In situ nitrogen fixation was investigated in a cyanobacterial mat growing on the bed of rocks of the Muga River, Spain. The filamentous non-heterocystous cyanobacterium Schizothrix dominated the mat, showing nitrogenase activity in the light at similar rates to those found in nearby heterocystous Rivularia colonies. N2 fixation in the light was significantly increased by an inhibitor of PSII and oxygen evolution, DCMU (3-[3,4-dichlorophenyl]-1,1-dimethylurea), and anaerobic conditions. However, no nitrogenase activity was found in the dark. Addition of fructose as a respiratory substrate induced nitrogenase activity in samples incubated under aerobic conditions in the dark but not in anaerobic conditions. Microelectrode oxygen profiles showed internal microaerobic microzones where nitrogen fixation might concentrate. Analyses of the 16S rRNA gene revealed only the presence of sequences belonging to filamentous non-heterocystous cyanobacteria. nifH gene diversity showed that the major phylotypes also belonged to this group. One of the three strains isolated from the Schizothrix mat was capable of fixing N2 and growing in the absence of combined N. This was consistent with the nifH gene analysis. These results suggest a relevant contribution of non-heterocystous cyanobacteria to nitrogen fixation in these mats.

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In order to estimate the potential utilization of N2-fixing (heterocystous) cyanobacteria as natural biofertilizers in the Valencian rice fields (Spain), the distribution and seasonal variation of these microorganisms in water and sediment samples were evaluated, and the relationships among cyanobacterial abundance and physical and chemical characteristics of soil and water were investigated. N2-fixing cyanobacteria were present in all the samples analyzed (25 sampling points sampled three times per year during two years). The relative cyanobacterial abundance in soil and water followed contrasting patterns, maximum presence in soil coincided with minimum abundance in water. Correlation analysis showed that cyanobacterial abundance in the two phases (water and sediment) was influenced more by water than by soil properties. Salinity, mineralization variables, and soluble reactive phosphate (SRP) correlated positively with heterocystous cyanobacteria presence. Furthermore, dissolved inorganic nitrogen (DIN) and the ratio DIN:SRP correlated negatively with cyanobacterial abundance. However DIN: SRP ratio better described the cyanobacterial distribution, with a threshold effect: below the Redfield ratio value (7.2 in mass units) cyanobacterial abundance was clearly higher.

The distribution of nitrogenase activity in the rice-soil system and the possible contribution of epiphytic cyanobacteria on rice plants and other macrophytes to this activity were studied in two locations in the rice fields of Valencia, Spain, in two consecutive crop seasons. The largest proportion of photodependent N 2 fixation was associated with the macrophyte Chara vulgaris in both years and at both locations. The nitrogen fixation rate associated with Chara always represented more than 45% of the global nitrogenase activity measured in the rice field. The estimated average N 2 fixation rate associated with Chara was 27.53 kg of N ha ؊1 crop ؊1 . The mean estimated N 2 fixation rates for the other parts of the system for all sampling periods were as follows: soil, 4.07 kg of N ha ؊1 crop ؊1 ; submerged parts of rice plants, 3.93 kg of N ha ؊1 crop ؊1 ; and roots, 0.28 kg of N ha ؊1 crop ؊1 . Micrographic studies revealed the presence of epiphytic cyanobacteria on the surface of Chara. Three-dimensional reconstructions by confocal scanning laser microscopy revealed no cyanobacterial cells inside the Chara structures. Quantification of epiphytic cyanobacteria by image analysis revealed that cyanobacteria were more abundant in nodes than in internodes (on average, cyanobacteria covered 8.4% ؎ 4.4% and 6.2% ؎ 5.0% of the surface area in the nodes and internodes, respectively). Epiphytic cyanobacteria were also quantified by using a fluorometer. This made it possible to discriminate which algal groups were the source of chlorophyll a. Chlorophyll a measurements confirmed that cyanobacteria were more abundant in nodes than in internodes (on average, the chlorophyll a concentrations were 17.2 ؎ 28.0 and 4.0 ؎ 3.8 g mg [dry weight] of Chara ؊1 in the nodes and internodes, respectively). These results indicate that this macrophyte, which is usually considered a weed in the context of rice cultivation, may help maintain soil N fertility in the rice field ecosystem.Rice fields are one of the most extensive freshwater ecosystems on Earth, covering about 150 ϫ 10 6 ha. In spite of the widespread dominance of rice plants, a conspicuous photosynthetic aquatic biomass composed of cyanobacteria, planktonic, filamentous, and macrophytic algae, and vascular macrophytes develops during different phases of rice growth and competes with rice for nutrients and light (23). Nitrogen is a major factor in rice production. However, the efficiency of N fertilizer is one of the lowest efficiencies of all plant nutrients due to large N losses from flooded soils (7). Native soil nitrogen is the main N source for rice, accounting for more than 50% of the N in the rice plant (9, 10, 17), and so long-term sustainability of rice cultivation depends on the use and effective management of internal resources that maintain soil N fertility. The soil N pool is believed to be maintained by biological nitrogen fixation (17,25). In fact, biological N 2 fixation has allowed a stable and moderate yield to be maintained in traditional low-input rice cul...

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