In-situ production of humic-like fluorescent dissolved organic matter during Cochlodinium polykrikoides blooms

Estuarine, Coastal and Shelf Science 203 (2018) 119e126

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In-situ production of humic-like fluorescent dissolved organic matter during Cochlodinium polykrikoides blooms Hyeong Kyu Kwon a, Guebuem Kim a, *, Weol Ae Lim b, Jong Woo Park b a b

School of Earth and Environmental Sciences/Research Institute of Oceanography, Seoul National University, Seoul 08826, Republic of Korea Ocean Climate and Ecology Research Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 November 2017 Received in revised form 31 January 2018 Accepted 7 February 2018 Available online 11 February 2018

We investigated phytoplankton pigments, dissolved organic carbon (DOC), and fluorescent dissolved organic matter (FDOM) during the summers of 2013 and 2016 in the coastal area of Tongyeong, Korea, where Cochlodinium polykrikoides blooms often occur. The density of red tides was evaluated using a dinoflagellate pigment, peridinin. The concentrations of peridinin and DOC in the patch areas were 15and 4-fold higher than those in the non-patch areas. The parallel factor analysis (PARAFAC) model identified one protein-like FDOM (FDOMT) and two humic-like FDOM, classically classified as marine FDOM (FDOMM) and terrestrial FDOM (FDOMC). The concentrations of FDOMT in the patch areas were 5fold higher than those in the non-patch areas, likely associated with biological production. In general, FDOMM and FDOMC are known to be dependent exclusively on salinity in any surface waters of the coastal ocean. However, in this study, we observed strikingly enhanced FDOMC concentration over that expected from the salinity mixing, whereas FDOMM increases were not clear. These FDOMC concentrations showed a significant positive correlation against peridinin, indicating that the production of FDOMC is associated with the red tide blooms. Our results suggest that FDOMC can be naturally enriched by some phytoplankton species, without FDOMM enrichment. Such naturally produced FDOM may play a critical role in biological production as well as biogeochemical cycle in red tide regions. © 2018 Elsevier Ltd. All rights reserved.

Keywords: Cochlodinium polykrikoides blooms Fluorescent dissolved organic matter Phytoplankton pigments Protein-like FDOM Humic-like FDOM Dissolved organic carbon

1. Introduction The fraction of dissolved organic matter (DOM) that emits absorbed UV radiation as fluorescence is called fluorescent DOM (FDOM). FDOM comprises a variable but significant fraction of the DOM pool and is capable of influencing the optical characteristics of the water column. Fluorescence spectroscopy by means of excitation-emission matrices (EEMs) has been widely used to characterize FDOM in coastal and oceanic environments (Coble, 1996; Stedmon and Markager, 2005; Chari et al., 2012; Mendoza et al., 2012; Kim and Kim, 2016). The intensity of emission (Em) fluorescence spectra collected at different excitation (Ex) wavelengths results in EEMs where different peaks (characteristics of humic-like and protein-like substances) are distinguished (Coble, 1996). Known fluorescence signatures are peaks C (Ex/Em 350/ 460 nm), M (Ex/Em 320/420 nm), and A (Ex/Em 260/460 nm),

* Corresponding author. E-mail addresses: [email protected] (H.K. Kwon), [email protected] (G. Kim), [email protected] (W.A. Lim), [email protected] (J.W. Park). https://doi.org/10.1016/j.ecss.2018.02.013 0272-7714/© 2018 Elsevier Ltd. All rights reserved.

