2.1 Cultures and culture conditions

Three algal species, E. huxleyi RCC1216 obtained from the Roscoff Culture Collection (http://roscoff-culture-collection.org/, last access: 21 October 2019), P. globosa PLY 575, and Chrysochromulina sp. PLY 307 obtained from the Marine Biological Association of the United Kingdom (https://www.mba.ac.uk/facilities/culture-collection, last access: 21 October 2019), were studied. In order to keep non-axenic algae cultures largely free of bacteria, the cultures were diluted regularly, resulting in quasi-constant exponential algal growth while minimizing bacterial cell density. All incubation experiments were carried out in controlled and sterile laboratory conditions under a 16 ∕ 8 h light–dark cycle at a light intensity of 350 µmol photons m⁻² s⁻¹ and a temperature of 20 ^∘C. All samples were taken at the end of the light cycle. Monoclonal cultures were grown in full-batch mode (Langer et al., 2013) in sterile filtered (0.2 µm ∅ pore size) natural North Sea seawater (sampled off Helgoland, Germany) enriched in nutrients according to F/2 medium (Guillard and Ryther, 1962). The initial dissolved inorganic carbon (DIC) of the F/2 medium was 2152±6 µmol L⁻¹ (measured by Shimadzu TOC-V CPH). The DIC value falls within the range of typical DIC concentrations of North Sea seawater.

2.2 Determination of cell densities

Cell densities were determined from four aliquots of each culture sample using either a Fuschs–Rosenthal or Neubauer counting chamber, depending on cell density.

2.3 Incubation with ¹³C-labeled hydrogen carbonate

To investigate CH₄ production by algal cultures, borosilicate glass bottles (Schott, Germany) filled with 2.0 L of 0.2 µm filtered F/2 medium and with 0.35 L headspace volume were used in our investigations of Chrysochromulina sp. and P. globosa. For the investigations of E. huxleyi 0.85 L medium and 0.4 L headspace volume were used (Schott, Germany). The vials were sealed airtight with lids (GL 45, PP, two-port, Duran Group) equipped with one three-way port for liquid and a second port fitted with a septum for gas sampling. For measurements of the mixing ratio and stable carbon isotope value of CH₄ (δ¹³C−CH₄) samples of headspace (20 mL) were taken from each vial. Afterwards, samples (2 mL) for determining cell densities were taken. In order to maintain atmospheric pressure within the vial, the surrounding air was allowed to enter via the three-way port and through a sterile filter to avoid biological contamination. The inflow of surrounding air was taken into consideration when CH₄ production was calculated. Cultures that were studied during the incubation were inoculated from a pre-culture grown in dilute-batch mode (Langer et al., 2009). To investigate algal-derived CH₄ formation six vials were inoculated with algae and another six vials contained medium only. In addition, three vials of each group were treated with ¹³C–hydrogen carbonate (${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$) to investigate CH₄ formation by measuring stable carbon isotope values of CH₄. Four different treatments were used: medium either with ${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$ (medium + ${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$) or without (medium, data not available) and cultures supplemented either with ${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$ (medium + culture + ${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$) or without (medium + culture). The different treatments and the number of replicates for the experiments with Chrysochromulina sp. and P. globosa are provided in Fig. 1. Please note that stable isotope measurements using ${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$ were not performed for E. huxleyi as evidence for the isotope labeling of CH₄ was already provided by Lenhart et al. (2016). To study the CH₄ formation of E. huxleyi by measuring headspace concentration, three replicates (culture and medium group, n=3) were used.

Figure 1Experimental setup for measuring CH₄ formation by Chrysochromulina sp. and P. globosa. Methane formation was investigated by concentration measurements within six vials containing either algae or medium only (left column). For stable isotope measurements of CH₄, ¹³C-labeled hydrogen carbonate (${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$) was added to three vials of both groups (right column).

