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

Parente T¹, Fernandes I¹, Vasconcelos V^1,2 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

Abstract

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)

Abbreviations

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

Introduction

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.

Table 1: Major findings of the cultivable cyanobiont A. azollae isolated from Azolla species and growth conditions.




  
    Growth    conditions 
    Cyanobiont 
    Reference 
  
  
    Culture medium:    BG-11₀
      Light    intensity: 5 W/m² (9 μmol/m².s)
      Temp: 26°C
      Supplements: 1)    nitrate at 125, 350 ppm
      2) ammonium at    10, 20, 40 ppm
      3) fructose,    sucrose and glucose at 2, 5, 10, 30 mM
    
      Nitrate at 350 ppm    stimulated growth, protein and chlorophyll contents; inhibition of    phycobiliprotein content and heterocyst frequency
      Ammonium at 20 ppm    stimulated growth, protein, chlorophyll and phycobiliproteins contents
      Fructose (2-30 mM)    stimulated nitrogen fixation
      Glucose and sucrose    (2-30 mM) decreased nitrogen fixation
    
    [4]
  
  
    Culture medium:    BG-11₀
      Light    intensity: 5 W/m² (9 μmol/m².s)
      Temp: 26°C
      Supplements:    Fructose, sucrose and glucose at 8 mM
    
      Fructose stimulated    higher growth than sucrose and glucose
      Fructose increased    chlorophyll a and glycogen content,    heterocyst frequency, nitrogen fixation and glucose-6-phosphate dehydrogenase    activity
    
    [5]
  
  
    Culture medium:    BG-11₀
      Light    intensity: 200 μmol/m².s
      Temp: 33°C
      Supplements: 1)    bubbled air and pH 9.3
      2) air + 1.5%    CO₂, pH 6.4
      3) air + 1.5%    CO₂ + 20 mM NaHCO₃, pH 8.2
      4) air + 1.5%    CO₂, 10 mM HEPES, pH 7.2
      5) air + 1.5%    CO₂ + 20 mM NaHCO₃, 15 mM NaNO₃, pH 8.3
      6) air + 1.5%    CO₂ + 20 mM NaHCO₃, 2.5 mM NaNO₃, pH 8.3
      7) air + 1.5%    CO₂ + 20 mM NaHCO₃, 5 mM NH₄Cl, pH 8.0
    
      Aeration with CO₂ and nitrogen source induced a little growth of A. azollae
      Phycocyanin    increased with the addition of HEPES and ammonium
    
    [6]
  
  
    Culture medium:    BG-11₀ (pH 7.4)
      Light    intensity: 135 μmol/m².s
      Temp: 22-25°C
      Agitation in a    water bath at 30°C
    
      Colonial, globular,    fimbriate and tubular growth
      Vegetative cells    and heterocysts size are not homogenous among cyanobiont
      Low heterocyst    frequency
    
    [7]
  
  
    Growth    conditions:
      1) BG-11₀,    0.1% agar, 30°C, 10000 lux (135 μmol/m².s), without initial    darkness
      2) BG-11₀ or AA, 30°C, 10000 lux (135 μmol/m².s), initial 6 days in darkness
      3) AA + 2 mM NH₄Cl    + 10 mM fructose, 4.5 days with agitation in darkness at room temperature; 3    days at 10000 lux (135 μmol/m².s) and 30°C
      4) AA + 2 mM NH₄Cl    + 10 mM fructose + 0.1% yeast extract (0.1% agar), pre-incubation in    darkness; agitation at room temperature for 15 days; growth at 5000 lux (68    μmol/m².s) after transfer to semi-solid media
      5) AA + 5mM NH₄Cl    + 10 mM fructose + 0.1% agar, pre-treatment in darkness; transfer to 30°C and    then increase radiation until 10000 lux (135 μmol/m².s)
      6) AA + 10 mM    fructose + 0.05% yeast extract + 0.05% casamino acid + 0.47 mM NaNO₃,    pre-treatment in darkness; transfer to 30°C and then increase radiation until    10000 lux (135 μmol/m².s)
      7) AA + 10 mM    fructose + 0.05% yeast extract + 2% agar + 0.05% casamino acid + 0.47 mM NaNO₃,    7 days incubation in darkness; under 1% O₂ and 99% N₂,    growth at 10000 lux (135 μmol/m².s) and 30°C
      8) AA + 10 mM fructose    + 0.05% yeast extract + 0.05% casamino acid + antibiotic mixture + 2% agar, 7    days incubation in darkness; under 1% O₂ and 99% N₂,    growth at 10000 lux (135 μmol/m².s) and 30°C
      9) AA + 10 mM    fructose + 0.05% casamino acids + 0.05% yeast extract + 0.35 mM NaNO₃+    2% agar, 7 days incubation in darkness; under 1% O₂ and 99% N₂,    growth at 10000 lux (135 μmol/m².s) and 30°C in Petri dishes
    
