A system to quantitatively recover bacterioplankton respiratory CO2 for isotopic analysis to trace sources and ages of organic matter consumed in freshwaters

We present a new system and method to measure the natural abundance isotopic (delta C-13, Delta C-14) values of respiratory CO2 produced by bacterioplankton, to directly assess the sources and ages of organic carbon (OC) respired by bacteria in aquatic ecosystems. The Respiratory Carbon Recovery System (ReCReS) and operating procedure were designed to reduce background dissolved inorganic carbon values by > 98% and then quantitatively recover the CO2 derived from bacterial respiration after incubation of freshwater samples. The 2-component ReCReS consists of an airtight incubation system ( 20 L), for short-term regrowth incubations of filtered water samples inoculated with ambient bacteria, and a harvest system to recover the respiratory CO2 produced during these incubations. The multistep operating procedure involves the following: ( 1) filling of incubation system with 0.2-mu m filtered sample water; ( 2) addition of 1 N HCl ( pH to similar to 2.8); ( 3) sparge with ultrahigh-purity (UHP) He to remove dissolved inorganic carbon; ( 4) sparge with UHP, volatile organic carbon-, CO2-free air to replenish oxygen; ( 5) neutralization ( 1 N carbonate free NaOH), reinoculation with the ambient bacterial assemblage, and incubation (80-132 h); ( 6) acidification to pH 2.8; and ( 7) UHP He sparge for > 12 h. The evolved CO2 is sent through 2 water traps ( dry ice slurries) before cryogenic trapping in liquid N-2. Control incubations were processed concurrently to evaluate extraction efficiencies, potential methodological contamination, and fractionation artifacts. Collectively, our results suggest that respiratory CO2 is quantitatively recovered and the isotopic fidelity (delta C-13, Delta C-14) between the OC respired and the CO2 harvested is retained. Moreover, the system allows the recovery of sufficient C to measure both delta C-13 and Delta C-14 values, even in the most oligotrophic systems.