Marine Chemistry 176 (2015) 83–95
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Dissolved organic nitrogen bioavailability indicated by amino acids during a diatom to dinoflagellate bloom succession in the Changjiang River estuary and its adjacent shelf Guicheng Zhang a,b, Shengkang Liang a,b,⁎, Xiaoyong Shi b,c, Xiurong Han a,b a b c
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China National Marine Hazard Mitigation Service, Beijing 100194, China
a r t i c l e
i n f o
Article history: Received 24 February 2015 Received in revised form 3 August 2015 Accepted 3 August 2015 Available online 5 August 2015 Keywords: Changjiang River estuary and its adjacent shelf Harmful algal blooms Dissolved organic nitrogen Amino acids Degradation index Bioavailability
a b s t r a c t This study investigates the bioavailability and potential nutritional role of dissolved organic nitrogen (DON) in the development and progression of a large-scale dinoflagellate bloom that occurred in the Changjiang River estuary and its adjacent shelf (CJREAS) in the East China Sea (ECS). Three cruises were conducted during the late spring and early summer following the bloom progression, i.e., the termination phase of the diatom bloom, and the initiation/growth and peak phases of the dinoflagellate bloom. Concentrations of dissolved organic carbon (DOC), DON, dissolved inorganic nitrogen (DIN), dissolved free amino acids (DFAA) and dissolved combined amino acids (DCAA) were measured. The degradation index (DI), based on the relative abundance of total dissolved amino acids (TDAA) and amino acids yields was applied to indicate the diagenetic alteration of dissolved organic matter (DOM). During the succession from a diatom to dinoflagellate bloom, a decrease in DON cooccurred with a decrease in DIN. Concentrations of DFAA and DCAA also clearly decreased. The yields of DFAA and the DCAA and DI values decreased concomitantly with an increase in the DOC/DON, indicating that bioavailability of DON was relatively higher during the end stage of the diatom bloom than during the peak of the dinoflagellate bloom. These results indicated that DON may provide a significant portion of the total N demand throughout the duration of the large-scale dinoflagellate bloom under conditions of low DIN availability in the CJREAS. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The increased intensity of anthropogenic perturbations, and the increased delivery of biologically available nitrogen to estuaries and coastal regions in recent decades have been linked to eutrophication (McIsaac et al., 2001), recognized as a classic triggering mechanism for micro-algal blooms in aquatic ecosystems (Glibert et al., 2005). Toxic algal outbreaks have thus emerged as a major environmental problem in many coastal waters worldwide. The Changjiang River (CJR), one of the largest rivers in China, discharges ~103 km3 yr−1 of freshwater with excess nutrients to the East China Sea (ECS) (Zhang et al., 1999). From the 1980s onward, the CJR estuary and its adjacent shelf (CJREAS) have been suffering from severe eutrophication, resulting in an increase in the frequency of harmful algal blooms (HABs) (Gao and Song, 2005; Li et al., 2009, 2010; Tang et al., 2006; Zhou et al., 2003, 2006). During the last decade, a typical succession of dominant phytoplankton species was observed during ⁎ Corresponding author at: Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China. E-mail address:
[email protected] (S. Liang).
http://dx.doi.org/10.1016/j.marchem.2015.08.001 0304-4203/© 2015 Elsevier B.V. All rights reserved.
blooms in the CJR estuary (Zhou et al., 2003), i.e., a small-scale diatom bloom dominated by Skeletonema costatum first broke out in early April, followed by a large-scale dinoflagellate bloom dominated by Prorocentrum donghaiense (or Karenia mikimotoi, or Alexandrium tamarense) that occurred in early May and lasted approximately one month. Shifts in the dominant phytoplankton species are likely caused by various physical, chemical and biological factors. The chemical composition and the nutrient levels, e.g., those of nitrogen, may affect aquatic primary productivity and the phytoplankton succession (Berg et al., 2003; Seitzinger and Sanders, 1999). In the CJREAS, the concentration of dissolved inorganic nitrogen (DIN) showed sharp depletion during the diatom blooms, while the DON concentration and the % contribution of DON to total dissolved nitrogen (TDN) increased, similar to the patterns of nitrogen availability in other coastal ecosystems, such as the Baltic Sea and Chesapeake Bay (Berg et al., 2001; Glibert et al., 1991). Studies on the relationship between nitrogen availability and phytoplankton blooms in the CJR estuary and its adjacent regions have mainly focused on inorganic species (Li et al., 2009), although DON amounts to a substantial portion of the nitrogen pool. Indeed, as the largest nitrogen pool in coastal aquatic systems, DON has been recognized as a potential nitrogen source for both bacteria and
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phytoplankton (Bronk, 2002; Bronk et al., 2007). Moreover, either directly or indirectly, some DON compounds were the main source of N supporting bloom development and growth, while the concentration of DON and the % contribution of DON to TDN greatly increased during the blooms (Bronk et al., 2007). The ability of some algal species to utilize DON in a nitrogen-deficient ecosystem may provide a competitive advantage that allows these species to alter or inhibit phytoplankton succession. Therefore, the chemical form of DON may affect its reactivity or bioavailability (Benner, 2002). As the largest identified component of the DON pool (~ 15%) (Dittmar et al., 2001; Keil and Kirchman, 1991; Sharp, 1983), amino acids have high bioavailability and can be utilized directly or indirectly by phytoplankton (Antia et al., 1991; Berg et al., 1997; Berman and Bronk, 2003; Berman and Chava, 1999; Bronk et al., 2007; Hu et al., 2012; Palenik and Henson, 1997; Varela et al., 2005; Wawrik et al., 2009). The relative abundance of individual amino acids in the dissolved organic matter (DOM) pool is known as the DOC or DON normalized yields of amino acids (%DOC or %DON) and can be used to describe the diagenetic state of organic matter (Colombo et al., 1998; Dauwe and Middelburg, 1998; Dauwe et al., 1999a, 1999b; Yamashita and Tanoue, 2003). Moreover, the degradation index (DI), based on the changes in amino acid composition (Dauwe and Middelburg, 1998; Dauwe et al., 1999b), and which describes a wide range of degradation states, has been successfully applied to estimate bioavailability and the degradation stage of DOM (Amon et al., 2001; Davis et al., 2009; Kaiser and Benner, 2009; McCarthy et al., 2004; Shen et al., 2012; Yamashita and Tanoue, 2003). While several studies have measured bulk DON concentrations, there is a paucity of information on the chemical composition and bioavailability of DON in ECS ecosystems (Li et al., 2009, 2010). There is thus still a need for greater appreciation and understanding of the potential role of DON in the succession from a diatom to a dinoflagellate bloom. In this study, the temporal and spatial variability of DON and its chemical composition dissolved free amino acids (DFAA) and dissolved combined amino acids (DCAA) were studied during the transition from a diatom bloom dominated by S. costatum to a dinoflagellate bloom dominated by P. donghaiense in the CJREAS. We focused on the detailed variability of dissolved amino acids including their concentration and composition, the diagenetic alteration of DOM based on the DI, yields of carbon- and nitrogen-normalized amino acids, combined with molar ratio of DOC to DON (DOC/DON) during this period. The primary objectives of this study were (1) to characterize the dynamics of DON and amino acids, as the latter are the most labile in the DON fraction; (2) to determine the bioavailability of DON based on the composition and concentration of amino acids; and (3) to identify the role of DON as a potential nutrient controlling bloom succession.