      Photobleaching in    cyanobiont cells
      Green color    maintenance depends on culture medium, supplementation and growth conditions -highest    days (119 and 183 days) in experiment 7 and 9
      Cell division only    in agar cultures
    
    [8]
  
  
    Culture medium:    BG-11₀
      Light    intensity: 15 μmol/m².s
      Temp: 28°C
      Supplements:    Fructose, glucose, sucrose, and maltose at 0.3 %
    
      Colonies flat and    smooth
      Filaments with    cylindrical vegetative cells and ellipsoidal heterocysts
      Presence of    hormogonia
      Photoheterotrophic    growth with fructose
      Cultured filaments    did not show chromatic adaptation
    
    [9]
  
  
    Culture medium:    N-free medium
      Supplements: 1)    NO₃^- and NO₂^- at 5 mM
      2) NH₄⁺ at 1 mM
    
      Cyanobionts only    grown with supplements
    
    [10]
  
  
    Culture medium:    BG-11₀
      Light    intensity: 52 μmol/m².s
      Temp: 25°C
      Photoperiod:    16/8 h (day/night)
    
      Lower content of    chlorophylls, phycobiliproteins, proteins, and sugars
      Low heterocyst    frequency
      Filaments with    small vegetative cells and heterocysts
      High nitrogen    fixation
    
    [11]
  
  
    Culture medium:    BG-11₀
      Light    intensity: 170 and 250 μmol/m².s
      Temp: 29°C
      Photoperiod:    16/8 h (day/night)
      Supplements:    Glucose and sucrose at 0.5%
    
      Dry mass increased    with high light intensity
      Protein,    chlorophyll, carotenoids and phycobiliproteins content increased with high    light intensity and supplements
      Nitrogen fixation    increased with high light intensity
    
    [12]
  
  
    Culture medium:    BG-11₀
      Light    intensity: 52 μmol/m².s
      Temp: 25°C
      Photoperiod:    16/8 h (day/night)
    
      Low heterocyst    frequency
      Coiled and straight    filaments
      Small sized    vegetative cells and heterocysts
      Dull green color
    
    [13]



Table 1: Major findings of the cultivable cyanobiont A. azollae isolated from Azolla species and growth conditions.

Figure 1: Morphological aspects of A. filiculoides. A) Round sporophyte with alternate ramifications with the chlorophyllous dorsal lobes. B) Cross-section of the cavity (C) of the dorsal lobes with filaments of the cyanobiont A. azollae (arrow).

    
    
    Figure 1:  Morphological aspects of A. filiculoides. A) Round sporophyte with
alternate ramifications with the chlorophyllous dorsal lobes. B) Cross-section
of the cavity (C) of the dorsal lobes with filaments of the cyanobiont A. azollae
(arrow).

The aim of the present study was to culture the cyanobiont A. azollae in three media (BG-11₀, AA and AA 1/8) supplemented with an aqueous extract of A. filiculoides, Potassium Nitrate (KNO₃), B₁₂ vitamin and three sugar sources (fructose, D-glucosamine, and N-acetyl-glucosamine). The cyanobiont fitness was assessed by quantification of the Phycobiliproteins Phycoerythrocyanin (PEC), Phycocyanin (PC) and Allophycocyanin (APC).

