Improving circular economy by biogas plants: valorization of agricultural feedstocks

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(1)Tesis Doctoral. Improving circular economy by biogas plants: valorization of agricultural feedstocks. Mirco Garuti Seville, Septiembre 2022. Directores: Antonia Jiménez Rodríguez Fernando Gonzalez Fermoso.

(2) Improving circular economy by biogas plants: valorization of agricultural feedstocks. La Directora. El Director. Fdo.: Antonia Jiménez Rodríguez. Fdo.: Fernando González Fermoso. Departamento de Sistemas Físicos, Químicos y Naturales. Universidad Pablo de Olavide. Departamento de Biotecnología de los Alimentos. Instituto de la Grasa (CSIC).. Memoria presentada por Mirco Garuti para optar al título de Doctor en Biotecnología, Ingeniería y Tecnología Química por la Universidad Pablo de Olavide, de Sevilla. Fdo.: Mirco Garuti Graduado en Biotecnologías Moleculares e Industriales. Facultad de Ciencias Matematicas, Fisicas, Naturales. Universidad de Bologna, Bologna, Italy..

(3) Este trabajo presentado para aspirar al Grado de Doctor por la Universidad de Pablo de Olavide ha sido influenciado por la pandemia generada por el coronavirus de tipo 2 causante del síndrome respiratorio agudo severo (SARS-CoV-2) COVID-19.

(4) Abstract Anaerobic digestion is a biotechnological process operating at the boundary between of livestock effluent and agro-industrial byproducts management, bioenergy generation and food production. Anaerobic digestion is an efficient solution to mitigate greenhouse gas emissions and to improve the circular economy of the agri-food sector via renewable energy generation from biogas/biomethane utilization and nutrient recycling using digestate as soil improver. An inspiring example of how food, feed and biomethane production can be coupled comes from a set of practices developed in Italy under the initiative Biogasdoneright®, introducing the double cropping system along with digestate fertilisation and minimum tillage of soil. The first crop is grown to supply the food and feed sector, while the second crop is grown for. biomethane production. More in particular, farmers introduce a second crop immediately following the first crop harvest, thereby producing additional feedstock for biomethane production on lands that would otherwise remain bare through the winter period. Biomethane is also produced using animal manure, lignocellulosic agricultural residues, and agro-industrial byproducts. For the application of the Biogasdoneright® model, the correct management of agricultural feedstock, the optimization of operational and biochemical parameters into the digesters, as well as the improvement of the global biological efficiency of the anaerobic digestion process are necessary. Starting from the presented challenges, the main goal of this PhD thesis is to assess biotechnological aspects to improve the anaerobic digestion of agricultural feedstock, generating additional knowledge to attain a fully sustainable and circular system to produce food, feed, and bioenergy. In Chapter 2 and Chapter 3, the cultivation of sorghum and triticale as promising second crops for biomethane production has been investigated..

(5) The several parameters affecting the methane yield per unit of cultivated area (methane hectare yield) such as the specific methane yield, the harvesting time, and the varieties of these crops has been studied. In Chapter 2, different sorghum phenotypes (forage, high-tonnage energy, sweet and grain with tall size) have been evaluated for biomethane production. In Chapter 3, nineteen varieties of triticale harvested at milk and dough development stages has been assessed for biomethane production. The lignocellulose in second crops, manures and agro-industrial byproducts acts as a barrier preventing anaerobic degradability.. In. Chapter 4, the effects on physical modifications, biomethane formation, and anaerobic degradability of agricultural feedstocks after different mechanical pretreatments. have. been. studied.. Four. commercials. mechanical. pretreatment technologies (knife milling, hammer milling, extrusion, shredding + hydrodynamic cavitation) commonly found at full-scale biogas plants have been investigated, including their electrical consumptions. To reach environmental goals and to optimize economic output, biogas plants should be operated at high biological efficiency, obtaining high biomethane yield per reactor volume, with a fast anaerobic conversion of agricultural feedstock, and resulting into a well-digested material that will decrease greenhouse gases emissions associated with digestate storage. In Chapter 5, the monitoring of 16 full-scale biogas plants fed with different agricultural feedstocks has been carried out to identify important parameters leading to the lowest residual methane potential of digestate. Among such parameters, the concentration of essential trace elements (selenium, nickel, cobalt, molybdenum) in relation to the hydraulic retention time and the organic loading rate has been investigated. Other metals of environmental interest have been also monitored due to the importance of a safe agronomic utilization of digestate on soil..

(6) Resumen La digestión anaerobia es un proceso biotecnológico cuya aplicación facilita el tratamiento y la gestión de residuos ganaderos y subproductos agroindustriales, a la vez que la generación de bioenergía y la producción de nutrientes. En particular, esta biotecnología permite reducir la emisión de gases de efecto invernadero y mejorar la economía circular del sector agroalimentario gracias a la generación de energía renovable en forma de biogás/biometano, así como el reciclado de nutrientes al utilizar el digestato como enmienda orgánica en la agricultura. Un buen ejemplo de cómo se pueden acoplar la producción de alimentos, piensos y biometano puede observarse en las actividades desarrolladas en Italia bajo la iniciativa Biogasdoneright®, que introduce el sistema de doble cultivo junto con la fertilización con digestato y una labranza mínima del suelo. En este sistema, el "primer cultivo" se destina al abastecimiento del sector alimentario y producción de piensos, mientras que el "segundo cultivo" se destina a la producción de biometano. En particular, el segundo cultivo se introduce inmediatamente después de la cosecha del primer cultivo, obteniéndose así una biomasa adicional para destinar a la producción de biometano en tierras que de otro modo habrían permanecido sin cultivar durante todo el periodo invernal. Así mismo, otras fuentes de biomasa orgánica del sector agroalimentario como el estiércol animal, y los subproductos agrícolas lignocelulósicos y agroindustriales pueden servir también para la generación de biometano. Para aplicar el modelo Biogasdoneright® es necesario tanto la correcta gestión de la biomasa agrícola, como la optimización de los parámetros operativos y bioquímicos en los digestores, y la mejora de la eficiencia biológica global del proceso de digestión anaerobia. En base a la situación y desafíos descritos, el objetivo principal de esta tesis de doctorado es evaluar los.

(7) aspectos biotecnológicos que permiten mejorar la digestión anaerobia de biomasas agrícolas, desarrollando al mismo tiempo el conocimiento necesario para mejorar la sostenibilidad y la economía circular de la producción de alimentos, piensos y bioenergía. En los Capítulos 2 y 3, se ha investigado el uso de los cultivos de sorgo y triticale como segundos cultivos destinados a la producción de biometano. En particular, se han estudiado diversos parámetros operacionales que afectan al rendimiento de biometano por hectárea cultivado, tales como el rendimiento específico de biometano de la materia prima, el tiempo de cosecha, o las variedades de los cultivos. En el Capítulo 2, se han evaluado diferentes fenotipos de sorgo (sorgo forrajero, sorgo de pastoreo, sorgo dulce y sorgo de grano de gran tamaño) para la producción de biometano. En el Capítulo 3, se ha estudiado la producción de biometano a partir de 19 variedades de triticale cosechadas en las etapas de desarrollo del cereal correspondientes al estado lechoso y pastoso del grano. Los compuestos lignocelulósicos presentes en los segundos cultivos, así como el estiercol y los subproductos agroindustriales pueden limitar la biodegradabilidad anaerobia de dichos cultivos. En el Capítulo 4, se ha estudiado el efecto de distintos tratamientos mecánicos sobre las características fisicoquímicas, generación de biometano y biodegradabilidad anaerobia de distintos sustratos agrícolas. En particular, se han estudiado 4 tecnologías comerciales de pretratamiento mecánico (fresado con cuchillas, triturado con martillo, extrusión, triturado + cavitación hidrodinámica) comúnmente utilizadas en plantas de generación de biogás a escala industrial, evaluando también los consumos eléctricos de las mismas. Para alcanzar los objetivos medioambientales y optimizar la producción económica, las plantas de biogás deben funcionar con una alta eficiencia biológica, obteniendo un alto rendimiento de biometano por volumen de.

(8) reactor, así como una rápida conversión de la biomasa agrícola, con el objetivo de reducir las emisiones de gases de efecto invernadero asociadas con almacenamiento del digestato. En el Capítulo 5, se llevó a cabo el seguimiento de 16 plantas de biogás a escala real alimentadas con diferentes tipos de biomasa agrícola, identificándose los parámetros más relevantes para lograr reducir el potencial de metano residual del digestato. Entre tales parámetros, se ha investigado la concentración de oligoelementos esenciales (selenio, níquel, cobalto, molibdeno) en relación con el tiempo de retención hidráulica y la velocidad de carga orgánica. También se ha llevado a cabo el seguimiento de otros metales de interés ambiental debido a su importancia para una utilización agronómica segura del digestato..

