MATERIALS AND METHODS

4.1 STUDY LAKE

Kuivajärvi (Fig. 3) is a typical Finnish humic and mesotrophic lake that usually has anoxic hypolimnion during the late summer stratification. The lake has a mean pH of 6.5 (Dinsmore et al.

2013). It is located in the boreal zone close to the Hyytiälä Forestry Field Station in Juupajoki (N 61°

50.845’, E 24° 17.686'). The lake is surrounded by managed Scots pine forest (Pinus sylvestris) that stand on a flat terrain (Hari and Kulmala 2005).

The area has mean annual temperature of 3.5 °C and precipitation of 711 mm (Pirinen et al. 2012).

The lake has a surface area of 61.3 ha, length of 2.6 km and maximum depth of 13 m. The size of the catchment area is approximately 940 ha and it consists of forests as well as peat- and agricultural land (Miettinen et al. 2015). Lake Kuivajärvi has four upstream lakes: Pirttijärvi, Mustalammi, Piikainlammi and Saarijärvi. The northern inlet of Lake Kuivajärvi is located downstream of Lake Saarijärvi and has a length of approximately 250 m (Dinsmore et al. 2013).

Figure 3. The map of Lake Kuivajärvi (National Land Survey, www.paikkatietoikkuna.fi). A red dot represents the location of the measuring platform.

4.2 WATER SAMPLING AND MEASUREMENTS

Water sampling was carried out 4 times between May and September in 2016 at the deepest point of the lake (~12 m). The sampling dates were chosen to follow the development of the summer stratification. The water sampling was done on the measuring platform of Kuivajärvi and the analyses were performed at the University of Eastern Finland (Kuopio) and University of Jyväskylä (Table 4).

Table 4. Sampling schedule of this study.

Date Analysis

May 25, 2016 Temperature, pH, O2, gas concentrations (CH4, CO2)

July 18, 2016 Temperature, pH, O2, gas concentrations (CH4, CO2), nutrient samples,

13CH4, ¹³CO2

August 15, 2016 Temperature, pH, O2, gas concentrations (CH4, CO2), nutrient samples, DOC, ¹³CH4, ¹³CO2, ¹³C-CH4 oxidation experiment

September 5, 2016

Temperature, pH, O2, gas concentrations (CH4, CO2), nutrient samples, DOC, Fe, ¹³CH4, ¹³CO2, ¹³C-CH4 oxidation experiment

4.2.1 Temperature, oxygen and pH

Vertical profiles of dissolved O2 concentration (mg l^-1), O2 saturation (%) and water temperature (°C) were measured from a Limnos water sampler with a field meter YSI ProODO Optical Dissolved Oxygen Instrument (Yellow Springs Instruments). The measurements were done at 0.5 m intervals, starting from the surface water and continuing close to the bottom (12 m) without disturbing the sediment. The pH was measured from Limnos at 1 m intervals using WTW ProfiLine pH 3110.

4.2.2 Nutrient samples

Nutrient samples were collected at 1 m intervals from the surface water close to the bottom (11.5-11.75 m) by using Limnos water sampler. The samples were filtered through a plankton net (mesh

size 25 µm) and a filter unit (pore size 0.22 µm, Millipore^® Sterivex), and stored frozen (-18 °C) until the further analysis with Ion Chromatograph (Dionex DX-120) for the sulphate (SO42-) concentrations, and colorimetric analysis (Wallac^® 1420 Victor^3TM) for the nitrate (NO3-) and ammonium (NH4+) concentrations. The NO3- concentration was determined according to Miranda et al. (2001), and the NH4+ concentration was determined applying the method by Fawcett and Scott (1960). The concentrations of each sample were calculated using a standard curve.

The DOC concentration was determined with Shimadzu TOC-Vcph at the Lammi Biological Station, University of Helsinki. The analysis was based on the standard method SFS-EN 1484.

The concentrations of Fe³⁺/Fe²⁺ and sulphide (S^2-) were determined with LCK320 cuvette test reagents and Spectrophotometer (Hach Lange DR2800).

4.2.3 Gas concentrations and stable isotope compositions

The samples for the concentrations of CH4 and CO2 and stable isotopic analyses of CH4 were collected at 1 m intervals from the surface water close to the bottom (11.5-11.75 m) by using Limnos water sampler and transferring the water from the sampler into 60 ml polypropylene syringes (Terumo) with Luer Lok^® tips and three-way stopcocks (Codan) (n=3 per depth).

