19 Wildfires produce large amounts of pyrogenic carbon (PyC), including charcoal, known 20 for its chemical recalcitrance and sorption affinity for organic molecules. PyC can be transported to fluvial networks. Here it may alter dissolved organic matter 22 (DOM) concentration and composition as well as microbial biofilm functioning. Effects of 23 PyC on carbon cycling in freshwater ecosystems remain poorly investigated. Employing 24 in-stream flumes with a control vs treatment design (PyC pulse

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... addition), we present 25 evidence that field-aged PyC inputs to rivers can increase dissolved organic carbon 26 (DOC) concentration, and alter DOM composition. DOM fluorescence components were 27 not affected by PyC. In-stream DOM composition was altered due to leaching of 28 pyrogenic DOM from PyC and possibly concurrent sorption of riverine DOM to PyC. 29 Decreased DOM aromaticity indicated by lower SUVA245 (-0.31 units), and higher pH 30 (0.25 units) were associated with changes in enzymatic activities in benthic biofilms, 31 including a lower recalcitrance index (β-glucosidase/phenol oxidase), suggesting 32 preferential usage of recalcitrant over easily available DOM by biofilms. Particulate PyC 33 deposition onto biofilms may further modulate the impacts of PyC due to direct contact 34 with the biofilm matrix. This study highlights the importance of PyC for in-stream 35 biogeochemical organic matter cycling in fire-affected watersheds. 36 Keywords 37 Charcoal, black carbon, dissolved organic carbon, dissolved organic matter, biofilm 38 functioning 39 Vegetation fires annually burn ~4 % of Earth's vegetated land surface, forming 41 approximately 256 Tg of pyrogenic carbon (PyC). 1 PyC is a continuum of thermally-42 altered organic materials, of which a considerable fraction is highly recalcitrant, 43 persisting in the environment for prolonged periods of time. 2 The chemical composition 44 of PyC is determined by fire conditions (e.g. temperature, charring duration, oxygen 45 availability) and fuel type (e.g. grassy vs woody). 3 In fire-affected landscapes fresh as 46 well as aged PyC, in both dissolved and particulate forms, is mobilized to fluvial 47 ecosystems via water erosion. 4-6 Indeed, significant charcoal presence in river bed 48 deposits in fire affected ecosystems have been the subject of investigation decades 49 ago 7 and hydrological events (e.g. stormwater runoff) can transport large quantities of 50 PyC into river ecosystems in short periods of time. 6,8 Coppola and colleagues 9 found 51 that globally 15.8 ± 0.9% of riverine particulate organic carbon is of pyrogenic origin. 52 Jones and colleagues 10 estimated that PyC accounts for 12 ± 5% of the riverine 53 dissolved organic carbon (DOC, i.e., filtered < 0.45 µm). PyC is, therefore, a 54 quantitatively substantial fraction of the organic matter component in many river 55 systems. 56 Rivers do not only transport but also transform organic matter on its way downstream 57 towards the ocean. These transformations can occur via photochemical, microbial, and 58 mechanical processes. 11-14 Photo-chemical reactions, which are strongly site 59 dependent 15 preferentially degrade highly aromatic dissolved organic matter (DOM) 60 deriving from PyC. 16 In-stream microbiota, especially biofilm communities which are 61 hotspots of microbial functioning, are central to the role of fluvial ecosystems as 62 bioreactors of terrestrial material, 17 but the microbial degradation of pyrogenic DOM 63 remains poorly understood. Bostick and colleagues 18 recently measured considerable 64 degradation of the labile DOC fraction leached from fresh PyC in laboratory-based 65 experiments, however, the metabolization of fresh as well as aged PyC under natural 66 conditions remains elusive. Finally, mechanical processes can physically disintegrate 67 PyC into smaller particles. 14 This can lead to leaching of particulate as well as dissolved 68 pyrogenic organic matter and metals. 14,19,20 69 The less aromatic, more labile fraction of PyC can be a relevant component of in-stream 70 carbon turnover. 21,22 Changes in DOC quantity and DOM composition induced by PyC 71 may alter microbial functioning, based on observations in non-fire affected aquatic 72 systems. 12,23,24 For example, Freixa and colleagues 24 showed that shifts in DOM 73 sources (i.e. allochthonous to autochthonous) along the river continuum were 74 accompanied by a change in extracellular enzymatic activities. In addition to pyrogenic 75 DOM, PyC particles can also affect DOM composition and its bioaccessibility by 76 interacting with riverine DOM via selective adsorption. This process has previously been 77 observed for other carbonaceous materials, including carbon-nanomaterials, 25,26 78 graphite, and biochar. 26 79 PyC is therefore a common component in limnic systems, with the potential to alter 80 riverine microbial DOM cycling. However, field-based experiments to elucidate specific 81 impacts and processes are still lacking. Here we carry out a field experiment in a natural 82 river to investigate the effects of wildfire-derived PyC inputs on in-stream DOM 83 properties and biofilm functioning. In addition, to our knowledge this study is the first to 84 analyze effects of PyC on in-stream biofilm enzymatic activities. We hypothesized that of riverine DOM and leaching of pyrogenic DOM leading to a net increase in DOC 87 concentration, and (ii) microbial functions, measured via enzymatic activities by altering 88 substrate composition and pH. 89 Methods 90 2.