Sensitive quantification of dipicolinic acid from bacterial endospores in soils and sediments

Endospore-forming bacteria make up an important and numerically significant component of microbial communities in a range of settings including soils, industry, hospitals and marine sediments extending into the deep subsurface. Bacterial endospores are non-reproductive structures that protect DNA and improve cell survival during periods unfavourable for bacterial growth. An important determinant of endospores withstanding extreme environmental conditions is 2,6-pyridine dicarboxylic acid (i.e. dipicolinic acid, or DPA), which contributes heat resistance. This study presents an improved HPLC-fluorescence method for DPA quantification using a single 10-min run with pre-column Tb³⁺ chelation. Relative to existing DPA quantification methods, specific improvements pertain to sensitivity, detection limit and range, as well as the development of new free DPA and spore-specific DPA proxies. The method distinguishes DPA from intact and recently germinated spores, enabling responses to germinants in natural samples or experiments to be assessed in a new way. DPA-based endospore quantification depends on accurate spore-specific DPA contents, in particular, thermophilic spores are shown to have a higher DPA content, meaning that marine sediments with plentiful thermophilic spores may require spore number estimates to be revisited. This method has a wide range of potential applications for more accurately quantifying bacterial endospores in diverse environmental samples.