Ecosystems (2013) 16: 1152–1164 DOI: 10.1007/s10021-013-9674-z  2013 Springer Science+Business Media New York

Variable Production by Different Pelagic Energy Mobilizers in Boreal Lakes Paula Kankaala,1* Jessica Lopez Bellido,2 Anne Ojala,2 Tiina Tulonen,3 and Roger I. Jones4 1 Department of Biology, University of Eastern Finland, P.O. Box 111, 80101 Joensuu, Finland; 2Department of Environmental Sciences, University of Helsinki, Lahti, Finland; 3Lammi Biological Station, University of Helsinki, Lammi, Finland; 4Department of Biological and Environmental Science, University of Jyva¨skyla¨, Jyva¨skyla¨, Finland

ABSTRACT total pelagic energy mobilization in the lakes, suggesting that only a minor fraction of allochthonous DOC became available for higher trophic levels. High MOB activity was detected in the water columns of the stratified lakes when the molar ratio of CH4:O2 varied between 0.5 and 12. In the small stratified lakes (area < 0.01 km2), MOB production contributed 13–52% of the total pelagic energy mobilization, being greatest during the autumn mixing period. Our results indicate that in small stratified lakes (area < 0.01 km2) bacteria, especially MOB, are potentially quantitatively important supplementary food resources for zooplankton. However, in larger lakes primary producers are the most important (>70%) potential food source for zooplankton.

We studied production by three key pelagic energy mobilizer communities, phytoplankton (PP), heterotrophic bacteria (HB), and methanotrophic bacteria (MOB), in five boreal lakes of varying size and concentration of dissolved organic carbon (DOC). Production by PP was responsible for most (>55%) of the total pelagic energy mobilization in all five lakes. Production by HB and PP estimated for the whole water column during the ice-free period were positively correlated, but with the exception of the clearest and most eutrophic lake PP apparently could not support the total carbon demand of bacteria. However, the DOC concentration did not explain the variability of heterotrophic bacterial production (HBP) within or between the lakes. Thus, our results provide circumstantial evidence for the ‘‘priming effect’’ whereby labile organic matter from autochthonous production enhances decomposition of allochthonous DOC. However, HBP was only 10–23% of the

Key words: primary production; bacterial production; methane oxidation; pelagic food web; priming effect; dissolved organic carbon.

INTRODUCTION As an extension to the fundamental grazing food chain, from phytoplankton (PP) to zooplankton, the modern view of lake pelagic food webs now includes a microbial food chain, whereby dissolved organic carbon (DOC) is made available via bacteria to protozoan and metazoan plankton consumers in the food web (for example, Weisse 2004; Jansson

Received 10 January 2013; accepted 25 March 2013; published online 16 May 2013 Author contributions: PK, AO, and RIJ designed the studies, PK analyzed the data, JLB and TT participated in field and laboratory studies, PK and RIJ were mainly responsible for writing the manuscript with contributions from the other authors. *Corresponding author; e-mail: [email protected]

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Pelagic Energy Mobilization and others 2007). Stable carbon, nitrogen, and hydrogen isotope analyses together with associated mixing model calculations have indicated that a high proportion of the DOC in lakes is actually often of terrestrial (allochthonous) origin, especially in brown-water lakes (for example, Wilkinson and others 2013), potentially stimulating the microbial food chain and providing a cross-system subsidy to lake food webs (for example, Jansson and others 2007; Carpenter and others 2005; Karlsson and others 2012). In addition, a pathway from methane (CH4), an end product of anaerobic decomposition of organic matter, made available to the food web via methane-oxidizing bacteria (MOB), is important for zooplankton diets, at least in small stratified lakes (Jones and others 1999; Kankaala and others 2006b, 2010; Taipale and others 2008). Hence, several different basal resources (energy mobilizers sensu, Jones 1992; Jansson and others 2007) can contribute to lake pelagic food webs, and the relative importance of each of these can be expected to vary according to key lake characteristics, such as size and DOC concentration. Primary production of phytoplankton (PPP) in lakes is strongly regulated by seasonal variation of light and its penetration into the water column, as well as by the availability of nutrients, especially of phosphorus. Stained humic substances reduce light penetration in the water column and thus the depth of the euphotic zone, and the concentration of allochthonous, humic DOC tends to be highest in small headwater lakes having peatlands and coniferous vegetation in their catchments (Kortelainen 1993; Xenopoulos and others 2003). Colored substances strengthen temperature stratification in lakes, but flagellate algae can undertake diel vertical migrations to take up nutrients from the nutrient-rich hypolimnion during the night for photosynthesis in the shallow euphotic zone during the day which is then reflected in an enhanced growth rate (Salonen and others 1984b; Arvola and others 1991; Ojala and others 1996). In oxic conditions heterotrophic bacteria (HB), utilizing mostly autochthonous and, to various degrees, allochthonous DOC as their carbon source, dominate in the microbial community (for example, Pe´rez and Sommaruga 2006; Taipale and others 2009). HB may compete with algae for inorganic nutrients (Kirchman 1994; Jansson and others 2001), but are also influenced by seasonal and depth-related variations in temperature (Tulonen 1993; Bergstro¨m and Jansson 2000). The origin and age of allochthonous DOC as well as conditions in the recipient aquatic ecosystem greatly influence the utilization of DOC as well as bacterial net

