Dark Carbon Fixation Measurements in the East Sea (Sea of Japan)

Abstract

The vertical distribution patterns of daily primary production and dark carbon fixation were investigated at three stations in the East/Japan Sea (hereafter East Sea), a semi-enclosed marginal sea in the northwest Pacific Ocean. Our results displayed consistent vertical patterns of daily primary production at two of the stations, while the third station exhibited a markedly different distribution pattern, highlighting localized variations in production dynamics. In contrast, dark carbon fixations displaying varying vertical patterns among the stations are not specific enough to have much meaning. Water column-integral values showed differences in the contribution of dark carbon fixation to total primary production, ranging from 4.5% to 27.1%. These variations may reflect environmental parameters such as nutrient concentrations. However, our study is limited by the lack of direct data on the microbial community structure and chemoautotrophic activities, which are crucial for a more comprehensive understanding of these patterns. Understanding the environmental drivers of dark carbon fixation is crucial for elucidating carbon cycling dynamics in the East Sea. Notably, dark carbon fixation could contribute up to one-third of primary production in the region as an additional source of newly produced organic matter, highlighting the need for further investigation into this previously overlooked process.

1. Introduction

Despite the deep ocean (depth > 200 m) constituting more than two-thirds of the global oceanic volume [1], our understanding of carbon cycling in this dark environment and the role of microbial communities remains limited [2,3]. Recent studies have demonstrated that dark carbon fixation in various oceanic deep basins as well as in coastal regions plays a significant role in carbon cycling and carbon sequestration [4,5,6,7]. Chemoautotrophs are considered the major contributors to dark carbon fixation [7,8,9]. However, non-autotrophic CO₂ fixation mediated by heterotrophic organisms has also been suggested as a mechanism for dark carbon fixation [10]. While it remains uncertain whether dark carbon fixation is primarily driven by autotrophic or heterotrophic microorganisms [8], recent research underscores the importance of dark carbon fixation in the twilight zone across various oceanic basins and coastal regions [4,5,6,7]. Studies conducted in the deep North Atlantic and the East China Sea have highlighted the significant contribution of dark carbon fixation to the carbon demand of mesopelagic heterotrophic prokaryotes [4,5,6]. In the deep North Atlantic, dark carbon fixation has been found to contribute up to 72% of daily carbon demand for mesopelagic heterotrophic prokaryotes [4,5]. Similarly, in the East China Sea, dark carbon fixation was estimated to contribute approximately 83% to the daily carbon demand of heterotrophic prokaryotes [6]. Extrapolations from these measurements suggest that dark carbon fixation may account for up to approximately 15% of global ocean primary production [7]. In the East China Sea, dark carbon fixation integrated from the 1500 m water depth has been observed to contribute up to approximately 400% of primary production locally, indicating its role as a significant source of newly produced organic matter [6]. Despite exploration in various oceanic basins and coastal regions, the dark carbon fixation in the twilight zone of the ocean (100 to 1000 m water depth) largely remains unassessed in open oceans, highlighting the need for further investigation into this overlooked carbon sink.

The East/Japan Sea (hereafter East Sea) is a semi-enclosed marginal sea in the northwest Pacific Ocean (Figure 1). Known for its high productivity, particularly in the southwestern part encompassing the Ulleung Basin, the sea is influenced by various eddies, subpolar fronts, and coastal upwelling [11,12,13,14]. These dynamic conditions sustain high productivity levels in the East Sea [12,15,16,17]. Recently, continuous field measurements for primary production have been conducted in the East Sea along with satellite observations through various Korean government programs [13,14,18,19]. However, no measurements for dark carbon fixation have been previously attempted in the East Sea to date. Thus, in this study, our primary objective was to measure the dark carbon fixation as an additional source of organic matter in the East Sea. We conducted vertical profiling of dark carbon fixation and primary production across three stations in the East Sea, examining their relationship with environmental parameters such as nutrient concentrations. Our results revealed the significant role of dark carbon fixation in carbon cycling within the twilight zone and the potential impact of environmental drivers on this process.

