Biochar: Woody Perennials Source and Process Impact on Crop Production

INTERNATIONAL DOCTORAL SCHOOL OF THE USC Subhi Salman PhD Thesis BIOCHAR: WOODY PERENNIALS SOURCE AND PROCESS IMPACT ON CROP PRODUCTION Lugo, 2024 Doctoral Programme in Agricultural and Forestry Research DOCTORAL THESIS BIOCHAR: WOODY PERENNIALS SOURCE AND PROCESS IMPACT ON CROP PRODUCTION Author Subhi Salman Supervisor: Prof. Maria Rosa Mosquera Losada Co-Supervisor: Dr. Nuria Ferreiro Domínguez PHD PROGRAMME IN AGRICULTURAL AND FORESTRY RESEARCH SANTIAGO DE COMPOSTELA / LUGO I ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest gratitude to my supervisor, Prof. Maria Rosa Mosquera Losada, whose expertise, guidance, and unwavering support have been invaluable throughout this journey. Her encouragement and wisdom have been the cornerstone of my academic growth and success. I am equally grateful to my co-supervisor, Dr. Nuria Ferreiro Domínguez, for her insightful feedback and constant motivation. Her meticulous attention to detail and constructive critiques have significantly shaped the quality of this thesis. A heartfelt thank you goes out to the dedicated laboratory team: Divina, Teresa, Pablo, and Manuel. Your tireless assistance in both the fieldwork and the laboratory was essential. Without your hard work, patience, and expertise, this project would have been an impossible challenge. Your contributions have made this experience manageable, enriching, and enjoyable. I extend my deepest appreciation to the soul of my late father, whose memory has been a source of strength and inspiration. To my mother, brothers, and sisters, your unwavering love and support have been my bedrock. Your belief in me has been a constant source of motivation, driving me to persevere even in the most challenging times. To my friends and colleges, who have stood by me through thick and thin, your encouragement and companionship have been invaluable. You have lifted my spirits and provided a sense of normalcy and joy amidst the rigors of research and writing. Finally, I am profoundly grateful to everyone who has contributed to this thesis. Your academic, emotional, or practical support has made this achievement possible. This work stands as a testament to the collective effort and dedication of all those who believed in me and supported me along the way. Thank you all Subhi Salman II TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................................... I LIST OF TABLES ................................................................................................................................ V LIST OF FIGURES ............................................................................................................................. VI RESUMO.............................................................................................................................................. XI RESUMEN ........................................................................................................................................ XIII ABSTRACT ...................................................................................................................................... XVI CHAPTER 1 . JUSTIFICATION AND OBJECTIVE ....................................................................... 1 Justification and Objectives ................................................................................................................. 2 CHAPTER 2 . INTRODUCTION ........................................................................................................ 4 2.1 Introduction ................................................................................................................................... 5 2.1.1 The forest and agricultural area in Galicia ............................................................................. 5 2.2 Biochar: Definition, and Benefits ................................................................................................... 6 2.3 Biochar and Literature ................................................................................................................... 7 2.4 Biochar Production ........................................................................................................................ 8 2.4.1 Pyrolysis Temperature and Residence Time .......................................................................... 8 2.4.2 Feedstock Selection ................................................................................................................ 9 2.4.2.1 Conifers as a biochar feedstock ..................................................................................... 10 2.4.2.2 Broadleaves as a biochar feedstock ............................................................................... 10 2.5 Biochar's physical, chemical and biological properties affect the soil ........................................ 10 2.5.1 Physical Properties Of Biochar In Soil ................................................................................. 11 2.5.2 Chemical Properties Of Biochar In Soil ............................................................................... 12 2.6 Biochar and Plant growth ............................................................................................................ 12 CHAPTER 3 . ASSESSING THE INFLUENCE OF TREE SPECIES, TREE FRACTION, AND PYROLYSIS TIME ON BIOCHAR'S TOTAL NITROGEN, CARBON, AND C/N RATIO CONTENT ........................................................................................................................................... 14 3.1 Abstract ....................................................................................................................................... 15 3.2 Introduction ................................................................................................................................. 15 3.3 Materials and methods ............................................................................................................... 16 3.4 Results ......................................................................................................................................... 17 3.5 Discussion .................................................................................................................................... 25 3.6 Conclusion ................................................................................................................................... 28 CHAPTER 4 . Exploring the Potential of the Biochar Derived from Select Conifers and Broadleaves Species as a MacroNutrient Amendment .................................................................... 29 4.1 Abstract ....................................................................................................................................... 30 4.2 Introduction ................................................................................................................................. 30 III 4.3 Materials and methods ............................................................................................................... 31 4.4 Results ......................................................................................................................................... 32 4.4.1 Phosphorous ......................................................................................................................... 33 4.4.2 Potassium ............................................................................................................................. 35 4.4.3 Calcium ................................................................................................................................ 37 4.4.4 Magnesium ........................................................................................................................... 39 4.5 Discussion .................................................................................................................................... 41 4.6 Conclusion ................................................................................................................................... 43 CHAPTER 5 . Exploring the Potential of the Biochar Derived from Select Conifers and Broadleaves Species as a Micronutrient Amendment ...................................................................... 44 5.1 Abstract ....................................................................................................................................... 45 5.2 Introduction................................................................................................................................. 45 5.3 Materials and Methods ............................................................................................................... 46 5.4 Results ......................................................................................................................................... 46 5.4.1 Iron ....................................................................................................................................... 47 5.4.2 Copper .................................................................................................................................. 49 5.4.3 Zinc ...................................................................................................................................... 51 5.4.4 Manganese ............................................................................................................................ 53 5.5 Discussion .................................................................................................................................... 55 5.6 Conclusion ................................................................................................................................... 56 CHAPTER 6 . Effect of biochar application on pasture production in a greenhouse experiment ............................................................................................................................................................... 58 6.1 Abstract ....................................................................................................................................... 59 6.2 Introduction................................................................................................................................. 59 6.3 Materials and methods ............................................................................................................... 60 6.4 Results ......................................................................................................................................... 60 6.5 Discussion .................................................................................................................................... 66 6.6 Conclusions.................................................................................................................................. 68 CHAPTER 7 . GENERAL Discussion ............................................................................................... 69 7.1 General Discussion ...................................................................................................................... 70 7.2 Carbon, Nitrogen, and C/N ratio ................................................................................................. 70 7.3 Macronutrients ........................................................................................................................... 72 7.4 Micronutrients ............................................................................................................................ 73 7.5 Production ................................................................................................................................... 75 CHAPTER 8 . GENERAL CONCLUSION ..................................................................................... 77 IV CHAPTER 9 . References ................................................................................................................... 79 V LIST OF TABLES Table 2-1. Biochar Definitions in Literature. .......................................................................................... 7 Table 2-2. Operating conditions and product yields of different technologies. ...................................... 9 Table 2-3. Feedstock Influence on Biochar Characteristics. ................................................................. 10 Table 6-1 Chemical properties of the biochar applied in this experiment. ........................................... 60 VI LIST OF FIGURES Figure 2-1. Uses of the territory in Galicia (Xunta de Galicia, 2018; Bruña-García and Marey-Pérez, 2018). ....................................................................................................................................................... 5 Figure 2-2. Percentage of the forest area in Galicia according to the species (Forest Statistics Yearbook, 2019). ....................................................................................................................................................... 6 Figure 2-3. Potential effects of biochar on Soils' physical, chemical and biological properties. .......... 11 Figure 2-4. Impact of biochar on plant height, dry shoot biomass, and dry root biomass (Kamara et al., 2015). ..................................................................................................................................................... 13 Figure 3-1. The total concentrations of carbon (C), nitrogen (N), and carbon-nitrogen ratio (C/N) in the biochar made of different conifers and broadleaves species. Lowercase letters indicate significant differences among tree species. ............................................................................................................. 17 Figure 3-2. The total carbon (C) (a), nitrogen (N) (b), and C/N ratio (c) content in tree fractions (branches, litterfall, and green leaves) of the conifers (left) and broadleaves (right). Lowercase letters indicate differences among tree fractions, while uppercase letters indicate significant differences among tree fractions between different tree species (conifers and broadleaves. ............................................... 18 Figure 3-3. The total carbon (C) (a), nitrogen (N) (b), and C/N ratio (c) content after three different pyrolysis times (30, 60, and 120 minutes) of the conifers (left) and broadleaves (right). Lowercase letters indicate differences among tree fractions, while uppercase letters indicate significant differences among pyrolysis time between different tree species (conifers and broadleaves)............................................. 19 Figure 3-4. The total C (%) in the biochar produced from branches, litterfall and green leaves of different tree species (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton, BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill) under different pyrolysis timings (30, 60 and 120 minutes). In each tree fraction, different lowercase letters indicate significant differences among tree species under the same pyrolysis time and different uppercase letters indicate significant differences among pyrolysis times within the same tree species. ............................................................................ 