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production and respiration (Berggren and others 2007, 2009; Lennon and Pfaff 2005; Lennon and Cottingham 2008). However, Guenet and others (2010) have argued that autochthonous labile organic matter causes a ‘‘priming effect’’ for mineralization of recalcitrant terrestrial organic matter in aquatic ecosystems, as reported for soils (Kuzyakov and others 2000). Hence, algal primary production could play an important role in stimulating bacterial decomposition of allochthonous organic matter in lakes, thus contributing to the availability of allochthonous carbon to upper trophic levels. In anoxic fresh-water environments, CH4, produced by methanogenic archaea is the major end product of the anoxic decomposition of organic matter (Capone and Kiene 1988). Methane-oxidizing bacteria (MOB) at the oxic–anoxic interface zone can then oxidize CH4 to CO2 and partly incorporate CH4-C into microbial biomass (Rudd and Hamilton 1978; Bastviken and others 2003; Kankaala and others 2006b), thus mobilizing the energy available in CH4 to the wider food web. In boreal lakes, the stratification/mixing pattern of the water column during the ice-free period (IFP) is strongly influenced by the size of the lake (Kankaala and others unpublished manuscript). In small humic lakes, absorption of light energy by stained organic substances in the surface layers promotes early water column stratification in spring (Salonen and others 1984a). Incomplete spring mixing and microbial organic matter decomposition often lead to summer hypolimnetic anoxia in small humic lakes, promoting conditions for mobilization of energy from CH4 into pelagic food webs. These conditions are less likely to develop in large lakes (see Jones and Grey 2011). The magnitude of these various energy mobilizing pathways across lake types and how the balance between them is determined is still poorly understood and quantified. Here, we present results of production by different energy mobilizer communities (PP, HB, and MOB) in five boreal lakes of different character. We predicted that: (1) production by HB is related to DOC concentration; (2) a ‘‘priming effect’’ through PP is detected in HB production; (3) pelagic CH4 oxidation activity is related to water column stability and lake size; and (4) the relative importance of total bacterial energy mobilization decreases with lake size, reflecting a diminishing impact of the terrestrial interface and reduced total DOC.

STUDY AREA

AND

METHODS

The five study lakes, with an areal range from 0.004 to 13.4 km2 and a DOC concentration

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varying between approximately 7 and 24 mg C l-1, are located in the boreal zone in the Kokema¨enjoki river basin of southern Finland (Table 1). We combined and analyzed measurements made using similar methods during several research projects (see Kankaala and others 2006a, b; Taipale and others 2008; Ojala and others 2011). The study years were 2002, 2007, and 2008 for Valkea-Kotinen, 2004 for Ormaja¨rvi and Pa¨a¨ja¨rvi, 2005 for Mekkoja¨rvi, and 2007 for Alinen Mustaja¨rvi. Of these, the summers 2004 and 2008 were more rainy than an average summer in the study area (413 mm in 2004, 268 mm in 2008, average June– August precipitation 200–220 mm, measured at Lammi Biological Station). The lakes were covered by ice for 4–6 months, the smallest ones usually from November to late April or early May, and the largest ones from mid-December to late April or early May. Temperature and oxygen concentration profiles of water columns were measured during every sampling occasion with a YSI 58 probe (accuracy ±0.3C, ±0.3 mg O2 l-1) at 0.5 or 1 m intervals. Water column stability during each sampling was calculated as squared Brunt–Va¨isa¨la¨ stability frequency (N2, s-2) (LakeAnalyzer program http:// lakeanalyzer.gleon.org/; Read and others 2011): N2 ¼

g @qw ; q w @z

ð1Þ

where g is the gravitational constant, qw is the density of water, and ¶qw/¶z is the density gradient. Water samples for chemical analyses and biological activity measurements were taken with a Limnos tube sampler (Mekkoja¨rvi 4.25 l, other lakes 2 l). The frequency of the measurements varied between 12 and 24 times during the IFP from late April or early May through October. DOC concentration of water, prefiltered through 0.2 lm Millipore filters, was measured with a Shimazdu TOC-5000 analyzer. Total nitrogen (TotN) and total phosphorus (TotP), and soluble reactive phosphorus (SRP) were analyzed with Finnish standard methods (see Arvola and others 1996, for details). Methane (CH4) and carbon dioxide (CO2) concentrations in the water column were measured from samples taken into 50-ml polypropylene syringes and brought to the laboratory under crushed ice. The analyses were done within 24 h with gas chromatography (Agilent 6890N equipped with FID and TCD) using a headspace equilibrium technique (see Ojala and others 2011, for details). PPP was measured according to Keskitalo and Salonen (1994) as 14C incorporation from Na14CO3 solution in light and dark incubations in situ,

