respectively. This can be taken as an indication that benzoic rings rather than furans dominate the aromatic network. Table 3.5. Relative distribution of the 15N intensity between the chemical shift region -150 to - 285 ppm of the solid-state 15N NMR spectra of primary (A_SS) and secondary (T_SS) sewage sludges and their respective hydrochars produced at 200 °C (_HTC_200) and 260 °C (_HTC_260) for 0.5 and 3 hours (_0.5, _3, respectively), as well as the pyrochars produced at 600 °C for 1h (_Py__600_1). Pyrrole N Amide N ppm -150 to -248 -248 to -285 A_SS 22.4 77.6 A_HTC_200_0.5 38.7 61.3 A_HTC_200_3 50.9 49.1 A_HTC_260_0.5 65.3 34.7 A_HTC_260_3 60.3 39.7 A_Py_600_1 75.4 24.6 T_SS 17.2 82.8 T_HTC_200_0.5 30.5 69.5 T_HTC_200_3 56.3 43.7 T_HTC_260_0.5 64.7 35.3 T_HTC_260_3 57 43 T_Py_600_1 58.2 41.8 The 15N NMR spectra of the hydrochars confirm the formation of N-heterocyclic aromatics during HTC already at 200 °C by a clear shoulder in the region of pyrroletype N (-150 and -248 ppm). Structures such as indoles, imidazole and pyrroles are contributing to the intensity in this region. Protonated pyridine-type N may be a further component giving rise to intensity around this chemical shift but considering the pH of the CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 36 Figure 3.3. Solid-state 15N NMR spectra of primary (A_SS) and secondary (T_SS) sewage sludges and their respective hydrochars produced at 200 °C (_HTC_200) and 260 °C (_HTC_260) for 0.5 and 3 hours (_0.5, _3, respectively), as well as the pyrochars produced at 600 °C for 1h (_Py__600_1). CHAPTER 3 Hydrothermal carbonization and pyrolisis of sewage sludges: what happen to carbon and nitrogen? 37 samples, such structures are unlikely. However, unprotonated pyridine-type N may contribute to the weak shoulder around -70 ppm, but the low signal-to-noise ratios of the spectra does not allow an unbiased differentiation of such a signal from the noise. In contrast to the observations derived from 13C NMR spectroscopy, the solidstate 15N NMR spectra indicate chemical changes, expressed as an augmentation of the pyrrole-type N, not only with increasing temperature but also with residence time. The change is also expressed in a shift of the peak of the main signal from - 260 ppm to -254 ppm. Note that pyrrole N has its chemical shift around -230 ppm and indole N around -245 ppm. The largest contribution of pyrrole-type N is evidenced in the samples obtained after heating at 260 °C for 30 min, with a 65 % for A_HTC_260_0.5 and T_HTC_260_0.5 (Table 3.5). The 15N intensity in the chemical shift region of amide N (-248 to -285 ppm) underwent a drastic decrease after HTC. This region dominates only the spectra of hydrochars produced at the mildest HTC conditions. Here, it has to be taken into account that carbazole N has its chemical shift around -262 ppm. Benzamide-type N has its resonance frequency at -275 ppm. Thus, such compounds are likely to contribute to the amide N region as well. Regarding dry pyrolysis, it resulted in a dominance of pyrrole-type N with a contribution of 75 and 58 % for A_600_1 and T_600_1, respectively (Table 3.5). Here, the main peak occurs at -240 ppm. 3.3.3 Fourier transform-infrared spectroscopy All FT-IR spectra (Figure 3.4) of the original SS, hydrochars and pyrochars exhibit several peaks around 3700-3600 cm-1, which are attributable to vibration of OH groups in the mineral matter (Hossain et al., 2011; Bernier et al., 2013). The peaks appearing from 3600 cm-1 to 3300 cm-1 are assigned to stretching OH bond in water, carboxyl and hydroxyl groups (Bernier et al., 2013). All spectra show these two bands but their intensities decrease after the HTC and dry pyrolysis processes. In addition, all spectra exhibit an intense signal at 1030 cm-1 which can be caused by Si-O-Si vibrations (Zielinska et al., 2015; Peng et al., 2016) or by C-O vibrations in polysaccharides. Silicate may derive from street run-offs which also enter the waste water systems or from residues of the sand used during the mechanical pretreatment of the water-cleaning process. The increase of this band with temperature supports such an assignment to Si-O-Si vibration since partial combustion of organic matter during heating leads to a relative enrichment of mineral matter of the sample. The FT-IR spectra of the untreated SS (A_SS and T_SS) indicate a pattern, which can be assigned to microbial biomass. 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Hydrothermal carbonization and pyrolysis of sewage sludges: effects on Lolium perenne germination and growth 48 4 HYDROTHERMAL CARBONIZATION AND PYROLYSIS OF SEWAGE SLUDGES: EFFECTS ON LOLIUM PERENNE GERMINATION AND GROWTH Abstract The pyrolysis and hydrothermal carbonization (HTC) of sewage sludge (SS) result in products free of pathogens, with the potential of increasing crops yields. However, more research is needed to determine the most beneficial production conditions for agricultural purposes. With this work, we evaluate the impact of pyrolysistreated (600ºC, 1h) and HTC-treated (200 ºC, 260 ºC; 0.5 h, 1h) SS on the germination, survival and growth of Lolium perenne during an 80-days greenhouse experiment. The hydrochars and pyrochars were applied on a Calcic Cambisol with doses of 5 and 25 t ha-1. Our results showed that the addition of pyrochars to the Cambisol had neither impact on Lolium germination and survival rates, nor on its biomass production. In comparison to the control, higher or equal biomass was yielded from hydrochar-amended pots, even though some hydrochars decreased plant germination and survival rates. Among all the evaluated char properties, only the organic and inorganic nitrogen (N) contents of the chars, along with their organic C values, correlated with increased total and shoot biomass production. Our work shows that low-temperature HTC of SS results in a soil amendment with Nfertilization potential whereas chars produced from HTC with higher temperatures and pyrolysis do not exhibit the same suitability. Keywords Hydrochar, pyrochar, nitrogen, fertilizer, greenhouse experiment, biosolids 4.1 INTRODUCTION The revised Waste Framework Directive (Council Directive 2008/98/EC) promotes prevention and minimization of organic waste as well as their reuse and recycling. This can be supported by its transformation into added-value products for use in agriculture. Traditionally, this is achieved by digestion and composting (Kelessidis and Stasinakis, 2012). Sewage sludge (SS) is a nutrient-rich organic waste, which is produced in increasing amounts. More than 10 million tons (dry weight) are produced annually in Europe (Millieu Ltd., WRc and RPA, 2010). Recycling of SS to agriculture can return nitrogen (N), phosphorus (P), other plant nutrients and organic matter to the soil and may help to reduce the dependency on fossil fuel-consuming synthetic N CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 49 fertilizer and non-renewable P sources. However, its application poses some environmental risks such as nutrient leaching, reduced soil biodiversity, increased greenhouse gas emissions (Millieu Ltd., WRc and RPA, 2010), and health risks if not pre-treated properly. One possibility for hygienization of SS before its application to soil represents composting. However this process consumes not only space and time but also releases greenhouse gases such as CO2 and volatile N. An alternative may be a thermal treatment of SS through pyrolysis or hydrothermal carbonization (HTC). These technologies allow efficient hygienization while concomitantly stabilizing organic C and N within a relative short process time. Of course, during thermal treatment greenhouse gases are also released, but this emission may be compensated by recycling the produced thermal energy for other energy requiring purposes. Both pyrolysis and HTC carbonize biomass in low-oxygen environments. Temperatures between 300 and 700 ºC are typically used during dry pyrolysis. Hydrochars are typically produced at temperatures between 180 and 250 ºC in the presence of water, which creates autogenous pressure. The advantage of transforming SS into hydrochar rather than pyrochar lies in lower energy costs due to lower process temperatures and the fact that pre-process drying of the feedstock is not necessary. On the other hand they are considered to be biochemically less stable than biochar (Lehmann and Joseph, 2015; Libra et al., 2011). Commonly, SS is characterized by a high ash content yielding in pyrolyzed products with organic matter contents which are too low to meet the requirements of the International Biochar Initiative or the European Biochar Certificate (International Biochar Initiative, 2015; European Biochar Foundation, 2016) to be called biochars (Bachmann et al., 2016). Therefore, we will refer to pyrolyzed SS as pyrochar. Despite the potential of pyrochars and hydrochars to increase the amount of stable carbon stored in soils, its use in agriculture will only be economically feasible if they provide additional benefits such as increasing crop production. Whereas pyrolysis of green waste and wood commonly results in products with a high porosity (De la Rosa et al., 2014; Bachmann et al., 2016) the pyrolysis of SS turns into carbonized residues with fertilizing potential (Chan et al., 2007; Chan et al., 2008; Paneque et al., 2017; Frišták et al., 2018). Solid-state 15N nuclear magnetic resonance (NMR) studies confirmed that most of the organic N in pyroand hydrochars from SS occurs as heterocyclic N, which represents an integrated part of the aromatic network of the chars (Paneque et al., 2017). Bearing in mind that this so-called black nitrogen (BN) (Knicker, 2010) is less bioavailable than inorganic N, a big advantage of applying such fertilizers lies CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 50 in the fact that N losses due to leaching can be reduced. However, this concept only works if the organic N is sufficiently bioavailable for maintaining plant health and growth. Thus, both, the biochemical resistance and the biodegradability of BN, determine the potential of N-rich chars as fertilizer. Previous studies indicated that BN is less biochemically recalcitrant as commonly assumed and that this N can be used for the build-up of new plant biomass (De la Rosa et al., 2011). A recent study demonstrated that Fusarium oxysporum is involved in the biochemical degradation of SS-derived pyrochar (De la Rosa et al., 2018). However, the biochemical degradability of BN in charred residues is still lower than that observed for peptides found in unburnt plant residues (LopezMartin et al., 2017). Thus, the observed co-occurrence of some quickly accessible inorganic N-forms in hydrochars (Paneque et al., 2017) may be advantageous for overcoming a possible N-deficit, which may restrict plant growth, particularly during the early stages of plant development. Pyrolysis and HTC of SS decreases P mobility (Huang and Tang, 2016). However, Frišták et al. (2018) revealed higher P contents in plants growing on pyrolyzed-SS amended soils than in those of the control, which demonstrates that at least part of the P was bioavailable. These data point out to the potential of HTC/pyrolysis treated SS to act both as slow-release P and N product. These promising results are counteracted by the fact that phytotoxic compounds may be formed during thermal treatments. Indeed, negative impacts on germination and seedling growth have been observed in other studies (Jandl et al., 2013; Busch et al., 2012; Bargmann et al., 2013; Thuille et al., 2015). They may be eliminated by well-designed pyrolysis conditions (Buss and Masek, 2014) and washing treatments (Bargmann et al., 2013). However, the knowledge about the most suitable conditions for converting SS into thermally treated products suitable for agriculture is still scarce. Bearing this in mind, the goal of the present work is to fill those knowledge gaps by complementing a former investigation on the chemical transformation of organic C and N forms during HTC and pyrolysis of two different SS (Paneque et al., 2107) with a 80-day greenhouse experiment. The focus of those experiments was to obtain insights on the impact of the application of the respective hydrochars and pyrochars on germination, survival and biomass production of Lolium perenne. In addition, the char properties were related to the growth of Lolium. CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 51 4.2 MATERIALS AND METHODS 4.2.1 Characteristics of the sample material Two different sewage sludges (SS) were collected at the Experimental Wastewater Treatment plant (CENTA), located in Carrion de los Céspedes, near Seville, Southern Spain. The first sample, further called “A_SS”, is a primary sludge produced by the settlement of suspended organic matter in a pond. The second sample, assigned “T_SS”, is a secondary sludge accumulated in an extended aeration treatment system and later stored in a thickener, in order to reduce its water content. In addition, “W” pyrochar was yielded from urban green waste, grape pomace, wood, Miscanthus and other suitable biomass. Paneque et al. (2017) found that the material collected from the pond (A_SS) was more humified, thus biochemically stabilized, than that derived from the thickener (T_SS). The total N content of A_SS and T_SS were 19 and 32 g kg−1, respectively (Table 4.1) and occurred mainly as peptides and in amino sugars (Paneque et al., 2017). Regarding heavy metal content, only Zn for A_SS and Cd for both A_SS and T_SS slightly exceeded the thresholds established in the Working document on sludge (European Comission, 2000) (data not shown). Hydrochars were produced from the primary (A) and secondary (T) SS at 200 ºC (_HTC_200) and 260 ºC (_HTC_260) for 0.5 and 3 h (_0.5, _3, respectively) as described by Paneque et al. (2017) in a 1L stirred pressure reactor (Parr reactor series 4520, IL, USA). The reaction water was removed through a filter and the material was rinsed with distilled water until the rinsing water turned clear. To obtain the A and T pyrochars, further called A_Py and T_Py, 200 g of dry SS were pyrolyzed in a close steel container in a preheated muffle oven at 600 ºC for 1h. W_Py carries the European Biochar Certificate and was produced at 600 °C for 20 min by Swiss Biochar, Lausanne, Switzerland. According to the International Biochar Initiative (IBI, 2015), the organic C content of the hydroand pyrochars (Table 4.1) allows their classification as class 3 biochar, however, only the pyrochars fulfill the second requirement of having an atomic H/C ratio < 0.7. A detailed characterization of the organic matter composition is given in Paneque et al. (2017). Due to the high N content of the source material all chars contained considerable amounts of N between 1 and 3%, most of which (>97%) occurred in an organic form (Table 4.1). In contrast to the HTC chars, the pyrochars did not concentrate inorganic N (Ni). Total phosphorus (PT) and potassium (KT) contents were determined after digestion with aqua regia (1:3 v/v conc. HNO3/HCl) in a microwave oven (Microwave Laboratory Station Mileston ETHOS 900, Milestone s.r.l., Sorisole, Italy) by inductively coupled plasma-optical CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 52 emission spectrometer (ICP-OES) spectrophotometer Varian ICP720-ES (Table 4.1). The pH of the samples was measured in distilled water (1:10, w/v) (Table 1). The heavy metals content of the chars tended to increase compared to the nontreated SS, however only Cd and Zn exceeded the limits (European Comission, 2000), similar to the non-treated SS. The concentrations of selective volatile organic compounds in process liquid (5-HMF, 2-furfural, phenol, catechol, cresol, and resorcinol) was measured using a modified ICS 3000 Dionex (Thermo Scientific) with a UV detector (wavelength 280 nm) and Knaur Eurosphere II (C 18) column. A 15% acetonitrile (85% DI water) was used as mobile phase in the IC. Column temperature was set at 23°C and flow rate was 1.0 ml min-1. No presence of these molecules was found in A_SS, T_SS or in their chars. CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 59 Figure 4.4. Biomass production per pot (mg per pot) of Lolium perenne for the control (soil without amendment), hydrochars and pyrochars amended pots at 5 and 25 t ha-1. Hydrochars were produced from primary (A_) and secondary (T_) sewage sludges at 200 ºC (_HTC_200) and 260 ºC (_HTC_260) for 0.5 and 3 h (_0.5, _3, respectively). Pyrochars were produced from primary (A_) and secondary (T_) sewage sludges (_Py). a) NS: no significant. Asterisks inside the bars show significant differences to the control according to t-test. The above-line asterisks indicate significant