Organisms incorporate stable isotopes from their growth environment into their biological molecules, thus stable isotope ratios can provide a link between an organism and its environmental origin. Stable isotope ratios have been used to trace origins of substances and organisms as diverse as cocaine, butterflies, and elephants. We tested whether they might also yield information about the culture environment of microorganisms.

The ¹⁸O and D values of precipitation are predictable based on topography, distance from an ocean, and climate, and maps based on measured values have been assembled. Although not unique for every geographic region, the ¹⁸O and D values nevertheless constitute a geographic signature for a given region that can often be used to distinguish waters from two such regions. Local tap water is usually isotopically similar to surface water. We hypothesized that the O and H stable isotope ratios of microorganisms might contain a potential geographic signature imparted through their culture water.

We grew Bacillus subtilis, a Gram-positive spore-forming soil bacterium, in liquid medium made with 4 isotopically distinct waters. We harvested both vegetative cells and spores and measured their O and H stable isotope ratios. The graph to the left shows the relationship between the ¹⁸O value of the microbes and their culture water. Each point represents the average of a minimum of three independent trials. The slope of 0.74 in the spore relationship indicates that the spores obtained about 70% of their oxygen atoms from the culture water

The relationships between the D values of spores or cells and culture water were also linear. Each point again represents the average of at least three independent trials. Spores obtained about 30% of their H atoms from water.

To test whether stable isotope ratios might be used to link spores to the geographic region in which they were grown, we sent dehydrated culture medium and genetically identical B. subtilis cultures to laboratories across the United States. Our colleagues produced spores on the medium we provided, and sent us the spores along with a sample of their laboratory water.

We were able to assign spore samples to their laboratories of origin based on O or H stable isotope ratio values. The samples from Columbus, OH, and Durham, NC could not be distinguished because the water from those laboratories was isotopically identical The graph shows the actual O and H isotope ratios of the spore cultures (the points) with the values predicted from our model (the line). In every case the actual values matched within 95% confidence limits.

The following three graphs show the relationship between C, N and H stable isotope ratio values of dehydrated culture media and spores produced in liquid media made with the powders. Each point in the graphs represents the value of three cultures grown in an individual medium. The graph to the right shows that the stable carbon isotope ratio value (¹³C) of spores was very close to the
¹³C value of the culture medium in which the spores were produced.

The relationship between stable N isotope ratio values (¹⁵N) of medium nutrients and spores is essentially 1:1 with a fairly constant offset (y-intercept) value. Since the spores obtain all their C and N atoms from nutrients in the culture medium, we expected the slopes of the lines of the C and N relationship to be 1.0, and this value was indeed within the 95% confidence intervals of the experimentally determined relationships.

In contrast to C and N atoms, which spores must obtain from the culture medium nutrients, spore H atoms can originate in either the nutrients or the water. The graph to the left shows the relationship between stable H isotope ratio values (D) in the powdered medium nutrients and spores. The slope of 0.68 indicates that about 70% of the H atoms in spores originate from the culture medium nutrients.

We also looked at the relationship between H stable isotope ratio values of the water used to make culture media and spores produced in the various media. To do this, we made four batches each of five different kinds of media, using isotopically distinct water for each batch. We then grew at least three cultures in each of the twenty batches of media and allowed them to sporulate. The graph at the right shows the relationship between ²H values of the water and spores. The slopes of all the lines are approximately 0.3, indicating that about 30% of the H atoms in spores originate from the culture medium water and complementing the 70% from nutrients.

To determine how much variation exists in stable isotope ratios of commercially available bacteriological culture media, we measured the carbon, nitrogen, oxygen and hydrogen stable isotope ratio values of over 500 samples of culture media collected across the United States. We found sufficient variation to produce readily distinguishable spore isotope ratios. (Kreuzer-Martin et al, 2004, Part 2)

These results extend the findings reported in Kreuzer-Martin et al 2003 that stable isotope ratio analysis of microbiological agents could provide useful forensic information. Given the isotopic variation present in culture media, the linear relationships between C, N and H stable isotope ratios of culture medium nutrients and spores should allow investigators to match seized agents to seized culture media from a suspect laboratory. Since the stable isotope ratio values of water carry a geographic signature, the linear relationships between culture water and spore D values can potentially provide geographic information about where a culture might have been produced. This information is very different from genetic information; thus stable isotope ratio analysis of microbial agents should provide a useful complement to genetic analysis.