Bacillus subtilis, a Gram-positive, endospore-forming soil bacterium, was grown in media made with water of varying oxygen (␦ 18 O) and hydrogen (␦D) stable isotope ratios. Logarithmically growing cells and spores were each harvested from the cultures and their ␦ 18 O and ␦D values determined. Oxygen and hydrogen stable isotope ratios of organic matter were linearly related with those of the media water. We used the relationships determined in these experiments to calculate the effective whole-cell fractionation factors between water and organic matter for B. O) that form the organic matter of those organisms. For example, isotope ratios have been used to trace the origins of migratory butterflies (1), birds (2, 3), and elephants (4, 5). Point-of-origin information can span several spatial scales. As an example, the physiological differences between C 3 and C 4 photosynthetic pathways, which result in large 13 C͞ 12 C differences, allow one to trace the flow of this organic carbon as differential dietary inputs to animals (6), the transport of carbon across ecosystems (7), and, ultimately, the movement of region-specific carbon back into the atmosphere (8-10). This sourcing aspect of stable isotopes has also been applied to forensic issues, such as determining the pointof-origin of illicit drugs (11, 12) and adulteration of foods and alcoholic beverages (13-15).Continentality, storm-track trajectories, and moisture origins result in substantial geographic gradients in the ␦ 18O and ␦D values of precipitation and, therefore, of local waters (16). Several maps have been produced that show the extent of isotopic variations in waters at the continental and global scales (17, 18). Again, organisms often record the isotopic composition of these source waters in their organic compounds. In trees, for example, the isotopic composition of cellulose is correlated with that of source water (19,20). Similar examples are found in animals where oxygen isotope ratios of bone and blood are closely related to that of the local water (21).We hypothesized that microorganisms, too, should show a record of their growth environment in their cellular components. If relationships between growth environment and cellular isotope composition could be elucidated, it might become possible to draw conclusions about the waters in which the microbes had been grown, particularly for genetically identical organisms cultured in different geographic regions. We used the growth of Bacillus subtilis in laboratory culture as a model system. B. subtilis is a nonpathogenic, endospore-forming, Gram-positive soil bacterium. Because B. subtilis can grow on a variety of nutrients, we focused first on the relationship between the ␦ 18O and ␦D values in the microbial products (cells or spores) and those ratios in the water used to make the culture media. We then tested the values predicted by laboratory model with cultures grown in different geographical regions across the United States. MethodsOur experimental organism was B. subtilis strain 6051 (American ...

The stable isotope ratios of a seized pathogen culture could potentially reveal information about the environment in which the agent was produced. In this paper we describe general relationships between stable isotopes of carbon, nitrogen, and hydrogen in bacteriological culture media and spores of Bacillus subtilis, an endospore-forming soil bacterium. In numerous media that varied both in nutrient composition and water stable isotope ratios, medium to spore enrichment in carbon isotopes was 0.3 ± 2.0‰ (parts per thousand), and in nitrogen, 4.5 ± 0.7‰. We achieved mass balance for the contribution of hydrogen isotopes from nutrients (70%) and water (30%) to spores in independent experiments by varying the isotope ratios of nutrients or water. A model was derived for predicting the isotope ratio values of spores from those in nutrients and water.

Since the anthrax attacks of 2001 the need for methods to trace the origins of microbial agents has become urgent. The stable isotope ratios of bacteria record information from both the nutrients and the water used to make their culture media (1) and could potentially be used to provide information about their growth environment. We present a survey of carbon (C), nitrogen (N), and hydrogen (H) stable isotope ratios in 516 samples of bacteriological culture media. The observed variation was consistent with expected isotopic variation in the plant and animal products upon which the media are based. The variation is sufficient to translate into substantial isotope variation in cultures grown on different batches of media, and thus to allow investigators to determine whether seized media could have been used to produce seized bioweapons agents.