Methane and CO2 production in the wetland Lake Podpeč (Slovenia)

This study deals with the identification of CH4 and CO2 sources in the high-carbonate wetland Lake Podpeč in the Ljubljana Marshes, Slovenia. Lake Podpeč is situated on the periphery of the Ljubljana Marshes in central Slovenia. A combination of chemical analysis and natural abundance analysis of stable carbon and hydrogen isotopes of CH4, along with analysis of dissolved inorganic carbon (DIC), was employed in an incubation experiment. The isotopic composition of dissolved inorganic carbon (δ13CDIC) suggests three main processes occurring during incubation: oxic degradation of organic matter (OM), anoxic OM degradation, and methanogenesis. During oxic degradation of OM, the δ13CDIC values slightly decrease from − 13.2 to − 14.5‰. However, after 50 days, the δ13CDIC values started to increase, reaching − 12.2‰ by the end of the experiment. 13C enrichment coincided with the formation of CH4, which began to increase simultaneously. The CH4 produced had an average δ13CCH4 value of − 67 ± 1‰ and δ2HCH4 value of − 389 ± 3‰, suggesting that CH4 is formed through acetate fermentation. The contribution of calcite dissolution to DIC increased during the degradation of OM by 53%. However, during methanogenesis, there was no significant change in the concentrations of Ca, and the estimated contribution to DIC was only 3%. This study enhances our understanding of methane production in wetland Lake Podpeč and its relevance in the context of other high-carbonate lakes. The findings offer insights into the complex interactions between OM degradation, methane production pathways, and carbonate dissolution, which has implications for the global carbon cycle and greenhouse gas emissions.


