Preservation of rhizobia by lyophilization with trehalose

Pedro Antonio Arraes Pereira,Fredrick Allen Bliss,John Crowe,Ann Oliver,Lois Crowe

doi:10.1590/s0100-204x2002000600012

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 beta (1-2) glucan, which may have a protective effect. beta (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 70ºC, 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.

Highlights

Symbiotic nitrogen fixation results from the interaction of a legume host with rhizobia, leading to root nodulation of the host plant
In order to isolate the β (1-2) glucan produced by the CIAT 899 strain, colonies were grown on YEM medium plates at 28oC for seven days. β (1-2) glucan was extracted by scratching the plates with a spatula to remove the bacterial colonies, and suspending them in sterile water
Fourier transform infrared (FTIR) spectroscopy was used to determine the gel to liquid crystalline phase transition temperatures of the membranes of rhizobial strains CIAT 899 and CFN 42

Summary

Introduction

Symbiotic nitrogen fixation results from the interaction of a legume host with rhizobia, leading to root nodulation of the host plant. Main problems of the inoculation industry is to keep rhizobial cells viable in large numbers in the inoculant. This is especially important with the recent tendency to shift from peat-based to alternative inoculants containing freeze-dried rhizobia. The disaccharide trehalose, which accumulates to extremely high levels in several dehydrationtolerant yeast strains, has been used with great success to protect bacteria from lyophilization-induced damage (Israeli et al, 1993; Leslie et al, 1995). Escherichia coli cells freeze dried in 100 mM trehalose showed as high as 75% survival after rehydration, compared to less than 10% survival after freeze drying in the absence of added sugar (Leslie et al, 1995)

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Conclusion