Co-cultivation of diazotrophic terrestrial cyanobacteria and Arabidopsis thaliana

Abstract

Diazotrophic cyanobacteria are able to fix N₂ from the atmosphere and release it as bioavailable nitrogen what other organisms can utilize. Thus, they could be used as living nitrogen supplier whereby the use of fertilizer could be reduced in agricultural industry what results in a decrease of laughing gas released during fertilizer production. The diazotroph cyanobacterium Desmonostoc muscorum (D. muscorum) was characterized in shake flasks cultivated in nitrogen-free and nitrogen-containing medium. Similar growth rates were reached in both cultivations and the release of ammonium by D. muscorum was detected under nitrogen depletion. Subsequently, D. muscorum was co-cultivated with Arabidopsis thaliana (A. thaliana) in nitrogen-free medium. Additionally, the plant was cultivated in nitrogen containing and nitrogen-free medium without D. muscorum as reference. A co-cultivation led to higher growth rates of the cyanobacterium and similar growth of A. thaliana with similar maximum photochemical efficiency of photosystem II compared to the growth of nitrogen containing medium. Further, accumulation of cyanobacterial cells around the roots of A. thaliana was detected, indicating a successfully induced artificial symbiosis. Based on these results, D. muscorum could be a promising cyanobacterium as living nitrogen supplier for plants.

Keywords: co‐cultivation; cyanobacteria; diazotrophic biofilms; phototrophic biofilms; plants.

FIGURE 1

Experimental set‐up of co‐cultivation. Agar plates were inoculated with three dots of cyanobacterial Cell Wet Weight (CWW). In each dot one sterilized plant seed was placed

FIGURE 2

Characterization of D. muscorum in BG‐11 and nitrogen free BG‐11‐0 medium in shake flasks over the cultivation time. (A) Cell dry weight (CDW) per liter. (B) EPS content per CDW. (C) C‐phycocyanin content per CDW. (D) Content of chlorophyll‐a (chla) and carotenoids (caro) per CDW. Cultivation parameters: cultivation time = 14 days, continuous illumination with a light intensity = 100 µmol_photons·m ⁻²·s^− 1, temperature = 30°C, BG‐11, respectively BG‐11‐0 (nitrogen free), 300 mL shake flasks without baffles, n_b = 3

FIGURE 3

Characterization of the most important media components of D. muscorum cultivations in BG‐11 and nitrogen free BG‐11‐0 in shake flasks over the cultivation time. (A) Nitrate, (B) Nitrite, (C) Phosphate, (D) Ammonium, (E) Magnesium. Cations and anions were determined with ion exchange chromatography. Cultivation parameters: cultivation time = 14 days, continuous illumination with a light intensity = 100 µmol_photons·m ⁻²·s^− 1, temperature = 30°C, BG‐11, respectively BG‐11‐0 (nitrogen free), 300 mL shake flasks without baffles, n_b = 3

FIGURE 4

Composition of phycobiliproteins over the cultivation time of D. muscorum cultivated in BG‐11 (A) and nitrogen free BG‐11‐0 (B) medium. Phycobiliprotein composition is given in percent. PE = Phycoerythrin. APC = Allophycphycocyanin. PC = C‐phycocyanin. Cultivation parameters: cultivation time = 14 days, continuous illumination with a light intensity = 100 µmol_photons·m ⁻²·s^− 1, temperature = 30°C, BG‐11, respectively BG‐11‐0 (nitrogen free), 300 mL shake flasks without baffles, n_b = 3

FIGURE 5

Areal growth of D. muscorum with and without A. thaliana on agar plates without nitrogen. Cultivation parameter: Cultivation time = 47 days, Light intensity = 90 µmol_photons m ⁻² s⁻¹; Day/Night Rhythm = 16/8 h; Temperature = 26°C, Murashige skoog medium without nitrogen (MS‐0). Areal growth was determined using PAM fluorometry, n_b = 3

FIGURE 6

Pictures of A. thaliana cultivated 14 days in MS and MS‐0 and in co‐culture with D. muscorum in MS‐0. Cultivation parameter: Cultivation time = 47 days, Light intensity = 90 µmol_photons m ⁻² s⁻¹; Day/Night Rhythm = 16/8 h; Temperature = 26°C

FIGURE 7

Root formation of A. thaliana in MS medium, nitrogen free medium (MS‐2) and MS‐0‐medium with D. muscorum. (A) Amount of rootedness of A. thaliana under different growth conditions over the cultivation period. (B) Microscopic image of D. muscorum on roots of A. thaliana after 47 days of cultivation. (C‐E) Microscopic images of rootedness of A. thaliana after 14 days of cultivation. (C) Roots of A. thaliana on MS‐0‐medium, (D) on MS‐0 medium in co‐cultivation with D. muscorum and (E) on MS‐medium. Cultivation parameter: Cultivation time = 47 days, Light intensity = 90 µmol_photons m ⁻² s⁻¹; Day/Night Rhythm = 16/8 h; Temperature = 26°C, on MS, MS‐0 medium with 0.8% plant agar, n_b = 3, microscopic evaluation n = 7