21 Figure 3-5. The total N concentration (%) in the biochar produced from branches, litterfall and green leaves of different tree species (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton, BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill) under different pyrolysis timings (30, 60 and 120 minutes). The initial total N concentration indicates the N concentration in each tree species and fraction before the pyrolysis process. In each tree fraction, different lowercase letters indicate significant differences among tree species under the same pyrolysis time and different uppercase letters indicate significant differences among pyrolysis times within the same tree species 23 XIII RESUMEN __________________________________________________________________________________ XIV RESAMEN En las últimas décadas, el creciente interés por las prácticas agrícolas sostenibles ha impulsado el estudio de enmiendas del suelo innovadoras que mejoren la fertilidad del suelo, promuevan el crecimiento de las plantas y contribuyan al secuestro de carbono. Una solución prometedora es el uso de biocarbón como enmienda del suelo, un material rico en carbono producido a partir de biomasa orgánica mediante pirólisis. Galicia, con sus extensas zonas boscosas, se enfrenta a un importante riesgo de incendios forestales, agravado por la acumulación de residuos forestales secos. La utilización de esta biomasa forestal como materia prima para la producción de biocarbón podría reducir el riesgo de incendios forestales, y proporcionar un método sostenible para la gestión de los residuos forestales. Al convertir en biocarbón distintas fracciones de los árboles como ramas, hojarasca y hojas verdes, podemos mitigar los riesgos medioambientales asociados a los incendios forestales al tiempo que creamos una valiosa enmienda para el suelo. Además, los suelos de Galicia presentan a menudo una baja fertilidad, debido a su acidez que limita la productividad de los sistemas. La aplicación de biocarbón puede mejorar la fertilidad del suelo, la retención de agua, la disponibilidad de nutrientes y la resiliencia de los cultivos, mejorando la productividad agrícola y la seguridad alimentaria. Este estudio pretende evaluar de forma exhaustiva el biocarbón derivado de diferentes especies arbóreas (coníferas y frondosas) y fracciones (ramas, hojarasca y hojas verdes) bajo diferentes tiempos de pirólisis (30, 60 y 120 minutos) y examinar sus efectos sobre la producción de ryegrass en experimentos establecidos en macetas en el invernadero. Los objetivos del estudio incluyen la determinación de la influencia combinada de estas variables sobre el carbono total, el nitrógeno y la relación C/N del biocarbón, la evaluación del contenido en macronutrientes (P, K, Mg, Ca), el análisis del contenido en micronutrientes (Fe, Cu, Zn, Mn) y el impacto de la aplicación de biocarbón sobre la producción de pasto. Esta investigación es significativa por su enfoque integral para comprender las interacciones entre el tipo de materia prima, las condiciones de pirólisis y las propiedades del biocarbón. Los resultados de este estudio tendrán implicaciones prácticas para los agricultores, los gestores de la tierra y los responsables políticos que pretendan implementar prácticas agrícolas sostenibles y contribuir al discurso científico más amplio sobre el biocarbón, ofreciendo nuevas perspectivas y llenando las lagunas de conocimiento existentes. Los resultados obtenidos revelaron que, independientemente de la fracción de árbol y del tiempo de pirólisis, la concentración total de carbono en el biocarbón elaborado a partir de coníferas era mayor que en el biocarbón procedente de frondosas; pero la concentración total de nitrógeno era mayor en las frondosas que en las coníferas. Debido a la mayor concentración de carbono en el biocarbón de coníferas, la relación C/N fue mayor en el biocarbón de coníferas que de frondosas. Por lo general, las ramas implicaban un biocarbón con una mayor concentración de carbono total que la hojarasca y las hojas verdes, mientras que las hojas verdes implicaban mayores niveles de nitrógeno. El tiempo de pirólisis no afectó significativamente a la concentración total de carbono y nitrógeno ni a la relación C/N en el biocarbón. Las concentraciones de macronutrientes fueron mayores en el biocarbón de frondosas que en el de coníferas, siendo las hojas verdes las que mostraron un mayor incremento. El contenido en micronutrientes se vio influido por el tipo de materia prima y el tiempo de pirólisis, siendo el biocarbón derivado de las frondosas generalmente más rico en manganeso y cobre, mientras que el derivado de las coníferas era más rico en hierro y zinc. La hojarasca presentó las mayores concentraciones de micronutrientes. La combinación de fertilizantes minerales con biocarbón afectó positivamente a la producción de pasto y a la altura de las plantas en suelos limosos, lo XV que demuestra el potencial del biocarbón para mejorar la fertilidad del suelo y el crecimiento de las plantas. XVI ABSTRACT __________________________________________________________________________________ XVII ABSTRACT In recent decades, the growing interest in sustainable agricultural practices has driven the study of innovative soil amendments that enhance soil fertility, promote plant growth, and contribute to carbon sequestration. One promising solution is the use of biochar as a soil amendment, a carbon-rich material produced from organic biomass through pyrolysis. Galicia, with its extensive forested areas, faces a significant risk of wildfires, exacerbated by the accumulation of dry forest residues. Utilizing this forest biomass as a feedstock for biochar production could reduce the risk of wildfires and provide a sustainable method for managing forest residues. By converting various tree fractions, such as branches, litterfall, and green leaves, into biochar, we can mitigate the environmental risks associated with wildfires while creating a valuable soil amendment. Additionally, Galicia's soils often have low fertility due to their acidity, which limits the productivity of the systems. The application of biochar can improve soil fertility, water retention, nutrient availability, and crop resilience, enhancing agricultural productivity and food security. This study aims to comprehensively evaluate biochar derived from different tree species (conifers and broadleaves) and fractions (branches, litterfall, and green leaves) under different pyrolysis times (30, 60, and 120 minutes) and examine their effects on ryegrass production in pot experiments established in the greenhouse. The study objectives include determining the combined influence of these variables on the total carbon, nitrogen, and C/N ratio of biochar, evaluating the macronutrient content (P, K, Mg, Ca), analysing the micronutrient content (Fe, Cu, Zn, Mn), and assessing the impact of biochar application on grass production. This research is significant for its comprehensive approach to understanding the interactions between raw material type, pyrolysis conditions, and biochar properties. The results of this study will have practical implications for farmers, land managers, and policymakers aiming to implement sustainable agricultural practices and contribute to the broader scientific discourse on biochar, offering new insights and filling existing knowledge gaps. The results revealed that, regardless of tree fraction and pyrolysis time, the total carbon concentration in biochar made from conifers was higher than in biochar from broadleaves; but the total nitrogen concentration was higher in broadleaves than in conifers. Due to the higher carbon concentration in conifer biochar, the C/N ratio was higher in conifer biochar than in broadleaves biochar. Generally, branches resulted in biochar with a higher total carbon concentration than litterfall and green leaves, while green leaves resulted in higher nitrogen levels. Pyrolysis time did not significantly affect the total carbon and nitrogen concentration or the C/N ratio in biochar. Macronutrient concentrations were higher in broadleaves biochar than in conifer biochar, with green leaves showing the highest increase. Micronutrient content was influenced by the type of raw material and pyrolysis time, with broadleaves derived biochar generally being richer in manganese and copper, while conifer derived biochar was richer in iron and zinc. Litterfall showed the highest concentrations of micronutrients. The combination of mineral fertilisers with biochar positively affected grass production and plant height in loamy soils, demonstrating the potential of biochar to improve soil fertility and plant growth. 1 CHAPTER 1 . JUSTIFICATION AND OBJECTIVE _____________________________________________________________________ 2 JUSTIFICATION AND OBJECTIVES In recent decades, the increasing interest in sustainable agricultural practices has driven the exploration of innovative soil amendments that enhance soil health meaning fertility to promote plant growth, while contributing to carbon sequestration. One promising solution is the use of biochar, a carbon-rich material produced from organic biomass through pyrolysis (Lehmann & Joseph, 2015). Biochar's potential benefits extend beyond soil fertility improvements to include climate change mitigation and waste management (Schmidt et al., 2012). Galicia, with its extensive forested areas, faces a significant risk of forest fires, exacerbated by the accumulation of dry forest waste. Utilizing this forest waste biomass as feedstock for biochar production not only reduces the fire risk but also provides a sustainable method for managing forest waste. By converting tree fractions such as branches, litterfall, and green leaves into biochar, we can mitigate the environmental risks associated with forest fires while creating a valuable soil amendment (Rigueiro-Rodríguez et al., 2009; Xunta de Galicia, 2009). The agricultural soils in Galicia often exhibit low fertility, which limits the productivity of both herbaceous and tree components in agricultural systems (Zas & Alonso, 2002). An interesting option to increase soil fertility is the use of biochar derived from forest waste as an organic amendment. Biochar is rich in organic matter, carbon (C), and phosphorus (P), which are essential nutrients for soil health and plant growth. Using biochar in agriculture not only enhances soil fertility but also offers a sustainable disposal method for forest waste (Glaser et al., 2002). Optimising the biochar produced to increase soil health is one of the most important steps to increase the soil improvement, based on the different characteristics that biochar has when is submitted to different processing. Understanding the process factors affecting biochar quality is essential for the effective and efficient production of biochar as soil health improver (Jeffery et al., 2015). The application of biochar in agriculture has the potential to enhance plant growth and yield. By improving soil fertility, biochar can increase agricultural productivity, which is vital for food security. Studies have shown that biochar can improve water retention, nutrient availability, and crop resilience to environmental stress (Atkinson et al., 2010). Considerable research has been conducted on biochar. However, significant knowledge gaps remain regarding the specific impacts of different feedstocks and pyrolysis conditions on biochar properties. This research seeks to fill these gaps by systematically evaluating the effects of different tree species, tree fractions, and pyrolysis times on biochar's chemical composition and its practical applications in soil amendment (Schmidt et al., 2012). Biochar's potential extends beyond theoretical benefits; it offers practical solutions for farmers and land managers. By providing detailed insights into how different production parameters affect biochar properties, this research aims to offer practical guidelines for biochar application in various agricultural contexts. This PhD is significant in its comprehensive approach to understanding the interactions between feedstock type, pyrolysis conditions, and biochar properties. It provides a detailed analysis that is critical for optimising biochar production and application. The findings will have practical implications for farmers, land managers, and policymakers aiming to implement sustainable agricultural practices. Moreover, this study will contribute to the broader scientific discourse on biochar, offering new insights and filling existing knowledge gaps. 3 More specifically, the objectives were:  To determine the combined influence of different tree species (conifers and broadleaves), tree fractions (branches, litterfall and green leaves), and pyrolysis time (30, 60, and 120 minutes) on biochar characteristics such as the total carbon, nitrogen, and C/N ratio of biochar,total and Mehlich macronutrient (P, K, Mg, Ca).  To analyse the micronutrient (Fe, Cu, Zn, Mn) content of biochar, including total and Mehlich concentrations, and assess how these vary with tree species (conifers and broadleaves), tree fractions (branches, litterfall, green leaves), and pyrolysis time (30, 60, and 120 minutes).  To investigate the impact of biochar application on pasture production in a controlled greenhouse experiment, focusing on plant dry matter and height under different doses of biochar and mineral fertiliser treatments.  To provide practical recommendations for the use of biochar as a soil amendment, based on the findings related to its chemical properties and effects on plant growth and soil fertility. 4 CHAPTER 2 . INTRODUCTION _____________________________________________________________________________ 11 Figure 2-3. Potential effects of biochar on Soils' physical, chemical and biological properties. 2.5.1 Physical Properties Of Biochar In Soil Biochar can play a vital role in the soil's physical characteristics, which can enhance soil structure, soil porosity, soil surface area, and water-holding capacity (WHC). Biochar amendment to the soil can increase soil aggregation and stability as well, leading to improved soil structure and reducing the risk of soil erosion (Situ et al., 2022). Being a biochar amendment, it plays a crucial role in allowing water infiltration and retention due to its porous structure, which facilitates the formation of a microenvironment within the soil (Chen et al., 2021). This increase in soil moisture not only provides drought resistance but also enhances plant growth and yield (Wu et al., 2023). Moreover, biochar treatment has been found to reduce leaching by accumulating soil particles and reducing soil volume, especially in hard soils (Chen et al., 2024) Such high volumes this reduction improves soil mobility and root penetration, further increasing plant growth and nutrient utilization (Basset et al., 2023). In addition, biochar amendment affects soil porosity, which plays an important role in controlling soil air and water dynamics (Wei et al., 2023). Wang et al. (2023) showed that the addition of biochar increases soil porosity, especially microporosity, thereby increasing air exchange and air diffusion in the soil. This improved soil aeration supports root growth and microbial activity, which contributes to overall soil health and productivity (Pathy et al., 2020). Furthermore, biochar analyses have been found to increase soil water holding capacity by increasing water content in soil moisture and reducing water loss through infiltration, especially in sandy soils (Razzaghi et al., 2020). These increased soil moisture levels in terms of water use efficiency are effective and buffers against drought stress, contributing to sustainable crop growth and yield (Li et al., 2021). Finally, biochar can increase soil surface area, thereby capturing nutrients in the soil and preventing leaching (Ghorbani et al., 2023). Overall, the addition of biochar soil systems has a positive effect on physical diversity, creating favourable conditions for plant growth and ecological activities. 12 2.5.2 Chemical Properties Of Biochar In Soil Biochar application significantly influences soil properties crucial for soil fertility and plant growth (El-Naggar et al., 2019). Increased soil carbon content improves soil fertility by promoting microbial activity, organic matter decomposition, and nutrient cycling, ultimately benefiting plant growth (Pacheco et al., 2024). Moreover, biochar amendments enhance the retention and availability of macronutrients such as nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sodium (Na), essential for plant nutrition (Jan et al., 2023). Increasing soil cation exchange capacity (CEC) through biochar application enhances nutrient retention and availability, ultimately promoting soil fertility (Lago et al., 2021). Incorporating biochar has been demonstrated to increase soil pH, especially beneficial in acidic soils, as it acts as a liming agent and helps buffer against soil acidity (Bolan et al., 2023). This pH adjustment improves nutrient solubility and availability, facilitating optimal nutrient uptake by plants (Wei et al., 2023). Additionally, biochar amendments affect the availability of sulfur (S) and micronutrients such as iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) in soil, impacting their bioavailability and plant uptake (Sarmah et al., 2023). Overall, biochar influences soil chemical properties, nutrient availability, and pH regulation, leading to enhanced soil fertility and healthy plant growth. 