Data Package Metadata   View Summary

Experimental Design

We quantified seasonal variation in biofilm composition and CO2 exchange in response to lowered and raised water-table position (relative to a control) during years with varying levels of background dissolved organic carbon (DOC). We then used nutrient-diffusing substrates to evaluate cause-effect relationships between changes in plant subsidies (i.e., leachates) and biofilm composition among water-table treatments.

Methods

FIELD: This study was conducted at the Alaska Peatland Experiment (APEX) site. To capture seasonal variation in biofilm composition among water-table treatments, we quantified the autotrophic and heterotrophic components of the biofilm weekly during each summer growing season. Microbial biofilm was sampled at six locations within each of the three water-table plots and each sample was a composite collected from four senesced Carex utriculata stems (each 10 cm in length) within a 1 m2 area (Kane et al., 2021). The microbial biofilm was removed from substrates with a toothbrush and the resulting slurry was homogenized and split for analysis of chlorophyll a and ash-free dry mass (AFDM). Gas flux (i.e., CO2) was measured within each water-table treatment (within plot n = 3) in the same location as, but prior to, harvesting stems for biofilm collection (described above). Flux measurements were collected only during flooded conditions (i.e., when there was a saturated photic zone for aquatic biofilm development) using a floating chamber (Kane et al., 2021). This resulted in 2 sampling campaigns in 2021, 10 in 2022, and 6 in 2023. Water depth was similar among water-table treatments at the time of sampling (i.e., flooded across all treatments) in either year. The floating chamber was constructed from a clear polycarbonate plastic bucket (18.9 L) with Styrofoam floatation around the base and one CPU fan inside for air circulation. This design allows for the measure of CO2 flux in the airspace above the water. The floating chamber was equipped with an airlock to equilibrate pressure. Temperature, relative humidity, and photosynthetically active radiation (PAR) were logged continuously with a PP Systems TRP-1 sensor mounted within the clear floating chamber. The CO2 flux rate (�g CO2 m-2 s-1) was calculated as the slope of the linear relationship between headspace CO2 concentration and time using a portable infrared gas analyzer (IRGA; PP Systems EGM-4, Amesbury, MA, USA). Net ecosystem exchange (NEE) was measured under ambient light conditions and positive NEE values indicated carbon release to the atmosphere while negative values indicated carbon uptake. For annual estimates of the biofilm contribution to CO2 flux, we converted NEE values to g CO2 m-2 y-1 by converting seconds to days assuming the average 21 h d-1 of sunlight available for biofilm photosynthesis (Hinzman et al., 2006). We then converted NEE from days to years based on the growing season length of 135 days for the study site (Hinzman et al., 2006). Outside of this timeframe, environmental conditions constrain biofilm production. Floating chamber measurements were conducted within water-table treatments from a small inflatable boat or by wading along the established boardwalk network. Nutrient diffusing substrates were constructed using 60 mL polyethylene canisters filled with agar + one of three plant leachates (3 g L-1; Carex, Potentilla, Equisetum) or a control with agar only. Leachate amendments were selected to emulate natural carbon and nutrient levels upon release (Rober et al., 2023). Canisters were topped with a fritted glass disc, providing an inorganic substrate for biofilm colonization (Tank et al., 2017). Inorganic substrates were used so that we could evaluate the influence of plant leachates, a source of organic carbon, on the biofilm community without the confounding effects of substrate composition. Each disc was held in place by a tight-fitting cap with a 2.5 cm-diameter circular hole cut from the center to allow for biofilm growth. Replicate NDS (n = 4) of each leachate treatment (Carex, Potentilla, Equisetum, or agar-only control) were secured to pieces of angle iron using all-purpose adhesive and one bar of each leachate treatment was submersed 10 cm below the water surface in each water-table treatment (Figure S1). The experiment was left for three weeks (beginning on June 3 2021, June 16 2022, and June 4 2023) to allow for biofilm colonization.Biofilm chlorophyll a, AFDM, and AI were used (as described above) to evaluate how the ratio of autotrophs to heterotrophs vary among NDS. Physiochemical conditions were measured within each water-table plot during biofilm collection and gas flux measurements. Water depth (cm) was measured with a meter stick and measurements of water temperature (�C), pH, conductivity (�S), and dissolved oxygen (DO; mg L-1) were made with a Hach model 40d multiprobe (Hach Company, Loveland, CO, USA). Photosynthetically active radiation (�mol m�2 s�1) was measured at approximately 10 cm below the water surface in each plot using a Li-Cor submersible quantum sensor and LI-250 light meter (Li-Cor, Lincoln, NE, USA) attached to a 1-m pole to prevent disturbance of macrophytes. Water samples for dissolved nutrient analysis and DOC were collected with a syringe and filtered through a 0.45 micrometer filter (Millipore Corporation, Bedford, MA, USA) into 60 mL acid-washed polyethylene bottles. Dissolved nutrient samples were stored on ice in the field and frozen until analysis for nitrate (NO3; mg L-1) and PO4 (mg L-1) using ion chromatography (Dionex Corporation, Sunnyvale, CA, USA). Dissolved organic carbon (mg L-1) and TDN (mg L-1) were analyzed using a Shimadzu TOC-V carbon analyzer with a TN unit (Shimadzu Scientific Instruments, Columbia, MD, USA). A subsample of filtered samples was analyzed for ultraviolet absorption at 254 nm using an Agilent Cary 60 UV-VIS spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Specific UV absorbance at 254nm (SUVA254) was calculated by dividing ultraviolet absorption at 254 nm by DOC concentration for an estimate of aromatic content and molecular weight. LAB: Biofilm chlorophyll a, AFDM, and autotrophic index (AI) were used to evaluate how the ratio of autotrophs to heterotrophs vary among water-table treatments over time (Steinman et al., 2006). Autotrophic biofilm colonization was quantified as chlorophyll a (a proxy for algal biomass) from a subsample collected on a 0.7 �m glass fiber filter (GF/F; Whatman, Maidstone, UK) following 24 h extraction with 90% ethanol in the dark. Chlorophyll a concentration was measured from the extract with a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) at 665 and 750 nm after acidification to correct for phaeopigments (APHA, 2005). A separate aliquot was poured into pre-weighed aluminum pans, dried at 105�C for 24 h and then ashed at 500�C for 1 h for measures of dry- and ash- mass, respectively, which were used to determine AFDM (APHA, 2005). An AI was used to quantify the ratio of autotrophs to heterotrophs among treatments. Autotrophic index is determined by dividing AFDM (a measure of the total autotrophic and heterotrophic biomass accumulated) by the concentration of chlorophyll a (a measure of algal biomass) using standard methods (APHA, 2005). Lower values of the index indicate a higher proportion of autotrophy in the microbial community (Bechtold et al., 2012).