Fig. 1. Sampling stations and the regional circulation system in the ECS (modified from Liu et al. (2007)). CDW: Changjiang Diluted Water; ZFCC: Zhe-Fu Coastal Current; TWWC: Taiwan Warm Current.
the various bloom phases: April 28 to May 2 (the termination of the S. costatum bloom, Phase I), May 5 to May 6 (the initiation of the P. donghaiense bloom, Phase II), and May 25 to May27 (the peak of the P. donghaiense bloom, Phase III) (Figs. 1 and 2). Transects were set between 28.1°N and 29.6°N, and all stations were set between the 20 m and 75 m isobaths (Fig. 1). The first cruise was conducted along ZA and ZB transects, and the other two cruises were conducted along ZA, ZB and ZC transects. 2.2. Sample collection During the three cruises, seawater was collected at standard depths of 0 m, 5 m, 10 m, 20 m, 30 m, 50 m and the bottom layer using Niskin bottles mounted on a rosette with a conductivity–temperature–depth (CTD) sensor. Samples were filtered through pre-combusted Whatman GF/F glass fiber filters (0.7 μm nominal pore size) and stored frozen at − 20 °C in 100 ml pre-combusted glass bottles until analyses of
2. Materials and methods 2.1. Study area and sampling sites The ECS is one of the largest shelf seas in the world and is also a site where HABs occur in every spring. The Changjiang diluted water (CDW) mass, which carries a large amount of nutrients, extends to the south of the CJR estuary. Surface currents in the ESC shelf consist of the southward flow of the cold and brackish Zhejiang-Fujian Coastal Current (ZFCC) and the relatively warm and saline Taiwan Warm Current (TWC) (Fig. 1). During the spring and early summer of 2011, a massive algal bloom broke out and the succession of the dominant species was observed in the CJREAS. Two diatom blooms dominated by S. costatum occurred from the end of March to the end of April; then, the dominant diatom species was gradually replaced by the dinoflagellate P. donghaiense in early May, and a large-scale dinoflagellate bloom developed and lasted approximately 1 month within a total area of 11,500 km2. To investigate the variations in physical, chemical and biological factors during bloom succession, three cruises were conducted that tracked the time series of
Fig. 2. Schematic of various bloom phase investigated as part of this study in the CJREAS. Phase I: the termination of diatom bloom; Phase II: the initiation/growth of the dinoflagellate bloom; Phase III: the peak of the dinoflagellate bloom (according to the phytoplankton data of the ‘973’ Program meeting in 2011).
Subsurface: 5 m or 10 m water layer. a
Bottom Subsurface
19.35 ± 1.19 32.11 ± 0.96 7.26 ± 11.15 6.09 ± 7.13 12.48 ± 2.84 111.19 ± 16.14 74.40 ± 17.58 9.19 ± 1.75 49.9 ± 45.1 0.18 ± 0.13 0.53 ± 0.41 474.91 ± 109.89 1.60 ± 0.38 4.38 ± 1.32 −1.18 ± 0.41 20.23 ± 0.99 31.85 ± 1.03 7.98 ± 12.44 5.94 ± 7.20 13.23 ± 3.05 115.32 ± 15.49 75.36 ± 16.13 9.02 ± 1.70 63.0 ± 47.1 0.23 ± 0.15 0.66 ± 0.43 461.62 ± 69.84 1.48 ± 0.21 4.05 ± 1.10 −1.14 ± 0.44 16.81 ± 1.41 33.43 ± 1.11 1.48 ± 1.68 8.10 ± 4.30 13.96 ± 4.38 89.00 ± 9.10 64.07 ± 12.04 6.81 ± 1.65 43.2 ± 30.5 0.20 ± 0.15 0.43 ± 0.29 438.90 ± 259.36 2.02 ± 1.34 4.05 ± 4.13 0.10 ± 0.51 17.72 ± 3.33 31.89 ± 1.92 7.22 ± 8.71 7.56 ± 5.00 14.78 ± 4.14 105.11 ± 10.61 68.31 ± 12.51 7.64 ± 2.12 66.0 ± 32.040 0.28 ± 0.15 0.63 ± 0.33 506.65 ± 156.59 1.98 ± 0.54 3.86 ± 1.17 0.57 ± 0.71 17.99 ± 3.09 31.57 ± 2.10 8.18 ± 14.75 7.49 ± 5.77 14.58 ± 3.73 105.86 ± 11.82 66.98 ± 21.59 7.49 ± 1.69 77.6 ± 60.4 0.36 ± 0.29 0.76 ± 0.75 691.23 ± 317.88 2.68 ± 1.03 5.45 ± 2.39 0.47 ± 0.90 16.13 ± 1.41 33.38 ± 1.22 0.98 ± 0.91 10.65 ± 7.23 13.71 ± 2.83 90.60 ± 12.97 58.97 ± 9.66 6.72 ± 0.98 30.9 ± 14.9 0.16 ± 0.07 0.28 ± 0.14 397.11 ± 94.82 1.83 ± 0.68 3.72 ± 1.30 0.43 ± 0.26 15.27 ± 0.92 31.52 ± 1.20 3.87 ± 1.95 12.46 ± 7.06 15.88 ± 3.41 102.1 ± 27.79 58.53 ± 12.04 6.62 ± 1.06 62.4 ± 15.3 0.29 ± 0.08 0.54 ± 0.19 558.44 ± 189.45 2.19 ± 0.74 4.25 ± 1.39 0.49 ± 0.53 15.56 ± 0.72 30.38 ± 0.82 5.94 ± 4.70 13.02 ± 8.57 17.26 ± 3.37 110.36 ± 10.53 60.54 ± 13.52 6.58 ± 1.16 96.6 ± 51.9 0.41 ± 0.19 0.79 ± 0.48 686.73 ± 187.33 2.59 ± 0.84 4.98 ± 1.77 0.82 ± 0.31
Phase III
Bottom Subsurface Phase II
Surface Bottom Subsurfacea Surface
Phase I Parameters
Table 1 Physicochemical characteristics (mean ± standard deviation) during the three bloom phases in the East China Sea.
+ Concentrations of ambient nitrate (NO− 3 ), ammonium (NH4 ) nitrite (NO− ), and soluble reactive phosphorus (SRP) were measured manual2 ly using the cadmium–copper reduction method, the indophenol method (Grasshoff, 1976), the Griess–Ilosvay method (Barnes, 1959), and the phosphorus–molybdenum blue colorimetric method (Strickland and Parsons, 1972), respectively. Dissolved inorganic nitrogen was the − − sum of NH+ 4 , NO2 , and NO3 . Chlorophyll a was extracted with 90% acetone in Milli-Q water for 24 h in the dark at 4 °C and analyzed using a fluorescence spectrophotometer (F-4500, Hitachi Co., Japan) with a detection limit of 0.01 mg·m−3 according to Aminot and Rey (2000). Zooplankton samples were examined under a microscope and the individuals were identified using keys provided in Newell and Newell (1963), Riedl (1983) and Todd and Laverack (1991). Dissolved organic carbon and TDN were analyzed using hightemperature catalytic oxidation (HTCO) (Spyres et al., 2000) that yielded CO2 and N2, respectively, and subsequently measured with an auto-analyzer. Briefly, aliquots of filtered (Whatman GF/F) water samples were acidified to pH ~ 2 with 2 mol·l−1 hydrochloric acid (HCl) for DOC and TDN analyses. Dissolved organic carbon and TDN concentrations were determined with a TOC-VCPH TOC/TN analyzer (Shimadzu Corp., Tokyo, Japan), based on HTCO/infrared and HTCO/ chemiluminescence detection methods (Spyres et al., 2000), respectively. Milli-Q water (as a blank) and reference standards (1 mg·l−1 potassium biphthalate and 0.5 mg·l− 1 potassium nitrate) were injected every 5th sample to check the accuracy of the measurements. Dissolved organic nitrogen was calculated by subtracting DIN from TDN. Simultaneous measurements of 14 individual amino acids [aspartic acid (Asp), glutamic acid (Glu), serine (Ser), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), valine (Val), methionine (Met, not detected in DCAA), phenylalanine (Phe), isoleucine (Ile), and leucine (Leu)] were performed on a highperformance liquid chromatography (HPLC) system (e2695, Waters Alliance, USA) equipped with an auto-sampler, a pump, an Agilent ZORBAX Eclipse AAA (4.6 × 150 mm, 5 μm particles) column and a 2475 type fluorescence detector, using a protocol modified from Keil and Kirchman (1991). Briefly, 1 ml (30% w/w) HCl and 10 μl (12 μmol·ml−1) ascorbic acid were added to 1 ml of filtered water samples in 5 ml pre-combusted ampoules. These were flushed with nitrogen gas, sealed, and placed in an oven at 110 °C for 22 h. After hydrolysis, 300 μl hydrolysate was dried using a stream of nitrogen. To ensure the removal of HCl, 200 μl of Milli-Q water was added and the sample was redried, and then re-dissolved with Milli-Q water prior to HPLC analysis. The TDAA concentrations were measured using o-phthaldialdehyde (OPA) derivatization methods described by Kaiser and Benner (2005). The separation of amino acids was performed on an Agilent ZORBAX Eclipse AAA column with a 4.6 × 12.5 mm guard column with 5 μm particles, at a column temperature of 40 °C. Eluent A was methanol/acetonitrile (67/33 v/v), and eluent B was 40 mmol·l−1 potassium di-hydrogen phosphate adjusted to pH 7.2 with 50 w/w% sodium hydroxide. Amino acid derivatives were separated with a linear binary gradient starting with 12% A to 27% A at 6 min, then 40% A at 20 min, and 54% A at 36 min; after 36 min, the system was returned to 12% A at 40 min and equilibrated for 5 min. The flow rate was 1.0 ml min−1. The signals of the OPA derivatives were determined at an excitation
Surface
2.3. Chemical analyses
Temperature (°C) Salinity (psu) Chl a (μg·l−1) DIN (μmol·l−1) DON (μmol·l−1) DOC (μmol·l−1) DON/TDN (%) DOC/DON DFAA (nmol⋅l−1) DFAA (% DOC) DFAA (% DON) DCAA (nmol⋅l−1) DCAA (% DOC) DCAA (% DON) DI (TDAA)
dissolved organic carbon (DOC), total dissolved nitrogen (TDN), total dissolved amino acids (TDAA), and dissolved free amino acids (DFAA) were conducted in the laboratory. Several hundred milliliters of water were filtered through GF/F filters, which were immediately wrapped in aluminum foil and stored at − 20 °C for chlorophyll a (Chl a) measurements. All filters, glass bottles and aluminum foil were combusted for 4 h at 450 °C before use. Zooplankton samples were collected with a 0.4 m diameter plankton net (500 μm mesh size) at 1 m depth and preserved with buffered 4% formalin (Unesco, 1968).
