**Temporal and Spatial Variations of the Biochemical Composition of Phytoplankton and Potential Food Material (FM) in Jaran Bay, South Korea**

**Jae Hyung Lee 1, Won-Chan Lee 2, Hyung Chul Kim 2, Naeun Jo 3, Kwanwoo Kim 3, Dabin Lee 3, Jae Joong Kang 3, Bo-Ram Sim 2, Jae-Il Kwon <sup>4</sup> and Sang Heon Lee 3,\***


Received: 22 September 2020; Accepted: 30 October 2020; Published: 4 November 2020

**Abstract:**Foodmaterial (FM)derived from biochemical components (e.g., proteins,lipids, and carbohydrates) of phytoplankton can provide important quantitative and qualitative information of the food available to filter-feeding animals. The main objective of this study was to observe the seasonal and spatial variations of the biochemical compositions of phytoplankton and to identify the major controlling factors of FM as a primary food source in Jaran Bay, a large shellfish aquaculture site in South Korea. Base d on monthly sampling conducted during 2016, significant monthly variations in the depth-integrated concentrations of major inorganic nutrients and chlorophyll *a* within the euphotic water column and a predominance (49.9 ± 18.7%) of micro-sized phytoplankton (>20 μm) were observed in Jaran Bay. Carb ohydrates were the dominant biochemical component (51.8 ± 8.7%), followed by lipids (27.3 ± 3.8%) and proteins (20.9 ± 7.4%), during the study period. The biochemical compositions and average monthly FM levels (411.7 <sup>±</sup> 93.0 mg m−3) in Jaran Bay were not consistent among different bays in the southern coastal region of South Korea, possibly due to differences in controlling factors, such as environmental and biological factors. Acco rding to the results from multiple linear regression, the variations in FM could be explained by the relatively large phytoplankton and the P\* (PO4 <sup>3</sup><sup>−</sup> <sup>−</sup> 1/16 <sup>×</sup> NO3 <sup>−</sup>) and NH4 <sup>+</sup> concentrations in Jaran Bay. The macromolecular compositions and FM, as alternatives food source materials, should be monitored in Jaran Bay due to recent changes in nutrient concentrations and phytoplankton communities.

**Keywords:** phytoplankton; biochemical compositions; carbohydrates; proteins; lipids; Jaran Bay

#### **1. Introduction**

Bays are important aquatic systems that provide food resources for fisheries and aquaculture since they provide habitats and prey for various marine organisms. Rece ntly, mollusk farming, including bivalves, has contributed greatly to global farming production [1]. The present study site, Jaran Bay, is one of the largest shellfish aquaculture regions for oysters and scallops in South Korea [2], and these filter-feeding oysters and scallops feed mainly on water-dwelling phytoplankton for their growth and reproduction [3,4].

The growth and physiological conditions of phytoplankton can vary depending on environmental conditions [5–7]. In particular, phytoplankton synthesize biochemical components through photosynthesis and are therefore highly dependent on light conditions and quality [8–10], temperature [11], species composition [12,13] and nutrient availability [5,8,14]. Rece ntly, Lee et al. [5] reported that dissolved inorganic nitrogen loading from river discharge is a major factor that controls the photosynthetic biochemical compositions (e.g., carbohydrates, proteins and lipids) of phytoplankton in Gwangyang Bay. More over, the community structure and, consequently, biochemical composition of phytoplankton can be altered by differences in nutrient inputs due to river discharge [7]. Diff erences in the biochemical compositions of phytoplankton can lead to differences in nutritional qualities for potential consumers [5,15–17]. Ther efore, the biochemical compositions of phytoplankton, as natural food resources, are very important for phytoplankton-grazing herbivores. In agreement with this finding, Yun et al. [16] reported a strong positive relationship between the lipid composition in phytoplankton and protein content in the mesozooplankton community in the northern Chukchi Sea, indicating that a high lipid content in phytoplankton can be important for protein synthesis for zooplankton growth.

Food material (FM) is represented as the sum of the concentrations of proteins, lipids and carbohydrates [18,19]. FM indicates the quantity of food that is available to potential consumers [19] and is also used as a food index of food quality [20]. Seas onal and spatial variations in the quantity and quality of the natural diet available to filter feeders could be important for their grazing characteristics [20]. Nava rro and Thompson [20] observed that the seasonal trends in FM dynamics are closely correlated with the trends of the chlorophyll *a* concentration in Logy Bay, southeast Newfoundland, Canada. Rece ntly, Kang et al. [21] found that small-sized cells of phytoplankton could assimilate higher amounts of FM per unit of chlorophyll *a* concentration compared to large-sized cells of phytoplankton in the East/Japan Sea based on size fractionation filtering methods. Simi lar results from Gwangyang Bay, Korea, were also in agreement with this consistent observation [7].

Previously, most biochemical composition studies have been conducted once a year or, at most, seasonally [5,7,21]. Cons idering the importance of phytoplankton as a primary food source for filter-feeding aquaculture animals, the present study aimed to observe monthly and spatial variations in biochemical compositions as a food quality indicator of phytoplankton and to determine the major environmental controlling factors of FM available to shellfish, such as oysters and scallops, growing in Jaran Bay as a large aquaculture site in South Korea.

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

#### *2.1. Water Sampling and Analysis*

Using a 5 L Niskin sampler (General Oceanics Inc., Miami, FL, USA), water samples for the determination of the nutrient and chlorophyll *a* concentrations were obtained from three different light depths (e.g., 100, 30 and 1% of photosynthetic active radiation (PAR), determined by using a Secchi disk) at seven different stations (Figure 1). The study area Jaran Bay is a relatively shallow coastal bay with an average water depth of 10 m [2]. Samp ling was conducted monthly from January to December 2016. The depth-averaged values were obtained from the three light depths (e.g., 100, 30 and 1% PAR), and monthly observed values were obtained from all depths and stations.

The water samples (0.2 L) used for determining the dissolved inorganic nutrient concentrations were filtered through a 47 mm GF/F filters (0.7-μm pore size, Whatman, Maidstone, UK), and the filtrates were stored at −20 ◦C for further analysis using an Auto Analyzer (Quaatro, Bran+Luebbe, Germany) at the National Institute of Fisheries Science (NIFS), Korea. For determining the total chlorophyll *a* concentration as a proxy for biomass, water samples (0.2 L) were filtered through 25-mm GF/F filters (0.7-μm pore size, Whatman, Maidstone, UK). The water samples (0.6 L) were filtered sequentially through 47-mm Nucleopore filters (20- and 2-μm) and 47-mm GF/F filters (0.7-μm pore size, Whatman, Maidstone, UK) to determine size-fractionated chlorophyll *a* concentrations of different cell-sized phytoplankton communities [22,23]. The filters retained chlorophyll *a* and were immediately frozen and preserved at −70 ◦C for chlorophyll *a* extraction at the home laboratory at Pusan National University, South Korea. The chlorophyll *a* concentrations were measured using a previously calibrated

10-AU fluorometer (Turner Designs, San Jose, CA, USA) after extraction (approximately 24 h, 4 ◦C) with 90% acetone and centrifugation at 4480 g for 20 min [24].

**Figure 1.** Sampling stations in Jaran Bay, South Sea of Korea.

The water samples that were used for determining the macromolecular compositions (e.g., carbohydrates, proteins and lipids) of particulate organic matter (POM) were filtered through 47 mm GF/F filters, and the filters were immediately preserved at−70 ◦C until further spectrophotometric analysis. The samples were filtered under a constant vacuum (<10 cm Hg) because live cells could be damaged during the strong vacuum filtration [23]. Carb ohydrate extraction was performed by following Dubois et al. [25]. The preground POM-retained filter paper was transferred to a polypropylene (PP) tube. Afte r the addition of 1 mL deionized water, 1 mL of a 5% phenol solution was added and allowed to rest for 40 min. Then , 5 mL of sulfuric acid (H2SO4) was added and allowed to stand for 10 min. Next , the solutions were centrifuged at 3430 g for 10 min. The absorbance of the supernatant was measured at 490 nm. A glucose solution (1 mg mL<sup>−</sup>1, Sigma Aldrich) was used as the standard for determining the carbohydrate concentration.

