C. Forni

Evaluation of the fern Azolla for growth, nitrogen and phosphorus removal from wastewater.

Water fern (Azolla filiculoides Lam.) has been assessed for nitrogen and phosphorus removal in outdoor experiments comparing sewage water (S) from an experimental aquaculture plant, well water (W) and mineral growth medium Hoagland (H). The experiments were undertaken during the spring and the summer. The yield of fern biomass and nitrogenase activity was higher in H than in W and S waters. The enzyme activity had a decreasing trend with significant differences (p < 0.05) in the three waters. Peroxidase (POD) activity in April decreased with significative differences in W and S waters (p < 0.05). The electrical conductivity and the concentrations of NO3- in the three waters decreased significantly (p < 0.05). The highest removal of nitrate from the media was obtained in July. In S water, NO2- concentration decreased, while it increased in W water. PO(4)3- concentration was very low in W and S waters and decreased in H medium. The results obtained confirm the ability of the fern to grow in sewage water.

Indole-3-acetic acid (IAA) production by Arthrobacter species isolated from Azolla

Arthrobacter species, isolated from the leaf cavities and the microsporocarps of the aquatic fern species Azolla pinnata and Azolla filiculoides, produced indole-3-acetic acid (IAA) in culture when the precursor tryptophan was added to the medium. No IAA production was detected in the absence of tryptophan. Maximum IAA formation was obtained in the first 2 d of incubation. Part of the tryptophan was transformed to N alpha-acetyl-L-tryptophan.

Effects of antibiotic treatments on Azolla-Anabaena and Arthrobacter

The leaf cavities and the sporocarps of the water fern Azolla contain bacteria belonging to the genus Arthrobacter Conn and Dimmick. The number of these bacteria can be decreased by treating the plants with novobiocin and erythromycin. The latter antibiotic kills the Anabaena, so Anabaena free plants can be obtained. In these plants the bacteria disappear almost completely. The novobiocin does not affect the heterocyst differentiation, but it decreases the N2-fixation activity by affecting the synthesis of the dinitrogenase reduciase.

Bacteria in the Azolla-Anabaena symbiosis

The leaf cavities of the fern Azolla contain the N2-fixing cyanobacterium Anabaena Azollae Strasb. and bacteria. These bacteria have been isolated from five Azolla species, grown in the Botanic Garden of Naples, Italy; they were aerobic, Gram-positive and with a rod-coccus life cycle. Moreover they neither fixed nitrogen nor produced gas, H2S or indole, and they were catalase positive. Nutritionally non-exacting strains grew on mineral salts medium with an ammonium salt and a carbon source. Most of the isolates produced mucilage. The bacteria were identified as Arthrobacter globiformis Conn and Dimmick, A. nicotianae Giovannozzi-Sermanni, A. aurescens Phillips and A. cristallopoietes Ensign and Rittenberg or A. pascens Lochhead and Burton. Among the species isolated A. globiformis is the most common. It is likely that the mucilage produced by A. globiformis and A. aurescens fulfils the function of retaining bacteria and Anabaena in the leaf cavities; moreover the bacteria, alone or together with the endophyte, may contribute to the formation of the envelope, which contains bacteria and algal cells.

The Hard Life of Prokaryotes in the Leaf Cavities of Azolla

Life of cyanobionts and bacteriobionts inside the leaf cavities of Azolla cannot be so easy. Fern shows many features of remedation plant, able to adapt itself to the change of the environmental conditions such as high light intensity, high temperature or water composition. Cyanobionts seem to modify their metabolism according to that of the fern, and whenever the host life conditions become stressing, the symbiont can be eliminated (Hill, 1975; Kitoh et al., 1991). Therefore speaking in term of symbiosis, it may be not so easy for the symbionts, since Azolla behaves at its convenience, forcing them to follow its benefit. Anyway, some authors consider the Azolla-Anabaena relationship like an happy marriage between lucky partners, where each of them contributes to the menage. Some considerations can be made on the different aspects of such unusual association.

Structure, Metabolism and Nitrogenase Regulation in the Azolla-Anabaena Association

Structural relations between the Azolla leaf cavity envelope, hair cells and the cyanobiont Anabaena azollae were tested with frozen hydrated preparations by scanning electron microscopy, exhibiting close interactions between the cyanobiont and the cavity envelope cells, and a mucilage layer covering parts of the cavity envelope. Hair cells contained starch granules and the cyanobiont exhibited amylase activity hence, both starch, fructose and sucrose can serve as carbon substrates of the cyanobiont. Light regulation of nitrogenase seems to involve the direct effect of light and the effect of oxygen as resolved by nitrogenase analysis and immuno-blot of dinitrogenase reductase. The respiratory activity of the cyanobiont is high and heterotrophic growth increased cytochrome oxidase activity.

Azolla: An efficient N2-fixing association with three components

Abstract Bacterial population is found together with the cyanobacterium Anabaena azollae in the leaf cavities and the sporocarps of Azolla. Different Arthrobacter species have been invariably isolated from the leaf cavities and sporocarps of several fern species. About the role of the bacteria in the association, it is probable that the bacteria affect the carbon and nitrogen metabolism of the cavity by competing with the Anabaena for the nutrients and in the meantime it is likely that their high respiratory activity favours the establishment of a microaerobic environment and consequently the nitrogenase activity. Furthermore Arthrobacter may contribute to the production of the mucilage present in the algal packets by excreting extracellular polysaccharides, which components can be involved in lectin binding with Azolla and Anabaena. Finally the IAA formation by Arthrobacter may influence the hormonal balance during the establishment of the association.