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The overall incubation time was 9, 11, and 6 d for Chrysochromulina sp., P. globosa, and E. huxleyi, respectively. Headspace and liquid samples were collected on a daily basis for E. huxleyi and in 2–3 d intervals from cultures of Chrysochromulina sp. and P. globosa. The incubation time and sampling intervals varied between species because of variations in the growth rate and the cell density in the stationary phase. Cell densities were plotted versus time, and the exponential growth rate (μ) was calculated from exponential regression using the natural logarithm (Langer et al., 2013). The exponential growth phase (from which μ was calculated) was defined by the cell densities which corresponded to the best fit (r²>0.99) of the exponential regression. This was done by using the first three (Chrysochromulina sp. and E. huxleyi) or four data points (P. globosa) of the growth curve. For stable carbon isotope experiments 48.7 µmol L⁻¹ NaH¹³CO₃ in final concentration was added to the F/2 medium. The added amount of NaH¹³CO₃ corresponds to 2 % of the DIC of the North Sea seawater (2152±6 µmol L⁻¹), resulting in a theoretically calculated δ¹³C value of DIC of $+\mathrm{2014}\pm \mathrm{331}$ ‰ . To determine the δ¹³C−CH₄ values of the source, the Keeling plot method was applied (Keeling, 1958). For a detailed discussion of the Keeling plot method for determination of the isotope ratio of CH₄ in environmental applications, please refer to Keppler et al. (2016). Oxygen concentration was monitored daily (using inline oxygen sensor probes; PreSens, Regensburg) at the end of the light cycle (Fig. S1 in the Supplement).

2.4 Determination of CH₄ production rates

Since the experiment in the Sect. 2.3 was not designed to obtain POC quotas (POC: particulate organic carbon), we conducted an additional experiment. To best compare the CH₄ formation rates of the three algae species it is necessary to obtain exponential growth to ensure constant growth rates and constant (at a given time of day) cellular POC quotas over the course of the experiment. Exponential growth is a prerequisite for calculating production on the basis of growth rate and quota (here CH₄ quota). The point is a general, technical one and is not confined to CH₄ production. The studies by Langer et al. (2012, 2013) discuss this point in the context of batch culture experiments. Briefly, production on this account is the product of growth rate and quota (e.g., CH₄, calcite, organic carbon). Production here is an integrated value, typically over many cell divisions. For this calculation of production to be meaningful a constant growth rate is required. The exponential growth phase fulfills this criterion, whereas the transition phase and the stationary phase do not. Therefore, production cannot be calculated meaningfully in the non-exponential phases. The problem can, however, be minimized by using small increments (1 d) because growth rate can be regarded as quasi-constant (see also Lenhart et al., 2016). The CH₄ production rates can be calculated by multiplying the growth rate μ with the corresponding cellular or POC–CH₄ quota that was measured at the end of the experiment. For this additional experiment the cultures were grown in 160 mL crimped serum bottles filled with 140 mL medium and 20 mL headspace (n=4). Oxygen concentration was monitored (using inline oxygen sensor probes; PreSens, Regensburg) at the end of each light and dark cycle (Fig. S2). The growth rate (μ) was calculated from cell densities of the beginning and end of the experiment according to Eq. (1):

where N₀ and N₁ are the cell densities at the beginning (t₀) and end of the experiment (t₁). The daily cellular CH₄ production rates (CH₄P_cell, ag CH₄ cell⁻¹ d⁻¹, ag = 10⁻¹⁸ g) were calculated according to Eq. (2):

where m(CH₄) is the amount of CH₄ that was produced at the end of the experiment.

To calculate POC-based CH₄ production rates the cellular organic carbon content (POC_cell) was derived from cell volume (V_cell) by using the Eq. (3) according to Menden-Deuer and Lessard (2000):

The cell volume was determined by measuring the cell diameter in light micrographs using the program ImageJ (Schindelin et al., 2012). According to Olenina (2006) a ball shape can be assumed for calculating the cell volume for the three species investigated here. The daily cellular CH₄ production rates (CH₄P_POC, µg CH₄ g⁻¹ POC d⁻¹) were calculated from growth rate and CH₄–POC quotas at the end of the experiment according to Eq. (4).

The CH₄ production potential (CH₄–PP) was used to translate differences in cellular production rates to the community level. According to Gafar et al. (2018), the CH₄–PP can be calculated for different periods of growth by calculating a cellular standing stock for each time period from a known starting cell density (N₀) (whereby constant exponential growth is assumed). The corresponding amount of produced CH₄ (CH₄PP) for each period of growth and standing stock is the product of the cellular standing stock and CH₄ quota (Eq. 5).

In the present study the CH₄–PP was calculated for a standing stock that is obtained after 7 d of growth starting with a single cell.