85 17.98 ± 0.39 32.95 ± 1.34 2.36 ± 3.66 9.09 ± 5.38 15.03 ± 3.22 96.98 ± 17.24 64.13 ± 14.72 6.71 ± 1.91 31.8 ± 41.4 0.12 ± 0.13 0.25 ± 0.33 410.10 ± 72.45 1.63 ± 0.44 3.21 ± 0.89 −1.44 ± 0.30
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wavelength of 330 nm and emission wavelength of 450 nm. The precision of the liquid-phase hydrolysis and chromatographic analysis was 3–5% (relative standard deviation, SD). The DFAA concentrations were determined directly without hydrolysis; DCAA was calculated by subtracting DFAA from TDAA.
Accordingly, the salinity increased gradually from inshore to offshore waters (Table 1 and Fig. 4).
2.4. Statistical analyses
Variation in the concentration and distribution of certain bulk parameters, including Chl a, DIN, DOC, DON, the molar ratio of DOC to DON (DOC/DON) and the percentage contribution of DON to TDN, is shown in Figs. 3 and 4. From Phase I to Phase III, the concentration of Chl a in the surface and subsurface layers clearly increased, while the concentration of DIN and DON gradually decreased from 13.02 ± 8.57 μmol·l−1 to 5.94 ± 7.20 μmol·l−1 and from 17.26 ± 3.37 μmol·l−1 to 13.23 ± 3.05 μmol·l−1, respectively (Table 1, Fig. 3a, b). Accordingly, the percentage contribution of DON to TDN increased from 59.35 ± 11.53% to 71.29 ± 16.63% in the whole water column (Fig. 3b), and the trend of increasing the percentage contribution of DON to TDN in surface and subsurface layers was more pronounced than that in the bottom layer. Overall, Chl a concentrations in the coastal area and the southwest area were higher than those in the offshore and northeast areas (Fig. 4), while the DIN and DON concentrations showed a regular, gradually decreasing trend from nearshore to offshore waters during the three phases. Due to the input from the CDW, the concentration of DIN in the northern area was significantly higher than that in the southern area (Fig. 4) (Chai et al., 2006). However, the concentration of DON in the northern area was lower than that in the southern area, a similar spatial distribution to that of Chl a. The percentage contribution of DON to TDN increased gradually from inshore to offshore waters (Fig. 4), thus showing an opposite tendency to the DIN distribution. DOC/DON varied from 6.58 to 9.02 and from 6.62 to 9.19 in surface and subsurface waters, respectively (Table 1) and showed a similar spatial distribution to that of the % contribution of DON to TDN (Fig. 4).
Statistical analyses were performed using SPSS 16.0 (IBM Statistical Package for the Social Sciences Inc.). Differences among samples in individual variables were tested for statistical significance with one-way analysis of variance (ANOVA) or a t test. If the significance level was p b 0.001 (two-tailed test), the differences were recorded as significant. The significance of correlations between DFAA or TDAA and environmental parameters was tested using Pearson correlation coefficients (R), with significant correlations reported when p b 0.001 (two-tailed test, R software package). Principal component analysis (PCA) was carried out using the % molar composition of individual amino acids as the original data matrix to calculate the degradation index (DI). The DI was calculated using the formula originally proposed by Dauwe and Midddleburg (1998): DI ¼
X vari −AVGvar i i
STDvari
f ac:coe f i
where vari is the original % molar concentration of the individual amino acid i, AVGvari, and STDvari are the mean % molar concentration and SD of each amino acid, respectively, and fac. coefi is the factor coefficient of amino acid i. 3. Results
3.2. Dynamics and distribution of bulk parameters during the succession from a diatom to a dinoflagellate bloom
3.1. Hydrographic properties during the surveys From Phase I to Phase III, the temperature of surface and subsurface waters gradually increased, while that of bottom seawater varied little (Table 1 and Fig. 4). In contrast, an obvious stratification of seawater salinity was observed in this region, which was mainly controlled by the low-salinity CDW and the high-salinity TWWC (Chai et al., 2006).
3.3. Dynamics and distribution of amino acid indicators during the succession from a diatom to a dinoflagellate bloom The DFAA concentration in surface water varied from 40.9 nmol·l−1 to 217.8 nmol·l−1, with an average of 96.6 ± 51.9 nmol·l−1 during termination of the diatom bloom. While the dinoflagellate blooms were
Fig. 3. Changes in a) the average concentration of DIN and the percentage contribution of DIN to TDN, b) the concentration of DON and the % contribution of DON to TDN, and c) the concentration of DOC and the percentage of DOC to DON during the three bloom phases. Error bars represent the standard deviation. S: surface layer; M: middle layer; B: bottom layer.
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ongoing, the average DFAA concentration decreased to 77.6 ± 60.4 nmol·l−1 during Phase II, and decreased further, to 63.0 ± 47.1 nmol·l−1, during Phase III (Table 1, Fig. 5a). The DCAA concentration was ~10-fold higher than that of the DFAA in the study area. The average of DCAA concentration remained about 690 nmol·l−1 during Phase I and Phase II, but decreased obviously to 416.62 ± 69.84
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nmol·l−1 during Phase III (Table 1, Fig. 5a). From Phase I to Phase III, the DFAA and DCAA concentrations at all depths decreased by 22.41% and 15.32%, respectively. In particular, there were marked decreasing trends in DFAA and DCAA concentrations in surface waters, i.e., reductions of 34.82% and 32.78%, respectively. During Phase Ι, DFAA showed a pattern of gradually decreasing concentrations from
Fig. 4. Spatial distributions of salinity, chlorophyll a, DIN, DON concentrations, and the percentage contribution of DON to TDN and DOC/DON during the three bloom phases in surface waters of the Changjiang River estuary and its adjacent shelf.
