Bioavailable DOC : reactive macronutrient ratios control heterotrophic nutrient assimilation - An experimental proof of the macronutrient-balance hypothesis
Authors: Graeber Daniel , Tenzin Youngdoung , Stutter Marc , Weigelhofer Gabriele , Shatwell Tom , von Tümpling Wolf , Tittel Jörg , Borchardt Dietrich 
Published: March 2021

&lt;p&gt;We assess the &amp;#8220;macronutrient-balance hypothesis,&amp;#8221; which we define as: &amp;#8220;Aquatic heterotrophic nutrient assimilation is controlled by the balance between the bioavailable DOC : reactive macronutrient stoichiometry and the microbial stoichiometric macronutrient demand.&amp;#8221; Here we define the reactive macronutrients as the sum of dissolved inorganic nitrogen, soluble-reactive phosphorus (SRP), and dissolved bioavailable organic N (bDON) &amp; P (bDOP). A global meta-analysis of monitoring data from various freshwaters suggests this hypothesis, yet clear experimental support is missing.&lt;/p&gt;&lt;p&gt;We assessed this hypothesis in a proof-of-concept experiment for waters from four different small agricultural streams. We used seven different bioavailable DOC (bDOC) : reactive N and bDOC : reactive P ratios, induced by seven different levels of alder leaf leachate addition. With these treatments and a stream-water specific bacterial inoculum, we conducted a separate 3-day experiment, with three independent replicates per combination of stream water, treatment and sampling occasion. Here, we extracted dissolved organic matter (DOM) fluorophores by measuring excitation-emission matrices with subsequent parallel factor decomposition (EEM-PARAFAC). We assessed the true bioavailability of DOC, DON, and the DOM fluorophores as solute concentration difference between the beginning and end of each experiment. Separately, we predicted bDOC and bDON concentrations based on the bioavailable fluorophores, which we compared to their true bioavailability measured before. Due to very low DOP concentrations, the DOP determination uncertainty was high, and we had to neglect DOP as part of the reactive P.&lt;/p&gt;&lt;p&gt;For bDOC and bDON, the bioavailability measurements agreed with the same fractions calculated indirectly from bioavailable DOM fluorophores (bDOC r&amp;#178;&amp;#8196;=&amp;#8196;0.96, p&amp;#8196;&lt;&amp;#8196;0.001; bDON r&amp;#178;&amp;#8196;=&amp;#8196;0.77, p&amp;#8196;&lt;&amp;#8196;0.001), hence we could predict bDOC and bDON concentrations based on the molecular composition of DOM. Moreover, we found that bDOC : reactive nutrient ratios at specific ranges (molar bDOC&amp;#8196;:&amp;#8196;reactive N&amp;#8196;=&amp;#8196;2&amp;#8197;&amp;#8722;&amp;#8197;17; molar bDOC&amp;#8196;:&amp;#8196;reactive P&amp;#8196;=&amp;#8196;50&amp;#8197;&amp;#8722;&amp;#8197;300) control microbial heterotrophic nutrient uptake.&lt;/p&gt;&lt;p&gt;In summary, the results of our simple laboratory experiment provide first proof that the bDOC : reactive macronutrient ratio strongly controls heterotrophic reactive macronutrient uptake. Combined with the previous large-scale monitoring evidence, our study implies that the &amp;#8220;macronutrient-balance hypothesis&amp;#8221; holds in many aquatic ecosystems. However, this hypothesis needs to be corroborated by further experiments with different DOC sources and assessments of changes in bDOC : reactive macronutrient ratios on freshwater carbon and nutrient cycles.&lt;/p&gt;