For protein extraction, each preground sample filter was transferred to a 12-mL glass tube with 1 mL deionized water (DH2O) and was added to 5 mL of an alkaline copper solution. Afte r the solution was well mixed, 0.5 mL of diluted Folin–Ciocalteu phenol reagent (1:1, *v*/*v*) were added and allowed to sit for 1 h 30 min at room temperature. Then , the solutions were centrifuged for 10 min at 2520 g. The absorbance of the supernatant was measured at 750 nm. Bovi ne serum albumin (2 mg mL−1, Sigma Aldrich) was used as the standard for determining the protein concentration based on previous works in various oceans [5–7,16,17,21].

Last, the filters used for lipid extraction were transferred into a 16-mL glass tubes, ground with 3 mL of chloroform-methanol (1:2, *v*/*v*) and stored at 4 ◦C for 1 h. Afte r the solution was homogenized with 4 mL of DH2O, the lower (chloroform) phase of the solution was dried at 40 ◦C for 48 h and then heated at 200 ◦C for 15 min with 2 mL of H2SO4. An additional 3 mL of DH2O was added to the chloroform phase in the glass tubes and then they were allowed to rest for 10 min. The absorbance of the supernatant was measured at 375 nm, and a tripalmitin solution (Sigma Aldrich) was used as the standard for determining the lipid concentration. Afte r each extraction process, the concentration of each biochemical component was determined using a UV spectrophotometer (Hitachi-UH5300, Hitachi, Tokyo, Japan).

#### *2.2. Statistical Analysis*

Principal component analysis (PCA) was performed on our field-obtained data of the chemical and biological variables (i.e., nutrient concentrations and phytoplankton biomass) for their relative significance and interrelationship patterns among the various biochemical conditions measured during our sampling period. Bart lett's sphericity tests were used to determine the validity of the PCA (*p* < 0.01) [26,27]. Fact or analysis was conducted to obtain various factors selected by the principal component method with varimax rotation [28]. Due to the strong dependency between PO4 <sup>3</sup><sup>−</sup> and NO3 <sup>−</sup> (*r* = 0.56, *p* < 0.01; Pearson's correlation coefficient), PO4 <sup>3</sup><sup>−</sup> was excluded but included P\* (PO4 <sup>3</sup><sup>−</sup> <sup>−</sup> <sup>1</sup>/<sup>16</sup> <sup>×</sup> NO3 <sup>−</sup>) in the PCA. P \* reflects the excess (or deficiency) of PO4 <sup>3</sup><sup>−</sup> versus NO3 − [29,30].

To determine the major factors controlling the macromolecular composition and FM of POM, multiple linear regression analysis was conducted in this study based on the PCA results. The multiple linear regression equation of Pedhazur [31] is as follows:

$$Y = a + b\_1 X\_1 + \dots + b\_k X\_k + e \tag{1}$$

where *Y* denotes a dependent variable and the FM of POM is estimated from the independent variables (predictors), *X*1··· *Xk*. Para meter α is a constant, *b*1··· *bk* are the regression coefficients for the predictors (FM in this study), and *e* is an error term.

Insignificant variables for the controlling the FM variation were stepwise eliminated from the model by stepwise variable selection after multiple linear regression analysis. Stat istical analysis was performed with IBM SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA). *t* statistics were conducted for testing the regression coefficients and values of the coefficient of determination (R2) were obtained for measure of goodness of fit for the FM in this study.

#### **3. Results**

#### *3.1. Monthly Concentrations of Nutrients and Chlorophyll a*

The monthly depth-integrated nutrient concentrations within the euphotic water column from 100 to 1% light depths during the present study period are summarized in Table 1. The ranges of the NH4 <sup>+</sup>, NO2 <sup>−</sup> + NO3 <sup>−</sup>, PO4 <sup>3</sup><sup>−</sup> and Si(OH)4 <sup>2</sup><sup>−</sup> concentrations were 4.0−47.5, 10.9−80.0, 0.5−6.0 and 20.9−166.9 μm, respectively, in Jaran Bay from January to December 2016. The concentration ranges varied significantly during the observation period, and the highest concentrations were detected in September, except for the Si(OH)4 <sup>2</sup><sup>−</sup> concentrations, which showed the largest peak in June and a secondary peak in September.


**Table 1.** Monthly variations in the water column-integrated major nutrient concentrations averaged from seven different stations in Jaran Bay.

*Water* **2020**, *12*, 3093

The total monthly chlorophyll *a* concentration averaged from the three light depths at seven stations ranged from 0.77 μg L−<sup>1</sup> in September to 4.89 μg L−<sup>1</sup> in October, with an average of 2.13 <sup>μ</sup>g L−<sup>1</sup> (S.D. = <sup>±</sup> 1.18 <sup>μ</sup>g L−1) (Figure 2). Base d on the different size-fractionated chlorophyll *a* concentrations (Figure 3), the compositions of the micro- (> 20 μm), nano- (2−20 μm) and pico-sized chlorophyll *a* concentrations (0.7−2 μm) varied significantly in Jaran Bay among the different months. The compositions of the micro-sized chlorophyll *a* concentrations ranged from the lowest value in April (23.8 ± 18.7%) to the highest value in January (77.8 ± 6.8%), whereas the nano-sized chlorophyll *a* compositions ranged from the lowest value in January (14.3 ± 7.0%) to the highest value in June (50.3 ± 21.3%). In comparison, the compositions of pico-sized chlorophyll *a* were lowest in January (7.9 ± 4.4%) and highest in April (46.0 ± 18.1%). Seas onally, the compositions of the micro-sized chlorophyll *a* concentrations steadily increased from spring (March−May) to winter (December−February), although significant monthly variations were present. In contrast, the compositions of the pico-sized chlorophyll *a* concentrations steadily decreased from spring to winter. The compositions of the nano-sized chlorophyll *a* concentrations were highest in summer (June−August) and lowest in winter. On average, micro-sized (>20 μm) cells contributed 49.9% (± 18.7%) of the total chlorophyll *a* concentration in Jaran Bay during our observation period. In comparison, the nano- and pico-sized chlorophyll *a* compositions contributed 28.5% (± 12.4%) and 21.6% (± 11.2%), respectively. A strong positive relationship was found between the micro-sized chlorophyll *a* concentrations and total chlorophyll *a* concentrations integrated from the euphotic water columns in this study (y = 1.31x + 5.74, r<sup>2</sup> = 0.82; Figure 4).

#### *3.2. Spatial and Temporal Variations of the Macromolecular Compositions of POM*

Figure 5 shows the average of three light depth values of each macromolecular composition of POM in Jaran Bay from January to December 2016. No distinctive spatial variations were detected in the macromolecular compositions among the different stations; however, they significantly varied among the different months. Carb ohydrates were the predominant biochemical component during our observation period from January to December, with monthly proportions of carbohydrates ranging from 40.9% to 66.4%. In comparison, the protein and lipid proportions were 11.1–31.0% and 22.5–35.1%, respectively. The lipid proportion appeared to decrease steadily from January to December. Seas onally, the carbohydrate proportion were relatively variable compared to the protein and lipid proportions. The carbohydrate proportion was lowest during summer (45.6 ± 1.4%) and highest during autumn (59.1 ± 10.9%). In comparison, the protein proportion was lowest during winter (17.5 ± 5.7%) and highest during summer (28.1 ± 2.7%), while the lipid proportion was lowest in autumn (23.1 ± 0.6%) and highest in winter (31.1 ± 3.7%).