In this paper, we outline detailed field sampling techniques, analytical methods, and data processing procedures employed to quantify dark carbon fixation and primary production. Then, we present a comprehensive analysis of the vertical distribution and spatial variability of both dark carbon fixation and primary production, alongside their correlations with key environmental parameters, discussing the potential mechanisms driving the observed patterns and their implications for regional carbon dynamics. Finally, we summarize the major findings and propose directions for future research to further elucidate the role of dark carbon fixation in marine ecosystems.

2. Materials and Methods

We investigated the vertical distribution patterns of daily primary production and dark carbon fixation at three stations (103-11, 104-11, and 105-07) in the East Sea (Figure 1), with Table S1 providing detailed information on the sampling times and locations of these stations. Vertical hydrographic measurements, including temperature and salinity, were conducted using an SBE9/11 CTD (Sea-Bird Electronics, Bellevue, WA, USA)/rosette sampler fitted with 8 L-Niskin bottles. During the cruise, water samples for inorganic nutrient analysis were obtained from six light depths (100%, 50%, 30%, 12%, 5%, and 1% penetration of the surface photosynthetically active radiation, PAR) within the euphotic zone, which extends from the water surface down to the depth where light intensity reaches 1% of surface PAR. Additionally, water samples were collected from four deeper water depths (75, 100, 200, and 500) within the aphotic zone, defined as the region below the euphotic zone where light levels fall below 1% of surface PAR, at all productivity measurement stations (Figure 2). Inorganic nutrient samples were filtered onboard immediately after collection using GF/F filters (Whatman, Maidstone, UK; 0.7 μM) to analyze dissolved inorganic nutrient concentrations. These samples were then transferred under frozen conditions (−80 °C) to the laboratory for analysis with an automatic analyzer (Quaatro, Bran + Luebbe, Norderstadt, Germany) at the National Institute of Fisheries Science (NIFS), Korea. Dissolved inorganic nitrogen (NH₄, NO₂, and NO₃), dissolved inorganic phosphorus (DIP), and silica (DSi) were determined in this study.

Similarly, water samples for Chl-a measurements were collected from the same six euphotic zone depths and five deeper aphotic zone depths (75, 100, 200, 500, and 750 or 800 m), following the same light penetration criteria. To measure Chl-a concentration, water samples (0.1–0.4 L) were filtered onboard onto GF/F filters (Whatman, 0.7 μm pore size) at low vacuum pressure (<150 mmHg). The filtered samples were immediately stored in a freezer at −80 °C. Pigment extraction and measurement were performed within a month following the same protocol from Jang et al. (2021) [19].

Water samples for primary production measurements were collected from six light depths (100%, 50%, 30%, 12%, 5%, and 1% PAR) within the euphotic zone. For dark uptake measurements, water samples were collected from eight depths: three within the euphotic zone (100%, 30%, and 1% PAR) and five from deeper water (75, 100, 200, 500, and 750 or 800 m) within the aphotic zone at all productivity stations (Figure 3). Polycarbonate bottles (Nalgene, Rochester, USA; 1 L) used for primary production measurements were covered with LEE light filter screens (LEE Filters, Hampshire, UK) [13] to match the light levels at each depth of collection. Bottles for dark uptake measurements were wrapped in dark LEE filters to ensure complete light exclusion. Samples from the euphotic zone were incubated on-deck in a large on-deck incubator with continuous circulating surface seawater to maintain in-situ temperature, while those from the aphotic zone were incubated in a dark refrigerator (4 °C) to simulate in-situ conditions. After a 24 h incubation, samples were filtered and immediately stored at −80 °C for later analysis. The analytical procedures for determining dark carbon fixation rates followed the same methods used for primary production [19].