(9) List of contents 1. General introduction. 1. 2. Biomethane production using double cropping system with various sorghum phenotypes. 28. 3. Biomethane production using triticale in a double cropping system. 59. 4. Mechanical pretreatments of agricultural feedstocks in full-scale biogas plants. 90. 5. Biochemical conditions for anaerobic digestion of agricultural feedstocks. 124. 6. Conclusion. 158. Acknowledgements. 166. Appendix. 167. List of scientific production. 179.

(10) 1. General Introduction Abstract Anaerobic digestion is a biotechnological process operating at the boundary between of livestock effluent and agro-industrial byproducts management, bioenergy generation and food production. It is an interesting solution to mitigate greenhouse gas emissions and to improve the circular economy in the agri-food sector via renewable energy generation from biogas/biomethane and nutrient recycling using digestate as soil improver. An inspiring example of how food, feed and biomethane production can be coupled comes from Biogasdoneright® model. For a correct application of Biogasdoneright® model, the better management of feedstock, the optimization of operational and biochemical parameters into the digesters, as well as the improvement of the global biological efficiency of the anaerobic digestion process is a need. The main goal of this thesis is to assess biotechnological aspects to improve the anaerobic digestion of agricultural feedstock generating additional knowledge to make it a sustainable and circular system to produce food, feed, and bioenergy..

(11) Chapter 1. 1.1. Biomethane: a biogenic molecule Methane (CH4) is the simplest of all hydrocarbon molecules, it is a colorless and odorless gas that is flammable over a range of concentrations (5.4–17%) in air at standard pressure. One of the first observations of methane combustion come from the will-. o -the-wisp phenomena around. BC; the observed light formed during will-. o -the-wisp phenomena is due to ignition of (bio)methane present above or in wet soil but the scientific explanation of this occurrence will be provided many years later. The discovery of the biogenic production of methane was attributed to Alessandro Volta when he collected gas from sediment of Lake Maggiore (Italy) in 1778. He noticed large bubbles of gas coming out to the surface sediments indicating as inflammable air from marshlands .. The correct composition of the gas was determined in 1805 by Thomas. Henry. The name "methane" was coined in 1866 by the German chemist August Wilhelm von Hofmann. Bechamp in 1868 and Popoff in 1875 reported that the formation of (bio)methane during the decomposition of organic materials was due to a microbiological process (Gunnerson and Stuckey, 1986). The microbiological process is known as anaerobic digestion and it. comprises. four. major. degradation. steps:. hydrolysis,. acidogenesis,. acetogenesis, and methanogenesis (Figure 1). Lipids, proteins, and carbohydrates are metabolized to long-chain fatty acids, glycerol, amino acids, monosugars, that are further mainly converted to C2-C7 volatile fatty acids (acetate, propionate, butyrate, etc.), hydrogen, and carbon dioxide. Volatile fatty acids and alcohols are converted mainly to acetate, hydrogen, and carbon. 2.

(12) Chapter 1 dioxide. These end products are finally metabolized to methane and carbon dioxide by the methanogenic archaea (Kougias and Angelidaki, 2018). Carbohydrates. Lipids. Proteins. Fatty Acids degradation. Aminoacidi. Alcohols. HYDROLYSIS. Polysaccharides Oligosaccharides Monosaccharides. C7 C6. ACIDOGENESIS C5 C4. VOLATILE FATTY ACIDS. Eptanic Acid Caproic Acid Valeric Acid. i-Caproic Acid. C3. i-Butyric Acid. CO2. NH3. C2. Acetic Acid. Acetic Acid. C4. C3. Propionic Acid. C2. C6 C5. i-Valeric Acid. Butyric Acid. H2 S C7. H2. ACETOGENESIS CO2. CH4. METHANOGENESIS. Figure 1: Anaerobic degradation of carbohydrates, lipids, and proteins and the microbiological steps commonly reported in anaerobic digestion. The figure is adapted from Kougias and Angelidaki (2018).. 1.2. Anaerobic digestion for bioenergy production The ability of anaerobic microorganisms to decompose complex organic matter and to produce biogas can be utilized for production of bioenergy through biotechnological process. Moreover, anaerobic digestion process leads nutrients recycling (nitrogen, phosphorous, and other trace elements), applying the digested effluent (digestate) on agricultural land as biofertilizer to replace chemical fertilizers.. 3.

(13) Chapter 1 In Europe, the biotechnological development of anaerobic digestion was limited until the beginning of the 20th century; however, with increasing industrial interest, research into anaerobic microbial processes was also enhanced aiming to identify fundamental process parameters and to explore the potential biomethane production of several residual organic matter, especially agricultural residues such as manure and slurry (Nikolausz and Kretzschmar, 2020). Agricultural feedstocks constitute the highest feedstock in Europe for their extensive use by Germany, Italy and the United Kingdom, three of the biggest biogas producers in Europe. Within these three countries, agri-based feedstocks (energy crops, livestock effluents, agricultural residues) are commonly used. Agricultural feedstock such as livestock effluent (i.e., pigs, cows, horses, poultry slurries and manure), energy crops (e.g. maize, sorghum, triticale, cereals, grass, sugar beet, etc.), agricultural residues (straw, stovers, etc.) and agro-industrial by-products obtained from manufacturing agro-food sector (e.g., molasses, straw, maize stalks, olive pomace, tomato peels, vegetable and fruits manufacturing residues, etc.). The biogas produced from anaerobic digestion of agricultural residues, manure, energy crops, organic fraction of municipal solid waste, sewage sludge can be valorised energetically in a combined heat and power units for the simultaneous production of heat and electricity, or it can be upgraded to natural gas purity and injected in the natural gas grid or liquefied and directly used as biofuel (Rafiee et al., 2021). At the end of 2019, Europe reached a total of 18,943 biogas plants (167 TWh or 15.8 bcm of biogas) and 725 biomethane plants (26 TWh or 2.43 bcm of biomethane). Agricultural feedstocks comprising livestock manure, farm residues, plant residues and energy crops are the driving force of the. 4.

(14) Chapter 1 European biogas sector with a roughly share of 65-70% of the total feedstock used (EBA, 2021). European countries have developed modern biogas technologies throughout decades of intensive research and technical development (Karki et al., 2021; Nikolausz and Kretzschmar, 2020). This has been possible by the support schemes and national policy, by the efforts of private companies and stakeholders and by the knowledge developed in Universities and Research Centers.. Figure 2: Agricultural biogas plant (photo credits CRPA). Support schemes are an element of European and national policy aimed at encouraging demand for renewable energy in the energy market and thus stimulating technical progress in Europe. The final goal is to make renewable energy technologies competitive with conventional technologies producing energy from fossil fuels (Menanteau, et al., 2003). The most used support 5.

(15) Chapter 1 schemes in Europe. for renewable energy are feed-in tariffs, feed-in. premiums, tax credits and a quota system which is used in conjunction with tradable green certificates (Pablo-Romero et al., 2017).. 1.3. Anaerobic Digestion as mitigating strategy for GHG emissions From environmental point of view, methane plays important roles in atmospheric chemistry (Whiticar, 2020). Absorption of solar radiation by water vapor, carbon dioxide (CO2), and other trace gases, like CH4 and nitrous oxide (N2O), in the atmosphere is responsible for the Earth surface temperature. One kilogram of CH4 has a global warming potential 28 and 84 times greater than 1 kg of CO2 greater than CO2 over a 100- and 20-year period, respectively (Whiticar, 2020). The residence time of methane in the atmosphere is about 9 years, where the main sink is the tropospheric reaction with OH radicals that produce H 2O and CO2; the increase of atmospheric methane concentrations bring to increase CO2 concentration as one of the most important driver of contemporary climate change (Canfield et al., 2005). Lal reviewed the impacts of agriculture and forestry on gaseous emissions into the atmosphere (Lal, 2021). Principal agricultural activities leading to greenhouse gases emissions are plowing, drainage of wetlands, enteric fermentation, fertilizer use, manure management, rice cultivation and fuel consumption (Figure 3). Combined, all agricultural activities may contribute about 25%, 65%, and 90% of total anthropogenic emissions of CO2, CH4, and N2O, respectively. Livestock remains a major source of greenhouse gases, especially of CH4. Many of the more stringent mitigation scenarios about GHG emissions. 6.