2.5.3 Biological Properties Of Biochar In Soil The biochar’s porous structure plays a vital role in providing a favourable habitat for soil microorganisms, including mycorrhizal fungi and actinobacteria (Palansooriya et al., 2019). These microorganisms serve as food for protozoa, mites, nematodes and other essential soil components, as well as converting the element from one form to another (Neemisha, 2020). By promoting a higher level of microbiological communities, biochar contributes to the simultaneous reduction of GHG emissions in the soil (Jiang et al., 2021) Adding biochar to increase microorganism activity should begin with studying other soil characteristics such as pH and soil type (Zhang et al., 2018). For instance, it was found that the addition of rice residue-derived biochar negatively affected earthworm populations in both the short and long term which can be attributed to the high pH of the biochar (Rahman et al., 2020). Pokharel et al. (2020) proposed that biochar enhances soil enzymatic activity, indicating improved soil health. Moreover, Dinesh et al. (2013) mentioned that there is a significant increase in dehydrogenase and urease activity by 19.0% and 44.0%, respectively. Additionally, the integration of biochar into soil has been associated with decreased soil acidity and elevated levels of nitrogen and organic carbon (Yu et al., 2017). 2.6 BIOCHAR AND PLANT GROWTH Plants cultivated in soils exhibiting nutrient deficiencies manifest a range of physiological and morphological symptoms, from an immediate cessation of root growth to disruptions in membranes, cell walls, and variations in cytosolic pH (Notununu et al., 2022). These alterations may induce oxidative stress, culminating in the breakdown of chloroplasts and the identifiable symptoms of chlorosis and necrosis (Hodges and Constable, 2010). Numerous studies confirm that integrating biochar into nutrient-deficient soils enhances plant growth across diverse crops, including maise (Uzoma et al., 2011), common bean (Rondon et al., 2007), rice (Yu et al., 2019), oat (Schulz et al., 2013), and lettuce (Carter et al., 2013, Ma et al., 2022). Additionally, research by Ullah et al. (2020) demonstrated that biochar application can positively affect plant growth parameters such as plant height, dry root weight, and dry shoot weight, as illustrated in Figure 2-4. The improved plant growth attributed to biochar can be ascribed to three key factors: 1) the nutrient content within biochar’s (Schulz et al., 2013); 2) enhanced efficiency in nutrient 13 utilization (Phares et al., 2022); and 3) the creation of a favourable rhizosphere environment (Sarfraz et al., 2019). Biochar possesses a high concentration of nutrients such as N, P, K, Ca, Mg, S, Mn, Cu, Zn, and B (Hossain et al., 2020; Rasuli et al., 2022), with specific element concentrations varying based on feedstocks and carbonization methods (Janu et al., 2021). Generally, lower pyrolysis temperatures (250–350 °C) are favoured for soil amendment as they help sustain higher levels of nutrient elements (Hassan et al., 2020). Applying biochar with inorganic or organic fertilizers enhances plant nutrient use efficiency (Arif et al., 2017). In a similar vein, Rafique et al. (2020) reported a substantial enhancement in both the fresh and dry weight of maize by 50–55% following soil amendment with biochar. Jabborova et al. (2021) have mentioned that the biochar application has improved root and shoot weights, with a 56% increase in root fresh weight and a 39% increase in shoot dry weight. Figure 2-4. Impact of biochar on plant height, dry shoot biomass, and dry root biomass (Kamara et al., 2015). These collective findings underscore the consistently positive impact of biochar on diverse crops, affirming its role in fortifying plant growth, yield, and physiological functions. The reported successes across different studies and crop systems highlight the versatility and efficacy of biochar as a beneficial soil amendment. Such evidence contributes to the growing body of support for using biochar to optimize agricultural productivity, especially amidst varying environmental challenges (Muhammad et al., 2023). The documented enhancements in plant performance following biochar incorporation underscore its potential as a sustainable and versatile tool in modern agricultural practices (Zubairu et al., 2023). Furthermore, biochar's inhibitory effects on the transformation of NO3− into unavailable forms after fertilizer release (Widowati et al., 2011), emphasize its potential to reduce nutrient leaching and enhance nutrient availability in soils (Ding et al., 2016). Overall, applying biochar to problematic soils not only improves soil physical and chemical properties but also establishes a symbiotic relationship among plant roots, biochar-amended soils, and microbes, fostering a healthy rhizosphere conducive to optimal plant growth (Dorak et al., 2022; Gu et al., 2022; Rivelli et al., 2022). 14 CHAPTER 3 . ASSESSING THE INFLUENCE OF TREE SPECIES, TREE FRACTION, AND PYROLYSIS TIME ON BIOCHAR'S TOTAL NITROGEN, CARBON, AND C/N RATIO CONTENT __________________________________________________________________________________ 15 3.1 ABSTRACT Carbon and nitrogen play a crucial role in soil by influencing its physical, chemical, and biological properties, ultimately shaping its overall fertility and health. Biochar is one carbonrich material with additional elements that can contribute to plant growth due to the biological, chemical and physical improvement of soil characteristics. However, biochar's total carbon and nitrogen concentration can vary significantly depending on factors such as the feedstock or pyrolysis time. This study aimed to assess the impact of tree species, tree fraction used, and pyrolysis time on the total carbon (C%) and N (N%) concentrations and C/N ratio of biochar. For biochar production, three conifer species (Pinus radiata D. Don (Ps), Pinus sylvestris L. (Ps), and Pinus pinaster Aiton (Pp)) and three broadleaves species (Betula alba L. (Ba), Quercus robur L. (Qr), and Castanea sativa Mill. (Cs)) were used. Three different fractions, including branches, litterfall, and green leaves, were obtained from each species for biochar production. The pyrolysis process was carried out for varying durations of 30, 60, and 120 minutes. The findings revealed that regardless of the tree fraction and pyrolysis time, the order of tree species according to total carbon concentration was Pp>Ps>Pr>Ba>Qr and Cs highlighting the higher proportion of conifers compared with broadleaves; the order of tree species according to total nitrogen concentration was Ps>Qr>Ba>Cs>Pp>Pr linked to broadleaves and Ps compared with conifers. However, the order of tree species according to the total C/N ratio was Pr>Pp>Ps, Ba, and Cs>Qr indicating the high level of carbon in conifers that are also associated with a lower level of nitrogen than broadleaves. Regardless of the tree species and pyrolysis time, branches generally contain a higher total carbon concentration than litterfall and green leaves. However, the nitrogen produced from the green leaves tree fraction was higher than the nitrogen from the other fractions. Independently of the tree species and tree fraction, the pyrolysis time did not significantly affect the total carbon and nitrogen concentration and C/N ratio. Keywords: charcoal, carbon source, pyrolysis, feedstock, conifers, broadleaves 3.2 INTRODUCTION Wood production in Europe is one of the most important productions, while in the European Union (EU-27) about 159 million hectares are approximately estimated to be planted as a forest (EC, 2020). In terms of wood production, the EU-27 generated an estimated 489.8 million cubic meters in 2018. Consequently, approximately 50.2 million tons of wood waste was produced annually across the 28 EU countries (Borzęcki et al., 2018). Unfortunately, only around 70% of this wood waste undergoes proper treatment and management (EC, 2020). In this context, producing biochar from different fractions (such as branches, litterfall, and green leaves) from various tree species, including those found in Galicia, presents an opportunity to promote forest waste management (Waqas et al., 2018). This perspective aligns with the European Parliament's objectives, as outlined in the Waste Framework Directive (2008/98/EC), which establishes a comprehensive legal framework for managing waste materials, including their recovery and disposal. Biochar is a carbon-rich material that forms through the pyrolysis process in the absence of oxygen, from various organic biomass, such as plant leftovers, animal manure, wastewater sludge, or wastewater sludge (IBI, 2015; Shoudho et al., 2024). An extremely stable carbon source that may persist for hundreds or perhaps thousands of years, biochar is generated (Kapoor et al., 2024). The presence of several additional micro and macro components in biochar, however, has a significant impact on the plant and its development (Wei et al., 2019). According several studies, biochar has a promising soil modification for its soil physical and 16 chemical properties (Zhang et al., 2014; Liu et al., 2019). The biochar contains a vital component of the soil organic matter that can improve soil fertility. Additionally, biochar can be a promising tool to decrease carbon dioxide (CO2) and offer a high capacity to sequester carbon in the soil which can be a valid method for mitigating climate change. Furthermore, the biochar contains some other macro and micro nutrients which have a vital role in plant growth such as nitrogen (N) which is responsible for many physiological roles such as protein formation, chlorophyll synthesis, DNA synthesis, and metabolic regulation (Xiong et al., 2021). Having the biochar as a high surface area amendment will help reduce nitrogen volatilization and reducing nitrogen leaching (Sun et al., 2024; Yang et al., 2024). However, the N in biochar can be influenced by various factors, including pyrolysis temperature and time (Angın, 2013; Wang et al., 2020). Moreover, the biochar application on the soil with a high C/N ratio can lead to a higher microbial nitrogen immobilization in the soil (Kirkby et al., 2014). The soil microbial activity resulting from the high C/N ratio in the biochar leads to decreased soil greenhouse gas fluxes and increased soil organic carbon (SOC) levels (Cleveland and Liptzin, 2007; Hu et al., 2024). The chemical composition of biochar can be significantly influenced by factors such as temperature and pyrolysis time (Balmuk et al., 2023). Moreover, the choice of feedstock plays a crucial role in nutrient enrichment and biochar quality (Anand et al., 2023). Research conducted by Park et al. (2019) demonstrated that different plant species yield biochar with varying proportions of essential elements for plant growth, thereby impacting soil nutrient availability. Consequently, careful consideration of the desired properties is necessary when selecting an appropriate feedstock for biochar production to achieve the intended outcomes (Wei et al., 2019; El Barkaoui et al., 2023). This study aims to investigate the total carbon and nitrogen concentrations and its relationship in biochar produced from different tree species (Pinus radiata D. Don, Pinus sylvestris L., Pinus pinaster Aiton, Betula alba L., Quercus robur L., Castanea sativa Mill.), tree fractions (branches, litterfall, and green leaves), and pyrolysis times (30, 60, and 120 minutes) at a temperature of 300°C in Galicia, NW Spain. 3.3 MATERIALS AND METHODS In September 2021, feedstock for biochar production was obtained from various forest plantations in Galicia, located in the northwest region of Spain. This period was selected to minimise the impact of on the main growing season of the tree species in Galicia. The feedstock consisted of six tree species. Three of these species were conifers: Pinus radiata D. Don (Pr), Pinus sylvestris L. (PS), and Pinus pinaster Aiton (Pp). The remaining three species were broadleaves: Betula alba L. (Ba), Quercus robur L. (Qr), and Castanea sativa Mill (Cs). Three different tree fractions were collected: branches, litterfall, and green leaves. After feedstock samples collection, the tree fractions were manually separated in the laboratory and dried in an oven at 45°C for four days. The pyrolysis process to produce biochar was then conducted using a muffle furnace with ceramic crucibles containing the feedstock for durations of 30, 60, and 120 minutes at a temperature of 300°C. The resulting biochar was ground using an agate mortar and pestle and stored in plastic-sealed bags (Gonzaga et al., 2017). To ensure the experiment's validity, three biochar replicates were produced for each type of feedstock. Following this, the C and N were determined in the biochar after pyrolysis for each tree species and fraction in each pyrolysis time using laboratory LECO CNS-2000 instrumentation (Kowalenko and Grant, 2001). The C/N ratio was also calculated for each of the fractions described above. 17 The total concentrations of carbon and nitrogen and the C/N ratio were analysed statistically using SAS software. The statistical analysis included both the LSD (Least Significant Difference) and ANOVA (Analysis of Variance) tests. The LSD test was specifically used to differentiate the means when the ANOVA test results were determined to be significant. 3.4 RESULTS Figure 3-1 illustrates the significant differences (p<0.05) in the total carbon (C), nitrogen (N), and the C/N ratio between broadleaves and conifers. Specifically, conifers exhibited a significantly higher total C content of 8.2% (p<0.05) than broadleaves. Conversely, broadleaves demonstrated a greater N content, approximately 10.6% (p<0.05) higher than conifers. Furthermore, the C/N ratio was around 31.6% (p<0.05) higher in conifers compared to broadleaves because the concentration of carbon was higher than nitrogen. Figure 3-1. The total concentrations of carbon (C), nitrogen (N), and carbon-nitrogen ratio (C/N) in the biochar made of different conifers and broadleaves species. Lowercase letters indicate significant differences among tree species. In Figure 3-2a, the total carbon (C) in conifers' branches, litterfall, and green leaves were significantly (p<0.05) higher than in broadleaves. Within conifers, total biochar carbon varied among branches, litterfall, and green leaves, ranging from 69.84 to 72.48%, 66.35 to 68.84%, and 68.02 to 71.22%, respectively. For broadleaves, concentrations ranged from 63.61 to 68.30%, 59.39 to 66.92%, and 61.70 to 66.83%, respectively. Biochar C levels in conifers and broadleaves were approximately 5% (p<0.05) higher in branches compared to litterfall and green leaves. The concentration of total nitrogen (N) in conifers' branches and green leaves was significantly smaller than in broadleaves (p<0.05), as shown in Figure 3-2b. Within conifers, biochar total N varied among branches, litterfall, and green leaves, ranging from 1 to 1.64%, 1.8 to 2.7%, and 2.12 to 3.14%, respectively. For broadleaves branches, litterfall, and green leaves concentrations the total N ranged from 1.57 to 2.16%, 1.92 to 2.81%, and 3.25 to 3.49%, 18 respectively. In conifers, biochar total N levels were around 100% (p<0.05) higher in litterfall and green leaves compared to branches. Additionally, in broadleaves, total N levels in green leaves were about 45.9% (p<0.05) higher than in litterfall and approximately 81.6% (p<0.05) higher than in branches. Regarding the C/N (Figure 3.2c) ratio of conifers' branches and green leaves was significantly higher than in broadleaves (p<0.05). C/N ratio in biochar varied among branches, litterfall, and green leaves, with ranges of 45.1 to 70.73%, 17.46 to 37.09%, and 22.39 to 33.95%, respectively, in conifers, and 32.44 to 40.54%, 21.16 to 34.79%, and 18.44 to 19.15%, respectively, in broadleaves. In conifers, the biochar C/N ratio was approximately 103% (p<0.05) higher in branches than in green leaves and litterfall. Similarly, in broadleaves, C/N levels in branches were roughly 89.8% (p<0.05) higher than in green leaves and about 27.4% higher than in litterfall. Additionally, litterfall exhibited approximately 48.9% (p<0.05) higher C/N ratio than green leaves. Figure 3-2. The total carbon (C) (a), nitrogen (N) (b), and C/N ratio (c) content in tree fractions (branches, litterfall, and green leaves) of the conifers (left) and broadleaves (right). Lowercase letters indicate differences among tree fractions, while uppercase letters indicate significant differences among tree fractions between different tree species (conifers and broadleaves. The total C (Figure 3-3a) levels in conifers after 30, 60, and 120 minutes significantly exceeded those in broadleaves (p<0.05). Biochar total C ranged within conifers after 30, 60, and 120 minutes from 68.04 to 70.53%, 67.42 to 71.13%, and 68.75 to 70.87%, respectively. For broadleaves, it ranged from 61.74 to 67.59%, 62.52 to 67.68 %, and 62.55 to 66.78%. No significant differences (p>0.05) were observed for total C among pyrolysis times for both conifers and broadleaves. The total N (Figure 3-3b) levels at 30, 60, and 120 minutes did not significantly differ between conifers and broadleaves (p > 0.05). Biochar total N ranged within conifers after 30, 19 60, and 120 minutes from 1.71 to 3.06%, 1.8 to 3.17%, and 1.69 to 2.58%, respectively. For broadleaves, it ranged from 2.39 to 2.65%, 2.35 to 2.64%, and 2.44 to 2.67%. It was observed that there is no significant effect of changing the pyrolysis time on the C/N ratio for both conifers and broadleaves (p>0.05), as illustrated in Figure 3-3c. In conifers, biochar C/N ratio ranged after 30, 60, and 120 minutes from 26.50 to 44.97%, 26.09 to 42.43%, and 31.74 to 48.62%, respectively, while in broadleaves, it ranged from 24.70 to 29.09%, 25.26 to 29.61%, and 24.77 to 29.05%. Changing the pyrolysis time did not significantly affect the C/N ratio of the conifers and broadleaves (p>0.05). Figure 3-3. The total carbon (C) (a), nitrogen (N) (b), and C/N ratio (c) content after three different pyrolysis times (30, 60, and 120 minutes) of the conifers (left) and broadleaves (right). Lowercase letters indicate differences among tree fractions, while uppercase letters indicate significant differences among pyrolysis time between different tree species (conifers and broadleaves). Figure 4, demonstrated that the total carbon concentration (C), the total nitrogen concentration (N), and the C/N ratio in the biochar were significantly affected (p<0.05) by the tree species, tree fraction and the pyrolysis time employed for biochar. The total carbon (C) concentration varied among the tree species in each tree fraction and pyrolysis time (p<0.05). The branches derived biochar total C ranged from 63.6 to 73%. After 30 minutes of pyrolysis, the total C from Pp, Ps, and Pr branches was around 10% (p<0.05) higher compared to Qr and Cs, with Ba branches also exceeded Cs total C by 8%. Moving on to a pyrolysis after of 60 minutes, the total C found in the branches tree fraction of Pp and Ps demonstrated about 9% higher concentration when compared to the total C in the biochar derived from Qr and about 15% (p<0.05) higher than Cs branches, while Pr branches also was about 10% (p<0.05) higher levels than Cs. At 120 minutes, Pp branches had higher total C (p<0.05) than Ba, Qr, and Cs, by 5, 10%, and 12%, respectively. Moreover, Ps and Pr branches were higher (p<0.05) than Qr and Cs, by 8 and 9%, respectively. 