differences between doses according to t-test. The letters show significant differences between treatments according to Tukey’s test. 4.3.3 Root-to-shoot ratios Pyrochars addition significantly increased the root-to-shoot (R:S) ratio of Lolium perenne whereas the presence of A and T hydrochars resulted in lower or equal values than the control (Figure 4.5). Within hydrochars, production conditions did not affect this parameter; however R:S ratio tended to decrease with increasing rate of hydrochar application. These changes were mainly due to a higher root biomass in the case of pyrochars and a higher shoot biomass in the case of hydrochars (Figure 4.3). CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 60 Figure 4.5. Root-to-shoot ratio of Lolium perenne for the control (soil without amendment), hydrochars and pyrochars amended pots at 5 and 25 t ha-1. Hydrochars were produced from primary (A_) and secondary (T_) sewage sludges at 200 ºC (_HTC_200) and 260 ºC (_HTC_260) for 0.5 and 3 h (_0.5, _3, respectively). Pyrochars were produced from primary (A_) and secondary (T_) sewage sludges (_Py). a) NS: no significant. Asterisks inside the bars show significant differences with the control according to t-test. The above-line asterisks indicate significant differences between doses according to t-test. 4.3.4 Relationship between plant response and chars properties The NMDS graph showed that carbonization type had an overall effect on all parameters since pyrochars were separated from hydrochars (Figure 4.6). Pyrochars were characterized by higher R:S ratios together with higher root biomass and, in a lower extent, by higher germination and survival rates than hydrochars. In contrast, hydrochars were characterized by larger shoot and total biomass per plant. Regarding the feedstock type, T hydrochars showed larger shoot and total plant biomass than A hydrochars. In addition, total biomass per plant and shoot biomass significantly correlated with increased Ni and Norg contents of the chars. To a lower extent, total biomass per plant and shoot biomass also correlated with increased Corg levels of the chars. The p values of the above mentioned correlations were less or equal to 0.001. The Corg/N ratio, PT, and KT parameters showed p values higher than 0.001 and hence were not considered significant variables. CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 61 Figure 4.6. NMDS ordination indicates how the different thermally treated SS impact on Lolium-measured parameters. R:S rat: Root-to shoot ratio; Roots: roots biomass per plant; Surv: survival rate; Germ: germination rate; Pot: total biomass per pot; Plant: total biomass per plant; Shoot: shoot biomass per plant. Char properties significantly fitting onto NMDS are shown as vectors (p<0.001). Ni: inorganic nitrogen; Norg: organic nitrogen; Corg: organic carbon. Goodness of fit was 0.05. 4.4 DISCUSSION 4.4.1 Germination and survival rates Our data did not allow an unbiased assignment of factors causing the observed differences in germination and survival rates between hydrochars and pyrochars. A decrease in germination after addition of hydrochars has also been observed by others (Jandl et al., 2012; Busch et al., 2012; Bargmann et al., 2013; Thuille et al., 2015) and was attributed to phytotoxic volatile organic compounds adsorbed on the surface of the hydrochar. Bargmann et al. (2013) demonstrated that these compounds were mostly water soluble and could be removed by washing the hydrochars with distillate water but soluble nutrients are expected to be at least partially lost during this process. However, analysis of the chars prior to incubation did not indicate the presence of HMF, furfural, resorcin, catechol, phenol and CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 62 kresol. Thus, either other non-measured phytotoxic compounds were present in our hydrochars or other factors are responsible. Previous works have also found diverse germination responses among hydrochars produced from different feedstocks (Jandl et al., 2012; Bargmann et al., 2013). Recently, the fermentation of hydrochars in an anaerobic biogas reactor has been proposed as an alternative method to eliminate toxic compounds since it avoids the loss of nutrients (Mumme et al., 2014; Lanza et al; 2018). Dry pyrolysis performed at temperatures greater than 500 ºC in appropriate pyrolysis reactors reduces the presence of volatile compounds, including polycyclic aromatic compounds and other phytotoxic (De la Rosa et al., 2016). Thus, the possible negative impact due to soil amendment with pyrochars on germination rates should be less prominent than with HTC chars. Previous works have shown both neutral and positive effects of pyrochars (Free et al., 2010; Busch et al., 2012; De la Rosa et al., 2014; Thuille et al., 2015). This is in agreement with our results indicating no major impact of pyrochar addition except for W_Py applied at 25 t ha-1. 4.4.2 Biomass production and chars properties Despite some hydrochars decreased the survival rate of Lolium perenne, the total biomass production per pot was always higher or equal in the hydrochar amended pots, compared to the control. This is best explained by the size of the remaining plants, since hydrochars tended to increased biomass production per plant. However, our pyrochars had no effect on biomass production. These results are in contrast to studies by Thuille et al. (2015) who obtained higher shoot biomass production of winter wheat in the presence of corn silage-derived pyrochar than in presence of corn silage-derived hydrochar. However, here it has to be taken into account that in the former study inorganic nitrogen fertilizer was added. Nitrogen availability is most likely also responsible for our observation, as it is indicated by the positive correlation between Ni and Norg contents of the chars with the total and shoot biomass production per plant. Accordingly, the pyrochars showed the lowest Norg contents along with absence of Ni which is in line with no increase of Lolium biomass production. The hydrochars produced at 260 ºC exhibited higher Norg than the pyrochars and contained some Ni which slightly affected plant biomass production. However, amendment of the hydrochars produced at 200 ºC, which contained the highest concentrations of Norg and Ni, yielded in the highest amount of biomass. Those results strongly indicate that biomass production is more related to the availability of N or other nutrients than to CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 63 physical properties of the char. Note that we are not using wood biochars but chars with very low porosity and that the N content of our soil is extremely low. Most of this nitrogen occurs in organic form. Thus, adsorption of Ni from the soil onto the char surface is unlikely. As a consequence, the processes limiting bioavailability of N in our systems is determined by the biochemical degradability of black nitrogen, rather than by adsorption of Ni. Fang et al. (2015) also found that greenwastederived hydrochars produced at 200ºC showed higher yields than those produced at higher temperatures. In contrast to N, PT and KT contents of the chars revealed no correlation with growth parameters. Either the soil contained sufficient plant available P and K already prior to char application or the P and K of the chars are less plant available. In both cases, our results indicate that other factors than PT and KT contents have a stronger impact on plant growth. The correlation of Corg contents of the chars with the total and shoot biomass production may be due to an improvement of some soil physical properties like a decrease in soil density, an increase of the soil water holding capacity or providing an habitat for soil microorganisms. Note that hydrochars exhibited higher Corg contents and thus higher biomass production than pyrochars. Since the latter are expected to exhibit a higher biochemical stability than hydrochars, one has to keep in mind that increasing the potential of soils to act as a C sink via addition of charcoal does not necessarily coincide with enhancement of biomass production. 