**Characteristics of the Biochemical Composition and Bioavailability of Phytoplankton-Derived Particulate Organic Matter in the Chukchi Sea, Arctic**

#### **Bo Kyung Kim, Jinyoung Jung, Youngju Lee, Kyoung-Ho Cho, Jong-Ku Gal, Sung-Ho Kang and Sun-Yong Ha \***

Division of Polar Ocean Sciences, Korea Polar Research Institute, 26 Songdomirae-ro, Yeonsu-gu, Incheon 21990, Korea; bkkim@kopri.re.kr (B.K.K.); jinyoungjung@kopri.re.kr (J.J.); yjlee@kopri.re.kr (Y.L.);

kcho@kopri.re.kr (K.-H.C.); jkgal@kopri.re.kr (J.-K.G.); shkang@kopri.re.kr (S.-H.K.)

**\*** Correspondence: syha@kopri.re.kr

Received: 21 July 2020; Accepted: 20 August 2020; Published: 21 August 2020

**Abstract:** Analysis of the biochemical composition (carbohydrates, CHO; proteins, PRT; lipids, LIP) of particulate organic matter (POM, mainly phytoplankton) is used to assess trophic states, and the quantity of food material is generally assessed to determine bioavailability; however, bioavailability is reduced or changed by enzymatic hydrolysis. Here, we investigated the current trophic state and bioavailability of phytoplankton in the Chukchi Sea (including the Chukchi Borderland) during the summer of 2017. Based on a cluster analysis, our 12 stations were divided into three groups: the southern, middle, and northern parts of the Chukchi Sea. A principal component analysis (PCA) revealed that relatively nutrient-rich and high-temperature waters in the southern part of the Chukchi Sea enhanced the microphytoplankton biomass, while picophytoplankton were linked to a high contribution of meltwater derived from sea ice melting in the northern part of the sea. The total PRT accounted for 41.8% (±7.5%) of the POM in the southern part of the sea, and this contribution was higher than those in the middle (26.5 ± 7.5%) and northern (26.5 ± 10.6%) parts, whereas the CHO accounted for more than half of the total POM in the northern parts. As determined by enzymatic hydrolysis, LIP were more rapidly mineralized in the southern part of the Chukchi Sea, whereas CHO were largely used as source of energy for higher trophic levels in the northern part of the Chukchi Sea. Specifically, the bioavailable fraction of POM in the northern part of the Chukchi Sea was higher than it was in the other parts. The findings indicate that increasing meltwater and a low nutrient supply lead to smaller cell sizes of phytoplankton and their taxa (flagellate and green algae) with more CHO and a negative effect on the total concentration of POM. However, in terms of bioavailability (food utilization), which determines the rate at which digested food is used by consumers, potentially available food could have positive effects on ecosystem functioning.

**Keywords:** particulate organic matter; biochemical composition; phytoplankton; Chukchi Sea; Arctic Ocean

#### **1. Introduction**

In terms of bottom-up controls, phytoplankton is key organism that serves as a primary producer and primary food source for organisms at higher trophic levels in the foodwebs of aquatic ecosystems. Climate change enhances the sea ice melting in the Arctic Ocean with increasing concerns about primary production and nutrient cycling. Sea ice loss reduces surface albedo and enhances light penetration, creating irregularities on the timing and the duration of phytoplankton blooms [1,2]. These conditions can create discontinuity between the available food resources and the nutritional demands of higher producers [1], including higher trophic level organisms [3], and thus affect the energy flow of the entire arctic food web.

In natural systems, however, the food value of phytoplankton cannot be adequately described by measuring their biomass (chlorophyll a) and primary production. Hence, measures of the main biochemical classes (proteins, PRT; lipids, LIP; carbohydrates, CHO) of organic compounds have been used by various authors to estimate the quality and quantity of food in organic pools (reviewed by Bhavya et al. [4]). It is assumed that other biochemical components comprise negligible weights [5] and that the three major biochemical constituents (PRT, LIP, and CHO) are easier to digest and assimilate [6–8]. In reality, LIP, PRT, and CHO play roles in the structural components and energy storage of marine organisms [9,10], accounting for up to 90% of the weight in algae [11].

Generally, particulate organic matter (POM) is composed of living and dead organisms and refractory organic matter. For POM that consists of mainly phytoplankton-derived materials, the biochemical composition of POM reflects the physiological state of the phytoplankton in response to environmental conditions and phytoplankton energy value [4,12–14]. For example, PRT synthesis is generally promoted in productive areas or the exponential growth phase of phytoplankton [13,15,16], while the biosynthesis of non-nitrogenous storage compounds, such as CHO and LIP, is enhanced under high light intensity [17], low temperatures [18] and low nitrogen conditions [19,20]. LIP contain more calories than PRT and CHO [21]. In addition, the labile fractions of POM are characterized by the activities of enzymes, and their degradation provides insight into how POM is bioavailable to consumer organisms [22–24]. Therefore, changes in the biochemical composition and hydrolysable fractions of phytoplankton-derived POM can be useful for determining the physiological and nutritional conditions of phytoplankton.

Our study area is the Chukchi Sea (including the Chukchi Borderland), which contains pathways of water from the Pacific Ocean that flow poleward through the narrow Bering Strait to the Arctic Ocean [25] and transfer freshwater, heat, and nutrients from the northern Bering Sea (Yang and Bai [26] and reference therein). The southern Chukchi Sea is one of the most productive areas globally (up to 4.7 g C m−<sup>2</sup> day<sup>−</sup>1; Korsak [27]), and has an especially high benthic productivity and biodiversity [28,29] because of the nutrients supplied by the inflow of water from the Pacific Ocean. A few studies have estimated the quantity and biochemical composition of POM in the Arctic Ocean [30–33]. However, bioavailable POM food resources created through enzymatic hydrolysis have not been investigated. Hence, the purpose of this study was (i) to investigate the spatial distribution and influence of physical (e.g., salinity, temperature, and meltwater) and chemical (major inorganic nutrients) properties on the biochemical composition of POM and (ii) to estimate potentially bioavailable food for higher trophic levels in the Arctic marine ecosystem using the labile fraction of POM obtained by enzymatic hydrolysis.

#### **2. Materials and Methods**

#### *2.1. Field Sampling and Measurements of the Environmental Variables*

This study was carried out at 12 stations in the Chukchi Sea onboard the R/V *Araon* icebreaker from 7 to 24 August 2017 (Figure 1A). The potential temperature, salinity, and photosynthetically active radiation (PAR) from the surface to a 100 m depth were measured by a rosette-mounted Sea-Bird conductivity-temperature-depth (CTD) system—1% PAR at the surface light level was defined as the euphotic layer [34] by a Secchi disc using the vertical attenuation coefficient (Kd = 1.7/secchi depth). The meltwater percentage (MW; %) was calculated from the salinity at each sampled depth (*Smeas*) and the greatest depth (either the bottom depth or 100 m in this study; *Sdeep*), assuming an average sea ice salinity of 6 [35,36] since the mean salinity at a melt pond in the western Arctic Ocean was 5.9 [37]:

$$\text{MW}(\%) = \left\{ 1 - \left[ \frac{(\mathcal{S}\_{\text{meas}} - \theta)}{(\mathcal{S}\_{\text{dep}} - \theta)} \right] \right\} \times 100 \tag{1}$$

**Figure 1.** (**A**) Location of sampling area in the Chukchi Sea, August 2017. Sea ice extent for the month of August in 2017 (red line) was obtained from the National Snow and Ice Data Center (NSIDC, Fetterer et al. [38]). (**B**) Temperature-salinity diagram from surface to 100 m depth (labeled with station numbers in bold black at the surface); (**C**) cluster analysis of the surface potential temperature and salinity data allowed identification of 3 types of the regions in the Chukchi Sea, (**D**) MW (%) distributions at surface water in the Chukchi Sea during a summer cruise in August 2017. (Ocean Data View (ODV) version 5.1.0) (AWI, Bremerhaven, Germany, Schlitzer, R.).

The water samples used to determine the dissolved inorganic nutrients, chlorophyll a (chl-a), photosynthetic pigments, and POM (from carbon isotope samples at the surface), were obtained from the surface to the euphotic layer (2–5 depths) using a CTD/rosette sampler with 10-L Niskin bottles (Ocean Test Equipment Inc., Fort Lauderdale, FL, USA). The dissolved inorganic nutrients (nitrate + nitrite, ammonium, silicate, and phosphates) were analyzed onboard using a 4-channel QuAAtro Auto Analyzer (Seal Analytical, Norderstedt, Germany). The concentrations of the nutrients were measured using standard colorimetric methods, and the reference material for nutrients in seawater (Lot. No. "BV", Kanso Technos Co., Ltd., Osaka, Japan) were used in addition to standards for every batch of runs to ensure accurate and comparable measurements during the cruise.

After prefiltration through a 200 μm mesh net to remove large zooplankton, the water samples used to determine total chlorophyll a (chl-a) and accessory pigments were filtered onto GF/F filters (precombusted at 450 ◦C for 4 h; Whatman, Port Washington, NY, USA) immediately after collection. The filters were stored at −80 ◦C until the analyses were performed. Size-fractionated chl-a was determined from samples passed sequentially through 20 μm (>20 μm; microphytoplankton), 2 μm (2–20 μm; nanophytoplankton) and Whatman GF/F filters (0.7–2 μm; picophytoplankton). All the chl-a concentrations were calculated by the methods described by Parsons et al. [39] using a Trilogy fluorometer (Turner Designs, San Jose, CA, USA). The phytoplankton community composition was determined with photosynthetic pigments measured by high performance liquid chromatography (HPLC; Agilent 1260 Infinity LC, Agilent Technologies Inc, Santa Clara, CA, USA)-CHEMTAX analyses. For the stable carbon isotope composition of POM, seawater was filtered onto precombusted (450 ◦C for 4 h) 25 mm GF/F (Whatman, 0.7 μm pore) filters. The filters were immediately stored at −80 ◦C until further analysis. Stable carbon isotope composition was determined using isotope ratio mass

spectrometry (IRMS; visION, Elementar UK, Manchester, UK) in the stable isotope laboratory at the University of Hanyang, Ansan, Korea, after HCl fuming overnight to remove the carbonate. The carbon isotope fractionation, δ13C (‰), was calculated using the following equation:

$$
\delta^{13}\mathcal{C}\left(\%\right) = \left[\frac{\left(\frac{^{13}\text{C}}{^{12}\text{C}}\right)\_{sample}}{\left(\frac{^{13}\text{C}}{^{12}\text{C}}\right)\_{standard}} - 1\right] \times 1000\tag{2}
$$

where the standard for δ13C is IAEA-CH-3 [40].

#### *2.2. Biochemical Composition and Enzyme-Hydrolysable Experiments Related to the POM*

The water samples (*n* = 51) used to assess the biochemical composition of the POM were obtained from two to six different depths at each site within the euphotic layer, and for each macromolecule (PRT, CHO, and LIP), 0.5–1 L of the seawater sample went through a precombusted 25 mm GF/F filter (at 450 ◦C for 4 h). The filter was immediately stored at −80 ◦C until analysis. Analysis of the PRT and CHO was performed using the methods described by Lowry et al. [41] and Dubois et al. [42], respectively. For the total PRT extraction, we added deionized water to a filter and, alkaline copper solution and Folin-Ciocalteu phenol regent to the sample tube. The CHO content was measured by a phenol–sulfuric acid reaction. The LIP were extracted from the filter with chloroform and methanol (1:2; *v*:*v*) [43], followed by sulfuric acid at 200 ◦C [44]. The absorbance of the samples, blanks and standards was determined at wavelengths of 750, 490, and 360 nm for the PRT, CHO, and LIP, respectively, using a spectrophotometer (Hitachi, Tokyo, Japan). The concentrations of the macromolecules were determined by comparison to the standard curve created with blank filters (procedural control filters, Whatman GF/F filter). The standard solutions for the PRT, CHO, and LIP were used a protein standard (2 mg mL<sup>−</sup>1, Albumin from bovine serum, CAS No. 9048-46-8, Sigma-Aldrich, St. Louis, MO, USA), glucose standard (1 mg mL<sup>−</sup>1, CAS No. 50-99-7, Sigma-Aldrich), and tripalmitin (50 mg in 100 mL chloroform, CAS No. 555-44-2, Sigma-Aldrich), respectively.

For enzyme-hydrolysable experiments, sampling was conducted by randomly selected samples of 35. Three enzymes were used in the enzyme-hydrolysable experiments: proteinase K derived from *Tritirachium album* (CAS No. 39450-01-6), β-glucosidase from almonds (CAS No. 9001-22-3), and lipase from *Rhizopus oryzae*(CAS No. 9001-62-1) (Sigma-Aldrich). Since these enzymes have hydrolytic activities similar to those of natural marine organisms, including autotrophs and heterotrophs [45], proteinase K, β-glucosidase, and lipase were chosen for the hydrolysis of PRT, CHO, and LIP, respectively [22,24,46–49]. The sample filters and blank filters were placed in enzyme solutions (100 mg L−<sup>1</sup> in 0.1 M sodium phosphate buffer) to react for 2 h (proteinase K),2h(β-glucosidase), and 30 min (lipase). After hydrolysis, each filter was rinsed with buffer and deionized water and the concentrations of PRT, CHO, and LIP were determined as previously described. The concentration of the hydrolyzed biochemical fractions was calculated by the difference between before and after treatment of enzyme for each fraction.