which are humic-like substances, while peaks B (Ex/Em 275/ 305 nm) and T (Ex/Em 275/340 nm) are considered to be proteinlike substances (Coble, 1996, 2007; Stedmon and Nelson, 2015). Fluorescence intensity measurements at these peaks can be used to understand the dynamics of different DOM pools. It has been well documented that phytoplankton is one of the major sources of DOM in the ocean (Biddanda and Benner, 1997). According to culture experiment results, many phytoplankton can directly contribute to the production of protein-like FDOM (FDOMT) (Romera-Castillo et al., 2010; Fukuzaki et al., 2014; Kinsey et al., 2018). Phytoplankton is known to release extracellular nitrogenous compounds such as amino acids, carbohydrates, and peptides as well as proteins (Biddanda and Benner, 1997). Among these substances, tryptophan in amino acids, which is the dominant substance during phytoplankton growth, may be detected as FDOMT (Yamashita and Tanoue, 2003; Kinsey et al., 2018). FDOMT has been considered to be a labile component (Coble, 1996, 2007; Stedmon et al., 2003). In general, marine humic-like FDOM (FDOMM) has been shown to be produced by the microbiological degradation of organic matter (Kinsey et al., 2018). Culture

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experiments using bacterial assemblages without phytoplankton confirmed the bacterial production of FDOMM (Shimotori et al., 2009). However, marine phytoplankton is also capable of producing FDOMM (Romera-Castillo et al., 2010; Fukuzaki et al., 2014). On the other hand, the production of humic-like FDOM (FDOMC), traditionally regarded as humic substances originating from terrestrial environments, is more complicated. In culture experiments using phytoplankton, FDOMC was produced by the diatoms Chaetoceros muelleri, Ditylum brightwellii, dinoflagellate Heterocapsa circularisquama, raphidophyte Heterosigma akashiwo, and chlorophyte Oltmannsiellopsis virdis, but not by the diatom C. curvisetus, dinoflagellate Alexandrium catenella, cryptophyte Rhodomonas ovalis, and haptophyte Pleurochrysis roscoffensis (Fukuzaki et al., 2014; Stolpe et al., 2014). FDOMC also can be produced by microbial transformation in culture experiments with bacteria growing on phytoplankton exudates (Romera-Castillo et al., 2011; Kinsey et al., 2018). In coastal environments, FDOM is derived by autochthonous and allochthonous processes. In general, FDOMT were found to be increased during phytoplankton bloom events (Suksomjit et al., 2009; Mendoza et al., 2012). The microbial degradation of plankton-derived DOM is known to be the most likely major source of the FDOMM (Yamashita et al., 2008) in addition to terrestrial sources of FDOMM (Murphy et al., 2008; Chari et al., 2012). Since FDOMM is transformed or consumed by marine bacteria that in turn produce FDOMC, FDOMC in the deep ocean is produced mainly by microbial remineralization of organic matter (Jørgensen et al., 2011; Kim and Kim, 2016). However, FDOMC in coastal waters is controlled primarily by conservative mixing of high terrestrial FDOMC concentrations in fresh waters as shown by strong negative correlations between salinity and FDOMC (Del Castillo et al., 1999; Stedmon and Markager, 2005; Kim and Kim, 2017). In this study, we investigated the distributions of phytoplankton pigments, dissolved organic carbon (DOC), and FDOM in red tide areas, dominated by the harmful dinoflagellate Cochlodinium polykrikoides, where biological production and remineralization are extremely high. Based on these observational results, we determined the behaviors of FDOM in red tide areas. We also attempted to look at the in-situ production characteristics of FDOM by C. polykrikoides and bacteria.

2. Materials and methods 2.1. Study area The study region is the coastal waters off Tongyeong, the southeastern coast of Korea (Supplementary Fig. 1S). In this region, the input of open-ocean seawater is affected by the Tsushima