2.5 Incubation with ¹³C labeled DMS, DMSO, and MSO

The sulfur-bonded methyl group(s) in DMS, DMSO, and MSO were investigated as precursors for algal-derived CH₄ in an incubation experiment with E. huxleyi. For all tested compounds only the C atom of the sulfur-bonded methyl group(s) was labeled with ¹³C (R–S–¹³CH₃, 99 %). A final concentration of 10 µM was used for each compound. The different treatments to investigate potential CH₄ formation by ¹³C₂–DMS, ¹³C₂–DMSO, ¹³C–MSO are provided in Fig. 2. Three independent replicates and repeated measurements over time were used. Headspace and vial size were analogous to the experiment described in Sect. 2.3 for E. huxleyi. Samples were taken daily during an overall incubation time of 6 d.

Figure 2Experimental setup to investigate potential precursor compounds of CH₄. Dimethyl sulfide (¹³C₂–DMS), dimethyl sulfoxide (¹³C₂–DMSO), and methionine sulfoxide (¹³C–MSO) were added to the vials containing either a culture of E. huxleyi or medium only. For all tested compounds only the carbon atom of the sulfur-bonded methyl group(s) was labeled with ¹³C.

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2.6 Determination of CH₄ mass

A 5 mL gas sample was collected from the headspace of the vials using a gastight Hamilton gas syringe. The sample was analyzed by a gas chromatographer (GC-14B, Shimadzu, Japan; column: 2 m, ∅=3.175 mm inner diameter, high-grade steel tube packed with molecular sieve 5A 60/80 mesh from Supelco) equipped with a flame ionization detector (FID). Quantification of CH₄ was carried out by comparison of the integrals of the peaks eluting at the same retention time as that of the CH₄ authentic standard using two reference standards containing 9837 and 2192 ppbv. Mixing ratios were corrected for headspace pressure, which was monitored using a pressure-measuring device (GMSD 1, 3 BA, Greisinger). The CH₄ mass ($m_{{\mathrm{CH}}_{\mathrm{4}}}$) was determined by its mixing ratio ($x_{{\mathrm{CH}}_{\mathrm{4}}}$) and the ideal gas law (Eq. 6):

where $M_{{\mathrm{CH}}_{\mathrm{4}}}$ is molar mass, p is pressure, T is temperature, R is the ideal gas constant, and V is volume.

The dissolved CH₄ concentration was calculated by using the equation of Wiesenburg and Guinasso (1979).

2.7 GC-C-IRMS measurements

Stable carbon isotope values of the CH₄ of headspace samples were analyzed by gas chromatography–stable isotope ratio mass spectrometry (GC-C-IRMS; Deltaplus XL, Thermo Finnigan, Bremen, Germany). All δ¹³C–CH₄ values were corrected using two CH₄ working standards (Isometric Instruments, Victoria, Canada) with values of $-\mathrm{23.9}\pm \mathrm{0.2}$ ‰ and $-\mathrm{54.5}\pm \mathrm{0.2}$ ‰. The results were normalized by two-scale anchor calibration according to Paul et al. (2007). The average standard deviation of the analytical measurements was in the range of 0.1 ‰ to 0.3 ‰ (based on three repeated measurements of CH₄ working standards). All δ¹³C–CH₄ values are expressed in the conventional δ notation, in per mille (‰) vs. Vienna Pee Dee Belemnite (VPDB), using Eq. (7).

For a detailed description of the δ¹³C–CH₄ measurements by GC-IRMS and technical details of the pre-concentration system, we refer the reader to previous studies by Comba et al. (2018) and Laukenmann et al. (2010).

2.8 Statistics

To test for significant differences in cell density, CH₄ formation, and CH₄ content between the treatments, two-way analysis of variance (ANOVA) (considering repeated measurements) and a post hoc test (Fisher least significant difference (LSD) test; alpha 5 %) were used.