beginning at about 10:00 and finishing 24 h later. The water samples and corresponding incubation depths were selected to represent the euphotic zone (depth of 1% surface light), based on light attenuation and previous PPP measurements in each lake: Mekkoja¨rvi, pooled 0–0.6 m; Alinen Mustaja¨rvi and Valkea-Kotinen, 0, 0.5, 1, and 2 m; Ormaja¨rvi, 0, 1, 2, 4, and 6 m; and Pa¨a¨ja¨rvi, 0, 1, 2, 3, and 4 m depths. Due to the 24 h incubations, we consider these results to approximate net primary production (compare Lizon and Lagadeuc 1998). Production of heterotrophic bacteria (HBP) was measured as incorporation of 14C-leucine (30 nmol) into bacterial cells according to the method of Kirchman and others (1985), modified by Tulonen (1993). For the measurements, pooled water samples were taken from the euphotic zone, metalimnion and hypolimnion (Ormaja¨rvi at 26 m, Pa¨a¨ja¨rvi at 45 m depth) and the vials were incubated for 1 h at the sampling depths or in vitro at the respective temperatures. Because the 14C-leucine incorporation is based on uptake by bacterial cells, we consider this method to measure bacterial net production. Bacterial growth efficiency (BGE) was assumed to be 10–25% of total organic carbon uptake (see del Giorgio and Cole 1998; Krizberg and others 2005; Berggren and others 2007). Thus, the range of bacterial carbon demand was estimated by multiplying HBP (mmol C m-2) by 10 and 4. In the three smallest lakes, the activity of methane-oxidizing bacteria (MOB) was measured as consumption of CH4 in water sampled from surface to bottom for every 1 or 2 m layers and incubated for 24 h in 50-ml glass syringes in vitro at the respective field temperatures according to Kankaala and others (2006a). A test using difluoromethane, a specific inhibitor of methanotrophic activity (Miller and others 1998), showed that the syringes were gas tight (see details in Kankaala and others 2006a). For the two larger lakes (Ormaja¨rvi and Pa¨a¨ja¨rvi), CH4 oxidation activity was estimated by calculating differences between predicted and observed diffusive flux of CH4 in different water layers (see details in Kankaala and others 2006b). The sampling depths were 0, 1, 2, 4, 6, 10, 20, and 26 m for Ormaja¨rvi and 0, 1, 2, 3, 4, 10, 20, 30, and 45 for Pa¨a¨ja¨rvi. The use of this method was justified because in Lake Valkea-Kotinen it gave CH4 oxidation estimates comparable with those obtained by in vitro CH4 oxidation measurements (Kankaala and others 2006a). Because both methods measure gross consumption of CH4, we assumed net growth efficiency (that is, carbon incorporation into MOB cells) to vary between 10 and 40% of the gross CH4 consumption (compare Rudd and Hamilton 1978;

6113¢N; 2508¢E 0.004 0.011 4 0.5–1 11.6–21.4 4.2–4.8 21.2 0.0141 0 5.7 ± 0.2 421 ± 20 23.8 ± 2.4 22.4 ± 2.4 0.32 ± 0.13 11.5 ± 3.3 78.5 ± 6.3 2.0 ± 0.8 16.5 ± 2.7 5,337 30,686 597 ± 39 1,394 ± 33 6.0 ± 3.6 596 ± 162 800 ± 120 30 ± 26 113 ± 40

Coordinates Area (km2) Volume (m3 9 106) Max depth (m) Euphotic zone thickness (m) Temperature (0.2 m) (C) Temperature (bottom–0.5 m) (C) Euphotic zone vol% total volume Stability frequency (N2 s-2) O2 saturation (bottom–0.5 m) (%) pH (epi) Color (epi) (mg Pt l-1) DOC (epi) (mg C l-1) DOC(hypo) (mg C l-1) POC (epi) (mg C l-1) TotP (epi) (lg l-1) TotP (bottom–0.5 m) lg l-1 SRP (epi) (lg l-1) SRP (bottom–0.5 m) (lg l-1) DOC/TotP (epi) molar ratio DOC/SRP (epi) molar ratio TotN (epi) (lg l-1) TotN (bottom–0.5 m) (lg l-1) Chl a (epi) (lg l-1) CO2 (1 m) (lmol l-1) CO2 (bottom–0.5 m) (lmol l-1) CH4 (1 m) (lmol l-1) CH4 (bottom–0.5 m) (lmol l-1)

nd = no data. *Samples from 45 m depth in Pa¨a¨ja¨rvi, **5 m depth in Valkea-Kotinen.

Mekkoja¨rvi (MJ)

Lake 6112¢N; 2506¢E 0.008 0.031 6.5 1.5–2 9.9–21.4 4.1–4.7 45.0 0.0106 0 5.1 ± 0.1 104 ± 13 10.1 ± 0.7 19.8 ± 2.5 0.54 ± 0.21 11.6 ± 3.6 126 ± 24 1.2 ± 0.6 76.9 ± 18.8 2,245 21,704 428 ± 90 2,482 ± 393 7.6 ± 4.9 82 ± 67 1,154 ± 162 2.1 ± 5.2 744 ± 96

Alinen Mustaja¨rvi (AM) 6114¢N; 2504¢E 0.042 0.103 6.5 1.5–2 14.5–23.4 6.1–7.0 61.7 0.0087 0 5.5 ± 0.1 108 ± 18 13.3 ± 1.8 nd nd 15.6 ± 2.3 26.8 ± 3.6** 1.0 ± 0.4 1.2 ± 0.5** 2,198 34,297 485 ± 55 875 ± 82 15 ± 6 78 ± 29 487 ± 228 0.47 ± 0.68 87.1 ± 73.1