#### *2.3. Statistical Analysis*

The statistical analyses (*t*-test, Pearson's correlation, and principal component analysis (PCA)) were performed with SPSS statistical software (version 12.0; SPSS Inc., Chicago, IL, USA) and R software (version 3.4). Cluster analysis was performed by using a hierarchical clustering algorithm with Ward's method to identify the groups of sampling stations. A t-test evaluates whether the means of two independent groups are significantly different from each other. The relationships between the depth, nutrients, chl-a, and biochemical components were tested using Pearson's correlation. PCA was used to evaluate the differences in the biochemical components and identify the significance of the environmental factors (e.g., salinity, temperature, density, phytoplankton size, MW (%), and major inorganic nutrient concentrations) among the groups and at each station. The average value of each

variable within the euphotic layer was used for PCA (Table S1). We adopted the principle that an eigenvalue >1.0 can be used to determine the number of principal components.

#### **3. Results**

#### *3.1. Physicochemical and Biological Characteristics During the Sampling Period*

The potential temperature and salinity diagram reveal different hydrodynamic conditions during the sampling periods (Figure 1B). Based on Gong and Pickart's work [50], the summertime water mass properties of Stations 2, 3, and 6 were mainly composed of Alaskan coastal water (potential temperature (T) ≥ 3 ◦C and salinity (S) ≥ 0). Chukchi summer water (−1 ◦C < T < 3 ◦C and 30 < S < 33.6), and Pacific winter water (T < −1 ◦C and S > 31.5) were found in the other stations (Figure 1B). Since sea-surface temperature and salinity are strongly affected by sea ice and related meltwaters, brine rejection, continental runoff, and the heat flux in the Arctic Ocean [51], we assumed that the temperature and salinity at the surface were representative of the ambient water conditions. As a result, the cluster analysis of the surface potential temperature and salinity data allowed the identification of the three types of regions in the Chukchi Sea: cluster 1 (hereafter, the southern part; Stations 2, 3, and 6) was located at a latitude of approximately 66–70 ◦N; cluster 2 (hereafter, the northern part; Stations 15, 17, 20, 23, 31, and 33) was located at a latitude of 74.7–78 ◦N and included the Chukchi Borderland; cluster 3 (hereafter, the middle part; Stations 12, 14, and 35) was located between two areas of the Chukchi Sea (the southern and northern parts) (Figure 1C and Table 1).

**Table 1.** Description of sampling stations in the Chukchi Sea, 2017. Euphotic depth is the depth of the 1% light level. All samples were collected from two to six different depths at each site within euphotic depth.


The potential temperature at the surface was approximately 8 ◦C in the southern part, while in the northern part, it fell further, to below 0 ◦C, ranging from −1.6 to −0.6 ◦C. The salinity at the surface in the southern Chukchi Sea (shallow continental shelf) was above 31.9, with the maximum value (32.5) recorded at Station 3, while the salinity in the northern part was below 30.3, with the minimum value (27.2) recorded at Station 33 (Figure 1B). Overall, the northern part of the Chukchi Sea is characterized by a relatively cold temperature and low salinity, while we found higher temperatures and salinities in the surface water in the southern part (Figure 1B). Hydrodynamic characteristics are subject to the considerable influence of sea ice. The meltwater percentage (MW; %) in the euphotic layer of the study area ranged from 0 to 21.1%, with large spatial variations. Such a situation is specific to the northern part, with an average MW (%) ranging from 4.6 to 18.4 and a mean of 12.8% (SD = ± 3.6). Based on the sea ice extent, the MW (%) accounted for <15% of the surface water at the inner stations (Stations 17, 20, and 23) while at the outer stations (Stations 15, 31, 33, and 35), the MW accounted for more than 15% of the surface water (*t*-test, *p* < 0.05; Figure 1D). This result suggests that the salinity was greatly influenced by the regional melting of sea ice.

The concentrations of the dissolved inorganic nitrate + nitrite + ammonium (DIN), silicate (DSi) and phosphate (DIP) are shown in Figure 2. In the sampling period, the DSi and DIP concentrations

from the surface to the euphotic layer ranged from 1.9 to 29.0 μM and 0.2 to 1.7 μM with means of 7.8 (SD = ±6.4 μM) and 0.8 (SD = ±0.3 μM), respectively (Figure 2A). The concentration of DIN, which was generally depleted (<1 μM) at the surface layer throughout our study area, was in the range of 0–13.2 μM, with an average of 1.5 μM (SD = ±3.0 μM) (Figure 2B). All the mean nutrient concentrations decreased from the southern to the northern parts of this region.

**Figure 2.** The stoichiometric (**A**) dissolved inorganic silicate (DSi) and dissolved inorganic (DIP) and (**B**) dissolved inorganic nitrate + nitrite + ammonium (DIN) and dissolved inorganic phosphate (DIP) from the surface to euphotic layer at sampling stations.

The average total chl-a concentration of phytoplankton from the surface to the euphotic depth ranged from 0.04 to 5.3 <sup>μ</sup>g L−<sup>1</sup> with a mean of 0.8 <sup>μ</sup>g L−<sup>1</sup> (SD <sup>=</sup> <sup>±</sup>1.3 <sup>μ</sup>g L<sup>−</sup>1) at all stations, decreasing northward (Figure 3A). The phytoplankton community was dominated by picophytoplankton, which accounted for 46.2% (SD = ±15.0%) of the total chl-a concentration, followed by nanophytoplankton (mean ± SD = 27.8 ± 10.0%) and microphytoplankton (mean ± SD = 26.0 ± 17.3%) in the northern part of the Chukchi Sea (Figure 3B). In the southern and middle parts, microphytoplankton were dominant (mean ± SD = 80.1 ± 5.9% for the southern part and mean ± SD = 35.0 ± 34.5% for the middle part) within the euphotic layer (Figure 3B).

**Figure 3.** Average chlorophyll a (chl-a; μg L<sup>−</sup>1) concentration of phytoplankton within euphotic layer (**A**) in the study stations of the Chukchi Sea. (**B**) Relative chl-a (%) for size fraction of phytoplankton (0.7–2 μm, 2–20 μm and >20 μm; i.e., pico-, nano- and micro-phytoplankton, respectively). Data were sorted by station depths and divided into southern, middle, and northern. Error bar indicated standard deviation (*n* = 2–5).

#### *3.2. Biochemical Composition (PRT, LIP, and CHO) of POM*

The LIP and PRT concentrations in the POM ranged from 5.4 to 169.1 μg L−<sup>1</sup> (mean <sup>±</sup> SD = 32.4 <sup>±</sup> 32.8 <sup>μ</sup>g L−1) and 9.7 to 573.8 <sup>μ</sup>g L−<sup>1</sup> (mean <sup>±</sup> SD = 61.6 <sup>±</sup> 101.0 <sup>μ</sup>g L−1) within the euphotic layer, respectively (Figure 4). The CHO concentration ranged from 29.9 to 406.4 <sup>μ</sup>g L−<sup>1</sup> with a mean of 86.6 <sup>μ</sup>g L−<sup>1</sup> (SD <sup>=</sup> <sup>±</sup> 67.9 <sup>μ</sup>g L<sup>−</sup>1) (Figure 4A). The vertical distribution of the LIP, PRT, and CHO concentrations did not show a specific trend (*p* > 0.05) but was characterized by significant spatial changes (Figure 4A). In the southern part of the Chukchi Sea, the average PRT concentration (198.8 μg L−1) was approximately 5.1 and 6.9 times higher than those of the stations in the middle (39.2 μg L−1) and northern parts (28.7 μg L−1) (*t*-test, *p* < 0.05), respectively. Similarly, at the southern stations, the average LIP (80.3 μg L<sup>−</sup>1) concentration was approximately 3.4 and 3.8 times higher than the average LIP concentrations in the middle and northern parts, respectively, while the average CHO (162.7 μg L−1) concentration was approximately 1.8 and 2.7 times higher than the average CHO concentrations in the middle and northern parts (Figure 4A).

**Figure 4.** Average total (*n* = 51) and hydrolysable (*n* = 35) (**A**) lipids, (**B**) proteins, and (**C**) carbohydrates concentration of particulate organic matter (POM) within euphotic layer. Error bar indicated standard deviation (*n* = 2–6).

The food material (FM) is represented by the sum of PRT, CHO, and LIP concentrations in POM ([13] and reference therein) and concentration of each biochemical constituent (PRT, CHO, and LIP) covaried with the FM, as expected. The FM ranged from 53.7 to 1074.2 μg L−1, with an average of 180.5 <sup>μ</sup>g L−<sup>1</sup> (SD <sup>=</sup> <sup>±</sup> 195.3 <sup>μ</sup>g L<sup>−</sup>1), and the FM decreased northward in this study.

#### *3.3. Hydrolysable Compounds of POM*

The concentrations of hydrolysable compounds (hydrolysable PRT, HPRT; hydrolysable LIP, HLIP; hydrolysable PRT, HPRT) in the POM were different among the groups (Figure 4A). In the southern part, the concentrations of HPRT ranged from 10.6 to 306.0 <sup>μ</sup>g L−<sup>1</sup> (mean <sup>±</sup> SD <sup>=</sup> 93.4 <sup>±</sup> 129.4 <sup>μ</sup>g L<sup>−</sup>1), and the concentrations of HLIP ranged from 33.4 to 132.2 <sup>μ</sup>g L−<sup>1</sup> (mean <sup>±</sup> SD = 64.5 <sup>±</sup> 36.7 <sup>μ</sup>g L−1) (Figure 4A). The HCHO ranged from 8.3 to 113.8 <sup>μ</sup>g L<sup>−</sup>1, with a mean of 56.5 <sup>±</sup> 36.3 <sup>μ</sup>g L−<sup>1</sup> (Figure 4A). HLIP represented 79.0% of the total LIP value, followed by HCHO, which represented 34.2% of the total CHO value and HPRT, which represented 31.0% of the total PRT value. In comparison, the HPRT concentrations in the middle and northern parts ranged from 4.3 to 59.3 μg L−<sup>1</sup> (mean±SD <sup>=</sup>25.5±20.4 <sup>μ</sup>g L<sup>−</sup>1) and from 0.1 to 44.2 <sup>μ</sup>g L−<sup>1</sup> (mean±SD <sup>=</sup>20.6±13.1 <sup>μ</sup>g L<sup>−</sup>1), respectively (Figure 4A). In the middle and northern parts, the HLIP concentrations ranged from 3.2 to 22.1 μg L−<sup>1</sup> (mean <sup>±</sup> SD = 8.6 <sup>±</sup> 7.0 <sup>μ</sup>g L−1) and 0.9 to 23.9 <sup>μ</sup>g L−<sup>1</sup> (mean <sup>±</sup> SD = 10.6 <sup>±</sup> 6.2 <sup>μ</sup>g L−1), respectively, and the HCHO concentrations ranged from 19.7 to 124.6 <sup>μ</sup>g L−<sup>1</sup> (mean <sup>±</sup> SD = 63.7 <sup>±</sup> 35.6 <sup>μ</sup>g L−1) and 28.0 to 114.3 <sup>μ</sup>g L−<sup>1</sup> (mean <sup>±</sup> SD <sup>=</sup> 52.8 <sup>±</sup> 24.7 <sup>μ</sup>g L<sup>−</sup>1), respectively. Consistent with this observation, HCHO accounted for 72.1% (middle part) and 89.3% (northern part) of the overall value, which was more than the contributions of HLIP or HPRT (Figure 4). The concentrations of the hydrolysable compounds except for HCHO were higher in the southern part than in the middle or northern parts. Overall, the average concentrations of HLIP, HPRT, and HCHO at all the stations were 22.5 μg L−<sup>1</sup> (SD <sup>=</sup> <sup>±</sup> 29.1 <sup>μ</sup>g L<sup>−</sup>1), 38.2 <sup>μ</sup>g L−<sup>1</sup> (SD <sup>=</sup> <sup>±</sup> 67.5 <sup>μ</sup>g L<sup>−</sup>1), and 55.9 <sup>μ</sup>g L−<sup>1</sup> (SD <sup>=</sup> <sup>±</sup> 29.2 <sup>μ</sup>g L<sup>−</sup>1), respectively. The contributions of the hydrolysable components of POM to the total value were 56.1 ± 25.5% for LIP, 54.0 ± 31.3% for PRT, and 73.2 ± 26.6% for CHO.

In this study, the bioavailable fraction of POM (BFM, as the sum of HPRT, HLIP, and HCHO concentrations) can be considered the actual nutritional constituents and/or potentially available food for consumers that are able to be digested. In FM, the remaining values (excluding BFM) are expressed as a non-bioavailable form (N-BFM). In our study, similar to FM, the BFM concentration was much higher (mean <sup>±</sup> SD <sup>=</sup> 214.4 <sup>±</sup> 194.5 <sup>μ</sup>g L<sup>−</sup>1) in the southern than in the middle (mean <sup>±</sup> SD <sup>=</sup> 97.8 <sup>±</sup> 52.6 <sup>μ</sup>g L<sup>−</sup>1) and northern (mean <sup>±</sup> SD = 84.1 <sup>±</sup> 36.7 <sup>μ</sup>g L<sup>−</sup>1) parts. Similarly, the average N-BFM (256.0 <sup>μ</sup>g L<sup>−</sup>1) at the southern stations was approximately 4.3 and 8.4 times greater than that at the middle and northern stations, respectively. These results show that the positive effect of a large amount of FM is influenced by the quantity of BFM and that the majority of POM is not actually composed of bioavailable PRT, CHO, and LIP.

#### *3.4. Multivariate Statistical Analysis*

PCA was performed to determine the similarity among the environmental variables between stations. The PCA ordination of the sampled stations according to the measured environmental parameters is plotted in Figure 5 with eigenvalues presented in Table 2. The first two principal components (PC1 and PC2) accounted for 60.2% and 25.0% of the total variability, respectively. The temperature, salinity, density, and microphytoplankton (%) (eigenvectors of 0.931, 0.882, 0.785, and 0.891, respectively) were differentiated from the MW (%) and picophytoplankton (%) (eigenvectors of −0.907 and −0.880, respectively) by PC1, while PC2 was positively correlated with the major inorganic nutrient variables (eigenvectors ≥ 0.8). The analysis indicated general latitudinal groupings of stations in terms of their physical, chemical, and biological characteristics. The southern part was distinguished from the northern part by relatively high nutrient concentrations, temperatures, salinity, densities and relative contribution (%) of microphytoplankton. The northern part was characterized by a high MW (%) and relative contribution (%) of picophytoplankton values. The diagonal trajectory of

the stations in the middle part within the ordination indicated that they represented a combination of PC1 and PC2.

