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1. Carbon stable isotopes are commonly used as a research tool in physiological ecology. When elements are consumed, they are naturally enriched or depleted as the consumer processes them. The difference in isotopic composition between the consumer and the diet is known as the discrimination factor (Δ¹³C). 2. Mixing models are used to estimate the contribution of multiple dietary components to a consumer's tissues and discrimination must be estimated in the model. Often, discrimination factors vary depending on multiple factors, yet in many models the discrimination factor is assumed to be constant for each dietary component. 3. Few studies have evaluated the mechanistic basis of stable isotope metabolism and discrimination during macromolecule biosynthesis, despite the potential to improve estimations of discrimination factors. We tested whether ¹³C discrimination depends on the dietary concentration of ¹³C by culturing the bacterium Bacillus subtilis in a gradient of broths ranging from a δ¹³C of -11·8[per thousand] to -25·3[per thousand]. We found an increase in discrimination in whole bacterial tissue, bulk lipid, and lipid-extracted fractions as dietary the concentration of ¹³C increased, with lipids showing the greatest discrimination ranging from 2·72[per thousand] in the low ¹³C broths to 15·5[per thousand] in the high¹³C broths. 4. These findings contrast with the majority of isotopic ecology data that typically show a moderate enrichment of ¹³C as trophic level increases. This discrepancy is attributed to the de novo biosynthesis of the majority of cellular components in this study as opposed to the effects of isotopic routing seen in more metabolically complex taxa.