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inshore to offshore waters, while the DCAA concentrations showed the opposite trend (Fig. 6). There was a similar distribution of DFAA and DCAA during Phases II and III. During the three bloom phases, there was a significant decline in both the average carbon-normalized and nitrogen-normalized DFAA and DCAA relative concentrations. There was a parallel trend of gradually decreasing values in both DFAA (% DON) and DCAA (% DON) with increasing water depth. Overall, the percentage of DFAA to DON in the bottom water was two-fold lower than that in the surface water. In contrast, the spatial distributions of DFAA (% DON) and DCAA (% DON) in surface water were similar to those of DFAA and DCAA (Fig. 6). The DI values of TDAA ranged from −2.11 to 1.95 during the three phases and significantly decreased from Phase I to Phase III (Table 1 and Fig. 5c). The distribution of DI also changed markedly during the three phases (Fig. 6). During Phase Ι, the DI showed a pattern of gradually decreasing values from inshore to offshore waters; a positive DI values indicated that DOM was more labile during this phase. The DI values in the northern and southern areas became negative during Phase II and became increasingly negative in the entire study area during Phase III (Fig. 6).
3.4. Variation in the composition of amino acids during the succession from a diatom to a dinoflagellate bloom In total fourteen individual DFAA and thirteen individual DCAAs (except methionine) were identified in TDAA pool, the dominant individuals of DFAA and DCAA were similar (Fig. 7). Glycine showed the highest % molar concentration in both the DFAA and DCAA pools, accounting for ~ 21% and 24%, respectively. The Δmol% is defined in order to distinguish the changes in individual amino acids during the three phases (Fig. 7b, d). The variation in Δmol% showed that glycine, histidine and phenylalanine were enriched while tyrosine and alanine were depleted in the DFAA pool during the succession from a diatom to a dinoflagellate bloom; glycine, serine, tyrosine and valine, however, were enriched and glutamic acid, histidine, alanine, arginine and phenylalanine were depleted in the DCAA pool (Fig. 7b, d). These results indicated that the biogeochemical behavior of DFAA did not agree with that of DCAA. It is worth noting that glycine was enriched while alanine
was depleted in both the DFAA and DCAA pools, suggesting that alanine is more available to bacteria and/or phytoplankton during the bloom succession.
4. Discussion 4.1. Concentration and composition of DFAA and DCAA Dissolved amino acids, which are the most-identified components of the DON pool (~ 15%, Dittmar et al., 2001; Keil and Kirchman, 1991; Sharp, 1983), probably play an important role in the cycling of nitrogen in coastal waters (Berman, 1997). Concentrations of DFAA (3.8 to 264.6 nmol·l−1) and DCAA (248.2 to 1537.6 nmol·l−1) found in our study are comparable to those reported for other coastal waters (Table 2). Our finding that DFAA and DCAA concentrations were higher during termination of the diatom bloom than those during the peak of the dinoflagellate bloom, may be attributable to the synthesis and release of amino acids by diatom cells (Meon and Kirchman, 2001). The dominant composition of DFAA and DCAA were also consistent with previous studies (Chen et al., 2014; Duan and Bianchi, 2007; Fuhrman, 1990; Jorgensen et al., 1993; Linares, 2006; McCarthy et al., 2004; Yang et al., 2009). During the succession of the phytoplankton assemblage, a number of bacteria attached to the aggregation of phytoplankton debris and released considerable amino acid through hydrolysis (Smith et al., 1992). Glycine, serine and threonine were enriched in algal cell walls, while the acidic amino acids (glutamic acid and aspartic acid) and the aromatic amino acids (tyrosine and phenylalanine) were enriched in cell contents (Hecky et al., 1973). These amino acids were conserved, due to the selectivity of amino acid utilization by heterotrophic bacteria (Ittekkot, 1982). Furthermore, the high mol% of glycine in TDAA indicated that this amino acid is more stable than other amino acids during the degradation of DOM by heterotrophic bacteria (Cowie and Hedges, 1992). However, a few studies have showed that DFAA are not really selectively consumed (Coffin, 1989; Simon and Rosenstock, 2007). Hence, the elevated mol% of glycine might just be a result of the high release rate and partitioning in the protein. In estuarine and coastal waters, the concentrations and spatial and temporal distributions of DFAA and DCAA are mainly influenced by
Fig. 5. Changes in absolute and relative TDAA parameters: a) TDAA concentrations; b) carbon- and nitrogen-normalized yields of TDAA, and c) the degradation index DI during the three bloom phases. Error bars represent the standard deviation except the DI (standard error shown). S: surface layer; M: middle layer; B: bottom layer.
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several factors, including land input, assimilation and active release by phytoplankton excretion, dissolution from fecal pellets and sloppy feeding by zooplankton, and bacterial active uptake and passive diffusion (Bronk, 2002). Among these factors, bacterial remineralization is generally considered the main pathway by which amino acids are utilized in aquatic ecosystems (Carlucci et al., 1984; Cherrier and Bauer, 2004; Coffin, 1989; Lemke et al., 2010; Simon and Rosenstock, 2007). Although the abundance of heterotrophic bacteria was not determined in our study, previous studies in this region during the spring of 2006 showed that a large number of colony-forming bacteria appeared following the diatom bloom, and a positive correlation between bacterial abundance and Chl a was described (R = 0.684, p b 0.05) (Xia et al., 2011), thus indicating that the increase in fresh, plankton-derived DOM with a high %TDAA can stimulate bacterial activity (Ittekkot,
89
1982). A few studies have also shown that bacteria can efficiently utilize DFAA and DCAA efficiently from DOM and DFAA associated with algal debris, and that DCAA can support a large fraction of bacterial growth in estuarine waters (Carlucci et al., 1984; Cherrier and Bauer, 2004; Coffin, 1989). Except for DCAA during Phase III, both DFAA and DCAA showed significant negative correlations with salinity (Fig. 8), which indicated that the CJR input is an important source of the DFAA and DCAA pools. Additionally, the DFAA concentration showed a significant positive correlation with Chl a during Phases I and II (R = 0.858, p b 0.001, Phase I; R = 0.787, p b 0.001, Phase II) (Fig. 8), indicating that the DFAA distribution was closely related to phytoplankton activity. In highly productive waters of the river-dominated plume, high levels of phytoplankton production and bacterial uptake of DFAA occur simultaneously, and DFAA,
Fig. 6. Spatial distributions of DFAA, DCAA concentrations, DFAA (% DON), DCAA (% DON) and DI during the three bloom phases in surface waters of the Changjiang River estuary and its adjacent shelf.
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Fig. 7. Average % molar contribution (mol%) and percent change (Δmol%) of both dissolved free (a and b, respectively) and combined (c and d, respectively) amino acids between bloom phases in the Changjiang River estuary and its adjacent shelf. Error bars represent the standard deviation of the mean amino acid mol%. Asp, aspartic acid; Glu, glutamic acid; Ser, serine; His, histidine; Arg, arginine; Gly, glycine; Thr, threonine; Ala, alanine; Tyr, tyrosine; Val, valine; Phe, phenylalanine; Ile, isoleucine; Leu, leucine.
as a labile component of the DON pool, was kept at low concentrations due to the rapid turnover by microbial activity (Carlucci et al., 1984; Crawford et al., 1974; Lu et al., 2014). A positive correlation between DCAA and Chl a was also found during Phase I. These results indicated that the release of excess photosynthates from phytoplankton may be a possible source of TDAA (Fogg, 1983). A significantly negative correlation between TDAA and zooplankton found during the present study (Fig. 8c), indicated that zooplankton may influence the concentration and distribution of TDAA by previously described mechanisms (Braven et al., 1995; Carlucci et al., 1984; Crawford et al., 1974; Lu et al., 2014). 4.2. Biochemical indicators of dissolved organic matter diagenesis Measurements of amino acid yield, DI and DOC/DON served to evaluate the diagenetic alteration of DOM during bloom succession. Amino acid yields measured in our study were higher than those in the Chukchi Sea and the Beaufort Sea (Shen et al., 2012), and similar to those in Ise Bay, Japan (Yamashita and Tanoue, 2003), likely because the Chukchi Sea and the Beaufort Sea are located in the Western Arctic Ocean and thus have low primary productivity, whereas Ise Bay is similar to the CJREAS in that they are characterized by high primary productivity and annual recurrence of HABs. The decrease in both the average value of the carbon- and nitrogen-normalized DFAA or DCAA concentrations documented during bloom succession in the CJREAS supported previous findings that the DOM undergoes extensive biological alteration (Davis et al., 2009; Yamashita and Tanoue, 2003). The increase in the mean value of DOC/DON in both surface and subsurface layers found in the present study during bloom succession may be due to a higher degradation of nitrogen-rich compounds and accumulation of carbon-rich compounds (Görs et al., 2007). In fact, there are several reports about uncoupling of the uptake of carbon and nitrogen by phytoplankton and/or bacteria. For example, Mulholland et al. (2004) reported uncoupled uptake of amino acids, i.e., N was taken up preferentially to C along an estuarine salinity gradient, resulting in increasing in the DOC/DON, which is consistent with the spatial distribution of DOC/DON obtained in the present study.