3. Results and Discussion

The vertical temperature patterns were similar between stations 103-11 and 104-11, whereas station 105-07 exhibited a different distribution with relatively lower temperatures within the euphotic layers (Figure 2a). Below the euphotic layers, stations 104-11 and 105-07 displayed similar profiles, while station 103-11 demonstrated a substantially different distribution compared to the other two stations. At stations 104-11 and 105-07, the water temperature rapidly decreased from the surface to 200 m depth and then decreased more gradually with depth. In contrast, the vertical salinity distributions were relatively similar among all stations, except at the surface (Figure 2b).

Chl-a concentrations were lower at the surface layers, indicating typical summer conditions in the East Sea [19]. However, substantially higher Chl-a concentrations were observed at 35 m water depth at station 103-11 and 104-11, and at 11 m depth at station 105-07, which is much shallower compared to the other two stations. These high Chl-a concentrations at subsurface depths are consistent with the subsurface Chl-a maximum (SCM) layers commonly observed in the East Sea during the summer period [20]. Daily primary production rates were 0.51–14.36 mg C m⁻³ d⁻¹, 0.56–9.19 mg C m⁻³ d⁻¹, and 0.09–147.7 mg C m⁻³ d⁻¹ at stations 103-11, 104-11, and 105-07, respectively (Figure 3a). The vertical patterns of daily primary production were similar between stations 103-11 and 104-11, while station 105-07 displayed a different distribution (Figure 3). Notably, daily primary production at 100%–30% light depths was substantially higher at station 105-07 compared to the other two stations. This can likely be attributed to several interrelated oceanographic processes specific to this region. One key factor is the presence of a subpolar front, which interfaces the North Korean Cold Current and the East Korean Warm Current. This front is known to create favorable conditions for enhanced primary production by promoting the mixing of nutrient-rich cold waters with warmer, less nutrient-rich waters [21]. Additionally, during the stratified summer season, the inflow of the Tsushima Warm Current (TWC) plays a crucial role. According to Ji et al. (2019) [22], the energy dissipation associated with the strong TWC inflow leads to surface stirring and mixing, resulting in a relatively less stable surface mixed layer in the southern part of the East Sea. This process alleviates nutrient limitation at the surface by bringing nutrients from deeper layers to the surface, further boosting primary production. These factors may create a highly favorable environment for phytoplankton growth at station 105-07, explaining the substantial disparity in primary production levels compared to the other two stations.

Daily primary productions were positively correlated with POC and negatively correlated with NO₂ and DIP (Figure 4). The strong positive correlation between daily primary productions and POC indicates biomass-driven primary production due to their similarly lower carbon uptake rates during the observation period.

Dark carbon fixation rates were 0.023–0.337 mg C m⁻³ d⁻¹, <0.001 to 0.538 mg C m⁻³ d⁻¹, and 0.007–1.155 mg C m⁻³ d⁻¹ at stations 103-11, 104-11, and 105-07, respectively (Figure 3). These values observed in this study fall within the range observed previously in various oceans [6], which display a wide difference among the observed stations. Dark carbon fixations exhibited varying vertical patterns above 500 m water depth among the three stations. Generally, dark carbon fixation rates within the euphotic zone were considerably higher than those within the aphotic zone, except at station 103-11, where the highest fixation rate was observed at 200 m water depth.

For comparison purposes, the averaged values for daily primary productions and dark carbon fixations were presented in Figure 5. Within the euphotic zone, both daily primary production and dark carbon fixation peaked at intermediate depths before decreasing toward deeper levels (Figure 5). In the aphotic zone (below approximately 50 m water depth), dark carbon fixation increased until 200 m water depth before declining toward the deepest measurement depth at around 800 m water depth (Figure 5).

Based on the vertical profiles of daily primary production and dark carbon fixation at each station, we computed water column-integral values within the euphotic zone (from 100% light depth to 1% light depth) using the trapezoidal rule.

