Sampling
Water was sampled from Cep lake in Czechia, at a regular sampling site of 10 m depth (48.944 °N, 14.877 °E). The lake originates from sand mining in the 1970–80s. It is a permanent meso-oligotrophic (chlorophyll-a concentrations ranged from 1.4–16.4 µg L−1) seepage reservoir filled with groundwater penetrating from the nearby river Lužnice. The lake area is about 1.16 km2, with the maximum depth about 11–12 m. These characteristics are representative for most of temperate and boreal lakes [16].
Samples were collected every four weeks from April till October in 2018, and from April till November in 2019. Ten liters of water were collected from 0.5 m depth using a Ruttner Water Sampler (model 11.003KC Denmark AS). Temperature and oxygen profiles were taken with an EXO1 multi-parameter probe (YSI Inc., Yellow Springs, USA). Water was transported to the laboratory within 2 h from the collection in a closed container made from high-density polyethylene, rinsed three times with the sampled water and stored in a cooled box.
Nutrients
Samples were filtered through glass fiber filters with 0.4 µm nominal porosity (GF-5, Macherey-Nagel, Düren, Germany). Concentrations of soluble reactive phosphorus (SRP) were determined spectrophotometrically [17, 18]. Concentrations of nitrate and ammonium were measured according to Procházková [19] and Kopáčkek and Procházková [20]. Dissolved organic carbon (DOC) and dissolved nitrogen (DN) were measured with a TOC 5000 A analyzer (Shimadzu, Kyoto, Japan).
Pigments
Seston from 1.43 to 3.65 L of water was collected onto GF-5 glass fiber filters (diameter 47 mm, Macherey-Nagel). The filters were dried of excess water by gently pressing in a paper towel, and flush frozen in liquid nitrogen. Pigments were extracted and analyzed by HPLC as described in Piwosz et al. [21].
Net primary production (NPP) and community respiration (oxygen measurements)
Oxygen concentration was measured with the Winkler method [22]. It was chosen because it allows O2 concentration to be measured directly in the water without the need to consider carbonic equilibrium, which is the case when changes in CO2 concentration is measured [23]. Samples were unfiltered to avoid the removal of particle-associated bacteria and also of free-living AAP bacteria, which tend to be larger than average freshwater bacteria [12]. Glass stoppered Winkler type oxygen bottles (115 mL nominal capacity, VTR glass, Prague, Czechia) were filled with the sampled water directly from the sampler via a rubber tube. Each bottle was first rinsed three times and then filled without the formation of air bubbles. Water was allowed to overflow the neck of the bottle for about 1 min, and the bottle was closed with a glass stopper to avoid air bubbles. The closed bottles were kept in the dark in a cooled box. On the shore, three bottles were selected as T0, and 1.2 mL of manganese (II) chloride solution (concentration 3 mol L−1) was addded, followed by the addition of 1.2 mL of a mixture containing 4 mol L−1 of sodium iodide solution and 8 mol L−1 of sodium hydroxide solution. These samples were processed in the laboratory within 3 h. The remaining bottles were incubated for 24 h at in situ temperature in the IR-box prepared from the MAKROLON IR polycarbonate sheet (4 mm thickness, Professional Plastics, Inc. Fullerton, CA, USA). These panels have a maximum transmittance of 90% in the infrared region 850–2000 nm, 50% at 780 nm, and 0% <740 nm. Dark bottles were wrapped in tinfoil to cut off all irradiance. Illumination was provided with 40 W incandescent (tungsten filament) lightbulbs delivering approx. 50 Wm−2 of IR irradiance, which according to our measurements corresponds to IR irradiation on a sunny day. Bottles for NPP were incubated in white light next to the IR-box. To ensure stable temperature and avoid overheating, the incubations were done in a 60 L water bath and the temperature was controlled with a CTB 06 C cryostat (LABIO a.s., Prague, Czechia).
The incubations were terminated by adding manganese (II) chloride and sodium iodide – sodium hydroxide solutions, as described above for the T0 samples. The bottles were incubated for 1 h in a fridge (4 °C). Subsequently, 2.4 ml of 50% H2SO4 was added, and samples were immediately titrated with 0.01 mol L−1 Na2S2O3 solution. The precise concentration of the Na2S2O3 solution was determined each time by titrating the KIO3 standard. For the samples collected in 2018, titrations were done manually: the samples were titrated until the solution turned pale yellow. Then, 1 mL of 0.1% starch solution was added, and the titration continued until full decolorization was achieved. In 2019, samples were titrated to a monotonic equivalence point using Metrohm 877 Titrino plus equipped with a double Pt-wire coulometric electrode (Herisau, Switzerland). Respiration rates were calculated as a difference between oxygen concentrations at the end of the incubation and T0 samples.