Introduction
Methane (CH 4 ) is a potent greenhouse gas that plays a significant role in the Earth's climate system.Natural sources of CH 4 include wetlands (102-179 Tg CH 4 year −1 ), freshwater systems (117-212 Tg CH 4 year −1 ), oceans (9-22 Tg CH 4 year −1 ), fauna (4-18 Tg CH 4 year −1 ), and other land sources (13-54 Tg CH 4 year −1 ), each of which will respond differently to a changing climate (Saunois et al. 2016(Saunois et al. , 2020)).Biogenic CH 4 , produced through the anaerobic decay of organic matter (OM), accounts for approximately one-third of global CH 4 production.In lakes, a significant fraction of biogenically produced CH 4 can accumulate in the anoxic part of the water column.However, the CH 4 can be rapidly emitted to the upper water column and ultimately to the atmosphere through ebullitive transport, plant-mediated flux, or storage flux via advective processes, particularly during periods of seasonal lake mixing (Borrel et al. 2011;Lehmann et al. 2015).Therefore, it is essential to acknowledge that significant uncertainties accompany current CH 4 budgets and emission estimates, and one of the most substantial knowledge gaps revolves around the rates of aquatic CH 4 formation and release (Rosentreter et al. 2021).
The production of CO 2 in wetland lakes is another critical aspect of their role in the carbon cycle.As wetlands are characterized by high organic content, low oxygen levels, and high-water table, they become conducive environments for both CH 4 and CO 2 production.Unlike other wetlands, carbonate wetland lakes such as Lake Podpeč are rich in calcium and magnesium carbonate minerals, which affect their water chemistry, alkalinity, and pH levels.According to Marcé et al. (2015), 57% of the Earth's surface area is occupied by lakes and reservoirs, particularly in tropical and temperate latitudes that exhibit higher alkalinity, which influences CO 2 production.The primary sources of CO 2 production in carbonate wetland lakes include biological and geochemical processes.Biological sources stem from the decomposition of organic matter, such as plants and algae, by microbial activity.Organic matter accumulates in the sediments and serves as a nutrient source for methanogens, which produce methane as an end-product of their metabolic processes.Simultaneously, the decomposition of organic matter also results in the release of CO 2 .On the other hand, geochemical sources arise from the interaction of lake water with the underlying carbonate bedrock, leading to the dissolution of carbonates and subsequent release of CO 2 into the water.Thus, understanding the factors controlling CH 4 and CO 2 turnover rates is vital, especially considering the significant contributions of aquatic sources and wetlands to greenhouse gas emissions (Dean et al. 2018).
Stable isotopes of CH 4 and dissolved inorganic carbon (DIC) can provide valuable information on the origin and biogeochemical processes of CH 4 and CO 2 in lake sediments.DIC comprises carbon dioxide (CO 2 ), bicarbonate ions (HCO 3 − ) and carbonate ions (CO 3 2− ) and can have distinct isotopic compositions depending on the sources and processes involved.During organic degradation in lake sediments, microbial respiration produces CO 2 , which is enriched in the heavier carbon isotope, 13 C.As a result, the remaining DIC pool in the water column becomes relatively depleted in 13 C. Calcite dissolution, particularly in carbonate-rich lakes, can release CO 2 into the water, decreasing the content of 13 C in DIC due to the preferential release of 12 C-enriched CO 2 .The process of methanogenesis often involves the incorporation of 12 C-enriched carbon from organic matter into CH 4 , resulting in DIC with a higher 13 C content.
Further carbon isotopes (δ 13 C-CH 4 ) can be used to distinguish between different sources of CH 4 , such as biogenic and thermogenic CH 4 .Both acetotrophic (Eq. 1) and hydrogenotrophic (Eq.2) methanogens (Conrad 2005) can be found in freshwater lakes, marine environments, and sediments. (1) The δ 13 C-CH 4 values ranged from − 65 up to − 30‰ if acetate fermentation is a predominant methanogenic pathway in sediments (Whiticar 1999), while δ 13 C-CH 4 values of methane resulting from the CO 2 reduction are lower ranging between − 110 and − 60‰ (Whiticar 1999).Hydrogen isotopes (δ 2 H-CH 4 ) can provide insights into the environmental conditions under which the CH 4 was produced.In lake sediments, δ 2 H-CH 4 values can be influenced by the sources of hydrogen, such as water or OM, as well as the isotopic fractionation that occurs during the production and consumption of CH 4 .For example, CH 4 production via CO 2 reduction typically results in higher δ 2 H-CH 4 values (typically from − 250 to − 150‰), while CH 4 production via acetate fermentation or other pathways can result in lower δ 2 H-CH 4 values, i.e., − 400 to − 300‰ (Whiticar 1999).
This study aimed to investigate and characterize the sources and biogeochemical processes of CH 4 and CO 2 production in the high-carbonate wetland Lake Podpeč in the Ljubljana Marshes, Slovenia.The primary objectives were to identify the dominant pathways of methane production by analyzing stable carbon isotopes (δ 13 C-CH 4 ) and hydrogen isotopes (δ 2 H-CH 4 ) of CH 4 in lake sediments.Additionally, the study contributed to understanding the sources of CO 2 production in the carbonate wetland lake by examining the isotopic composition of DIC and distinguishing between biological and geochemical sources.By investigating these isotopic signatures, this research aimed to understand the underlying environmental conditions and microbial processes responsible for methane and carbon dioxide emissions in Lake Podpeč.Thus, it contributes to a deeper understanding of greenhouse gas dynamics in this critical high-carbonate wetland ecosystem.

Study site
This study was conducted in Lake Podpeč, located at the southern edge of Ljubljana marsh, under the Northern slopes of Krim hills (45° 58' 7.63" N, 14° 25' 58.04" E; Fig. 1).The lake is a small, circular, karstic lake with a longer diameter of 135 m, a shorter diameter of 120 m, and a surface area of approximately 1.2 ha.A depth of up to 51 m makes this lake one of the deepest lakes in Slovenia.Water drains to the lake from seven karst springs from the white limestone and dolomite hillsides at the southeastern side of the plain.The lake waters fill the northwestern part of an 800-m-long and 400-m-wide depression, which is stretched in the Dinaric direction (NW-SE).This depression is filled with thick layers of alluvium and separated from the Ljubljana Marsh basin by a low rock barrier.The peat and alluvium deposits surrounding the lake are thick, most likely reaching the rock base more than 10 m below.The water level in the lake remains consistently at the same height, although it occasionally floods the surrounding swampy plain.In winter, the temperature of the lake remains above 7 °C, while in summer, it can reach up to 20 °C.The entire lake, along with its surrounding marsh plain, is protected due to the presence of a unique flora and fauna that are greatly threatened by human activities.These include a variety of river mussels and fish (pike, perch, chub, and catfish).The organic carbon (OC) concentration is high (51.1 wt%) in the upper 16 cm of the surrounding marsh soil.The C/N ratio was determined to be 49.1, indicating a predominantly terrestrial plant origin of OM, while δ 13 C OC of the soil was − 26.1‰ (Andrič per.com).