20 The litterfall derived biochar total C ranged from 59 to 68.84%. Within the litterfall tree fraction, after 30 minutes of pyrolysis, Ps, Pr, and Pp had around 15% higher total C levels than in Qr and Cs. After 60 minutes of pyrolysis time, the total C level produced from Pp was around 6% (p<0.05) higher than the total C level produced from Ba, Cs and Ps, and around 16% higher than in the Qr. Moreover, Ps, Ba, and Cs litterfall had around 12% (p<0.05) higher total C than Qr. After 120 minutes, Pp litterfall showed around 3% (p<0.05) higher total C than Ps, and Ba, while it was around 10 % (p<0.05) higher total carbon than in Cs, and Qr. The total C level obtained from Pr, Ps, and Ba litterfall biochar was (p<0.05) higher than the total C level from Cs and Qr, by 9 and 13%, respectively. While the total C in the Cs litterfall biochar was 6% (p<0.05) higher than in Qr. The pyrolysis residence time effect was evident only in the case of Qr litterfall, with the total C level being around 2% (p<0.05) higher at 30 minutes compared to 120 minutes. The green leaves derived biochar total C ranged from 61.7 to 71.2%. After 30 minutes of the pyrolysis time, the total C obtained from Pp green leaves biochar was around 7% (p<0.05) higher compared to that obtained from Ps and Ba, and 17% higher than in Qr and Cs. Additionally, the biochar derived from Pr, Ba, and Ps green leaves exhibited 6% (p<0.05) higher total C compared to Qr and Cs. After 60 minutes of pyrolysis, the total C level in Pr, Ps, and Pp green leaves biochar surpassed (p<0.05) that of Qr green leaves biochar, by 17%. The total C level in Ba is higher (p<0.05) than in Qr, by 9%. After 120 minutes, Pp and Ps green leaves biochar had around 5% higher total C than Ba, and around 15% (p<0.05) higher total C than Qr, and Cs. Moreover, Pr and Ba green leaves biochar were around 9% (p<0.05) higher than Qr and Cs. 27 observed order was Ps > Qr and Ba > Cs > Pp > Pr. This particular order can be attributed to the presence of specific amino acids, such as arginine, which contain amide groups that readily convert into gaseous by-products like NH3. As nitrogen-containing components accumulate, they contribute to the formation of additional N-functional groups, ultimately leading to higher nitrogen content in the produced biochar (Leng et al., 2020). Nitrogen has a vital role in plant growth (Ye et al., 2022), and a higher N level in the biochar produced can reduce the nitrogen artificial fertilizers use (Solaiman et al., 2020). Ba is a broadleave pioneer and fast-growing species compared with chestnut and oaks and all conifers. The Ba is the first species losing the leaves after summer, which may be related to the high protein content of the leave, unable to translocate the nitrogen so fast at the end of the season. On the other hand, Pinus sylvestris L., the conifer with the lowest growth rate, has the higher litterfall content in any of the processing biochar temperature compared with other broadleaves and conifers, which can be associated with the fact of been evergreen and slow-growth species. The level of nitrogen in the green leaves is always lower in the Pinus pinaster Aiton, the species with a high value of carbon and therefore lignin, therefore providing a major role to the structure than to the growth. In terms of the C/N ratio, conifer species exhibited a significantly wider range of C/N ratios, varying from 16.1 to 68.6%, which can be associated to the fact that conifer had in general a higher carbon levels and a lower nitrogen values. In contrast, broadleaves species displayed a narrower range of C/N ratios, ranging from 18.1 to 40.8%. The C/N for biochar varies widely and can be much higher, ranging from C/N ratios ranging from approximately 10 to 250 (Phillips et al, 2022). However, when used as a soil amendment, it is suggested to adjust the C/N ratio of the materials being added to fall within the range of 20-30 (USDA, 2020), being the optimal for mineralization the value of 10. Furthermore, a general trend in the C/N contents of biochar, derived from various feedstock sources, was observed, regardless of the tree fraction, with the order being Pr > Pp > Ps, Ba, and Cs > Qr. This can be attributed to the change in the C and N total concentration in the biochar. High C/N values lead to rapid nitrogen immobilisation and a longer stay of the carbon in the soil which in turn avoids nitrogen leaching in the soil and its volatilisation (Prasad et al., 2016) Additionally, regardless of the specific tree species and pyrolysis duration, when examining the three tree fractions utilised (branches, litterfall, and green leaves), it was evident that the C in the resulting biochar varied in the following order: 68.6% for branches, 65.5% for litterfall, and 66.4% for green leaves. Likewise, the C/N ratio followed a similar trend with values of 45.01% for branches, 27.4% for litterfall, and 23.5% for green leaves. Notably, the branches exhibited a higher carbon content and C/N ratio compared to both litterfall and green leaves. The literature demonstrates a significant lack of information specifying the C, N and C/N ratio in each plant fraction, which is essential to deliver a high biochar value for specific purposes. Konaka et al. (2021) noted that biochar derived from tree branches fraction generally exhibits a higher C and C/N than the leaves tree fraction. This could be attributed to the harder tissues (lignin) found in the branches which are harder to decompose compared to the litterfall and green leaves (Li et al., 2023). The increase in C of branches-derived biochar is probably due to the increasing degree of carbonization (Keiluweit et al., 2010; Al-Wabel et al., 2013; Wei et al., 2017). However, the green leaves tree fraction exhibited a higher N% compared to the litterfall and branches. With values of 1.64% for branches, 2.58% for litterfall, and 2.97% for green leaves. this could be attributed to the higher arginine found in the leaves compared to the other tree fractions (Yang et al., 2020). The impact of processing time on the C, N level and C/N ratio was found to be significant in the case of C produced from the Qr litterfall, N produced from Pr branches and Pp litterfall, and C/N ratio from the Pr and Pp branches. The Qr litterfall C content significantly decreased 28 after 120 minutes compared with the C after 30 minutes of pyrolysis time. This could be attributed to that the organic matter in the feedstock has been decomposed at 300 ºC and 30 minutes. As a result, longer pyrolysis times are not required. However, in the case of the Qr litterfall, it may be got affected by the residence time because of attributed to greater losses of C-bound (Wang et al., 2019). The biochar produced from Pr branches after 120 minutes of pyrolysis decreased the N . However, in the biochar produced from the Pp litterfall, the N increased with increasing the pyrolysis time from 30 minutes to 60 minutes. This could be attributed to the decomposition level of the feedstock used for biochar production (Gaskin et al., 2008). He et al. (2023) has mentioned that excessive pyrolysis residence time could lead to a decrease in the nitrogen content in the biochar due to nitrogen volatility. The C/N ratio was influenced by changing the pyrolysis residence time in the case of the Pr branches, while the N level after 120 minutes was higher than after 60 minutes. In the biochar produced from Pp branches the N level demonstrated a decreasing trend with increasing the time from 30 minutes to 120 minutes. These results reflect the importance of processing time and feedstock origin in the potential use of biochar coming from different resources. 3.6 CONCLUSION The results of this study demonstrate that the C, N level and C/N ratio in biochar were influenced by several factors. These factors include the type of tree species, with three different conifer species (Pinus radiata D. Don, Pinus sylvestris L., Pinus pinaster Aiton) with a higher level of C and a lower level of N than the three broadleaves species evaluated (Betula alba L., Quercus robur L., Castanea sativa Mill.). The carbon sequestration potential of biochar derived from different species and fractions (branches, litterfall, and green leaves), and the duration of residence time during pyrolysis (30, 60, and 120 minutes) affected the biochar quality. The study found that optimizing feedstock selection is crucial for producing biochar suitable as soil amendment and carbon sequestration. It is recommended to use both conifers and broadleaves species to balance the C/N ratio to fulfil the requests from the biochar to be used, with more branches and conifers if the soil structure is the persecuted and more broadleaves and leaves if nitrogen is the aim for the biochar to be used as an amendment in the soil. Branches and litterfall from Ba, Ps, and Pr trees are recommended as carbon sources, while Ba and Qr branches, along with Ps green leaves and litterfall, can provide nitrogen. The capacity of the tree to translocate nutrient sources like nitrogen confers different quality to the litterfall. Maintaining biochar quality requires a pyrolysis time not exceeding 60 minutes. When using biochar as a soil amendment, it is advised to adjust the C/N ratio to fall within the 20-30 range. This can be achieved by using Cs, Qr, and Pp from litterfall, along with Pr and Ps green leaves. Using biochar from these sources improves nutrient retention, water-holding capacity, plant growth, microbiological activity, and carbon sequestration. Further studies should focus on studying and knowing the C, N level and C/N ratios of different species mix can impact soil characteristics. 29 CHAPTER 4 . EXPLORING THE POTENTIAL OF THE BIOCHAR DERIVED FROM SELECT CONIFERS AND BROADLEAVES SPECIES AS A MACRONUTRIENT AMENDMENT ______________________________________________________________________________ 30 4.1 ABSTRACT Macronutrients play a vital role in soil health, fertility, and supporting plant growth. Biochar holds great perinatal as an organic amendment enriched with carbon and various essential macro and micronutrients. However, the concentration of macronutrients in biochar depends on several factors such as the source material used, the duration of pyrolysis, and the temperature applied during the process. The objective of this study is to determine whether biochar derived from different tree species and fractions can effectively serve as a soil amendment by evaluating the overall concentration of key macronutrients, including phosphorus (P), potassium (K), magnesium (Mg), and calcium (Ca). To produce the biochar, we employed three conifer species (Pinus radiata D. Don, Pinus sylvestris L., and Pinus pinaster Aiton) and three broadleaves species (Betula alba L., Quercus robur L., and Castanea sativa Mill.). Each species provided three distinct fractions, namely branches, litterfall, and green leaves, which were used in the biochar production process. Pyrolysis was conducted at 300 oC for different durations of 30, 60, and 120 minutes. The findings revealed that the concentration of macronutrients in the broadleaves was consistently higher than in conifers, regardless of the tree faction. Additionally, among the three fractions, the green leaves tree fraction showed the highest increase in macronutrient levels, regardless of the tree species. This study provides further confirmation that the recommended feedstock has a significant impact on macronutrient enhancement. Keywords: charcoal, birch, pines, pyrolysis, organic amendment 4.2 INTRODUCTION In the northwestern region of Spain known as Galicia, approximately 60.5% of the entire area is covered by forests (MAPA, 2020). Within these forested areas, the available tree species include Pinus pinaster Aiton, Quercus robur L., and Eucalyptus globulus Labill, then less dominant Pinus radiata D. Don, Pinus sylvestris L., Betula alba L., and Castanea sativa Mill. (Anuario de Estadística Forestal, 2019). Biochar is a carbon-rich product obtained through pyrolysis, a process that converts organic biomass into char in the absence of oxygen (IBI, 2015). Its potential as a green technology has garnered attention from the European Union, particularly for its role in soil amendment, waste management, and environmental management, aligning with Europe's goal to become the first climate-neutral continent by 2050 (EC, 2010). Extensive research has highlighted the agricultural and environmental benefits of biochar. In this context, studies by Laird et al. (2010) and Lehmann et al. (2021) have emphasized the importance of biochar in enhancing water infiltration rates, water retention capacity, ion exchange processes, and nutrient availability in the soil. By improving nutrient availability and reducing the reliance on synthetic fertilizers in agriculture, biochar holds significant potential. The chemical and physical properties of biochar are influenced by the pyrolysis conditions and the type of feedstock used. Factors such as residence time are critical in determining biochar’s cost and chemical composition (Tomczyk et al., 2020; Leng et al., 2021). Biochar contains a wide range of micro and macronutrients that play a significant role in plant growth and physiology (Ippolito et al., 2015). For example, phosphorus is essential for energy transfer and nucleic acid synthesis (Malhotra et al., 2018), potassium is vital for osmoregulation and enzyme activation (Demidchik et al., 2002), calcium contributes to cell structure and signalling mechanisms (Hou et al., 2019), and magnesium is a central component of chlorophyll (Cakmak et al., 2010). Evaluating the concentration and availability of these macronutrients in biochar is 31 crucial for assessing its potential as a soil amendment. Moreover, the biochar of plant-based biomass can reduce the leaching potential of heavy metals unlike the biochar produced from sewage sludge which has the risk of heavy metals (Huang et al., 2017). The aim of this study is to investigate the potential of biochar derived from various tree species and fractions as an effective soil amendment. The study focuses on evaluating the overall concentration of essential macronutrients, including phosphorus (P), potassium (K), magnesium (Mg), and calcium (Ca), within the biochar produced from three conifer species (Pinus radiata D. Don, Pinus sylvestris L., and Pinus pinaster Aiton) and three broadleaves species (Betula alba L., Quercus robur L., and Castanea sativa Mill.). Each tree species offered distinct fractions, namely branches, litterfall, and green leaves, which were employed in the biochar production process. The pyrolysis decomposition process was carried out at a consistent temperature of 300°C for varying durations of 30, 60, and 120 minutes. By examining these factors, this study aims to gain insights into the effectiveness of biochar derived from different tree species and fractions as a valuable soil amendment. 