The focus of WP3 will be on the influence of the DOM quality on the growth and activity of benthic microorganisms and on the utilization of the DOM, including the production of greenhouse gases.
WP 3.1. Laboratory experiments about the bacterial response to different DOM quality:
Based on the results of WP2, we will perform short-term flume experiments with sediments exposed to different DOM sources. Small PVC flumes (0.4×0.03 m) will be filled with pre-cleaned sand up to a sediment depth of 1 cm. A peristaltic multi-channel pump will provide a permanent flow through the system with unfiltered, oligotrophic stream water. Half of the flumes will be continuously supplied with nutrients via a 2 cm thick, nitrate- and phosphate-enriched Agar layer underneath the sand to mimic conditions in agricultural streams. The experiments will be carried out in the dark at 18°C in the climate chamber. Oxygen will be continuously monitored in the flumes via Fi-Box oxygen sensors. After a pre-conditioning phase of 2 weeks, DOM will be added to both enriched and non-enriched flumes to reach concentrations similar to in-situ DOC concentrations of agricultural streams. Water will be analyzed daily for DOM quality and quantity changes between day 1 and day 7 after the DOM addition. Sediments will be analyzed before the addition and on day 7 for bacterial abundances, bacterial production, and extracellular leucine aminopeptidase activities. Experiments will be repeated for different DOM sources according to the results of WP 2.
WP 3.2. Incubation experiments for greenhouse gas production:
Short-term incubations will be performed to measure greenhouse gas production in response to different DOM additions to stream sediments. Sediments of the HOAL stream will be incubated in airtight 250 mL Schott bottles in 10 mL stream water enriched with different DOM under both oxic and anoxic conditions in the dark at 18°C over 4 hours. At the beginning and the end of the experiments, gas samples from the headspace will be analyzed for CO2 (oxic treatment) and CH4 (anoxic treatment) via gas chromatographie (HP5890II GC) at the Institute for Soil Research of the University of Natural Resources and Life Sciences.
WP 3.3. Analyses of DOM processing in the HOAL stream:
Parallel to the water samples in WP2.2, we will take water and sediment samples along the HOAL stream and analyze them for a) bacterial abundances in the water and sediments via Flow cytometer (Beckman-Coulter), b) microbial respiration in the water and sediments (analog to BSB5), c) bacterial secondary production (3H-leucine incorporation centrifuge method) and d) extracellular leucine aminopeptidase activity via addition of a fluorogenic substrate (Leucin-AMC) and measurement in a microplate reader. Extracellular leucine aminopeptidase activity can cleave N-terminus amino acids from peptide chains for microbial carbon and nitrogen uptake and, thus, be used as surrogate for DOM bioavailability. Data will be correlated to DOM changes along the study reach.