Valkea-Kotinen (VK) 6106¢N; 2458¢E 6.53 67 30 5–6 12.1–20.3 6.3–7.1 47.0 0.0023 23–91 7.6 ± 0.2 25 ± 10 8.0 ± 0.6 7.0 ± 0.9 0.58 ± 0.13 16.4 ± 6.2 16.6 ± 5.2 1.0 ± 0.5 7.3 ± 2.5 1,258 20,629 741 ± 60 823 ± 112 7.3 ± 2.9 30 ± 16 179 ± 107 0.06 ± 0.03 0.23 ± 0.57

Ormaja¨rvi (OJ)

6104¢N; 2508¢E 13.4 206 87 3.5–4 12.8–19.7 4.8–5.2 21.7 0.0019 74–93 7.2 ± 0.1 117 ± 35 12.9 ± 2.6 10.6 ± 0.2* 0.31 ± 0.09 12.7 ± 0.6 8.3 ± 1.0* 1.0 ± 0.6 1.4 ± 0.7* 2,619 33,265 1,575 ± 103 1,535 ± 67* 4.2 ± 1.1 42 ± 17 107 ± 30* 0.04 ± 0.02 0.02 ± 0.01*

Pa¨a¨ja¨rvi (PJ)

Table 1. Locations of the Five Study Lakes with Morphometric Characteristics, and Some Physical and Chemical Properties (mean ± SD or Range) in June–August

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Dedysh and others 1998; Templeton and others 2006). All results of PPP, HBP, and MOB activity were calculated per lake area (mmol C m-2 d-1) by weighting the results for the euphotic zone and meta- and hypolimnion (mmol C m-3 d-1) by the volume of the respective layers of each lake, and we integrated the results over time by interpolating between-sample dates for the whole IFP. We note that localized high abundances of other energy mobilizer communities (photoautotrophic and chemoautotrophic bacteria) have been reported in the anoxic zone of the small lakes (Taipale and others 2009; Peura and others 2012). However, only a few measurements of inorganic 14C-uptake by Chlorobi have been made in Mekkoja¨rvi (KuuppoLeinikki and Salonen 1992), and no measurements of the activity or net production of these bacteria are available for the other lakes. Therefore, we have not attempted to include these other microbial producers in our evaluation of energy mobilization. Statistical analyses were done with IBM SPSS 19 or SigmaPlot 12.3 programs.

RESULTS Physical and Chemical Properties of the Lakes All the study lakes have a dimictic mixing pattern. However, in the three smallest lakes the spring mixing is typically incomplete and the lakes stratify already in early May. The stability frequency of the water column (N2) during June–August decreased with lake size (Table 1). In the three smallest lakes, anoxia prevailed in the hypolimnion during the summer stratification and the concentrations of CH4 and CO2 were high in the hypolimnion. The water columns of the two largest lakes were oxic down to the bottom, although during the summer stratification the oxygen saturation close to the bottom was lower in Ormaja¨rvi (23–91%) than in Pa¨a¨ja¨rvi (74–93%). The concentration of DOC was highest in the smallest lake Mekkoja¨rvi (mean 24 mg C l-1) and lowest in Ormaja¨rvi (7– 8 mg C l-1). The other three lakes had intermediate DOC concentrations in the epilimnion varying between 10 and 13 mg C l-1. These differences were reflected in water color and in the depth of the euphotic zone. According to total phosphorus concentration in the epilimnion, the lakes are mesotrophic (TotP 11–16 lg l-1). The concentration of SRP was at the detection limit (1–2 lg l-1) in the epilimnion of all the lakes. However, in the three smallest lakes with an anoxic hypolimnion,

high concentrations of TotP, SRP, and TotN were measured close to the bottom.

Primary Production Production of PP was highest in the clearest lake Ormaja¨rvi peaking (50 mmol C m-2 d-1) immediately after ice-out in early May and varying between 5 and 36 mmol C m-2 d-1 from mid May to late September (Figure 1). The lowest PPP was measured in the polyhumic lake Mekkoja¨rvi with small peaks (8–11 mmol C m-2 d-1) in May–early June, but less than 5 mmol C m-2 d-1 during the later open-water season. In the three mesohumic lakes (Alinen Mustaja¨rvi, Valkea-Kotinen, Pa¨a¨ja¨rvi), the daily variation of PPP from late May to mid-September was from 3 to 16 mmol C m-2 d-1 and PPP declined during late September and October. The annual PPP over the IFP in the lakes varied between 0.43 and 3.89 mol C m-2 IFP-1 (Table 2).