Material and Methods

Growth of Azolla filiculoides

The sporophytes of A. filiculoides (accession FI1001) obtained from the germplasm collection at International Rice Research Institute (IRRI) were grown in H-40 medium, pH 6.1-6.2, in controlled conditions [18].

Extraction of aqueous compounds from Azolla filiculoides

In a sterile flow hood, the sporophytes of A. filiculoides were washed in sterile tap water, immersed in sterile distilled water and ground with a sterile pestle. The homogenate was centrifuged at 4546 xg, for 30 min, at 4°C. The supernatant was filtered through filter paper (Whatman N° 1) to eliminate remaining debris and stored at -20°C.

Isolation of the cyanobiont Anabaena azollae

In a sterile hood, roots were cut and the rootless sporophytes were disinfected twice in an aqueous solution of sodium hypochlorite (1 ml NaClO:10 ml distilled water, v/v) for 20 min, followed by three washes in sterile ultrapure water. The cyanobiont was isolated from sporophytes of A. filiculoides using the gentle roller method described by Rai and Rai [19]. The isolated cyanobiont was inoculated in each culture medium.

Experimental design

The cyanobionts were cultured in three media - AA, AA 1/8 fold strength [20] and BG-11₀ [21]. The added supplements were the aqueous extract from A. filiculoides (filtered through a sterile syringe filter 0.45 μm pore diameter prior to their adding to each culture media) in1:10 (v:v, extract: culture medium), 8 mM D-(-)-fructose [5], 5 mM D-(+)-glucosamine, 5 mM N-acetyl-D-glucosamine, 1 mg/L B₁₂ vitamin [22] and 5 mM KNO₃ [9] (Figure 2). The addition of 100 mg/L of cycloheximide [23] to all the media inhibited the eukaryotic growth. The cyanobionts freshly isolated from the foliar cavities of A. filiculoides was regarded as the control. Due to a shortage of filaments of A. azollae, it was only made one replica for each medium and supplements. Following three days of darkness to prevent photobleaching [8], the cyanobionts were grown at 24°C, photoperiod of 16 h/8 h (day/night) and light intensity of 40μmol/ m²s provided by one fluorescent lamp Phillips Cool White TLD 36W. The medium was changed every 15-20 days. After 3 months, the medium was centrifuged at 4542 xg, for 5 min, at 4°C and the pellet was stored at -20°C. An aliquot of the non-frozen pellet was observed under an Olympus BX41 (Olympus, Lisbon, Portugal) light microscope.

Figure 2: Diagram of the experimental design. Legend: W/AE - without aqueous extract; W/SS - without sugar source; W/B₁₂ - without B₁₂ vitamin; W/KNO₃ - without potassium nitrate; SS - sugar source (Fructose or D-glucosamine or N-acetyl-D-glucosamine); B₁₂- B₁₂ vitamin; KNO₃ - potassium nitrate.

    
    
    Figure 2:  Diagram of the experimental design. Legend: W/AE - without
aqueous extract; W/SS - without sugar source; W/B₁₂ - without B₁₂
vitamin; W/KNO₃ - without potassium nitrate; SS - sugar source (Fructose
or D-glucosamine or N-acetyl-D-glucosamine); B₁₂- B₁₂ vitamin; KNO₃ -
potassium nitrate.

Extraction and quantification of phycobiliproteins

The phycobiliproteins from each assay (except the assay supplemented by an aqueous extract of A. filiculoides) and a freshly isolated A. Azollae sample were extracted following Pereira et al. [24]. The absorbance was measured at 572, 612 and 647 nm in a Synergy HT spectrophotometer (BioTek, Madrid, Spain) equipped with Gen5 2.00 software. The content of PEC, PC, and APC was calculated according to Kaplan et al. [25].

Data analysis

Prior to the analysis of variance, data of the three phycobiliproteins were transformed as a percentage to control. This data was analyzed by two-way ANOVA without replication. The significance level (a) was 0.05. Statistical analyses were made using the Microsoft^® Office Excell^® 2007.

