Closing the loop of soilless cultivation residues through biochar production: its use in microgreen substrates and its role in plant growth promotion

Abstract

The number of the world population has surpassed 8 billion, and is estimated to rise above 9.7 billion by 2050, accompanied by an increase in the demand for nutritious foods. This rise in urbanization together with climate change result in decreases in arable land and resources for producers. Alternative farming strategies, like greenhouse, vertical or urban farming are required to complement the current industrial practices. Also, the enhancement of the nutritional quality of crops can contribute to the limitation of malnutrition and food insecurity. Here, microgreens are a crop of interest to easily explore cultivation techniques resulting in the sustainable production of crops, potentially increasing the nutritional values. Despite the increased interest in microgreens as a crop, research into its cultivation practices is still limited. The choice of (substrate-based) growing medium is labelled an important variable affecting microgreen growth and quality, and it has an impact on the sustainability of the cultivation practice. Here, soilless cultivation systems heavily rely on the use of peat-based substrates. However, as the need for growing substrates increases, legislation on the use of peat will become more stringent. Research into more environment-friendly alternatives is needed, and in addition, the sustainable management of spent substrate or other residual streams requires consideration. Here, biochar production from residual (waste) streams has proven itself a promising technology that implements circularity principles in the cultivation practice and, as an added-value product, contributes to a circular bioeconomy. The used pyrolysis parameters for the production of biochar, like the feedstock composition or pyrolysis temperature, can affect its potential for agronomic applications. Biochar-induced effects on eventual plant growth and crop yield in soils and substrates is already well-characterized. However, there is still a knowledge gap on the mode-of-actions of biochar-induced plant growth-stimulating mechanisms. The central theme of the current thesis was to use substrate-based cultivation derived residues (crop or spent substrates), use it as a feedstock for biochar production, and re-implement it into the subsequent (substrate) cultivations of those crops. Chapter 3 considered the use of wheat crop residue-derived biochar, and its re-use in the hemp-based cultivation of wheatgrass and the hydroponic cultivation of mature wheat. In Chapter 4, broccoli microgreens were cultivated on coconut coir amended with coconut coir-derived biochar. As a more sustainable alternative to peat and coconut coir, Chapter 5 considered miscanthus straw as an alternative substrate for wheatgrass microgreen cultivations, and also as a feedstock for biochar production that was afterwards amended to the substrate. Chapters 3 and 4 used and compared biochars from varying pyrolysis conditions (including temperature and feedstock composition). To quickly screen the produced biochars, and compare those produced using varying pyrolysis conditions, a fast in-house Arabidopsis thaliana screening was conducted using plant growth as a first indicator for the agronomic potential. In complement, to better understand the biochar-induced effects on plant growth through changes in the physicochemical characteristics of the growing medium, static leaching experiments were conducted to detect induced differences in nutrient composition, pH and EC. Here, when biochar was amended to the medium in increasing concentrations, the pH, EC and potassium (K) concentration consistently increased. This observation was also pyrolysis temperature-dependent, in which biochars produced at higher temperatures caused the same effect regarding the characteristics of the growing medium. The A. thaliana-based screening experiments always considered at least the root length and fresh weight of seedlings, as the stimulating, neutral or inhibiting effects of biochar on plant growth could be dependent on the plant parameter studied. In addition, a wide range of biochar concentrations were used to get a complete overview of the agronomic potential of the used biochars. In general, these screenings resulted in a rise-and-fall pattern regarding plant growth when increasing concentrations of biochar were used. In chapter 3, wheatgrass cultivated on hemp, or mature wheat cultivated hydroponically, also showed concentration-dependent, yield-stimulating responses to biochar. For mature wheat, the vegetative reproduction (number of spikes and grains) was also significantly affected. For broccoli microgreens in Chapter 4, biochar application did not consistently cause increases in fresh weight, as their growth remained largely unaffected. Here, the used seed priming