Heterotrophic Bacterial Production HBP showed less seasonal variation than that of PPP (Figure 1). In Alinen Mustaja¨rvi, a clear peak in HBP was observed in mid-September (5.3 mmol m-2 d-1) and in Pa¨a¨ja¨rvi in early August and in mid-October (6.1 and 4.2 mmol C m-2 d-1). In general, the lowest HBP was measured in the polyhumic Mekkoja¨rvi (0.3–1.5 mmol C m-2 d-1) and the highest in the clearest lake Ormaja¨rvi (1.7–4.8 mmol C m-2 d-1), although volume-based bacterial production showed much lower variation between lakes (Table 2; Figure 2). The daily variation of HBP in the whole water column (mmol C m-2 d-1) or in the euphotic zone (mmol C m-3 d-1) was not correlated with that of PPP or with any measured physical or chemical variable in any of the lakes or in the combined data set from all lakes (data not shown). However, the depth distribution of HBP showed within-lake differences (Figure 2); in all lakes HBP in the hypolimnion was lower than in the epilimnion and in the metalimnion (ANOVAR, Holm-Sidak method for pairwaise multiple comparisons, P < 0.05, n = 41–50). During the IFP, the total HBP in the lakes varied between 0.14 and 0.50 mol C m-2 IFP-1 (Table 2), being lowest in the most humic Mekkoja¨rvi and highest in the clearest lake Ormaja¨rvi. Across the five lakes, total HBP in the whole water column was positively correlated with PPP (Figure 3A). However, no significant relationship was observed between mean HBP and PPP values for lake euphotic zones, calculated per unit volume. Bacterial carbon demand estimates for the lakes,

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Figure 1. Seasonal variation of primary production (PPP), heterotrophic bacterial production (HBP), and activity of methaneoxidizing bacteria (MOB) (mmol C m-2 d-1) during the ice-free period (IFP) in five boreal lakes presented in order of increasing area from 0.04 to 13.4 km2. The data for Mekkoja¨rvi are from Taipale and others (2008), for HBP and MOB in Valkea-Kotinen from Kankaala and others (2006b), and for PPP in Ormaja¨rvi and Pa¨a¨ja¨rvi from Ojala and others (2011). Note different y-axis for Ormaja¨rvi.

Table 2. Summary of Energy Mobilization by Different Pelagic Communities During the Ice-Free Period (IFP) in the Five Boreal Lakes

-2

-1

PPP mol C m (IFP) PPP mol C m-3 (IFP)-1 (mean of the euphotic zone) HBP mol C m-2 (IFP)-1 HBP mol C m-3 IFP-1 (mean of the euphotic zone) BCD mol C m-2 (IFP)-1 HBP % of mean DOC pool (m-2) Net MOB mol C m-2 (IFP)-1

MJ 2005

AM 2007

VK 2002

VK 2007

VK 2008

OJ 2004

PJ 2004

0.43 0.43

1.73 0.86

1.10 0.55

1.06 0.53

0.74 0.37

3.89 0.65

1.59 0.40

0.14 0.06

0.33 0.10

0.25 0.07

0.30 0.18

0.26 0.15

0.49 0.06

0.43 0.06

0.57–1.43 2.2 0.17–0.69

1.34–3.35 9.7 0.24–0.97

1.00–2.50 9.0 0.02–0.09

1.19–2.99 11.7 nd

1.05–2.63 10.6 nd

1.94–4.86 7.0 0.06–0.23

1.73–4.32 2.7 0.002–0.01

nd = no data. Primary production of phytoplankton (PPP) and heterotrophic bacterial production (HBP) per unit area (m-2) and volume (m-3); bacterial carbon demand (BCD assuming 10–25% growth efficiency of HBP); HBP as percentage of mean DOC pool; and net production of CH4-oxidizing bacteria (MOB), assuming a range of 10–40% growth efficiency. Year from which data derived is shown after the lake abbreviation (see Table 1).

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Figure 2. Mean production of heterotrophic bacteria (HBP) (lmol C l-1 d-1) during the ice-free period (IFP) in the euphotic zone and meta- and hypolimnion of five boreal lakes presented in order of increasing area. The bars denote SE of the mean values.

assuming 10–25% BGE, indicated that autochthonous carbon from PP could not be the main carbon source for bacteria. Even though the proportion of bacterial carbon demand as a percentage of PPP tended to decrease with increasing PPP (Figure 3B), only in the clearest and most eutrophic lake (Ormaja¨rvi) and assuming 25% or greater BGE, could PPP have supported HBP during the whole IFP. HBP during the IFP was not related to mean DOC concentration, DOC/TotP or DOC/SRP ratios of the lakes, expressed either as volumetric or areal units. However, HBP as a percentage of the total DOC pool (mean pool during IFP, mol C m-2) in the water column was lowest in Mekkoja¨rvi (2.2%) and Pa¨a¨ja¨rvi (2.7%) and highest in Valkea-Kotinen (9–12%), thus increasing with the proportionally greater volume of euphotic zone of the total lake volume (Figure 3C).

Methane Oxidation Activity and Net MOB Production In the three smallest lakes with anoxic hypolimnia, the highest activities of MOB were measured at the metalimnetic oxycline, and then during the autumnal mixing through the whole water column (see Kankaala and others 2006a, b, 2007 for details in Mekkoja¨rvi and Valkea-Kotinen). In the whole combined dataset from the lakes, the MOB activity in the water column tended to increase when the CH4:O2 molar ratio increased from about 0.0001 to about 0.5 (Figure 4). Although estimated with a different method than that used in the small lakes (see ‘‘Study Area and Methods’’), MOB activity in Ormaja¨rvi and Pa¨a¨ja¨rvi was also related to the CH4:O2 ratio (Figure 4). The highest MOB activities

Figure 3. A Production of heterotrophic bacteria (HBP) and B bacterial carbon demand (assuming 10 and 25% bacterial growth efficiency BGE) in relation to primary production (PPP) during the ice-free period. C HBP as percentage of the available DOC pool in the water column during the ice-free period in relation to the proportion of euphotic zone volume to total lake volume. For lake symbols see A and Table 1.