3.1 Algal growth and CH₄ formation

To investigate CH₄ production by algal cultures, incubations with ¹³C-labeled hydrogen carbon were applied as described in Sect. 2.3. The growth curves during incubation of the three algal species are presented in Fig. 3 (panels a, b, c). The initial cell densities were $\mathrm{26.9}\pm \mathrm{4.0}\times {\mathrm{10}}^{\mathrm{3}}$ cells mL⁻¹ for Chrysochromulina sp., $\mathrm{25.6}\pm \mathrm{1.2}\times {\mathrm{10}}^{\mathrm{3}}$ cells mL⁻¹ for P. globosa, and $\mathrm{17.5}\pm \mathrm{2.0}\times {\mathrm{10}}^{\mathrm{3}}$ cells mL⁻¹ for E. huxleyi. The exponential growth rate µ was highest for E. huxleyi (1.71±0.04 d⁻¹), i.e., 3 or 5 times higher than for P. globosa and Chrysochromulina sp. (with 0.33±0.08 d⁻¹ and 0.52±0.07 d⁻¹, respectively). The dotted lines in Fig. 1a, b, and c mark the time points of exponential growth. Maximum cell densities were lowest for Chrysochromulina sp. with $\mathrm{0.18}\pm \mathrm{0.01}\times {\mathrm{10}}^{\mathrm{6}}$ cells mL⁻¹, followed by E. huxleyi with $\mathrm{1.70}\pm \mathrm{0.09}\times {\mathrm{10}}^{\mathrm{6}}$ cells mL⁻¹, and highest for P. globosa with $\mathrm{1.77}\pm \mathrm{0.15}\times {\mathrm{10}}^{\mathrm{6}}$ cells mL⁻¹. Significant CH₄ formation was observed in all three cultures over the whole incubation period of 5 to 11 d (Fig. 3d, e, f), whereas no increase in CH₄ over time was observed in the control groups. For all species the increase in headspace CH₄ was significant (p≤0.05) at second time point of measurement and at all following time points (p≤0.001). At the end of the incubation period the amounts of produced CH₄ were 34.9±7.3, 99.3±8.2, and 45.0±3.1 ng for Chrysochromulina sp., P. globosa, and E. huxleyi, respectively. A linear correlation was found between the absolute number of cells and the amount of produced CH₄ of Chrysochromulina sp., P. globosa, and E. huxleyi (Fig. 3g, h, i).

Figure 3Cell growth (a, b, c) and CH₄ production (d, e, f) in the course of time and the correlation between the total number of cells and produced CH₄ (g, h, i) from three algae species. Chrysochromulina sp. (a, d, g), P. globosa (b, e, h), and E. huxleyi (c, f, i). Please note that the cell numbers of Chrysochromulina sp. are presented in 10⁵ and those of P. globosa and E. huxleyi are in 10⁶. Mean values of six (Chrysochromulina sp., P. globosa) and three (E. huxleyi) replicated culture experiments are shown, and error bars mark the SD.

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3.2 Stable carbon isotope values of CH₄ during incubation with ¹³C–hydrogen carbonate

Stable carbon isotope values of CH₄ (δ¹³CH₄ values) for Chrysochromulina sp. and P. globosa are presented in Fig. 4a, c. We observed the conversion of ¹³C carbon (provided by ¹³C–hydrogen carbonate) to ¹³CH₄ in cultures of both species, indicated by increasing δ¹³CH₄ values over time. Stable isotope values increased from initial atmospheric (laboratory air) levels of $-\mathrm{48.7}\pm \mathrm{0.3}$ ‰ and $-\mathrm{48.4}\pm \mathrm{0.10}$ ‰ up to $+\mathrm{30.1}\pm \mathrm{10.2}$ ‰ and $+\mathrm{245}\pm \mathrm{16}$ ‰ for Chrysochromulina sp. and P. globosa, respectively, whilst the δ¹³CH₄ values of the control groups (algae without ¹³C–hydrogen carbonate or ¹³C–hydrogen carbonate in medium without culture) did not change over time. The increase in δ¹³CH₄ values in the headspace CH₄ depended on the amount of released CH₄ that was added to the initial (atmospheric) background level. To calculate the δ¹³CH₄ values of the CH₄ source that raised the CH₄ quantity above background level, the Keeling plot method (Keeling, 1958; Pataki et al., 2003) was used (Fig. 4b, d). The calculated δ¹³CH₄ values of the CH₄ source were $+\mathrm{1300}\pm \mathrm{245}$ ‰ (Chrysochromulina sp.) and $+\mathrm{1511}\pm \mathrm{35}$ ‰ (P. globosa) and thus close to the theoretical calculated ¹³C value of the DIC (2014±331 ‰ ) resulting from the addition of ¹³C–hydrogen carbonate. Please note that ¹³C–hydrogen carbonate stable isotope labeling experiments with E. huxleyi were already performed by Lenhart et al. (2016) and were not repeated in this study. This is why δ¹³CH₄ values and the respective Keeling plot of E. huxleyi are not shown in Fig. 4.