**Figure 5.** The principal component analysis performed from sampling stations. The environmental variables taken into consideration are temperature, salinity, density, nutrients (DIP; dissolved inorganic phosphate, DSi; dissolved inorganic silicate, DIN; dissolved inorganic nitrogen, nitrite + nitrate + ammonium), MW (meltwater, %), relative contribution of phytoplankton size classes (microphytoplankton, picophytoplankton, and nanophytoplankton), and relative contribution of biochemical pools (carbohydrates, CHO; proteins, PRT; lipids, LIP). Rotated eigenvectors for each parameter are indicated by arrows.



#### **4. Discussion**

#### *4.1. Origin and Quantity of POM*

Our POM samples were collected by filtration and consisted of a variety of complex mixtures of compounds. Many studies have reported that chemical markers, such as chl-a, natural abundance of the stable isotopes of carbon (δ13C), and the C:N ratio, can be used to distinguish phytoplankton, as live components, from POM [9,12,30,52]. In our samples, the respective concentrations of PRT, LIP, and CHO in the POM had a linear relationship to the chl-a concentration (r = 0.689, 0.714, 0.724, *n* = 47, *p* < 0.01 for PRT, LIP, and CHO, respectively), which was used as a proxy for phytoplankton biomass. The <sup>δ</sup>13C value of the POM ranged from <sup>−</sup>28.5 to <sup>−</sup>22.1‰ (mean <sup>±</sup> SD = <sup>−</sup>26.2 <sup>±</sup> 2.8‰); our values were within the range previously reported in phytoplankton samples (Kim et al. [30]; Ahn et al. [33]; reference therein). Kim et al. [30] and Ahn et al. [33] reported that POM is mainly derived from phytoplankton during summer in the Arctic Ocean based on δ13C and the C:N ratio. In addition, during the sampling period, the low DIN concentration (<1 μM) and salinity distribution in the surface water suggest that the POM was greatly influenced by regional sea ice rather than a riverine source (terrigenous). Therefore, in our study, the POM was considered to have mainly come from a marine phytoplankton origin.

A field study has shown a large spatial variability in the concentration of FM in the Chukchi Sea. Kim et al. [30] reported that FM concentrations ranged from 80.5 to 698.8 μg L−1, with an average of 294.4 <sup>μ</sup>g L−<sup>1</sup> (SD = <sup>±</sup> 228.1 <sup>μ</sup>g L−1) in the euphotic layer of the Chukchi Sea, a value that was approximately 1.6 times higher than that in this study (mean <sup>±</sup> SD <sup>=</sup> 180.5 <sup>±</sup> 195.3 <sup>μ</sup>g L<sup>−</sup>1). Yun et al. [31] also found concentrations of FM similar to results from a previous study in this area in this area that similar to results from a previous study in this area, ranged from 89.7 to 362.4 μg L−<sup>1</sup> with an average of 156.4 μg L−<sup>1</sup> in the euphotic layer during summer. These variations are thought to result from spatial and temporal variations in the biomass, composition, and productivity of phytoplankton which are common in the Arctic Ocean [53].

Early studies indicated that primary production is higher in the southern Chukchi Sea than in the northern Chukchi Sea, which is consistent with chl-a abundance [27,54–57]. Based on 13C uptake in the southern Chukchi Sea, Lee et al. [54] estimated a daily production of 0.6 g C m−<sup>2</sup> day−<sup>1</sup> (0.1 to 1.5 g C m−<sup>2</sup> day<sup>−</sup>1). In comparison, the estimated averages of the daily primary production rates for the southern Chukchi Sea are 1.6 g C m−<sup>2</sup> day−<sup>1</sup> and 1.7 g C m−<sup>2</sup> day−<sup>1</sup> from Zeeman [58] and Korsak [27], respectively. The mean daily production in the northern Chukchi Sea measured by Yun et al. [56] was somewhat lower (mean <sup>±</sup> SD <sup>=</sup> 0.14 <sup>±</sup> 0.10 g C m−<sup>2</sup> day<sup>−</sup>1) than the rate (0.66 <sup>±</sup> 0.62 g C m−<sup>2</sup> day<sup>−</sup>1) in the southern region, which is consistent with the findings from Lee et al. [54] (0.16 <sup>±</sup> 0.16 g C m−<sup>2</sup> day<sup>−</sup>1) and Lee et al. [55] (mean <sup>±</sup> SD = 0.18 <sup>±</sup> 0.07 g C m−<sup>2</sup> day<sup>−</sup>1). Similarly, the mean chl-a concentration (2.0 μg L−1) in the southern part of the Chukchi Sea during the summer of 2017 was approximately one order of magnitude higher than the average value (0.2 μg L−1) in the northern part (Figure 3). These results suggest that the regional differences in quantitative POM may have resulted from the different levels of phytoplankton biomass in the Chukchi Sea.

#### *4.2. Biochemical Composition in Relation to Environmental Parameters*

Overall, CHO accounted for 53.3% of the POM for all the survey stations, followed by PRT (29.2%) and LIP (17.5%) (Figure 6A), which led to a low PRT:CHO ratio (0.6). Consistent with this observation, the DIN:DIP (mean ± SD = 1.3 ± 2.0) molar ratio within the euphotic layer was also low compared with the N:P Redfield ratio of 16 [59], indicating substantial nitrogen limitation in this region (Figure 2B). However, interestingly, the PCA revealed that there were significant differences in the compounds among the groups (Figure 5). More specifically, the biochemical composition of the POM was dominated by PRT (41.8%); in the southern part, there was PRT:CHO ratio of 1.2 despite a low DIN:DIP ratio (mean ± SD = 2.7 ± 3.0), while a CHO-dominant (>50%) system was found in the northern Chukchi Sea with a PRT:CHO ratio of 0.5. In general, the PRT fraction was greater than the CHO and LIP

fractions under sufficient nitrogen conditions and growth stages of phytoplankton, which could lead to a high (>1) PRT:CHO ratio [13,15,16]. Fogg and Thake [60] and Hu [61] reported that as prolonged stressful conditions (such as nitrogen limitation) occur, metabolic changes in synthesizing enzyme systems can convert CHO into LIP synthesis. Thus, our results suggest that at least in the southern region, nitrogen limitation was not severe enough to limit phytoplankton growth. The northern part has not been exposed to nitrogen stress for a long time.

**Figure 6.** Spatial distribution of (**A**) specific biochemical compositions (PRT, CHO, and LIP) and (**B**) percentage non-bioavailable (N-BFM; non-hydrolysable) and bioavailable (BFM; hydrolysable, sum of hydrolysable PRT (HPRT), hydrolysable CHO (HCHO), and hydrolysable LIP (HLIP) concentration) fractions in each group (southern, middle, and northern part) and all samples (Chukchi Sea). Composition of the hydrolysable pool was deduced by subtraction of the non-hydrolysable pools from those of the total POM.

In addition, the results of the biplots (Figure 5) based on PCA revealed that microphytoplankton were influenced by relatively nutrient-replete conditions and had maximum chl-a and PRT values, while the picophytoplankton were more sensitive to nutrient deficiency and the MW (%), and were

characterized by a high CHO contribution. These conditions were situated between two distinct areas (the southern and the northern parts) (Figure 5). Similar phenomena have been described by Jin et al. [62] and Li et al. [63] in relation to dominant species, nutrient depletion, and ice cover conditions in the western Arctic Ocean. Li et al. [63] suggested that small cells (<2 μm) thrive as a result of low nitrate availability and a strong stratification since pico-sized cells have a large surface area to volume ratio compared to that of larger cells, which allows effective nutrient and photon acquisition. CHEMTAX pigment analysis revealed that changes in temperature (caused by the timing of sea ice retreat) influence phytoplankton community structure [64]. Thus, it seems that the variation in biochemical compounds discovered among the two different groups (i.e., the southern and northern groups) could be the result of environmentally (such as the level of nutrients and freshwater) induced differences in the size classes and communities of phytoplankton in the Chukchi Sea.

Generally, the analysis of photosynthetic marker pigments (e.g., fucoxanthin, diadinoxanthin and diatoxanthin for diatoms, zeaxanthin for cyanobacteria, chlorophyll b and prasinoxanthin for green algae, 19 butanoyloxy fucoxanthin and 19 hexanoyloxy fucoxanthin for flagellates) can be useful biomarkers for phytoplankton biomass and species [65]. In our study, thirteen pigments except chl-a were identified through the (HPLC)-CHEMTAX analyses (Figure S1). As shown by the abundance of specific phytoplankton groups based on their corresponding biomarker pigments, the southern part was dominated by diatoms (88%), whereas pigments associated with flagellates and green algae made up approximately 44% of the total accessory pigment concentration and diatoms (53%) were observed in the northern Chukchi Sea (Figure S1). Several studies of phytoplankton have documented that species-specific characteristics, such as the cell wall structure and functional characteristics, play a significant role in the variation in biochemical components of phytoplankton [10,33,66–69]. Haug et al. [66] found that in diatoms, the concentration of PRT was generally much higher than that of CHO and LIP, whereas dinoflagellates have abundant CHO within their cell walls. Yun et al. [69] also reported that there were significantly close relationships between flagellates and the LIP fraction and diatoms and the PRT proportion in the Chukchi Sea. According to Shifrin and Chisholm [67], green algae and diatoms contained an average of 17.1% and 24.5% LIP (% of total dry weight), respectively, during log-phase growth in 30 cultured phytoplankton species. Therefore, the distributions and the relative contribution of these different dominant species and/or taxa of phytoplankton might also largely affect changes in the biochemical composition in the region.

#### *4.3. Bioavailability of POM*

Even now, the FM concentration, is used to represent the quantity of food in POM in relation to indicators of energy and material transfer to higher trophic levels [8,13,14,30]. However, FM is ideal when POM is made only of bioavailable constituents. In reality, POM contains bioavailable and non-bioavailable (refractory or less labile) fractions.

Bioavailability is a pivotal term for nutritional effectiveness, and the contribution of BFM to FM (%; nutritional efficiency) was used to assess POM bioavailability in this study. The nutritional efficiency ranged between 33.1 and 89.7%, with an average of 64.1% in the Chukchi Sea. More interestingly, the nutritional efficiency in the northern Chukchi Sea (74.0%) was approximately 1.2 times higher (60.0%) than it was in the middle part, while a lower mean value (42.7%) was observed in the southern part (Figure 6B). These results may have contributed to the different hydrolysis rates among the components, for which a greater presence is also an important factor. For example, the POM in the southern Chukchi Sea had a high contribution from PRT (41.8%) but a low level of HPRT (approximately 31.0% of their total pool), whereas a high level of HLIP (approximately 79% of their total pool) were observed despite a low contribution of LIP to the POM (20.5%). In the northern part, a large contribution from the HCHO (>80% of their total pool) was observed, with CHO accounting, for more than 50%, on average, of the POM in the northern part. In the middle part, HCHO accounted for 72.1% of the total CHO pool, followed by HPRT (55.4% of their total pool) and HLIP (40% of their total pool).

However, our findings, except for the middle part, are contradictory to the conclusions of Handa and Tominaga [70] and to the results obtained by Dawson and Liebezeit [71], Christian and Karl [72], and Fabiano et al. [8]. These reports suggested that cellular and proteinous amino acids were lost more rapidly than extractable sugars and particulate CHO. Such contrasting results suggest that different sizes [62] and species [73] of phytoplankton likely influence bioavailability. In our study, we found that the bioavailable contribution was negatively correlated with the relative amount of microphytoplankton (r = −0.652, *p* < 0.05, *n* = 20) and positively correlated with the relative amount of picophytoplankton (r = 0.668, *p* < 0.05, *n* = 20) (Figure S2); this result is consistent with the results from Jin et al. [62], who reported that picophytoplankton is more likely to mineralize and degrade in the upper ocean layers. In addition, CHO and amino acids are more enriched in intracellular materials than in cell wall materials [74]. Liebezeit [73] showed somewhat lower CHO degradation (38%) at stations dominated by diatoms than at stations dominated by Haptophyceae (86%) in the upper 100 m of the water column in the Bransfield Strait. Diatoms are characterized by silica shells (frustules) that are resistant to acid conditions (reviewed in DeNicolar [75]) and crushing forces [76]. In this sense, inherent structural differences in phytoplankton might also affect enzymatic hydrolysis, because phytoplankton was the major source of organic matter in our study. Taken together, although these results cannot be explained simply, different enzymatically hydrolysable efficiencies among the three different regions in the Chukchi Sea resulted from a selective loss of labile compounds and different communities of phytoplankton. Therefore, the higher POM bioavailability in the northern part of the Chukchi Sea could be caused by the different biochemical structures of the dominant picophytoplankton community compared to those of the microphytoplankton and diatom dominated community in the southern part of the Chukchi Sea. Clearly, a higher POM bioavailability provides more effective food materials for potential consumers in the northern part of the Chukchi Sea despite their lower biomass and lower primary productivity.