4.3 MATERIALS AND METHODS In September 2021, feedstock for biochar production was obtained from various forest plantations in Galicia, located in the northwest region of Spain. The feedstock consisted of six tree species. Three of these species were conifers: Pinus radiata D. Don (Pr), Pinus sylvestris L. (PS), and Pinus pinaster Aiton (Pp). The remaining three species were broadleaves: Betula alba L. (Ba), Quercus robur L. (Qr), and Castanea sativa Mill (Cs). Three different tree fractions were collected: branches, litterfall, and green leaves. After feedstock samples collection, the tree fractions were manually separated in the laboratory and dried in an oven at 45°C for four days. The pyrolysis process to produce biochar was then conducted using a muffle furnace with ceramic crucibles containing the feedstock for durations of 30, 60, and 120 minutes at a temperature of 300°C as shown in Figure 4-1. The resulting biochar was ground using an agate mortar and pestle and stored in plastic-sealed bags (Gonzaga et al., 2017). To ensure the experiment's validity, three biochar replicates were produced for each type of feedstock. Figure 4-1. Schematic representation of the biochar production, made of different conifers and broadleaves, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. 32 In the laboratory, the total P concentration in the feedstock samples before the pyrolysis process and in the biochar produced was analysed using micro-Kjeldahl digestion (Castro et al., 1990) in the AA3 AUTOANALYZER using method G-189-97 (multitest) of Seal Analytical (2011). Moreover, the Total K, Ca, and Mg concentrations in the feedstock samples were analysed using a VARIAN 220FS spectrometer (Varian, Walnut Creek, CA, USA). However, the available P, K, Ca, and Mg were measured after extraction with Mehlich (1985) with the Varian 220 FS Spectrophotometer using atomic absorption. In this study, statistical analyses were performed using both the Least Significant Difference (LSD) and Analysis of Variance (ANOVA) tests, utilising SAS software. When the ANOVA test results were found to be significant, the LSD test was employed to distinguish between the means. 4.4 RESULTS Nutrients The total and Mehlich concentration of macronutrients (P, K, Ca and Mg) in the biochar was significantly higher in broadleaves compared with conifers tree species (p < 0.05) as shown in Figure 4-2. The total concentration of P, K, Ca, and Mg in broadleaves species was about 72, 87, 108, and 93% higher than in conifers, respectively. Similarly, the Mehlich concentration of P, K, Ca, and Mg in broadleaves species was about 73, 106, 259, and 246% higher than in conifers, respectively. 33 Figure 4-2. The total (left) and Mehlich (right) concentration of macronutrients (phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)) in the biochar made of different conifers and broadleaves, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. Lowercase letters indicate significant differences among tree species. 4.4.1 Phosphorous The total and Mehlich phosphorous (Mehlich-P) concentration in the branches and green leaves of broadleaves trees were significantly higher than in conifers (p < 0.05), as shown in Figure 4-3. The conifer biochar total P concentrations ranged for branches, litterfall, and green leaves fractions from 0.05 to 0.1 %, 0.07 to 0.15%, and 0.09 to 0.25 %, respectively. For broadleaves, the total P concentrations ranged from 0.19 to 0.23 %, 0.07 to 0.15 %, and 0.25 to 0.35 %, respectively. The biochar Mehlich phosphorus concentration varied within conifers branches, litterfall, and green leaves, ranging from 100.12 to 157.85, 158.82 to 258.38, and 226.2 to 333.3 mg kg-1, respectively, this means that around 20 to 24%, 17.3 to 22.8%, and 13.2 to 25.5% percentage of the total P is available, respectively. For broadleaves trees, the Mehlich phosphorus concentrations ranged from 60.58 to 287.67, 156.77 to 319.1 and 404.7 to 733.1 34 mg kg-1, respectively, meaning a percentage of P availability range of 3.2 to 22.8%, 21 to 22.8%, and 16 to 20.8%, for the branches, litterfall and green leaves Total P fraction. The biochar total phosphorous (P) concentration of conifers and broadleaves was around 97 and 47% (p < 0.05) higher in green leaves than in the branches fraction, respectively. The litterfall total P concentration was similar in green leaves and branches fractions in the case of conifers, while in broadleaves, the biochar made of litterfall total P concentration was significantly lower than the biochar made of green leaves and branches. Similarly, the biochar Mehlich phosphorus concentration of the conifers and broadleaves was around 119 and 84 % (p< 0.05) higher in the green leaves than in the branches tree fraction, respectively. The litterfall Mehlich phosphorus concentration was similar to green leaves Mehlich phosphorus concentration in the case of the conifers but significantly lower than the biochar made of green leaves for the broadleaves. Notably, the total P concentration and Mehlich-phosphorus in the green leaves and branches of broadleaves trees was significantly higher than that in conifers. However, there was no significant difference in the total or Mehlich phosphorus content of the litterfall between conifers and broadleaves. Figure 4-3. Total (left) concentration and Mehlich (right) phosphorus (Mehlich-P) in the biochar made of different conifers (up) and broadleaves (down) species branches, litterfall, and green leaves, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. Lowercase letters indicate significant differences among tree fractions, while uppercase letters indicate significant differences among tree fractions between different tree species (conifers and broadleaves). The impact of pyrolysis time on the total phosphorus (P) concentration in both conifer and broadleaves biochar was not significant (p < 0.05), being the conifer biochar total P concentration range after 30, 60, and 120 minutes of pyrolysis from 0.10 to 0.17%, 0.08 to 0.13%, and 0.08 to 0.15%, respectively (Figure 4.4). Broadleaves biochar showed total P concentration ranging from 0.18 to 0.25%, 0.15 to 0.27%, and 0.1 to 0.203%, respectively, over the same time intervals. Conversely, The Mehlich phosphorus in conifer biochar was 61% higher (p < 0.05) after 60 minutes than after 30 minutes. However, after 120 minutes, the Mehlich P was similar to 35 both 30 minutes and 60 minutes. Regarding the Mehlich phosphorus concentration in the biochar derived from broadleaves, there was a significant effect (p > 0.05) in response to variations in pyrolysis time. The Mehlich phosphorus concentrations in conifer biochar varied from 139.53 to 179.53, 223.35 to 297.68, and 150.9 to 272.3 mg kg-1 after 30, 60, and 120 minutes of pyrolysis, this indicates that the available percentage ranges from approximately 10.6 to 15%, 23 to 27.5%, and 18 to 20% of the total phosphorus concentration after 30, 60, and 120 minutes of pyrolysis time, respectively and shows an increase of availability of P when the processing time is enlarged. For broadleaves biochar, the Mehlich phosphorus concentrations ranged from 205.22 to 361.83, 47.78-589.88, and 369-492.9 mg kg-1 for the same times, which means that around 11.5 to 14.4 %, 3.1 to 21.9 %, and 24.6 to 37 % percentage of the total P concentration, respectively. Total P concentration in broadleaves biochar was significantly higher than in conifers after 30 and 60 minutes (p < 0.05), but no significant differences were observed after 120 minutes of pyrolysis time. Moreover, Mehlich phosphorus concentration was significantly greater in broadleaves biochar after 30 and 120 minutes compared to conifers, with no significant difference at 60 minutes, as shown in Figure 4-4. Figure 4-4. Total (left) concentration and Mehlich (right) phosphorus (Mehlich-P) in the biochar made of different conifers (up) and broadleaves (down) species after 30, 60, and 120 minutes of the pyrolysis time, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. Lowercase letters indicate significant variation among tree fractions, while uppercase letters indicate significant differences among pyrolysis times between the different tree species (conifers and broadleaves). 4.4.2 Potassium The concentration of total and Mehlich Potassium (Mehlich-K) in the branches, litterfall, and green leaves of broadleaves were significantly higher than in conifers (p < 0.05), as demonstrated in Figure 4-5. Notably, the total K and Mehlich potassium concentration in the broadleaves branches, litterfall, and green leaves was significantly higher than that in conifers. The conifer derived biochar total potassium (K) concentration was around 34% (p < 0.05) higher in litterfall than in the branches and green leaves tree fraction. However, for broadleaves, total potassium was around 121% higher (p < 0.05) in green leaves than in branches and 36 litterfall. The biochar total K concentrations varied within conifers branches, litterfall, and green leaves, ranging from 0.17 to 0.38%, 0.22 to 0.59%, and 0.92 to 1.28%, respectively. For broadleaves trees, the total K concentrations ranged from 0.81 to 1.23%, 0.51 to 1.29%, and 1.36 to 1.93%, respectively. Regarding Mehlich potassium, concentrations in conifers and broadleaves biochar were two and five times higher (p < 0.05) in green leaves than in branches and litterfall, respectively. The biochar’s Mehlich Potassium concentrations varied within conifers branches, litterfall, and green leaves, ranging from 1127.6 to 1574.2 mg kg-1, 988.7 to 2771.1 mg kg-1, and 4049 to 9354 mg kg-1, respectively. The available potassium content represents approximately 42 to 64%, 45 to 47%, and 43.9 to 73% of the total potassium concentration, respectively. For broadleaves trees, the Mehlich potassium concentrations ranged from 2706 to 7818.6 mg kg-1, 954.1 to 10501 mg kg-1, and 7936 to 18905 mg kg-1 for branches, litterfall and green leaves, respectively. Meaning, the available potassium content accounts for roughly 42 to 64%, 45 to 47%, and 43.9 to 73% of the total potassium concentration, respectively. Figure 4-5. Total (left) concentration and Mehlich (right) potassium (Mehlich-K) in the biochar made of different conifers (up) and broadleaves (down) species branches, litterfall, and green leaves, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. Lowercase letters indicate significant differences among tree fractions, while uppercase letters indicate significant differences among tree fractions between the different tree species (conifers and broadleaves). The concentration of both total and Mehlich potassium (Mehlich-K) in the biochar produced from broadleaves trees after 30, 60, and 120 minutes was significantly higher compared to that in conifers (p < 0.05), as shown in Figure 4-6. Both total K and Mehlich Potassium concentrations were significantly greater in broadleaves biochar after 30, 60, and 120 minutes compared to conifers. For conifer biochar, the total K concentrations varied after 30, 60, and 120 minutes of pyrolysis time, ranging from 0.36 to 0.50%, 0.58 to 0.93%, and 0.63 to 0.76%, respectively. In broadleaves biochar, total K concentrations ranged from 1.14 to 1.28%, 1.08 to 1.39%, and 0.72 to 1.26% over the same time intervals. The biochar's total potassium (K) concentration in conifers was approximately 64% higher (p < 0.05) after both 120 and 60 minutes compared to 30 minutes of pyrolysis time. 43 that the sewage sludge biochar could serve as a more effective amendment for P and Ca, however, the Pinus sylvestris L., Pinus taeda L., cotton straw, and rice husk biochar contained a higher K and Mg (Hossain et al., 2011; Yue et al., 2014; Zin et al., 2021). However, it is important to note that sewage sludge biochar carries a higher potential risk of heavy metal contamination and has a higher cost (Gherghel et al., 2019). On the other hand, compost can exhibit a higher or lower macro depending on the input feedstock, for instance, the compost produced from grass and hardwood residues, grass and coniferous residues, a mixture of food and park-wood wastes, contained lower macronutrients compared to this study biochar. However, the compost produced from herbal plant residues, and sugar cane plant residues contained higher macronutrients compared to this study biochar. Moreover, the compost can be added as a Ca amendment (Prasad et al., 2013; Khater et al., 2015; Mladenov et al., 2018). Liu et al. (2012), suggested that mixing the biochar with the compost can enhance the soil fertility and chemical properties. 4.6 CONCLUSION This study demonstrates that mostly the type of feedstock and pyrolysis time significantly influence the macronutrient content of biochar. Biochar derived from broadleaves, particularly from green leaves, shows higher total and Mehlich available concentrations of phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) compared to conifer-derived biochar, making the former more suitable for enhancing soil fertility. Increasing pyrolysis time generally increases these macronutrient concentrations, except for calcium. Given the higher nutrient content and better nutrient uptake capacity of broadleaves biochar, it is recommended for applications requiring significant soil nutrient enhancement. Conifer-derived biochar can be used where lower nutrient release rate is acceptable. Combining biochar with compost or other macronutrient resource rich (ie. Manure) could further improve soil fertility and chemical properties. Thus, for sustainable soil management, broadleaves derived biochar, especially from green leaves, is the preferred choice. 44 CHAPTER 5 . EXPLORING THE POTENTIAL OF THE BIOCHAR DERIVED FROM SELECT CONIFERS AND BROADLEAVES SPECIES AS A MICRONUTRIENT AMENDMENT __________________________________________________________________________________ 45 5.1 ABSTRACT Biochar is a carbon-rich material with significant potential as an organic amendment, enriched with essential macro and micronutrients. However, the concentration of these micronutrients in biochar varies based on factors such as the source material, pyrolysis duration, and temperature. Micronutrients are crucial for soil health, fertility, and plant growth. This study aims to evaluate the effectiveness of biochar derived from different tree species and fractions as a soil amendment by analysing the concentrations of key micronutrients, specifically Fe, Cu, Zn, and Mn. To produce the biochar, we employed three conifer species (Pinus radiata D. Don, Pinus sylvestris L., and Pinus pinaster Aiton) and three broadleaves species (Betula alba L., Quercus robur L., and Castanea sativa Mill.). Each species provided three distinct fractions, namely branches, litterfall, and green leaves, which were used in the biochar production process. Pyrolysis was conducted at 300 oC for different durations of 30, 60, and 120 minutes. The findings revealed that feedstock type and pyrolysis time significantly influence biochar's micronutrient content. Broadleaves derived biochar generally has higher manganese and copper levels, while conifers derived biochar was richer in iron and zinc. Litterfall showed the highest micronutrient concentrations among tree fractions. Longer pyrolysis times typically increase micronutrient levels. For enhancing soil micronutrients, broadleaves derived biochar from litterfall was preferable, whereas conifers derived biochar is better for higher iron and zinc. Keywords: charcoal, birch, pines, pyrolysis, heavy metals, composition 5.2 INTRODUCTION Biochar is a material with a high content of carbon, which is generated by the pyrolysis process the composition of the biochar is changeable according to the materials added and the temperature that will be applied throughout the burning process, the preferable temperature that should be sited while biochar is between 300-1000 ºC (Santos et al., 2020; Cao et al., 2022; Do et al., 2023). The scaling up of the temperature during the process seems to scale down the pH, magnesium and potassium content, carbon, and cation exchange capacity, and increase specific surface area (Kisiki et al., 2015; Seow et al., 2022). It was reported that the biochar from the animal residues (manure and other products) was more valuable and had higher nutrients than the wood and plant residues biochar, but it has the risk of heavy metals (Ndirangu et al., 2019; Su et al., 2022). Biochar is effective in increasing the availability of iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn) in soil (Hailegnaw et al., 2020). Iron is crucial for chlorophyll synthesis and is vital for photosynthesis and respiration processes, directly impacting plant health and productivity (Rout et al., 2015). Copper is important for photosynthesis, respiration, and the formation of lignin in plant cell walls, which contributes to structural strength and disease resistance (Printz et al., 2016). Zinc plays a significant role in enzyme activation, protein synthesis, and growth hormone production, which are essential for plant growth and development (Mousavi et al., 2012). Manganese is involved in photosynthesis, nitrogen assimilation, and the synthesis of some enzymes, which are necessary for plant metabolism. The concentration of zinc can vary greatly depending on the type of biochar used (Jia et al., 2019). Manganese concentrations in soil tend to rise after two to three months of biochar application, likely due to its association with organic and inorganic forms in plant-based material (Mitzia et al., 2023). Moreover, increased application rates have been shown to boost the concentrations of Zn, Cu, Mn, and Fe (Nemgay et al., 2010; Liu et al., 2016). Additionally, biochar-treated soils exhibit enhanced microbial activity, which is crucial for nutrient cycling and organic matter decomposition (Palansooriya et al., 2019). The application of biochar can 46 lead to increased dry biomass and promote the growth of grass crops, thereby enhancing soil fertility and agricultural productivity (Alkharabsheh et al., 2021). The goal of this research is to provide a comprehensive understanding of how feedstock selection and pyrolysis time influence the quality of biochar, particularly in terms of its micronutrient (Fe, Cu, Zn, Mn) content. Such insights are crucial for developing targeted biochar applications that enhance soil fertility, promote sustainable agriculture, and contribute to environmental conservation. 