CELL‐CELL INTERACTION IN THE EUKARYOTE‐PROKARYOTE MODEL OF THE MICROALGAE CHLORELLA VULGARIS AND THE BACTERIUM AZOSPIRILLUM BRASILENSE IMMOBILIZED IN POLYMER BEADS¹

Abstract

Cell-cell interaction in the eukaryote-prokaryote model of the unicellular, freshwater microalga Chlorella vulgaris Beij. and the plant growth-promoting bacterium Azospirillum brasilense, when jointly immobilized in small polymer alginate beads, was evaluated by quantitative fluorescence in situ hybridization (FISH) combined with SEM. This step revealed significant changes, with an increase in the populations of both partners, cluster (mixed colonies) mode of colonization of the bead by the two microorganisms, increase in the size of microalgae-bacterial clusters, movement of the motile bacteria cells toward the immotile microalgae cells within solid matrix, and formation of firm structures among the bacteria, microalgae cells, and the inert matrix that creates a biofilm. This biofilm was sufficiently strong to keep the two species attached to each other, even after eliminating the alginate support. This study showed that the common structural phenotypic interaction of Azospirillum with roots of higher plants, via fibrils and sheath material, is also formed and maintained during the interaction of this bacterium with the surface of rootless single-cell microalgae.

Interaction of Azospirillum spp. with Microalgae: A Basic Eukaryotic–Prokaryotic Model and Its Biotechnological Applications

“…Over time (6-48 h), depending on the pairing of microalgae and bacteria, both microorganisms are found in the same cavity within the bead, mainly just beneath the surface. Small parts of the internal structure of the bead matrix dissolve or split and separate as microcolonies develop and enlarge (Covarrubias et al 2012 ;Lebsky et al 2001 ;de-Bashan et al 2011 ). The bacteria mainly excrete indole-3-acetic acid (IAA) and other undefi ned signal molecules that reach the nearby microalgal cells (de-Bashan et al 2008a ).…”

Accumulation of intra-cellular polyphosphate in Chlorella vulgaris cells is related to indole-3-acetic acid produced by Azospirillum brasilense

“…1d). The period of interaction of the two partners in this synthetic mutualistic interaction was 72 h, where IAA was added after an initial acclimation period of 24 h. It was shown in earlier studies that, during this period, C. vulgaris is in its logarithmic phase of growth [13,41,44]. This period of exposure leads to optimum removal of phosphate from wastewater [50,69,75].…”

“…Control beads without microorganisms were used. Because growth of C. vulgaris fluctuated among different repetitions of the same experiment [59], C. vulgaris populations were routinely counted in each experiment, and the calculated values were derived from specific experiments, but each population count in each experiment are not shown because they were previously published [41,42], and doubling time under the influence of IAA was calculated from the raw data (http://www. doubling-time.com/compute.php, accessed March 9, 2015).…”

“…To combine both species in the same beads, a similar procedure was performed, using 20 mL of each culture in a mixture (40 mL total). After the second incubation, the beads were rinsed three times in saline solution (0.85% NaCl), placed in 500 mL Erlenmeyer flasks (40 g of beads per flask) containing 200 mL synthetic growth medium (SGM) containing 25 mg L À1 NH 4 Cl (described in de-Bashan et al [41]). The flasks were placed on an orbital shaker for 2 d under the same conditions for culturing Chlorella.…”

Mass Production of Microalgae

“…Light supply is an important variable in autotrophic culture. The consortium between microalgae and bacteria has been extensively studied for wastewater treatment (Cruz et al, 2013;de-Bashan and Bashan, 2010;de-Bashan et al, 2004de-Bashan et al, , 2011. To maximize the biomass production, light intensity should be supplied in saturation levels to the entire cultivation system.…”