Culture of the Cyanobiont Anabaena azollae Strasburger: Is it Possible?

Special Article - Microalgae & Cyanobacteria

Austin Biol. 2017; 2(1): 1023.

Culture of the Cyanobiont Anabaena azollae Strasburger: Is it Possible?

Parente T¹, Fernandes I¹, Vasconcelos V1,2 and Pereira AL¹*

¹Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Portugal

²FCUP - Department of Biology, University of Porto, Portugal

*Corresponding author: Pereira AL, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/ CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450-208, Porto, Portugal

Received: September 14, 2017; Accepted: November 13, 2017; Published: November 28, 2017


The culture of the nitrogen-fixing cyanobacterium Anabaena azollae, the cyanobiont of the fern Azolla, is a long-time debate. Therefore, on the present research, the cyanobiont was cultured in three culture media (AA, AA1/8, BG- 110) supplemented with an aqueous extract of Azolla filiculoides, three sugar sources (fructose, D-glucosamine, N-acetyl-glucosamine), KNO3 and B12 vitamin and their fitness was assessed by the analysis of the Phycobiliproteins (PBP) content. The results of the present research showed that the addition of a fern aqueous extract to the culture media AA, AA 1/8 and BG-110 induced the loss of A. azollae cells. Also, in all culture media without any supplements and media complemented with N-acetyl-glucosamine, KNO3, and B12 vitamin, the cyanobiont is formed by very short filaments or isolated vegetative cells and heterocysts. Regarding the PBP, the phycocyanin content almost disappeared from the cyanobiont followed by a sharp decrease in the allophycocyanin and phycoerythrocyanin in a lesser extent. The cyanobionts growing in the media AA, AA 1/8 and BG-110 without sugar source and supplements, medium AA 1/8+N-acetyl-glucosamine+B12 vitamin, and medium BG-110+N-acetylglucosamine+ B12 vitamin has phycoerythrocyaninin a higher amount than the freshly isolated cyanobiont. These changes can be due to nutritional and/or light limitations that are not adequate to this cyanobiont. Also, the culture conditions probably do not imitate the existing environment in the foliar cavities of the fern. However, N-acetyl-glucosamine seems to hinder a drastic decrease of allophycocyanin and phycoerythrocyanin, pointing to that this amino sugar can be beneficial to the growth of A. azollae.

Keywords: Anabaena azollae; Azolla filiculoides; Aqueous extract; Sugar source (fructose, D-glucosamine, N-acetyl-glucosamine); Supplements (B12 vitamin, KNO3)


AA: Medium Allen and Arnon; AA 1/8: Medium Allen and Arnon at 1/8 strength; APC: Allophycocyanin; BG-110: Medium Blue-Green without Nitrogen; H-40: Medium Hoagland at 1/4 strength; KNO3: Potassium Nitrate; NaClO: Sodium Hypochlorite; PC: Phycocyanin; PEC: Phycoerythrocyanin; v/v: volume/volume


The nitrogen-fixing cyanobacteria can form symbioses with plants in which both partners get benefits from the association. The cyanobiont has a supply of nutrients, and protection against herbivores, while the plant obtains all or almost all of the nitrogen needed for its development [1].

The fern Azolla (Figure 1A) is a unique plant because it is the only fern that has a never lasting symbiosis throughout the entire life cycle (both sexual and asexual) with a heterocystous nitrogenfixing cyanobacterium Anabaena azollae Strasburger which occupy a narrow space at the periphery of the foliar cavity leaving the centre empty (Figure 1B). The filaments of the cyanobiont contain vegetative cells, heterocysts and occasionally akinetes. The cyanobacterial cells have a Gram-negative cell wall type, inclusions in the cytoplasm (carboxysomes, cyanophycin granules, and others) and a rudimentary thylakoid system. The heterocysts are specialized cells for nitrogen fixation, with two polar nodes of cyanophycin, honeycomb-like thylakoids, thick cell wall [2] and a proteinaceous extra-sheath [3]. The high heterocyst frequency (30-40%) and high nitrogen fixation rate led several researchers to the isolation and growth of several presumptive cultivable cyanobionts from all Azolla species using different growing conditions (Table 1). The most widely used culture medium is BG-110, combined with a wide range of conditions such as light intensity (from 9 to 200 μmol/m².s), temperature (22-33°C) and supplements (different nitrogen sources, sugar sources, pH among others) turning the data comparison very difficult [4-13]. However, Tang et al. [8] using a broad array of culture conditions pointed to the difficulty in maintaining A. azollae in culture, especially due to photo bleaching and no cell multiplication. The maintenance of the green colour of the cyanobiont cells in complex culture conditions by 119 days (medium AA + 10 mM fructose + 0.05% yeast extract + 2% agar + 0.05% casamino acid + 0.47 mM NaNO3; 7 days incubation in darkness; 1% O2 and 99% N2, 10000 lux, 30°C) and 183 days (medium AA + 10 mM fructose + 0.05% casamino acids + 0.05% yeast extract + 0.35 mM NaNO3 + 2% agar; 7 days incubation in darkness; 1% O2 and 99% N2, 10000 lux, 30°C in Petri dishes) was used as a parameter to assess the viability of the cyanobiont cells [8] Compared to freshly cyanobiont isolated from Azolla cavities, the presumptive cultivable cyanobiont has low heterocyst frequency, small vegetative cells, and heterocysts, and dissimilar contents of chlorophyll a, proteins and phycobiliproteins [6-7,11-13]. The genome sequencing of A. azollae supported this difficulty due to the existence of pseudogenes or gene loss involved in the replication, repair, glycolysis and nutrient uptake [14] pointing that the presumptive cultivable cyanobiont was probably a resilient contaminant [15]. Given that the nitrogen fixation is a highly energy-demanding process and that the cyanobiont multiply inside the foliar cavities, the fern seems to be the main provider of carbohydrates for the cyanobiont [16]. But amino sugars as N-acetylglucosamine and D-glucosamine [17] can also be provided by the fern.