Figure 4δ¹³CH₄ values (a, c) and respective Keeling plots (b, d) from Chrysochromulina sp. (a, b) and P. globosa (c, d) after the addition of ${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$. Panels (a, c) show the δ¹³CH₄ values of three investigation groups (“culture + ${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$”, “culture”, and “${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$”), whereas each data point presented is the mean value of three replicated culture experiments with error bars showing SD. Panels (b, d) show the Keeling plots for the treatments “culture + ${\mathrm{H}}^{\mathrm{13}}{\mathrm{CO}}_{\mathrm{3}}^{-}$” from each replicated culture experiment (n₁, n₂, n₁), where f(0) refers to the ¹³C value of the CH₄ source.

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3.3 CH₄ production and production potential

Since the experiment in the section above (isotope measurements) was not designed to obtain POC quotas, we conducted an additional experiment to estimate the CH₄ production rates of the three algal species. From an initial cell density of $\mathrm{22.5}\pm \mathrm{3.1}\times {\mathrm{10}}^{\mathrm{3}}$, $\mathrm{80.9}\pm \mathrm{11.5}\times {\mathrm{10}}^{\mathrm{3}}$, and $\mathrm{29.0}\pm \mathrm{5.5}\times {\mathrm{10}}^{\mathrm{3}}$ cells mL⁻¹ cultures were grown up to $\mathrm{37.0}\pm \mathrm{9.2}\times {\mathrm{10}}^{\mathrm{3}}$, $\mathrm{219}\pm \mathrm{24.1}\times {\mathrm{10}}^{\mathrm{3}}$, and $\mathrm{283}\pm \mathrm{15.6}\times {\mathrm{10}}^{\mathrm{3}}$ cells mL⁻¹ for Chrysochromulina sp., P. globosa, and E. huxleyi, respectively. These cell densities corresponded to the cell densities of the exponential growth phase obtained from the experiment in Sect. 3.1. The POC-normalized daily CH₄ production rate was highest in E. huxleyi, followed by P. globosa and Chrysochromulina sp. However, the cellular or POC-normalized daily production rates of the three algal species were of the same order of magnitude (Table 1). We calculated the CH₄ production potential (CH₄PP), which is the amount of CH₄ produced within a week of growth (Gafar et al., 2018), to translate the cellular production rates (µ × CH₄ cell⁻¹) of each species to the community level. The CH₄–PP was 2 orders of magnitude higher for E. huxleyi than the other two species. This is a consequence of the higher growth rate of E. huxleyi. We furthermore observed the oxygen concentrations during the light and dark periods to ensure oxic conditions. The measured oxygen concentrations were always saturated or supersaturated relative to equilibration with ambient air (Fig. S2).

Table 1Growth rate, cellular POC, CH₄ production rates, and CH₄–PP_(7 d) of Chrysochromulina sp. (n=4), P. globosa (n=4), and E. huxleyi (n=4). Values are the mean of four replicated culture experiments with SD.

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3.4 CH₄ formation from ¹³C-labeled methyl thiol ethers

The three methylated sulfur compounds MSO, DMSO, and DMS were tested for potential CH₄ formation in incubation experiments with E. huxleyi. The treatments were initiated in parallel from a batch culture by inoculating $\mathrm{17.5}\pm \mathrm{2.0}\times {\mathrm{10}}^{\mathrm{3}}$ cells mL⁻¹, and cultures were grown to final cell densities of $\mathrm{1.77}\pm \mathrm{0.08}\times {\mathrm{10}}^{\mathrm{6}}$ cells mL⁻¹ (Fig. 5a). Cell densities and CH₄ formation correlated in all treatments, while no difference in cell growth pattern or CH₄ formation was observed when isotope-labeled methyl thioether and sulfoxides were added to the culture (Fig. 5a, b, c). Differences between treatments were found in δ¹³CH₄ values of headspace CH₄. The initial δ¹³CH₄ value of headspace ($-\mathrm{47.9}\pm \mathrm{0.1}$ ‰, laboratory air) increased slightly over time in untreated cultures (without isotope treatment) to $-\mathrm{46.8}\pm \mathrm{0.3}$ ‰ (Fig. 6b). In contrast, experiments in which ¹³C₂–DMS, ¹³C₂–DMSO, and ¹³C–MSO were applied to cultures of E. huxleyi, δ¹³CH₄ values increased to $-\mathrm{31.0}\pm \mathrm{1.1}$ ‰, $-\mathrm{45.7}\pm \mathrm{0.1}$ ‰, and $+\mathrm{18.3}\pm \mathrm{7.7}$ ‰, respectively, over a time period of 6 d (Fig. 6a, b, c) and differed significantly from control groups (p<0.05). The results unambiguously show that a fraction of the ¹³C-labeled methyl groups of the added substances was converted to ¹³C–CH₄ in cultures of E. huxleyi. Much smaller changes in δ¹³CH₄ values were observed for controls of sterile filtered media to which only ¹³C₂–DMS and ¹³C–MSO were added ($-\mathrm{42.8}\pm \mathrm{1.7}$ ‰ and $-\mathrm{43.9}\pm \mathrm{0.2}$ ‰, respectively; Fig. 6a, c; day 6), whereas δ¹³CH₄ values did not change over time in the seawater controls (no addition of isotopic-labeled compounds) or in the seawater controls treated with ¹³C₂–DMSO (Fig. 6b). Based on the initial amount of ¹³C-labeled substance that was added to the cultures and the total amount of ¹³CH₄ at the end of the incubation period, 9.5±0.2 pmol (¹³C₂–DMS), 3.0±3.2 pmol (¹³C₂–DMSO), and 30.1±3.6 pmol (¹³C–MSO) of 8.5 µmol were converted to CH₄.