Sediment cores and pore water extraction
Sediment cores were collected in the anoxic hypolimnion in the deepest part of the lake in November 2005 (Fig. 1).The bottom water temperature was 9.5 °C.Once collected, the sediment cores were promptly transported to the laboratory for pore water extraction and subsequent geochemical analysis.Additionally, the overlaying water, approximately 5 cm in depth, was collected for further examination.Sediment cores were extruded and sectioned in 1-, 2-, or 4-cm intervals in an N 2 -filled glove bag.Pore water was extracted by centrifugation at 5000 rpm and filtered through 0.45µm Millipore HA membrane filters inside the glove bag.The extracted pore water was then analyzed for various parameters, including pH, total alkalinity (TA), isotopic composition of dissolved inorganic carbon (δ 13 C DIC ), and cation analysis (Ca, Mg, Fe, Mn).The sectioned samples were freeze-dried and finely ground into powder form for the determination of δ 13 C organic carbon (δ 13 C OC ) and carbonates (δ 13 C Ca ).

Incubation experiment
In November 2005, during autumn, sediment samples for the incubation experiment were collected following the methodology described in Hall et al. (1989).A SCUBA diver inserted an acrylic chamber (25-cm height, 30-cm ID, 0.6-cm wall thickness) into the sediment, with an acrylic plate attached to the bottom of the chamber and the top hole sealed underwater.The chamber was then carefully transported to the laboratory and stored in the dark in a refrigerator for 111 days at a temperature (9.5 °C) similar to that at the bottom of the lake during sampling.
At the start of incubation, the water above the sediment was replaced with water collected from the bottom of the lake.The sediment depth inside the chamber was 10 cm, with an overlying water volume of 10 L. Periodic (daily) samples were taken using a syringe for chemical and isotopic analysis.The fluxes of dissolved species across the sediment-water interface were determined by linear regression of the solute concentration variation with incubation time, and the fluxes were adjusted for dilution.

Sample collection
Sample aliquots of pore water and incubation experiment collected for chemical analysis were passed through a 0.45µm nylon filter into bottles and refrigerated until analyzed.Samples for cation (pre-treated with HNO 3 ) and total alkalinity analyses were collected in HDPE bottles.Samples for δ 13 C DIC analyses were stored in glass bottles filled to leave no headspace.The pH was determined using an Orion™ 3-star pH meter with Orion™ 8102BNUWP ROSS Ultra™ pH combination electrode (Thermo Fisher Scientific Inc., Fig. 1 Location of Lake Podpeč (wetland lake, Ljubljana Marshes), Slovenia USA) and the concentration of dissolved oxygen using a YSI model 58 m and a YSI 5239 DO probe with high sensitivity membranes.

Chemical analysis
Total alkalinity (TA) was determined on refrigerated water samples using weak hydrochloric acid titration using a Mettler Toledo DL15 auto-titrator, and the data were reanalyzed using the Gran alkalinity method (Stumm and Morgan 1996).Analytical uncertainty for alkalinity was estimated to be 10%.Ten milliliters of water was used to determine the concentration of CH 4 .The samples were injected into N 2 -flushed ampoules (20 ml) and treated in an ultrasound bath at 70 °C for 20 min.Headspace gas samples were directly analyzed by gas chromatography (HP 5840 A GC/ FID) with an analytical error of ± 5%.
Major cations (Ca, Mg, Fe, Mn-precision ± 2%) in water samples were determined using a PerkinElmer inductively coupled plasma optical emission spectrometry (ICP-OES, DV5300) .The precision of the measurements was ± 3%.Saturation indices (SIs) of calcite and dolomite were calculated from TA, pH, and temperature using the PHREEQC for the Windows program (Appelo and Postma 2005).