4.4.3 Root-to-shoot ratios and char properties Although pyrochars addition had no impact on Lolium biomass production, their presence increased their R:S ratios, compared to the control. Considering that increased R:S ratios have been observed when growth is limited by N or P supply (Andrews et al., 1999), our observation may be related to the absence of Ni in our pyrochars. This deficiency may have been increased by adsorption of NO3– and NH4+ from the soil solution to the char surface as it was suggested to occur for some biochars (Spokas et al., 2012; Nelissen et al., 2012). Hydrochars applied at 25 t ha-1 showed lower R:S ratios than the control whereas the 5 t ha-1 application rate produced no significant impact or a lower decrease of this parameter. In general, when nutrient availability increases, plants can develop their aboveground vegetation in detriment of their subsoil part because less effort is required to acquire nutrients (Ågren and Franklin, 2003). This is in agreement with our results, since char amendment with 25 t ha-1 delivered higher nutrients levels than addition of 5 t ha-1. Comparably, Thuille et al. (2015) observed a very short root system of winter wheat in the presence of a corn silage derived hydrochar, CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 64 whereas with the respective pyrochar, root development was comparable to that of the plants grown without corn silage treatment. 4.4.4 Fertilization potential of thermally treated SS Our work indicates that low-temperature HTC of SS results in a product with Nfertilization potential whereas chars yielded from HTC with higher temperatures and pyrolysis do not exhibit the same suitability. This potential depends on the amount of Ni contained in the chars together with the bioavailability of their organic N. Whereas Ni provides fast and immediate N fertilization, the degradation rate of the organic N compounds determines the slow-release N fertilization potential of the amendments. Paneque et al. (2017) showed that organic N in SS occurred mainly in peptide-like structures, part of which may be easily degradable. During both HTC and pyrolysis this compounds were partially transformed into N-heterocyclic aromatic entities, which are less microbiologically accessible. This transformation was more efficient for pyrolysis than for HTC which suggested a quicker degradation of the organic N in hydrochars than in pyrochars. Therefore, not only the total nutrient content values but also their speciation should be considered since their bioavailability from chars alters with processing conditions. In addition, it also has to be considered that our hydrochars and pyrochars may exhibit a different crop response depending on the crop type as well as on the soil properties and the climatic conditions (Sakrabani et al., 2017). 4.5 CONCLUSIONS Although the problem of reduced germination after HTC application still has to be solved, our study indicates a higher potential of SS-derived hydrochars as soil amendment with immediate fertilizing effect than the respective pyrochars. Bearing in mind that plant growth parameters correlated better with N than with P and K contents, the higher impact of hydrochars on plant growth is best explained by the concomitant presence of inorganic N and easily available organic N forms. However, further experiments have to show if the disadvantage of less microbially accessible N in pyrochars may turn into an advantage for crops which need low but constant N fertilization. From an energetic and thus economic point of view, HTC seems to be a better choice than pyrolysis since drying of the SS prior to its thermal treatment can be avoided. Lower temperatures and shorter residence times are needed which considerably reduce the costs for energy. Our studies reveal further that for the CHAPTER 4 Hydrothermal carbonization and pyrolysis of sewage sludge-effects on lolium perenne germination and growtth 65 production of a slow-release fertilizer from SS, HTC at 200 ºC is more suitable than 260 ºC, which allows even less energy consumption. In addition, the composition of the preserved N seems to be favorable for plant growth. Thus, aside from being advantageous with respect to economic considerations, the application of lower temperatures during HTC is also of environmental interest since the use of mineral fertilizer can be reduced and the release of greenhouse gases into the atmosphere occurring during disposal of untreated SS can be avoided. 4.6 REFERENCES Ågren G.I., Franklin O. (2003) Root:shoot ratios, optimization and nitrogen productivity. Annals of Botany 92: 795–800. Andrews M., Sprent J.I., Raven J.A., Eady P.E (1999) Relationships between shoot to root ratio, growth and leaf soluble protein concentration of Pisum sativum, Phaseolus vulgaris and Triticum aestivum under different nutrient deficiencies. Plant, Cell and Environment 22, 949–958. 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Vegan: community ecology package version 2.4-4. http://cran.r-project.org/. Paneque M., De la Rosa J.M., Kern J., Reza M.T., Knicker H. (2017) Hydrothermal carbonization and pyrolysis of sewage sludges: What happen to carbon and nitrogen? Journal of Analytical and Applied Pyrolysis 128, 314–323. Reza, M.T., Becker, W., Sachsenheimer, K., Mumme, J. (2014) Hydrothermal carbonization (HTC): Near infrared spectroscopy and partial least-squares regression for determination of selective components in HTC solid and liquid products derived from maize silage. Bioresource Technology 161, 91–101. Sakrabani R., Kern J., Mankasingh U., Zavalloni C., Zanchettin G., Bastos A.C., Tammeorg P., Jeffery S., Glaser B., Verheijen F.G.A. (2017). Representativeness of European biochar research: part II – pot and laboratory studies. Journal of Environmental Engineering and Landscape Management 25, 152–159. Spokas K.A., Novak J.M., Venterea R.T. (2012) Biochar's role as an alternative Nfertilizer: ammonia capture. Plant and Soil 350, 35–42. Thuille A., Laufer J., Höhl C., Gleixner G. (2015) Carbon quality affects the nitrogen partitioning between plants and soil microorganisms. Soil Biology & Biochemistry 81, 266–274. Veihmeyer F.J., Hendrickson A.H. (1949) Methods of measuring field capacity and wilting percentages of soils. Soil Sci 68, 75–94. CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 74 Table 5 .1. pH, EC, PT, P recovery and extr-P values for primary (A_SS) and secondary (T_SS) sewage sludges and for their respective hydrochars and pyrochars pH ECa PTb P recovery Extr-Pc mS cm-1 g kg-1 (%) mg kg-1 A_SS 7.4 15.0 12.3 100 510 A_HTC_200_0.5 6.5 15.0 13.9 97.9 183 A_ HTC_200_3 6.5 12.8 14.5 90.4 139 A_ HTC_260_0.5 6.4 3.0 15.4 87.8 181 A_ HTC_260_3 6.6 8.5 14.9 79.1 145 T_SS 7.5 14.5 16.7 100 651 T_ HTC_200_0.5 6.7 25.5 20.0 99.7 278 T_ HTC_200_3 6.2 24.8 20.8 82.3 234 T_ HTC_260_0.5 6.3 18.3 21.6 78.5 200 T_ HTC_260_3 6.4 21.3 21.6 73.3 70 A_Py 9.3 6.3 17.0 81.2 1017 T_Py 10.0 9.3 25.2 75.9 668 W_Py 9.3 1.1 0.7 - 51 a Electrical Conductivity b Total phosphorus c Extractable phosphorus 5.3.1.2. Phosphorus characterization of non-treated SS The non-treated SS showed PT contents of 12 and 17 g kg-1 for A_SS and T_SS, respectively (Table 5.1). The availability of this PT was around 4% since the extractable P of A_SS and T_SS was 510 and 651 mg kg-1, respectively (Table 5.1; Figure 5.1). Both the PT contents and the percentages of available P are below the average values found for SS produced in Europe (Millieu Ltd., WRc and RPA, 2010). The amount of extr-P was also low compared to the average SS produced in Spain (Spanish Government, 2009). This is likely due to the fact that, in contrast to common SS, our sludges derive from a wastewater treatment plant (WWTP) which processes the domestic waste water of a small village with at most 3000 residents, whereas most of the common WWTPs process the wastewater of larger populations. In addition, considering that aluminum (Al) and calcium (Ca) are the predominant metals of A_SS and T_SS (Table 5.2), Ca and Al phosphates are likely to dominate in our samples since the type of metals-phosphate complexes are highly influenced by the composition of the feedstock. These results are in agreement with Wang et al. (2012). CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 75 Considering these data, the potential of our SS as a source of P for soils is likely lower than that of the average SS and this has to be taken into account when it comes to the extrapolation of the results. Table 5.2. Calcium (Ca), magnesium (Mg), iron (Fe) and aluminum (Al) contents of the primary (A_SS) and