Results and Discussion

One of the particularities of the Azolla-A. azollae symbiosis is the synchronous development of both partners [15] indicating that probably Azolla might have a chemical or nutritional control regarding the development, differentiation, and multiplication of the cyanobiont. In the present research and after three months in culture conditions there were no filaments or isolated cyanobacterial cells in all the three media tested to which was added an aqueous extract of A. filiculoides. Probably water-soluble compound(s) from A. filiculoides can impair the multiplication of the cyanobiont or may induce its necrosis, but nothing is known about it.

A. azollae growing in artificial conditions showed straight filaments with cylindrical vegetative cells and ellipsoidal heterocysts [7,9,11-13]. However, at the present research, in all the media (AA, AA 1/8 and BG-11₀) lacking any sugar source and supplements (KNO₃ and B₁₂ vitamin), the cyanobiont did not had green colour, did not grew and had very short filaments formed by cylindrical vegetative cells and ellipsoidal heterocysts with full-size inclusions (Figure 3A) or isolated vegetative cells and heterocysts (Figure 3B). The addition of a nitrate source stimulated the A. azollae growth [4,6,8,10], but in the present research, the addition of potassium nitrate as an additional nitrogen source did not induce growth of A. azollae. Moreover, since the nitrate transport gene in A. azollae is a pseudogene [13] the KNO₃ is not transported to the cyanobiont cells and thus not necessary for the cyanobiont growth. Regarding the B₁₂ vitamin, there are no studies about their use for A. azollae growth, but since after 3 months very few filaments of A. azollae were observed probably their addition is not necessary.

Figure 3: A. azollae growth under a light microscope. A) Filaments with heterocysts (H) and vegetative cells (arrow) grown in BG-11₀ withKNO₃ and without sugar source and B₁₂ vitamin. B) Isolated cells of the cyanobiont (arrow) grown in medium AA without sugar source, KNO₃, and B₁₂ vitamin. C) A short filament with heterocysts (H) and vegetative cells (arrow) of the cyanobiont grown in medium BG-11₀ supplemented with fructose and KNO₃ and without B₁₂ vitamin. D) Filaments formed by vegetative cells (arrow) and heterocysts (H) grown in medium AA supplemented with N-acetylglucosamine and KNO₃ and without B₁₂ vitamin.

    
    
    Figure 3:  A. azollae growth under a light microscope. A) Filaments with
heterocysts (H) and vegetative cells (arrow) grown in BG-11₀ withKNO₃ and
without sugar source and B₁₂ vitamin. B) Isolated cells of the cyanobiont
(arrow) grown in medium AA without sugar source, KNO₃, and B₁₂ vitamin.
C) A short filament with heterocysts (H) and vegetative cells (arrow) of the
cyanobiont grown in medium BG-11₀ supplemented with fructose and KNO₃
and without B₁₂ vitamin. D) Filaments formed by vegetative cells (arrow)
and heterocysts (H) grown in medium AA supplemented with N-acetylglucosamine
and KNO₃ and without B₁₂ vitamin.

The present research showed that the addition of fructose to the medium BG-11₀ without KNO₃ and B₁₂ vitamin, medium BG- 11₀+KNO₃ and medium AA+KNO₃ did not induce A. azollae growth. Instead, short filaments formed by vegetative cells and heterocysts with cytoplasm inclusions and without the typical green colour were observed (Figure 3C). On the remaining combinations of culture media with fructose, the cyanobiont cells were rare and contained many bacteria. These results are in disagreement with Rosen et al. [5] and Vagnoli et al. [9], which showed that the addition of fructose increased the photo heterotrophic growth of A. azollae. This dissimilarity could be due to the different temperatures and/ or different light intensities used in the present research (40 μmol/ m².s and 24°C) and by Rosen et al. [5] (9 μmol/m².s and 26°C ) and Vagnoli et al. [9] (15 μmol/m².s and 28°C). On the other hand, Tel-Or and Sandovsky [4] showed that fructose had not induced an increase in A. azollae growth rate, which agrees with the results of the present research. In the assays made in the present research with the three media (AA, AA 1/8 and BG-11₀) to which was added D-glucosamine and with or without KNO₃ and B₁₂ vitamin, the cyanobiont cells were rare, did not exists filaments of the cyanobiont pointing to its almost disappearance of culture media. Thus, D-glucosamine seems to be not useful for the cyanobiont growth. However, in all the assays at the present research with the N-acetyl-glucosamine with or without KNO₃ and B₁₂ vitamin, the cyanobiont had a few short filaments formed by vegetative cells and heterocysts with inclusions and no green colour (Figure 3D), which may indicate that this amino sugar may be used as sugar source in more complex formulations of culture media.