Figure 5Cell growth (a), CH₄ production (b), and the relation between the total number of cells and produced CH₄ (c) from E. huxleyi treated with ¹³C₂–DMS, ¹³C₂–DMSO, and ¹³C–MSO or without any treatment. Mean values of three replicated culture experiments are shown, and error bars mark the SD.

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Figure 6δ¹³CH₄ values of headspace CH₄ in cultures of E. huxleyi supplemented with (a) ¹³C₂–DMS, (b) ¹³C₂–DMSO, and (c) ¹³C–MSO. Mean values of three replicated culture experiments are shown, and error bars mark the SD.

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4.1 CH₄ formation from ¹³C-labeled methyl thioethers

4.2 POC-normalized production

For all three algal species significant correlations between CH₄ mass and cell density were found (r²>0.95 for all species; Fig. 3g, h, i), suggesting that CH₄ formation occurred over the entire growth curve. However, since CH₄ production can only be determined in the exponential phase (Langer et al., 2013) we additionally ran dilute batch cultures to determine CH₄ production. All three species displayed similar CH₄ production ranging from 1.9±0.6 to 3.1±0.4 µg CH₄ g⁻¹ POC d⁻¹, with Chrysochromulina sp. and E. huxleyi showing the lowest and highest rates, respectively. The CH₄ production for E. huxleyi was found to be 2-fold higher than rates reported for the same strain and comparable culture conditions by Lenhart et al. (2016) (0.7 µg CH₄ g⁻¹ POC d⁻¹). The lower production reported by Lenhart et al. (2016) may be explained by the fact that CH₄ production was not obtained from exponentially growing cultures. We also compared the cellular CH₄ production rates of E. huxleyi reported by Scranton (1977) with those of our study. Scranton (1977) reported a production rate of $\mathrm{2}\times {\mathrm{10}}^{-\mathrm{10}}$ nmol CH₄ cell⁻¹ h⁻¹. This value is close to the production rate of $\mathrm{1.6}\times {\mathrm{10}}^{-\mathrm{10}}$ nmol CH₄ cell⁻¹ h⁻¹ in our study. Scranton (1977) concluded from observed CH₄ production rates in laboratory experiments that natural populations might be adequate to support the widespread supersaturation of CH₄ observed in the open ocean. However, we suggest that the CH₄ production of various algae might differ substantially under changing environmental conditions, as already shown for terrestrial plants (Abdulmajeed and Qaderi, 2017; Martel and Qaderi, 2017). Moreover, the cellular concentrations of potential precursor compounds such as methylated sulfur compounds might vary greatly between species and cultures. The investigated algal species can reach millimolar intracellular concentrations of DMS and DMSP (Sunda et al., 2002; Liss et al., 1994; Keller, 1989), and even if the conversion rate of methylated sulfur compounds to CH₄ in algal cells might be low, they could be sufficient to explain a substantial fraction of the CH₄ production rates by marine algae.