(16) Chapter 1 strongly depend on a large-scale development of bioenergy with carbon dioxide capture and storage (BECCS), a technology that produces bioenergy and removes carbon (Peters and Geden, 2017).. Figure 3: Impacts of agriculture and forestry on gaseous emissions into the atmosphere (Lal, 2021). Anaerobic digestion of manure and slurries produce biomethane reducing CH4 emission in manure management and obtaining digestate to use as natural fertilizer for soils. Within the food chain, also the correct by-products management processes contribute to reduce the overall environmental impact of agri-food sector. Among the different mitigation strategies, several studies highlighted that 7.

(17) Chapter 1 anaerobic digestion is an effective solution to reduce GHG emissions from food processing. For instance, tomato processing involves a significant production of residues, mainly constituted by discarded tomatoes, skins, seeds and pulp. When these residues are discarded as wastes could represent an added cost for manufacturing companies, with environmental issues due to such kind of management concentrated in few months. Bacenetti et al evaluated the tomato byproducts utilization for biogas production as a strategy to reduce the environmental impact of tomato purée. Such solution implies lower impacts with respect to send back to the fields the tomato byproducts as organic fertilizers. The energetic valorization of tomato byproducts is carried out by anaerobic digestion allowing a saving of GHG emissions that, over the whole year, is equal to 1567 tons of CO2eq (Bacenetti et al., 2015). Recovering heat from combined heat and power unit as well as covering of digestate tank would significantly improve the environmental sustainability of electricity generation from biogas (Bacenetti et al., 2016). Bacenetti and co-workers proposed that the results of their study could be upscaled to the agri-food industries with high amount of vegetables and fruits to process and with high thermal energy consumption. Such kind of feedstock could be used for biogas production, optimizing the agri-food industry, and adding further benefits, under environmental and commercial point of view. Leong et al., reviewed the utilization of fruit byproducts or fruit and vegetable byproducts as feedstocks for anaerobic digestion (Kit Leong and Joshu, 2022). The mono-digestion of fruits and vegetables is challenging for their high concentration of soluble simple sugar, which can lead volatile fatty acids (VFAs) accumulation, causing rapid acidification and subsequently the inhibition of methanogenic activity. Different strategies to overcome this. 8.

(18) Chapter 1 problem could be employed, including co-digestion with other feedstocks, optimization of organic loading rate (OLR) and hydraulic retention time, and optimal substrate management. Other studies demonstrate how an integrated planning process for costeffective mitigation of both GHG emissions and eutrophication of water, taking in considerations the total environment into the design and decision processes, can support the co-digestion feasibility of beach-cost seaweed with food industry residues and manure (Kaspersen, et al., 2016).. 1.4. The contribute of anaerobic digestion to Circular Economy The circular economy is defined by The Ellen MacArthur Foundation as a global economic model that aims to decouple economic growth and development from the consumption of finite resources; unlike a linear economy, it is about optimizing systems rather than components. This includes the appropriate management of materials flows in both biological and technical cycles. (Ellen MacArthur Foundation, 2012). Anaerobic digestion is a biotechnological process operating at the boundary between of livestock effluent and agro-industrial byproducts management, bioenergy generation and food production. It is an interesting solution to mitigate greenhouse gas emissions and to improve the circular economy in the agri-food sector via renewable energy generation from biogas/biomethane and nutrient recycling using digestate as soil improver. Commonly, biogas plants produce electricity and heat by the combustion of biogas in combined heat and power units, but anaerobic digestion can also be a source of biomethane and used as a biofuel for transportation. Only the CH4 component of biogas produces bioenergy (transport fuel, electricity, heat). 9.

(19) Chapter 1 while the CO2 stream is often unused. Separation and capture of such biogenic CO2 could further lead to produce a carbon negative biogas. The CO2 can be used to add gaseous bubbles to beverage in food industry, as a carrier in cooling systems, and as a raw chemical building block. The CO2 could be converted in additional biomethane by reacting with green hydrogen via methanation reaction (Fagerström et al., 2018). The digestate is the effluent of the anaerobic digestion process and it is used as biofertilizer for the soil, recirculating the nutrients into the ground (Jurgutis et al., 2021; Koszel and Lorencowicz, 2015). Considering just the carbon balance, about. ‒. % of the carbon fed into an agricultural biogas. plant is transformed into biogas. The residual carbon is contained in the digestate and incorporated into the soil increasing the soil carbon content. Digestate can also contain a significant amount of plant nutrients, such as nitrogen, phosphorus, potassium, sulphur, calcium, magnesium, sodium and micronutrients/trace elements (i.e.: Fe, Mn, Zn, B, Cu, Co, Se, Ni, Mo). During anaerobic digestion, the nitrogen is partially mineralized into ammonium and taken up by vegetable crops. Effluent digestate usually undergoes solid-liquid separation before soil application. This post-treatment technology leads to concentrate solids and organic matter in the solid fraction whereas most of the mineral nitrogen is kept in the liquid fraction. The agrochemical value of the digestate depends on the composition of the input feedstock and on the efficiency of the biological process. The high nutrients and organic matter content makes the digestate a valuable fertilizer and soil conditioner to increase the organic content of lands. In fact, both digestate fractions can be used to fertilize soils, to reduce the use of chemical fertilizers optimizing farm management and promoting circular economy benefits. The opportunity to. 10.

(20) Chapter 1 apply digestate as biofertilizers to the land, creating closed-loop systems is extremely important for nutrient recycle. Anaerobic digestion is considered as one of the most correct approaches to a circular bioeconomy across the agri-food sector catalysing many efforts in research and innovation projects (Toop et al., 2017).. 1.5. Biogasdoneright® The European continent is pioneering in the Circular Economy development suggesting several business models, especially in agriculture, water reuse, and wastes management (Sillanpää and Ncibi, 2019). Sillanpää and Ncibi reviewed selected European case studies adopting circular economy principles. Biogasdoneright® is addressed as Italian flagship initiative to foster a greener and efficient low carbon farming practices (Figure 4).. Figure 4: The Biogasdoneright model (Dale et al., 2020). 11.

(21) Chapter 1 This initiative integrates anaerobic digestion with other industrial and agricultural practices increasing the net primary production of the farm producing bioenergy and managing new organic carbon and nutrients using digestate on the soil. The fundamental pillars of Biogasdoneright® are showed in Figure 5.. Figure 5: Pillars and effects of the Biogasdoneright® model (Dale et al., 2020).. Since the first decade of 2000s, a group of farmers in the Northern Italy supported by the Italian Biogas Consortium (CIB, Consorzio Italiano Biogas) decided to exploit a new agricultural model based on farm-anaerobic digestion plants to produce renewable bioenergy (biogas) and natural fertilizers (digestate). In this model, biogas production must not negatively interfere with food/feed production and must not to increase the rate of indirect land use change (iLUC) avoiding potential criticism about such issues (Thompson et al., 2012). To move beyond food versus fuel as an either/or. choice, it is necessary the investigation on managed farming and grazing operations. Biogasdoneright® does not to support for land use dedicated. 12.

(22) Chapter 1 exclusively. to. biomethane. production. with. large-scale. bioenergy. monocultures, but rather to promote an integrated and regenerative food– feed–bioenergy production system on land (Schulte et al., 2022). The fundamental pillars of Biogasdoneright® are following described. Valorization of livestock manure, agricultural residues and agrondustrial byproducts. Livestock manure was recognized to be an interesting and very cost-effective feedstock for biogas production, combining renewable energy production, nutrient recycling and greenhouse gases (CH4 and N2O) and ammonia reduction compared to conventional manure management. High concentration of nitrogen and phosphorus in livestock manures could be an environmental threat if not properly handled and disposed. Moreover, livestock manure contains smell compounds that could contribute to create odour nuisance when slurry is stored or applied as fertiliser on the soil. After anaerobic digestion, many of these compounds are degraded, contributing to reduce of odour nuisance during digestate spreading (Fagerström et al., 2018). Cereal straw and maize straw are often considered agricultural residues. Straw is often left in the fields to increase the soil carbon stock or used for the livestock bedding, in horticultural applications, or in composting. Straw and other lignocellulosic agricultural residues represent also a suitable feedstock for anaerobic digestion, although mechanical or thermochemical or biological pretreatments are necessary to achieve the highest methane yields possible (Iskalieva et al., 2012; Moset et al., 2015; Theuretzbacher et al., 2015; Zhong et al., 2011). In Europe, the largest potential for biogas production could be obtained from agriculture (manure, straw and cutting/prunings from permanent crops). The quantities of total solids forecasted for the year 2030 in the EU. 13.

