Formulating bacterial endophyte: Pre-conditioning of cells and the encapsulation in amidated pectin beads.

Despite the benefits of bacterial endophytes, recent studies on the mostly Gram-negative bacteria lack of regard for formulation strategies. The encapsulation into biopolymeric materials such as amidated pectins hydrogels is a suitable alternative. Here, this research aimed at supporting the capability of the plant growth-promoting bacteria Kosakonia radicincitans DSM16656T to endophytically colonize plant seedlings. In this approach, the pre-conditioned cells through osmoadaptation and hydroxyectoine accumulation were used. In general, pre-osmoadapted and hydroxyectoine-supplemented bacteria cells formulated in amidated pectin dried beads increased the endophytic activity by 10-fold. Moreover, plant promotion in radish plants enhanced by 18.9% and 20.7% for a dry matter of tuber and leaves. Confocal microscopy studies with GFP-tagged bacteria revealed that bacterial aggregates formed during the activation of beads play an essential role in early colonization stages. This research encourages the integration of fermentation and formulation strategies in a bioprocess engineering approach for exploiting endophytic bacteria.

Anhydrobiotic engineering for the endophyte bacterium Kosakonia radicincitans by osmoadaptation and providing exogenously hydroxyectoine.

This study presents an anhydrobiotic engineering approach aiming at conferring a high degree of desiccation tolerance to the Gram-negative endophyte Kosakonia radicincitans. In particular, pre-conditioning of bacteria under high salinities provides a remarkable positive influence on drying survival. The endophytic bacteria accumulate exogenous hydroxyectoine > 500 µmol g-1 dry weight cells exerted by osmotic stress at 4% NaCl. Microfermentation research demonstrated that hydroxyectoine provides positive effects on reducing the lag phase duration and alleviates the dissolved oxygen consumption under high salinity conditions. Beyond the amassing of hydroxyectoine, this work provides evidence supporting the notion that hydroxyectoine can produce significant changes in the endogenous bacterial metabolome during the exponential growth phase at high-osmolarity. Metabolome changes include alterations on tricarboxylic acid cycle, novo-synthesis of specific intracellular metabolites such as mannitol, myo-inositol and trehalose, and fold changes on amino acids such as L-leucine, L-asparagine, L-serine, L-methionine and L-proline. The significant fold change of L-aspartate suggests a potential acidic proteome at high-osmolarity environments, extending the knowledge of salt-stressed bacterial endophytes. Thus, these findings place the metabolic salt stress response and the hydroxyectoine accumulation by K. radicincitans into a physiological context, paving the way into the interaction between cellular phenotype associated with salt stress tolerance and drying survival capacity of Gram-negative endophytes.

Salt stress and hydroxyectoine enhance phosphate solubilisation and plant colonisation capacity of Kosakonia radicincitans.

Gram-negative bacterial endophytes have attracted research interest caused by their advantageous over epiphytic bacteria in plant nutrition and protection. However, research on these typically Gram-negative endophytes has deficiencies concerning the role of cultivation and pre-formulation strategies on further plant colonisation capabilities. Besides, the influence of cultivation conditions and osmotic stress within bacterial endophytes on their phosphate solubilising ability has not yet been addressed. By pre-conditioning cells with an osmoadaptation and a hydroxyectoine accumulation approach, this research aimed at enhancing the capability of the plant growth promoting bacterium Kosakonia radicincitans strain DSM 16656T to both solubilise phosphate and colonise plant seedlings. The results showed that halotolerant bacterial phenotypes increased the root-colonising capability by approximately 3-fold and presented growth-promoting effects in radish plants. Interestingly, findings also demonstrated that salt stress in the culture media along with the accumulation of hydroxyectoine led to an increase in the in vitro phosphate-solubilising ability by affecting the production of acid phosphatases, from 1.24 to 3.34 U mg-1 for non-salt stressed cells and hydroxyectoine-added cells respectively. Thus, this approach provides a useful knowledge upon which the salt stress and compatible solutes in bacteria endophytes can confer phenotypic adaptations to support the eco-physiological performance concerning phosphate-solubilising abilities and endosphere establishment.

Selección de un sistema de atomización para la formación de micropartículas de Eudragit S100 en lecho fluido / Selecting a spray system for the formation of Eudragit S100 microparticles in a fluid bed

La elaboración de micropartículas en lecho fluido es de gran interés en la industria farmacéutica, alimentaria y agrícola, ya que este tipo de formulación permite controlar la liberación de ingredientes activos y su estabilidad y funcionalidad mediante la formación de pequeñas partículas sólidas. El lecho fluido es el equipo comúnmente usado en la industria para realizar el proceso y éste puede tener dos sistemas de atomización: superior (Top spray) e inferior (Bottom spray). En este trabajo se evaluaron los dos sistemas de atomización con miras a la formación de micropartículas de Eudragit® S100. Se realizaron experimentos para ajustar los factores críticos del proceso y los niveles de éstos utilizando un diseño factorial multinivel y trabajando en un lecho fluido marca Glatt GmbH D – 01277. Los factores evaluados fueron la temperatura de entrada, la presión interna de la cámara y la velocidad de flujo. El material de recubrimiento consistió en un polímero del ácido metacrílico denominado Eudragit® S100 y como núcleo se empleó talco malla 325. El sistema seleccionado para la microencapsulación fue el de atomización superior (Top spray). Las condiciones de atomización establecidas fueron velocidad de flujo de 4,12 mL/min (flujo 8 rpm), temperatura de entrada de80ºC y presión interna de la cámara de 1 y 3 bares. Las micropartículas presentaron formas y tamaños homogéneos, menores de 100 μm.Las condiciones fijadas para el sistema de atomizaciónsuperior se pueden aplicar al desarrollo de procesos de microencapsulación de diferentes principios activos utilizando como polímero Eudragit® S100.