The decrease in DI values during the three bloom phases indicated that DOM experienced marked changes during the seasonal succession from a diatom to a dinoflagellate bloom. This conclusion was also supported by the variation of the DOC/DON and the amino acid yields. In fact, DI showed a significantly positive correlation with the carbonand nitrogen-normalized amino acids in the TDAA pool (Fig. 9b), in accordance with previous studies (Davis et al., 2009; Yamashita and Tanoue, 2003). However, the variations in DI values were more highly significant (p b 0.001, Student's t-test) than those of amino acid yields (p N 0.001, Student's t-test). These results indicated that the DI was a more sensitive parameter than the amino acid yields for diagnosing minor alterations in DOM decomposition (Davis et al., 2009). In fact, the DI was sufficiently sensitive to reflect diagenetic alterations of DOM in the Chukchi/Beaufort Seas and the western Canada Basin occurring on a time scale of days (Davis and Benner, 2005). In addition, decreasing DI values with increasing water depth described in our study are also in agreement with previous studies (Cowie and Hedges, 1994; Davis et al., 2009; Keil and Kirchman, 1999; Yamashita and Tanoue, 2003), indicating that the DOM degradation state became more pronounced from surface to bottom layers of the water column. The relative abundance of serine, glycine, valine and isoleucine in the TDAA pool decreased with increasing DI, while that of aspartic acid, glutamic acid, arginine and leucine increased with increasing DI (Fig. 10). Similar results were found in previous studies (Wheeler et al., 1974; Yamashita and Tanoue, 2003, 2004). In fact, some amino acids such as glutamic acid, aspartic acid, isoleucine, valine, tyrosine, and phenylalanine that are generally concentrated in diatom cell plasma (Dauwe and Middelburg, 1998), are very susceptible to degradation, and are abundant in freshly derived marine OM. These amino acids show strong depletion with an increasing state of decomposition. In summary, variable dynamics of biological indicators, including the DOC/DON, the amino acid yields, and the DI showed that DOM exhibited pronounced diagenetic alterations by phytoplankton (and presumably by bacteria) during the diatom to dinoflagellate bloom succession in the CJREAS.
)
This study
Lu et al. (2014)
Chen et al. (2013)
Shen et al. (2012)
Yang et al. (2009)
440–1180 – 640 ± 450 (8.2 ± 7.3) – 550–4200 (0.6–4.2% DON) TDAA: 250–520 (~10% DON) 120–1940 TDAA: 149–1694 (0.9–6.9% DOC) – – – – Avg.: 323 (0.39–4.23% DOC) Avg.: 186 (0.47–3.29% DOC) 780–5940 (2.3–14.0% DOC) 1390–7160 (1.5–14.3% DOC) – – 239.1–1537.6 (0.94–5.99% DOC; 1.6–18.12% DON)
(nmol⋅l )
April 2006
July to August 2002, 2004 July 2008, 2009 April 2010
April 2011 October 2011 April and May, 2011
Yellow Sea, China
Chukchi Sea Beaufort Sea, Arctic Sea Bohai Sea, China
Coastal Georgia, USA
East China Sea, China
February 1975 March to April 1976 June, September and October 1987 February, June and August 1985; April 1986 May to November 1996 Summer 2000 February and May 1999; May 2000 April 2001 May to June 2005 Biscayne Bay, USA Northern North Sea Ohtsuchi Bay, Japan Chesapeake Bay, USA Gulf of Riga, Baltic Sea Laptev Sea Stony Brook Harbor and Caribbean Sea Northwestern Pacific East China Sea, China
(nmol⋅l
17–47 100–300 170 ± 90(1.9 ± 0.8) 6.5–23 40–350 (0.5–9.1% DON) – 20–3500 – 0.24–1.12 μmol N l−1 0.18–0.48 μmol N l−1 940 ± 80 570 ± 50 – – 320–2350 200–860 13.2–77.5 54.3–420.0 3.8–264.6 (0.01–1.26% DOC; 0.03–3.35% DON)
−1
DCAA
−1
DFAA Depth Date Location
Table 2 Comparative review of the concentrations of DFAA and DCAA and their percent contribution to DOC and DON in global coastal waters.
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4.3. Implications for the role of DON as a potential nutrient supporting the outbreak of the dinoflagellate bloom
0– N 6 m b145 m Surface Surface 3–30 m 0– N 500 m Microlayer 0–400 m Surface 10 m Microlayer Subsurface 0–180 m 0–180 m Microlayer Subsurface 0–17 m 0–17 m 0–72 m
Reference
Lee and Bada (1977) Hammer and Kattner (1986) Tupas and Koike (1990) Fuhrman (1990) Jørgensen et al. (1999) Dittmar et al. (2001) Kuznetsova and Lee (2002) Yamashita and Tanoue (2003) Li et al. (2009)
G. Zhang et al. / Marine Chemistry 176 (2015) 83–95
In estuarine and coastal ecosystems, phytoplankton uptake of DON is dependent on the presence of other nitrogenous forms, particularly when the DIN pool is exhausted or near exhaustion (Bronk, 2002; Lomas and Glibert, 1999). In turn, the relative use of organic N by autotrophs and heterotrophs will potentially affect the plankton community composition and may stimulate HABs (Andersson et al., 2006). Generally, a high concentration of NO− 3 tends to promote the growth of diatoms, whereas dinoflagellates appear to prefer reduced N substrates, including DON, relative to diatoms (Berg et al., 2003; Paerl, 1991). In the present study, during the dissipation phase of the diatom bloom (Phase I), DON contributed to ~60% of the TDN pool, and DFAA also comprised a significant fraction of the DON pool and thus contributed a potential nitrogen source. During the development/growth and peak stages of the dinoflagellate bloom (Phases II and III, respectively), both the DIN concentration and the contribution of DIN to TDN decreased sharply, indicating that the status of DIN depletion and limitation were more severe. The contribution of DON to TDN increased while the concentration of DON (DFAA and DCAA) in surface and subsurface water layers showed a remarkable decrease, indicating that the DON components were directly or indirectly utilized during bloom succession. Variations in DI values during bloom succession also illustrated that DON underwent severe diagenesis and that DON may be partially decomposed into small molecules or DIN which can be readily taken up by phytoplankton. This observation is in agreement with findings of Collos et al. (2014) based on a monthly study in the Thau lagoon, southern France, where the outbreak of the diatom bloom was due to the inorganic nitrate and phosphate nutrient pulses, while that of the dinoflagellate (A. tamarense) bloom was due to organic nutrient enrichment over the last 20 years (1990−2011). Both heterotrophic and autotrophic uptake are recognized as the main sinks for DON; however, direct uptake of DON by phytoplankton, without bacterial mediation, was considered to be relatively minor in the nitrate-rich Thames estuary (Middleburg and Nieuwenhuize, 2000). A few studies have also demonstrated that bacterial degradation of DON is followed by phytoplankton uptake of the released compounds (Berman et al., 1991; Lisa et al., 1995). The recycling of organic and inorganic forms of N through bacterial pathways was also shown to be important in river-dominated plume (Gardner et al., 1996). The capacity and rate of DON uptake are known to be taxon- and composition-specific (Berman and Chava, 1999; Fan et al., 2003; Herndon and Cochlan, 2007). Nutrient conditions characterized by low DIN and high DON concentrations are expected to simulate mixotrophic assimilation of organic nutrients by dinoflagellates (Stoecker et al., 1997). Moreover, dinoflagellates exhibit a wide range of nutritional capabilities (Gaines and Elbrächter, 1987). There is increasing evidence that DFAA or DCAA can be utilized as a source of carbon or nitrogen by some phytoplankton species, particularly dinoflagellates, in a low inorganic N environment (Collos et al., 2014; Li et al., 2009, 2010; Mulholland et al., 2002). Although conventional bioassay experiments to measure the bioavailability of DON were not conducted in this study, several previous experimental studies have also demonstrated the ability of P. donghaiense to utilize and assimilate organic N compounds such as amino acids and fluvial DON as the sole source of nitrogen (Hu et al., 2012; Ou et al., 2014). Moreover, variable dynamics of biological indicators based on amino acid composition and yields showed that DON is bioavailable during the diatom to dinoflagellate bloom succession. 5. Conclusions The outbreak of a large-scale dinoflagellate bloom in the CJREAS, was accompanied by a concomitant decrease in DON (DFAA and DCAA) concentrations. Variable dynamics of biological indicators used in the
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Fig. 9. Correlations between a) carbon- and nitrogen-normalized yields, b) the degradation index (DI) and carbon-normalized yields, and c), DI and DOC/DON in the Changjiang River estuary and its adjacent shelf.