The monthly FM concentrations ranged from 297 to 630 mg m<sup>−</sup>3, with an average of 411.7 mg m−<sup>3</sup> (S.D. = <sup>±</sup> 93.0 mg m<sup>−</sup>3), in this study (Table 2). No noticeable monthly variations were observed for the FM concentrations. Spat ial variations in the FM concentrations were not noticeable for the seven stations during the observation period except for March, April and June, which had considerably higher FM concentrations at several stations (Figure 6). The monthly calorific values and FM contents of FM averaged from the three light depths at the seven stations did not vary significantly and ranged from 5.5–6.3 Kcal g−<sup>1</sup> and 1.7–3.7 Kcal m<sup>−</sup>3, respectively, in Jaran Bay (Table 2).

**Figure 4.** Relationship between the euphotic depth-integrated micro-sized chlorophyll *a* concentrations and the integrated total chlorophyll *a* concentrations in Jaran Bay.

**Figure 5.** Biochemical compositions of POM relative to the total FM at the sampling stations in Jaran Bay.


**Table 2.** Monthly averaged

compositions

 of different sized chlorophyll *a*

concentrations

 and biochemical

concentrations

 and

compositions

 of POM averaged from

*Water* **2020** , *12*, 3093

**Figure 6.** Water column-integrated the total FM concentrations at the sampling stations in Jaran Bay.

#### *3.3. Principal Component Analysis (PCA)*

The PCA results for our field-observed biochemical parameters are summarized in Table 3. Thre e PCs were selected for multiple linear regression analysis in this study. The variables shown in bold indicate the highest correlations among the 12 variables and the corresponding components. The nano-sized chlorophyll *a* concentrations, carbohydrates, proteins, lipids and FMs had the highest correlations with PC1, whereas the concentrations of NH4 <sup>+</sup>, NO3 <sup>−</sup>, P\* and Si(OH)4 <sup>2</sup><sup>−</sup> were highest correlated with PC2. For PC3, temperature and the micro- and pico-sized chlorophyll *a* concentrations showed the highest correlations. Base d on the PCA results in Table 3, multiple linear regression analysis was performed to obtain the major controlling factors for the variation in the FM in Jaran Bay (Table 4). The nano- and micro-sized chlorophyll *a* concentrations and P\* and NH4 <sup>+</sup> concentrations were found to be the major factors for controlling the FM in Jaran Bay during our observation period (Table 4). The concentrations of nano- and micro-sized chlorophyll *a* and NH4 <sup>+</sup> had positive effects whereas the P\* concentration had a negative impact on the FM in Jaran Bay during the study period. In other word, a total increase in the concentrations of nano- and micro-sized chlorophyll *a* and NH4 + could bring an increase in the FM. On the other hand, an increase in P\* concentration could lead to a decrease in the FM.




 **R2 (%)**

**Variables** Constant

Nano-chlorophyll

Micro-chlorophyll

 *a*

P\*

> NH4+

concentration

 *a*

concentration

concentration

 337.872

 112.476

 20.115 −230.321

 19.321

 12.08

 8.16

 5.412

 49.425

 5.362

 0.617

 0.156 −0.305

 0.225

 27.969

 13.784

 3.716 −4.66

 3.603

 0.000 \*\*

 0.000 \*\*

 0.000 \*\*

 0.000 \*\*

 0.000 \*\*

 0.544

 0.57

 0.582

 0.602

#### **4. Discussion**

The monthly-averaged concentrations of the depth-integrated nutrient concentrations measured were within the ranges previously reported from regions near Jaran Bay [32–35]. The present study indicates that each nutrient concentration showed significant seasonal variations. For example, the DIN concentrations were relatively higher during the period from September to December, whereas the silicate concentrations were higher in June−September compared to other months (Table 1).

The monthly depth-integrated total chlorophyll *a* concentrations within the euphotic water column from 100% to 1% light depths ranged from 5.2 to 36.7 mg m−<sup>2</sup> (mean <sup>±</sup> S.D. <sup>=</sup> 17.0 <sup>±</sup> 9.2 mg chl-*<sup>a</sup>* <sup>m</sup><sup>−</sup>2) during the study period from January to December 2016. The largest peak was observed in October immediately, followed by the nutrient peaks observed in September (Table 1). Howe ver, the seasonal chlorophyll *a* concentrations did not vary greatly and ranged from 15.8 to 17.8 mg m−2. Gene rally, the spatial variation of the total chlorophyll *a* concentrations appeared to be low among the seven stations in Jaran Bay during the observation period except for March (Figure 2). Over all, the phytoplankton community was dominated by micro-sized phytoplankton based on the size-fractionated chlorophyll *a* concentration results during our observation period. Prev ious studies have reported that the predominant species in this area consisted of diatoms [23,36]. In general, the spatial and seasonal variations of the total chlorophyll *a* concentrations were strongly related to the micro-sized (> 20 μm) chlorophyll *a* concentrations (Figure 4). This finding suggests that micro-sized cells greatly contributed to the total chlorophyll *a* concentration in Jaran Bay. In other words, 49.9% (± 18.7%) of total chlorophyll *a* was from micro-sized cells (Figure 3) during our observation period.

The overall dominant macromolecular composition of POM was carbohydrates (51.8 ± 8.7%), followed by lipids (27.3 ± 3.8%) and proteins (20.9 ± 7.4%), during our observation period (Figure 5). The macromolecular compositions obtained from the present study fell in a similar range to those obtained from Geoje-Hansan Bay by Kim et al. [6], in which their study area was close to our research site. Howe ver, the compositions in Jaran and Geoje-Hansan bays were considerably different from those in Gwangyang Bay. The mean compositions in Gwangyang Bay were 26.4% (± 9.4%), 37.8% (± 16.1%), and 35.7% (± 13.9%) carbohydrates, proteins, and lipids, respectively [5]. Thes e differences may have been due to the influence of river-borne nutrients. The protein and lipid proportions are largely dependent on the input of dissolved inorganic nitrogen from the Seomjin River in Gwangyang Bay [5]. In comparison, there are no large river inputs in the Jaran and Geoje-Hansan bays. For coastal management plans, e.g., artificial dam construction, the potential influence of river inputs on the dominant cell size and photosynthetic end-products of phytoplankton should be considered [7].

