CURRENT MICROBIOLOGY Vol. 20 (1990), pp. 387-389 Current Microbiology © Springer-Verlag New York Inc. 1990 Longevity of Bacteria: Considerations in Environmental Release J.M. Lynch AFRC Institute of Horticultural Research, Littlehampton, West Sussex, UK Abstract. Microbial growth theory has been developed primarily for laboratory culture. With increased opportunity for release of beneficial organisms, including those that have been geneti- cally engineered, into the environment, it is important to understand microbial behavior under natural conditions in which there is usually severe nutrient limitation. By investigation of the survival of three beneficial soil bacteria in distilled water, the surprising observation was made of cell longevities in excess of a year without substrate input. It is suggested that this could result from the utilization of dead cells within the population and from the viable cells' having a very low maintenance energy requirement, thus placing the cell in a state of arrested metabo- lism. Beneficial microorganisms could be released into the environment for crop protection and improve- ment of soil structure and nutrient status. While they should be persistent enough to be effective, they should not get out of control and cause unex- pected side effects. Therefore, for both economic and environmental reasons, a better understanding of microbial behavior under conditions of starvation is needed. Microbial growth theory predicts that without an input of maintenance energy in the soil, which is deficient in readily available carbon sub- strate, the introduced organisms will rapidly die [14]. As a test of this theory, the longevities and metabolic state of some useful soil bacteria have been investigated in conditions of extreme starva- tion by suspension of washed cells in distilled water on a shaking incubator at 25°C for periods up to 1 year. Two strains of Enterobacter cloacae were used, C2/4 having been isolated from decomposing wheat straw [6] and MOI from wheat roots. The species is of agricultural interest because it is a rhi- zosphere colonist [8], and various strains can fix dinitrogen [12], stabilize soil structure [6], and con- trol Pythium root disease [13]. To test whether the behavior of E. cloacae was typical of other soil bacteria, similar starvation ex- periments were set up with Pseudomonas putida WCS 358, a potential biocontrol agent [17] that pro- duces siderophores, and Azospirillum brasilense ATCC 29729, which fixes dinitrogen but can also stimulate plant growth by modifying root morphol- ogy [7]. Materials and Methods Bacteria were grown in nutrient broth (25 ml) contained in 250-ml conical flasks shaken at 100 rev min ~ for 48 h. They were har- vested by centrifugation at 5000 rev min ~for 10 min. They were then resuspended in sterile distilled water and recentrifuged. This procedure was repeated three times to obtain clean cells. After the final harvest, the cells were suspended in sterile dis- tilled water (25 ml) in a clean 250-ml conical flask in a shaking incubator at 25°C for periods up to 1 year. Four flasks per organ- isms were used. Sampling. Periodically, samples (0.5 ml) were withdrawn and dilutions made. Samples (0.5 ml) of the dilutions of 10-5, 10 6, and 10-v were spread on nutrient agar plates. The number of colonies appearing on plates after incubation for 24 h at 30°C were recorded to determine culture viability. Samples (0.5 ml) were also withdrawn to measure adenylates (AMP, ADP, and ATP) and total adenylate charge (AEC) [2] with the LUMAC A.E.C. Kit (Sonco Ltd, Batley, W. Yorkshire) and the LKB Wallac (Uppsala, Sweden) Model 1250 luminometer. ATP gener- ates bioluminescence in the luciferin-luciferase system, ADP is converted to ATP in the PEP-pyruvate kinase system, and AMP with ATP is converted to ADP with myokinase. The following formula relates the various measurements: AEC = (ATP + 0.5 ADP)/(ATP + ADP + AMP) At each sampling the adenylates and AEC were also mea- sured for a fresh bacterial culture to allow for any variation in reagent quality in the test. Loss of water by evaporation (but not by sampling) in the flasks was corrected by periodic addition of sterile distilled water. Address reprint requests to: Professor J.M. Lynch, AFRC Institute of Horticultural Research, Littlehampton, West Sussex, BN17 6LP, UK.