#### **5. Conclusions**

The biochemical composition of POM in the regions of the Chukchi Sea studied was due to differences in both environmental variables and the structure of the phytoplankton community. We also expect the observed results of the biochemical composition of POM to influence the nutritional quality of the available food. For instance, changes in the size, quantity and bioavailability of prey (phytoplankton) could affect the feeding, growth, reproduction and survival of predators [1,77,78]. In particular, in the Arctic Ocean, recent studies have indicated warming and decreased salinity of the water, with concomitant small phytoplankton sizes and decreased primary production [56,63]. If the sea ice continues to melt, then the quantity, quality, and labile level of POM will change, and consequently, the ecosystem structure, such as the trophic chain and microbial loop efficiency, will change in Arctic ecosystems. Therefore, further studies are needed to better understand the recent potential food materials under rapidly changing environmental conditions in the Arctic Ocean and picophytoplankton trophic roles in the microbial foodweb process.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4441/12/9/2355/s1, Figure S1: Relative contribution of accessory pigments to total accessory pigment (wt:wt) in euphotic layer of southern, middle, and northern part of the Chukchi Sea, Figure S2: The relationship between relative contribution of micro (red dot) and picophytoplankton (green dot) fraction to total phytoplankton biomass (chl-a) and POM bioavailability. Solid lines indicate the fitted regression lines of the raw data points, Table S1: Average environmental parameters (± SD) within euphotic layer at each station in the Chukchi Sea.

**Author Contributions:** S.-Y.H. conceived of the study, participated in its design and helped to draft the manuscript; B.K.K. drafted the manuscript and performed the field and laboratory experiments; J.J. and Y.L. carried out the analysis of the nutrients, chl-a, δ13C, and pigments; K.-H.C. processed the CTD data; J.-K.G. critically reviewed the manuscript; S.-H.K. was the leader of the Korean Arctic Research Program and provided scientific advice. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by Ministry of Oceans and Fisheries (MOF) and undertaken part of "Korea-Arctic Ocean Observing System (K-AOOS; 20160245)".

**Acknowledgments:** We thank the captain, officers, and crew of the R/V *Araon* for their valuable assistance at field work.

**Conflicts of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Contribution of Small Phytoplankton to Primary Production in the Northern Bering and Chukchi Seas**

**Jung-Woo Park 1,\*, Yejin Kim 2, Kwan-Woo Kim 2, Amane Fujiwara 3, Hisatomo Waga 1,4, Jae Joong Kang 2, Sang-Heon Lee 2, Eun-Jin Yang <sup>5</sup> and Toru Hirawake 1,6**


**Abstract:** The northern Bering and Chukchi seas are biologically productive regions but, recently, unprecedented environmental changes have been reported. For investigating the dominant phytoplankton communities and relative contribution of small phytoplankton (<2 μm) to the total primary production in the regions, field measurements mainly for high-performance liquid chromatography (HPLC) and size-specific primary productivity were conducted in the northern Bering and Chukchi seas during summer 2016 (ARA07B) and 2017 (OS040). Diatoms and *phaeocystis* were dominant phytoplankton communities in 2016 whereas diatoms and Prasinophytes (Type 2) were dominant in 2017 and diatoms were found as major contributors for the small phytoplankton groups. For size-specific primary production, small phytoplankton contributed 38.0% (SD = ±19.9%) in 2016 whereas 25.0% (SD = ±12.8%) in 2017 to the total primary productivity. The small phytoplankton contribution observed in 2016 is comparable to those reported previously in the Chukchi Sea whereas the contribution in 2017 mainly in the northern Bering Sea is considerably lower than those in other arctic regions. Different biochemical compositions were distinct between small and large phytoplankton in this study, which is consistent with previous results. Significantly higher carbon (C) and nitrogen (N) contents per unit of chlorophyll-*a*, whereas lower C:N ratios were characteristics in small phytoplankton in comparison to large phytoplankton. Given these results, we could conclude that small phytoplankton synthesize nitrogen-rich particulate organic carbon which could be easily regenerated.

**Keywords:** Bering Sea; Chukchi Sea; HPLC; small phytoplankton; primary productivity

#### **1. Introduction**

The biologically productive northern Bering Sea and the Chukchi Sea are important conduit of water masses and organic matters from the North Pacific Ocean transported into the Arctic Ocean and biologically productive regions [1–5]. Over the past few decades, many environmental changes have been reported in the regions [4,6–8]. Unprecedented high sea surface temperature was reported in the Bering Sea in 2014 and persisted in 2018 and 2019 [8; refs therein]. The Pacific origin freshwater flux with increasing northward volume transport into the Arctic Ocean had been increased over the 1991–2015 period [9]. Moreover, seasonal sea ice cover has been retreating earlier and forming later in the Pacific Arctic region over the last decade [10]. These current and ongoing changes in environmental conditions could subsequently cause changes in biogeochemical processes and consequently alter marine ecosystem structure in the northern Bering and Chukchi

**Citation:** Park, J.-W.; Kim, Y.; Kim, K.-W.; Fujiwara, A.; Waga, H.; Kang, J.J.; Lee, S.-H.; Yang, E.-J.; Hirawake, T. Contribution of Small Phytoplankton to Primary Production in the Northern Bering and Chukchi Seas. *Water* **2022**, *14*, 235. https:// doi.org/10.3390/w14020235

Academic Editor: Michele Mistri

Received: 30 November 2021 Accepted: 8 January 2022 Published: 14 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

seas [11,12]. Indeed, the prior studies indicate that the variation in primary productivity of phytoplankton is mainly governed by freshwater content variability in the Pacific Arctic region [13,14]. Moreover, the seasonal sea ice cover could largely influence phytoplankton community composition [15], phytoplankton bloom period [16] and primary productivity [17].

Refs. [13,18–20] reported that pico-phytoplankton increased whereas larger cells declined in the Arctic Ocean because of stronger stratification and consequently lower nutrient supply into the upper water column caused by freshening surface waters. Based on the phytoplankton size classes derived from satellite ocean color data in the northern Bering and southern Chukchi seas [21], observed increasing trends in pico-phytoplankton in the Chirikov and St. Lawrence Island Polynya regions whereas an increasing trend in micro-phytoplankton in the southeastern Chukchi Sea from 1998 to 2016. The physiological conditions and subsequently photosynthetic end-products of phytoplankton affected by the recent environmental conditions were also previously reported in the northern Bering and southern Chukchi seas [21–23]. Phytoplankton as important primary producers in marine ecosystems can be a good indicator of environmental changes. These long-term changes in the functional phytoplankton group are strongly related to increasing annual sea surface temperature [13]. Therefore, monitoring the phytoplankton community responses such as shifts in dominant phytoplankton species and biomass to the current environmental changes is crucial to observe marine ecosystem alterations in the northern Bering and Chukchi seas [12,18–20].

Especially, the contribution of small phytoplankton could be necessary to understand potential impacts on the total primary production and, thus, whole marine ecosystems [12,20,21]. Moreover, the biochemical characteristics of phytoplankton such as C:N ratio are critical for understanding marine biogeochemical processes responding to environmental conditions. Ref. [24] reported higher C:N ratio related with low chlorophyll-*a* concentration and lower C:N ratio to high chlorophyll-*a* concentration in the Arctic Ocean. The C:N ratio could differ in various environmental conditions related to nutrients. However, little information on the small phytoplankton contribution to the total primary production and their biochemical traits such as C:N ratio is currently available in the northern Bering and Chukchi seas.

In this study, our objectives are to investigate the dominant phytoplankton communities and to assess the relative contribution of small phytoplankton (0.7–2.0 μm; picophytoplankton) to the total primary production and their biochemical characteristics (e.g., C:N ratio) in the northern Bering and Chukchi seas.

#### **2. Materials and Methods**

#### *2.1. Study Area and Water Sampling*

The ARA07B cruise was conducted in the northern Bering Sea and the Chukchi Sea during 5–19 August, 2016 onboard the Icebreaker R/V *Araon* (Figure 1; Table 1). As a total of 16 stations during the ARA07B cruises, only one station (st. 1) was located in the northern Bering Sea and 15 stations were in the Chukchi Sea. Water was sampled by Niskin bottles on conductivity-temperature-depth (CTD)/rosette sampler for the total chlorophyll-*a* and size-fractionated chlorophyll-*a* concentration. Euphotic depths were measured by a Secchi disk [25]. The OS040 cruise was executed mostly in the northern Bering Sea (8 stations) and partly in the southern Chukchi Sea (2 stations) during 9–21 July, 2017 onboard T/S *Oshoro-Maru* (Figure 1; Table 1). Physical properties and water samples were collected by CTD/rosette with Niskin bottles. The euphotic depths were calculated by comparing downward irradiance and surface irradiance measured by compact optical profiling system (C-OPS; Biospherical instrument Inc., San Diego, CA, USA).

**Figure 1.** Sampling locations of (**a**) ARA07B and (**b**) OS040 cruises.


**Table 1.** Sampling locations in the Northern Bering and Chukchi Seas.

#### *2.2. Chlorophyll-a Analysis*

The water samples were obtained from 6 different light depths (100%, 50%, 30%, 12%, 5% and 1% of the surface photosynthetically active radiation (PAR) for measuring the chlorophyll-*a* concentration. For the total chlorophyll-*a* concentration, 300 mL of seawater was filtered through 25 mm glass fiber filter (GF/F; Whatman). To obtain size-fractionated

chlorophyll-*a* concentration, 500 mL seawater was filtered through 20 μm and 2 μm pore size membrane filters and then 47 mm GF/F sequentially. After the filtration was done, the filters were wrapped with aluminum foil and stored at −80◦C freezer until analysis at the home laboratory. Chlorophyll-*a* extractions were followed by [26] and the concentrations were measured with a fluorometer (Turner Designs 10AU).

#### *2.3. High-Performance Liquid Chromatography Analysis for Accessory Pigment Concentration*

For high-performance liquid chromatography (HPLC) analysis, the water from 3 light depths (100%, 30% and 1%) were sampled during the ARA07B and OS040 cruises. Seawater (0.8–2.5 L) was passed through 2 μm membrane filter and 47 mm diameter GF/F filters to measure pigments concentration of small size phytoplankton (<2 μm) under gentle vacuum pressure (<100 mmHg). Seawater (0.5–1.5 L) was filtered onto GF/F for pigments of total phytoplankton during the ARA07B. For the OS040, samples were obtained only for total phytoplankton. For avoiding degradation, the filters for HPLC analysis were immediately frozen and stored in liquid nitrogen at −80◦C freezer until analysis at home laboratory. In the laboratory, the filter samples were broken into small pieces and then soaked in 3 mL of N'N-dimethylformamide (DMF) with canthaxanthin served as an internal standard. After 20 min of sonication, the filters were extracted at 4◦C in dark for 24 h and then extracts were filtered through a 0.45 μm pore membrane filter to remove GF/F particles. For minimizing photo-degradation of pigments, all the procedures were conducted under a low light condition. Pigments were analyzed using HPLC (Agilent Infinite 1260 in operation by JAMSTEC, Mutsu, Japan) with a ternary linear gradient system to separate each pigment. The pigment concentrations were calculated by the function of peak area, standard response factors and peak area of the internal standard following [27]. All the standards for each pigment were purchased from DHI in Denmark.

The CHEMTAX software based on a factorization program was used for estimating the relative contributions of different phytoplankton communities to the total chlorophyll-*a* concentration [28]. The ratios of accessory pigments to chlorophyll-*a* for each phytoplankton taxon for the CHEMTAX program were based on marker pigment concentrations of algal groups present in the Arctic Ocean [13,29] (Table 2). Since our two research cruises were in different periods and years, the final ratio matrix was separated for phytoplankton communities (Table 2). The contributions of Diatoms. Dinoflagellates, Cryptophytes, Pelagophytes, Prasinophytes (Type 2 and 3), Chlorophytes, Haptophytes and Phaeosystis were estimated by the CHEMTAX program. Small phytoplankton community was estimated from HPLC results by the equations described in the literature [28,29]. The relative proportions of the three size classes are derived from the concentrations of phytoplankton diagnostic pigments for the Chukchi and Bering seas using the equations described in [30,31].


**Table 2.** Pigment:chlorophyll-*a* ratios for nine algal groups referred to [32]. CHEMTAX initial ratio matrix and final pigment ratios obtained by CHEMTAX on the pigment data.


**Table 2.** *Cont.*

Abbreviations: chlorophyll-*b* (chl-b), chlorophyll-*c*3 (chl-c3), fucoxanthin (fucox), peridinin (period), alloxanthin (allox), 19 -butanoyloxyfucoxanthin (19butfu), 19 -hexanoyloxyfucoxanthin(19hexfu), chlorophyll-*c*1+*c*2 (chl-c), neoxanthin (neox), prasinoxanthin (prasinox), lutein (lut). Chrysophytes and Pelagophytes (Cryso-pelago). Prasinophytes type 2 (Prasino-2), Prasinophytes type 3 (Prasino-3), Haptophytes (Hapto-7).

#### *2.4. Particulate Organic Carbon and Primary Productivity*

The water samples for particulate organic carbon (POC) and primary productivity were obtained from 6 light depths (100, 50, 30, 12, 5 and 1% of PAR). 300 mL of seawater was filtered through 0.7 μm GF/F (pre-combusted at 450 ◦C for 4 h) for total POC and 500 mL was passed through 2 μm pore size membrane filter and then filtered onto GF/F filter for small POC (0.7–2 μm). Carbon and nitrogen uptake experiments were conducted using a 13C-15N dual isotope tracer technique previously reported from the Chukchi Sea [3,33]. After a 4 h incubation on deck, 300 mL water was filtered onto pre-combusted GF/F for total primary productivity and 500 mL water was filtered through 2 μm pore size membrane filter and sequentially onto GF/F filter for small phytoplankton productivity (0.7–2 μm). The filters were immediately preserved and stored in a freezer (−20 ◦C) until further mass spectrometric analysis using a Delta V+ Isotope Ratio Mass Spectrometers of Alaska Stable Isotope Facility at the University of Alaska Fairbanks, USA for ARA07B samples and using a 20–22 Isotope Ratio Mass Spectrometer (SERCON) at Japan Agency for Marine-Earth Science and Technology (JAMSTEC, Mutsu, Japan) for OS040 samples after HCl fuming overnight to remove carbonate. The carbon and nitrogen uptake rates were calculated based on [34].