Stable isotope analysis
The samples for determining the stable isotopic composition of CH 4 (δ 13 C CH4 ) were transferred to glass septum tubes previously purged with pure He.After treatment in an ultrasound bath at 70 °C, the isotopic composition was determined directly from the headspace by an Isotope Ratio Mass Spectrometer (IRMS) (Europa Scientific 20-20) with an ANCA-TG preparation module for trace gas samples equipped with a Gilson autosampler.The isotopic composition of H in CH 4 , δ 2 H CH4 , was measured in the GCA-geochemical laboratory in Iltnu, Germany.The analyses were performed with a GC-C-Finningan Mat Delta plus isotope mass spectrometer.The components were separated on a Poraplot Q column (ID 0.32 mm, 25-m length).The hydrocarbons were reduced to H 2 in a reduction oven at 1470 °C (Dumke et al. 1989).
The gas evolution method was used for isotope analyses of dissolved inorganic carbon (δ 13 C DIC ) in water (Atekwana and Krishnamurthy 1998).Briefly, phosphoric acid (105%) was added (200 µl) into glass septum tubes (Exetainer Septum Tubes, Labco Limited, UK) and purged with helium.The water (5 ml) samples were introduced using a syringe, and the acid-water reaction began immediately upon injection.The isotope ratio of CO 2 was determined directly from the headspace gas using a continuous-flow IRMS Europa 20-20 with ANCA TG trace gas separation module.The optimal extraction procedure was checked using standard solutions with Na 2 CO 3 with known δ 13 C values of − 10.8 ± 0.2‰ and − 4.12 ± 0.2‰, respectively.
Sediment samples for the determination of δ 13 C OC were first treated with 1 M HCl, and an aliquot of dry sample was wrapped in a tin capsule and analyzed after combustion in an O 2 atmosphere in a quartz reactor.The C isotopic composition of the CO 2 produced was determined using a Europa 20-20 ANCA-SL mass spectrometer.The method's reproducibility was checked using IAEA-CH-7 polyethylene and NBS22 oil as reference materials.The δ 13 C Ca values were then obtained using a standard method based on the reaction with phosphoric acid.Samples were prepared by grinding into a powder in an agate mortar; 2 mg of the sample was then placed into ampoules, which were capped, flushed with helium, and then reacted with anhydrous H 3 PO 4 for 1 day at 25 °C.The released CO 2 was then measured using IRMS with an ANCA-TG preparation module for trace gas samples.NBS 18 and NBS 19 were used as reference materials.All stable isotope results are reported with the conventional delta (δ) notation in per mil (‰) using the general formula (Brand et al. 2014). 1 Reproducibility of the measurements was ± 0.1‰ for δ 13 C Ca , ± 0.2‰ for δ 13 C DIC and δ 13 C OC , ± 0.5‰ for δ 13 C CH4 , and ± 3 ‰ for δ 2 H CH4 .