(23) Chapter 1 member states were estimated to range from 83 to 122 Mt y-1 from manure where the share of cattle manure, pig manure, and poultry manure represented in total solids about 74%, 21, and 9%, respectively (Meyer et al., 2018). Co-digestion of livestock manure with agricultural residues (grass, maize stalks, and straw) is addressed as a possibility to increase European biogas production to 2030 and to 2050 but technological developments may be necessary for an efficient anaerobic degradation of such agricultural feedstock. However, several studies provided an economic analysis regarding biogas and biomethane production using livestock manures and they conclude the key-role of subsidies and policy guidance for the development of farm-scale biogas projects (Cucchiella et al., 2019; Stürmer et al., 2021; Venus et al., 2021).. Introduction of sequential cropping increases the efficiency of land use and economic stability of the farm. Sequential cropping is a rotation cultivation system in which a new winter crop is cultivated after the existing summer on the same plot of land in the same year. The first crop o main. crop traditional crop is grown to supply the food and feed while the second crop or double-crop is grown to feed the anaerobic digestion. In such. model, farms continue to produce food and feed from the first crop as they have always done avoiding negative impact on the food/feed markets.. Farmers introduce a second crop immediately following the first crop harvest, thereby producing feedstock for biogas purpose on lands that would have remained bare through the winter period (Dale et al., 2016; McCabe et al., 2020). According to iLUC theory, the carbon footprint of bioenergy should account for the greenhouse gases emissions that are released when additional land is used to produce agricultural feedstocks used for bioenergy production. 14.

(24) Chapter 1 (McCabe et al., 2020). The main advantage of Biogasdoneright® system is exactly the avoided utilization of land for solely bioenergy purpose. Valli and co-workers calculated the marginal lifecycle greenhouse gas emission of biomethane potentially produced from application Biogasdoneright® model ranged from 10 to 36 grams CO2 per megajoule (MJ) while the corresponding emission factor for conventional biogas from anaerobic mono-digestion of. maize silage, for natural gas and for fossil fuel generated 27 g CO2 MJ-1, 72 g CO2 MJ-1 , and 115 g CO2 MJ-1, respectively (Valli et al., 2017). Pirelli et al., investigated the environmental sustainability of the Biogasdonerigh® in Italy through the methodology of the Global Bioenergy Partnership concluding that the land use and land use changes caused by the implementation of the biogas/biomethane value chain in Italy do not represent, as of today, a relevant issue (Pirelli et al., 2021).. Increase and maintenance of organic matter in the soil by utilization of digestate as natural fertilizer, abandonment of deep ploughing and application of minimum tillage. Considering just the carbon balance, about ‒. % of the carbon fed into an agricultural biogas plant is transformed into. biogas. The residual carbon is contained in the digestate and incorporated into. the soil increasing the soil carbon content (Dale et al., 2016). The whole digestate usually undergoes to solid-liquid separation after anaerobic digestion. The solid-liquid separation allows to concentrate the coarse solids and organic matter in the solid fraction whereas most of the mineral nitrogen is kept in the liquid fraction. Slepetiene and co-workers described as the soil carbon and the soil fertility is enhanced by digestate solid fraction utilization. Moreover, the content of mobile humic substances tended to increase in grassland and crop rotation field in soil treated with digestate (Slepetiene et. 15.

(25) Chapter 1 al., 2022). Soils which are rich in humus are also more resilient to climate change. Building humus in soil is possible when the soil is covered with plants all year long, adopting sequential cropping for example. More plants on the field also increase the natural photosynthesis activity and CO2 removal from the atmosphere. Soil carbon levels are further enhanced by the second crop, primarily by decomposition of roots of the second crop into the soil and secondly by the additional undigested carbon intake present in the digestate. Soil organic carbon sequestration, described as long-term or permanent (i.e. 100 years) removal of CO2 from the atmosphere into the soil may be further enhanced by adoption of best management practices such as conservation tillage (Béghin-Tanneau et al., 2019; Stockmann et al., 2013). In such way, the farm in which Biogasdoneright® is applied represents a system for bioenergy with carbon capture and storage (BECCS) via natural photosynthesis. Pirelli et al. showed the sustainability of practices to produce efficiently biogas from agricultural biogas plants as bioenergy systems and BECCS technology, consisting in sequential cropping that continuously covers the soil, utilisation of manure and agricultural residues for biogas production, and soil fertilization with digestate (Pirelli et al., 2021). Style and co-workers applied LCA studies to anaerobic digestion deployment scenarios in United Kingdom to understand how environmental performance of anaerobic digestion might to evolve becoming more circular and to move towards climate neutrality. Transport biomethane is currently the most effective final use for greenhouse gases mitigation, but strategic investment in anaerobic digestion infrastructure to allow flexible switching of biomethane use from transport to large scale combustion in BECCS systems could maximise greenhouse gas mitigation efficacy through time (Styles et al., 2022).. 16.

(26) Chapter 1 Implementation of conservative agriculture by overall reduction of inputs (chemical fertilizers, pesticides, water, etc.). The most rational model for the agronomic use of digestate depends on the specific characteristics of the farm considering the availability of land for spreading and total potential receptivity of nitrogen and other nutrients, the distance from the storage location (in general the biogas plant) and land of distribution, the features of the soil and land suitability for crops grown, the vegetable crops and crop rotation system, and the availability of water irrigation systems. Fallow soil is continuously subjected to unprotected conditions causing higher soil erosion rates resulting in higher CO2 emission (Lal, 2021). On the contrary, the double cropping system can be introduced to reduce soil erosion, to protect soils, and to captures mobile soil nutrients, and to protect water supplies, in addition to a diversification of conventional food-based cropping system (Parenti et al., 2022; Reeves, 1997). The distribution of the solid fraction of digestate is usually carried out at the time of the preseeding processing, while the spreading of the whole digestate or the liquid fraction is generally carried out at the optimal times for crop uptake. The liquid fraction of digestate should be applied using innovative technologies, such as band spreaders, and then incorporated rapidly into the soil, or shallow/deep injectors, to minimize emissions of ammonia into the atmosphere. The digestate liquid fraction can be also applied with irrigation water on the soil. fertigation , thereby recycling. mineral nutrients and providing irrigation water improving water use efficiency (Guido et al., 2020).. The soil erosion hazards depend on the different amounts herbicides, pesticides, fertilizers, irrigation, and seeding rates and its impact on CO 2. 17.

(27) Chapter 1 emissions is related to tillage intensity (conventional tillage, minimum tillage, no-till, conservation tillage). Then, the positive effect in terms of carbon sequestration due to soil fertilization with digestate can be further enhanced by practices derived from conservation agriculture such as minimum tillage, strip tillage and sod seeding (Lal, 2021).. 1.6. Motivation and purpose of this thesis One of the most important challenges of today s society regarding all. economical sector is related to natural resources management and environmental impact of production systems. Waste prevention and reduction, renewable energy despite fossil fuels utilization, reduction of greenhouse gases emissions to mitigate global climate changes have becoming priorities. All of these challenges can be addressed to a large extent through innovative agriculture fostering the circular economy across the agri-food sector. Among the different mitigation strategies, several studies highlighted that anaerobic digestion is an effective solution to reduce greenhouse gas emissions of agricultural sector and to produce bioenergy. The anaerobic digestion of agricultural feedstocks produces renewable electricity and heat by the combustion of biogas on-site in combined heat and power units. Biogas can be purified after upgrade step obtaining biomethane to use as a biofuel for transportation. Biogas and biomethane are storable energy sources and they can balance the intermittent supply of other renewable energy sources such as wind and solar power. An inspiring example of how food, energy and wealth production can be coupled comes from Biogasdoneright® initiative. In such innovative. 18.

(28) Chapter 1 agricultural systems, the food production continues as before, during the regular growing season. Second crops during periods when cropland would otherwise be left unplanted are cultivated for biogas/biomethane purpose and co-digested with manure, agricultural residues, and agro-industrial byproducts. The effluent digestate represents the unconverted residue from the anaerobic digestion process and it is spread on fields as a valuable soil amendment. Among organic materials, agricultural feedstocks are already dominating the biogas production, but the utilization of maize silage will be progressively reduced and replaced. Agricultural feedstocks will represent the most abundant organic material available in the future for anaerobic digestion, higher than potential from food waste or sludge. Nowadays, the co-digestion of second crops, manure, lignocellulosic agricultural residues and agroindustrial by-products still represent a challenge for biogas sector due to recalcitrant lignocellulose, inhibitory substances such as ammonia, and incompatible feedstock mixing ratio resulting in organic overloading, acidification, or system failure. Mastering anaerobic digestion process will be necessary to reduce operational costs and maximize gas incomes. Efforts in research and development program will be focused on the better management of feedstock, the optimization of operational and biochemical parameters into the digesters, as well as the improvement of the global biological efficiency of the anaerobic digestion process, as show in Figure 6.. 19.

