Abstract Salinity and drought induce osmotic stress in plants and nodulating bacteria. The introduction of soybean in areas with higher soil salt contents or periods of drought pose a challenge for the rhizobial inoculants used to improve nodulation and enhance nitrogen fixation. Bradyrhizobium japonicum is a slow- growing rhizobium used for soybean inoculation that was previously regarded as salt-sensitive. We tested the survival ability of cultures of B. japonicum E109 at the exponential and stationary phases of growth in liquid culture medium against different concentrations of NaCl. We found that stationary-phase cells could tol- erate higher levels of salt than exponential-phase cells. This result suggested that the physiological manipula- tion of the cultures could improve the salt tolerance of this strain. Nonetheless, we also found that exponen- tial-phase cells adapted significantly better to two key situations that a commercial product must face, sur- vival in liquid formulations and survival in soil micro- cosms resembling conditions of drought. These results suggest that the use of actively growing cells could be an improvement in the production of inoculants. However, it is not cost-effective, because bacteria should be harvested at a time when cell density is lower than that of early stationary-phase cultures, which are normally used in the industry. To overcome this drawback we proved that a fed-batch system can pro- duce exponential-phase cultures with higher cell den- sities and able to produce liquid inoculants with acceptable survival rates. Keywords Bradyrhizobium japonicum Æ Fed-batch Æ Inoculants Æ Microcosms Æ Salt stress Æ Soybean Æ Stationary growth phase Introduction Rhizobia are able to fix atmospheric nitrogen when they are symbiotically associated with legumes. The inoculation of legume crops with specific rhizobia is a cost-effective and environmentally friendly practice that enhances N availability for the crop and reduces the pollution caused by chemical fertilizers. Commercial rhizobial inoculants must have an ade- quate number of active cells, frequently determined by national regulations. Some countries also set require- ments regarding the accepted levels of microbial con- taminants and the minimum expected shelf-life. One of the factors that will determine the success of a product is its capacity to overcome stressful situations along its life cycle. There are a number of factors that will determine product stability, among others, long- term survival of cells in the formulation and in soil, tolerance to temperature changes and tolerance to desiccation upon application to the seeds. In the course of evolution, microorganisms have developed sophis- ticated mechanisms to sense the environment and respond to stress. The adaptive responses are one of those strategies for survival, and they have an inter- esting potential for technological applications (Saarela et al. 2004; van de Guchte et al. 2002). The basic M. A. Soria (&) Æ F. E. Pagliero Æ O. S. Correa Æ N. L. Kerber Æ A. F. Garcia Facultad de Agronomı ´a, Microbiologı´a Agrı´cola, Universidad de Buenos Aires and IByF-CONICET, Av. San Martı´n 4453, 1417 Buenos Aires, Argentina e-mail: soria@agro.uba.ar Tel.: +54-11-4524-8061 Fax: +54-11-4514-8741 World J Microbiol Biotechnol (2006) 22:1235–1241 DOI 10.1007/s11274-006-9166-9 123 ORIGINAL PAPER Tolerance of Bradyrhizobium japonicum E109 to osmotic stress and the stability of liquid inoculants depend on growth phase Marcelo A. Soria Æ Fabiola E. Pagliero Æ Olga S. Correa Æ Norma L. Kerber Æ Augusto F. Garcia Received: 3 February 2006 / Accepted: 19 March 2006 / Published online: 21 April 2006 Ó Springer Science+Business Media B.V. 2006