To calculate the balance between the NPP and respiration measured in the dark and in the IR light, we assumed that one mole of O2 produced or consumed was equivalent to one mole of CO2. Such assumption may not be always accurate for respiration measurements, but considering the high variability of a respiratory quotient in freshwaters, we decided to adopt the most frequent choice [3]. Values of daily NPP were calculated based on the length of light time from sunrise to sunset on the sampling day (Equation 1 in Supplementary File S1). Values of daily dark respiration were taken directly from the measurements, while values of the daily IR-respiration were calculated as an average weighted for the length of light time to take into account that the effect of light was only during the light time (Equation 2 in Supplementary File S1). Rates were integrated over the duration of the season with water temperatures >10 °C according to our measurements (180 days, Equation 3 in Supplementary File S1). Subsequently, the differences in the Cep Lake’s carbon budget for the surface layer (down to 0.5 m depth) were calculated by multiplying the integrated values by the volume of this layer (Equation 4 and 5 in Supplementary File S1).
HCO3
− incorporation
Triplicated water samples (32 mL) were incubated for 3.2–5.2 h in the IR light and the dark at in situ temperature, as described for respiration. Total activity added to each bottle was measured from 1 mL aliquot of the incubated sample that was transferred to a scintillation vial containing 20 μl of 5 mol L−1 NaOH (to prevent a loss of 14C-bicarbonate). Thirty mL of sample was filtered through 2.5 μm nitrate cellulose filters (Pragopor, Prague, Czechia, diameter 25 mm). Five mL of the filtered water was collected and subsequently filtered through a 0.17 μm nitrate cellulose filter. The resulting cell-free filtrate, which contained 14C-DOC was collected. The filtration was done at a low vacuum (0.02 MPa) to avoid cell breakage. The total CO2 assimilation rate was calculated as the sum of all these fractions.
The filters were kept in an HCl-saturated atmosphere for 24 h at room temperature in a fume hood. They were placed in scintillation vials and dissolved in 1 mL of ethyl acetate (Penta, Prague, Czechia). Then, 5 mL of Ultima Golt LLT scintillation cocktail (PerkinElmer, Waltham, MA, USA) was added. Five mL of cell-free filtrates were acidified by adding 100 μL 5 mol L−1 HCl to volatilize non-incorporated H14CO3 and incubated 24 h at room temperature in a fume hood. Then, 10 mL of the scintillation cocktail was added. Finally, 5 mL of the scintillation cocktail was added to the total activity samples. Subsequently, the samples were gently mixed and left in the dark for 48 h. The radioactivity in the samples was measured using a Tri-Carb 2810 TR scintillation counter (PerkinElmer).
To estimate carbon fluxes (μmol C L–1 h–1), a fraction of the added H14CO3 incorporated or released was corrected for the incubation time and multiplied by the concentration of total dissolved inorganic carbon (DIC). The DIC concentration was calculated based on temperature, pH, and alkalinity measurements (Inolab pH 720, WTW Xylem Inc. Rye Brook, NY, US) determined by Gran titration.
Assimilation of organic monomers
The difference between microbial activity in the IR light and dark was also estimated based on assimilation rates of radiolabeled glucose, pyruvate, leucine and thymidine (American Radiolabeled Chemicals, St. Louis, MO, USA). Tritiated glucose (specific activity (SA): 2220 GBq mmol−1), leucine (SA: 4440 GBq mmol−1) and thymidine (SA: 2275.5 GBq mmol−1) were added to 5 mL samples to a final concentration of 5 nmol L−1. 14C-pyruvate (SA: 2.035 GBq mmol−1) was added to a final concentration of 10 nmol L−1. Trichloroacetic acid (TCA) was added to the killed controls to a final concentration of 1%. Samples were incubated for 1 h in the dark and IR light as described for respiration. The incubations were terminated as the killed controls and kept at 4 °C in the dark until processed within <4 h. Biomass was collected onto 0.17 μm nitrate cellulose filters as described for HCO3− incorporation. The filters were washed twice with 2.5 mL of ice-cold 5% TCA, and then twice with 2.5 mL of ice-cold 80% ethanol [24]. They were placed in the scintillation vials and air-dried overnight. Dried filters were dissolved in 1 mL of ethyl acetate, and 5 mL of Ultima Golt LLT scintillation cocktail (PerkinElmer) was added. Samples were gently mixed and left in the dark for 48 h. The radioactivity in the samples was measured using a Tri-Carb 2810 TR scintillation counter (PerkinElmer).
