Turning the aquatic weed Azolla into a sustainable crop

Abstract

Growing worldwide demands for food, energy and chemicals threatens natural ecosystems and global climate. Plants are crucial for food production, but may also be used to produce sustainable energy and materials. Hereto novel crops are sought with high productivity per hectare, whilst requiring minimal fossil fuel or fertilizer input. The aquatic fern Azolla is a potential novel crop due to its rapid growth, symbiosis with a nitrogen fixing cyanobacteria and favorable chemical composition. The aim of this thesis is to provide the knowledge and methods necessary to turn the aquatic weed Azolla into a sustainable crop. To provide reliable starting material for cultivation we developed methods to collect, store, fertilize and germinate Azolla spores. Drying for 7 days followed by freezing in liquid nitrogen (cryopreservation) allowed to preserve Azolla spores at -80°C for up to 7 months, as well as the cyanobacteria symbiont residing inside the spores. To better understand the mechanism leading to the formation of spores (sporulation), a first transcriptome database of Azolla was constructed, revealing that ferns have many genes alike those regulating sexual reproduction in seed plants. Growth potentials of A. filiculoides and A. pinnata were determined at 35.5 and 32.8 t dry weight (dw) ha-1 year-1, respectively. Addition of CO2 up to 800 ppm boosted biomass productivity to 48.3 t dw ha-1 year-1. Under ambient CO2, N2-fixation by the fern’s cyanobacterial symbionts completely supported Azolla’s rapid growth, fixing 1200 kg N ha-1 annually. Nitrogen was predominantly fixed during the day by the phototropic Nostoc Azollae. Analysis of diel transcript profiles and microscopic investigations revealed several adaptations to this form of N-supply in Azolla’s metabolism, leaf architecture and transcriptional regulation of nutrient transport. Rapidly growing Azolla contained between 17.6-20.8% protein, valuable as feed due to a high content of essential amino acids compared to soybean meal. While elevated CO2 concentrations boosted biomass yield, protein content and amino acid composition remained similar. Azolla biomass further contained 7.9-10.0 % lipids of which 41±13% consisted of fatty acids that could be converted into biodiesel. Azolla biodiesel meets requirements set by European standards on fuel density, cetane number and iodine value. However, a fractionation step will be required to decrease the high Cold Filter Plugging Point. Azolla was shown to have a relatively high content of soluble (poly)phenols, including 4.0-5.8% of condensed tannins, that are known to limit protein digestibility. Various polyphenol biosynthesis genes were identified in the A. filiculoides genome, including a highly expressed Leuco-Anthocyanidin Reductase (AzfiLAR-like), likely essential for the synthesis of condensed tannins. Digestibility of Azolla biomass may be enhanced by manipulating (poly)phenol biosynthesis, e.g. by knocking out the AzfiLAR-like gene, or by extracting protein separately from the (poly)phenols. Since the formation of protein-tannin complexes limited the yield of alkaline protein extraction we developed two methods to produce protein extracts with reduced (poly)phenol content: pre-extraction using aqueous acetone and competitive binding of tannins to polyethylene glycol during extraction. Both methods produced a protein precipitate containing 38% of total biomass nitrogen, while considerably reducing (<-67%) (poly)phenol content.