Although the macromolecular compositions between Jaran and Geoje-Hansan Bays [6] in south Korea are similar, the monthly FM concentrations were relatively lower in Jaran Bay and ranged from 297 to 630 mg m−<sup>3</sup> with an average of 411.7 mg m−<sup>3</sup> (S.D. <sup>=</sup> <sup>±</sup> 93.0 mg m<sup>−</sup>3), than in Geoje-Hansan Bay, which had a range of 346−1280 mg m−<sup>3</sup> (615.5 <sup>±</sup> 291.7 mg m<sup>−</sup>3; Table 5). Howe ver, the average monthly FM concentration (411.7 <sup>±</sup> 93.0 mg m<sup>−</sup>3) of POM in Jaran Bay during our observation period was similar to that in Gwangyang Bay (434.5 <sup>±</sup> 175.5 mg m<sup>−</sup>3) [5] despite the large difference in macromolecular compositions between the two bays. Base d on the fact that FM concentrations are derived from the total concentrations of carbohydrates, proteins and lipids [18,19] and that their relative compositions can be affected by various environmental and biological factors [5,8–14], different macromolecular compositions are unlikely to be strongly related to the FM concentrations of POM. Inst ead of the compositions of the chlorophyll *a* concentrations, which are often used to represent phytoplankton biomass, would be more appropriate for comparisons. Howe ver, no strong relationship between the FM concentrations and total chlorophyll *a* concentrations was found in the present study, although a strong correlation was found in Gwangyang Bay by [7]. Simi larly, no significant linear relationship was observed between the FM and total chlorophyll *a* concentrations among the different bays in South Korea (Table 5). The average chlorophyll *a* concentrations were 2.13 <sup>μ</sup>g L−<sup>1</sup> (S.D. = <sup>±</sup> 1.18 <sup>μ</sup>g L−1, this study), 4.34 μg L−<sup>1</sup> [6] and 3.45 μg L−<sup>1</sup> [5] in the Jaran, Geoje-Hansan and Gwangyang Bays, (Table 5). Thes e bays are all in the South Sea of South Korea. In the Garolim-Asan Bay, Yellow Sea [37], the average chlorophyll *<sup>a</sup>* concentration (2.81 <sup>±</sup> 2.12 <sup>μ</sup>g L<sup>−</sup>1) was within the low range (2.13−4.34 <sup>μ</sup>g L<sup>−</sup>1) among the three bays, but the average FM concentration (781.4 <sup>±</sup> 228.2 mg m<sup>−</sup>3) was highest among the bays in this study. The chlorophyll *a* concentration has been used as a proxy for biomass, but may not be completely representative of phytoplankton biomass since the chlorophyll *a* concentration is greatly influenced by light and nutrient conditions, physiological status and species composition of phytoplankton [38–41]. Inst ead of the chlorophyll *a* concentration, Lee et al. [5] and Kim et al. [7] suggested that the FM concentration of POM, mainly phytoplankton, could be an alternative proxy for food sources available to higher trophic levels in bay or coastal marine ecosystems. Ther efore, the FM concentration could have a quantitatively complementary value for the amount of various food material sources available to potential consumers in estuarine or bay ecosystems [7,21]. With respect to energy aspects, the calorific content, which depends on the different macromolecular compositions of the FM concentration, should be considered as representative of the physiological or ecological conditions of higher trophic levels of consumers [5,7,21].

**Table 5.** Comparison of the total chlorophyll *a* concentrations and FM concentrations of POM among different Korean bays.


According to the PCA results, spatiotemporal variations in FM are primarily governed by the nano-sized chlorophyll *a* concentrations, carbohydrates, proteins and lipids since FM is the sum of the concentrations of the three different macromolecules. Howe ver, the positive relationship between the nano-sized chlorophyll *a* concentration and FM would not be predictable. In Jaran Bay, the spatiotemporal change of the total chlorophyll *a* concentration was primarily controlled by the micro-sized chlorophyll *a* concentrations because of their high contribution to the total chlorophyll *a* concentration. In comparison, nano-sized chlorophyll *a* compositions contributed 28.5% (± 12.4%) of the total chlorophyll *a* concentration in this study, although their monthly contributions varied somewhat broadly and ranged from 14.3% (±7.0%) in January to 50.3% (±21.3%) in June (Figure 3). In PC2, the positive correlations among the major inorganic nutrient concentrations (e.g., NH4 +, NO3 <sup>−</sup>, P\* and Si(OH)4 <sup>2</sup>−) were reasonable. Temp erature and the micro- and pico-sized chlorophyll *a* concentrations in PC3 indicate positive correlations among the three variables in Jaran Bay (Table 3). PCA was used in this study for ranking their relative significance (Table 3) among our field-observed biochemical parameters for multiple linear regression analysis and deriving major controlling factors (Table 4) of the FM in our study site. In this approach, we could predict the FM in our study site based on the multiple linear regression analysis. Acco rding to the multiple linear regression, approximately 60% of the variation in FM could be explained by the nano- and micro-sized chlorophyll *a* concentrations and P\* and NH4 <sup>+</sup> concentrations in Jaran Bay (Table 4). With this approach, the four major controlling factors were determined for the observed FM variations in Jaran Bay during our observation period from January to December 2016. Howe ver, the somewhat low prediction of up to 60% suggests that other potential factors in addition to our observed parameters should be investigated to improve the spatiotemporal variation in the FM in Jaran Bay. Sinc e this study was a pilot study, some of important parameters were not considered. For example, grazing effects from predators, such as aquaculture shellfish and zooplankton, could be highly correlated with FM, which is a main food source available to them.

#### **5. Conclusions**

A detailed spatiotemporal evaluation of the biochemical compositions and FM of POM of phytoplankton communities and a set of multiple linear regression analyses were conducted in Jaran Bay to understand their major controlling factors. Base d on this research, the variations in FM representing food source materials could be explained by large-cell-sized phytoplankton (>2 μm) and major inorganic nutrient concentrations. Kim et al. [42] observed progressive decreases in dissolved inorganic nutrients in the southern coastal region of South Korea in recent decades. A progressive decline of the chlorophyll *a* concentration has been consistently reported in several regions in the southern coastal region of South Korea [43]. At this point, we cannot assume that the changes of the species compositions or size compositions of phytoplankton are correlated with the decreases of the concentrations of nutrients and chlorophyll *a*. Howe ver, we may expect greater numbers of small-sized phytoplankton cells than of large cell-sized phytoplankton cells under these conditions. Thes e changes in nutrient concentrations and dominant phytoplankton communities could cause changes in FM and further alterations in potential consumers. Jara n Bay is one of the largest shellfish aquaculture sites in the South Sea of Korea. Furt her studies on the spatial and temporal variations in the macromolecular compositions and FM of POM in regard to various environmental conditions are needed to better understand the quality and quantity of the primary food source available to higher trophic animals.

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

**Funding:** This research was supported by a grant (R2020048) from the National Institute of Fisheries Science (NIFS) and partly by a project titled 'Improvements of ocean prediction accuracy using numerical modeling and artificial intelligence technology' which are funded by the Ministry of Oceans and Fisheries, Republic of Korea.

**Acknowledgments:** We thank the anonymous reviewers who greatly improved an earlier version of manuscript.

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

#### **References**


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### *Article* **Spatial Patterns of Macromolecular Composition of Phytoplankton in the Arctic Ocean**

**Keyseok Choe 1,2, Misun Yun 3,\*, Sanghoon Park 1, Eunjin Yang 4, Jinyoung Jung 4, Jaejoong Kang 1, Naeun Jo 1, Jaehong Kim 1, Jaesoon Kim <sup>1</sup> and Sang Heon Lee 1,\***


**Abstract:** The macromolecular concentrations and compositions of phytoplankton are crucial for the growth or nutritional structure of higher trophic levels through the food web in the ecosystem. To understand variations in macromolecular contents of phytoplankton, we investigated the macromolecular components of phytoplankton and analyzed their spatial pattern on the Chukchi Shelf and the Canada Basin. The carbohydrate (CHO) concentrations on the Chukchi Shelf and the Canada Basin were 50.4–480.8 μg L−<sup>1</sup> and 35.2–90.1 μg L<sup>−</sup>1, whereas the lipids (LIP) concentrations were 23.7–330.5 μg L−<sup>1</sup> and 11.7–65.6 μg L−1, respectively. The protein (PRT) concentrations were 25.3–258.5 μg L−<sup>1</sup> on the Chukchi Shelf and 2.4–35.1 μg L−<sup>1</sup> in the Canada Basin. CHO were the predominant macromolecules, accounting for 42.6% on the Chukchi Shelf and 60.5% in the Canada Basin. LIP and PRT contributed to 29.7% and 27.7% of total macromolecular composition on the Chukchi Shelf and 30.8% and 8.7% in the Canada Basin, respectively. Low PRT concentration and composition in the Canada Basin might be a result from the severe nutrient-deficient conditions during phytoplankton growth. The calculated food material concentrations were 307.8 and 98.9 μg L<sup>−</sup>1, and the average calorie contents of phytoplankton were 1.9 and 0.6 kcal m−<sup>3</sup> for the Chukchi Shelf and the Canada Basin, respectively, which indicates the phytoplankton on the Chukchi Shelf could provide the large quantity of food material and high calories to the higher trophic levels. Overall, our results highlight that the biochemical compositions of phytoplankton are considerably different in the regions of the Arctic Ocean. More studies on the changes in the biochemical compositions of phytoplankton are still required under future environmental changes.