5.3 MATERIALS AND METHODS In September 2021, biochar feedstock was sourced from various forest plantations in Galicia, NW Spain. The feedstock included six tree species: three conifers (Pinus radiata D. Don (Pr), Pinus sylvestris L. (PS), and Pinus pinaster Aiton (Pp)) and three broadleaves (Betula alba L. (Ba), Quercus robur L. (Qr), and Castanea sativa Mill (Cs)). Three different tree fractions were collected: branches, litterfall, and green leaves. After collection, the tree fractions were manually separated in the laboratory and dried in an oven at 45°C for four days. Biochar was produced through pyrolysis in a muffle furnace with ceramic crucibles containing the feedstock for 30, 60, and 120 minutes at 300°C. The resulting biochar was ground with an agate mortar and pestle and stored in plastic-sealed bags (Gonzaga et al., 2017). To ensure validity, three biochar replicates were produced for each feedstock type. In the laboratory, total Fe, Cu, Zn, and Mn concentrations in the feedstock samples were analysed using a VARIAN 220FS spectrometer (Varian, Walnut Creek, CA, USA). The available Fe, Cu, Zn, and Mn were measured after extraction with Mehlich (1985) using the Varian 220 FS Spectrophotometer with atomic absorption. Statistical analyses were performed using Least Significant Difference (LSD) and Analysis of Variance (ANOVA) tests with SAS software. When ANOVA results were significant, the LSD test was used to differentiate the means. 5.4 RESULTS Nutrients Figure 5.1 illustrates the significant differences (p<0.05) in micronutrients (Fe, Cu, Zn, and Mn) total and Mehlich concentrations between the biochar derived from broadleaves and conifers. The total concentration of Fe and Zn in the broadleaves species was about 72 and 85% higher than in conifers, respectively. However, conifers had about two times more total Mn than broadleaves. The total Cu levels showed no significant differences (p>0.05) between broadleaves and conifers. Opposite to what happened with the total Zinc, the Mehlich zinc concentration in the broadleaves was approximately 27% higher than in the conifers. Similarly, the Mehlich manganese concentration in the broadleaves was three times higher than in the conifers. In contrast, the Mehlich iron and copper concentrations showed no significant difference (p>0.05) between broadleaves and conifers. 47 Figure 5-1. The total (left) and Mehlich (right) concentration of macronutrients (iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn)) in the biochar made of different conifers and broadleaves, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill). Lowercase letters indicate significant differences among tree species. 5.4.1 Iron The concentration of the biochar total iron (Fe) (Figure 5.2) varied within conifers' branches, litterfall, and green leaves, ranging from 330 to 630 mg kg-1, 710 to 1430 mg kg-1, and 210 to 450 mg kg-1, respectively. For broadleaves, total Fe concentrations in the branches, litterfall, and green leaves ranged from 100 to 260 mg kg-1, 400 to 610 mg kg-1, and 250 to 400 mg kg-1, respectively. The concentration of total Fe in conifers' branches and litterfall was significantly higher than in broadleaves (p<0.05). Biochar's total Fe concentration was about one (p<0.05) time higher in conifers' litterfall compared to branches and green leaves. Similarly for broadleaves, the total Fe concentration in litterfall was approximately two and one (p< 0.05) times higher compared to branches and green leaves, respectively. Moreover, the total Fe in green leaves of broadleaves exceeded that in branches by 99%. 48 Mehlich iron concentrations (Figure 5.2) varied within conifers' branches, litterfall, and green leaves, ranging from 14.4 to 17.3 mg kg-1, 16.3 to 24.4 mg kg-1, and 15.2 to 26.9 mg kg- 1, respectively. Moreover, for the broadleaves branches, litterfall, and green leaves, it ranged from 12.2 to 16.3 mg kg-1, 16.3 to 22.1 mg kg-1, and 19.2 to 30.4 mg kg-1, respectively. Meaning, that the percentage of the available Fe in the conifers' branches, litterfall, and green leaves ranged from 4.36 to 5.24%, 1.14 to 3.44%, and 5.24 to 12.81% of the total Fe concentration, respectively. Moreover, the available percentage of Fe available in broadleaves branches, litterfall, and green leaves ranged from12.2 to 16.3%, 2.67 to 5.53%, and 4.8 to 12.16% of the total Fe concentration, respectively. Mehlich iron in conifers’ green leaves and litterfall was around 40.8% (p<0.05) higher than in branches, while in broadleaves, it was 58.4 and 29.2%(p<0.05) higher in green leaves than in branches and litterfall, following therefore the same tendency in both types of species, respectively. Additionally, broadleaves litterfall was approximately 31% (p<0.05) higher compared to branches tree fractions. Figure 5-2. Total (left) concentration and Mehlich (right) iron (Mehlich-Fe) in the biochar made of different conifers (up) and broadleaves (down) species branches, litterfall, and green leaves., while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. Lowercase letters indicate significant differences among tree fractions, while uppercase letters indicate significant differences among tree fractions between the different tree species (conifers and broadleaves). Biochar total Fe within conifers (Figure 5.3) after 30, 60, and 120 minutes ranged from 430 to 900 mg kg-1, 400 to 880 mg kg-1, and 380 to 730 mg kg-1, respectively, while for broadleaves, it ranged from 260 to 400 mg kg-1, 160 to 450 mg kg-1, and 310 to 500 mg kg-1, respectively. The total Fe concentration after 30 and 60 minutes in biochar derived from conifers fraction was significantly higher compared to those in broadleaves (p < 0.05). The biochar total Fe in conifers was around two and three (p<0.05) times higher after 60 minutes compared to 30 and 120 minutes, respectively, while no significant differences were observed in broadleaves among the different pyrolysis times. Mehlich iron (Figure 5.3) in broadleaves was 24% (p<0.05) higher after 120 minutes than after 30 minutes, while after 60 minutes, it showed a similar concentration to both pyrolysis times. Within conifers after 30, 60, and 120 minutes of pyrolysis time, Mehlich iron concentrations ranged from 12.6 to 21.6 mg kg-1, 15.6 to 25.2 mg kg-1, and 17.8 to 22.9 mg kg- 1, respectively, indicating available iron percentages ranging from 1.4 to 5.02%, 1.77 to 6.3%, 49 and 3.08 to 6.03% of the total Fe concentration, respectively. For broadleaves after 30, 60, and 120 minutes of pyrolysis time, Mehlich iron ranged from 16.1 to 19.3 mg kg-1, 17.3 to 24.1 mg kg-1, and 19 to 25.3 mg kg-1, respectively, constituting approximately 6.19 to 7.42%, 5.56 to 15.06%, and 5.08 to 8.16% of the total Fe content after 30, 60, and 120 minutes of the pyrolysis time, respectively. Figure 5-3. Total (left) concentration and Mehlich (right) iron (Mehlich-Fe) in the biochar made of different conifers (up) and broadleaves (down) species after 30, 60, and 120 minutes of the pyrolysis time, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. Lowercase letters indicate significant variation among tree fractions, while uppercase letters indicate significant differences among pyrolysis times between the different tree species (conifers and broadleaves). 5.4.2 Copper Biochar total copper (Cu) (Figure 5.4) varied within conifers' branches, litterfall, and green leaves from 4.60 to 8.05 mg kg-1, 3.43 to 27.6 mg kg-1, and 5.24 to 7 mg kg-1, respectively. For broadleaves branches, litterfall, and green leaves, the total Cu concentration ranged from 6.05 to 9.68 mg kg-1, 5.36 to 7.71 mg kg-1, and 10.03 to 16.79 mg kg-1, respectively. In conifers' litterfall, total Cu levels were significantly (p<0.05) higher than in broadleaves. Conversely, broadleaves green leaves had significantly greater (p<0.05) total Cu than conifers. The biochar total Cu concentration in conifers' litterfall was around 2.5 (p<0.05) times higher than in branches and green leaves, while total Cu in green leaves of broadleaves was 65% (p<0.05) higher than both in branches and litterfall. For conifers' branches, litterfall, and green leaves, the Mehlich copper (Figure 5.4) ranged from 2.44 to 2.78 mg kg-1, 2.34 to 2.99 mg kg-1, and 2.54 to 2.95 mg kg-1, respectively. Meaning that the available copper concentration ranged from 53.0 to 60.4%, 8.48 to 68.5%, and 36.2 to 56.3% of the total Cu concentration in the conifers' branches, litterfall, and green leaves, respectively. In the broadleaves branches, litterfall, and green leaves, the Mehlich copper varied within the range from 2.67 to 3.12 mg kg-1, 2.57 to 2.86 mg kg-1, and 2.79 to 3.03 mg kg-1 for the same fractions, representing around 27.6 to 51.6%, 33.3 to 53.4%, and 16.6 to 27.9% of the total Cu concentration, respectively. In conifers litterfall, the total Cu concentration significantly (p<0.05) exceeded that in broadleaves. The biochar Mehlich copper in conifers and broadleaves showed no significant difference (p>0.05) among tree fractions. 50 Figure 5-4. Total (left) concentration and Mehlich (right) copper (Mehlich-Cu) in the biochar made of different conifers (up) and broadleaves (down) species branches, litterfall, and green leaves. Lowercase letters indicate significant differences among tree fractions, while uppercase letters indicate significant differences between the two species of each tree fraction. The biochar total copper (Cu) (Figure 5.5) concentrations after 30, 60, and 120 minutes of pyrolysis time ranged from 5.66 to 10.38 mg kg-1, 4 to 12.17 mg kg-1, and 4.30 to 16.9 mg kg-1 in conifers, respectively, and from 5.66 to 7.26 mg kg-1, 6.52 to 10 mg kg-1, and 11.61 to 18.19 mg kg-1 in broadleaves, respectively. The total Cu concentration in the conifers exceeded that in the broadleaves after 60 minutes. Conversely, after 120 minutes, the total Cu concentration in the conifers decreased compared to the broadleaves (p<0.05). The biochar total Cu concentration of the conifers and broadleaves increased by around 2.5 (p<0.05) times after 120 minutes, compared to after 30 and 60 minutes. Mehlich copper (Figure 5.5) concentrations after 30, 60, and 120 minutes ranged from 2.32 to 2.50 mg kg-1, 2.59 to 2.96 mg kg-1, and 2.64 to 3.22 mg kg-1 in conifers, and from 2.29 to 2.65 mg kg-1, 2.74 to 3.08 mg kg-1, and 3 to 3.29 mg kg-1 in broadleaves, respectively. This means that the available copper percentage of conifers after 30, 60, and 120 minutes ranged from 40.99 to 44.17%, 21.28 to 64.21%, 15.62 to 74.88%, respectively, and 31.54 to 46.82%, 27.4 to 47.24%, 16.5 to 25.34% for the broadleaves, of the total Cu concentration, respectively. After 120 minutes, the Mehlich copper concentration in conifers increased by around 22.6% (P<0.05) compared to that after 30 minutes, while after 60 minutes, the Mehlich copper concentration was similar to the concentrations detected after 30 and 120 minutes. In the broadleaves, the Mehlich copper increased by about 56% (P<0.05) after 60 and 120 minutes than after 30 minutes. 51 Figure 5-5. Total (left) concentration and Mehlich (right) copper (Mehlich-Cu) in the biochar made of different conifers (up) and broadleaves (down) species after 30, 60, and 120 minutes of the pyrolysis time, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. Lowercase letters indicate significant variation among tree fractions, while uppercase letters indicate significant differences among pyrolysis times between the different tree species (conifers and broadleaves). 5.4.3 Zinc Biochar total zinc (Zn) (Figure 5.6) varied within conifers branches, litterfall, and green leaves, ranging from 57.88 to 90.05 mg kg-1, 57.46 to 141.52 mg kg-1, and 51.89 to 76.68 mg kg-1, respectively. For broadleaves the total Zn varied within branches, litterfall, and green leaves from 43.59 to 103.27 mg kg-1, 22.14 to 35.25 mg kg-1, and 27.81 to 55.43 mg kg-1, respectively. The total Zn concentration in conifers' litterfall and green leaves was significantly higher than that in broadleaves (p<0.05). The biochar total Zn in conifers' litterfall was approximately 41% (p<0.05) higher than in branches and green leaves, while in broadleaves branches, the total Zn concentration exceeded (p<0.05) that in litterfall and green leaves by around 71%. Mehlich zinc (Zn) (Figure 5.6) in conifers' branches, litterfall, and green leaves, ranged from 5.48 to 7.97 mg kg-1, 6.81 to 10.33 mg kg-1, and 4.87 to 8.06 mg kg-1, respectively, while the available zinc percentage is about 9.47 to 9.08%, 11.85 to 7.30%, and 9.38 to 10.51% of the total Zn concentration, respectively. Biochar Mehlich zinc varied within broadleaves branches, litterfall, and green leaves, ranging from 5.68 to 13.43 mg kg-1, 6.64 to 8.61 mg kg-1, and 4.84 to 13.29 mg kg-1, respectively, this indicated that the available zinc concentration is about 9.47 to 9.08%, 11.85 to 7.30%, 9.38 to 10.51% of the total Zn level. In broadleaves branches, litterfall, and green leaves Mehlich zinc was around 40% (p<0.05) higher in branches than in litterfall, with the green leaves containing a similar Mehlich zinc concentration to the branches and litterfall. 52 Figure 5-6. Total (left) concentration and Mehlich (right) zinc (Mehlich-Zn) in the biochar made of different conifers (up) and broadleaves (down) species branches, litterfall, and green leaves, while the conifers (PR: Pinus radiata D. Don, PS: Pinus sylvestris L., PP: Pinus pinaster Aiton) and broadleaves (BA: Betula alba L., QR: Quercus robur L., CS: Castanea sativa Mill. Lowercase letters indicate significant differences among tree fractions, while uppercase letters indicate significant differences among tree fractions between the different tree species (conifers and broadleaves). Biochar total zinc (Zn) (Figure 5.7) varied within conifers after 30, 60, and 120 minutes, ranging from 49.23 to 62.33 mg kg-1, 71.15 to 120.63 mg kg-1, and 71.65 to 109.78 mg kg-1, respectively. For broadleaves, total Zn after 30, 60, and 120 minutes were ranged from 33.33 to 7.98 mg kg-1, 21.01 to 48.25 mg kg-1, and 35.68 to 80.2 mg kg-1, respectively. The conifers total Zn concentration after 60 minutes significantly exceeded that in broadleaves (p<0.05). The biochar total Zn in conifers was around 36% (p<0.05) higher after 30 minutes compared to 120 minutes, with similar concentrations after 60 minutes after both pyrolysis times 30 and 120 minutes. Conversely, in broadleaves, no significant difference in total Zn concentration was observed among pyrolysis times (p>0.05). Mehlich zinc (Zn) (Figure 5.7) varied within conifers after 30, 60, and 120 minutes, ranging from 5.33 to 7.25 mg kg-1, 7.46 to 9.56 mg kg-1, and 4.69 to 9.39 mg kg-1, respectively. Meaning that the percentage of the available zinc after 30, 60, and 120 minutes ranged from 5.33 to 11.64%, 6.19 to 7.93%, and 6.67 to 8.56% from the total Zn, respectively. In broadleaves, Mehlich zinc after 30, 60, and 120 minutes ranged from 5.58 to 7.98 mg kg-1, 9.38 to 11.71 mg kg-1, and 9.79 to 10.58 mg kg-1, respectively. Indicating that the available zinc percentage after 30, 60, and 120 minutes was around from 5.58% to 18.36, 42.34 to 33.4%, and 35.21 to 19.1% from the total Zn concentration, respectively. While conifers showed no significant difference in Mehlich zinc concentration across pyrolysis time changes. However, biochar from broadleaves exhibited approximately 65% (p<0.05) higher levels after 60 and 120 minutes compared to 30 minutes. 