#### *2.5. Statistical Analysis*

Student's t-test was applied to verify correlations among factors and differences between the mean values of POC:chlorophyll-*a* ratio, PON:chlorophyll-*a* ratio, C:N ratio of each cruise and size group. The agglomerative hierarchical clustering (AHC) with Ward's method (XLSTAT software, Addinsoft, Boston, MA, USA) was performed to calculate the dissimilarity in observed 20 variables; temperature and salinity), size-fractionated primary productivity, particulate organic carbon of each size, size-fractionated chlorophyll-*a* and accessory pigments, among stations.

#### **3. Results and Discussion**

#### *3.1. Spatial Distribution of Temperature and Salinity*

The temperature and salinity ranged from −1.5 ◦C to 9.2 ◦C (mean ± standard deviation (SD) = 0 ± 2.7 ◦C) and from 26.5 to 32.3 (mean ± SD = 29.9 ± 1.6) during the ARA07B cruise (Figure 2). The temperature during the OS040 were from –1.1 to 13.3 ◦C (mean ± SD = 6.2 ± 3.6 ◦C) and the salinity ranged from 28.9 to 32.9 (mean ± SD = 31.7 ± 0.9). Water mass at the most stations in the northern Chukchi corresponded to melting glacier water, which called Ice melt water (IMW; temperature < 2.0 ◦C and salinity < 30.0) and Bering Chukchi winter water (BCWW; −2–0 ◦C and <30–33.5 for temperature and salinity; [35]) during the ARA07B cruise. Other stations during the ARA07B were influenced by Bering shelf water (BSW; 0.0–10.0 ◦C and 31.8–33.0 for temperature and salinity). During the OS040 cruise, the relatively warm and low salinity Alaskan coastal water (ACW; 2.0–13.0 ◦C and <31.9 for temperature and salinity) and the warm and saline Bering shelf water (BSW) were predominant (Figure 2). The Bering shelf Anadyr water (BSAW; −1.0–2.0 ◦C and 31.8–33.0 for temperature and salinity), which is a mixed BSW with cold/saline Anadyr water (AW; [36,37]), was observed at some stations for the OSO40 cruise.

**Figure 2.** T–S diagram in the ARA07B (Red) and OS040 (Green). Alaskan coastal water (ACW), Bering Shelf water (BSW), ice melt water (IMW), Bering–Chukchi winter water (BCWW), Bering Sea Anadyr water (BSAW).

#### *3.2. Chlorophyll-a Concentration and Different Size Chlorophyll-a Compositions in the Northern Bering and Chukchi Seas*

The average euphotic depths were 45.6 m (SD = ±22.2 m) for the ARA07B cruise and 23.8 m (SD = ±9.1 m) for the OS040 cruise, respectively. In ARA07B, Chlorophyll-*a* concentrations were 0.02–1.3 mg chl-*<sup>a</sup>* <sup>m</sup>−<sup>3</sup> (mean ± SD = 0.2 ± 0.3 mg chl-*<sup>a</sup>* <sup>m</sup><sup>−</sup>3) at surface, 0.02–15.0 mg chl-*<sup>a</sup>* <sup>m</sup>−<sup>3</sup> (mean ± SD = 1.0 ± 2.5 mg chl-*<sup>a</sup>* <sup>m</sup>−3) for euphotic layer. In OS040, Chlorophyll-*<sup>e</sup>* concentrations were 0.002–5.5 mg chl-*<sup>a</sup>* <sup>m</sup>−<sup>3</sup> (mean ± SD = 0.7 ± 1.4 mg chl-*<sup>a</sup>* <sup>m</sup><sup>−</sup>3) at surface, 0.002–5.5 mg chl-*<sup>a</sup>* <sup>m</sup>−<sup>3</sup> (mean ± SD = 1.6 ± 2.2 mg chl-*<sup>a</sup>* <sup>m</sup>−3) for euphotic layer. Within the euphotic zone, integral chlorophyll-*a* concentrations were 3.2–172.1 mg chl-*a* m−<sup>2</sup> (mean ± SD = 34.2 ± 48.0 mg chl-*<sup>a</sup>* <sup>m</sup><sup>−</sup>2) during the ARA07B and 12.3–107.8 mg chl-*<sup>a</sup>* <sup>m</sup>−<sup>2</sup> (mean ± SD = 45.4 ± 34.1 mg chl-a m<sup>−</sup>2) for the OS040, respectively (Figure 3). The average euphotic-depth integral chlorophyll-*a* concentrations in this study are within the range reported previously in the northern Bering Sea and the Chukchi Sea [3,14,21].

**Figure 3.** Spatial distributions of column-integrated chlorophyll-*a* concentration of (**a**) ARA07B and (**b**) OS040.

The chlorophyll-*a* contributions of each size phytoplankton (pico-, nano- and microphytoplankton) to the total phytoplankton were plotted in Figure 4 for the three different depths (100, 30 and 1% of light depths) at every station of the ARA07B and only surface for the OS040. The contributions of small phytoplankton to the total chlorophyll-*a* concentrations were found largely variable among the stations during both cruises.

The contributions of small phytoplankton to the total chlorophyll-*a* concentrations ranged from 2.9% to 71.1% with a depth-integrated average of 32.2% (SD = ±23.1%) during the ARA07B. In the ARA07B, the dominant size group of phytoplankton was microphytoplankton (mean ± SD = 43.5 ± 29.7% of chlorophyll-*a* concentration) followed by pico-phytoplankton (32.1 ± 23.1%) and nano-phytoplankton (24.3 ± 9.1%) during the observation period. In the Chukchi Sea, large phytoplankton are generally dominant although the areal distribution of their contribution mostly depends on local water masses in different nutrient conditions [3,21]. Normally, large phytoplankton growing under nutrient-enriched conditions are predominant in AW or BSW, whereas small phytoplankton are dominant in nutrient-depleted ACW [3,21]. Our average contribution of small phytoplankton is relatively higher than that (24.8 ± 23.0%) previously reported by [21] in the Chukchi Sea during the middle of August to early September, 2004. By contrast, our average contribution of small phytoplankton is relatively lower than that (55.1 ± 26.8%) from the study by [38] that was conducted in the northern Chukchi Sea during mid-July–mid-August, 2012. This difference among the studies could be caused by different regions with non-homogeneous nutrient conditions and different observation periods with a seasonal phytoplankton succession. The relative contribution of small phytoplankton could be caused by freshwater content in the Chukchi Sea since the nutrient concentrations and primary production rates of phytoplankton are largely governed by the nutrient-depleted freshwater content in the Chukchi Sea [14,39].

In comparison to the Chukchi Sea, the contributions of small phytoplankton were 0.7–80% (mean ± SD = 37.2 ± 31.0%) to the total chlorophyll-*a* concentration in the northern Bering Sea for the OS040 in this study. The proportions of different size chlorophyll-*a* were 40.2% (±35.4%), 22.5% (±10.5%) and 37.2% (±31.3%) for micro-, nano- and picophytoplankton, respectively, during our observation period in 2017. In the northern Bering Sea, the dominant size groups of phytoplankton are generally nano- and microphytoplankton based on phytoplankton size class results derived from satellite ocean color data from 1998 to 2016 [12]. The overall dominant size of phytoplankton is composed of nano-phytoplankton (49.0 ± 9.6%), followed by micro-phytoplankton (34.9 ± 8.0%) and pico-phytoplankton (16.1 ± 7.3%) in the Chirikov Basin of the northern Chukchi Sea [12]. However, the chlorophyll-*a* contributions of small phytoplankton are largely variable

among different seasons [40]. The average contribution of small phytoplankton was 14.8% in late May to early June during the phytoplankton bloom period and largely increased up to 50.0% in middle June after the bloom [41]. Consistently, [13] found a seasonal increasing contribution of small phytoplankton in the northern Bering Sea (around Chirikov Basin) from May (5.2%) to July (31.8%). In addition to the seasonal variation, spatially the biochemical environmental conditions in the northern Bering Sea are also generally influenced by northward advection of AW, BSW and ACW [3,5]. Over recent decades, several environmental changes have been reported in the northern Bering Sea [4,5]. A steady increasing trend in the annual contribution of small phytoplankton is distinct in the Chirikov Basin from 1998 to 2016, although no significantly strong relationship was observed between the annual contribution of small phytoplankton and sea surface temperature [12]. Long-term changes in dominant phytoplankton communities should be monitored for Arctic marine ecosystems under ongoing environmental changes. Especially, the contribution of small phytoplankton could be used as one of indicators for changing marine ecosystems.

**Figure 4.** Total chlorophyll-*a* concentration of (**a**) ARA07B and (**b**) OS040.

*3.3. Pigment Composition and Major Dominant Phytoplankton Groups*

The euphotic depth-integral concentrations of marker pigments from the two cruises are shown in Figure 5. Fucoxanthin (a marker pigment of diatoms), chlorophyll-*c*1+*c*2 and chlorophyll-*b* (a marker pigment of chlorophytes) were major accessory pigments during the ARA07B, although the pigment compositions spatially varied significantly across the stations. Among the pigments, fucoxanthin was the most dominant pigment with an average value of 12.58 ± 21.8 mg m−<sup>2</sup> and the second and third predominant pigments were chlorophyll-c1+c2 (4.04 ± 4.83 mg m−2) and chlorophyll-b (2.64 ± 2.53 mg m<sup>−</sup>2). Previous studies reported that fucoxanthin dominating the Chukchi shelf is a typical characteristic during fall [13,31]. For the small phytoplankton group for the ARA07B (data not shown), major predominant pigments were chlorophyll-*<sup>b</sup>* (1.59 ± 1.83 mg m−2), fucoxanthin (1.46 ± 1.47 mg m<sup>−</sup>2) and chlorophyll-*c*1+*c*2 (0.65 ± 0.62 mg m<sup>−</sup>2). In comparison, fucoxanthin, chlorophyll-c1+c2 and peridinin (a marker pigment of dinoflagellates) were major accessory pigments for the OS040. Fucoxanthin was the most dominant pigment with an average value of 23.03 ± 19.89 mg m−2, followed by chlorophyll-c1+c2 (9.35 ± 7.23 mg m<sup>−</sup>2) and peridinin (7.54 ± 9.89 mg m<sup>−</sup>2) for the OS040. High proportions of diatom-related pigments (fucoxanthin, chlorophyll-*c*1+*c*2) were observed in both cruise periods. Small diatoms appeared to be major phytoplankton communities for the small phytoplankton group during the ARA07B, based on the high proportions of chlorophyll-b and fucoxanthin. No pigment data were available for the small phytoplankton during the OS040 cruises.

**Figure 5.** Pigment compositions of total phytoplankton in the (**a**) ARA07B (**b**) OSO040.

Based on the CHEMTAX results, eight major phytoplankton communities were identified in the study area (Figure 6). Diatoms (43.1% ± 31.5%) and Phaeocystis (33.2% ± 14.9%) were co-dominated during the ARA07B. In comparison, Diatoms were the most dominant community (46.1 ± 17.3%) and the second dominant community was Prasinophyte (Type 2) (11.8% ± 5.3%) for the OS040. Micro-phytoplankton communities were most dominant (59.7 ± 30.5%), followed by nano-phytoplankton (11.5 ± 9.7%) and pico-phytoplankton (28.9 ± 23.5%) during the ARA07B. For the OS040, micro-phytoplankton contributed 51.5% (±18.2%) of the total chlorophyll a concentration. In comparison, nano-phytoplankton and pico-phytoplankton contributed 11.0% (±10.5%) and 37.5% (±15.7%), respectively. The relative proportions of the three size classes based on the diagnostic pigments from HPLC are different from those of the size-fractionated chlorophyll-*a* concentrations (Figure 4). This is probably due to a simple assumption that diatom-related pigments belong to the micro-phytoplankton although small diatoms (<2 μm) could contribute to the phytoplankton group.

#### *3.4. Primary Production Contribution of Small Phytoplankton and Their Ecological Roles*

The daily primary productivities of total phytoplankton which were integrated over the euphotic zone at each station were 33.9–811.8 mg C m−<sup>2</sup> <sup>d</sup>−<sup>1</sup> (mean ± SD = 142.6 ± 205.7 mg C m−<sup>2</sup> <sup>d</sup><sup>−</sup>1) for the ARA07B and 202.1–3100.1 mg C m−<sup>2</sup> <sup>d</sup>−<sup>1</sup> (mean ± SD = 942.1 ± 969.9 mg C m−<sup>2</sup> <sup>d</sup><sup>−</sup>1) for the OS040 (Figure 7). In comparison, the daily primary productivities of small phytoplankton ranged from 4.9 to 227.7 (mean ± SD = 42.3 ± 53.1 mg C m−<sup>2</sup> <sup>d</sup><sup>−</sup>1) and 56.1 to 322.2 mg C m−<sup>2</sup> <sup>d</sup>−<sup>1</sup> (mean ± SD = 152.8 ± 85.2 mg C m−<sup>2</sup> d<sup>−</sup>1) for the ARA07B and the OS040, respectively (Figure 8). The contribution of small phytoplankton to the total primary productivity ranged from 8.1 to 71.7% (mean ± SD = 38.0 ± 19.9%) for the ARA07B and from 6.0 to 40.3% (mean ± SD = 25.0 ± 12.8%) for the OS040 (Figure 9).