(29) Chapter 1. Figure 6: Main stages of an optimized anaerobic co-digestion process (adapted from Brémond et al., 2021).. Starting from the presented challenges, the main goal of this thesis is to assess biotechnological aspects to improve the anaerobic digestion of agricultural feedstock generating additional knowledge to make it a sustainable and circular system to produce food, feed, and bioenergy. In Chapter 2 and Chapter 3 the cultivation of Sorghum and Triticale as promising second crops for biomethane purpose has been investigated. The several parameters affecting the methane yield per unit of cultivated area (methane hectare yield) such as the specific methane yield of the feedstock, the harvest time, the varieties of the crops due to their genotype has been studied. In Chapter 2, different sorghum phenotypes (forage, high-tonnage energy, sweet and grain with tall size) when cultivated as first crop and second crop has been evaluated for biomethane production. In Chapter 3, nineteen varieties of triticale cultivated as second crop and harvested at milk and dough development stages has been assessed for biomethane production. For both agricultural feedstocks, agronomical field trials have been carried out to obtain biomass yields. The chemical components characterization (i.e.: cellulose, hemicellulose, lignin, starch, sugars, proteins, fats) has been performed by the near infrared reflectance spectroscopy (NIRS), while the biomethane production has been measured carrying out Biochemical. 20.

(30) Chapter 1 Methane Potential (BMP) tests for each sorghum and triticale sample. The relation between chemical composition, biomass yield and methane production has been investigate using Principal Component Analysis (PCA) and discussed. The anaerobic degradability and the biogas production of agricultural feedstocks even depend on their content of cellulose, hemicellulose, and lignin. When cellulose and hemicellulose are strictly associated with lignin, it acts. as. a. barrier. preventing. anaerobic degradability.. Mechanical. pretreatments technologies act enhancing the microbial attack aiming to improve the anaerobic degradation and the biomethane production.. In. Chapter 4, the effects on physical modifications and anaerobic degradability of agri-based feedstocks after different mechanical pretreatments have been studied. Four commercials mechanical pretreatment technologies (knife milling, hammer milling, extrusion, shredding + hydrodynamic cavitation) that effectively run under real operational conditions at full-scale biogas plants have been investigated, including their electrical consumptions. Methane yields and methane production rates of untreated and treated agricultural feedstock have been evaluated by BMP tests. Scanning Electron Microscope (SEM) analysis and particles size distribution analysis have been used to evaluate the morphological and structural changes of agricultural feedstocks surface before and after each mechanical pretreatment. To reach environmental goals and to optimize economic output, biogas plants should be operated at high biological efficiency, obtaining high biomethane yield per reactor volume, with a fast anaerobic conversion of agricultural feedstock producing a well-digested material will decrease greenhouse gases emissions associated with digestate storage. In Chapter 5, the monitoring of existing 16 full-scale biogas plants fed with different. 21.

(31) Chapter 1 agricultural feedstocks has been carried out to identify important parameters to achieve the lowest residual methane potential (RMP). Among such parameters, the concentration of important trace elements (Selenium, Nichel, Cobalt, Molybdenum) in relation to Hydraulic Retention Time (HRT) and Organic Loading Rate (OLR) has been studied using Principal Component Analysis (PCA). Other metals of environmental interest have been also monitored due to the importance of a safe agronomic utilization of digestate on soil. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has been used to measure the concentration of metals in the digestate. The Specific Methane Residual Yield (SMRY) of the effluent digestate from biogas plants has been measured with laboratory BMP test while the calculation of RMP as indicator of biological efficiency of biogas plants has been calculated on the basis of mass balance.. References Bacenetti, J., Duca, D., Negri, M., Fusi, A., Fiala, M., 2015. Mitigation strategies in the agro-food sector: The anaerobic digestion of tomato purée byproducts. An Italian case study. Sci. Total Environ. 526, 88–97. https://doi.org/10.1016/j.scitotenv.2015.04.069 Bacenetti, J., Sala, C., Fusi, A., Fiala, M., 2016. Agricultural anaerobic digestion plants: What LCA studies pointed out and what can be done to make them more environmentally sustainable. Appl. Energy 179, 669–686. https://doi.org/10.1016/j.apenergy.2016.07.029 Béghin-Tanneau, R., Guérin, F., Guiresse, M., Kleiber, D., Scheiner, J.D., 2019. Carbon sequestration in soil amended with anaerobic digested matter. Soil Tillage Res. 192, 87–94. https://doi.org/10.1016/j.still.2019.04.024 Brémond, U., Bertrandias, A., Steyer, J.P., Bernet, N., Carrere, H., 2021. A vision of European biogas sector development towards 2030: Trends and. 22.

(32) Chapter 1 challenges. J. Clean. https://doi.org/10.1016/j.jclepro.2020.125065. Prod.. 287.. Canfield, D.E., Kristensen, E., Thamdrup, B., 2005. The Methane Cycle, Advances in Marine Biology. https://doi.org/10.1016/S00652881(05)48010-4 Cucchiella, F., D Adamo, )., Gastaldi, M., 2019. An economic analysis of biogasbiomethane chain from animal residues in Italy. J. Clean. Prod. 230, 888– 897. https://doi.org/10.1016/j.jclepro.2019.05.116 Dale, B.E., Bozzetto, S., Couturier, C., Fabbri, C., Hilbert, J.A., Ong, R., Richard, T., Rossi, L., Thelen, K.D., Woods, J., 2020. The potential for expanding sustainable biogas production and some possible impacts in specific countries. Biofuels, Bioprod. Biorefining 14, 1335–1347. https://doi.org/10.1002/bbb.2134 Dale, B.E., Sibilla, F., Fabbri, C., Pezzaglia, M., Pecorino, B., Veggia, E., Baronchelli, A., Gattoni, P., Bozzetto, S., 2016. BiogasdonerightTM: An innovative new system is commercialized in Italy. Biofuels, Bioprod. Biorefining 10, 341–345. https://doi.org/10.1002/bbb.1671 EBA, 2021. European Biogas Association Statistical Report: 2020 European Overview. Brussels, Belgium. Ellen MacArthur Foundation, 2012. Towards the circular economy. Fagerström, A., Al Seadi, T., Rasi, S., Briseid, T., 2018. The Role of Anaerobic Digestion and Biogas in the Circular Economy. IEA Bioenergy Task 37 37, 1–24. Guido, V., Finzi, A., Ferrari, O., Riva, E., Quílez, D., Herrero, E., Provolo, G., 2020. Fertigation of maize with digestate using drip irrigation and pivot systems. Agronomy 10. https://doi.org/10.3390/agronomy10101453 Gunnerson, C.G., Stuckey, D.C., 1986. Anaerobic digestion: principles and practices for biogas systems [WWW Document]. Bank Tech. Pap. URL https://www.osti.gov/biblio/5582401-anaerobic-digestion-principlespractices-biogas-systems-world-bank-technical-paper (accessed 7.4.22). Iskalieva, A., Mbouyem, B., Gogate, P.R., Horvath, M., Horvath, P.G., Csoka, L., 2012. Ultrasonics Sonochemistry Cavitation assisted delignification of 23.