**Keywords:** macromolecules; phytoplankton; Chukchi Shelf; Canada Basin; food material

#### **1. Introduction**

The Arctic Ocean is one of the most affected geographical locations in the world due to global climate change. In the Arctic, there has been a rapid decline of sea ice for several decades [1,2], which can be visualized by the downward trend of sea ice range through continuous satellite observations over the past decade [3–5]. In the last 30 years, the density of sea ice has decreased by about 9% every 10 years, and the sea ice thickness has also decreased [6]. With the disappearance of sea ice, various physico-chemical processes are likely to be altered [7–9].

Recent and rapid changes in the marine environment in the Arctic Ocean have been revealed to have significant impacts on the phytoplankton community [10–12]. For example, Kahru et al. [13] revealed that early phytoplankton blooms were caused by a decrease in sea

**Citation:** Choe, K.; Yun, M.; Park, S.; Yang, E.; Jung, J.; Kang, J.; Jo, N.; Kim, J.; Kim, J.; Lee, S.H. Spatial Patterns of Macromolecular Composition of Phytoplankton in the Arctic Ocean. *Water* **2021**, *13*, 2495. https:// doi.org/10.3390/w13182495

Academic Editor: Arantza Iriarte

Received: 4 August 2021 Accepted: 9 September 2021 Published: 11 September 2021

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**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/).

ice in the Arctic Ocean. According to Ardyna et al. [14], the lengthening of the open water season in the Arctic Ocean was correlated with the increasing occurrence of the autumn bloom. In the areas of the Arctic Ocean where sea ice was absent, satellite observations have shown a significant increase in annual net primary production (NPP) [15–17]. Other than the quantitative changes of phytoplankton, the physiological conditions of phytoplankton appear to be affected by the recent environmental conditions [18–20].

In general, the organic matter produced by phytoplankton is composed of carbohydrates (hereafter, CHO), proteins (hereafter, PRT), and lipids (hereafter, LIP). The composition and synthesis of these major macromolecules of phytoplankton can provide important clues to the physiological status under the environment in which phytoplankton grow since they reflect the rapid adjustment of environmental conditions [21–23]. Furthermore, the relative amount of each macromolecular component in phytoplankton indicates the quality, or nutritional value, of phytoplankton as a food source [24]. The determination of the energy content for phytoplankton can be important since it could be transferred to the marine herbivores and the higher trophic levels and consequently determine the growth of higher trophic levels.

Previously, some studies on the macromolecular composition of phytoplankton were reported to understand their physiological conditions in the Polar Oceans [23,25–32]. The comparison of macromolecular compositions between phytoplankton and microzooplankton was conducted in the Arctic Ocean [27]. Kim et al. [28] revealed that the Antarctic phytoplankton indicated high protein composition under sustained high nutrient conditions, whereas the Arctic phytoplankton produced more lipids [19,20,27] or carbohydrates [30,31]. These studies mainly focused on the macromolecular compositions of or their vertical distributions within the euphotic zone or related environmental factors. Although the macromolecular composition of phytoplankton could be largely affected by environmental conditions in the regions, very little information is available on the spatial pattern of macromolecular concentration, composition, and nutritional value within the regions of the Arctic Ocean.

In this study, we examined the composition and concentration of the macromolecular pool (CHO, PRT, and LIP) of phytoplankton to understand the energy content of Arctic phytoplankton. In addition, we investigated how the spatial variation of the macromolecular composition is linked to physicochemical and biological parameters in different domains. Finally, the energy content of phytoplankton was calculated to estimate the nutritional value, which could be transferred to the organisms in the higher trophic levels in the Arctic ecosystem.

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

#### *2.1. Research Area and Sampling*

From 31 July to 23 August 2014, 21 survey stations on the Chukchi Shelf and the Canada Basin were occupied onboard R/V *Araon*. The samples for the macromolecular components were collected from 10 stations of shelf area (hereafter, Chukchi Shelf) and 11 stations of basin area (hereafter, Canada Basin) during the cruise period (Figure 1). The physical data were obtained through CTD (a Sea-Bird 911+) at each station, and seawater samples were obtained by using rosette samplers. A vertical irradiance profile (photosynthesis active irradiance (PAR), 400–700 nm) was obtained using an LI-COR underwater optical sensor mounted on a CTD/rosette sampler to determine the light depth.

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

Seawater samples for nutrient measurements were obtained from different photic depths (100%, 30%, and 1% depths of surface PAR) determined from the underwater PAR sensor. Samples were collected from all of the corresponding light depths, and the surface water collected was used for 100% light treatment. The nutrient concentrations were measured immediately using an automatic nutrient analyzer (SEAL, QuAAtro, Norderstedt, Germany).

Total chlorophyll-a concentrations (Whatman GF/F filter, ø = 24 mm) were analyzed using the method in [33]. The filters (Whatman GF/F filter, ø = 24 mm) were immediately frozen at −80 ◦C in each petri dish wrapped in aluminum foil until chlorophyll-a extraction at Pusan National University, South Korea. All the samples for chlorophyll-a concentrations were extracted with 90% acetone at −5 ◦C for 24 h, and the concentrations were measured using a fluorometer (Turner Designs, 10-AU, San Jose, CA, USA), which was calibrated before the analysis.

**Figure 1.** Map of the study area with functional regions highlighted in different colors. The red and blue colors indicate the sampling stations of the Chukchi Shelf and Canada Basin, respectively.

#### *2.3. Macromolecular Concentration Analysis*

Seawater samples (1L) were obtained from 100%, 30%, and 1% depths of surface PAR for the macromolecular concentration in the euphotic zone. The water sample was filtered through a 47-mm Whatman GF/F filter and then immediately stored at −80 ◦C until further analysis was performed at the home laboratory of Pusan National University. To extract CHO, PRT, and LIP, the phenol-sulfuric acid method [34], modified PRT method [35], and column method [36,37] were used, respectively. The detailed methods are available in [38].

#### *2.4. Caloric Content Calculation*

The Winberg [24] formula was used to calculate the calorie content (Kcal m<sup>−</sup>3) of food material (FM; the sum of PRT, LIP, and CHO concentrations [39]).

Calorie content (Kcal m<sup>−</sup>3) = Kcal g FM−<sup>1</sup> <sup>×</sup> g FM m−<sup>3</sup>

#### **3. Results**

#### *3.1. Temperature and Salinity Properties*

Figure 2 shows the distributions of water temperature and salinity in the upper ocean. The range of water temperature and salinity of the entire study area varied from −1.8 to 9.7 ◦C and 26.7 to 32.4 psu, respectively. Regionally, the range of water temperature is from −1.4 to 9.7 ◦C (1.8 ± 3.4 ◦C) on the Chukchi Shelf and from −1.8 to 0.2 ◦C (−0.9 ± 0.5 ◦C) in the Canada Basin, showing a large difference between the Chukchi Shelf and the Canada Basin. The salinity ranges were 27.2–32.3 psu (30.1 ± 1.9 psu) on the Chukchi Shelf and 26.7–32.4 psu (30.1 ± 1.8 psu) in the Canada Basin, respectively. According to the distribution of salinity, a decreasing tendency was found to the north across the Chukchi Sea (Figure 2).

