BACKGROUNDIt is generally accepted that recalcitrant dissolved organic carbon (RDOC) concentrations in the ocean are in the region of 42 μmol/L. However, the concentrations of RDOC in the karst river systems remain unidentified and there is little evidence about these compounds unavailable to microbial degradation.

OBJECTIVESTo reveal the existence of RDOC and its concentrations in the karst river systems, by collecting water samples at different depths (0 m, 5 m and 10 m) from three typical sites (city-river section, reservoir area and outflow area) in the dammed Liu River in 2006.

METHODSWater samples (3 L) were pre-filtered in-situ using 3 μm filter membranes, and then filtered through 0.22 μm pore-size filter membranes. The 0.22 filters were used for DNA extraction following the manufacturer's instructions of the Power Water DNA Isolation Kit (Mobio Laboratories, Inc., Carlsbad, CA, USA). The real-time PCR assay of the 16S rRNA gene, as a marker of phytoplankton-bacteria, was carried out in a volume of 25 μL. The assay mixture contained 12.5 μL Green-2-Go qPCR Mastermix (Sangon Biotech Co., Ltd, China), 1 μL of primer (10 μmol/μL), 9.5 μL of distilled water, and 1 μL of template DNA (at 5 ng/μL). Thermal cycling conditions for 16S rRNA gene were as follows:an initial cycle of 95℃ for 3 min, 39 cycles of 95℃ for 60 s, 56℃ for 60 s, 72℃ for 60 s, and 72℃ for 5 min. The primers for 16S rRNA gene were designated as 338f (5'-CCTACGGGAGGCAGCAG-3') and 518r (5'-ATTACCGCGGCTGCTGG-3'). Thermal cycling, fluorescent data collection, and data analysis were carried out with CFX96 Touch? Real-time PCR Detection System (Bio-Rad, USA) according to the instruction manual. The filtrates after through 3 μm filter membranes were transferred to sterile glass bottles. The bottles were incubated in the dark room at 30℃ for 180 days. Dissolved organic carbon (DOC) concentrations of all water samples were determined at 30 days interval on a total organic carbon analyzer (Multi N/C^© 3100) at the Institute of Karst Geology, CAGS. The obtained data was used for correlation analyses using the Pearson correlation method (one-tailed t test) with significance defined as p<0.05 by using SPSS 13.0 software for Windows XP (IBM, Armonk, NY, USA).

RESULTSThe relationship between C_t and log starting concentration is linear (R²>0.99). The amplification efficiencies are 90%-105%. 24.63 μmol/L RDOC is stored in the dammed Liu River systems, supporting the hypothesis that they are resistant to microbial degradation and the dilution limits dissolved organic carbon utilization.

CONCLUSIONSThese findings demonstrate the mechanism for the long-term storage of RDOC in karst hydrosphere, which has hitherto been largely ignored. The advanced techniques (Fourier-transform Ion Cyclotron Resonance Mass Spectrometry) provide new evidence to test the initial hypotheses for existence of RDOC in the deep karst reservoir waters.