Daily integrated primary productions were 533.8 mg C m⁻² d⁻¹ at station 103-11, 330.9 mg C m⁻² d⁻¹ at station 104-11, and 1274.8 mg C m⁻² d⁻¹ at station 105-07 (Figure 6). These values fall within the range observed in the Ulleung Basin, known as a biological hotspot with high primary production and minimal inter-annual variation in the East Sea [11,12,13,14]. Various mechanisms have been suggested to explain this high productivity, including different types of subpolar fronts and frequent eddies and coastal upwelling events [17]. Kwak et al. (2013) [17] also suggested that a strong upward flux of nitrate through a shallower pycnocline and euphotic depth, driven by hydrographic conditions, sustains high production in the Ulleung Basin. Previous studies have reported that primary production in the East Sea is negatively correlated with temperature and positively correlated with Chl-a concentration in the East Sea based on principal component analysis (PCA) results [19]. In this study, the surface temperature at station 105-07 was notably lower compared to the other two stations, although we did not find a direct relationship between primary production and temperature. Additionally, the euphotic water column-integrated Chl-a concentration was substantially higher at station 105-07 compared to the other two stations.

In comparison, daily integrated dark carbon fixation displayed a relatively narrower range compared to daily primary production among the stations (Figure 6). Specifically, daily dark carbon fixations integrated up to 800 m water depth were 95.5 mg C m⁻² d⁻¹, 89.5 mg C m⁻² d⁻¹, and 57.6 mg C m⁻² d⁻¹ at 103-11, 104-11, and 105-07 station, respectively (Figure 6). These rates respectively represent 17.9%, 27.1%, and 4.5% of the daily primary production at the specific stations. Notably, these values are comparable to previously reported ranges in the Subtropical North Atlantic Ocean (5–16%) [5], although they are relatively higher than those documented in the Indian Ocean (2–13%) [7], and markedly lower than those recorded in the Central Mediterranean Sea (52–94%) [23] and the South China Sea (384%) [6].

In this study, the spatial variation in the contribution of dark carbon fixation to primary production is evident. Dark carbon fixation exhibits a strong association with nutrient conditions in this study, which is consistent with previous studies [6]. Furthermore, microbial communities in the dark ocean display diverse mechanisms for assimilating dissolved inorganic carbon as part of their metabolic processes [24,25,26]. Generally, dark carbon fixation is linked with both chemoautotrophs and heterotrophs and their roles in this process exhibit spatial and temporal variations [5,6]. Therefore, the differences in the contribution of dark carbon fixation among stations may reflect variations in environmental parameters such as nutrient concentrations, water column stability, and microbial community structure. Understanding the environmental drivers of dark carbon fixation can provide valuable insights into the factors regulating carbon cycling in the East Sea. In this study, dark carbon fixation rates showed strong positive correlations with water temperature, NH₄ concentration, Chl-a, and POC, while displaying negative correlations with NO₃, DIP, and DSi concentrations (Figure 4). The strong positive correlation between dark carbon fixation rates and POC, which represents microorganism biomass, suggests that dark carbon fixation rates are primarily driven by their existing biomass rather than specific uptake rates. Furthermore, this positive correlation indicates that dark carbon fixation could play an important role in supporting the food web, carbon cycling, and carbon sequestration by providing new organic carbon in the twilight zone of the East Sea [4,5,6,7]. On average, the integrated values from the three measurement stations were 713.2 mg C m⁻² d⁻¹ (±496.9 mg C m⁻² d⁻¹) for daily primary production and 80.9 mg C m⁻² d⁻¹ (±20.4 mg C m⁻² d⁻¹) for dark carbon fixation. This dark carbon fixation accounted for an average of 16.5% (±11.3%) of the daily primary production across all stations in this study. Notably, the relatively high contribution of dark carbon fixation to total primary production at station 104-11 in this study, amounting to as much as one-third of the primary production, underscores the importance of this process as a previously unaccounted source of newly produced organic matter in the East Sea. Actually, the daily primary production in this study is significantly (t-test, p < 0.01; Supplementary Figure S1) higher than that in the East Sea, 2018 (106.3 ± 77.2 mg C m⁻² d⁻¹) [19]. Then, the average dark carbon fixation could correspond to 76.1% of the daily primary production in 2018 if the dark carbon fixations in this study were adopted. However, seasonal and annual variations in dark carbon fixation and its annual contribution to primary production remain uncertain in the East Sea, highlighting the need for further investigation into this previously overlooked organic matter. Further investigation into whether dark carbon fixation in the East Sea is primarily driven by autotrophic or heterotrophic microorganisms is crucial, particularly in terms of the energy balance in the twilight zone. The negative correlations between the dark carbon fixation rate and nutrient concentrations imply nutrient consumption by chemoautotrophs during their dark carbon fixation [7]. In the twilight zone, various chemoautotrophs assimilate dissolved inorganic carbon and use reduced compounds as electron donors to generate energy [7]. Various studies indicate that dark carbon fixation rates are higher in suboxic oxygen minimum zones, serving as niches for diverse chemoautotrophs, largely driven by the activity of ammonium oxidizers and anammox bacteria [27,28,29]. Zhou et al., 2017 [6], suggested that most dark carbon fixation can be attributed to heterotrophs, based on the observed decoupling between low ammonium concentration and high dark carbon fixation in deep waters during their study period. However, in our study, we observed a strong positive correlation between dark carbon fixation and NH₄ concentration, indicating a different dynamic. The specific contributions and dynamics of various chemoautotrophs in the East Sea remain poorly understood. Therefore, further investigation into the microbiome and chemoautotrophic activities in these regions is necessary. Currently, there is no strong evidence suggesting that the net result of dark carbon fixation by heterotrophic bacteria is an increase in energy stored in the water column, as is the case with photosynthesis or chemoautotrophic activity. Understanding these processes will enhance our knowledge of the energy dynamics and carbon cycling in this marine environment.