Current, which is a tributary of the Kuroshio Current. This region is one of the regions where C. polykrikoides blooms often occur. In 2013, C. polykrikoides blooms in this region were first observed in July 17, with a maximum cell density of 3.5
104 cells mL1 (NIFS, 2015). The blooms lasted for 51 days. On the other hand, C. polykrikoides blooms did not occur in 2016. 2.2. Sampling Sampling was performed at the coastal waters off Tongyeong in August 2013 following the outbreak of massive C. polykrikoides blooms (Supplementary Fig. 1S). Although sampling was conducted at the same area in July 2016, the blooms did not occur (Supplementary Fig. 1S). Seawater samples were collected from the surface layer (0.5 m depth) using a submersible pump on shipboard for the analyses of phytoplankton pigments, DOC, and FDOM. Salinity was measured using a portable sensor (Orion star A329, Thermo Scientific). During the C. polykrikoides blooms in 2013, brown-colored patches were visually identified in the surface water. Thus, samples were collected from both patch and non-patch areas for comparison (Supplementary Fig. 1S). 2.3. Phytoplankton pigment analysis The concentrations of pigments were determined using the method described by Zapata et al. (2000). Pigments were extracted using 95% CH3OH at 20  C for 24 h in the dark. After sonification and centrifugation, the extract was filtered through a nylon syringe filter (0.2 mm pore size and 13 mm diameter) in order to remove filter and cellular debris. An aliquot of the extracts (1 mL) was transferred to auto-sampler vial containing 300 mL of deionized water. The extracts were analyzed using high performance liquid chromatography (HPLC, Waters 2695) with a C8 column (Waters Symmetry, 4.6
150 mm, 3.5 mm particle size, 100 Å pore size). The marker pigments were identified by retention times and spectral properties in comparison with those given in the literature (Zapata et al., 2000). 2.4. DOC analysis Seawater samples were filtered through pre-combusted (500  C for at least 4 h) GF/F filters (0.7 mm pore size) under low vacuum pressure (<100 mm Hg) to prevent destruction of phytoplankton cells. In order to prevent microbial activity, the filtered samples were acidified using 6 M HCl, and then hermetically sealed in precombusted 20 mL glass ampoules (Wheaton Scientific, Millville, NJ). The concentrations of DOC were measured using a TOC analyzer (TOC-VCPH, Shimadzu, Japan). The accuracy of the DOC

Table 1 Spectral characteristics of excitation and emission maxima of the three fluorescent components identified by PARAFAC modelling, compared with previous identified sources. Component

Exmax/Emmax

Compare with previous studies

Possible sources

1

325/404

Marine humic-like substance (biological degradation)

2

285/336

3

385/468

Peak M: 290e310/370-420a C2: 315/418b C4: 325(250)/416c Peak T: 275/340a C5: 270/332d Peak C: 320e360/420-480a C4: 250(360)/440e C8: 250(380)/416b

a b c d e

Coble, 1996. Murphy et al., 2008. Stedmon et al., 2003. Kowalczuk et al., 2009. Stedmon and Markager, 2005.

Autochthonous protein-like substance Terrestrial humic-like substance

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Fig. 1. Distributions of (a) salinity, (b) chlorophyll a, (c) peridinin, (d) fucoxanthin, (e) DOC, (f) FDOMT, (g) FDOMC, and (h) FDOMM in surface waters off Tongyeong in 2013. The star marked stations denote the patch areas of C. polykrikoides blooms.

concentrations was verified daily using deep-sea reference samples (41-44 mM, University of Miami). 2.5. FDOM analysis In order to analyze the FDOM, the seawater samples were

filtered using pre-combusted GF/F filters (0.7 mm pore size) under low vacuum pressure (<100 mm Hg). The filtered samples were transferred into pre-combusted vials and kept in a refrigerator at a temperature less than 4  C until spectral analysis. The filtrate was re-filtered using Nucleopore polycarbonate filters (0.2 mm pore size) to remove fine particles and microorganisms. Optical

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measurements of FDOM fluorescence were analyzed using a fluorescence spectrometer (FluoroMate FS-2, SCINCO, Korea) within one week of sampling. The scanning wavelengths of the excitation and emission spectra ranged from 250 to 600 nm (2 nm intervals) and 250 to 500 nm (5 nm intervals), respectively. Rayleigh and Raman scatters were eliminated and interpolated based on Delaunay triangulation, which is developed by Zepp et al. (2004), to prevent any problems with the blank subtraction method (Zepp et al., 2004; Nelson and Coble, 2009). The inner filter effect was not corrected because the influence of this artifact was negligible (less than 3% of the fluorescence intensity) for seawater using the same fluorescence spectrometer for various coastal seawaters, groundwaters, river waters, and deep-sea waters (Kim et al., 2012; Kim and Kim, 2016, 2017). The parallel factor analysis (PARAFAC) model was applied to a combined dataset of 45 EEMs. An appropriate number of components were evaluated statistically through the split-half test using the DOMFluor toolbox for MATLAB (Stedmon and Bro, 2008). The concentrations of FDOM were normalized daily using the values at a specific wavelength (Ex/Em 350/450 nm) of quinine sulfate (QS) dihydrate diluted in 0.1 N H2SO4. The concentrations are expressed