secondary (T_SS) sewage sludges and of their respective hydrochars and pyrochars Ca Mg Fe Al g kg-1 g kg-1 g kg-1 g kg-1 A_SS 73.3 8.6 15.8 36.5 A_HTC_200_0.5 80.7 9.0 18.5 42.0 A_ HTC_200_3 84.4 9.3 19.4 44.0 A_ HTC_260_0.5 87.4 10.0 19.7 45.7 A_ HTC_260_3 86.5 9.6 19.1 44.2 T_SS 62.2 9.2 19.1 32.2 T_ HTC_200_0.5 72.8 10.1 23.1 39.6 T_ HTC_200_3 76.1 10.9 24.0 41.0 T_ HTC_260_0.5 78.5 11.6 24.4 42.2 T_ HTC_260_3 78.7 11.7 26.1 42.3 A_Py 100.0 10.9 21.7 50.1 T_Py 92.5 13.6 28.2 48.5 5.3.1.3. Effect of the thermal treatments on P contents of SS The data of the recovered P show that most of the P contained in the non-treated SS remained after both pyrolysis and HTC (Table 5.1). Recovery P values ranged from 99 to 76%. Within hydrochars, this parameter decreased as temperature and residence time increased. In addition, the “A” chars showed slightly higher values than “T” chars, which is in accordance with the higher yields of the former compared to the latter (data not shown). Paneque et al. (2017) found that the losses of carbon (C), nitrogen (N) and hydrogen (H) were always higher than the P losses for the same chars. This explains the observed increase of PT contents after both thermal treatments with a relative enrichment of this element. Considering that the losses of C, N and H were higher during pyrolysis than during HTC, the PT contents were higher in the pyrochars than in the hydrochars derived from the same SS. In addition, the process water of the HTC must contain part of the water CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 76 soluble P. Thus, the A_Py and T_Py pyrochars exhibited PT contents of 17 and 25 g kg-1 respectively; whereas their hydrochars showed lower PT values (Table 5.1). The high preservation and enrichment of P after both pyrolysis and HTC of SS have been previously reported (Wang et al., 2012; Wang et al., 2017; Frišták et al., 2018). Despite both thermal treatments preserved most of the P of the non-treated SS, the availability of this element decreased after HTC. Thus, the extr-P values of the hydrochars were between 278 and 70 mg kg-1 (Table 5.1). Pyrolysis, however, did not demonstrate a consistent effect since A_SS and A_Py showed comparable extr-P values whereas T_Py exhibited double the amount of extr-P than T_SS. This points out to different P compounds in A_SS and T_SS and hence to a different speciation/complexation during pyrolysis. The W_Py had the lowest extr-P value due to its low PT content. The observed decrease of the P availability after HTC resulted, logically, in a lower portion of labile P, comprising between 1.4 and 0.3% (Figure 5.1). In general, both the labile P fraction of the hydrochars and their extr-P contents decreased with intensifying the production conditions. Regarding the pyrochars, less labile P was found in T_Py than in T_SS whereas A_Py showed a higher labile P fraction than A_SS. This supports the above-mention conclusion of different P compounds in starting A_SS and T_SS, which then leads to different speciation/complexation during pyrolysis. In general, these results indicate that the P immobilization was higher for HTC than for pyrolysis. However, the underlying mechanisms for this behavior still have to be explored. Huang and Tang (2016) observed an immobilization of P after both HTC and pyrolysis which was attributed to an increase of metal/P complexes. This is likely to have happened in our chars, too, since a relative enrichment of Ca, Mg, Fe and Al occurred after both thermal treatments (Table 5.2). However, physical constraints such as embedment of P species into the char structure may also play a role. Huang and Tang (2016) found that different Al and Ca phosphates as well as phytic acid were extracted by H2O or NaHCO3 in non-treated SS but they could only be extracted by NaOH or HCl in the chars. The mechanisms which explain the results of A_Py, however, must be further investigated. CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 77 Figure 5.1. Labile fractions of P extracted by the Olsen method of the primary (A_SS) and secondary (T_SS) sewage sludges and their respective hydrochars and pyrochars. Summarizing our results, the higher PT and extr-P contents of the SS-derived pyrochars compared to the hydrochars suggest that pyrolysis may be more suitable than HTC for producing SS-derived chars useful as P-fertilizer. 5.3.2 Greenhouse experiment 5.3.2.1. pH The Calcic Cambisol used as soil matrix for the incubation experiment exhibits a pH of 8.4 (Table 5.3), which is within the range expected for such soils in the sampled region (Paneque et al., 2016). Most likely due to the lower pH of the hydrochar, its addition resulted in decreased soil pH values at the end of the experiment. The extent of this effect depended on the dose applied. Amendment of 5 t ha-1 lowered the pH values of the soil between 0.3 and 0.5 units while 25 t ha-1 decreased this parameter by 0.8 and 1.4 units. However, plant growth may have contributed to the pH decrease. In spite of the high pH of the pyrochars, its addition at 5 t ha-1 caused minor to no changes on soil pH, whereas A_Py and T_Py lowered the soil alkalinity when applied at 25 t ha-1. The presence of roots exudates, which acidify the soil, together with their inherent buffering capacity may explain these results. Bruun et CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 78 al. (2017) also found minor changes after the addition of pyrochar to a low clay acidic soil. The impact on soil pH of both hydrochars and pyrochars at 5 t ha-1 is slight and may not have a beneficial effect on soil nutrients availability. However, the soil pH at the highest application dose was closer to neutrality and hence nutrients availability may have been favored. Table 5.3. Soil pH and EC of the control (soil without amendment), hydrochars and pyrochars amended pots at 5 and 25 t ha-1 at the end of the greenhouse experiment. pH EC µS cm-1 Control 8.4 ± 0.02 ± 0.02 100.6 ± 2.0 ± 2 Amended soils 5 t ha-1 25 t ha-1 5 t ha-1 25 t ha-1 A_HTC_200_0.5 8.0 ± 0.03 7.4 ± 0.07 116.4 ± 7.7 280 ± 18 A_ HTC_200_3 8.2 ± 0.03 7.0 ± 0.02 112.6 ± 7.5 380.3 ± 40 A_ HTC_260_0.5 8.2 ± 0.05 7.5 ± 0.09 111.0 ± 14 231.8 ± 11 A_ HTC_260_3 8.0 ± 0.12 7.3 ± 0.09 103.5 ± 1.9 256.5 ± 38 T_ HTC_200_0.5 8.0 ± 0.11 7.3 ± 0.06 111.9 ± 2.2 644.3 ± 22 T_ HTC_200_3 8.2 ± 0.05 7.0 ± 0.04 109.0 ± 1.2 524.8 ± 11 T_ HTC_260_0.5 8.2 ± 0.03 7.5 ± 0.04 117.0 ± 2.4 554.0 ± 27 T_ HTC_260_3 8.1 ± 0.07 7.6 ± 0.05 117.3 ± 5.4 378.5 ± 8.1 A_Py 8.2 ± 0.04 7.3 ± 0.09 115.4 ± 2.1 236.0 ± 15 T_Py 8.2 ± 0.04 7.8 ± 0.03 129.5 ± 3.9 223.3 ± 9 W_Py 8.4 ± 0.04 8.5 ± 0.07 92.4 ± 0.1 108.5 ± 5.2 5.3.2.2. Electrical conductivity (EC) The Calcic Cambisol used for the incubation experiment exhibited an EC value of 101 μS cm-1 (Table 5.3), which is in the range of this soil-type of the region. Both hydrochars and pyrochars added at 5 t ha-1 produced minor to no changes on soil EC. However, hydrochars applied at 25 t ha-1 did increase the EC of the soil in an extent which depended on the EC value of the char. Thus, T hydrochars applied at 25 t ha-1 ranged from 378 to 644 μS cm-1, whereas for A hydrochars this parameter was between 231 and 380 μS cm-1. The A_Py and T_Py addition increased soil EC to a lower extent than hydrochars, which is in accordance with their lower EC values, whereas W_Py had no impact on this parameter. Most of the crops grow up properly in soils with EC values between 100 and 1000 μS cm-1. However, soils with more than 2000 μS cm-1 are considered saline (Soil survey division staff, 1993). Despite the EC of the hydrochars and pyrochars exceeded the CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 79 above mentioned limits, their application at 5 and 25 t ha-1 is not expected to affect the crop since all the treatments showed soil EC values below 2000 μS cm-1. 