Fig. 10. Plots of the degradation index (DI) and relative percent molar abundance (mol%) of individual dissolved amino acids for all stations and depths. Correlation coefficients for individual dissolved amino acids vs. DI: aspartic acid, R = 0.572, p b 0.001, n = 126; glutamic acid, R = 0.823, p b 0.001, n = 126; serine, R = −0.888, p b 0.001, n = 125; histidine, R = 0.515, p b 0.001, n = 126; glycine, R = −0.566, p b 0.001, n = 126; leucine, R = 0.508, p b 0.001, n = 95.
present study, including the DOC/DON, the amino acid yields, and the degradation index, showed that DOM underwent marked diagenetic alteration by phytoplankton (and presumably bacteria) during the diatom to dinoflagellate bloom succession in the CJREAS. These results indicated that the recycling pathway of DON increased in importance
over time during the study period and that the components in the DON pool likely provide a significant portion of the total nitrogen demand during a dinoflagellate bloom under conditions of low inorganic nitrogen availability. Given that DON transport was probably not conservative, DON production and consumption were difficult to quantify.
Fig. 8. Correlations among salinity, chlorophyll a and zooplankton abundance with DAA (● DFAA, ○ DCAA) during the three bloom phases (a, Phase I; b, Phase II and c, Phase III) in the Changjiang River estuary and its adjacent shelf.
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Therefore, in future, it is necessary to trace the biogeochemical cycle of DON in the CJREAS using isotopically-labeled compounds. Acknowledgments The authors gratefully acknowledge colleagues from the laboratory of Marine Chemistry Theory and Technology, Ocean University of China, the captain and crews of R/V Run Jiang for their help in sampling and sample processing. The authors also thank Dr. Y. Shen and two anonymous referees for their helpful comments that greatly improved the manuscript. This study is funded by the National Science Foundation of China (NSFC)-Shandong Joint Fund for Marine Science Research Centers with Grant No. U1406403 and the National Key Basic Research Program of China with Grant No. 2010CB428701. References Aminot, A., Rey, F., 2000. Standard procedure for the determination of chlorophyll a by spectroscopic methods. Tech. Mar. Environ. Sci. 1–17. Amon, R.M., Fitznar, H., Benner, R., 2001. Linkages among the bioreactivity, chemical composition, and diagenetic state of marine dissolved organic matter. Limnol. Oceanogr. 46 (2), 287–297. Andersson, M.G., Van Rijswijk, P., Middelburg, J.J., 2006. Uptake of dissolved inorganic nitrogen, urea and amino acids in the Scheldt estuary: comparison of organic carbon and nitrogen uptake. Aquat. Microb. Ecol. 44, 303–315. Antia, N.J., Harrison, P.J., Oliveira, L., 1991. The role of dissolved organic nitrogen in phytoplankton nutrition, cell biology and ecology. Phycologia 30 (1), 1–89. Barnes, H., 1959. Apparatus and methods of oceanography. Part One: Chemical. George Allen and Unwin Ltd, London, p. 341. Benner, R., 2002. Chemical composition and reactivity. In: Hansell, D.A., Carlson, C.A. (Eds.), Biogeochemistry of Marine Dissolved Organic Matter. Academic Press, San Diego, pp. 56–90. Berg, G.M., Glibert, P.M., Lomas, M.W., Burford, M.A., 1997. Organic nitrogen uptake and growth by the chrysophyte Aureococcus anophagefferens during a brown tide event. Mar. Biol. 129 (2), 377–387. Berg, G.M., Glibert, P.M., Jørgensen, N.O., Balode, M., Purina, I., 2001. Variability in inorganic and organic nitrogen uptake associated with riverine nutrient input in the Gulf of Riga, Baltic Sea. Estuaries 24 (2), 204–214. Berg, G.M., et al., 2003. Plankton community composition in relation to availability and uptake of oxidized and reduced nitrogen. Aquat. Microb. Ecol. 30 (3), 263–274. Berman, T., 1997. Dissolved organic nitrogen utilization by an Aphanizomenon bloom in Lake Kinneret. J. Plankton Res. 19 (5), 577–586. Berman, T., Bronk, D.A., 2003. Dissolved organic nitrogen: a dynamic participant in aquatic ecosystems. Aquat. Microb. Ecol. 31 (3), 279–305. Berman, T., Chava, S., 1999. Algal growth on organic compounds as nitrogen sources. J. Plankton Res. 21 (8), 1423–1437. Berman, T., Chava, S., Kaplan, B., Wynne, D., 1991. Dissolved organic substrates as phosphorus and nitrogen sources for axenic batch cultures of freshwater green algae. Phycologia 30, 339–345. Braven, J., Butler, E.I., Chapman, J., Ecens, R., 1995. Changes in dissolved free amino acids composition in sea water associated with phytoplankton populations. Sci. Total Environ. 172, 145–150. Bronk, D.A., 2002. Dynamics of DON. In: Hansell, D.A., Carlson, C.A., Hansell, D.A., Carlson, C.A. (Eds.), Biogeochemistry of Marine Dissolved Organic Matter. Academic Press, San Diego, pp. 153–249. Bronk, D.A., See, J.H., Bradley, P., Killberg, L., 2007. DON as a source of bioavailable nitrogen for phytoplankton. Biogeosciences 4, 282–296. Carlucci, A.F., Craven, D.B., Henrichs, S.M., 1984. Diel production and microheterotrophic utilization of dissolved free amino acids in waters off Southern California. Appl. Environ. Microbiol. 48 (1), 165–170. Chai, C., Yu, Z., Song, X., Cao, X., 2006. The status and characteristics of eutrophication in the Yangtze River (Changjiang) estuary and the adjacent East China Sea, China. Hydrobiologia 563, 313–328. Chen, Y., Yang, G., Wu, G., Gao, X., Xia, Q., 2013. Concentration and characterization of dissolved organic matter in the surface microlayer and subsurface water of the Bohai Sea, China. Cont. Shelf Res. 52, 97–107. Chen, C., et al., 2014. Accumulation of free amino acids in marine diatom resting cells during rejuvenation. J. Sea Res. 85, 483–490. Cherrier, J., Bauer, J.E., 2004. Bacterial utilization of transient plankton-derived dissolved organic carbon and nitrogen inputs in surface ocean waters. Aquat. Microb. Ecol. 35, 229–241. Coffin, R.B., 1989. Bacterial uptake of dissolved free and combined amino acids in estuarine waters. Limnol. Oceanogr. 34, 531–542. Collos, Y., et al., 2014. Comparing diatom and Alexandrium catenella/tamarense blooms in Thau lagoon: importance of dissolved organic nitrogen in seasonally N-limited systems. Harmful Algae 37, 84–91. Colombo, J.C., Silerberg, N., Gearing, J.N., 1998. Amino acids biogeochemistry in the Laurentian Trough: vertical fluxes and individual reactivity during early diagenesis. Org. Geochem. 29 (4), 933–945. Cowie, G.L., Hedges, J.I., 1992. Sources and reactivities of amino acids in a coastal marine environment. Limnol. Oceanogr. 37 (4), 703–724.