Based on nearly 30 years of data from the longest available oceanic time series at ALOHA and BATS, Baltar and Herndl (2019) [30] suggest that incorporating dark carbon fixation rates into global oceanic primary production estimates could increase these estimates by 5–22%, highlighting the significance of dark carbon fixation in the total production of organic matter. They emphasize that this incorporation may become even more crucial if the observed trend of increasing dark to total primary production ratios continues. Changes in the contribution of dark carbon fixation to total primary production over time or in response to environmental perturbations may indicate shifts in ecosystem dynamics and function. Monitoring these changes can help assess the resilience of marine ecosystems to environmental stressors and guide management and conservation efforts in the East Sea. Overall, these findings contribute to our understanding of carbon cycling dynamics in the East Sea and underscore the significance of dark carbon fixation as a previously overlooked component of marine ecosystems. This information is crucial for modeling carbon fluxes and predicting ecosystem responses to environmental changes in the region. The data obtained from this study provide valuable insights into the potentially additional source of newly produced organic matter in a dark environment that has not previously been considered in the East Sea ecosystem, offering a basis for further research into the spatial and temporal variabilities of biological processes in this marine environment.

4. Summary

This study offers a comprehensive analysis of primary production and dark carbon fixation in the East Sea, focusing on their vertical distributions, spatial variations, and relationships with environmental parameters. Our findings reveal that dark carbon fixation, traditionally considered negligible in the photic zone, is a significant process in the East Sea, particularly within the twilight zone. The observed positive correlations between dark carbon fixation rates and variables such as NH₄ concentration, Chl-a, and POC suggest that this process is closely linked to microbial biomass and nutrient availability. These correlations suggest that dark carbon fixation is not merely a background process but a critical component of the carbon cycle, potentially supporting the microbial food web and contributing to carbon sequestration in the region.

The study also highlights that the negative relationships between dark carbon fixation rates correlations with NO₃, DIP, and DSi concentrations imply active nutrient consumption by chemoautotrophs during carbon fixation. This nutrient uptake underscores the role of dark carbon fixation in shaping nutrient dynamics in the twilight zone, which could have broader implications for understanding carbon and nutrient cycling in other similar marine environments. Our research contributes to a deeper understanding of marine carbon dynamics, emphasizing the need for further investigation into the role of chemoautotrophic processes in different oceanic regions.