in quinine sulfate equivalent units (QSU) (i.e., in mg eq QS L1). The fluorescence of QS (average: 135±4) was almost constant throughout the measurements. The PARAFAC model identified two humic-like fluorescence peaks and one protein-like fluorescence peak from the data of the 45 EEMs (Table 1 and Supplementary Fig. 2S). The maximum peaks of component 1 were located at Exmax 325 nm and Emmax 404 nm, which is similar to those of FDOMM reported by Coble (1996). The maximum peaks of component 2 were located at Exmax 285 nm and Emmax 336 nm, which is similar to those of FDOMT (Coble, 1996; Coble et al., 1998). The maximum peaks of component 3 were located at Exmax 385 nm and Emmax 468 nm, which is similar to those of FDOMC (Coble, 1996). 3. Results and discussion 3.1. Characteristics of red tide areas In August 2013, the salinities ranged from 33.06 to 33.90 (average: 33.44±0.22), with the slightly lower values for stations farther from Tongyeong (Fig. 1a), perhaps due to the influence of

Fig. 2. Averages and standard deviations of (a) DOC, (b) FDOMT, (c) FDOMC, and (d) FDOMM in the patch and non-patch areas in surface waters off Tongyeong in 2013 and 2016.

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Changjiang Diluted Water (CDW). In general, CDW is defined as the water mass with salinity <32, although the upper limit of salinity differs slightly (Chang and Isobe, 2003). During the summer, the contribution of CDW was about 30% in this region, which is approximately 800 km from the mouth of Chanjiang River (Lee et al., 2014). Thus, it is common to observe CDW in this region during this period. The concentrations of chlorophyll a (chl. a) and peridinin ranged from 0.17 to 11.3 mg L1 (average: 2.1±2.3 mg L1) and 0.04 to 10.7 mg L1 (average: 1.4±2.6 mg L1), respectively (Fig. 1b and c). The concentrations of fucoxanthin ranged from 0.02 to 2.6 mg L1 (average: 0.80±0.68 mg L1) (Fig. 1d). Phytoplankton pigments have been shown to be valuable diagnostic markers of phytoplankton groups (Jeffrey et al., 1997). In particular, peridinin and fucoxanthin

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are diagnostic markers for dinoflagellates and diatoms, respectively. The concentrations of chl. a and peridinin in the patch areas were 5- and 15-fold higher than those in the non-patch areas, respectively, while the concentrations of fucoxanthin in the patch areas were lower than those in the non-patch areas (Supplementary Table S1). Such a trend was also found during the outbreak of red tides in this region in 2007 (Lee et al., 2010). In 2007, the concentrations of peridinin in the patch areas (average: 1.7 mg L1) were 2-fold higher than those in the non-patch areas (average: 0.83 mg L1). However, the concentrations of peridinin in the patch areas in 2013 were 3-fold higher than those in 2007. This difference is thought to be due to difference in the intensity of red tides between 2007 (~8.4
103 cells mL1) and 2013 (~3.5
104 cells mL1).

Fig. 3. Plots of salinity versus (a) DOC, (b) FDOMT, (c) FDOMC, and (d) FDOMM in surface waters off Tongyeong in 2013 and 2016. The dark circles denote patch areas of C. polykrikoides blooms in 2013. The solid lines represent the relationship between salinity and DOC or FDOMT or FDOMC or FDOMM only for the non-patch areas.

