# Sustaining Rice Production through Biofertilization with N\({}_{2}\)-Fixing Cyanobacteria
## 1 Introduction  
One of the challenges facing agriculture today is producing enough food for an ever-growing population. Rice is the most important cereal crop in the world and the major food source for millions of households in dozens of countries [1]. In 2019, the world dedicated more than 150 million hectares and almost a third of the Earth's fresh water ([[http://www.fao.org/faostat/en/#data/QC](http://www.fao.org/faostat/en/#data/QC)]([http://www.fao.org/faostat/en/#data/QC](http://www.fao.org/faostat/en/#data/QC)), accessed on 10 April 2021) to rice cultivation. Rice plants require large amounts of water and mineral nutrients, including N and P, for their growth, development, and grain production [2]. Around 16-17 kg N/ton of N fertilizer, generally urea, are used to meet the crop's demands [3]. Wetland areas provide a perfect place for rice cultivation, but flooded conditions call for the usage of large amounts of inorganic N fertilizers to account for its loss into adjacent water bodies. In fact, only30-40% of the fertilizer dose, even less in some cases, is utilized by the crop [4]. Large amounts of N fertilizers are lost into surface waters, polluting the environment and causing crop health issues due to ammonia volatilization, denitrification, and leaching [5]. Nitrate that is leached from agricultural systems causes serious eutrophication of ecosystems. To date, more than 150 eutrophic and hypoxic coastal areas have been identified in Europe, mostly at the delta of important European rivers, including zones where rice is cultivated [6]. One of them occurs in the Guadalquivir marshes, located in the Donana National Park in southern Spain. This space contains the largest area in Spain that is dedicated to rice cultivation, producing an average of 370,000 tons of this cereal over 40,000 ha divided into plots of 10 and 12 ha. This region is principally located in the municipalities of Isla Mayor and La Puebla del Rio, which are economically reliant on the cultivation of this cereal that is farmed by family farmers, most of whom have a few intensive farming plots [7]. At the same time, this region is of great ecological and environmental value due to its rich fauna, especially since it houses migratory birds in their journey between Europe and Africa [8]. However, intensive farming in the region is increasing the levels of N in the water bodies, enhancing the proliferation of phototrophic microorganisms [9]. Some of them that produce hepatotoxins, such as _Microystis aeruginosa_, are responsible for the cases of massive intoxication of waterfowls in the region [9;10]. The use of plant-growth-promoting bacteria (PGPB) to enhance crop yield and control disease is gaining worldwide acceptance as a sustainable agricultural practice while reducing costs by supplanting the use of expensive (and polluting) agrochemicals. These bacteria can facilitate plant growth either directly, by providing essential nutrients (nitrogen, phosphorus, and essential minerals); modulating plant hormones; and development, or indirectly, by suppressing the inhibitory effects of various plant pathogens, improving soil structure, and bio-remediating polluted soils [3]. The paddy field ecosystem represents a unique aquatic-terrestrial habitat, which provides a favorable environment for the growth of cyanobacteria. From the different groups of cyanobacteria, the N\({}_{2}\)-fixing _Nostoc_ genera are naturally found in floating assemblages, attached to the root surface, and can colonize the roots of rice [11;12]. These cyanobacteria, which are of great relevance in the global carbon and nitrogen cycles, are the main contributors to the natural deposition of nitrogen in paddy fields [13;14]. Their N\({}_{2}\)-fixing ability, which provides a natural nitrogen source for the plant, can potentially be exploited in crop agriculture. However, this biotechnological potential remains utterly unstudied. In this work, native cyanobacterial strains from the southern Spanish paddies were isolated and used as bioinoculants for rice crops.