The distinct amino acid carbon isotope fingerprint of obligate anaerobic bacteria allows estimation of gut bacteria as protein sources for herbivorous mammals.

Herbivores evolved mutualisms with bacteria that break down plant fiber and provide usable energy. Bacteria may also provide a source of dietary protein, but to differing extents: foregut fermenters digest rumen bacteria in addition to plant proteins, whereas hindgut fermenters cannot, but may recoup bacterial protein by consuming cecal pellets (coprophagy). Carbon isotope fingerprinting of essential amino acids (EAAs) is a recently refined approach that uses characteristic patterns of carbon isotope ratios among EAAs synthesized by major lineages of life (e.g., bacteria, fungi, and plants) to identify protein sources in consumers. Here, we use carbon isotope fingerprinting to 1) answer whether obligate anaerobic bacteria (i.e., gut bacteria) have the same carbon isotope fingerprint as previously measured aerobic or facultatively anaerobic bacteria; and 2) evaluate this method as a tool in differentiating the contribution of bacterial protein to herbivores with foregut (moose Alces alces and muskox Ovibos moschatus) versus hindgut fermentation (horse Equus ferus caballus) and hindgut fermentation with coprophagy (snowshoe hares Lepus americanus), represented by blood serum/plasma EAAs. We found that obligate anaerobic bacteria had a carbon isotope fingerprint distinct from obligate aerobic and facultative anaerobic bacteria. Moose, muskox, and snowshoe hares had approximately equal proportions of EAAs from diet and gut bacteria, while horses had two-thirds of EAAs from diet and one-third from gut bacteria. Carbon isotope fingerprinting provides a biologically sensitive and field adaptable measure for understanding bacterial contribution to protein nutrition in herbivores with different digestive strategies.