The phycobiliproteins content can be related to chromatic adaptation or nutrient starvation such as a lower influx of carbohydrates or nitrogen [26]. In the present study, the cyanobionts growing in the medium AA with fructose and N-acetyl-glucosamine without supplements (KNO₃ and B₁₂ vitamin), N-acetyl-glucosamine + B₁₂ vitamin and N-acetyl-glucosamine + KNO₃ showed low levels of phycocyanin (Figure 4A). The most severe decrease or disappearing of phycocyanin occurred with media AA 1/8 (Figure 4B) and BG- 11₀ (Figure 4C), but adding N-acetyl-glucosamine appeared to hinder the total loss of phycocyanin. Probably, this may indicate that phycocyanin could be the first phycobiliprotein to be degraded or their synthesis repressed regardless medium, sugar source or other supplements added. But, the two-way ANOVA indicated that the sugar source added to the all the culture media induced statistical significant differences on the PC content (F=17.44, P<0.05) but not the type of culture media (F=0.47, P>0.05) pointing to the sugar source as an important parameter in the culture media formulation due to their possible influence on the phycocyanin content in the cyanobiont. Relating to allophycocyanin, the addition of fructose to medium AA seemed to protect against their loss from A. azollae (Figure 4D). The cyanobionts that grew in the media AA 1/8 without added sugar source and with N-acetyl-glucosamine showed a smaller loss of allophycocyanin in comparison to other growth media (Figure 4E). As to the medium BG-11₀, the content of allophycocyanin in A. azollae growing with N-acetyl-glucosamine without KNO₃ and B₁₂ vitamin induced a more rapid loss of allophycocyanin (Figure 4F). The analysis of variance (two-way ANOVA) showed that the sugar source added to the media induced statistical significant differences on the APC content (F=5.30, P<0.05) but not the type of culture media (F=0.80, P>0.05) again pointing to the sugar source as an important constraint in the culture media formulation and in the APC content of the cyanobiont. The content of phycoerythrocyanin surpassed the 100% in six assays - medium AA without sugar source and supplements, medium AA +N-acetyl-glucosamine + KNO₃ (Figure 4G), medium AA 1/8 without sugar source and supplements, AA 1/8 + N-acetylglucosamine + B₁₂ vitamin (Figure 4H), medium BG-11₀ without sugar source and supplements, BG-11₀ + N-acetyl-glucosamine and B₁₂ vitamin (Figure 4I) - which means that the cultured cyanobionts have higher amounts of phycoerythrocyanin than the freshly isolates of A. azollae. The two-way ANOVA pointed to the sugar source as an important parameter in the culture media formulation and the maintenance of APC in the cyanobiont since it induced statistical significant differences on the PEC content (F=21.18, P<0.05) but not the type of culture media (F=0.83, P>0.05). The amino sugar D-glucosamine has induced almost the total loss of allophycocyanin (Figure 4D,4E,4F) and phycoerythrocyanin (Figure 4G,4H,4I) from the cyanobionts. Nitrogen starvation induced the degradation of phycobiliproteins in A. azollae [25], but Tel-Or and Sandovsky [4] showed that the phycobiliprotein content was inhibited by nitrate and Zimmerman [6] showed that phycocyanin was stimulated by the presence of ammonium. However, the phycobiliprotein data from the present research seems to indicate that the cyanobiont in culture probably suffered from a severe carbohydrate and/or nitrogen starvation. Also, the type of sugar source added to the medium and not the type of culture medium seems to have a major influence on the PBP content of the cyanobiont. Moreover, the data of the present research seems to point to that the amino sugar N-acetylglucosamine can be included in more complex formulations since it seems to hinder the total loss of phycocyanin and prevented a more drastic decrease of allophycocyanin and phycoerythrocyanin and also seems to favour the presence of cyanobiont cells but not the loss of the green colour.