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3.2. DOC and FDOM in red tide areas The DOC concentrations ranged from 45 to 1330 mM (average: 172±235 mM) (Fig. 1e). The concentrations of DOC in the patch areas were 4-fold higher than those in the non-patch areas (Fig. 2a). In general, the variations of DOC in river-dominated coastal waters are dependent on salinities (Cifuentes and Eldridge, 1998; Fichot and Benner, 2011). However, in this region, DOC concentrations were independent of salinity variations (Fig. 3a), indicating that biological production of DOC resulted in DOC concentrations an order of magnitude higher in the patch areas relative to the normal coastal waters. This is evidenced by a significant correlation (r2¼0.73, p<0.001, n¼40) between DOC and peridinin concentrations (Fig. 4b). The concentrations of FDOMT, known as a protein-like component, ranged from 1.3 to 25 QSU (average: 3.2±4.4 QSU) (Fig. 1f). The FDOMT concentrations in the patch areas were 5-fold higher than those in the non-patch areas (Fig. 2b). Previous studies with field observations and culture experiments showed that phytoplankton could release protein compounds, detected as FDOMT (Yamashita and Tanoue, 2003). Romera-Castillo et al. (2010) and Fukuzaki et al. (2014) reported a corresponding peak in culture experiments of the various phytoplankton species. In addition, Suksomjit et al. (2009) observed that 6-fold increased FDOMT during the blooming periods in Yashima Bay, Japan. Similar results have been reported for coastal waters, such as the northwest Mediterranean Sea (France) and southwest coast of Florida (USA) (Romera-Castillo et al., 2010; Mendoza et al., 2012). In this study, a positive relationship (r2¼0.62, p<0.001, n¼40) between FDOMT and peridinin suggests that FDOMT was produced efficiently by C. polykrikoides (Fig. 4c). This nitrogen-containing organic substance may serve as a nutrient source of C. polykrikoides, which can utilize organic nutrients to maintain the growth under inorganic nutrient-limited environments (Gobler et al., 2012). The concentrations of FDOMC ranged from 0.48 to 2.7 QSU (average: 0.81±0.42 QSU) (Fig. 1g). The FDOMC concentrations in the patch areas were 2-fold higher than those in the non-patch areas (Fig. 2c). In most surface waters of the coastal sea, the major source of FDOMC is river-discharged terrestrial humic-like substances, and consequently, there are strong negative correlations between salinity and FDOMC (Del Castillo et al., 1999; Stedmon and Markager, 2005; Kim and Kim, 2017). On the other hand, Mendoza et al. (2012) observed 1.5-fold increased concentrations of FDOMC, together with FDOMM, in red tide area caused by the harmful dinoflagellate Karenia brevis. However, they attributed this enhanced humic-like component to low-salinity waters in southwest coast of Florida (USA) based on a significant correlation between FDOMC and salinity (Mendoza et al., 2012). However, FDOMC also can be produced by autochthonous biological production and microbially transformed planktonic materials in culture experiments using phytoplankton with bacteria (Kinsey et al., 2018). In this study, we observed much higher concentrations of FDOMC in the patch areas than those expected from the fresh water mixing (Fig. 3c). The concentrations of FDOMC showed a good correlation with peridinin (r2¼0.67, p<0.001, n¼40), indicating that there is a significant in-situ production of FDOMC by C. polykrikoides in red tide areas (Fig. 5a). The concentrations of FDOMM ranged from 1.01 to 2.17 QSU (average: 1.28±0.26 QSU) (Fig. 1h). There were no significant

Fig. 4. Plots of (a) peridinin versus chlorophyll a, (b) DOC versus chlorophyll a and peridinin, and (c) FDOMT versus chlorophyll a and peridinin in surface waters off Tongyeong in 2013. The solid lines represent the relationships between peridinin and DOC or FDOMT. The dashed lines represent the relationships between chlorophyll a and DOC or FDOMT.