**Figure 2.** Vertical profiles of salinity (**left panel**) and temperature (**right panel**) in the stations of the study area.

#### *3.2. Dissolved Inorganic Nutrients*

The vertical patterns of dissolved inorganic nutrients showed spatial variations depending on the stations on the Chukchi Shelf, while they were relatively uniform in the Canada Basin (Figure 3). On the Chukchi Shelf, the concentration ranges of PO4, NO2 + NO3, NH4 and SiO2 were 0.1–2.1, 0–14.3, 0–3.3, and 0.1–51.0 μM, respectively (Figure 3 upper panel). The average concentrations of PO4, NO2 + NO3, NH4, and SiO2 are 0.7 ± 0.4, 1.7 ± 4.0, 0.5 ± 0.8, and 8.7 ± 10.1 μM, respectively. NO2 + NO3 was depleted at the upper layers on the Chukchi Shelf, and NH4 showed low concentration rather than being depleted. In comparison, the concentration ranges of PO4, NO2+NO3, NH4, and SiO2 in the Canada Basin were 0.5–1.6, 0–12.3, 0, and 1.6–31.5 μM, respectively (Figure 3 lower panel). The average concentrations of PO4, NO2 + NO3, NH4, and SiO2 were 0.8 ± 0.3, 2.3 ± 3.8, 0 ± 0, and 7.3 ± 6.6 μM, respectively. Similar to what was observed on the Chukchi Shelf, NO2+NO3 was depleted at the surface of the Canada Basin. Furthermore, NH4 was depleted in the entire water column.

**Figure 3.** Vertical profiles of dissolved inorganic nutrients in stations of the Chukchi Shelf (**upper panel**) and the Canada Basin (**lower panel**).

#### *3.3. Total Chlorophyll-a Concentration*

During the cruise, the integrated chlorophyll-a concentration from the surface to 1% light depth in the entire study area was 66.3 ± 84.3 mg m<sup>−</sup>2, with the total chlorophylla concentration showing a large regional variation as it ranged from 5.5 (station 27) to 376.2 mg m−<sup>2</sup> (station 1) (Figure 4). The average concentration of chlorophyll-a on the Chukchi Shelf was 98.6 ± 104.3 mg m−2, which was approximately three times that (30.3 ± 31.5 mg m<sup>−</sup>2) in the Canada Basin.

**Figure 4.** Spatial distribution of the chlorophyll-a concentration integrated from the surface to 1% light depth.

#### *3.4. Vertical Distribution of Macromolecular Concentration and Composition on the Chukchi Shelf*

Quantitative concentrations and relative ratios of CHO, PRT, and LIP on the Chukchi Shelf are summarized in Tables 1–3. The range of CHO, PRT, and LIP concentrations of phytoplankton was 48.2–409.3, 35.4–123.3, and 25.7–326.7 μg L−1, respectively, at the surface of the Chukchi Shelf (Table 1). The average CHO concentration at each station was 114.7 ± 106.8 <sup>μ</sup>g L−<sup>1</sup> with a 43.6% contribution, being the dominant macromolecule found in phytoplankton. LIP (86.4 ± 93.1 <sup>μ</sup>g L−1) and PRT (60.2 ± 26.5 <sup>μ</sup>g L−1) contributed to 29.3% and 27.1% of the total compositions, respectively. The range of FM concentration was 117.6–859.3 <sup>μ</sup>g L−<sup>1</sup> (261.3 ± 221.2 <sup>μ</sup>g L−1). At 30% light depth (Table 2), the range of CHO concentration was determined to be 43.8–919.4 <sup>μ</sup>g L−<sup>1</sup> (167.3 ± 267.9 <sup>μ</sup>g L−1), in which the macromolecule accounted for 42.4% of the total composition. The range and contribution of LIP concentration were 19.2–546.4 <sup>μ</sup>g L−<sup>1</sup> (116.5 ± 161.3 <sup>μ</sup>g L<sup>−</sup>1) and 30.1%, respectively, and the range and contribution of PRT concentration were 27.2–586.8 μg L−<sup>1</sup> (107.6 ± 170.8 <sup>μ</sup>g L<sup>−</sup>1) and 27.5%, respectively. Both the average concentration and contribution of LIP and PRT tended to increase compared to those at the surface layer. The range of FM concentration was 90.1–2052.6 <sup>μ</sup>g L−<sup>1</sup> (391.5 ± 597.0 <sup>μ</sup>g L−1), which was considerably higher than that at the surface. Compared to 30% light depth, the average composition of CHO concentration was slightly reduced to 41.7%, and its range was 34.9–259.0 μg L−<sup>1</sup> (101.4 ± 64.0 <sup>μ</sup>g L−1) at 1% euphotic depth (Table 3). At this depth, the ranges of LIP and PRT concentrations were found to be 12.0–256.7 (90.5 ± 79.7 <sup>μ</sup>g L<sup>−</sup>1) and 10.0–185.8 <sup>μ</sup>g L−<sup>1</sup> (78.7 ± 52.8 <sup>μ</sup>g L<sup>−</sup>1). Consequently, the LIP contributed to 29.7% of the overall composition while PRT contributed to 28.6%, respectively. The CHO synthesis continued to decrease, whereas the PRT synthesis increased with depth. The range of FM concentration was 56.9–701.6 <sup>μ</sup>g L−<sup>1</sup> (270.6 ± 183.2 <sup>μ</sup>g L<sup>−</sup>1) at the 1% depth.


**Table 1.** Concentrations and compositions of macromolecular components (carbohydrates: CHO, proteins: PRT, and lipids: LIP), food material (FM), and calorie content of phytoplankton at 100% light depth on the Chukchi Shelf.

**Table 2.** Concentrations and compositions of macromolecular components (carbohydrates: CHO, proteins: PRT, and lipids: LIP), food material (FM), and calorie content of phytoplankton at 30% light depth on the Chukchi Shelf.


**Table 3.** Concentrations and compositions of macromolecular components (carbohydrates: CHO, proteins: PRT, and lipids: LIP), food material (FM), and calorie content of phytoplankton at 1% light depth on the Chukchi Shelf.