**Figure 6.** Phytoplankton community compositions of (**a**) ARA07B and (**b**) OS040.

**Figure 7.** Primary production of total phytoplankton during the (**a**) ARA07B and (**b**) OS040.

**Figure 8.** Primary production of small phytoplankton during the (**a**) ARA07B and (**b**) OS040.

**Figure 9.** Primary production of small phytoplankton of (**a**) ARA07B and (**b**) OS040.

Overall, the primary productions of total and small phytoplankton communities during the study period were different depending on the sea area. Indeed, agglomerative hierarchical clustering (AHC) analysis based on 25 stations and phytoplankton size-related variables sorted stations into four distinct groups (Figure 10; Table 3). Cluster 1 include station 1 of OS040 that was high primary productive region (1992.9 mg C m−<sup>2</sup> d−1) near Bering strait. Cluster 1 had a relative low contribution of small phytoplankton in primary productivity (5.9%) and surface chlorophyll-*a* (2.9%). Cluster 2 was station 7 of OS040 that was an extremely high productive station (3100.0 mg C m−<sup>2</sup> d−1) and represented the lowest contribution of small phytoplankton among clusters. Small phytoplankton contribution to primary production was 6.9% and the contribution to surface chlorophyll-*a* concentration was only 0.7% for clusters 2. The physical properties of Cluster 1 (3.5 ◦C and 32.7 psu) and 2 (5.5 ◦C and 32.7 psu) were similar. These two clusters are influence by BSW [3,21]. Cluster 3 contains all the stations of the northern Chukchi Sea and two stations of the Bering Sea. The stations form Cluster 3 had a lower productivity and lower concentration of surface chlorophyll-*a*. In Cluster 3, small phytoplankton contribution was the highest among the clusters. 40.5% of primary production, 39.1% of surface chlorophyll-*a* and 40.9% of POC were contributed by small phytoplankton. Dominant water mass, IMW can explain the high contribution of small phytoplankton in Cluster 3 because IMW has nutrient-depleted water from sea ice melting [34]. Cluster 4 includes most of the stations in the Bering Sea and 3 stations of the southern Chukchi sea in ARA07B. Cluster 4 had a lower productivity (559.2 mg C m−<sup>2</sup> d−1) than cluster 1 and 2 but higher than cluster 3. Cluster 4 seems to be affected by nutrient-depleted ACW but not too low productivity for Cluster 4. This suggests that water masses that had an effect on Cluster 4 were not only ACW but also other source such as mixed water of AW, ACW and BSW.



**Figure 10.** Dendrogram stands for sampling stations were divided into four clusters by agglomerative hierarchical clustering (AHC).

The primary production contributions of small phytoplankton are rather different from their chlorophyll-*a* contributions in this study. Normally, the contributions of small phytoplankton are higher to primary production in comparison to those in chlorophyll-a concentrations in the polar oceans [21,41] and temperate oceans [42]. This is probably due to the considerably higher POC contribution of small phytoplankton (and consequently higher production contributions of small phytoplankton) than the chlorophyll-*a* contribution [21,40,42]. We also observed the higher POC:chlorophyll-*a* ratio in small phytoplankton than large phytoplankton during both cruises (Figure 11) as discussed later. However, the case in the northern Bering Sea in this study is against the general pattern previously reported. The lower contribution of small phytoplankton was observed in the primary production rather than chlorophyll-*a* concentration in the northern Bering Sea. This indicates higher standing stock (represented by chlorophyll-*a* concentrations) of small phytoplankton but their significantly lower contribution to the primary productions in the northern Bering Sea during this study than in other studies. Ref. [20] argued that seasonal

increasing contribution of small phytoplankton is not caused by their increasing biomass and photosynthetic rate but caused by relative declining in biomass and photosynthetic rate of large phytoplankton in the Amundsen Sea, Antarctic Ocean. Based on these results, the biomass of large phytoplankton could have had decreased faster than their photosynthetic rate in the northern Bering Sea during our observation period.

**Figure 11.** Comparison of (**a**) POC:chlorophyll-*a* ratios, (**b**) PON:chlorophyll-*a* ratios and (**c**) C:N ratios between small and large phytoplankton in the northern Bering and Chukchi seas. Only POC:chlorophyll-*a* data available for the OS040. (**d**) C:N ratio and chlorophyll-a of each size group.

The regional contributions of small phytoplankton to the primary production are summarized at various regions in the Arctic Ocean (Table 4). The average contribution of small phytoplankton in this study is comparable to the previous results in the Chukchi Sea. However, it is considerably lower than those (average ± SD = 56.7 ± 20.0%) in the Kara, Laptev and East Siberian Seas [43]. Similarly, reference [41] found a similar contribution (average ± SD = 60 ± 7.9%) of small phytoplankton in the high northern Chukchi Sea and Canada Basin. Because of no data in the northern Bering Sea, the small phytoplankton contribution to the primary production in this study could not be compared. Regionally, the primary production contribution of small phytoplankton in the northern Bering Sea (average ± SD = 25.0 ± 12.8%) is considerably lower than those in others (Table 4). At this point, we do not know whether this is a latitudinal pattern (i.e., increasing contribution of small phytoplankton in higher latitude) or simply seasonal difference among the different regions in the Pacific Arctic Ocean. Indeed, [12] found a seasonal patterns of different phytoplankton size compositions with increasing contribution of small phytoplankton in the northern Bering Sea. Since the seasonal contribution of small phytoplankton to the primary production would be different, further seasonal observations on the small phytoplankton contribution to the primary production will be warranted for better understanding their ecological roles in the Bering and Chukchi Seas.

**Table 4.** Small phytoplankton contributions to the total primary production in the Arctic Ocean.


Biochemical compositions (POC:chlorophyll-*a*, PON:chlorophyll-*a* and C:N ratios) were compared between small and large phytoplankton in Figure 11. Large phytoplankton group has relatively lower POC:chlorophyll-*a* ratios (*t*-test, *p* < 0.01) which were 78.0–3549.0 (mean ± SD = 1358.6 ± 1170.8) for the ARA07B and 41.4–340.2 (mean ± SD = 173.8 ± 110.4) for the OS040 (Figure 9a). In comparison, POC:chlorophyll-*a* ratios of small phytoplankton were 408.5–6547.4 (mean ± SD = 2590.2 ± 1523.0) for ARA07B and 274.9–2303.6 (mean ± SD = 623.4 ± 639.2) for the OS040. The PON:chlorophyll-*a* ratio of large phytoplankton was 1.9–184.2 (mean ± SD = 62.4 ± 48.7) whereas the ratio of small phytoplankton ranged from 50.0 to 328.7 (mean ± SD = 211.9 ± 88.3) for the ARA07B (no data for OS040). The C:N ratios were 7.5–251.9 (mean ± SD = 34.1 ± 58.9) for large phytoplankton and 7.0–19.9 (mean ± SD = 11.9 ± 3.8) for small phytoplankton during the ARA07B cruise. Small phytoplankton showed a comparatively higher POC:chlorophyll-*a* ratio than large phytoplankton during both cruises (Figure 11). This result is consistent with the previous result in the Chukchi Sea, which suggests that higher carbon contents per unit of chlorophyll-*a* concentration in small phytoplankton in comparison to large phytoplankton [21]. In the Antarctic Ocean, [41] observed the consistent results in non-polynya and polynya regions in the Amundsen Sea. A similar pattern was observed for the PON:chlorophyll-*a* ratio in this study. However, the C:N ratios of small phytoplankton were lower than those of large phytoplankton in this study. Similarly, the overall C:N assimilation ratio of small

phytoplankton was previously reported as significantly lower than that of large phytoplankton [21]. These results are consistent with the result in the Gulf of St. Lawrence, Canada [44]. In the Antarctic Ocean, the similar result was obtained in the Amundsen Sea [41]. The C:N ratios were negatively correlated with chlorophyll-*a* concentrations for small and large phytoplankton in this study (R<sup>2</sup> > 0.6). However, there was no statistically significant difference in the relationship between small and large phytoplankton (*p* > 0.05; Figure 11).

#### **4. Summary and Conclusions**

For determining the dominant phytoplankton communities and the relative contribution of small phytoplankton (<2 μm) to the total primary production, two arctic research cruises were conducted in the Chukchi Sea onboard the icebreaker R/N *Araon* in 2016 (ARA07B) and mainly in the northern Bering Sea onboard T/S *Oshoro-Maru* in 2017 (OS040) for this study. The dominant phytoplankton communities were diatoms and phaeocystis during the ARA07B, whereas diatoms and Prasinophyte (Type 2) during the OS040. Based on the AHC analysis, the primary productions of total and small phytoplankton communities were different depending on the sea area. Overall, high primary productions and low contributions of small phytoplankton during both study periods were distributed in the Bering Strait region which was affected by nutrient-enriched BSW. Different biochemical compositions between small and large phytoplankton were observed in this study. The small phytoplankton group had a higher POC:chlorophyll-*a* (*t*-test, *p* <0.01) and PON:chlorophyll-*a* ratio than large phytoplankton in this study, which suggests that small phytoplankton have higher carbon and nitrogen contents per unit of chlorophyll-a concentration [21]. In addition, small phytoplankton had lower C:N ratios than large phytoplankton in this study. Together with these results, we could conclude that small phytoplankton incorporate more nitrogen in relation to carbon into their bodies and thus produce nitrogen-rich organic matters [43] which could be relatively faster regenerated than carbon-rich organic matters such as carbohydrates [46]. Therefore, the study for small phytoplankton which could be an important basic food source in the Arctic ecosystem should be further conducted under the current warming ocean scenario.

**Author Contributions:** Conceptualization, J.-W.P., T.H. and S.-H.L.; methodology, J.-W.P., A.F., Y.K., K.-W.K. and H.W.; software, J.-W.P.; validation, J.-W.P. and T.H.; formal analysis, J.-W.P., A.F., J.J.K. and T.H.; investigation, K.-W.K., J.-W.P. and E.-J.Y.; writing—original draft, J.-W.P.; writing—review and editing, T.H. and J.J.K.; visualization, J.-W.P., Y.K. and K.-W.K.; supervision, T.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Arctic Challenge for Sustainability (ArCS; JPMXD1300000000) and Arctic Challenge for Sustainability II (ArCS-II; JPMXD1420318865) Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and by the Global Change Observation Mission-Climate (GCOM-C; 17RSTK001714) project of the Japan Aerospace Exploration Agency (JAXA). This research also was part of the projected titled "Korea-Arctic Ocean Warming and Responses of Ecosystem (K-AWARE, KOPRI, 1525011760)", funded by the Ministry of Oceans and Fisheries, Korea.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data Availability Statements in section "MDPI Research Data Policies" at https://www.mdpi.com/ethics, accessed on 7 January 2022.

**Acknowledgments:** The austhors thank the captains and crew members for our field observations. In addition, We thanks to ArCS-II project for funding this publication.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Picocyanobacterial Contribution to the Total Primary Production in the Northwestern Pacific Ocean**

**Ho-Won Lee 1, Jae-Hoon Noh 1, Dong-Han Choi 1, Misun Yun 2, P. S. Bhavya 3, Jae-Joong Kang 4, Jae-Hyung Lee 5, Kwan-Woo Kim 4, Hyo-Keun Jang <sup>4</sup> and Sang-Heon Lee 4,\***

	- Tianjin 300457, China; misunyun@tust.edu.cn

**Abstract:** Picocyanobacteria (*Prochlorococcus* and *Synechococcus)* play an important role in primary production and biogeochemical cycles in the subtropical and tropical Pacific Ocean, but little biological information on them is currently available in the North Pacific Ocean (NPO). The present study aimed to determine the picocyanobacterial contributions to the total primary production in the regions in the NPO using a combination of a dual stable isotope method and metabolic inhibitor. In terms of cell abundance, *Prochlorococcus* were mostly dominant (95.7 ± 1.4%) in the tropical Pacific region (hereafter, TP), whereas *Synechococcus* accounted for 50.8%–93.5% in the subtropical and temperate Pacific region (hereafter, SP). Regionally, the averages of primary production and picocyanobacterial contributions were 11.66 mg C m−2·h−<sup>1</sup> and 45.2% (±4.8%) in the TP and 22.83 mg C m−2·h−<sup>1</sup> and 70.2% in the SP, respectively. In comparison to the carbon, the average total nitrogen uptake rates and picocyanobacterial contributions were 10.11 mg N m−2·h−<sup>1</sup> and 90.2% (±5.3%) in the TP and 4.12 mg N m−2·h−<sup>1</sup> and 63.5%, respectively. These results indicate that picocyanobacteria is responsible for a large portion of the total primary production in the region, with higher contribution to nitrogen uptake rate than carbon. A long-term monitoring on the picocyanobacterial variability and contributions to primary production should be implemented under the global warming scenario with increasing ecological roles of picocyanobacteria.

**Keywords:** cyanobacteria; *Prochlorococcus*; *Synechococcus*; primary production; northwestern Pacific Ocean

#### **1. Introduction**

Phytoplankton are major biological components as primary producers in marine ecosystems. Marine phytoplankton not only account for a significant proportion of global primary production, but also are an important food source in marine ecosystems and a potential moderator of global carbon cycle at the ocean–atmosphere interface [1]. Distribution, abundance, and diversity of phytoplankton differ greatly among dominant water masses in the various oceanic regions, which are closely related to physiochemical properties. In addition, long-term research on the limiting factors (e.g., temperature, nutrients, and light regime) of phytoplankton has reported that biological and ecological changes resulted from variations of these factors such as increasing of seawater temperature and reinforcement of stratification [2,3]. Primary production is widely used as one of key biological factors for

**Citation:** Lee, H.-W.; Noh, J.-H.; Choi, D.-H.; Yun, M.; Bhavya, P.S.; Kang, J.-J.; Lee, J.-H.; Kim, K.-W.; Jang, H.-K.; Lee, S.-H. Picocyanobacterial Contribution to the Total Primary Production in the Northwestern Pacific Ocean. *Water* **2021**, *13*, 1610. https://doi.org/ 10.3390/w13111610

Academic Editor: Maria Moustaka-Gouni

Received: 16 April 2021 Accepted: 4 June 2021 Published: 7 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

understanding the regional differences in basic environmental and biological conditions such as thermohaline properties, nutrients, chlorophyll-*a,* etc. [4–8].

