Spatial variance in river bed methane cycling &#8211; measurement and interpretation of geochemical profiles linked with quantitative PCR and 16S rRNA sequencing
Authors: Michaelis Tamara , Orsi William , Wunderlich Anja , Baumann Thomas , Einsiedl Florian 
Published: March 2022

&lt;p&gt;Rivers and streams are often supersaturated in methane (CH&lt;sub&gt;4&lt;/sub&gt;) and emit significant amounts of this potent greenhouse gas to the atmosphere. Methane is produced by methanogenic archaea in anaerobic sediments where energetically more favorable redox processes are substrate-limited. Diffusing up towards the sediment surface, methane can be oxidized in the hyporheic zone (HZ) aerobically with oxygen or anaerobically with nitrate, nitrite, sulfate or iron and manganese oxides as electron acceptors. Yet, knowledge about net carbon emissions from streams is restricted, because high spatial heterogeneities in production and consumption zones make bottom-up global estimations particularly challenging.&lt;/p&gt;&lt;p&gt;In this study we want to increase process understanding of riverine methane cycling, in particular production and oxidation in hyporheic stream sediments. Several studies have investigated predictors for potential methane production and oxidation in river sediments using incubation experiments. We chose a different approach by measuring high-resolution depth-dependent geochemical profiles at five different locations across a stream bed. An in-situ equilibrium dialysis sampler (peeper) was used to obtain pore-water samples with a 1 cm depth-resolution for the measurement of dissolved oxygen, relevant anion and cation concentrations as well as methane concentrations and stable carbon isotopes (&amp;#948;&lt;sup&gt;13&lt;/sup&gt;C) of methane. In the methanogenic zone stable carbon isotopes may provide information about the most relevant methane production pathway, while an isotopic enrichment in &amp;#948;&lt;sup&gt;13&lt;/sup&gt;C-CH&lt;sub&gt;4&lt;/sub&gt; towards the sediment surface linked with decreasing methane concentrations may indicate microbial degradation. Production and oxidation rates were estimated using inverse numerical modeling of measured concentration gradients. Additionally, the concentration of 16S rRNA genes (a measure of bacteria biomass) was quantified from one of the locations using quantitative PCR, which revealed an increase in microbial biomass at the nitrate-methane transition zone.&amp;#160; Sequencing of the 16S rRNA genes shows clear shifts in microorganisms driving the streambed methane cycle at and below the nitrate-methane transition zone.&lt;/p&gt;&lt;p&gt;The measured &amp;#948;&lt;sup&gt;13&lt;/sup&gt;C-CH&lt;sub&gt;4&lt;/sub&gt; values between -75 &amp;#8240; and -70 &amp;#8240; indicate that hydrogenotrophic methanogenesis is the dominant production pathway. However, we found that methane fluxes into the surface water were low. Mainly responsible for a strong decrease in methane concentrations towards the sediment surface were most likely diffusive processes. Decreasing methane concentrations linked with a significant enrichment in &amp;#948;&lt;sup&gt;13&lt;/sup&gt;C towards heavier isotopes was only observed at one of the sampling locations. The isotopic shift together with modeling results and microbiological analyses may reveal microbially driven aerobic and anaerobic methane oxidation in the HZ, but in most locations methane is only oxidized at low rates where the environment is already methane depleted by diffusion. Limiting for both aerobic and anaerobic methane oxidation was supposedly the low methane concentration in zones with available electron acceptors.&lt;/p&gt;