59 6.1 ABSTRACT Grasslands, which contribute approximately 30% of the world's soil organic carbon reserve, play a vital role, both environmentally and agriculturally, in achieving "net-zero" carbon emissions by 2050. The use of biochar, a carbon-rich material produced from organic matter, as a soil amendment can help achieve the “net-zero” carbon aim and enhance soil fertility and therefore pasture production. The objective of this study was to assess the effect of different doses of biochar produced from low-quality grass on pasture production in an experiment established in Galicia (NW Spain). A pot experiment was placed in a greenhouse using two types of substrates (sand and silt loam soil). On both substrates, two doses of biochar were applied (50 and 100 kg N ha-1) with and without mineral fertiliser (8:24:16) at a rate of 500 kg ha-1. Moreover, two control treatments were established on both substrates (mineral fertilisation without biochar and substrate alone). All pots were sown with 25 kg ha-1 of Lolium perenne L. The results showed a positive effect of combining mineral fertiliser with biochar, regardless of the biochar dose, on pasture dry weight. The biochar amendment to the sand substrate did not affect the plant height. However, in silt loam soil substrate the the results indicated that combining mineral fertiliser with biochar positively impacted plant height, irrespective of the biochar dosage. Keywords: grassland, charcoal, mineral, pot experiment 6.2 INTRODUCTION In Galicia (NW Spain), soils are characterised by their acidity, which implies a high percentage of Al saturation in the soil exchange complex, as well as a low availability of cations (Palleiro et al., 2018). This acidic nature often poses a challenge for agricultural productivity, necessitating innovative soil management practices. the addition of biochar to acidic soils in Galicia is expected to mitigate the negative impacts of high aluminium saturation and low cation availability, thus improving plant growth parameters (Villagra-Mendoza et al., 2021). In Europe, organisations such as the EBC and IBI promote the use of biochar as a soil amendment to improve soil characteristics and enhance plant production (IBI, 2015; EBC, 2023). Biochar, a carbon-rich material produced from organic matter through pyrolysis, has the capacity to absorb and store carbon, thus contributing to the “net-zero” carbon aim by reducing carbon footprints (Minkosse, 2023). However, biochar properties vary depending on the feedstock and pyrolysis conditions, which can affect its efficacy as a soil amendment (Břendová et al., 2012). This variability necessitates careful consideration of biochar's characteristics and application rates to optimise its benefits. Many researchers in the literature have shown that biochar can enhance soil microbial activity, water retention, and nutrient availability, leading to improved crop yields (Tomczyk et al., 2020; Leng et al., 2021). Specifically, biochar amendments have been observed to increase the dry weight of pasture plants, indicating better overall health and higher productivity (He et al., 2021). Biochar can enhance soil structure and nutrient availability, leading to increased plant biomass and growth (Olmo et al., 2016). Additionally, the height of the plants is positively influenced by biochar, particularly when combined with mineral fertilisers (Geo et al., 2021). Biochar's ability to sequester carbon also makes it a promising tool in climate change mitigation strategies (McDonald et al., 2019). However, the interaction between biochar and mineral fertilisers significantly impacts nutrient dynamics and plant growth, highlighting the need for tailored application strategies (Van Zwieten et al., 2010). The objective of this study was to assess the effect of different doses of biochar (50 kg and 100 kg N ha-1) produced from low-quality grass on pasture production in a greenhouse 60 experiment established in Galicia. By understanding these interactions, the potential of biochar to contribute to sustainable agricultural practices and achieve carbon sequestration goals can be better harnessed. 6.3 MATERIALS AND METHODS In May 2023, a greenhouse experiment was established at the High Polytechnic School of the University of Santiago de Compostela (Lugo, NW Spain) using 36 pots (12 cm in height × 12 cm in width) which were filled with two different types of substrates. Half of the pots were filled with washed sand and the remaining pots were filled with silt loam soil. On both substrates, two doses of biochar were applied (50 kg and 100 kg N ha-1) with and without mineral fertiliser (8:24:16) at a rate of 500 kg ha-1. Moreover, two control treatments were established on both substrates (mineral fertilisation without biochar and substrate alone). After the substrate preparation, all pots were sown with 25 kg ha-1 of Lolium perenne L. Therefore, the experimental design was a random block design with 12 treatments and three repetitions. In this experiment, the biochar used as an amendment was produced from low-quality grass and their chemical properties can be observed in Table 6.1. In all pots, this biochar was mixed with the substrates. Table 6-1 Chemical properties of the biochar applied in this experiment. pH (H2O) N (%) P (%) K (%) Na (%) Ca (%) Mg (%) Fe (%) Cu (mg kg -1 ) Zn (mg kg -1 ) 10.33 0.45 0.29 1.67 0.44 1.26 0.6 0.1 23.1 100.04 In July and November 2023, pasture production in each pot, consisting of Lolium perenne L. and spontaneous species, was assessed by harvesting the total plant biomass. The harvested samples were then dried in the laboratory at 60ºC for 72 hours to determine their dry matter content. Plant height measurements were taken using a measuring ruler during the same months. The dry weight data were analysed using ANOVA, followed by the LSD test for pairwise comparisons when the ANOVA indicated significant differences. All statistical analyses were performed at a 5% significance level using the SAS software package (2001). For plant height, the data were analysed using ANOVA and the Tukey test in SPSS to determine significant differences among the means. 6.4 RESULTS The total P leached into the water from the sand substrate was approximately four times higher (p<0.05) when Min was used, either alone or in combination with biochar, as illustrated in Figure 6-1. For the soil substrate treatments, the P concentration leached in the Min, Bio100, and Bio100+Min treatments was about 50% higher (p<0.05) than in the other treatments, except for Bio50+Min, which had a similar P leaching concentration to all other treatments. In the sand substrate, the nitrate leached in the Min treatment was approximately twice as high (p<0.05) compared to all other treatments. For the soil substrate, nitrate leached in the Min treatment was around 40% (p<0.05) higher compared to the Bio100 and Bio100+Min treatments. Furthermore, nitrate leaching in the control, Bio50, and Bio50+Min treatments were similar to both the Min and the Bio100 and Bio100+Min treatments, as demonstrated in Figure 6-1. 61 Figure 6-1. The total phosphorus (P) (mg P l-1) (left) and nitrate (mg N-nitrate l-1) (right) concentration in the leached water under various treatments from May to November 2023. Min: mineral fertilisation, Bio50: 50 kg N ha-1 of biochar and Bio100: 100 kg N ha-1 of biochar. Different letters indicate significant differences between treatments in each type of substrate (sand and silt loam soil). The leaching of phosphorus (P) into the water was significantly affected by the treatments applied (p<0.05), as shown in Figure 6-2. In the sand substrate, the leached phosphorus (P) in the water in May was about fifteen times higher (p<0.05) in the Min, Bio50+Min, and Bio100+Min treatments compared to the control, Bio50, and Bio100. In June, the P concentration in the leached water was approximately 80% higher (p<0.05) in the Bio100 and Bio100+Min treatments compared to all other treatments, except for Bio50+Min, which had a similar P concentration to Bio100, Bio100+Min, Min, and Bio50, but was 89% higher than the control. In July, there was no significant difference in the P leached across the different treatments (p>0.05). In August, the P leached was about three times higher (p<0.05) in the Bio100+Min treatment compared to the control and Min, while Bio50 and Bio100 had similar levels to Bio100+Min and the control. In September, Bio100+Min and Bio100 showed approximately two and a half times higher P leaching (p<0.05) compared to the control, Min, and Bio50, while Bio50+Min was similar to all treatments. In October, the leached P was about one to two times higher (p<0.05) in Bio100+Min compared to all other treatments except for Min. Additionally, Bio100+Min was about 50% higher (p<0.05) compared to Bio50+Min. In November, Bio100+Min showed around two times higher P leaching (p<0.05) compared to the control, Min, and Bio50. Bio50+Min had approximately 70% higher P leaching (p<0.05) compared to Min. In the soil substrate, there was no significant difference in P leaching concentration among the different treatments in May. In June, the P leaching in Bio100+Min was approximately three to four times higher (p<0.05) compared to all other treatments except for Bio100, which was similar to Bio100+Min and Bio50. The P leaching in Bio100 was around three times higher (p<0.05) compared to Min and the control. In July, the P leaching was about twice as high (p<0.05) in the Bio100+Min and Bio100 treatments compared to the control and Min, while the P leaching in Bio50 and Bio50+Min was similar to that in all other treatments. In August, the P leaching in Bio100+Min was about one to five times higher (p<0.05) compared to all other treatments. In September, P leaching was around twice as high (p<0.05) in Bio100+Min and Bio100 compared to all other treatments. In October, P leaching in the Min treatment was higher than in the control, Bio100+Min, Bio50+Min, and Bio50 and Bio100, by one, three, four, and four times, respectively. Moreover, the P leaching in Bio100+Min was about twice as high (p<0.05) compared to Bio50 and Bio100 treatments. 62 Figure 6-2. The phosphorus (P) (mg P l-1) leaching (mg P l⁻¹) from June to October (left) and significant differences each month from May to November (right) for concentrations in leached water under various treatments from May to November 2023. Treatments include mineral fertilization (Min), 50 kg N ha⁻¹ of biochar (Bio50), and 100 kg N ha⁻¹ of biochar (Bio100). Different small letters indicate significant differences between treatments in each type of substrate: sand (up) and silt loam soil (down) 63 The leaching of nitrate into the water was significantly affected by the treatments applied (p<0.05), as shown in Figure 6-3. In the sand substrate, on 22/05/2023, the Min treatments showed four times higher nitrate leaching compared to all other treatments, except for Bio50+Min and Bio100+Min. On 25/05/2023, the Min treatments exhibited eleven times higher nitrate leaching compared to all other treatments, except for Bio50+Min. By 29/05/2023, nitrate leaching in the Min treatment was around four times higher (p<0.05) than in all other treatments. On 05/06/2023, the leached nitrate in the Bio100+Min treatment was higher than in all other treatments, except for the control, while the nitrate concentration in Min, Bio50, Bio100, and Bio50+Min was almost zero. There was no significant difference (p>0.05) in nitrate leaching among all treatments on 18/05/2023, 01/06/2023, 08/06/2023, and 12/06/2023. It is significant to note that the measurement of nitrate leaching stopped after 12/06/2023 since the values were zero or close to zero. In the soil substrate, there was no significant difference in nitrate leaching among all treatments across the different measuring dates, except for 29/05/2023. On this date, the Min treatment had significantly higher nitrate leaching compared to all other treatments, except for the control. 64 Figure 6-1. The nitrate (mg N-nitrate l-1) from 18/05 to 08/06/2023 (left) and significant differences each month from 18/05 to 18/06/2023 (right) for concentrations in leached water under various treatments from 18/05 to 18/06/2023. Treatments include mineral fertilization (Min), 50 kg N ha⁻¹ of biochar (Bio50), and 100 kg N ha⁻¹ of biochar (Bio100). Different small letters indicate significant differences between treatments in each type of substrate: sand (up) and silt loam soil (down 65 Figure 6-4 shows that in July 2023, in the sand substrate, the Min+Bio50 and Min+Bio100 treatments resulted in about three times (p<0.05) higher Lolium perenne L. dry matter (DM) compared to all other treatments. In the soil substrate, Min+Bio50 and Min+Bio100 also showed about 2.5 times (p<0.05) higher DM compared to the Bio50, Bio100, and control treatments (sand alone). Additionally, the Min treatment resulted in two times (p<0.05) higher DM compared to Bio50, Bio100, and the control treatment (sand). Regarding to the spontaneous species, Amaranthus retroflexus L. DM was about 32% (p<0.05) higher in Min+Bio100 compared to Min and Min+Bio50, and about two times (p<0.05) higher than in Bio100, bio50, and control treatment (soil alone). Adding Min to the soil resulted in about 88% (p<0.05) higher Amaranthus retroflexus L. DM compared to the control treatment. In November 2023, the different treatments did not significantly (p>0.05) affect Lolium perenne L. DM in the sand substrate. However, in the soil substrate, Min+Bio100 showed around three times (p<0.05) greater Lolium perenne L. DM compared to all other treatments except for Min+Bio50 and Bio50, which had higher DM compared to the control treatment (sand alone). Moreover, the spontaneous species in both substrates did not exhibit any significant differences (p>0.05). Figure 6-4. Production of Lolium perenne L. and weed species (Amaranthus retroflexus L., Trifolium repens L. and Cerastium glomeratum Thuill.) (Mg dry matter (DM) ha-1) under the different treatments established in July and November 2023. Min: mineral fertilisation, Bio50: 50 kg N ha-1 of biochar and Bio100: 100 kg N ha-1 of biochar. Different letters indicate significant differences between treatments in each type of substrate (sand and silt loam soil). In July 2023 and November 2023, in the sand substrate, there were no significant differences in Lolium perenne L. height across the different treatments applied (p>0.05), as shown in Figure 6-5. However, in July 2023, in the soil substrate, Lolium perenne L. was approximately 45% taller (p<0.05) under Min+Bio100, Min+Bio50, and Min treatments compared to Bio100 and the control treatment (soil alone). Furthermore, in November 2023, in the soil substrate, Lolium perenne L. was about twice as tall (p<0.05) under Min+Bio100, Min+Bio50, and Bio50 treatments compared to the control treatment. 66 Figure 6-5. Production of Lolium perenne L. (Plant height (cm)) under the different treatments established in July and November 2023. Min: mineral fertilisation, Bio50: 50 kg N ha-1 of biochar and Bio100: 100 kg N ha-1 of biochar. Different letters indicate significant differences between treatments in each type of substrate (sand and silt loam soil). 