#### *3.5. Vertical Distribution of Macromolecular Concentration and Composition in the Canada Basin*

The range and contribution of CHO concentration of phytoplankton at the surface layer in the Canada Basin were dominant, being 28.8–177.2 <sup>μ</sup>g L−<sup>1</sup> (70.9 ± 42.3 <sup>μ</sup>g L<sup>−</sup>1) and 61.9%, respectively (Table 4). LIP concentration contributed to 29.7% of the total composition with a concentration range of 10.9–81.0 <sup>μ</sup>g L−<sup>1</sup> (34.2 ± 26.3 <sup>μ</sup>g L<sup>−</sup>1), and the range of PRT concentration was 3.2–13.6 <sup>μ</sup>g L−<sup>1</sup> (8.0 ± 3.2 <sup>μ</sup>g L<sup>−</sup>1) with a contribution of 8.4% at the surface layer. The range of FM concentration was 55.5–250.2 <sup>μ</sup>g L−<sup>1</sup> (113.1 ± 51.5 <sup>μ</sup>g L<sup>−</sup>1). At 30% euphotic depth (Table 5), CHO contributed to 57.6% of the total macromolecular composition and had a concentration range of 35.0–77.1 <sup>μ</sup>g L−<sup>1</sup> (48.2 ± 13.1 <sup>μ</sup>g L<sup>−</sup>1). Compared to the surface layer, the concentration of CHO was significantly reduced, and its contribution was somewhat decreased. On the other hand, the contribution of LIP concentration was 32.7%, which was increased compared to that at the surface, with a range of 10.4–58.7 μg L−1. However, the average concentration of LIP was reduced to 29.3 μg L−<sup>1</sup> (±18.7 <sup>μ</sup>g L<sup>−</sup>1) and smaller compared to that at the surface. The range and content of PRT concentration were 2.9–15.4 <sup>μ</sup>g L−<sup>1</sup> (8.4 ± 3.8 <sup>μ</sup>g L−1) and 9.8%, respectively, and it was identified to be greater than what was found at the surface. The range of FM concentration was between 56.0 and 110.0 <sup>μ</sup>g L−<sup>1</sup> (85.9 ± 19.1 <sup>μ</sup>g L−1). At 1% light depth (Table 6), both the range (33.3–92.9 μg L−1) and content (62.2%) of CHO concentrations increased. The range and contribution of LIP concentration were determined to be 9.0–92.5 μg L−<sup>1</sup> (32.2 ± 28.6 <sup>μ</sup>g L<sup>−</sup>1) and 29.9%, respectively. A decrease in the range and contribution of PRT concentration was observed. The range of PRT concentration was 0.7–83.8 μg L−<sup>1</sup> (11.4 ± 24.1 <sup>μ</sup>g L−1) with a total contribution of 7.9% and the range of FM concentration was 46.8–269.2 <sup>μ</sup>g L−<sup>1</sup> (97.5 ± 63.1 <sup>μ</sup>g L<sup>−</sup>1) at 1% light depth in the Canada Basin.

**Table 4.** Concentrations and compositions of macromolecular components (CHO, PRT, and LIP), food material (FM), and calorie content of phytoplankton at 100% light depth in the Canada Basin.


**Table 5.** Concentrations and compositions of macromolecular components (CHO, PRT, and LIP), food material (FM), and calorie content of phytoplankton at 30% light depth in the Canada Basin.



**Table 5.** *Cont.*

**Table 6.** Concentrations and compositions of macromolecular components (CHO, PRT, and LIP), food material (FM), and calorie content of phytoplankton at 1% light depth in the Canada Basin.


*3.6. Temperature, Salinity, Nutrients, and Macromolecular Concentration along the Shelf/Basin Gradient*

The transect from station 10 to station 15 was run to understand the variability of environmental conditions and macromolecular concentrations along the shelf/basin gradient (Figure 5). Due to sea ice, the water column was in a freezing condition toward the basin. Based on the salinity and temperature distributions, station 12 was determined as the sea-ice edge. The dissolved inorganic nutrients at the depths around the sea-ice edge were distinctly high and decreased toward the basin. The chlorophyll-a distribution gradually decreased toward the basin. The chlorophyll-a concentration at the shelf was lowest at the surface and increased with depth.

The highest CHO concentration was observed at station 10 (>200 μg L−1). Similarly, the LIP was highest at station 10. The upper layer of the basin showed a high LIP concentration. The distribution of PRT concentration was also similar to those of CHO or LIP, but PRT concentration at the ice-covered stations of the basin (from station 13 to station 15) was distinctly low. Interestingly, CHO and PRT concentrations were two or three times lower at the stations of the basin compared to the shelf area.

**Figure 5.** The temperature (**a**), salinity (**b**), DIN concentration (**c**), PO4 concentration (**d**), SiO2 concentration (**e**), chlorophyll-a concentration (**f**), CHO concentration (**g**), PRT concentration (**h**), and LIP concentration (**i**) distributions in the vertical section from the Chukchi shelf to Canada Basin in the Arctic Ocean.

#### *3.7. Spatial Distribution of the Macromolecular Composition*

No statistically significant difference in the relative percentage of each macromolecular component was found among the different light depths (*t*-test, *p* > 0.05). Thus, each component was averaged from three different light depths. Figure 6 shows a spatial distribution of the macromolecular composition over the euphotic zone during this study period. The contribution of CHO was the lowest at station 2 and highest at station 15. The LIP component accounted for 18.5–62.6% of the total macromolecular composition among the stations. The PRT contributed to 2.3–34.6% of the total macromolecular composition. Over the entire study area, CHO was identified as the biggest contributor (52.0%) to the overall average composition of phytoplankton, which was followed by LIP (30.3%) and PRT (17.8%). Regionally, the CHO contributed to 42.6% and 60.5% of the total macromolecular compositions for the Chukchi Shelf and the Canada Basin, respectively. The LIP contributions were 29.7% on the Chukchi Shelf and 30.8% in the Canada Basin, respectively. The average PRT composition was significantly low (*t*-test, *p* < 0.05) in the Canada Basin (8.7% of total) compared to the Chukchi Shelf (27.7% of total) (Figure 6).

**Figure 6.** Spatial distribution of the macromolecular compositions of phytoplankton during the 2014 expedition.

#### **4. Discussion**

#### *4.1. Major Controlling Factors for the Spatial Variation in Macromolecular Composition*

In this study, the major macromolecule contributing to the overall average composition of phytoplankton was determined to be CHO on the Chukchi Shelf and the Canada Basin (Figure 7). It is interesting compared to those previously reported from the other regions of the polar oceans. For example, Yun et al. [27] observed a higher rate of LIP (58%) compared to CHO or PRT in the phytoplankton in the northern Chukchi Sea. In the Antarctic Ocean, Fabiano et al. [40,41] reported a high contribution, of 50% or more, of PRT to the total FM. Kim et al. [28] also found the high contribution of PRT (67%) to the macromolecular composition of phytoplankton in the Amundsen Sea due to the high concentrations of nitrate + nitrite. In general, the composition of PRT in the phytoplankton increases under nitrogen saturation conditions [40,42]. When the nitrogen or phosphorus is limited, triglycerides, which are energy stores, increase and are converted from PRT metabolism to LIP or CHO metabolisms [23,43,44]. Since CHO and LIP are not nitrogen-derived substrates, the accumulation of these storage compounds can be a reaction mechanism under nitrogen-deficient conditions [45]. In particular, LIP acts as a secondary storage material for the survival of long-term nitrogen conditions due to the fat synthetase system [46]. Thus, photosynthesis products are converted from CHO to LIP synthesis in a nitrogen-depleted environmental condition for an extended period [45,46], even though preferred accumulation in CHO or LIP compounds as a reservoir appears to be specific to species [45]. Some oily diatom species assimilate LIP as a major storage component under nitrogen or silicon restrictions [47,48]. Indeed, Ahn et al. [31] observed a sharp increase in LIP concentration with an increase in micro-phytoplankton in the Arctic Ocean. However, Harrison et al. [49] and Wear et al. [50] reported that diatoms have a relatively constant LIP under nitrogen deficiency while rapidly increasing CHO content and decreasing PRT. Therefore, the high CHO and moderate LIP compositions in the present study might have been due to the deficient nutrient conditions, which is consistent with previous findings [49,51], while PRT production predominates under nitrogen-rich conditions [40,42]. Consequently, it implies that major inorganic nutrients, especially nitrogen, are important controlling factors for the macromolecular composition of phytoplankton.

**Figure 7.** Contribution of each macromolecular component at three light depths on the Chukchi Shelf (**a**) and in the Canada Basin (**b**).

