Development of a Fatty Acid and RNA Stable Isotope Probing-Based Method for Tracking Protist Grazing on Bacteria in Wastewater

作者：Kuppardt Steffen; Chatzinotas Antonis; Kaestner Matthias

来源：Applied and Environmental Microbiology, 2010, 76(24): 8222-8230.

DOI：10.1128/AEM.01632-10

Removal of potential pathogenic bacteria, for example, during wastewater treatment, is effected by sorption, filtration, natural die-off, lysis by viruses, and grazing by protists, but the actual contribution of grazing has never been assessed quantitatively. A methodical approach for analyzing the grazing of protists on C-13-labeled prey bacteria was developed which enables mass balances of the carbon turnover to be drawn, including yield estimation. Model experiments for validating the approach were performed in closed microcosms with the ciliate Uronema sp. and C-13-labeled Escherichia coli as model prey. The transfer of bacterial C-13 into grazing protist biomass was investigated by fatty acid (FA) and RNA stable isotope probing (SIP). Uronema sp. showed ingestion rates of similar to 390 bacteria protist(-1) h(-1), and the temporal patterns of C-13 assimilation from the prey bacteria to the protist FA were identified. Nine fatty acids specific for Uronema sp. were found (20:0, i20:0, 22:0, 24:0, 20:1 omega 9c, 20:1 omega 9t, 22:1 omega 9c, 22:1 omega 9t, and 24:1). Four of these fatty acids (22:0, 20:1 omega 9t, 22:1 omega 9c, and 22:1 omega 9t) were enriched very rapidly after 3 h, indicating grazing on bacteria without concomitant cell division. Other fatty acids (20:0, i20:0, and 20:1 omega 9c) were found to be indicative of growth with cell division. The fatty acids were found to be labeled with a percentage of labeled carbon (atoms percent [atom%]) up to 50. Eighteen percent of the E. coli-derived C-13 was incorporated into Uronema biomass, whereas 11% was mineralized. Around 5 mol bacterial carbon was necessary in order to produce 1 mol protist carbon (y(x/s) approximate to 0.2), and the temporal pattern of C-13 labeling of protist rRNA was also shown. A consumption of around 1,000 prey bacteria (similar to 98 atom% C-13) per protist cell appears to be sufficient to provide detectable amounts of label in the protist RNA. The large shift in the buoyant density fraction of C-13-labeled protist RNA demonstrated a high incorporation of C-13, and reverse transcription-real-time PCR (RT-qPCR) confirmed that protist rRNA increasingly dominated in the heavy RNA fraction.

• 出版日期2010-12