Cowie, G.L., Hedges, J.I., 1994. Biochemical indicators of diagenetic alteration in natural organic matter mixtures. Nature 369. Crawford, C.C., Hobbie, J.E., Webb, K.L., 1974. The utilization of dissolved free amino acids by estuarine microorganisms. Ecology 551–563. Dauwe, B., Middelburg, J.J., 1998. Amino acids and hexosamine as indicators of organic matter degradation state in North Sea sediments. Limnol. Oceanogr. 43 (5), 782–798. Dauwe, B., Middelburg, J.J., Rijswijk, P.V., Sinke, J., Herman, P.M.J., Heip, H.R., 1999a. Enzymatically hydrolyzable amino acids in North Sea sediments and their possible implication for sediment nutritional values. J. Mar. Res. 57, 109–134. Dauwe, B., Middelbury, J.J., Herman, P.M.J., Heip, C.H.R., 1999b. Linking diagenetic alteration of amino acids and bulk organic matter reactivity. Limnol. Oceanogr. 44 (7), 1809–1814. Davis, J., Benner, R., 2005. Seasonal trends in the abundance, composition and bioavailability of particulate and dissolved organic matter in the Chukchi/Beaufort Seas and western Canada Basin. Deep-Sea Res. II Top. Stud. Oceanogr. 52 (24), 3396–3410. Davis, J., Kaiser, K., Benner, R., 2009. Amino acids and amino sugar yields and compositions as indicators of dissolved organic matter diagenesis. Org. Geochem. 40, 343–352. Dittmar, T., Fitznar, H.P., Kattner, G., 2001. Origin and biogeochemical cycling of organic nitrogen in the eastern Arctic Ocean as evident from D- and L-amino acids. Geochim. Cosmochim. Acta 65 (22), 4103–5114. Duan, S., Bianchi, T.S., 2007. Particulate and dissolved amino acids in the lower Mississippi and Pearl Rivers (USA). Mar. Chem. 107, 214–229. Fan, C., Glibert, P.M., Burkholder, J.M., 2003. Characterization of the affinity for nitrogen, uptake kinetics, and environmental relationships for Prorocentrum minimum in natural blooms and laboratory cultures. Harmful Algae 2 (4), 283–299. Fogg, G.E., 1983. The ecological significance of extracellular products of phytoplankton photosynthesis. Bot. Mar. 26 (1), 3–14. Fuhrman, J., 1990. Dissolved free amino acid cycling in an estuarine outflow plume. Mar. Ecol. Prog. Ser. 66, 197–203. Gaines, G., Elbrächter, M., 1987. Heterotrophic Nutrition. The Biology of Dinoflagellates. In: Taylor, F.J.R. (Ed.)Blackwell Scientific Publications, Oxford, pp. 224–268. Gao, X., Song, J., 2005. Phytoplankton distributions and their relationship with the environment in the Changjiang Estuary, China. Mar. Pollut. Bull. 50 (3), 327–335. Gardner, W.S., Benner, R., Amon, R.M.W., Cotner, J.B., Cavaletto, J.F., Johnson, J.R., 1996. Effects of high-molecular weight dissolved organic matter on nitrogen dynamics in the Mississippi River plume. Mar. Ecol. Prog. Ser. 133, 287–297. Glibert, P.M., Garside, C., Fuhrman, J.A., Roman, M.R., 1991. Dependent coupling of inorganic and organic nitrogen uptake and regeneration in the plume of the Chesapeake Bay estuary and its regulation by large heterotrophs. Limnol. Oceanogr. 36 (5), 895–909. Glibert, P.M., Anderson, D.M., Gentien, P., Graneli, E., Sellner, K.G., 2005. The global, complex phenomena of harmful algal blooms. Oceanography 18 (2), 130–141. Görs, S., Rentsch, D., Schiewer, U., Karsten, U., Schumann, R., 2007. Dissolved organic matter along the eutrophication gradient of the Darβ-Zingst Bodden Chain, Southern Baltic Sea: I. Chemical characterisation and composition. Mar. Chem. 104 (3), 125–142. Grasshoff, K., 1976. Methods of Seawater Analysis. Verlag Chemie, Weinheim, New York, pp. 276–281. Hammer, K.D., Kattner, G., 1986. Dissolved free amino acids in the marine environment: a carbon to nitrogen ratio shift during diatom blooms. Mar. Ecol. Prog. Ser. 31, 35–45. Hecky, R.E., Mopper, K., Kilham, P., Degens, E.T., 1973. The amino acid and sugar composition of diatom cell-walls. Mar. Biol. 19 (4), 323–331. Herndon, J., Cochlan, W.P., 2007. Nitrogen utilization by the raphidophyte Heterosigma akashiwo: growth and uptake kinetics in laboratory cultures. Harmful Algae 6 (2), 260–270. Hu, Z., Mulholland, M.R., Duan, S., Xu, N., 2012. Effects of nitrogen supply and its composition on the growth of Prorocentrum donghaiense. Harmful Algae 13, 72–82. Ittekkot, V., 1982. Variations of dissolved organic matter during a plankton bloom: qualitative aspects, based on sugar and amino acid analysis. Mar. Chem. 11, 143–158. Jorgensen, N.O.G., Kroer, N., Coffin, R.B., Yang, X., Lee, C., 1993. Dissolved free amino acids, combined amino acids, and DNA as sources of carbon and nitrogen to marine bacteria. Mar. Ecol. Prog. Ser. 98, 135–148. Jørgensen, N.O., Tranvik, L.J., Berg, G.M., 1999. Occurrence and bacterial cycling of dissolved nitrogen in the Gulf of Riga, the Baltic Sea. Mar. Ecol. Prog. Ser. 191, 1–18. Kaiser, K., Benner, R., 2005. Hydrolysis-induced racemization of amino acids. Limnol. Oceanogr. Methods 3, 318–325. Kaiser, K., Benner, R., 2009. Biochemical composition and size distribution of organic matter at the Pacific and Atlantic time-series stations. Mar. Chem. 113, 63–77. Keil, R.G., Kirchman, D.L., 1991. Dissolved combined amino acids in marine waters as determined by a vapor-phase hydrolysis method. Mar. Chem. 33, 243–259. Keil, R.G., Kirchman, D.L., 1999. Utilization of dissolved protein and amino acids in the northern Sargasso Sea. Aquat. Microb. Ecol. 18 (3), 293–300. Kuznetsova, M., Lee, C., 2002. Dissolved free and combined amino acids in nearshore seawater, sea surface microlayers and foams: influence of extracellular hydrolysis. Aquat. Sci. 64 (3), 252–268. Lee, C., Bada, F.L., 1977. Dissolved amino acids in the equatorial Pacific, the Sargasso Sea, and Biscayne Bay. Limnol. Oceanogr. 22, 502–510. Lemke, A., Lunau, M., Badewien, T.H., Simon, M., 2010. Short-term and seasonal dynamics of bacterial biomass production and amino acid turnover in the water column of an intertidal ecosystem, the Wadden Sea. Aquat. Microb. Ecol. 61, 205–218. Li, J., Glibert, P.M., Zhou, M., Lu, S., Lu, D., 2009. Relationships between nitrogen and phosphorus forms and ratios and the development of dinoflagellate blooms in the East China Sea. Mar. Ecol. Prog. Ser. 383, 11–26. Li, J., Glibert, P.M., Zhou, M., 2010. Temporal and spatial variability in nitrogen uptake kinetics during harmful dinoflagellate blooms in the East China Sea. Harmful Algae 9 (6), 531–539.
