Remote effects (occurring without physical contact) of two plant growth-promoting bacteria (PGPB)Azospirillum brasilense Cd and Bacilus pumilus ES4 on growth of the green microalga Chlorella sorokiniana UTEX 2714 were studied. The two PGPB remotely enhanced the growth of the microalga, up to six-fold, and its cell volume by about three-fold. In addition to phenotypic changes, both bacteria remotely induced increases in the amounts of total lipids, total carbohydrates, and chlorophyll a in the cells of the microalga, indicating an alteration of the microalga's physiology. The two bacteria produced large amounts of volatile compounds, including CO 2 , and the known plant growth-promoting volatile 2,3-butanediol and acetoin. Several other volatiles having biological functions in other organisms, as well as numerous volatile compounds with undefined biological roles, were detected. Together, these bacteria-derived volatiles can positively affect growth and metabolic parameters in green microalgae without physical attachment of the bacteria to the microalgae. This is a new paradigm on how PGPB promote growth of microalgae which may serve to improve performance of Chlorella spp. for biotechnological applications.In vitro culturing at laboratory or mass industrial scales is the most common way by which the biotechnological industry is producing products from microalgae 1,2 and inoculants of plant growth-promoting bacteria (PGPB) for agricultural and environmental applications 3 . The current paradigm of how PGPB enhance plant growth is through attachment of the bacteria to plant roots. PGPB first establish a stable physical interaction with its host and subsequently colonize the root system, which results in beneficial effects on plants 4,5 When the PGPB move toward its host before attachment, the chances of successful colonization improve 6 . In the absence of attachment and colonization to plant surface, no effect of PGPB on higher plants are recorded 7 . Attachment of the PGPB Azospirillum spp. to roots of many plant species has been demonstrated since the emergence of the field some 40 years ago 4,[8][9][10][11][12] . A. brasilense has also been observed to attach to, and form stable aggregates with, the microalga Chlorella vulgaris 13 . Similar interactions have also been proposed for Gram-positive bacteria, such as Bacillus spp.14 . While attachment to roots has been documented as the major requirement for a beneficial association of PGPB with plants 7 , it is also well documented that the PGPB

The plant-growth-promoting bacterium Azospirillum brasilense is able to associate with the microalgae Chlorella sorokiniana. Attachment of A. brasilense increases the metabolic performances of the microalgae. Recent genome analyses have revealed that the A. brasilense Az39 genome contains two complete sets of genes encoding type VI secretion systems (T6SS), including the T6SS1 that is induced by the indole-3-acetic acid (IAA) phytohormone. The T6SS is a multiprotein machine, widespread in Gramnegative bacteria, that delivers protein effectors in both prokaryotic and eukaryotic cells. Here we show that the A. brasilense T6SS is required for Chlorella-Azospirillum synthetic mutualism. Our data demonstrate that the T6SS is an important determinant to promote production of lipids, carbohydrates and photosynthetic pigments by the microalgae. We further show that this is likely due to the role of the T6SS during the attachment stage and for the production of IAA phytohormones. Finally, we demonstrate that the A. brasilense T6SS provides antagonistic activities against a number of plant pathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia species in vitro, suggesting that, in addition to promoting growth, A. brasilense might confer T6SS-dependent bio-control protection to microalgae and plants against bacterial pathogens.

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