During the cruise, the phytoplankton in the Canada Basin were observed to have a significantly lower PRT (8.7%) compared to that of the Chukchi Shelf. This might be related to different conditions of nutrient limitation. Normally, nutrient deficiency can be indicated following as; nitrogen limit condition with N/P ratio with < 10 and Si/N ratio > 1, phosphorus restriction under N/P ratio > 22 and Si/P ratio > 22, and silicon limitation in Si/N ratio < 1 and Si/P ratio < 10 [52]. In terms of macromolecular composition, values with a higher PRT/CHO ratio (>1) are observed in the areas with high productivity or nitrogen-rich blooms [40], while the lower ratios (<1) are in nitrogen deficiency conditions [39]. Thus, low N/P ratios, PRT/CHO ratios, and PRT/LIP ratios indicate that nitrogen is particularly limited in the environment. In this study, the average molar ratio of (NO3 + NO2 + NH4):PO4 and SiO2:(NO3 + NO2 + NH4) in the euphotic zone was 3.0:1 and 3.9:1 on the Chukchi Shelf, respectively, and 3.4:1 and 2.8:1 in the Canada Basin, respectively. Overall, the ratio of N/P was significantly lower than the Redfield ratio [53]. The PRT/CHO ratio and PRT/LIP ratio of the Canada Basin are 0.16 and 0.33, respectively, which are significantly lower (*t*-test, *p* < 0.05) than the PRT/CHO ratio (0.68) and PRT/LIP ratio (1) of the Chukchi Shelf. These results elucidate that the deficiency of nitrogen in the study area was present during the cruise, and it was especially noticeable that the nitrogen utilization by the phytoplankton in the Canada Basin was severely restricted. Therefore, the substantially low PRT composition of phytoplankton in the Canada Basin could be caused by the result of severe nitrogen deficiency during phytoplankton growth.

According to Suárez et al. [54], the light condition could act as an important factor for determining different macromolecular compositions of phytoplankton. The macromolecular composition of phytoplankton can vary depending on the amount of light [55,56]. For example, an increase in PRT is observed with a decrease in light intensity because of the lower illuminance saturation level of PRT in comparison to other macromolecules [54,57]. In contrast, the productions of CHO and LIP as storage materials can be observed under an excessive energy supply condition [54,58]. Suárez et al. [54] also reported that lower irradiance was more relevant in PRT synthesis than in LIP synthesis. However, no distinct pattern of macromolecular compositions was observed among three different light depths in this study (mentioned in Section 3.7), although the relatively higher protein concentrations were observed at deeper depths (30% and 1% light levels) than at the surface. In addition, the stations in the ice-covered Canada Basin showed significantly low PRT composition, even though it was thought to be a low light condition (Figure 5). Thus, we could conclude that the light condition might be insignificant in controlling the macromolecular composition during this study period.

#### *4.2. The Implication of Macromolecular Composition as Energy Content Aspect*

In this study, the concentration ranges of CHO, LIP, and PRT were substantially higher on the Chukchi Shelf than in the Canada Basin (Table 7). As a result, the average FMs were 307.8 μg L−<sup>1</sup> on the Chukchi Shelf and 98.9 μg L−<sup>1</sup> in the Canada Basin, respectively. The average calorie content of phytoplankton for the Chukchi Shelf and the Canada Basin

during the cruise was 1.9 kcal m−<sup>3</sup> and 0.6 kcal m<sup>−</sup>3, respectively (Table 7). The overall FM concentration and calorie content of phytoplankton were three times higher on the Chukchi Shelf than in the Canada Basin, which implies that the phytoplankton on the Chukchi Shelf could provide higher FM and calories to the upper trophic levels in the Arctic ecosystem.

According to previous studies, the average calorie contents were 1.0 and 1.2 kcal m−<sup>3</sup> in the northern Chukchi Sea of the Arctic Ocean [59,60]. Fabiano et al. [41] reported the calorie content of 1.6 kcal m−<sup>3</sup> in the Ross Sea of the Antarctic Ocean. Recently, Kim et al. [32] observed the different calorie contents of phytoplankton between the two different periods in the Ross Sea of the Antarctic Ocean, indicating 1.3 kcal m−<sup>3</sup> during the ice-free period and 0.6 kcal m−<sup>3</sup> during the ice-covered season. Although Kim et al. [29] reported the exceptionally high-calorie content in the productive polynyas of the Amundsen Sea, the calorie content from our study is in a similar range with the previous studies in the polar oceans (Table 7). According to Kim et al. [32], the PRT concentration during the ice-free period in the Ross Sea of the Antarctic Ocean was 20 times increased than that during the ice-covered period, even though CHO or LIP concentrations showed a slight increase (Table 7). If it can be applied in the Arctic Ocean, the PRT concentration than other components might be largely increased under a decrease in sea ice conditions in the Arctic Ocean. Subsequently, the PRT composition that predominates as sea ice decreases could lead to a potential change in the calorie content from an energy point of view. In particular, the macromolecular composition or calorie content of the phytoplankton in ice-covered regions, such as the Canada Basin, might be anticipated to be changed. Consequently, the rich protein-containing FM might be transferred to the upper trophic levels under ongoing and future sea-ice decrease conditions. Thus, the potential effects of the different macromolecular compositions of phytoplankton on the upper trophic levels need to be further evaluated. Above all, in terms of sea ice change, the variability of macromolecular concentration, composition, and calorie content could be important in the Arctic Ocean under ongoing environmental changes.


**Table 7.** Comparison of carbohydrates (CHO), proteins (PRT), lipids (LIP), food material (FM) concentrations, and calorie content of phytoplankton at different regions of the Polar Oceans. Given were range or mean values.

#### **5. Conclusions**

This study reported the spatial distributions of macromolecular concentrations, compositions, and energy contents of phytoplankton on the Chukchi Shelf and in the Canada Basin. CHO was the major macromolecular component of phytoplankton in the study area, accounting for 41.5% on the Chukchi Shelf and 58.4% in the Canada Basin. The LIP was moderate in both regions. Interestingly, the PRO composition was significantly different between the two regions, showing a low contribution in the Canada Basin (8.7%) and a relatively high contribution on the Chukchi Shelf (27.7%). Severe nutrient-deficient conditions for phytoplankton growth appear to be a major reason for the low PRT composition of phytoplankton in the Canada Basin. In terms of FM concentration and calorie content, a large quantity of high-calorie content food is available in the productive Chukchi Shelf compared to the Canada Basin.

Under the ongoing changes in Arctic environments, the concentration or composition of macromolecules of phytoplankton would be expected to change significantly. Since the different concentrations and compositions determine phytoplankton energy content and consequently regulate the growth and/or nutritional structure of upper trophic levels in the Arctic ecosystem, it is needed to monitor the variability of macromolecular concentration and compositions of phytoplankton. In particular, the macromolecular measurements of phytoplankton with different ocean conditions should be required to better understand how phytoplankton's energy content and its transfer to higher trophic levels are different in the regions of the Arctic Ocean. Recently, using satellite data, Roy et al. [62] tried to estimate the concentrations of CHO, PRT, and LIP and energy values of phytoplankton in the world's oceans. In situ measurement data in various oceans would be useful for improving the global scale estimation based on satellite-derived data.

**Author Contributions:** Conceptualization, K.C., E.Y., J.J. and S.L.; methodology, K.C., M.Y. and S.L.; validation, K.C., M.Y. and S.L.; formal analysis, K.C., S.P., J.K. (Jaejoong Kang), N.J., J.K. (Jaehong Kim), J.K. (Jaesoon Kim) and S.L.; investigation, K.C., E.Y. and J.J.; writing—original draft preparation, K.C.; writing—review and editing, M.Y. and S.L.; visualization, S.P., J.K. (Jaejoong Kang), N.J., J.K. (Jaehong Kim) and J.K. (Jaesoon Kim); supervision, S.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was a part of the project 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:** Not applicable.

**Acknowledgments:** We thank the captain and crew of the *ARAON* for their outstanding assistance during the cruise.

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

#### **References**