G. Zhang et al. / Marine Chemistry 176 (2015) 83–95 Linares, F., 2006. Effect of dissolved free amino acids (DFAA) on the biomass and production of microphytobenthic communities. J. Exp. Mar. Biol. Ecol. 330, 469–481. Lisa, T., Piedras, P., Cardenas, J., Pineda, M., 1995. Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells. Plant Cell Environ. 18, 583–588. Liu, J.P., et al., 2007. Flux and fate of Yangtze River sediment delivered to the East China Sea. Geomorphology 85 (3), 208–224. Lomas, M.W., Glibert, P.M., 1999. Temperature regulation of nitrate uptake: a novel hypothesis about nitrate uptake and reduction in cool-water diatoms. Limnol. Oceanogr. 44 (3), 556–572. Lu, X., et al., 2014. Temporal dynamics and depth variations of dissolved free amino acids and polyamines in coastal seawater determined by high-performance liquid chromatography. Mar. Chem. 163, 36–44. McCarthy, M.D., Benner, R., Lee, C., Hedges, J.I., Fogel, M.L., 2004. Amino acids carbon isotopic fractionation patterns in oceanic dissolved organic matter: an unaltered photoautotrophic source for dissolved organic nitrogen in the ocean? Mar. Chem. 92, 123–134. McIsaac, G.F., David, M.B., Gertner, G.Z., Goolsby, D.A., 2001. Eutrophication: nitrate flux in the Mississippi River. Nature 414 (6860), 166–167. Meon, B., Kirchman, D.L., 2001. Dynamics and molecular composition of dissolved organic material during experimental phytoplankton blooms. Mar. Chem. 75, 185–199. Middleburg, J.J., Nieuwenhuize, J., 2000. Nitrogen uptake by heterotrophic bacteria and phytoplankton in the nitrate-rich Thames estuary. Mar. Ecol. Prog. Ser. 203, 13–21. Mulholland, M.R., Gobler, C.J., Lee, C., 2002. Peptide hydrolysis, amino acid oxidation, and nitrogen uptake in communities seasonally dominated by Aureococcus anophagefferens. Limnol. Oceanogr. 47 (4), 1094–1108. Mulholland, M.R., Boneillo, G., Minor, E.C., 2004. A comparison of N and C uptake during tide (Aureococcus anophagefferens) blooms from two coastal bays on the east coast of the USA. Harmful Algae 3, 361–376. Newell, G.E., Newell, R.C., 1963. Marine Plankton. A Practical Guide. 5th ed. Hutchinson ed., London, p. 244. Ou, L., Lundgren, V., Lu, S., Graneli, E., 2014. The effect of riverine dissolved organic matter and other nitrogen forms on the growth and physiology of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller. J. Sea Res. 85, 499–507. Paerl, H.W., 1991. Ecophysiological and trophic implications of light-stimulated amino acid utilization in marine picoplankton. Appl. Environ. Microbiol. 57 (2), 473–479. Palenik, B., Henson, S.E., 1997. The use of amides and other organic nitrogen sources by the phytoplankton Emiliania huxleyi. Limnol. Oceanogr. 42 (7), 1544–1551. Riedl, R., 1983. Fauna e flora del Mar Mediterraneo. Omega Ed, Barcelona. Seitzinger, S.P., Sanders, R.W., 1999. Atmospheric inputs of dissolved organic nitrogen stimulate estuarine bacteria and phytoplankton. Limnol. Oceanogr. 44 (3), 721–730. Sharp, J.H., 1983. The Distributions of Inorganic Nitrogen and Dissolved and Particulate Organic Nitrogen in the Sea. Academic Press, New York, pp. 1–35. Shen, Y., Fichot, C.G., Benner, R., 2012. Dissolved organic matter composition and bioavailability reflect ecosystem productivity in the Western Arctic Ocean. Biogeosci. Discuss. 9 (7), 9571–9601.
95
Simon, M., Rosenstock, B., 2007. Different coupling of dissolved amino acid, protein, and carbohydrate turnover to heterotrophic picoplankton production in the Southern Ocean in austral summer and fall. Limnol. Oceanogr. 52, 85–95. Smith, D.C., Simon, M., Alldredge, A.L., Azam, F., 1992. Intense hydrolytic enzyme activity on marine aggregates and implications for rapid particle dissolution. Nature 359, 139–142. Spyres, G., Nimmo, M., Worsflod, P.J., Achterberg, E.P., Miller, A.E.J., 2000. Determination of dissolved organic carbon in seawater using high temperature catalytic oxidation techniques. Trends Anal. Chem. 19 (8), 498–506. Stoecker, D.K., Li, A., Coats, D.W., Gustafson, D.E., Nannenl, M.K., 1997. Mixotrophy in the dinoflagellate Prorocentrum minimum. Mar. Ecol. Prog. Ser. 152, 1–12. Strickland, J., Parsons, T., 1972. A practical handbook of seawater analysis. Fish Res. Board Can. Bull. 167–311. Tang, D., Di, B., Wei, G., Ni, I., Wang, S., 2006. Spatial, seasonal and species variations of harmful algal blooms in the South Yellow Sea and East China Sea. Hydrobiologia 568 (1), 245–253. Todd, C.D., Laverack, M.S., 1991. Coastal Marine Zooplankton. A Practical Manual for Students. Cambridge University Press. Tupas, L., Koike, I., 1990. Amino acid and ammonium utilization by heterotrophic marine bacteria grown in enriched seawater. Limnol. Oceanogr. 35 (5), 1145–1155. Unesco, 1968. Zooplankton Sampling. Monographs on Oceanographic Methodology. The Unesco Press, Paris, p. 177. Varela, M.M., et al., 2005. Nitrogen uptake and dissolved organic nitrogen release in planktonic communities characterised by phytoplankton size-structure in the Central Atlantic Ocean. Deep-Sea Res. I 52, 1637–1661. Wawrik, B., Callaghan, A.V., Bronk, D.A., 2009. Use of inorganic and organic nitrogen by Synechococcus spp. and diatoms on the West Florida Shelf as measured using stable isotope probing. Appl. Environ. Microbiol. 75 (21), 6662–6670. Wheeler, P.A., North, B.B., Stephens, G.C., 1974. Amino acids uptake by marine phytoplankters. Limnol. Oceanogr. 19 (2), 249–259. Xia, W.J., Wang, J.T., Tan, L.J., Wang, Q.L., 2011. Vatiation of bacteria biomass and its possible controlling factors in the East China Sea. J. Ocean Univ. China 10 (2), 135–141. Yamashita, Y., Tanoue, E., 2003. Distribution and alteration of amino acids in bulk DOM along a transect from bay to oceanic waters. Mar. Chem. 82, 145–160. Yamashita, Y., Tanoue, E., 2004. Chemical characteristics of amino acids-containing dissolved organic matter in seawater. Org. Geochem. 35, 679–692. Yang, G., Chen, Y., Gao, X., 2009. Distribution of dissolved free amino acids, dissolved inorganic nitrogen and chlorophyll a in the surface microlayer and subsurface water of the Yellow Sea, China. Cont. Shelf Res. 29, 1737–1747. Zhang, J., et al., 1999. Human impacts on the large world rivers: would the Changjiang (Yangtze River) be an illustration? Glob. Biogeochem. Cycles 13 (4), 1099–1105. Zhou, M., Yan, T., Zhou, J., 2003. Preliminary analysis of the characteristics of red tide areas in Changjiang River estuary and its adjacent sea. J. Appl. Ecol. 14 (7), 1031–1038. Zhou, W., Yin, K., Zhu, D., 2006. Phytoplankton biomass and high frequency of Prorocentrum donghaiense harmful algal bloom in Zhoushan sea area in spring. J. Appl. Ecol. 17 (5), 887–893.