(33) Chapter 1 wheat straw: A review. Ultrason. - Sonochemistry 19, 984–993. https://doi.org/10.1016/j.ultsonch.2012.02.007 Jurgutis, L., Šlepetienė, A., Šlepetys, J., Cesevičienė, J., . Towards a full circular economy in biogas plants: Sustainable management of digestate for growing biomass feedstocks and use as biofertilizer. Energies 14. https://doi.org/10.3390/en14144272 Karki, R., Chuenchart, W., Surendra, K.C., Shrestha, S., Raskin, L., Sung, S., Hashimoto, A., Kumar Khanal, S., 2021. Anaerobic co-digestion: Current status and perspectives. Bioresour. Technol. 330, 125001. https://doi.org/10.1016/j.biortech.2021.125001 Kit Leong, Y., Jo-shu, C., 2022. Valorization of fruit wastes for circular bioeconomy: Current advances, challenges , and opportunities. Bioresour. Technol. 359, 127459. https://doi.org/10.1016/j.biortech.2022.127459 Koszel, M., Lorencowicz, E., 2015. Agricultural Use of Biogas Digestate as a Replacement Fertilizers. Agric. Agric. Sci. Procedia 7, 119–124. https://doi.org/10.1016/j.aaspro.2015.12.004 Kougias, P.G., Angelidaki, I., 2018. Biogas and its opportunities—A review. Front. Environ. Sci. Eng. 12. https://doi.org/10.1007/s11783-018-10378 Lal, R., 2021. Climate change and agriculture. Clim. Chang. Obs. Impacts Planet Earth, Third Ed. 661–686. https://doi.org/10.1016/B978-0-12-8215753.00031-1 McCabe, B., Kroebel, R., Pezzaglia, M., Lukehurst, C., Lalonde, C., Wellisch, M., Murphy, J.D., 2020. Integration of Anaerobic Digestion into Farming Systems in Australia, Canada, Italy, and the UK, IEA Bioenergy Task 37. https://doi.org/10.1002/bbb.2134 Meyer, A.K.P., Ehimen, E.A., Holm-Nielsen, J.B., 2018. Future European biogas: Animal manure, straw and grass potentials for a sustainable European biogas production. Biomass and Bioenergy 111, 154–164. https://doi.org/10.1016/j.biombioe.2017.05.013. 24.

(34) Chapter 1 Moset, V., Almeida, C. De, Xavier, N., Bjarne, H., 2015. Optimization of methane yield by using straw briquettes- influence of additives and mold size. Ind. Crop. Prod. 74, 925–932. https://doi.org/10.1016/j.indcrop.2015.05.075 Nikolausz, M., Kretzschmar, J., 2020. Anaerobic digestion in the 21st century. Bioengineering 7, 1–3. https://doi.org/10.3390/bioengineering7040157 Pablo-Romero, M. del P., Sánchez-Braza, A., Salvador-Ponce, J., SánchezLabrador, N., 2017. An overview of feed-in tariffs, premiums and tenders to promote electricity from biogas in the EU-28. Renew. Sustain. Energy Rev. 73, 1366–1379. https://doi.org/10.1016/j.rser.2017.01.132 Parenti, A., Zegada-lizarazu, W., Pagani, E., Monti, A., 2022. Soil organic carbon dynamics in multipurpose cropping systems. Ind. Crop. Prod. 187, 115315. https://doi.org/10.1016/j.indcrop.2022.115315 Peters, G.P., Geden, O., 2017. Catalysing a political shift from low to negative carbon. Nat. Clim. Chang. 7, 619–621. https://doi.org/10.1038/nclimate3369 Pirelli, T., Chiumenti, A., Morese, M.M., Bonati, G., Fabiani, S., Pulighe, G., 2021. Environmental sustainability of the biogas pathway in Italy through the methodology of the Global Bioenergy Partnership. J. Clean. Prod. 318, 128483. https://doi.org/10.1016/j.jclepro.2021.128483 Rafiee, A., Khalilpour, K.R., Prest, J., Skryabin, I., 2021. Biogas as an energy vector. Biomass and Bioenergy 144, 105935. https://doi.org/10.1016/j.biombioe.2020.105935 Reeves, D.W., 1997. The role of soil organic matter in maintaining soil quality in continuous cropping systems. Soil Tillage Res. 43, 131–167. https://doi.org/10.1016/S0167-1987(97)00038-X Schulte, L.A., Dale, B.E., Bozzetto, S., Liebman, M., Souza, G.M., Haddad, N., Richard, T.L., Basso, B., Brown, R.C., Hilbert, J.A., Arbuckle, J.G., 2022. Meeting global challenges with regenerative agriculture producing food and energy. Nat. Sustain. 5, 384–388. https://doi.org/10.1038/s41893021-00827-y Sillanpää, M., Ncibi, C., . A circular world, The Circular Economy. https://doi.org/10.1016/b978-0-12-815267-6.00005-0 25.

(35) Chapter 1 Slepetiene, A., Kochiieru, M., Jurgutis, L., Mankeviciene, A., Skersiene, A., Belova, O., 2022. The Effect of Anaerobic Digestate on the Soil Organic Carbon and Humified Carbon Fractions in Different Land-Use Systems in Lithuania. Land 11. https://doi.org/10.3390/land11010133 Stockmann, U., Adams, M.A., Crawford, J.W., Field, D.J., Henakaarchchi, N., Jenkins, M., Minasny, B., McBratney, A.B., Courcelles, V. de R. de, Singh, K., Wheeler, I., Abbott, L., Angers, D.A., Baldock, J., Bird, M., Brookes, P.C., Chenu, C., Jastrow, J.D., Lal, R., Lehmann, J., O Donnell, A.G., Parton, W.J., Whitehead, D., Zimmermann, M., 2013. The knowns, known unknowns and unknowns of sequestration of soil organic carbon. Agric. Ecosyst. Environ. 164, 80–99. https://doi.org/10.1016/j.agee.2012.10.001 Stürmer, B., Leiers, D., Anspach, V., Brügging, E., Scharfy, D., Wissel, T., 2021. Agricultural biogas production : A regional comparison of technical parameters. Renew. Energy 164, 171–182. https://doi.org/10.1016/j.renene.2020.09.074 Styles, D., Yesufu, J., Bowman, M., Prysor Williams, A., Duffy, C., Luyckx, K., 2022. Climate mitigation efficacy of anaerobic digestion in a decarbonising economy. J. Clean. Prod. 338, 130441. https://doi.org/10.1016/j.jclepro.2022.130441 Theuretzbacher, F., Lizasoain, J., Lefever, C., Saylor, M.K., Enguidanos, R., Weran, N., Gronauer, A., Bauer, A., 2015. Steam explosion pretreatment of wheat straw to improve methane yields: Investigation of the degradation kinetics of structural compounds during anaerobic digestion. Bioresour. Technol. 179, 299–305. https://doi.org/10.1016/j.biortech.2014.12.008 Thompson, P., Thompson, B., P., 2012. The Agricultural Ethics of Biofuels: The Food vs. Fuel Debate. Agriculture 2, 339–358. https://doi.org/10.3390/agriculture2040339 Toop, T.A., Ward, S., Oldfield, T., Hull, M., Kirby, M.E., Theodorou, M.K., 2017. AgroCycle - Developing a circular economy in agriculture. Energy Procedia 123, 76–80. https://doi.org/10.1016/j.egypro.2017.07.269 Valli, L., Rossi, L., Fabbri, C., Sibilla, F., Gattoni, P., Dale, B.E., Kim, S., Ong, R.G., Bozzetto, S., 2017. Greenhouse gas emissions of electricity and biomethane produced using the Biogasdoneright® system: four case 26.

(36) Chapter 1 studies from Italy. Biofuels, Bioprod. Biorefining 11, 847–860. https://doi.org/10.1002/bbb.1789 Venus, T.E., Strauss, F., Venus, T.J., Sauer, J., 2021. Understanding stakeholder preferences for future biogas development in Germany. Land use policy 109, 105704. https://doi.org/10.1016/j.landusepol.2021.105704 Whiticar, M.J., 2020. The Biogeochemical Methane Cycle, Hydrocarbons, Oils and Lipids: Diversity, Origin, Chemistry and Fate. https://doi.org/10.1007/978-3-319-90569-3_5 Zhong, W., Zhang, Z., Luo, Y., Sun, S., Qiao, W., Xiao, M., 2011. Bioresource Technology Effect of biological pretreatments in enhancing corn straw biogas production. Bioresour. Technol. 102, 11177–11182. https://doi.org/10.1016/j.biortech.2011.09.077. 27.

(37) 2. Biomethane production using double cropping system with various sorghum phenotypes Abstract In order to achieve a sustainable energy crops production, energy crops should not compete for land against feed and food crops production. One option towards sustainable energy crop cultivation is the use of double cropping systems with minimum tillage use and digestate as natural fertilizer, where in the same growing season a second crop for biomethane production is planted after a first crop used for feed/food. Different sorghum phenotypes have been evaluated in the present study as first and second crop in a double cropping system. The Principal Component Analysis of the various sorghum phenotypes showed that starch content positively affects the methane production. However, sorghum chemical composition did not influence the profitability of bioenergy production as much as the total solids biomass yields of the different sorghum phenotypes. The most total solids biomass productive sorghum phenotype led to the highest methane hectare yield..