Bacterial and AAP abundance
Samples of 50 mL were fixed with buffered, sterile-filtered paraformaldehyde (Penta, Prague, Czechia) to a final concentration of 1%, and 0.5 mL was filtered onto white polycarbonate filters (pore size 0.2 µm, Nucleopore, Whatman, Maidstone, UK). Cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) at concentration of 1 mg L−1 [25]. Total and AAP bacterial abundances were determined using an epifluorescence Zeiss Axio Imager.D2 microscope equipped with Collibri LED module illumination system (Carl Zeiss, Jena, Germany). Ten microphotographs were taken for every sample under 325–370 nm excitation and 420–470 nm emission wavelengths for DAPI fluorescence (total bacteria), 450–490 nm excitation and 600–660 nm emission wavelengths for autofluorescence from Chl-a (algae and cyanobacteria), and combined 325–370 nm, 450–490 nm, 545–565 nm and 615–635 nm excitation and 645–850 emission wavelengths for autofluorescence from BChl-a (AAP bacteria). As some part of Chl-a autofluorescence is also visible in the infrared spectrum, only the IR-positive cells that did not show any autofluorescence from Chl-a were counted as AAP bacteria [26].
DNA extraction
Between 300 and 1460 mL of water were filtered through sterile 2 µm and 0.2 µm Nucleopore Track-Etch Membrane filter units (Whatman). The filters were put inside sterile cryogenic vials (Biologix Group Limited, Jinan, Shandong China) containing 0.55 g of sterile zirconium beads, flash-frozen in liquid nitrogen and stored at −80 °C. Total nucleic acids were extracted within a month following the protocol by Nercessian et al. [27]. Lysis buffer (75 µl of 10% sodium dodecyl sulfate (Tokyo Chemical Industry CO, LTD., Tokyo, Japan), 75 µl of 10% N-Lauroylsarcosine (Sigma–Aldrich, St. Louis, USA), 750 µl of phenol-chloroform-isoamyl alcohol (25:24:1; AppliChem GmbH, Darmstadt, Germany) and 750 µl of 10% hexadecyltrimethylammonium bromide (CTAB; Sigma-Aldrich) in 1.6 M NaCl and 240 mM potassium-phosphate-buffer, pH = 8) was added to the vials and they were vortexed for 10 min. After centrifugation for 10 min at 4 °C and 16,000 x g, supernatant was mixed carefully with the equal volume of chloroform (PENTA s.r.o., Prague, Czechia). After the second centrifugation, the supernatant was mixed with two volumes of 30% Poly(ethylene glycol) (PEG; Sigma–Aldrich) in 1.6 M of NaCl, and incubated for 2 h in the dark at 4 °C, followed by centrifugation for 90 min at 4 °C and 17,000 x g. The pellet was washed with 70% ethanol (VWR International S.A.S., Fontenay-sous-Bois, France) and centrifuged again for 1 min. Extracted DNA was re-suspended in 35 µl of DNase and RNase-free water (MP Biomedicals, Solon, OH, USA) and stored at −20 °C. Concentration and quality of the extracts were checked using NanoDrop (Thermo Fisher Scientific).
Bacterial community composition
The V3-V4 region of bacterial 16S rRNA gene was amplified using 341F and 785R primers [28]. PCR was performed in triplicate 20 μL reactions using Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific, USA) with the following reaction conditions: 98 °C for 3 min, 25 cycles at 98 °C for 10 s, 60 °C for 20 s, 72 °C for 20 s, and a final extension at 72 °C for 3 min. The triplicate product reactions for each sample were pooled andpurified from the gel using the kit Wizard SV Gel and PCR Clean-Up System (Promega, USA), and sequenced on Illumina MiSeq (2 × 250 bp) platform of the Genomic Service of the Universitat Pompeu Fabra (Barcelona, Spain).
Initial analysis, performed as described below, indicated that the communities in both fractions were similar for each sampling day (Bray-Curits similarity >65% except for the samples from 9th May and 29th Aug 2018 (10% each), 1st Aug 2018 (21%), 25th Nov 2018 (1%), and 14th Aug 2019 (22%), Supplementary Fig. S1A). Thus, we decided to concatenate the fastaq files and analyze both fractions together as the total community. This also facilitated statistical analysis, as the activity rates were measured for the whole community without fractionations.