Calculations
A carbon isotope mass balance calculation was employed to understand better the CO 2 and CH 4 production.The total DIC flux is formed from the remineralization of OM (J rem ) and the carbonate dissolution flux (J Ca ) during the oxic degradation of OM (Boehme et al. 1996;Ogrinc et al. 2008): Consequently, the isotope mass balance equation could be written in the form: δ 13 C tot,ox is the isotopic composition of the total flux during the oxic phase of incubation, δ 13 C J-DIC is the isotopic composition of the DIC flux, while δ 13 C ca is the where E is the element (H, C) and R is the isotope ratio between the heavier "i" and the lighter "j" isotope ( 2 H/ 1 H, 13 C/ 12 C) in the sample and relevant internationally recognized reference standard.The delta values are multiplied by 1000 and expressed in units "per mil" (‰).For hydrogen the Vienna Standard Mean Ocean Water (VSMOW), while for carbon the Vienna Pee Dee Belemnite (VPDB) are used as a reference standard.mean isotope value of carbonate of − 8.2‰.The CH 4 flux (J CH4 ) also contributes to the general isotope mass balance during methanogenesis.Therefore, Eqs. 3 and 4 could be written in the form: The isotopic composition of DIC (δ 13 C J-DIC ) and the CH 4 (δ 13 C J-CH4 ) fluxes were calculated using the analogous approach applied in other studies (Jahnke et al. 1997;Ogrinc et al. 2002Ogrinc et al. , 2008)).The δ 13 C J-DIC value was determined for different periods from the slope of the regression line when ([DIC] ti / [DIC] t0 ) − 1 was plotted against δ 13 C DIC,i × ([DIC] ti / [DIC] t0 ), where [DIC] ti is the concentration of DIC at time "i" and [DIC] t0 represents the concentration of DIC at the beginning of the experiment.δ 13 C DIC,i is the measured δ 13 C DIC at time ti.Similarly, δ 13 C J-CH4 can be calculated from the slope of the line when ([CH 4 is the initial concentration of CH 4 determined after 50 days of incubation.δ 13 C CH4 is the measured isotopic composition during the incubation.The unknown isotopic value of degradable sedimentary OM (δ 13 C rem ) was then calculated from these estimates.

Sediment and pore water
The pore water pH decreased from 7.8 to 6.7 at a depth of 18 cm.At the same time, the levels of TA, Ca, and Mg remained constant within the depth profile with an average value of 4.08 ± 0.11 mmol L −1 , 1.10 ± 0.14 mmol L −1 , and 0.64 ± 0.25 mmol L −1 , respectively (Fig. 2).In contrast, δ 13 C DIC values displayed a high positive gradient from − 11.7 to 3.7‰ from the bottom water layer to the sediment depth of 18 cm, which is typical of methanogenic sediments.Also, Mn decreased from 3.82 to 0.55 μmol L −1 , while Fe increased from 5.18 to 27.5 μmol L −1 , indicating reducing conditions in the pore water profile (Fig. 2).The study observed low Fig. 2 Depth profiles of total alkalinity (TA),isotopic composition of dissolved inorganic carbon (δ 13 C DIC ), Ca, Mg, Mn, and Fe in pore water of Lake Podpeč sulfate concentration typical for lake environments with an average value of 0.86 ± 0.20 mmol L −1 .The sediments exhibit an average δ 13 C OC value of − 29.7 ± 0.6‰ and a low δ 13 C Ca average value of − 8.2 ± 0.4‰.

Incubation experiment
The temporal variations of the studied solutes during incubation are presented in Figs. 3 and 4, while the estimation of the isotopic composition of DIC (δ 13 C J-DIC ) and CH 4 (δ 13 C J-CH4 ) fluxes is presented in Fig. 5.During incubation, linear changes in the concentrations of various electron acceptors and total alkalinity (TA) were observed with time.The regression lines (Figs. 3 and 4) represent the basis for calculating the benthic fluxes of solutes presented in Table 1.Dissolved O 2 was present in the incubation chamber for 11 days (Fig. 3) and was related to the oxic degradation of OM.Increases of soluble Mn and Fe were observed during the anoxic phase, present for 50 days, while during methanogenesis, concentrations of Mn decreased and Fe slightly increased (Fig. 3).The levels of Ca and Mg during incubation (Fig. 4) increased in oxic phase due to the dissolution of carbonates also indicated by the calculated saturation indices (SIs) of calcite and dolomite.In parallel, an increase in TA occurred.In the anoxic phase, the levels of Ca and Mg remained relatively stable and TA only slightly increased.After 50 days, CH 4 levels began to increase, and the end products of methanogenesis were 13 C-and 2 H-depleted CH 4 and 13 C-enriched DIC.The average δ 13 C CH4 and δ 2 H CH4 values of CH 4 at the end of the experiment were − 67 ± 1‰ and − 389 ± 3‰, respectively.