The Pacific Ocean, due to its vastness extending from tropical regions to both the boundaries of polar oceans, is subjected to have distinctive climatic conditions at its various regions [4]. In the northwestern Pacific Ocean (NPO), the physico-chemical conditions are mainly influenced by North Equatorial Current, Kuroshio Current, Tsushima Warm Current, and pelagic/coastal water intrusions at the coastal zones in the East China Sea (ECS). In terms of phytoplankton community, autotrophic picoplankton communities were more abundant in the NPO than large-sized phytoplankton and heterotrophic bacteria [9,10]. Lee et al. [10] reported that autotrophic plankton (mainly pico-sized phytoplankton) comprised up to 80% of the total phytoplankton biomass in the euphotic zone, whereas the contribution of heterotrophic bacteria was 6–21% of phytoplankton biomass in the NPO. Furthermore, a few research works reported that picoplankton including pico-sized cyanobacteria (*Prochlorococcus* and *Synechococcus*) have been a significant component of biomass and primary production in the subtropical and tropical Pacific Ocean [11–17].

In general, *Prochlorococcus* exhibits a wide adaptation for the variability in light or nutrient conditions, whereas they are often observed to be limited by high temperature in the water column [16,18–23]. Other cyanobacterial species such as *Synechococcus* have relatively eurythermal characteristics and extend to low salinity waters. Hence, *Synechococcus* are widely distributed around the world ocean from tropical to polar waters with a high biomass in the upper euphotic zone [22,24]. Recently, it was reported that abundance and distribution of the small-sized autotrophic plankton communities including cyanobacteria, *Prochlorococcus,* and *Synechococcus*, increase in various oceans with global warming, which indicates that this issue is not limited to a local scale anymore [25,26].

Normally, the primary production by picophytoplankton (i.e., picocyanobacteria and picoeukaryotes) is estimated through filter fractionation [7,27–31]. However, it is difficult to distinguish carbon and/or nitrogen uptake rates between picocyanobacteria (*Prochlorococcus* and *Synechococcus*) and picoeukaryotes. Moreover, the fractionation in natural samples makes it difficult to physically separate picophytoplankton from heterotrophic bacteria, in case of nitrogen uptake [32]. Previous studies used metabolic inhibitors to partition the relative contributions of eukaryotes and prokaryotes in marine systems [33–35]. For example, cycloheximide inhibits the function of the 80-S ribosome of eukaryotes [36], whereas streptomycin specifically inhibits protein synthesis on the 70-S ribosome in bacteria [37]. Thus, these metabolic inhibitors could be effective in separating target organisms from non-target organisms [32]. Middelburg and Nieuwenhuize [34,35] successfully partitioned autotrophic and heterotrophic activity using metabolic inhibitors. Fouilland et al. [32] also applied metabolic inhibitors to partition the uptakes of nitrate, ammonium, and urea between prokaryotic and eukaryotic phytoplankton. As a result, they quantitatively reported the contribution of heterotrophic bacteria to nitrogen uptake [32].

In present study, a metabolic inhibitor (cycloheximide) based on the method of Fouilland et al. [32] was applied to measure picocyanobacterial contribution to the total primary production, since the inhibitor can remove the eukaryotes and directly determine only carbon and nitrogen uptake rates by picocyanobacteria in the samples. The objectives of this study were as follows: (1) to determine carbon and nitrogen uptake rates by picocyanobacteria and (2) to evaluate picocyanobacterial contribution to the primary production in the regions (subtropical-temperate Pacific region and tropical Pacific region) in the NPO.

#### **2. Materials and Methods**

#### *2.1. Study Area and Sample Collection*

The present study was conducted at 9 stations in the NPO during the POSEIDON cruise (13 May–4 June 2014) (Figure 1). In order to understand characteristics of primary productivity under the different environmental conditions in the NPO, our productivity stations were divided into two regions; the subtropical and temperate Pacific region (SP; A89 and A50), mainly affected by the coastal water of the ECS and tropical Pacific region

(TP; F10, F06, F03, F01, P03, P07, and P11), which are influenced by the Tsushima Warm Current, North Equatorial Current, and Kuroshio Current. Seawater samples from water column up to 1% light depths were collected using 10 L Niskin sampling bottles on the R/V Onnuri of the Korea Institute of Ocean Science and Technology (KIOST, Busan, Korea). The physical parameters (temperature and salinity) were determined using a Sea-Bird 911plus system (Sea-Bird, Inc., Brooklyn, NY, USA).

**Figure 1.** Sampling station in the two sampled regions of the northwestern Pacific Ocean; TP: tropical Pacific, SP: subtropical and temperate Pacific.

#### *2.2. Measurements for Biomass and Abundance of Phytoplankton and Nutrient Concentrations*

Chlorophyll-*a* (Chl-*a*) and phytoplankton abundance, as well as nutrient concentrations were measured at the 9 productivity stations. One liter of seawater for Chl-*a* concentrations presenting for phytoplankton biomass was filtered onto 25 mm GF/F filters. The filters were stored in a deep freezer and extracted within a month using 6 mL of 95% acetone by the method of Parsons et al. [38]. The final extracts were analyzed using a 10 AU fluorometer (Turner Design Inc., San Jose, CA, USA). Seawater samples for the enumeration and identification of major pico-sized phytoplankton groups (<2 μm) were counted by flow cytometry (BD Accury C6, BD Biosciences Inc., Mountain View, CA, USA) after staining with mixture of yellow-green and UV beads by the method of Olson et al. [39]. Nutrient data were provided by KIOST based on the standard colorimetric procedure [38].

#### *2.3. Carbon and Nitrogen Uptake Rate Measurements*

Total carbon and nitrogen uptake rates were measured at the 9 different stations using a 13C-15N dual isotope tracer technique that has been applied in various oceans [27,40–43]. Seawater samples at 6 light depths (100%, 50%, 30%, 12%, 5%, and 1% of light intensity at surface) were collected from Niskin samplers to 1 L polycarbonate bottles covered with different LEE film screens (LEE Filters, Inc., Hampsire, UK) that corresponded to the different light levels. Further, the water samples were injected with enriched solutions of 13C (NaH13CO3) and 15N (K15NO3 or 15NH4Cl) (less than 10% of the ambient concentrations) followed by deck incubation for 4 h. Hourly picocyanobacterial carbon and nitrogen uptake rates were measured at all the stations except station A50 in the SP using the dual isotope technique. For measuring the picocyanobacterial carbon and nitrogen uptake rates, the autotrophic eukaryotes were inhibited by a metabolic inhibitor (cycloheximide), which blocks

the cytoplasmic protein biosynthesis in 80-S ribosome of phytoplankton (eukaryotes) [32]. All the bottles were incubated in deck incubators along with primary productivity sample bottles for 4 h.

After incubation, seawater samples (0.5 L) for the carbon and nitrogen uptake rates were filtered onto the pre-combusted 25 mm GF/F filters. The filters were immediately frozen in the deep freezer until the analysis. Prior to the mass spectrometric analysis, samples were thawed, dried overnight, and packed in tin capsules. Particulate organic carbon (POC)/nitrogen (PON) and the amount of 13C and 15N were determined by Finnigan Delta + XL mass spectrometer at the Stable Isotope Facility, University of Alaska Fairbanks (UAF), USA after HCl fuming during 24 h for removing carbonate. Samples of analyzed total carbon and nitrogen uptake rates were calculated by using the methods of Hama et al. [44] and Dugdale and Goering [45]. Dark carbon uptake rates were subtracted for considering the heterotrophic bacterial process [46]. Because the carbon uptake rates from dark bottles were subtracted from the light bottles for removal of heterotrophic productivity without light, we assumed that only the contributions of autotrophic bacterial (i.e., picocyanobacterial) communities were obtained for the primary productivity.

#### **3. Results**

#### *3.1. Physiochemical Structures in Water Column*

Vertical profiles of temperature and salinity from all the stations in the NPO are shown in Figure 2. Surface temperature and salinity at the stations in the TP were higher than those in the SP. The average temperature and salinity in the upper water column were 17.3 ◦C and 33.2 psu in the SP, respectively, whereas they were 29.1 ◦C (S.D. = ±0.92 ◦C) and 34.8 psu (S.D. = ±0.52 psu) for TP, respectively.

**Figure 2.** Vertical profiles of temperature (**a**), salinity (**b**) in the northwestern Pacific Ocean. Solid line represents average temperature and salinity in the TP and SP regions, respectively.

Generally, nutrient concentrations were depleted in both TP and SP regions except for 1% light depths (Figure 3). The mean nitrate concentrations within the euphotic zone were 0.13 (S.D. = ±0.35 μM) and 0.84 μM (S.D. = ±1.80 μM) in the TP and the SP, respectively. Ammonium concentrations were consistently low at euphotic zones of all the stations. The mean ammonium concentrations in the TP and the SP were 0.14 (S.D. = ±0.07 μM) and 0.18 μM (S.D. = ±0.03 μM), respectively. The euphotic depths at the stations in the TP were deeper than those in the SP (*t*-test, *p* < 0.05). The mean euphotic depths were 127.4 m (S.D. = ±16.5 m) and 35.0 m in the TP and the SP, respectively (Table 1).

**Figure 3.** Vertical profiles of nitrate (**a**) and ammonium (**b**) concentrations averaged from each region (TP, closed circles; SP, open circles). SDs are shown by bars. Data courtesy of KIOST.

**Table 1.** The environmental conditions in the TP and SP regions of the northwestern Pacific Ocean.


#### *3.2. Distribution of Phytoplankton in Water Column*

The average euphotic depth-integral total Chl-*a* concentrations were 15.0 (S.D. = ±6.6 mg Chl-*a* m−2) and 18.1 mg Chl-*a* m−<sup>2</sup> in the TP and SP, respectively (Table 2). Although the integral total Chl-*a* concentrations were not significantly different between the TP and SP locations (Table 2), the vertical distributions of Chl-*a* were obviously different between the two locations (Figure 4). Deep chlorophyll maximum (DCM) layers, in which the Chl-*a* concentrations were significantly (*t*-test, *p* < 0.01) higher compared to those at the surface, were observed at the bottom (1% light depth) of the euphotic zone in the TP. However, no substantial DCM layers were found in the SP (Figure 4).

The cell abundance of autotrophic plankton, including picocyanobacteria (*Synechococcus* and *Prochlorococcus*) and picoeukaryotes, were different between the TP and the SP (Figure 5). The average depth-integral abundance *of Synechococcus*, *Prochlorococcus*, and picoeukaryotes in the TP were 1.85 × 1011 (S.D. = ±0.64 × 1011 cells m−2), 0.64 × 1013 (S.D. = ±0.10 × <sup>10</sup><sup>13</sup> cells m<sup>−</sup>2), and 0.96 × 1011 cells m−<sup>2</sup> (S.D. = ±0.31 × 1011 cells m<sup>−</sup>2), respectively (Figure 5). In the SP, the cell abundance of *Synechococcus* and picoeukaryotes were 14.4 × 1011 and 4.28 × <sup>10</sup><sup>11</sup> cells m<sup>−</sup>2, respectively. No *Prochlorococcus* were generally found in the SP except some at 46 m of A89 (Figure 5). Consequently, *Prochlorococcus* (mean ± S.D. = 95.7 ± 1.4%), *Synechococcus* (mean ± S.D. = 2.8 ± 1.0%), and picoeukaryotes (mean ± S.D. = 1.4 ± 0.4%) contributed the plankton community in the TP. In contrast, *Synechococcus* accounted for 93.5% and 50.8%, whereas picoeukaryotes were 5.2% and 49.2% at A89 and A50 in the SP, respectively.


**Table 2.** Chlorophyll-*a*, C/N ratio, *f*-ratio, carbon, and nitrogen (nitrate and ammonium) uptake rates by total phytoplankton communities in the TP and SP regions of the northwestern Pacific Ocean.

#### *3.3. Total Carbon and Nitrogen Uptake Rates in the NPO*

The largest carbon uptake rate was at 100% light depth at each station in the SP, whereas in the TP, the largest rate was observed at 30–50% light depths (Figure 6a). The lowest carbon uptake rate was found at the chlorophyll-maximum layer corresponding to 1% light depth in the SP. The average rates of carbon uptake at each light depth were significantly higher in the SP (*t*-test, *p* < 0.05) than in the TP. The ranges of depth-integrated carbon uptake rates in the TP and SP were 3.29–16.89 mg C m−2·h−<sup>1</sup> with an average of 11.66 mg C m−2·h−<sup>1</sup> and 9.17–32.54 mg C m−2·h−<sup>1</sup> with an average of 20.85 mg C m−2·h<sup>−</sup>1, respectively (Figure 7a and Table 2). Based on our dark carbon uptake rates in this study, the heterotrophic contributions to the total primary productions were 1.5% (S.D. = ±0.7%) and 8.7% (S.D. = ±12.8%) for the SP and the TP, respectively.

**Figure 5.** Integrated abundance of *Prochlororoccus* (**a**), *Synechococcus* (**b**), and picoeukaryotes (**c**) in the euphotic zone. Biomass of *Prochlororoccus*, *Synechococcus*, and picoeukaryotes in the euphotic zone (**d**) in the euphotic zone.

**Figure 6.** Vertical profiles of the total carbon and nitrogen absolute uptake rates (TP, closed triangles; A89, closed circles; A50, closed squares) and picocyanobacterial carbon and nitrogen absolute uptake rates (TP, open triangles; A89, open circles) in the TP and SP regions in the North Pacific Ocean. SDs are shown by bars. Carbon uptake rates (**a**), Nitrate uptake rates (**b**), and Ammonium uptake rates (**c**).

**Figure 7.** Regional distribution of total carbon and nitrogen uptake rates ((**a**), left) and picocyanobacterial carbon and nitrogen uptake rates ((**b**), right) in the northwestern Pacific Ocean. Bars with diagonal stripes indicate carbon and nitrogen uptake rates of picocyanobacterial communities.