Reads quality was evaluated using FastQC v0.11.7 (Babraham Bioinformatics, Cambridge, UK). After primer sequences trimming using Cutadapt [29] (v1.16), the number of reads per sample ranged from 49,354 to 188,942. Subsequent analyses were done in the R/Bioconductor environment using the dada2 package (version 1.14.1) [30]. Forward and reverse reads were truncated to 225 bp and low quality sequences were filtered out with the filterAndTrim function (truncLen = c(225, 225), maxN = 0, maxEE = c(2, 2), truncQ = 2), which reduced the number of reads per sample to range from 30,190 to 143,552. After merging and chimera removal using the removeBimeraDenovo function, 4,893 amplicon sequence variants (ASV) were obtained. Rare ASVs (not seen >3 times in at least 20% of the samples) were removed, which reduced the number of ASVs to 658, and the number of reads to 14,613–69,046 per sample. Taxonomic assignment was done using SILVA 138.1 database [31, 32] released on August 27, 2020. ASVs identified as Chloroplast or Cyanobacteria were excluded from the analyses, giving the final number of 546 ASVs and from 10,819 to 54,799 reads per sample. The bacterial community composition graphs were done using phyloseq [33] and ggplot2 [34] packages.
AAP community composition
The composition of AAP community was analyzed by amplicon sequencing of pufM gene encoding the M subunit of bacterial type-2 reaction centers. This gene is routinely used for diversity studies of AAP bacteria [35].
PufM gene amplicons (approx. 245 bp) were prepared using pufM_UniF (5′-GGN AAY YTN TWY TAY AAY CCN TTY CA-3′) and pufM_WAW (5′-AYN GCR AAC CAC CAN GCC CA-3′) primers [36]. PCR was performed in triplicate 20 μL reactions using Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific, USA) with the following reaction conditions: 98 °C for 3 min, 27 cycles at 98 °C for 10 s, 58 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. The triplicate product reactions for each sample were pooled and gel purified using the kit Wizard SV Gel and PCR Clean-Up System (Promega, USA). The sequencing was performed on the Illumina MiSeq platform (2 × 150 bp) at Macrogen, South Korea.
The fastq files were concatenated as described for bacteria communities. The Bray-Curtis similarity between two fractions for each sampling day was >70%, except for the samples from 1st Aug 2018 (47%) and 14th Aug 2019 (18%, Supplementary Fig. S1B).
The samples were analyzed as described for bacterial communities. The number of reads per sample ranged from 192,360 to 239,418 after the cutadapt trimming. Forward and reverse reads were truncated to 130 bp, and the number of reads per sample after the quality filtering and denoising ranged from 189,432 to 235,311. Merging the forward and reverse reads with mergePairs function created 12,692 ASVs and reduced the number of reads to 183,136–221,281 per sample. The chimera removal lowered the number of ASVs to 1816, and the number of reads to 159,451–208,679. Rare ASVs (not seen >3 times in at least 20% of the samples) were removed, which resulted in the final 566 ASVs, and a number of reads ranging from 155,915 to 203,021 per sample. A manually curated taxonomic database was used for taxonomic assignment following the naïve Bayesian classifier method [37]. It contained 1580 unique pufM sequences, downloaded from the Fungene repository on May 16, 2019 (http://fungene.cme.msu.edu [38]), from metagenomes from the Římov Reservoir [39, 40] and from the Genome Taxonomy database accessed on September 16, 2020 [41].
Statistical analysis
Linear mixed-effects models were calculated in R (version 3.6.2) using lme function from the nlme package (version 3.1.143) on untransformed activity data and log10 transformed environmental variables [42]. Models’ parameters were estimated using maximum likelihood method and their significance was tested with ANOVA. Relationships between the activity measures, the environmental variables and the composition of AAP communities were investigated with distance-based linear models (DistLM) [43, 44] in Primer (version 7.0.13) with PERMANOVA + 1 add on (e-Primer, Plymouth, UK) [45]. The sequence reads were transformed with the varianceStabilizingTransformation function of the DESeq2 package [46] (version 1.14.1, blind = FALSE, fitType = “mean”).
Data accessibility
The sequences of 16S and pufM amplicons that support the findings of this study have been deposited in the EMBL database as the BioProject with the accession number PRJEB41596, together with most of the environmental metadata. The scripts and the remaining data supporting the results are included in the Supplementary Material.
Source: Ecology - nature.com