6.5 DISCUSSION The addition of biochar significantly affected the leaching of phosphorus (P) and nitrate. Using biochar in the sand substrate reduced P leaching to one-fourth of the levels observed in the Min and Min with biochar treatments. However, even with Bio50+Min and Bio100+Min, P leaching was similar to the Min treatment alone, indicating that biochar did not reduce P leaching in these cases. Applying biochar alone showed a slow release of P over time. For instance, in the Bio50 treatment, P leaching decreased from 35.7 mg l-1 in June to 0.92 mg l-1 in November, a trend also observed in the Bio100 treatment. These results are consistent with Madiba et al. (2016), who reported that wheat chaff biochar reduced P leaching by two to three times at 1% and 2% per pot. Lehmann et al. (2003) also found that biochar increased nutrient retention and decreased leaching, likely due to enhanced water-holding capacity in biochar-amended soil. Additionally, Borchard et al. (2012) observed that 15.0 g kg-1 of beech wood biochar decreased P leaching and increased retention in sandy soil. Biochar application in the soil substrate demonstrated lower total P leaching with Bio50, similar to the control treatment. However, total P leaching increased with Bio50+Min and Bio100, matching levels observed in the Min treatment. This suggests biochar did not significantly affect total P leaching compared to the Min treatment, likely due to soil type and structure (Bornø et al., 2018). These results align with findings that biochars can intensify soil P leaching (Troy et al., 2014; Pratiwi et al., 2016). For example, Pratiwi et al. (2016) reported that husk biochar increased phosphate leaching by 72% in loamy soil compared to no-biochar controls. Some studies, however, mentioned that biochar addition does not significantly affect P leaching (Saarnio and Kettunen, 2020). Biochar alone can provide a slow release, beneficial for long-term soil and crop health, with Bio50 showing a gradual decrease in P leaching from May (8.1 mg l-1) to November (0.6 mg l-1). This is consistent with Freitas et al. (2020), who noted that biochar might require more time to interact with soil and effectively capture nutrients. The application of biochar on the sand substrate positively affected total nitrate leaching, with all biochar treatments showing nitrate leaching levels half of those in the Min treatment. Yang et al. (2021) reported similar findings with beech wood biochar. The Min treatment consistently showed higher leaching rates across measurement dates compared to biochar or control treatments, aligning with Madiba et al. (2016), who attributed increased P leaching to both fertilizers and biochar. Nitrate leaching peaked on 25/05/2023 for Min, Bio50+Min, and Bio100+Min treatments, and on 22/05/2023 for control, Bio50, and Bio100 treatments, before eventually decreasing to nearly zero. 67 The results from July 2023 demonstrated that in the sand substrate, the combined treatments of mineral fertiliser with biochar (Min+Bio50 and Min+Bio100) significantly increased the dry matter (DM) of Lolium perenne L., achieving approximately three times higher DM compared to other treatments (p<0.05). This aligns with findings by Van Zwieten et al. (2000), who reported that the combination of biochar and mineral fertilisers can enhance nitrogen-use efficiency, thereby promoting plant growth. However, the results also indicated that biochar alone, irrespective of the dose, did not significantly boost Lolium perenne L. production compared to the control treatments, as the biochar nutrient release is slow, in spite of the physical characteristics of the soil are improved. This observation supports Knicker (2010), who suggested that the nitrogen in biochar is largely unavailable because it is present in forms that are not easily accessible to plants, such as heterocyclic aromatic nitrogen. Additionally, the initial application of biochar might result in net nitrogen immobilisation due to the mineralizable fraction of organic carbon in biochar (Bruun et al., 2012). In November 2023, the sand substrate did not show significant differences in Lolium perenne L. DM across different treatments (p>0.05). This outcome suggests that the sand substrate's capacity to retain nutrients and support plant growth might be limited, regardless of the treatment applied. However, in the soil substrate, the Min+Bio100 treatment still resulted in about three times greater Lolium perenne L. DM compared to most other treatments, except for Min+Bio50 and Bio50, which also showed increased DM compared to the control (p<0.05). These findings match Ye et al. (2020), who noted that biochar works better with other nutrients in soil rather than in sand. For the spontaneous species Amaranthus retroflexus L., the results from July 2023 showed a 32% higher DM in Min+Bio100 compared to Min and Min+Bio50, and about twice as high compared to Bio100, Bio50, and the control treatment in the soil alone (p<0.05). This increase can be attributed to the higher nitrogen availability from mineral fertilisation, as Amaranthus retroflexus L. growth is known to be significantly enhanced by increased soil nitrogen (Ghorbani et al., 1999). This highlights the importance of adequate nitrogen fertilisation to manage weed interference in crops effectively. Regarding plant height, the results from July 2023 indicated that in the soil substrate, Lolium perenne L. was approximately 45% taller under Min+Bio100, Min+Bio50, and Min treatments compared to Bio100 and the control treatment (p<0.05). This suggests that the combined use of biochar and mineral fertilisers significantly enhances plant height, likely due to improved nutrient availability and soil structure. These results are in line with previous research carried out by Chen et al. (2021), who found that increased soil nitrogen supply significantly boosts plant growth, including height. In November 2023, Lolium perenne L. in the soil substrate was about twice as tall under Min+Bio100, Min+Bio50, and Bio50 treatments compared to the control treatment (p<0.05). This continued increase in plant height shows the strong effect of combining biochar with mineral fertilisers (Glaser et al., 2015). The lack of significant height differences in the sand substrate across all treatments (p>0.05) again suggests the limitations of sand in supporting nutrient retention and plant growth. Applying biochar in the Bio100 treatment reduced nitrate leaching by 40% compared to the Min treatments. Nitrate leaching in the control, Bio50, and Bio50+Min treatments was similar to that in Min, Bio100, and Bio100+Min treatments. These results are consistent with Sika et al. (2014), who found wood biochar decreased nutrient leaching and increased retention by 12 to 50%. Nitrate leaching did not vary significantly with different treatments except on 29/05/2023 when biochar application resulted in lower nitrate concentrations compared to the Min treatment. Nitrate release was observed during the first three measurement dates in all cases, indicating that biochar amendments did not retain nitrate longer than Min treatments, 68 aligning with Haider et al. (2017), who noted that biochar did not affect nitrate leaching behaviour throughout the experimental period. These findings suggest that combining biochar with mineral fertilisers significantly boosts Lolium perenne L. growth in both dry matter and height, especially in soil. However, using biochar alone does not have the same effect, highlighting the need for combined treatments to improve plant growth and soil health. Moreover, it is recommended to apply biochar either alone or in combination with mineral amendments. Specifically, applying biochar at a dose of 50 kg t-1 with Min can achieve better nutrient capture and reduce nitrate and phosphorus leaching. 6.6 CONCLUSIONS The combination of mineral fertiliser with biochar, regardless of the biochar dose, significantly enhances pasture production in both sand and soil substrates by optimising nitrogen fertiliser use efficiency. This study strongly recommends integrating biochar with nitrogen fertilisers to achieve sustainable and efficient pasture production in both the short and long term. The results demonstrate that such integration not only boosts the growth of Lolium perenne L. but also enhances overall soil health and nutrient availability. Additionally, the increased dry matter of Amaranthus retroflexus L. under treatments with mineral fertilisers highlights the importance of adequate nitrogen fertilisation for effective weed management. Applying Bio100+Min in the soil gave the best results overall, boosting plant production and reducing total nitrate leaching. Moreover, adding biochar at any dose along with mineral fertiliser to the sand soil reduced nitrate leaching, increased plant production, and further reduced nitrate leaching. 75 species, tree fractions, pyrolysis time, and micronutrient concentrations in biochar could serve as a guiding framework for targeted soil amendments, thereby contributing to the establishment of sustainable soil production practices. 7.5 PRODUCTION The addition of biochar significantly affected the leaching of phosphorus (P) and nitrate. In the sand substrate, biochar reduced P leaching to one-fourth of the levels observed in the Min treatments. However, in Bio50+Min and Bio100+Min treatments, P leaching was similar to the Min treatment alone, indicating that biochar did not always reduce P leaching. Biochar alone showed a slow release of P over time, with a notable decrease in P leaching from June to November in both Bio50 and Bio100 treatments. In the soil substrate, Bio50 demonstrated lower total P leaching similar to the control, while Bio50+Min and Bio100 showed increased P leaching, matching the Min treatment levels. This suggests that biochar did not significantly affect total P leaching compared to Min treatments. Biochar can provide a slow release, beneficial for long-term soil and crop health, with Bio50 showing a gradual decrease in P leaching from May to November. The reduction in P leaching with the addition of biochar is primarily due to its high surface area and its water-holding capacity (Lehmann et al., 2011; Jeffrey et al, 2011). In cases where biochar did not affect P leaching, it might be attributed to the biochar needing more time to activate (Sakhiya et al., 2020). The application of biochar on the sand substrate positively affected nitrate leaching, with all biochar treatments showing nitrate leaching levels half of those in the Min treatment. The Min treatment consistently showed higher leaching rates across measurement dates compared to biochar or control treatments. Nitrate leaching peaked on specific dates for different treatments but eventually decreased to nearly zero. Biochar in the Bio100 treatment reduced nitrate leaching by 40% compared to Min treatments. Overall, biochar amendments did not retain nitrate longer than Min treatments, as nitrate release was observed during the first three measurement dates in all cases. The reduction of nitrate leaching can be attributed to the same factors responsible for reducing phosphorus (P) leaching, such as biochar's high surface area and water-holding capacity. Other studies, such as Yao et al. (2012), have found similar results. Yao's study on thirteen different biochar samples indicated that some biochar effectively reduced both P and nitrate leaching, while others did not. The study from July 2023 showed that combining mineral fertilisers with biochar (Min+Bio50 and Min+Bio100) significantly increased the dry matter (DM) of Lolium perenne L. in a sand substrate, tripling the DM compared to other treatments due to improved nutrient availability, better soil structure, increased microbial activity, and higher cation exchange capacity (Hafeez et al., 2022). Moreover, biochar alone did not significantly boost production, likely due to the nitrogen in biochar being largely unavailable to plants (Alkharabsheh et al., 2021). By November 2023, no significant differences in DM were observed in sand, indicating its limited nutrient retention capacity. However, in soil, the Min+Bio100 treatment resulted in about three times greater DM compared to most treatments, confirming that biochar works better with other nutrients in soil. For Amaranthus retroflexus L., the July 2023 results showed a 32% higher DM in Min+Bio100 compared to Min and Min+Bio50, and about double compared to Bio100, Bio50, and control in soil, due to higher nitrogen availability. Plant height measurements indicated that in soil, Lolium perenne L. was approximately 45% taller under Min+Bio100, Min+Bio50, and Min treatments compared to Bio100 and control in July 2023, and twice as tall in November 2023, showing the combined treatment's effectiveness in enhancing growth. The study concludes that combining biochar with mineral fertilisers significantly boosts growth in both DM and height, especially in soil, 76 while biochar alone is insufficient, emphasising the need for combined treatments to improve plant growth and soil health. Rahayu et al., (2022) reported similar findings, noting that applying biochar produced from rice husk at a rate of 15 t ha-1 significantly influenced corn plant growth overall, particularly increasing plant dry weight and height. 77 CHAPTER 8 . GENERAL CONCLUSION ____________________________________________________________________ 78 GENERAL CONCLUSIONS For biochar production, the species category influences the biochar amendment capacity. Conifer species, particularly their branches, should be used to enhance carbon sequestration and improve long-term soil health due to their higher carbon content. In contrast, broadleaves species, especially their green leaves and litterfall, are recommended for boosting soil fertility and providing immediate nutrient supply owing to their higher nitrogen content. Broadleaves raw material generates higher P and cations content than conifers, which are therefore recommended for applications requiring higher total and available macronutrient content, particularly phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg), due to their greater nutrient uptake ability. Conifers may be used for those used that have a low nutrient content is sufficient, and there is a focus on long-term soil health without the need for an immediate nutrient supply. Conifer and broadleaves derived biochar were found to be safe for soil amendment use, without exceeding heavy metal toxicity thresholds. Broadleaves, especially their litterfall, are ideal for biochar aimed at enhancing soil total micronutrient content, whereas conifers may be preferable for applications requiring higher Fe and Zn, while broadleaves are better if Mn and Cu is needed. Generally, broadleaves biochar, particularly from litterfall, exhibited higher micronutrient availability compared to conifer biochar. Time processing more causes changes in biochar quality. Shorter pyrolysis times (30 to 60 minutes) are effective for maintaining balanced carbon and nitrogen levels, especially in broadleaves derived biochar, while longer times may be needed for specific feedstocks like conifer branches to achieve desired carbon levels. Increasing pyrolysis time generally raises macronutrient concentrations, except for P and Ca, which remains unaffected. Thus, broadleaves derived biochar, especially from green leaves and litterfall, are more suitable for enhancing soil fertility due its higher macronutrient content, while conifers derived biochar can be used for soil structure improvement and carbon sequestration purposes. Increasing pyrolysis time generally increased micronutrient concentrations, except for Fe, which remained unaffected. The recommended pyrolysis time is generally 60 minutes to achieve the highest levels of both macro and micronutrients. Biochar shown a higher capacity to reduce nutrient leaching, increase ryegrass and above all clover production, therefore optimizing the sustainable grassland production and the nutrient (P and N) use by the mixed sward at long term. Applying Bio100+Min to the soil yielded the best overall results, enhancing plant production and significantly reducing total nitrate leaching. 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Lehmann J, da Silva JP, Steiner C, Nehls T, Zech W, Glaser B (2003) Nutrient availability and leaching in an archaeological Anthrosol and a Ferralsol of the Central Amazon basin: fertiliser, manure and charcoal amendments. Plant Soil 249:343–357Return to ref 2003 in article Lehmann, J., & Joseph, S. (2015). Biochar for Environmental Management: Science, Technology and Implementation. Routledge. Lehmann, J., Cowie, A., Masiello, C. A., Kammann, C., Woolf, D., Amonette, J. E., Cayuela, M. L., Camps-Arbestain, M., & Whitman, T. (2021). Biochar in climate change mitigation. Nature Geoscience, 14, 883– 892 In recent years, biochar as a soil amendment has gained attention for its ability to enhance soil fertility, promote plant growth, and sequester carbon, particularly in regions like Galicia, which faces wildfire risks due to accumulated forest residues. This study comprehensively evaluates biochar produced from different tree species (conifers and broadleaves) and fractions (branches, litterfall, green leaves) under varying pyrolysis times, assessing their effects on ryegrass production. Results indicate that conifer-derived biochar has higher carbon content and C/N ratios, while broadleave biochar is richer in nitrogen and macronutrients. Combining biochar with mineral fertilisers significantly improved grass production, highlighting its potential to enhance soil fertility and agricultural productivity.