(38) Chapter 2. 2.1. Introduction Intensive soil tillage and external inputs such as synthetic fertilizers and pesticides to maximize crop productivity has been often used in cereals monoculture. These agronomic practices lead to soil physical erosion, recalcitrance of agricultural chemicals on the land, the need for weed control, nutrient leaching, and loss of biodiversity with a resulting considerable use of fossil fuels (Zegada-Lizarazu and Monti, 2011). It is general accepted that the biomethane valorization from anaerobic digestion obtaining heat, electricity or transport biofuel contributes towards the targets for renewable energy production and greenhouse gas mitigation in many countries. Within the available alternatives of organic feedstock suitable to be digested in biogas plants, energy crops show many advantages due to their energetic content (Mahmood et al., 2013). Maize had represented for many years the most predominant energy crop for biogas production in Central Europe but its cultivation was often carried out increasing the arable land areas (Mahmood et al., 2013; Wannasek et al., 2017). Additionally maize utilization in biogas plants was roughly criticized in relation to the food versus fuel debate for its possible disturbance with food/feed production (Thompson et al., 2012). The sustainable production of food and feed must be a priority and energy crops cultivation should not impair it. Double cropping consists in growing two crops in succession, in the same arable land and in the same growing season where the second crop is planted after the first crop is harvested (Hexem and Boxley, 1986). Double cropping has been proposed as a sustainable energy crops production system (Graß et al., 2013). Dale et al. (2016) proposed the Biogasdoneright® concept in which in a biogas oriented. 29.

(39) Chapter 2 double cropping system, food/feed production as first crop was not only affected by the cultivation of the second crop for biogas production but promoted chemical fertilizer reduction. Considering that just about. ‒. %. of the carbon fed to the digesters is converted into biogas, a part of this residual carbon, in addition to other nutrients, is incorporated into the soil in stable form by digestate valorization as natural fertilizer. Combing the digestate utilization with reduced or no tillage practices the soil carbon levels can be increased with a global improvement of soil fertility. Sorghum has been assessed and studied in the last years as energy crop for biomethane production (Mayer et al., 2014; Valli et al., 2017). Sorghum is a C4 plant with high photosynthetic efficiency that exhibits a higher drought resistance due to the extensive root system that allows to an efficient use of water from deep soil layers and minor fertilization requirement in comparison to other crops for bioenergy production (Shoemaker and Bransby, 2011; Theuretzbacher et al., 2013; Zegada-Lizarazu and Monti, 2011). Wannasek et al. (2017) showed that the cultivation of sorghum is a promising option for biomethane production especially with regard to sustainable increase in land use efficiency. There are different varieties of sorghum available on the market but detailed information about their chemical composition, biomass yield and methane hectare yield are limited, and especially scarce when sorghum is cultivated as second crop as feedstock in anaerobic digestion. The aim of this study is to evaluate different sorghum phenotypes for biomethane production when cultivated as first crop and second crop in a double cropping system. Correlation between chemical composition, biomass yield and methane production will be evaluated and discussed.. 30.

(40) Chapter 2. 2.2. Material and methods 2.2.1. Sorghum cultivation, harvest, and sampling The agronomical trials were carried out at two experimental field sites placed in Emilia Romagna region in Italy named STUARD Experimental Farm (San Pancrazio - Parma) and BETA Scarl (Baura and Contrapò - Ferrara). The trials examined the sowing of different varieties of sorghum as first and second crop in both experimental sites. The choice of commercial sorghum varieties was done on the basis of their maturation cycle, location of experimental field site and climate in order to be able to harvest the crops at four different growth stages up to the physiological maturity of plants (Zadocks et al., 1974). The commercial sorghum varieties used in the present study were classified in four group on the basis of their phenotype, as reported in Shoemaker and Bransby (2011). Each phenotype was identified by abbreviation from S1 to S4: forage (S1), high-tonnage energy (S2), sweet (S3) and grain (tall size) (S4). The sorghum phenotypes are showed in Figure 7. The field experiments were carried out as a randomized block design with four replicates for each variety of sorghum cultivated as first crop and second crop at four growth stages in the two experimental sites, as shown in Table 1. It is important to remark the crops previously used in the two experimental field sites to give an exhaustive overview of the rotation cycles. The previous crops in STUARD experimental farm were cabbage with sorghum as first crop and forage barley with sorghum as second crop. The previous crops in BETA were sequentially triticale and sorghum with sorghum as first crop and triticale with sorghum as second crop.. 31.

(41) Chapter 2. Forage sorghum (S1). High-tonnage energy sorghum (S2). Sweet sorghum (S3). Grain (tall size) sorghum(S4). Figure 7: The sorghum varieties used in the present study were classified in four phenotypes: forage (S1), high-tonnage (S2), sweet (S3) and grain (tall size) (S4).. 32.

(42) Chapter 2. Table 1 - Commercial sorghum varieties used in the two experimental field sites (BETA and STUARD) and harvest time. Grey colors indicate conditions that has been tested for biomethane production. Phenotype. S1. S2. Forage. Hightonnage. Sweet. Genetic background*. Variety. Supplying Experimental Cropping company field site Società Italiana Sementi (SIS). First. Sugar Graze II. Padana Sementi. Second. S. bicolor X S. bicolor. Bulldozer. KWS. First. S. bicolor X S. bicolor. Hannibal. S. bicolor X S. saccharatum. Super Sile 20. Caussade Semences. First. S. bicolor X S. sudanense. Freya. KWS. Second. S. bicolor X S. bicolor. PR845F. S. bicolor X S. bicolor. PR845F. S. bicolor X S. sudanense (BMR). BMR 333. S. bicolor X S. sudanese X S. saccharatum. Harvest 1st. 2nd. 3rd. 4th. BETA STUARD BETA STUARD BETA STUARD BETA. KWS. Second STUARD BETA STUARD. S3. S4. BETA STUARD BETA Pioneer. First STUARD. Grain (tall size). BETA Pioneer. Second STUARD. * Manufacturer specification. In STUARD Experimental Farm the sowing was on May 23rd and on June 13rd when sorghum was cultivated as first crop and second crop respectively. In such experimental site the distance between the single rows in each plot was 0.45 m in both cases.. 33.

(43) Chapter 2 In BETA the sowing was on May 16th and on May 22nd when sorghum was cultivated as first crop and second crop respectively. In such experimental site the distance between the single rows in each plot was 0.75 m in both cases. The depth of the sowing was 0.02-0.03 m. All experimental plots showed a silty clay loam soil texture. Tillage by plough and subsoiler and secondary tillage were used with sorghum cultivated as first crop while minimum tillage by harrow and grubber were performed with sorghum cultivated as second crop. STUARD and BETA experimental field sites were characterized by an. average cumulative rainfall within the sorghum vegetation period of 208 and 214 mm, respectively. The abundant rainfall and the optimal temperature monitored daily during the experimental activity established a sufficient water supply and for this reason irrigation during the growing of sorghum was not necessary. In STUARD Experimental Farm the fertilization was carried out on growing crops with 100 kg N/ha of urea when sorghum varieties were cultivated both as first crop and second crop. In BETA the fertilization was carried out on growing crops with 90 kg N/ha of urea when sorghum varieties were cultivated both as first crop and second crop. In BETA, sorghum as second crop was additionally fertilized in pre-sowing with 20 m3/ha of liquid fraction of digestate. In BETA, 6 liters ha-1 of CICLON® (Glyphosate 360) was used as herbicide before the sowing of sorghum cultivated as first crop. The harvesting of sorghum took place in four moments at different growth stages depending on the variety. When sorghum was cultivated as first crop the first harvest was carried out between 94 and 96 days after the sowing, the second harvest between 111 and 112 days after the sowing, the third harvest between 126 and 132 days after the sowing while the fourth and. 34.

(44) Chapter 2 latest harvest was done after 145 days after the sowing. When sorghum was cultivated as second crop, the first harvest was carried out between 94 and 96 days after the sowing, the second harvest between 107 and 112 days after the sowing, the third harvest between 116 and 126 days after the sowing while the fourth and latest harvest was done after 136 days after the sowing. The plants height from the soil surface to the base of the tallest leaf was measured on three plants per harvested area immediately before each harvest. At the moment of harvest the growth stage, the biomass yield, i.e. harvested wet weight per harvested area, were also determined and recorded. The identification and the description of sorghum growth stage were done adapting the general Zadocks Growth Stages (Zadocks GS) scale for cereals to sorghum. Sorghum was harvested and crushed to an average particle size of 2.5 cm by a plot forage harvester and the net size of each harvested area varied from 7 to 15 m2 depending on the experimental site (STUARD or BETA) and operational conditions. The harvested samples were dried within 24 hours at 65°C until constant weight and grounded at 0.5 mm texture using a mill to determine the total solids of biomass. The dried and milled sorghum samples were store in airtight zip-lock bags at room temperature until laboratory analysis. Within the overall experimental time, 64 conditions were collected and processed for chemical composition determination by near infrared reflectance spectroscopy (NIRS) while in 56 of them the specific methane yields and methane production rate were additionally measured by biochemical methane potential (BMP) tests (Table 1).. 35.

