We demonstrated that CH4 production by Emiliania huxleyi is correlated with light-dependent photosynthesis by ruling out possible disturbance of methanogenic archaea and heterotrophic bacteria, and E. huxleyi cultures did not produce CH4 in the dark. Subsequently, we quantified the counteractive impacts of the microalga’s CO2 fixation and subsequent release of CH4, showing that the molar ratio of CH4 released to CO2 fixed for E. huxleyi (CH4 production quotient) ranges between 1.5×10− 5-6.6×10− 5 (Fig. 4d), translating to up to 13% attenuation of E. huxleyi’s contribution to the biological carbon pump.
Photosynthesis-associated CH4 production is likely a result of the Fenton reaction driven by reactive oxygen species (ROS) 43 with methylated intracellular metabolites serving as substrates. In the process of photosynthesis, NADPH, relevant to electron transport, is produced during the light reaction and oxidized during the dark reaction 44. ROS could be produced during the oxidization of NADPH during the dark reaction 45 and Mehler reaction 46 or more simply generated due to the transfer of electrons to oxygen 47 in the light reaction. Therefore, when the light intensity increased above 260 µmol photons m− 2 s− 1 (optimal growth light), it is possible that enhanced accumulation of ROS under high light stress 48, 49, 50 could promote CH4 formation 43. This explains the observed liner correlation of rETRmax and POC-normalized CH4 production rate (Fig. 5c). E. huxleyi cells increase their size under illumination (Fig. 4c), indicating accumulation of organic matter during the light period. Thus, methylated metabolites produced via photosynthesis likely serve as an internal substrate, explaining the positive correlation between CH4 production, C fixation and cell size (Fig. S3 and Fig. S4), the higher POC-normalized CH4 production rate under high light intensities (Fig. 5b), and the increased CH4 production quotient during the light period (Fig. 4d). In E. huxleyi RCC1216, the POC-normalized CH4 production rate was not inhibited at high light levels 27. In contrast in the subarctic strain used in this study, isolated off the coast of Bergen, Norway (~ 61 °N), CH4 production was saturated at 440 µmol photons m− 2 s− 1 (Fig. 5b) somewhat inhibited at higher light intensities (Fig. 2c). It is therefore likely that different E. huxleyi strains, naturally adapted to different light regimes, display varying differences between CH4 production and light.
Calcification in E. huxleyi has been suggested to act as an electron sink51 and is known to increase with enhanced light intensities52. Strain PMLB 92 − 11, used in the present work, did not calcify (Fig. S2), although previous studies showed it calcifies at a rate lower than other strains (3.6 pg C cell− 1 d− 1 52 for PMLB 92 − 11 vs 8.96 pg C cell− 1 d− 1 for RCC1216 53). It is not clear whether the calcification process correlates with the CH4 release in E. huxleyi, however, CH4 production rate in the non-calcifying strain PMLB 92 − 11(Fig. 2B) was higher than that of the calcifying strain RCC1216 under similar light levels 27. As calcification acts as an electron sink51 and since the POC-normalized CH4 production rate is highly dependent on photosynthetic electron transfer (Fig. 5c), the calcification in E. huxleyi likely down-regulates CH4 production, which needs to be experimentally tested.
Emiliania huxleyi occurs in oceans worldwide with the most extensive known temperature range for phytoplankton between 1–31 ℃, and it can form blooms in many oceanic regions 30, 54. The light intensities we used in this study fall within the typical daytime mean light intensities during E. huxleyi blooms which range from 200–800 µmol photons m− 2 s− 1 55. We showed that CH4 production is light-dependent; therefore, higher light intensities in the field likely result in higher CH4 production. In addition to light, other environmental factors, including UV radiation, temperature, pCO2, pH, and salinity may also influence CH4 production by phytoplankton, although little has been documented on these aspects 27, 56.
It is evident that CH4 produced from autotrophs could offset their contribution via CO2 fixation to the marine biological carbon pump (BCP) and consequently enhance global warming (Fig. 6). To provide a quantitative estimate of this offsetting potential for E. huxleyi (Table S1), we utilized a CH4 production quotient of 6.6×10− 5 (Fig. 4d) from our results. Subsequently, we estimated that CH4 production by E. huxleyi could counteract its contribution to the BCP by up to 13% in a strain-specific manner (Table S1) based on the warming potential of CH4 (84 times that of CO2 over 20 years) and an estimated range of 5–30% of fixed carbon being sequestered into the ocean interior 42, 57, 58, 59. A similar methodology has been applied previously to assess the global warming potential (CO2 fixed by angiosperms vs. CH4 released from their sediment) of various coastal ecosystems 60. To our best knowledge, this study is the only one so far to measure both photosynthetic CO2 fixation and CH4 release simultaneously. In the case of the tropical strain RCC 1216, estimates of this attenuation range between 0.13–1.59%, whereas for the subarctic strain PMLB 92 − 11, attenuation could reduce the BCP contribution by 1.69–13.48%. Some studies suggest that only 2–3% of the fixed carbon reaches the ocean sediment 58, in which case the above attenuation values may be underestimated. Although the CH4 production quotients may vary among different phytoplankton species, all phytoplankton species tested so far release CH4 61 with cyanobacteria producing CH4 at rates 10–100 times higher than eukaryotic phytoplankton 28. Consequently, the CH4 released by phytoplankton is likely to exacerbate global warming (Fig. 6), stimulating more blooms, and thus creating both a negative feedback loop 28 as well as potentially resulting in the formation of anoxic dead-zones which may further enhance classical methanogenesis 62.