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The samples were filtered through 3-��m nitrocellulose membrane filters (Millipore, Carrigtwohill, Ireland) to eliminate most grazing protozoa (Weinbauer and Hofle 1998). Fresh pig manure (age http://www.selleck.cn/products/azd4547.html (COD) 51?g O2 per l. Microcosms, which consisted of 100-fold dilution of pig manure in filtered river water, were prepared in sterile 2-l or 5-l flasks. Four treatments were tested combining two levels of temperature with two oxygen conditions: 4��C or 20��C under an aerobic (5-l flasks with 2?l of river water with pig manure) or a microaerophilic (2-l flasks with 2?l of river water with pig manure) atmosphere. Microaerophilic flasks were hermetically sealed with silicone caps. Aerobic conditions were established by flushing the river water microcosms with 0��22-��m-filter-sterilized air (Millipore, St Quentin-en-Yvelines, France) to obtain dissolved oxygen saturation. Microaerophilic conditions http://www.selleckchem.com/products/MK-1775.html were established by flushing the river water microcosms with filter-sterilized nitrogen (0��22?��m) at the beginning of the experiment and after each sampling. In these microaerophilic conditions, oxygen concentrations ranged from 0��4 to 1��9?mg oxygen per l (corresponding to 1��5�C16��9% of oxygen saturation). The four river water microcosms and their respective control river water microcosms (same conditions without manure) were kept in the dark and gently stirred with a magnetic barrel. Fourteen samples were collected in duplicate over a 43-day period from each microcosm. Sampling was carried out daily for the first 5?days, then every 2?days until day 11 (D11) and then every 4?days until D26. Additional samples were collected on D34 and D43. At each sampling point, 55?ml of water were collected in duplicate either with a sterile pipette, for aerobic conditions, or with a 20-ml sterile syringe, for microaerophilic conditions and transferred to sterile vials for molecular and cultural analyses. After sampling, microaerophilic microcosms were flushed with filter-sterilized nitrogen for at least 15?min. For each duplicate http://www.selleckchem.com/products/AZD6244.html sample collected per river microcosm, 5?ml were kept for the enumeration of faecal indicators and 50?ml were filtered through 0��22-��m Nuclepore membrane filters (Whatman, Brumath, France). The filters were then placed in 1��6?ml of Stool Lysis (ASL, Qiagen, Courtaboeuf, France) buffer and frozen at ?20��C until DNA extraction. DNA was extracted using the QIAamp DNA stool kit (Qiagen) following the manufacturer��s instructions. The final elution volume was 50?��l. For each sample, a volume of 5?ml of water was used for the enumeration (in duplicate) of faecal indicators.