4.3 Implication for the marine environment and algal blooms

In general, the distribution of chlorophyll has not shown a consistent correlation with CH₄ distributions in field studies. There are studies in which no correlation was observed (e.g., Lamontagne et al., 1975; Foster et al., 2006; Watanabe et al., 1995) or a correlation was found within a few depth profiles (Burke et al., 1983; Brooks et al., 1981). Many field measurements in oxygenated surface waters in marine and limnic environments have shown examples of elevated CH₄ concentrations spatially related to phytoplankton occurrence (e.g., Conrad and Seifer, 1988; Owens et al., 1991; Oudot et al., 2002; Damm et al., 2008; Grossart, et al., 2011; Weller et al., 2013; Zindler et al., 2013; Tang et al., 2014; Bogard et al., 2014; Rakowski et al., 2015). Taken together these studies suggest that phytoplankton is not the sole source of CH₄ in oxygenated surface waters, but importantly, they also suggest that phytoplankton is one of the sources of CH₄. We therefore compared the CH₄ production rates of our cultures with two field studies for the Pacific Ocean (Weller et al., 2013) and the Baltic Sea (Schmale et al., 2018) to evaluate the potential relevance of algal CH₄ production. It was estimated that the gross CH₄ production in a southwest Pacific Ocean mesoscale eddy is 40–58 pmol CH₄ L⁻¹ d⁻¹ (Weller et al., 2013). Using reported phytoplankton cell densities (1.7×10⁸ to 2.9×10⁸ cells L⁻¹, Weller et al., 2013), we calculated a maximal cellular production of 5.5 ag CH₄ cell⁻¹ d⁻¹ for this eddy. The species investigated in this study showed ca. 3–11 times higher cellular production (Table 1). Hence, each of the three haptophyte algae studied here could account for the CH₄ production reported by Weller et al. (2013).

Schmale et al. (2018) reported CH₄ enrichments that were observed during summer in the upper water column of the Gotland Basin, central Baltic Sea. Furthermore, they found that zooplankton is one but not the only CH₄ source in the oxygenated upper waters. While the authors ruled out a major contribution of algae to the observed sub-thermocline CH₄ enrichment because of the low sub-thermocline phytoplankton biomass, they considered primary-production-associated CH₄ formation to be one likely source in the phytoplankton-rich mixed layer. The average phytoplankton carbon biomass of the mixed layer was approximately 600 µg L⁻¹ (averaged from Fig. 9 in Schmale et al., 2018). For the reported average net CH₄ production rate in the mixed layer (95 pmol CH₄ L⁻¹ d⁻¹), we calculated that a production rate of 2.5 µg g⁻¹ POC d⁻¹ is required if the CH₄ is produced by the algal biomass. This rate would be within the range of CH₄ production rates observed in our study. These calculations should be considered as a first rough estimate to assess whether the CH₄ production rates of laboratory-grown cultures can significantly contribute to CH₄ supersaturation associated with phytoplankton. We did not distinguish between species and did not take into account environmental factors or the complexity of microbiological communities.

Judging from cellular production, the species studied here are of similar importance for oceanic CH₄ production in biogeochemical terms. Regarding the highest cellular production, with that of E. huxleyi as 100 %, P. globosa produces 27 % and Chrysochromulina sp. 71 % (Table 1). However, several recent studies have emphasized that production potential (PP), as opposed to cellular production, is a biogeochemically meaningful parameter (Gafar et al., 2018; Marra, 2002; Schlüter et al., 2014; Kottmeier et al., 2016). The concept of production potential goes back at least to the first half of the 20th century (Clarke et al., 1946). Briefly, the production potential of substance X is the amount of X which a phytoplankton community or culture produces in a given time. For details, see Sect. 2 and the references above. Cellular production, by contrast, is the rate of production of X of a single cell, and therefore the cellular production is ill-qualified to express community-level production. We calculated the CH₄–PP (Sect. 2) for our three species, and when the one of E. huxleyi is considered to be 100 %, P. globosa has a CH₄–PP of 0.9 % and Chrysochromulina sp. 0.8 % (Table 1). In terms of CH₄ production in the field, therefore, E. huxleyi outperforms the other two haptophytes by 2 orders of magnitude. It can be concluded that the CH₄–PP under given environmental conditions is species-specific, and therefore community composition will have an influence on algal sea surface water CH₄ production.

It can be hypothesized that changing environmental conditions might drastically affect algal CH₄ production, which has to be taken into account when calculating annual averages. The effect of dominant environmental parameters such as light intensity and temperature on algal CH₄ production will therefore be the subject matter of future studies.