Nitrogen uptake rates did not show any significant pattern with light depths as carbon uptake rates (Figure 6b,c). The depth-integrated nitrogen (nitrate+ammonium) uptake rates in the TP and SP ranged from 6.52 to 17.96 mg N m−2·h−<sup>1</sup> with an average of 10.11 mg N m−2·h−<sup>1</sup> and from 2.98 mg N m−2·h−<sup>1</sup> to 6.50 mg N m−2·h−<sup>1</sup> with an average of 4.74 mg N m−2·h−1, respectively (Figure 7b and Table 2). In detail, the mean of nitrate and ammonium uptake rates in the TP were 1.06 mg N m−2·h−<sup>1</sup> and 9.05 mg N m−2·h<sup>−</sup>1, respectively, whereas those in the SP were 0.69 mg N m−2·h−<sup>1</sup> and 4.05 mg N m−2·h−1, respectively. Ammonium uptake rates were substantially higher than nitrate uptake rates in both regions.

#### *3.4. Picocyanobacterial Carbon and Nitrogen Uptakes in the NPO*

The average rates of picocyanobacterial carbon uptakes showed similar trends like vertical abundance profiles of these predominant species (Figure 8). Vertical profiles of picocyanobacterial carbon, nitrate, and ammonium uptake rates showed similar trends as those of the uptake rates by total phytoplankton community at each light depth (Figure 6). Picocyanobacterial carbon uptake rates integrated from the euphotic depths were 5.31 mg C m−2·h−<sup>1</sup> (S.D. = ±2.16 mg C m−2·h<sup>−</sup>1) in the TP, whereas the integrated carbon uptake rates by picocyanobacteria at the A89 (SP) was 22.8 mg C m−2·h−<sup>1</sup> (Figure 9a). The average rates of picocyanobacterial carbon uptake at each light gradient were significantly higher in the SP (Table 3; *t*-test, *p* < 0.05). Integrated hourly picocyanobacterial nitrogen uptake rates were 6.32–16.16 mg N m−2·h−<sup>1</sup> with an average of 9.10 mg N m−2·h−<sup>1</sup> in the TP and 4.12 mg N m−2·h−<sup>1</sup> at the A89 in the SP (Figures 7b and 9b). The average nitrate uptake rates by picocyanobacterial communities in the TP and A89 (SP) were 0.21 mg N <sup>m</sup>−2·h−<sup>1</sup> (S.D. = ±0.20 mg N m−2·h−1) and 0.40 mg N m−2·h−1, respectively, whereas the average ammonium uptake rates of picocyanobacterial communities were 8.89 mg N <sup>m</sup>−2·h−<sup>1</sup> (S.D. = ±3.18 mg N m−2·h−1) and 3.72 mg N m−2·h−1, respectively (Table 3). Picocyanobacterial ammonium uptake rates were more than the nitrate uptake rates in the NPO (Figure 9c,d).

**Figure 8.** Comparison of the uptake rates for total (closed circles) and bacterial (open circles) carbon uptake with the abundances of the predominant species (closed triangles) in the northwestern Pacific Ocean. TP (**a**) and SP (**b**). SDs are shown by bars.

**Figure 9.** Picocyanobacterial contribution to total carbon and nitrogen uptake rates (primary productivity) in the TP and SP regions of the northwestern Pacific Ocean. Unicolor bars indicate total uptake of each uptake rate, whereas other bars with diagonal stripes indicate picocyanobacterial uptake rates. SDs are shown by bars. Integrated nitrogen uptake rates (**a**), Integrated carbon uptake rates (**b**), Integrated nitrate uptake rates (**c**), and Integrated ammonium uptake rates (**d**).


**Table 3.** Carbon and nitrogen (nitrate and ammonium) uptake rates by picocyanobacterial communities in the TP and SP regions of the northwestern Pacific Ocean.

#### **4. Discussion**

In this study, the abundance of picophytoplankton was different between the TP and the SP (Figure 5). *Prochlorococcus* were not found but *Synechococcus* and picoeukaryotes co-occurred in the SP, whereas *Prochlorococcus* were the dominant picophytoplankton population in the TP. The difference in abundance of dominant population observed in the TP and the SP might be due to different physico-chemical properties as the result of the major currents. Because distribution and abundance of phytoplankton in the euphotic zone can be altered by the hydrological conditions of the seawater, these physiochemical properties are determined by the major currents [47–50]. In fact, the TP is directly influenced by North Equatorial Current, whereas the SP is influenced mainly by the Kuroshio Current, Tsushima Warm Current, and coastal fresh water, respectively [16,51]. According to Choi et al. [24], the picocyanobacterial distribution in the NPO was distinguished along the physical and chemical properties of the water masses. In this study, the water depth in the SP was shallow and had lower temperature and salinity than the TP (Figure 2), whereas the TP was a typical high-temperature oligotrophic water. Since *Prochlorococcus* have been found to be more abundant in the oligotrophic conditions because of their ecological plasticity with respect to requirements of nutrients and light [18,52–55], *Prochlorococcus* could be dominant under temperature and oligotrophic TP. According to previous studies [12,55], *Synechococcus* are usually dominant in the mesotrophic seawater or shallow waters. Thus, *Synechococcus* and picoeukaryotes could be abundant in relatively mesotrophic and shallow SP, which is consistent with previous study from the western Pacific Ocean [54].

In terms of carbon biomasses estimated from the average carbon contents [56,57], *Prochlorococcus* contributed 66.1% to the total phytoplankton in the TP (Figure 5d). In the SP, *Synechococcus* were 76.4% at A89 and picoeukaryotes were 84.0% at A50, respectively. Especially, the carbon biomass contribution of picoeukaryotes was higher than that of *Synechococcus* at the A50, although picoeukaryotes had lower cell abundances than *Synechococcus*, because picoeukaryotes have higher carbon contents compared to *Synechococcus*.

Based on the hourly carbon uptake rates by total phytoplankton, which were estimated in this study, the average daily primary productivities were 0.15 g C m−2·d−<sup>1</sup> (S.D. = ±0.06 g C m−2·d−1) and 0.29 g C m−2·d−<sup>1</sup> in the TP and SP, respectively (Table 4). Our daily primary productivities fell within the range of previous studies in both regions [4,5,51]. In the TP, Taniguchi [4] reported 0.09 g C m−2·d−<sup>1</sup> in the North Equatorial Current (Table 4). Kwak et al. [5] observed a relatively higher range of daily primary productivity from 0.17 to 0.23 g C m−2·d−<sup>1</sup> in the western Pacific Ocean (Table 4). For the SP, the average daily primary productivity obtained from this study is comparable with those from other previous studies [5,51]. Gong et al. [51] reported 0.31 ± 0.16 g C m−2·d−<sup>1</sup> and 0.52 ± 0.32 g C m−2·d−<sup>1</sup> during early spring and summer, respectively (Table 4). Our daily primary productivity is nearly identical to the daily production (0.28 g C m−2·d−1) reported by Kwak et al. [5] (Table 4).


**Table 4.** Comparison of daily primary productivity with previous studies in the northwestern Pacific Ocean.

Daily total ammonium uptake rates were calculated by multiplying hourly nitrogen uptake rates and each photoperiod [58] in this study. The average daily total ammonium uptake rates were higher than total nitrate uptakes in the euphotic zone of both regions. The average daily total ammonium and nitrate uptake rates were 0.16 g N <sup>m</sup>−2·d−<sup>1</sup> (S.D. = ±0.06 g N m−2·d<sup>−</sup>1) and 0.01 g N m−2·d−<sup>1</sup> (S.D. = ±0.01 g N m−2·d<sup>−</sup>1) in the TP, respectively (Table 4). In the SP, the daily total ammonium and nitrate uptake rates were 0.07 g N·m−<sup>2</sup> <sup>d</sup>−<sup>1</sup> and 0.01 g N m−2·d−<sup>1</sup> at A89, respectively (Table 4). Accordingly, average *f*-ratios ([nitrate uptake rate]/[nitrate+ammonium uptake rate], [59]) were 0.10 (S.D. = ±0.03) and 0.13 in the TP and SP (Table 2), respectively, as a result of prominent ammonium uptakes. This indicates that a main nitrogen source for growth of total autotrophic plankton was mainly supported by regenerated ammonium in this region during our observation period.

In this study, the average picocyanobacterial contributions to the total primary productivity accounted for 45.2% (S.D. = ±4.8%) in the TP and 70.2% in the A89 (SP) (Figure 9a). Glover et al. [12] reported that contribution of *Synechococcus* to the total primary production, which varies from 6% to 46% in different water masses in the northwestern Atlantic Ocean. In contrast, Liu et al. [15] observed a high contribution of *Prochlorococcus* up to 82% to the primary production at Station ALOHA in the subtropical North Pacific Ocean.

Based on each nitrate and ammonium uptake rate, the average picocyanobacterial *f*-ratios were 0.02 (S.D. = ±0.01) and 0.10 in the TP and A89 (SP), respectively (Table 3). This result suggests that picocyanobacterial communities strongly depended on a regenerated nitrogen source (i.e., ammonium) or N2 fixation in our study area during the observation period. From the comparison of *f*-ratios between the total phytoplankton and picocyanobacterial communities, we found that picocyanobacterial *f*-ratios were substantially lower compared to those of the total phytoplankton communities in the two regions (Tables 2 and 3).

Depth integrated hourly nitrogen uptake rates of picocyanobacterial communities were 9.10 mg N m−2·h−<sup>1</sup> (S.D. = ±3.73 mg N m−2·h−1) and 4.12 mg N m−2·h−<sup>1</sup> in the TP and the A89 (SP), respectively (Figure 9). The total nitrogen uptake rates at the same regions were 10.11 mg N m−2·h−<sup>1</sup> and 6.50 mg N m−2·h<sup>−</sup>1, respectively. Given the nitrogen uptake rates, the average picocyanobacterial contributions to the total nitrogen uptake rates were 90.2% (S.D. = ±5.3%) and 63.5% in the TP and the A89 (SP), respectively, in this study. These picocyanobacterial contributions to the total nitrogen uptake rates

are substantially higher than those to the total carbon uptake rates of the total plankton communities in TP. However, the nitrogen utilization by heterotrophic bacteria can be important since the heterotrophic bacteria account for a large fraction of nitrogen uptake in the global ocean including the Arctic Ocean [32,60,61]. Although we are incapable of distinguishing each contribution for nitrogen uptake between heterotrophic bacteria and picocyanobacteria from this study using a metabolic inhibitor (cycloheximide) blocking only photosynthetic eukaryotes, we need to consider the heterotrophic bacterial nitrogen utilization from the nitrogen contributions in future studies. Apart from this, the potential N2 fixation by cyanobacteria can vary with environmental conditions, particularly nutrient stoichiometry [62]. When the NH4 <sup>+</sup> concentration is relatively higher than phosphorous, the nitrogenase activity can be stopped and photosynthesis can be activated. On the other hand, if the NH4 +:P ratio is lower than the Redfield's ratio, N2 fixation can be a more major process than primary production. So, the contribution of picocyanobacteria towards the total primary production can be underestimated in that case. Furthermore, when autotrophic primary production is stopped by the inhibitor, the competition for nutrients in the samples may be lesser than one with autotrophic activity and, hence, the primary production rates by picocyanobacteria could be overestimated. Currently, there are some uncertainties for estimating picocyanobacterial contributions to the primary production and nitrogen uptake rates. Therefore, the combined approaches using several different applications are highly important for further future studies on cyanobacterial ecological roles in various oceans.

#### **5. Summary and Conclusions**

In this study, we measured picocyanobacterial contribution to the carbon and nitrogen uptake rates by total phytoplankton in the regions of the NPO. There are different abundances and biomasses of dominant species in the TP and the SP regions. *Prochlorococcus* and *Synechococcus* were abundant in the TP and the SP regions, respectively. The picocyanobacterial contributed 45.2% (S.D. = ±4.8%) to primary production by total picophytoplankton in the TP, whereas the picocyanobacterial contribution was about 70.2% in the SP. The picocyanobacterial community is believed to be more important in terms of nitrogen uptake rates since they could contribute about 90.2% (S.D. = ±5.3%) to the total nitrogen uptake rates by picophytoplankton in both regions.

The importance of picoplankton including cyanobacteria has been raised continuously in research regarding the global ocean [25,63,64]. In particular, the picocyanobacterial *Prochlorococcus* and *Synechococcus* have significant ecological positions in the biomass and production in the ocean, but the relative contributions of these organisms to primary productivity are different under various environmental conditions [22]. Under the global warming scenario, picoplankton contribution relative to large plankton will increase in the strongly stratified upper ocean [3]. This climate change will result in increasing distribution, abundance, and contributions to primary production of picocyanobacteria, especially in tropical and subtropical oceans and, consequently, will cause large impacts on the global ocean ecosystem and biogeochemical cycles [26]. Therefore, more measurements under various environmental conditions are needed to better understand the role of picocyanobacterial in the ecosystem.

**Author Contributions:** Conceptualization, H.-W.L., J.-H.N. and S.-H.L.; methodology, H.-W.L., P.S.B. and S.-H.L.; validation, H.-W.L., J.-H.N. and S.-H.L.; formal analysis, H.-W.L., J.-H.N., D.-H.C., J.-J.K., J.-H.L. and K.-W.K.; investigation, H.-W.L., J.-H.N. and D.-H.C.; writing—original draft preparation, H.-W.L.; writing—review and editing, M.Y., P.S.B. and S.-H.L.; visualization, J.-J.K., J.-H.L., K.-W.K. and H.-K.J.; supervision, S.-H.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT; NRF-2019R1A2C1003515) and partly by the Korea Institute of Ocean Science and Technology (KIOST; PE99923).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank the captain and crew members of the R/V *Onnuri* for their outstanding assistance. Especially, we very much appreciate the KIOST for providing CTD and nutrient data. Finally, we thank the anonymous reviewers who greatly improved an earlier version of manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

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