Discussion
The incubation experiment provides valuable information on the temporal dynamics of solutes and isotopic signatures during different phases of biological and geochemical processes (Figs. 3 and 4).Methanogenesis produced CH 4 with δ 13 C CH4 and δ 2 H CH4 values corresponding predominantly to acetotrophic methanogenesis (Whiticar 1999).The data are comparable to the latest studies on boreal lakes and wetlands (Thompson et al. 2016;Schenk et al. 2021).However, in the study performed by Schenk et al. (2021), isotopic ratios of CH 4 in deeper sediments were consistent with mixing/transition between CH 4 production pathways, indicating a higher contribution of the CO 2 reduction pathway.The study further indicates that the isotopic composition of CH 4 sources is consistently higher in littoral sediments than in deep waters across boreal and subarctic lakes.They explain these differences by the variability in organic matter substrates across depths.However, the predominant pathway should be verified using labeled compounds or selective inhibitors (Conrad 2005).In addition, the δ 2 H CH4 values could not be interpreted conclusively without considering the δ 2 H CH4 values of coexisting H 2 O since it was found that the δ 2 H CH4 value depends on the δ 2 H values in water in sulfate-poor freshwater environments (Waldron et al. 1999;Wand et al. 2006;Douglas et al. 2021).It was estimated that δ 2 H-H 2 O explain approximately 42% of the observed variation in δ 2 H CH4 globally.
The acetotrophic methanogenesis in Lake Podpeč is further supported by the previous microcosm experiments on soil slurries in the Ljubljana Marshes (Jerman et al. 2009,  In those experiments, Methanosarcinaceae were detected in samples with high CH 4 production.The results also showed the most prolonged lag phase for CH 4 production in anaerobic soil slurries, suggesting that OM is extensively processed (refractory) and that only a small proportion can be readily mineralized by microorganisms (Stres et al. 2008;Jerman et al. 2009).In addition, iron oxides help to form soil aggregates with OM, reducing the contact between microorganisms and the organic substrate and lowering the rate of its degradation (Roden and Wetzel 2003).In our study, the CH 4 production began after 50 days of incubation and can be related to the poorly degradable OM and the presence of only a small portion of easily accessible OM in the sediment.Unfortunately, the concentrations of OC and TN in sediments were not determined.However, we assume that the OC and C/N ratio is similar to the surrounding soil.The concentration of OC in the upper 16 cm of the marsh soil is high, amounting to 51.1 wt% with a C/N ratio of 49.1, indicating a predominantly plant-derived origin of the OM.
Further, a carbon isotope mass balance calculation was employed to understand better organic matter (OM) degradation, carbonate dissolution, and CH 4 production .
Table 2 presents the calculated fluxes and the isotopic value of degradable sedimentary OM (δ 13 C rem ) for all three periods.Changes in J DIC flux were observed during the three different processes and are associated with the consumption of electron acceptors during the degradation of the OM.It is seen that the contribution of carbonate dissolution to DIC changed during incubation.Initially, 53% of the DIC was derived from carbonate dissolution, while the contribution was only 3% during methanogenesis.The δ 13 C rem of − 28.1‰ during oxic degradation is comparable with the δ 13 C OC value of − 29.7 ± 0.6‰ determined in sediments, while during anoxic conditions, the δ 13 C rem of − 18.1‰ is higher, indicating that other OM sources contribute to the C pool.A relatively low δ 13 C rem value of − 41.2‰ was determined during methanogenesis.There are two possible explanations.The first is related to the oxidation of 13 C-depleted biogenic methane that can contribute to 13 C-depleted dissolved CO 2 to the dissolved inorganic carbon (DIC) reservoir.The assimilation of this DIC would lead to the production of 13 C-depleted biomass.Alternatively, 13 C-depleted biogenic methane formed in the sediment could enable the expansion of methanotrophic organisms that produced 13 Cdepleted biomass (Hinrichs et al. 1999;Orphan et al. 2001;Hollander and Smith 2001).
Drawing comparisons with other impacted high-carbonate lakes, such as the eutrophic subalpine Lake Bled (Ogrinc et al. 2002;Mandič-Mulec et al. 2012) and high-mountain Lake Planina (Ogrinc et al. 2008), provides valuable insights into the eutrophication processes and the sources of greenhouse gases on a global scale.In both lacustrine environments, hydrogenotrophic methanogenesis emerges as the dominant process responsible for CH 4 production.The average δ 13 C CH4 value for CH 4 produced in Lake Bled is approximately − 69.5 ± 1.2‰, which closely aligns with the values obtained in Lake Planina with δ 13 C CH4 value of − 70.1 ± 1.1‰ and Lake Podpeč − 67 ± 1‰.However, the difference in δ 2 H CH4 determined in Lake Planina compared to Lake Podpeč, i.e., − 208 ± 10‰ vs − 389 ± 3‰, respectively, shows that different processes are responsible for methanogenesis.Unfortunately, it was not possible to measure the δ 2 H CH4 in Lake Bled; however, the archaeal community supports the hypothesis that hydrogenotrophic methanogenesis is the dominant pathway in the sediment of Lake Bled (Mandič-Mulec et al. 2012).It is also interesting to note that the CH 4 flux in Lake Bled was twice as high, averaging 2.2 mmol m −2 day −1 , compared to Lake Planina's J CH4 of 1.1 mmol m −2 day −1 and nine times higher compared to J CH4 obtained in Lake Podpeč (Table 1).These data indicate that in both eutrophic lakes, OM remineralization occurs faster than in Lake Podpeč and is due to the presence of fresh and highly degradable OM in Lake Bled and Lake Planina sediments primarily originating from microalgae and deposited phytoplankton.Further, it was also observed that methanogenesis inhibits calcite dissolution in all three carbonate-rich systems.For example, the contribution of calcite dissolution to DIC decreased during incubation, i.e., from 20 to 7% in Lake Planina (Ogrinc et al. 2008) and from 53 to 3% in Lake Podpeč.