5.3.2.3. Extractable phosphorus (Extr-P) In general, most of the chars were able to increased soil extr-P; however, the impact of SS-derived pyrochars was higher than that of the hydrochars (Figure 5.2). This result was expected since the hydrochars showed lower labile-P fractions than their respective pyrochars (Figure 5.1). The extrP content of the control soil was 13 mg kg-1 at the end of the incubation. The A_Py and T_Py pyrochars applied at 5 t ha-1 increased the soil extr-P up to 59 and 49 mg kg-1 respectively. These values were between 3 and 4 times higher at the highest application rate. However, the addition of W_Py to the soil did not affect this parameter at any dose, which is explained by the low total and extr-P contents of this pyrochar. The behavior of W_Py compared to A_Py and T_Py is in accordance with the low nutrient value of greenwaste pyrochars (Chan et al., 2007). Within hydrochars, the impact on soil extr-P reduces when production condition increases. Thus, the hydrochars obtained at 200ºC after 30 min charring raised soil extr-P the most whereas those produced at 260ºC for 3h had no impact on this parameter. Both A and T hydrochars showed comparable behaviors in this sense. However, T hydrochar addition resulted in slightly higher soil extr-P values than the amendment of the A hydrochars, which is due to the higher PT and extr-P of the former compared to the latter. In addition, an augmentation of the application rate from 5 to 25 t ha-1 increased the soil extr-P values both for hydrochars and pyrochars. These results confirm the results obtained after the characterization P indicating that pyrolysis is apparently more appropriate than HTC to convert SS into a P fertilizer. CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 80 Figure 5.2. Extractable P in the control (soil without amendment), hydrochars and pyrochars amended pots at 5 and 25 t ha-1 at the end of the greenhouse experiment. The above-line asterisks indicate significant differences between doses. The letters show significant differences between treatments 5.3.2.4. Phosphorus release The Pdiff represents the difference between the amount of soil extr-P at the end of the experiment corresponding to the char and the extr-P added to the soil with each char and dose at the beginning of the experiment (Figure 5.3 A). The pots amended with the hydrochars produced at 260ºC and the W_Py pyrochar showed almost no or even negative Pdiff. Thus, it can be assumed that almost all of extr-P within the chars was used by the plants during the experiment. In contrast, the hydrochars produced at 200ºC and SS-derive pyrochars showed positive Pdiff values, ranging between 35 and 0.3 mg kg-1 for the former and 146 and 30 mg kg-1 for the latter. This means that the amount of soil extr-P was higher at the end of the experiment than at the beginning and hence that part of the originally nonextractable P must have turned into extractable forms during the incubation. Thus, changes in P availability seems to occur once thermally treated SS are applied to soil, which has to be considered for evaluating their potential as a P source for plant growth. A similar behavior was observed for pyrochars derived from digested (Brunn et al., 2017) and from pig slurry (Christel et al., 2014). CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 81 Figure 5.3. Amount of P release by the different hydrochars and pyrochars when applied at 5 and 25 t ha-1 at the end of the greenhouse experiment. A) Phosphorus difference between the amount of soil extr-P at the end of the experiment and the respective amount at the beginning; B) Phosphorus release per kg of char at the end of the experiment. The aboveline asterisks indicate significant differences between doses. The letters show significant differences between treatments. In order to obtain more insights into the potential of these chars to release P, the amount of P released per kg of char was calculated (Figure 5.3 B). In accordance with the higher Pdiff in pyrochars compared to hydrochars, the P release per kg of char was also higher for pyrochars than for hydrochars. Thus, the A_Py and T_Py CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 82 pyrochars released between 18 and 10 g of P kg-1, whereas the amount of mobilized P of the hydrochars produced at 200ºC was between 10 and 1.3 g kg-1. Note that the T hydrochars showed a higher P release than the A ones, which is best explained with the higher PT contents of the former compare to the latter. Generally, increasing the application dose from 5 to 25 t ha-1 raised the Pdiff values (Figure 5.3 A). This is a logical result since a higher amount of PT per pot was added. In contrast, the amount of P release per kg of char is not expected to depend on the application dose. However, in our experiments, this parameter decreased when the application rate increased (Figure 5.3 B). Commonly, such an observation is explained with changing pH. The phosphorus in the soil is at maximum availability when soil pH is between 6 and 7.5. Considering that the 25 t ha-1 amended pots showed soil pH values closer to this range than the 5 t ha-1 amended ones, the pH seems to have only a low impact on our results. Alternatively, our results may be related to an equilibrium between dissolved P and adsorbed/precipitated forms in the soil. Thus, at 25 t ha-1 saturation of the dissolved P fraction may have occurred. 5.4 Summary and general discussion Previous characterization studies indicated that an immobilization of P occurs after both HTC and pyrolysis of SS (Huang and Tang, 2016). Except for A_Py, this is in agreement with our results since a decreased of the labile P fraction was observed. However, both the pyrochars and the hydrochars produced at 200ºC were able to increase the amount of soil ext-P compared to the control. This increase was due to the amount of extr-P within the chars but also to transformation of originally non-extractable P of the chars into extractable forms during the incubation. This explains the apparent contradiction between the observed immobilization of P after both HTC and pyrolysis determined through characterization techniques (Huang and Tang, 2016) and works which demonstrate that SS-derived pyrochars can act as an efficient source of P for plants (Faria et al., 2017; Frišták et al., 2017). The results obtained in this work should be carefully extrapolated. Differences among different SS types have been observed both in this work and in literature (Huang et al., 2016). In the same way, the application of the same chars to different soils may result in a different P behavior (Brunn et al., 2017; Xu et al., 2014). However, as previously mentioned, the two SS used in this study were especially low in P compared to the average in the EU. Thus, the potential of thermally treated SS as a source of P for soils is expected to be generally higher. CHAPTER 5 Hydrochar and pyrochar derived from sewage sludge can increase the available phosphorus in amended soils. A greenhouse study. 83 5.5 CONCLUSIONS Both dry-pyrolysis and, in a lower extent, low-temperature HTC seems to be useful techniques to produce treated-SS with P fertilizing properties. The initial immobilization of P after both thermal treatments may reduce P losses by leaching. In addition, the subsequent P release once the chars were applied to the soil indicates their suitability as slow-release P fertilizers. 5.6 REFERENCES Bargmann I., Rillig M.C., Buss W., Kruse A., Kücke M. (2013) Hydrochar and biochar effects on germination of spring barley. J. Agro. Crop. Sci. 199, 360–373. Bargmann I., Martens R., Rillig M.C., Kruse A., Kücke M. (2014) Hydrochar amendment promotes microbial immobilization of mineral nitrogen. J. Plant Nutr. Soil Sci. 177, 59–67. Bruun S., Harmer S.L., Bekiaris G., Christel W., Zuin L., Hu Y., Jensen L.S., Lombi E. (2017) The effect of different pyrolysis temperatures on the speciation and availability in soil of P in biochar produced from the solid fraction of manure. Chemosphere 169, 377-386. Chan K.Y., Van Zwieten L., Meszaros I., Downie A., Joseph S. (2007) Agronomic values of greenwaste biochar as a soil amendment. Australian Journal of Soil Research, 45, 629–634. Christel W., Bruun S., Magid J., Jensen L.S. (2014) Phosphorus availability from the solid fraction of pig slurry is altered by composting or thermal treatment. Bioresource Technology 169, 543-551. Cordell D., Drangert J.O., White S. (2009) The story of phosphorus: Global food security and food for thought. Global Environmental Change 19, 292-305. Dai L., Tan F., Wu B., He M., Wang W., Tang X., Hu Q., Zhang M. (2015) Immobilization of phosphorus in cow manure during hydrothermal carbonization. Journal of Environmental Management 157, 49-53. Faria W.M., de Figueiredo C.C, Rodrigues Coser T.R., Vale A.T, Schneider B.G. (2017). Is sewage sludge biochar capable of replacing inorganic fertilizers