(ca. 2–12 lmol C l-1 d-1) were observed when the CH4:O2 ratio varied between about 0.5 and 12. The data presented in Figure 4 include only those measurements in which MOB activity was detected. In the three smallest lakes, no MOB activity was detected in approximately 30% of all in vitro measurements made either from the anoxic hypolimnion or the oxic epilimnion. In the two

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Table 3. Seasonal Variation in the Range of Percent Contribution (%) of Primary Production (PPP), Heterotrophic Bacterial Production (HBP), and Net Production of CH4 Oxidizing Bacteria (MOB) to Total Pelagic Energy Mobilization in the Five Lakes

Mekkoja¨rvi

Alinen Mustaja¨rvi

Valkea-Kotinen Figure 4. CH4 oxidation activity in the study lakes related to CH4:O2 molar ratio. The results are from different seasons and depths in which MOB activity could be detected. For lake symbols see Table 1.

largest lakes, no MOB activity was detected in the whole water column from July to September. The MOB activity estimated for the whole water column was highest in the two smallest lakes Mekkoja¨rvi (2–41 mmol C m-2 d-1) and Alinen Mustaja¨rvi (2–19 mmol C m-2 d-1, Figure 1). In Lake Ormaja¨rvi, a peak of MOB activity was detected during spring mixing in May (10–13 mmol C m-2 d-1) and low activity in June (<1 mmol C m-2 d-1), but again a peak (20–34 mmol C m-2 d-1) was detected during the autumn mixing in October. In Pa¨a¨ja¨rvi, very low MOB activity was detected in May–June (<0.8 mmol C m-2 d-1) and in October (<0.2 mmol C m-2 d-1). Assuming that the net production of MOB varies between 10 and 40% of the observed CH4 consumption, carbon incorporation into MOB cells during the IFP varied between 0.17 and 0.69 mol C m-2 IFP-1 in Mekkoja¨rvi, thus exceeding that of HBP (Table 2). In Alinen Mustaja¨rvi, the estimated net MOB production was 0.24– 0.97 mol C m-2 IFP-1, thus of the same order of magnitude as that of HBP. In the other three lakes, the net production of MOB during the whole IFP was estimated to be lower than that of HBP.

Proportions of Primary Producers and Bacteria in the Total Pelagic Energy Mobilization PPP was responsible for most (>55%) of the pelagic energy mobilization in spring (from late April to the end of May) in all five lakes (Table 3). In the three largest lakes, greater than 70% of the energy mobilization was due to PPP during the whole IFP (Figure 5). The proportion of MOB in

Ormaja¨rvi

Pa¨a¨ja¨rvi

Season

PPP

HBP

MOB

Spring Summer Autumn Spring Summer Autumn Spring Summer Autumn Spring Summer Autumn Spring Summer Autumn

56–77 47–69 15–33 75–86 60–78 32–53 73–74 76–79 70–76 80–88 87 74–82 79–80 79 73

7–10 10–15 11–26 9–10 9–12 15–25 25 19 29–21 8 12 12–14 20 21 27

13–37 15–42 42–74 5–16 10–30 22–53 1–3 1–5 3–11 3–12 <0.1 4–14 0.1–0.3 0.1–0.2 0.1–0.2

the total energy mobilization varied between the lakes and seasons. In the two smallest lakes, the proportion of MOB was already significant in spring and increased through summer (June– August) to autumn (September–October) reaching 42–74% in Mekkoja¨rvi and 22–53% in Alinen Mustaja¨rvi. In Ormaja¨rvi, the proportion of MOB was also high during spring and autumn (3–14%). Over the whole IFP the proportion of MOB in the total energy mobilization was 24–52% in Mekkoja¨rvi but only less than 0.5% in Pa¨a¨ja¨rvi, with the other lakes showing a range of intermediate values (Figure 5). HB was responsible for 10–23% of the total energy mobilization in the lakes, the proportion being greatest in the deepest and largest lake, Pa¨a¨ja¨rvi (seasonally varying between 20 and 27%, mean 23%).

DISCUSSION Despite increased interest in alternative basal resources for lake food webs, supplementing the phytoplankton production, so far as we are aware ours is the first study to present the magnitude and variation of several basal resources from a suite of contrasting lakes. We found that in larger lakes (>0.01 km2) PP is more important (>70%) than bacteria in the total energy mobilization. The contribution of HB to the total energy mobilization did not vary greatly between the lakes, the highest proportion being 23% in the mesohumic, deep lake Pa¨a¨ja¨rvi. In contrast, in small (<0.01 km2) stratified

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Figure 5. Proportions of primary production (PPP), heterotrophic bacterial production (HBP), and net production of CH4 oxidizing bacteria (MOB) in the pelagic energy mobilization of five boreal lakes presented in order of increasing area. The bars denote mean percentage range during the ice-free period. For lake symbols see Table 1.

