Compound-specific 13C stable isotope probing confirms synthesis of polyhydroxybutyrate by soil bacteria

Rationale: Many bacteria synthesize carbon (C) and energy storage compounds, including water-insoluble polyester lipids composed mainly or entirely of poly(3-hydroxybutyrate) (PHB). Despite the potential significance of C and energy storage for microbial life and C cycling, few measurements of PHB in soil have been reported. Methods: A new protocol was implemented, based on an earlier sediment extraction and derivatization procedure, with quantification by gas chromatography/ mass spectrometry (GC/MS) and C-13-isotopic analysis by GC/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Results: The PHB content was 4.3 mu g C g(-1) in an agricultural soil and 1.2 mu g C g(-1) in a forest topsoil. This was an order of magnitude more PHB than obtained by the existing extraction method, suggesting that native PHB in soil has been previously underestimated. Addition of glucose increased the PHB content by 135% and 1,215% over 5 days, with the largest increase in the relatively nutrient-poor forest soil. In the agricultural soil, 68% of the increase was derived from added( 13)C-labeled glucose, confirming synthesis of PHB from glucose for the first time in soil. Conclusions: The presence and responsiveness of PHB in both these contrasting soils show that PHB could provide a useful indicator of bacterial nutritional status and unbalanced growth. Microbial storage could be important to C and nutrient cycling and be a widespread strategy in the life of soil bacteria. The presented method offers new insight into the significance of this compound in soil.