After returning to the laboratory, a headspace was created into the sampling syringes by replacing 30 ml of water with 30 ml of N2 gas. The syringes were placed into the water bath for 30 minutes (T=20

°C) and then shaken vigorously for 3 minutes. The headspace from the syringe was transferred into a evacuated 12 ml gas vial (Labco Exetainer^®). The vials were filled to a volume of 20 ml to ensure the overpressure and were stored upside down at +4 °C until the further analysis.

CH4 and CO2 concentrations were measured using Agilent 7890B Gas Chromatograph (equipped with Gilson liquid handler GX271 autosampler) at the University of Eastern Finland, Kuopio. The

13C-CH4 stable isotopic analyses were done with Isoprime100 IRMS coupled to an Isoprime TraceGas pre-concentration unit at the University of Jyväskylä.

Water samples for the natural abundance of ¹³C-DIC were collected at 1 m intervals from the surface water close to the bottom (11.5-11.75 m) by injecting 3 ml of sample into pre-evacuated 12 ml Labco Exetainers^® (over-pressure released before injection). Exetainers contained 300 µL of H3PO4 (85 %) to ensure the transformation of bicarbonate ions to CO2. The samples were then stored upside down

at +4 °C until the analysis. The samples of July were analyzed with Delta Plus XP GC-IRMS (Thermo Co.) at the University of Eastern Finland, Kuopio, and the samples of August and September were analyzed with Isoprime100 IRMS at the University of Jyväskylä.

4.2.4 ¹³C-CH4 oxidation experiments

The samples for ¹³CH4 oxidation experiment were collected at the oxic-anoxic interface and below that in August and September (Table 4). Based on the depth profiles of O2, in August the samples were collected at 6 m, 11.5 m and the sediment surface, and in September the samples were collected at 8 m, 10 m and 11.5 m. The water samples were transferred from Limnos sampler to 12 ml Labco Exetainers^® without a headspace. After 12 h pre-incubation at +4 °C, 0.1 ml of ¹³CH4 trace gas mixture (140 ml N2/10 ml CH4) was injected to the samples. The estimated final concentration of

13CH4 in the incubation was approximately 25 µmol l^-1. Since CH4 was added above ambient levels, these are considered as potential CH4 oxidation rates. The incubations were terminated at 8-hour intervals (0, 8, 16 and 24 h). In August, there were four replicates and two background samples per each sampling depth and time point. In September, each sampling depth had two replicates for 0 h time point, six replicates for 8, 16 and 24 h time points, and one background sample for each time point. The incubations were finished by injecting 3 ml of sample into evacuated 12 ml Labco Exetainers^® (over-pressure released before injection) that had 300 µL of H3PO4 (85 %) in the bottom.

The samples were analyzed for ¹³CO2 with Isoprime100 IRMS at the University of Jyväskylä.

4.3 CALCULATIONS

4.3.1 Calculation of the oxidation rates

The potential CH4 oxidation rates were determined from the ¹³C-CO2 values of the incubation samples. First the difference (Atom %) between the incubated sample and background sample was calculated for each sampling depth and time point. Then the difference between the ¹³C-CO2

concentrations of incubated sample and background sample was calculated to determine the actual concentration of ¹³C in the incubated sample. After that the ¹³C-CO2 concentration (nmol l^-1) at certain time point (8, 16 and 24 h) was compared to the ¹³C-CO2 concentration at 0 h. The ¹³C-CO2

concentrations of each sampling depth were then plotted against time, and the slope of the curve described the potential CH4 oxidation rate.

4.3.2 Calculation of methanogenic pathway and fraction oxidized

To estimate methanogenic pathway in CH4 production, a carbon isotope fractionation factor αCH4-CO2

was calculated between average δ¹³C-CO2 and δ¹³CH4 in the deepest sampling depth above sediment (formula 12; Whiticar et al. 1986).

𝛼

=

δ13C-CH4+1000 (12)

A fraction of available CH4 oxidized in the water column was calculated from formula 13 (Kankaala et al. 2007):

f

=

1000 x (𝛼-1) (13)

where f^ox = fraction oxidized, δS = δ¹³C-CH4 in the surface water, δb= δ¹³C-CH4 in the deepest sampling depth above sediment and α = carbon isotope fractionation factor by methanotrophy (assumed to be 1.037; Kankaala et al. 2007).

4.4 STATISTICAL ANALYSIS

Statistical analyses were performed with IBM SPSS Statistics 23. Spearman’s rank correlation coefficient was chosen based on the normality test results (non-parametric data). The Spearman correlations (2-tailed) were calculated between the gas concentrations/stable isotope compositions and variables such as depth, O2, temperature, pH, DOC, NO3-, NH4+, SO42-, and Fe³⁺.

4.5 WEATHER CONDITIONS

Data on air temperature and precipitation were obtained from the measuring station of the Finnish Meteorological Institute in Hyytiälä.