lakes bacteria, and especially MOB, can contribute a major part of the total pelagic energy mobilization and hence can be considered potentially important food resources for zooplankton. A key finding from our study was how the importance of pelagic CH4 mobilization by MOB differed between lakes, apparently being regulated by the CH4:O2 ratio, which was related to lake size. We measured intensive CH4-oxidation activity in the water column of the three smallest lakes when the CH4:O2 molar ratio was 0.5–12 (Figure 4), suggesting a wider optimal range of this ratio than that obtained in laboratory experiments with culturable methanotrophs (around 0.9, see Amaral and Knowles 1995; Dedysh and others 1998). In small stratified polyhumic boreal lakes, the highest CH4 concentrations in the hypolimnion are typically measured during late summer (Kankaala and others 2006a; Taipale and others 2008; Juutinen and others 2009). Convective mixing plays an important role for gas transfer in small lakes (Read and others 2012). Despite steep stratification, mixed layer deepening during late summer effectively contributes to transfer of gases from the anoxic zone to the meta- and epilimnion (Huotari

and others 2009); increasing late summer CH4 oxidation activity in the small lakes (Figure 1) was related to this mixed layer deepening. However, the highest CH4 oxidation activities occurred during the autumn mixing when both CH4 and O2 were available for methanotrophs through the whole water column (Kankaala and others 2007). In the larger and more eutrophic lake Ormaja¨rvi, peaks of methanotrophic activity were observed during spring and autumn mixing, when CH4 from the sediment was mixed into the water column. However, CH4 oxidation in the water column was insignificant during summer stratification when the whole water column was oxic down to the bottom. A similar situation prevailed in the largest lake Pa¨a¨ja¨rvi, with an oxic water column throughout the year. Our results from lakes in southern Finland with a DOC range 7–24 mg C l-1 indicated that, despite high DOC concentration, PP exceeded HBP in the whole water column during the IFP. Hence, our results are the opposite of those reported by Jansson and others (2000) and Karlsson and others (2012), who measured PPP less than HBP in the pelagic zone of lakes with DOC concentration greater than 10 mg C l-1 in northern Sweden. Differences in methods do not explain the discrepancy between these findings. In the Swedish studies, a 4-h incubation around noon was used and daily values were estimated by relating the obtained 4-h PP values to light according to Wetzel and Likens (1991), whereas we used a 24-h incubation which usually gives lower daily PPP estimates than extrapolation from shorter incubations (compare Lizon and Lagadeuc 1998). In the 14 C-leucine uptake HBP measurements, we used carbon conversion factors obtained by Tulonen (1993) for lake Pa¨a¨ja¨rvi, and later studies by Maanoja (2008) confirmed that these conversion factors are valid for two of the other lakes (ValkeaKotinen and Alinen Mustaja¨rvi). Application of the leucine-to-protein and protein-to-carbon conversion factors of Simon and Azam (1989), as used in the study of Karlsson and others (2012), would have yielded still lower HBP estimates in our lakes, accentuating the discrepancy. In fact, the HBP results per unit volume in the epilimnia and hypolimnia of the lakes are of the same order of magnitude in both areas (compare for example, Tulonen 1993; Bergstro¨m and Jansson 2000). Thus, one possible explanation for the differences in the PPP:HBP ratio is the greater mean depth and relatively shallower euphotic zone in the Swedish humic lakes, which may also influence available food web resources.

Pelagic Energy Mobilization In our study, lakes with a DOC range of 7–24 mg C l-1, we found no correlation between DOC and HBP, calculated either on a volumetric or on an areal basis, or by using daily values or estimates integrated for the whole IFP. Instead, we found that HBP integrated for the whole water column of the lakes during the IFP was correlated with that of PPP. This correlation, indicating a direct coupling between PP and bacteria, has been reported in many previous studies both in fresh-water and marine environments around the world [see reviews by Cole and others (1988) and Fouilland and Mostaijr (2010)]. Our findings thus contrast with those of Jansson and others (2000), who observed a significant positive correlation between mean epilimnetic DOC concentration (5–19 mg C l-1) and HBP in June–September in five lakes in northern Sweden. Despite the significant positive correlation between HBP and PPP in our study lakes, the estimated total bacterial carbon demand compared with PPP suggests that most carbon for HB in the study lakes originated from allochthonous sources (see Figure 3), consistent with many previous reports (for example, Salonen and others 1983; Tranvik 1988; Bergstro¨m and Jansson 2000). However, we recognize that this inference is sensitive to the value of BGE used to estimate bacterial carbon demand. We estimated bacterial carbon demand in our study lakes by using a BGE range of 10–25% derived from a similar BP range for the lakes studied by Krizberg and others (2005). However, BGE can vary considerably with resource quality (del Giorgio and Cole 1998; Lennon and Cottingham 2008) and temperature (Biddanda and Cotner 2002). In experimental conditions, the BGE when bacteria are using autochthonous DOC, especially PP exudates (Kroer 1993; Middelboe and Søndergaard 1993), and fresh forest soil extracts (Lennon and Cottingham 2008), is much higher, up to 60%, than when using aged allochthonous DOC (<10%, Berggren and others 2009). Therefore, we consider our choice of BGE range to be appropriate, and that our overall inference of substantial reliance of bacteria in these lakes on allochthonous carbon is most likely broadly valid. In their review, Guenet and others (2010) suggested that autochthonous labile organic matter induces a ‘‘priming effect’’ for mineralization of recalcitrant terrestrial organic matter in aquatic ecosystems, as reported for soils (Kuzyakov and others 2000). Such a priming effect would be consistent with the significant correlation between HBP and PPP in our study lakes and the greater BGE with autochthonous than allochthonous sources. The increasing proportion of DOC incorporated by HB

