Preservation of rhizobia by lyophilization with trehalose

Abstract

-The objective of this study was to determine if trehalose can effectively enhance the viability and storage lifetime of Rhizobium tropici and Rhizobium etli after lyophilization as compared to the traditional protective combination of sucrose and peptone. Two strains of bean-nodulating rhizobia were effectively preserved in the lyophilized state by 100 mM trehalose, and survived for at least 12 days, even when stored under non-ideal conditions. Trehalose provided better protection than the sucrose/peptone combination. When protective excipients were introduced inside the cells, strain CFN 42 was more sensitive to the type of additive used than strain CIAT 899, probably because CIAT 899 produces β (1-2) glucan, which may have a protective effect. β (1-2) glucan combined with sucrose protected better than trehalose against leakage from freeze-dried liposomes, but by itself it had no protective capability. There was a high level of unfrozen water associated with the hydrated glucan, in addition to a thermal event at approximately 70 o C, corresponding to an observed gel-sol transition in the glucan. These findings may shed light on the mechanism by which the glucan contributes to the protection of dried cells and liposomes, and may lead to further improvements in rhizobial inoculants.Index terms: storage, inoculation, symbiosis, glucan. Preservação de rizóbio por liofilização com trealoseResumo -O objetivo deste estudo foi determinar se a trealose pode aumentar a viabilidade e o tempo de preservação de estirpes de Rhizobium tropici e Rhizobium etli após a liofilização, comparada com a proteção proporcionada pela combinação tradicional de sacarose e peptona. Duas estirpes de rizóbio para feijão foram preservadas liofilizadas com 100 mM de trealose e sobreviveram por pelo menos 12 dias, mesmo quando armazenadas sob condições não-ideais. A trealose proporcionou uma proteção superior à combinação de sacarose peptona. Quando agentes protetores foram introduzidos dentro da célula, a estirpe CFN 42 foi mais sensível em relação ao tipo de agente usado do que a estirpe CIAT 899, provavelmente porque a estirpe CIAT 899 produz β (1-2) glucano, que parece ter também um efeito protetor. β (1-2) glucano combinado com sacarose protege mais os lipossomas liofilizados de perdas do que a trealose. Entretanto, o β (1-2) glucano, isoladamente, não tem capacidade de proteção. Foi observado um alto nível de água descongelada associado com o glucano hidratado; além disso, o evento termal foi de, aproximadamente, 70ºC, o que correspondeu à transição gel-sol do glucano. Essas descobertas são importantes para o entendimento dos mecanismos com que o glucano contribui para a proteção de células e lipossomas liofilizados e para aumentar a sobrevivência do rizóbio nos inoculantes.Termos para indexação: armazenamento, inoculação, simbiose, glucano.

Choosing source of microorganisms and processing technology for next generation beet bioinoculant

“…In the case of sugar beet the above mentioned positive sucrose impact was enhanced by trehalose addition. Similar effect was observed for rhizobial strains, where trehalose worked better than sucrose/peptone mixture 58 . In general, 16S rRNA gene sequencing results considering diversity of endophytes associated with sea and sugar beet root did not show any effect of applied osmoprotectants neither on alpha nor beta diversity of bacteria.…”

Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry

“…However, the foot current also increases from 18 to 30 °C, but no increase in foot frequency is observed. The role of membrane lipids might be important here, and the range of temperature from 6 to 18 °C might cross a gel (solid) to liquid–crystalline transition stage in plasma membrane, − perhaps explaining the change from the low to middle versus to high temperature. Interestingly, the trend of the change of t foot with temperature contrasts that of the duration of whole spike, including t 1/2 , t rise , and t fall .…”

Shelf Life of Azospirillum brasilense in Alginate Beads Enriched With Trehalose and Humic Acid

“…Trehalose, in turn, is a disaccharide that can be used as source of energy and as protector against dehydration. Trehalose can increase the viability of freeze-drying cells as 70% of them survived after drying (Leslie, Israeli, Lighthart, L. Crowe, & J. Crowe, 1995), compared to other adjuvants (Pereira, Oliver, Bliss, L. Crowe, & J. Crowe, 2002). Nevertheless, the effect of trehalose and humic acid on the survival and viability of alginate encapsulated cells, in a short period of storage, remains unknown.…”