for corn production? Evidence from a two-year field experiment. Archives of Agronomy and Soil Science, DOI: 10.1080/03650340.2017.1360488. Frišták V., Pipíska M. and Soja G. (2018) Pyrolysis treatment of sewage sludge: A promising way to produce phosphorus fertilizer. J Clean Prod. 172, 1772-1778. CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 89 6.2.4 Greenhouse experiment A 10-month greenhouse experiment was conducted with the following treatments: only soil (control), soil amended with the three labeled materials (SS, Hyd, Py) and soil with K15NO3 (KNO3). The soil matrix used for the greenhouse incubation experiment derived from the Ah horizon of a Cambisol (IUSS WorkingGroup WRB 2014) (N 37° 32′ W 6° 15′) from the Sierra de Aznalcóllar, southern Spain. A brief characterization of the soil is given by Lopez-Martin et al. (2015). After removing visible root and plant residues, the soil was sieved through a 2-mm sieve and ovendried at 40 °C. The soil contained 38.7 g C kg−1 of which all was attributed to organic carbon and 3.4 g N kg−1 of which 27.5 mg corresponded to NH4+ and 1.87 mg to NO3-. Its pH in water is 5.7 and its water holding capacity (WHC) is 22%. The used pots were 250 ml-plastic containers (16 cm height) with a hole at the bottom part. For each treatment 9 replicates were prepared (n = 9). The control pots were filled with 230 g of soil. For the SS, Hyd and Py treatments 190 g of soil was covered with a 40 g amendment:soil mixture (5%, w:w). The enrichment degree of the amendments and the amount of CT, NT, 13C and 15N added per pot is shown in table 6.1. Finally, 25 certified grass seeds (Lolium perenne-ILURO seeds company, Spain) were sowed in each pot and soil moisture was adjusted to 60% of the maximum water holding capacity (WHC). The pots were placed into trays and put into a greenhouse at 25 °C under natural solar light conditions. No nutrient solution was added during the experiment. The WHC was checked three times per week and adjusted by weighing the pots and adding the mass difference as declorated water. The position of the pots on the tray was changed after adjusting the WHC to assure comparable light and growing conditions. Table 6.1. Relative atomic abundance of the stable isotopes in the amendments and the amount of CT, NT, 13C and 15N added per pot. A* Amount of added CT Amount of added 13C A Amount of NT added Amount of 15N added (%13C) (mg per pot) (mg per pot) (%15N) (mg per pot) (mg per pot) SS 2.8 581.8 16.5 1.0 83.2 0.8 Hyd 1.9 476.9 9 0.6 38.8 0.2 Py 2.2 372.9 8.2 0.7 47.6 0.3 *relative atomic abundance After 1, 3 and 10 months of incubation, three replicates of each treatment were destructively sampled. For each pot, the shoots were cut, dried (72 h at 60 °C) and CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 90 weighed. The first 3-4 cm of the soil column were separated from the rest and named topsoil. The remaining soil was called subsoil. The roots were manually separated from both the topand the subsoil, rinsed with distilled water, dried (72 h at 60 °C) and weighed. The soil nitrate (NO3-) and ammonium (NH4+) contents were measured both in the topand subsoil. The total C, N, 13C and 15N contents of both the topand subsoil as well as of the plant shoots were measured with a Flash 2000 HT combustion elemental microanalyzer and a Flash HT Plus elemental analyzer coupled to a Delta-V advantage isotopic ratio mass spectrometer (IRMS) via ConFlo IV interfase (Thermo Scientific, Bremen, Germany), with an analytical measurement error of ± 0.2‰. The proportion of total C derived from the amendments (%Camend) in the topand subsoil for each amendment and sampling time was calculated by using a two component isotopic mixing model (Bernoux et al., 1988): %Camend = δ13C(amend+soil)− δ13C(soil) δ13C(amend)−δ13C(soil) (1) where the δ13C(amend+soil) represents the δ13C value of a SS, Hyd or Py amended pot at a specific sampling; the δ13C(soil) represents the δ13C value of the nonamended; and the δ13C(amend) represents the δ13C value of the SS, Hyd or Py. The proportion of total N derived from the amendments (%Namend) in the topand subsoil for each amendment and sampling time was calculated by substituting the δ13C values for δ15N values in the equation (1). In addition, the amount of N derived from the amendments uptake by the plants was studied by applying the following enrichment factor (EF), modified from Michener and Lajtha, 2007: EF = δ15N(amend)−δ15N(control) 𝑚𝑔 15𝑁𝑎𝑑𝑑𝑒𝑑 ∗ 𝑑𝑎𝑦𝑠 𝑜𝑓 𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 Here, δ15N(amend) represents the δ15N value of the shoots growth in presence of the SS, Hyd or Py at a specific sampling; the δ15N(control) represents and the δ15N value of the shoots growth only with soil at a specific sampling. CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 91 6.2.5 Statistical analysis Data corresponding to the percentage of total C and N derived from the amendments in the soil were submitted to Shapiro-Wilk and Levene tests to test for normality and homoscedasticity, respectively. After that, an analysis of variance (one-way ANOVA) followed by a comparison of means (Tukey’s test) were performed to test for significant differences along the time for each char. Differences were considered as significant at p ≤ 0.05. Data were analyzed using SPSS version 17.0 (SPSS, Chicago, IL, USA). 6.3 RESULTS AND DISCUSSION 6.3.1 Characterization of the amendments The total C content of the non-treated SS was 290.9 g kg-1 whereas that of the Hyd and Py was 238.5 and 186.5 g kg-1 respectively (Table 6.2). The SS and Hyd exhibited an atomic H:C ratio of 1.7 and 1.5 which indicates low aromaticity. The lower H:C ratio of 0.5 for Py, on the other hand, is in line with a high content of aromatic structures in this sample of which every second C is protonated. Table 6.2. Carbon (C), N, H:Cat ratio and Corg/N ratio of the soil, sewage sludge (SS) and its hydrochar (Hyd) and pyrochar (Py). C N H:C Corg/N g kg-1 g kg-1 atomic ratio Soil 3.8 3.4 11.4 SS 290.9 41.6 1.7 7.0 Hyd 238.5 19.4 1.5 12.3 Py 186.5 23.8 0.6 7.8 The solid-state 13C NMR spectra are in accordance with the atomic H:C ratios (Figure 6.1). The Corg in SS occurred mainly as peptides (N-alkyl C, amide C and part of the alkyl C ) and carbohydrates (O-alkyl C) followed by lipids, which account for approximately 10%. Aromatic compounds contribute with less than 8% (O/Naryl C and aryl C) to the total Corg (Table 6.3). After HTC, the proportion of aromatic C increased to 20% of the total Corg in detriment of carbohydrates and peptides whereas pyrolysis transformed almost all of the OM into aromatic structures. CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 92 Table 6.3. Intensity distribution (%) in the solid-state 13C NMR spectra of the sewage sludge (SS), its hydrochar (Hyd) and its pyrochar (Py). Carboxyl / Amide C O/N -Aryl C Aryl C OAlkyl C N-Alkyl/ Methoxyl C Alkyl C ppm 225-160 160-140 140-110 110-90 90-60 60-45 45-0 SS 13.9 2.0 5.9 5.8 25.2 13.98 33.6 Hyd 8.6 4.6 16.0 4.4 12.5 11.17 42.9 Py 4.2 11.5 63.0 6.8 3.9 1.92 8.7 Figure 6.1. Solid-state 13C NMR spectra of the sewage sludge (SS), its hydrochar (Hyd) and its pyrochar (Py) The N content of the non-treated SS was 41.6 g kg-1 (Table 6.2). The Hyd and Py exhibited lower values, with 19.4 and 23.8 g kg-1, respectively. The higher loss of N during pyrolysis than during HTC is not common but Bargmann et al. (2014a) reported the same behavior in HTC-treated and pyrolyzed spent brewer’s grains. Paneque et al (2017) showed that most of the N in non-treated and thermally treated SS occurs in organic forms, although, some Ni is expected. They showed further that peptides and, to a lower amount, amides and amino sugars, which are the main Norg forms in SS are transformed into to N-heterocyclic aromatics during thermal treatment. During pyrolysis considerably more heterocyclic N was formed than during HTC: The non-treated SS and its derived hydrochar and pyrochar exhibited Corg/N ratios of 7.0, 12.3 and 7.8 respectively (Table 6.2) indicating that indeed a considerable amount of N was incorporated into the charred organic fraction. Note that the Corg/N values of the isotopically enriched SS and chars are CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 93 lower than those observed for the SS obtained from ponds in Paneque et al. (2017). This is best