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cells when the euphotic zone is a higher proportion of the total lake volume (Figure 3C) also supports the priming effect hypothesis. The low BGE with allochthonous DOC is reflected in high community respiration exceeding PPP (Salonen and others 1983; del Giorgio and Peters 1994), as also measured in our study lakes (Arvola and others 1996; Ojala and others 2011). Thus, the increased mineralization of allochthonous DOC results in greater supersaturation and atmospheric fluxes of CO2 from humic lakes compared with those from clear-water lakes (Ojala and others 2011; Huotari and others 2011; Larsen and others 2011), although this does not necessarily mean a greater availability of allochthonous sources to higher trophic levels. In a dataset from 12 boreal lakes, including the five present study lakes, the level of CO2 supersaturation at lake surfaces during the summer stratification was positively correlated with DOC concentration and negatively with lake size (Kankaala and others unpublished manuscript). The production of MOB and HB, and the carbon potentially transferred from them to higher trophic levels, does not necessarily originate mainly from allochthonous sources. In the smallest lakes, allochthonous sources for heterotrophic bacterial carbon presumably dominated (see Figure 3B). The very low d13C values (around -80&) of CH4 close to the bottom of the smallest lake (Mekkoja¨rvi) suggest that the H2/CO2 pathway predominates in CH4 production (Kankaala and others 2007) and the CO2 presumably originates from decomposition of allochthonous organic matter. In the more eutrophic Lake Ormaja¨rvi, the high proportion of DOC to HBP likely originated from autochthonous organic matter (see Figure 3B) and the bulk of carbon transferred via CH4 to methanotrophs presumably also originated from anaerobic decomposition in sediments of recently produced and deposited autochthonous organic matter. This inference is supported by the findings of Bastviken and others (2008) from experiments in two clear-water lakes, where a clear enrichment of 13C-CH4 was observed after 13C enrichment of pelagic particulate organic matter. Significantly lower HBP in the hypolimnion than in the euphotic zone and in the metalimnion of all our lakes (Figure 2) was presumably due to both lower availability of autochthonous carbon and lower temperatures in the hypolimnion. On the other hand, in the three smallest lakes with an anoxic hypolimnion, taxa other than heterotrophs (Chlorobi, Methylobacteria, and undefined operational taxonomic units) dominate the microbial community (Taipale and others 2009; Peura and others 2012). For these microbial groups, DOC may not be the primary carbon source, so that

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C-leucine uptake does not adequately reflect their productivity. For example, many photoautotrophic bacteria use CO2 as their primary carbon source (Van Gemerden and Mas 1995). Our results for seasonal variation in the relative proportions of PP, HB, and MOB in the net pelagic energy mobilization of the smallest lake, Mekkoja¨rvi (Table 3), correspond well with results obtained by Taipale and others (2008) for the respective diet source proportions of Daphnia longispina in the lake. Those results were obtained from an ecosystemscale 13C-DIC enrichment experiment and analyses of stable carbon and nitrogen isotopes. The proportion of PP in the diet of Daphnia decreased from 37– 71% in spring to 31–56% in the autumn and the proportion of MOB increased from 9–10 to 26–50%. Thus, Daphnia utilized the different food resources according to their availability. In accordance with the production measurements of this study, the stable carbon and nitrogen values of cladocerans, sampled in May and October 2006 from Alinen Mustaja¨rvi and Valkea-Kotinen, showed a higher contribution of MOB to their diets in autumn than in spring (Kankaala and others 2010). However, a bacterial diet deficient in polyunsaturated fatty acids (PUFA) and sterols cannot alone support somatic growth and reproduction of crustacean zooplankton (Ojala and others 1995; Martin-Creuzburg and others 2011; Taipale and others 2012). Nevertheless, the evidence is that all the energy mobilizers included in this study, and not just PP, can contribute to carbon flux through pelagic webs, and the controls of their production and the extent to which it is utilized, need to be better understood. In conclusion, our results from five boreal lakes with contrasting DOC concentration and size do not indicate a predominance of HB in the energy mobilization for pelagic food webs. Although our measurements do not provide any direct evidence, the results appear circumstantially to support the ‘‘priming effect’’ hypothesis, whereby autochthonous PP significantly contributes to allochthonous carbon decomposition and CO2 supersaturation in the lakes. In small stratified lakes (area < 0.01 km2) bacteria, especially MOB, are potentially quantitatively important supplementary food resources for zooplankton. However, in larger lakes, irrespective of DOC concentration, primary producers are the most important (>70%) potential food source for zooplankton.

and Grant 201623 to AO. AO was also supported by Nordic Centre of Excellence for Studies of Ecosystem Carbon Exchange (NECC). We thank two anonymous reviewers for their suggestions to improve an earlier version of the manuscript.

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