(45) Chapter 2 2.2.2. Near infrared reflectance spectroscopy (NIRS) Quadruplicates of all 64 conditions were analyzed for chemical components. characterization. using. the. near. infrared. reflectance. spectroscopy NIRS (Table 1). In particular, total solids (TS), ash, crude protein (CP), starch, sugars, lipids, neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL) were predicted. The predicted values of NDF, ADF, and ADL concentration were used to calculate hemicellulose (HCEL) by subtracting ADF from NDF and cellulose (CEL) by subtracting ADL from ADF. The samples enclosed in spinning ring cups (36 mm inside diameter) were scanned on Foss NIRSystem 5000 monochromator in the 1100-2498 nm spectral region. Spectra recorded as log 1/R, where R is the reflectance, were taken at 8 nm intervals using 173 wavelength. Mathematical treatments of spectral data were performed with the WinISI II V1.5 software (Infrasoft International, Port Matilda, PA, USA), using first order derivative with gaps and smoothing each 4 data point and standard normal variate and detrend procedure (SNVD) was applied for scatter correction. The Modified Partial Least Squares (MPLS) regression technique was used to develop the NIRS calibrations. The statistics used for the accuracy assessment of calibration equation were the standard error of calibration (SEC) and the coefficient of determination for the calibration (R2); the statistic used for the accuracy assessment of prediction equation were the standard error of cross-validation (SECV) and the coefficient of determination for cross validation (1-Vr). 2.2.3. Biochemical methane potential (BMP) test A total of 31 sorghum phenotype cultivated as first crop and 25 sorghum phenotypes cultivated as second crop were analyzed for the. 36.

(46) Chapter 2 determination of methane yield and methane production rate in order to have 6-8 conditions for each sorghum phenotype and distributed among the four harvests (Table 1). Methane yields and methane production rate were evaluated by BMP tests according to standard UNI EN ISO 11734:2004 and described in Langone et al (2018). The inoculum used in BMP tests was collected from an agricultural biogas plant (Reggio Emilia, Italy) and incubated at 38°C for 7 days prior to use. Glass bottles with 1.35 L working volume with 0.5 ratio between volatile solids (VS) from substrate and volatile solids from inoculum were used. BMP tests were run at 38°C in a thermostatic room for about 27 days. Biogas production was quantified by manometric system measuring the increase of headspace pressure increasing in the bottle headspace. Productivity of inoculum is calculated separately by inoculum-only bottle (blank). The quality of biogas is determined by gas analyzer (Model Gasboard3200 biogas analyzer, Wuhan Cubic Optoelectronics Co., Ltd) based on NonDispersive Infrared technology for methane (CH4) and carbon dioxide (CO2) gases and electrochemical sensors for O2 and H2S gases. The net methane production was obtained from the difference between the methane measured in the bottle with sorghum as substrate and the blank one. Specific methane yield was calculated by dividing the methane volume (LN) by the quantity of the sample added to each bottle (kgVS added) and it was expressed as normal litres of methane per kilogram of the volatile solids (LN kgVS−1). Specific. methane production rate is calculated by dividing the methane yield by the. corresponding time unit (day) and it is expressed as normal litres of methane per kilogram of the volatile solids produced in the time unit (LN kgVS−1 day-1).. The maximum value of specific methane production rate (Rmax) reached during the BMP test was used to describe the kinetic of the biological process.. 37.

(47) Chapter 2 The methane hectare yield for each sorghum phenotype was calculated relating the specific methane yield measured by BMP tests with the biomass yield data obtained from field experiments and the chemical characteristics evaluated by NIRS analysis. 2.2.4. Principal Component Analysis (PCA) Principal Component Analysis (PCA) was performed to investigate how chemical parameters could affect the methane production reducing the interrelated effect of the variables. Principal Component Analysis was applied to the dataset of 56 conditions where both NIR spectroscopy analysis and BMP tests were carried out to disclose the relations among the variables, particularly between chemical components, methane yields and methane production rate (SPSS Statistic software, SPSS Inc., Chicago, USA).. 2.3. Results and Discussion 2.3.1. Biomass yield -Sorghum cultivated as first crop When sorghum was used as first crop, the highest biomass yields were obtained with the last harvest at 145 days after sowing (Figure 8A). At that time S1 and S2 were in the early dough growth stage (Zadocks GS 83) in which nutrients uptake from soil is almost complete and lower leaves lose their functionality due to remobilization of nutrients to the upper part of plant. In this early dough growth stage, S1 and S2 reached a biomass yield of 99±16 and 97±25 Mg ha-1, respectively (Figure 8A). At the same time of harvest, i.e. 145 days, S3 and S4 showed a biomass yield of 82±10 and 59±17 Mg ha -1, respectively (Figure 8A), reaching the physiological maturity growth stage (Zadocks GS 89) in which grain should achieve its maximum dry weight.. 38.

(48) Chapter 2 S1 (A). S2. S3. Second crop. First crop. S4. 140. 140. (E). 120 Biomass yield (Mg ha-1). Biomass yield (Mg ha-1). 120 100 80 60 40. 100 80 60 40. 20. 20. 0. 0. 40. 40. (F). 35. Biomass TS yield (MgTS ha-1). Biomass TS yield (MgTS ha-1). (B). 30 25 20 15 10 5. 35 30 25 20 15. 10. 0. 0. 6. 6. (G). 5. 5. 4. 4 Height (m). Height (m). (C). 3. 2. 1. 1 0. 40. 40. (H). 35. 35. 30. 30. 25. 25 TS (%). TS (%). 3. 2. 0. (D). 5. 20. 20. 15. 15. 10. 10. 5. 5 0. 0 55 55 61 73. 61 61 77 83. 73 73 83 87. 61 49 61 61. 83 83 89 89. Zadocks Growth Stages 1st (94-96). 2nd (111-112). 3rd (126-132). 73 55 73 73. 77 61 83 83. 89 73 89 89. Zadocks Growth Stages 4th (145). 1st (94-96). Harvest (days after sowing). 2nd 3rd (107-112) (116-126) Harvest (days after sowing). 4th (136). Figure 8: Biomass yield, biomass total solids yield, height and total solids at time of harvest of S1-S4 Sorghum phenotypes as a function of growth stages. Data are showed as average of four replicates in each experimental field sites (STUARD and BETA). Zadock Growth Stage: 49= booting; 55= inflorescence emergence; 61= anthesis; 73=early milk; 77= late milk; 83=Early dough; 87=Hard dough; 89=physiological maturity.. 39.

(49) Chapter 2 The biomass total solids yield of harvested S1-S4 sorghum ranged from 10.9±1.8 to 29.0±9.0 MgTS ha-1 within the different growth stages (Figure 8B). The highest biomass total solids yield was obtained during the last harvest of S2, which was in its early dough growth stage while the lowest biomass total solids yield was obtained during the first harvest of S4 in its early milk growth stage (Zadocks GS 73) (Figure 8B). The highest biomass total solids yield obtained with S2 was 16%, 33%, 75% higher than those obtained with the cultivation of S1, S3, S4 respectively (Figure 8B). Low temperatures and drought during early growth stages could induce several physiological dysfunctions in sorghum carrying in many cases to limited biomass yield in terms of both wet weight and total solids (Tuinstra et al., 1997). In this study, the rainy summer period and the mild temperature in both field sites played an important role to facilitate the vegetative growth of all S1-S4 sorghum as first crop reaching biomass yields comparable with other studies (Mahmood et al., 2013; Wannasek et al., 2017). When sorghum was cultivated as first crop, the shortest height was observed in S4 in the interval from 1.94 to 2.03 m without significantly increasing from early milk to physiological maturity growth stages (Zadocks GS 73 to 89, respectively) (Figure 8C). The tallest height was reached in S2 in the anthesis, early milk, early dough maturity growth stage (Zadocks GS 61, 73, 83, respectively) when sorghum plants grown up over 4.5 m (Figure 8C). The big size of S2 permits to produce a huge amount of biomass yield with reduced grain quantity and this characteristic is typically observed in several high-tonnage sorghum phenotypes. High-tonnage sorghum (S2) is taller than grain sorghum (S4) that normally shows short panicles and panicle branches (Harlan and de Wet, 2010). S2 was leafier and late maturing in comparison to S4 as confirmed in other studies where forage sorghum (S1) and grain. 40.