explained with the fact that the enriched material was produced in the laboratory and was mostly composed of microbial residues whereas the pond material contained additional ingredients supplied with the wastewater. However, comparing the NMR spectra of the chars from the pond SS with those derived from the waste water reactor, one can assume that in spite of this difference the latter can be taken as being representative of chars derived from SS. 6.3.2 Greenhouse incubation experiment 6.3.2.1. Fate of the C derived from the amendments during the incubation experiment The percentage of C derived from isotopically enriched SS (CSS) decreased after 1 month of incubation (Figure 6.2), which points to a faster degradation of CSS if compared to the soil organic carbon (SOC) during this time. Most likely, this is due to the fact that SOC represented already well humified material, whereas CSS still contained considerable amounts of easily degradable residues. The relative contribution of C derived from Hyd (CHyd) decreased, too, but to a lower extent than CSS. Thuille et al. (2015) found comparable results after a 39-day greenhouse experiment with soils amended with a silage-derived hydrochar or with the nontreated silage. Between the first and the 10th incubation month, the relative proportion of CSS and of CHyd in the soil did not change, suggesting that either none of the C pool (SOC and Camend) decomposed further or, which is more likely, that both C pools degraded with a similar rate. CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 94 Figure 6.2. Percentage of the total C derived from the sewage sludge (CSS), hydrochar (CHyd) and pyrochar (CPy) in the topsoil at the different sampling times. The proportion of C derived from Py (CPy) did not change during the first incubation month. This suggests that CPy and SOC were degraded at a similar speed. However, the percentage of CPy increased between the 3rd and 10th month of incubation, allowing the conclusion that SOC suffered a preferential degradation relative to CPy. This would support a higher biochemical recalcitrance of pyrogenic organic matter relative to the SOC of the used soils. With respect to the subsoil, the relative contribution of Camend to the total Corg was lower than 0.5% for all treatments (Figure 6.3) revealing that leaching of the soil amendment or its degradation residues was negligible. CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 95 Figure 6.3. Percentage of the total C derived from the SS (CSS), its hydrochar (CHyd) and pyrochar (CPy) in subsoil at the different sampling times. 6.3.2.2. Soil nitrogen evolution during the incubation experiment - Nitrate (NO3-) During the first 3 months, the SS-amended pots exhibited between two and five times more topsoil NO3concentrations than the control (Figure 6.4). This is most likely due to NO3addition with the amendment. Despite that SS was only applied to the topsoil, the subsoil showed between 3 and 4 times more NO3than the control. This is explained by a leaching of this compound promoted by irrigation. The lack of NO3in both the top and subsoil after the 10th month of incubation is in line with the uptake of this nutrient by plants and microorganisms. However, loss due to volatilization cannot be excluded. Both addition of Hyd and Py decreased the topsoil NO3contents compared to the control after the first incubation month, which is in accordance with Bargmann et al. (2014a; 2014b). They observed that hydrochars promoted microbial immobilization whereas biochars seemed to increase N adsorption to particle. In addition, Haider et al. (2017) provide evidence that a “nitrate capture” may occur frequently after biochar addition. To which extend this can explain our results, however, has to be approached in future studies. After 3 and 10 months of incubation, NO3was found neither in the Hyd and Py treatments nor in the control. CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 96 In addition, these treatments had no impact on NO3content of the subsoil at any incubation time. Figure 6.4. Topand subsoil contents of nitrate (NO3-) and ammonium (NH4+) in the control, SS, Hyd and Py amended pots at the different sampling times. - Ammonium (NH4+) None of the treatments contained mentionable NH4+ concentrations, neither in the topnor in the subsoil after the 1st incubation month (Figure 6.4). However, after 3 and 10 months their abundance increased to 2.6 and 7.9 mg kg-1, which is in line with the degradation and desamination of organic N compounds. The lower NH4+ contents in the Py-amended pots than in the control after 3 months, may be due to adsorption of the nutrient onto the biochar surface and/or to an increase in the NH4+ consumption rates (Nelissen et al., 2012). After 10 months, all the amended pots and the control exhibited similar NH4+ contents CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 97 6.3.2.3. Fate of the N contained in the amendments during the incubation experiment - Changes after 1 month The relative contribution of N derived from SS (NSS) to the total N of the topsoil decreased after 1 month of incubation (Figure 6.5), indicating a preferential use of NSS with respect to soil N. Although CSS was also reduced during this time, the decrease of the N was stronger, which is related to the fact that the uptake and volatilization of NO3occurs independently from the C pool. The proportion of N derived from Hyd (NHyd) decreased to a lower extent than that of SS, which points to a lower N availability of the former compared to the latter during this time. This is likely due to the lack of Ni in Hyd together with a slightly higher content of heterocyclic N compounds. The degradation rate of the CHyd and the NHyd pools seems to be similar in this case. Figure 6.5. Percentage of the total N derived from the SS (NSS), hydrochar (NHyd) and pyrochar (NPy) in topsoil at the different sampling times. Despite the percentage of CPy was not altered after 1 month of incubation, the proportion of N derived from the pyrochar (NPy) decreased during this time. This result needs to be confirmed with further analyses before going into a deeper discussion. CHAPTER 6 Degradability of organic C and N of a 13C and 15N enriched hydrochar and a pyrochar derive from sewage sludge in a soil and the availability of their N for plant growth 98 Bearing in mind that the amount of N derived from the amendments (Namend) in the subsoil was less than 1% in all cases (Figure 6.6), the N loss is best explained with removal by N-uptake in plants and volatilization. The design of this experiment does not allow distinguishing between the pristine Namend (still non-degraded) and the Namend already degraded and immobilized by soil microorganisms. Considering that the Namend decreased during this first month, immobilization does not seem to play an important role. Figure 6.6. Percentage of the total N derived from the SS (NSS), hydrochar (NHyd) and pyrochar (NPy) in subsoil at the different sampling times. - Changes between the 1st and the 10th month of incubation The proportion of NSS of the total N in the soil slightly increased between the first and 10th incubation month, suggesting that soil N was preferentially lost compared to NSS during this time (Figure 6.5). Thus after degradation of the more labile Ncontaining compounds of SS during the first month, the remaining organic NSS exhibited a higher biochemical recalcitrance than soil N. The fact that, in contrast to NSS, the percentage of CSS did not increase during this time, may indicate that N containing components had a higher biochemical recalcitrance than those without N. Alternatively, our observation may be interpreted with deamination and subsequent recycling of the released N for the build-up of new microbial biomass whereas the remaining C-skeleton was metabolized to CO2. 104 On the other hand, further research is needed to evaluate to which extent a broad application is economically feasible. HTC seems to be a better choice than pyrolysis from an energetic and thus economic point of view. HTC avoids the needed for drying the SS prior the pyrolysis and lower temperatures are applied, which considerably reduce the costs for energy. Aside from being advantageous with respect to economic considerations, our study revealed that HTC at 200 ºC was more suitable than 260 ºC for plant growth (Chapter 4).