Conclusions
This study focused on CH 4 and CO 2 production in Lake Podpeč, a high-carbonate wetland lake in Ljubljana Marshes, Slovenia.The investigation involved a laboratory-based incubation experiment conducted at in situ temperatures, covering the transition from oxic to anoxic conditions.The experiment revealed three distinct processes: oxic degradation of organic matter (OM), anoxic degradation of OM, and methanogenesis.Further, the CH 4 produced had a measured average δ 13 C CH4 value of − 67 ± 1‰ and δ 2 H CH4 value of − 389 ± 3‰, indicating that, contrary to alpine lakes, CH 4 is formed through acetate fermentation.Moreover, based on carbon isotope mass balance calculations, different biological and geochemical processes influencing the DIC production were evaluated.For instance, OM degradation contributed around 50% to DIC, while methanogenesis contributed about 45%.Interestingly, the contribution of carbonate dissolution to DIC changed significantly during the incubation, from 53 to 3%.This finding highlights the dynamic nature of these geochemical processes and implies that methanogenesis inhibits calcite dissolution.
Overall, this study provides valuable insights into mechanisms of CH 4 and CO 2 production in high-carbonate wetland lakes providing information for further research on similar ecosystems and their role in the global carbon cycle.

Fig. 3
Fig. 3 Concentrations of dissolved oxygen, Mn, and Fe as a function of time in the laboratory-incubated flux chamber experiment in the dark in November 2005.Solid lines represent the linear regression

Fig. 4
Fig. 4 Concentrations of Ca, Mg, TA, CH 4 , δ 13 C DIC , and δ 13 C CH4 as a function of time in the incubation experiment performed in November 2005.Solid lines represent the linear regression equations used to

Table 1
Summary of fluxes of different solutes from laboratory-incubated flux chamber experiments in the dark during oxic degradation of organic matter (OM), anoxic degradation of OM, and methanogenesis.The fluxes were calculated from linear regression of the variation of solute concentration with incubation time

Table 2
Carbon isotope mass balance calculation in an incubation experiment of oxic degradation of organic matter, anoxic degradation ("transition"), and methanogenesis performed in November 2005.