strategies caused larger effects regarding crop yield. For the miscanthus-based cultivation of wheatgrass microgreens, miscanthus-based biochar did significantly stimulate plant growth. Throughout these chapters, the microgreen crop quality was also investigated using the determination of minerals in harvested materials, as well as a spectrophotometric determination of total antioxidative capacity (TAC), total flavonoid (TFC) or total polyphenol contents (TPC) and pigment concentrations. Across the different microgreen crops, TAC, TFC, TPC or pigments were largely unaffected by biochar application. However, the observed increases in microgreen yield were accompanied by increases in mineral concentrations, potentially attributed to the biochar-induced, higher nutrient availability. The A. thaliana-based biochar screening system was always accompanied by the analysis of the nuclear DNA content of seedlings using flow cytometry, to better understand biochar-induced effects on plant growth and development. In Chapter 3 and 4, it became evident that biochar generally increased the proxy (indicative of cell division), and did not affect or lowered the endoreplication index (EI, indicative of the amount of endocycles). Here, the organ or tissue-dependency of these responses to biochar were questioned. Therefore, in Chapter 5, these biochar-induced effects in full seedlings were compared to just rosettes, harvested at 5, 7 and 10 days after sowing (DAS), instead of the regular 7 DAS in Chapter 3 and 4. Here, the observed tissue-dependent effects of biochar on these indicators of nuclear DNA content were complemented by the observation that responses could be dependent on the time point of harvest. In Chapter 5, the influence of biochar on plant growth through the gene expression regulation of the cell cycle was also confirmed. In addition, this chapter further explored potential physiological processes behind biochar-induced plant development and growth promotion through the use of a multispectral imaging (MSI) platform. Here, plants could be non-invasively followed up throughout time. Consistent biochar-induced improvements on the photosynthetic efficiency and seedling health were observed, though other parameters (e.g. the anthocyanin index, non-photochemical quenching, reflectance ratio of red to far-red light) showed less consistent biochar-induced responses. Combined, these first MSI-derived results suggested a potential role for biochar in photomorphogenesis, further explored in Chapter 6. The experimental set-up in Chapter 6 allowed for the investigation of a potential dual role of biochar in plant growth stimulation mechanisms. Through the use of karrikin-insensitive mutant lines (kai2-1 and kai2-2), distinctive roles for the fertilizing function (dissolution of nutrients and changing the physicochemical characteristics of the medium) or the biostimulant role of biochar (through dissolvable organic compounds, like karrikins) were hypothesized and investigated. In addition, the gene expression levels of transcription factors related to photomorphogenesis (GLK1, BBX16) and their photosynthesis-related downstream genes, were measured in WT and kai2-2 seedlings exposed to miscanthus-based biochar. From the results of Chapter 6, the involvement of biochar in the improvement of plant health, photosynthetic efficiency and the regulation of photomorphogenesis-related genes was confirmed. In addition, across the paramters measured using the multispectral imaging (photosynthetic efficiency, non-photochemical quenching, chlorophyll or anthocyanin indices) and the gene expression analysis, the karrikin-insensitive mutant seedlings needed higher biochar concentrations to reach similar biochar-induced responses, compared to WT seedlings exposed to the same concentration. This indicates the potential role for KAI2 mediated signaling at lower biochar concentrations, whilst at higher biochar concentrations, the potential growth-stimulating effect through fertigation was observed in all genotypes. The miscanthus-derived biochar lacked the presence of karrikins, and was therefore screened for other organic compounds potentially fitting the binding pocket of the KAI2 receptor, as there is still some unclarity on the specifics of its ligands. Through a molecular docking analysis, several candidate ligands with a potential biostimulant function were found. To conclude, the use of biochar as a strategy to close waste loops in the soilless cultivation of microgreens has been proven as a promising strategy to increase the sustainability of these practices. Beneficial effects on plant growth and mineral contents were observed across several cultivation systems. In addition, the current PhD research provides proof of the simultaneous fertilizer and biostimulant role for biochar. More research is required, as this might have impact on its marketability within the legal framework of the European Union.