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3.3. Plausible mechanisms for the occurrence of FDOMC and FDOMM in red tide areas In order to examine whether or not our hypothesis for the observational results is reasonable, we roughly evaluated the relative contributions of C. polykrikoides production and bacterial activities to FDOMC and FDOMM concentrations. We assumed the production rate of FDOMC and FDOMM by C. polykrikoides to be 1.8
106 QSU cell1 d1 for FDOMC and 1.1
106 QSU cell1 d1 for FDOMM, which were the averages reported for four marine phytoplankton species, such as diatom D. brightwelli, dinoflagellate H. circularisquama, raphidophyte H. akashiwo, and chlorophyte O. virdis, by Fukuzaki et al. (2014). If the cell density of 2.0
104 cells mL1 reported for these patch areas (NIFS, 2015) is applied, the production rates of FDOMC and FDOMM by C. polykrikoides were 0.036 QSU d1 and 0.021 QSU d1, respectively. Romera-Castillo et al. (2010) documented that FDOMC can be produced, whereas FDOMM is consumed by bacteria. We used the average “production” rate of FDOMC and “consumption” rate of FDOMM by bacteria (4.7
103 QSU mg C1 L d1 for FDOMC and 5.0
103 QSU mg C1 L d1 for FDOMM) reported by RomeraCastillo et al. (2010). If the carbon content of 5.8
102 pg C cell1 and bacterial abundance of 2.9
106 cells mL1 are applied for the patch areas (Seong et al., 2006; Park et al., 2015), the production rate of FDOMC and consumption rate of FDOMM by bacteria were estimated to be 0.028 QSU d1 and 0.030 QSU d1, respectively. Based on this estimate, the “production” of FDOMC by C. polykrikoides was 1.3-fold higher than that by bacteria, whereas the “consumption” of FDOMM by bacteria was 1.4-fold higher than the production of FDOMM by C. polykrikoides. Thus, the different behaviors of FDOMC and FDOMM in the observed red tide areas are explained reasonably by the dominant production of FDOMC by C. polykrikoides and subsequent FDOMM consumption by bacteria.

4. Conclusions

Fig. 5. Plots of peridinin and fucoxanthin versus (a) FDOMC and (b) FDOMM in surface waters off Tongyeong in 2013. The solid lines represent the relationships between peridinin and FDOMC or FDOMM.

differences between patch areas and non-patch areas (Fig. 2d). In general, FDOMM can be produced either by biological production (Romera-Castillo et al., 2010) or bacterial activities (Romera-Castillo et al., 2011). However, in our field observations, we found no increase of FDOMM concentrations in the C. polykrikoides bloom areas (Fig. 5b). Changes in the bacteria abundances paralleled those of C. polykrikoides (Park et al., 2015). Romera-Castillo et al. (2011) reported that bioavailable FDOM released by phytoplankton can be preferentially taken up by bacteria. They reported that the consumption rates of FDOMM by bacteria (~12
103 QSU mg C1 L d1) were 2.4-fold higher than the production rates by phytoplankton (~5
103 QSU mg C1 L d1) (Romera-Castillo et al., 2010, 2011). Thus, no increase of FDOMM in the patch areas seems to be associated with the preferential consumption of FDOMM by bacteria in the C. polykrikoides bloom areas.

In the coastal waters off Tongyeong, where C. polykrikoides blooms occur, we observed the production characteristics of different components of FDOM. The concentrations of FDOMT and FDOMC increased 5- and 2-fold, respectively, in the red tide patch areas, relative to ambient seawaters due to biological production. However, the concentrations of FDOMM did not increase in the red tide patch areas, perhaps due to consumption by enhanced bacterial activities. Our results agree very well with previous laboratory studies, although such increases of FDOMC, without FDOMM increases, were observed for the first time in the natural environments. More extensive studies are necessary to determine the production and role of FDOM in the development of red tides in the future.

Acknowledgments We thank the members of the Environmental and Marine Biogeochemistry Laboratory (EMBL) for their assistance in field observations and laboratory analyses. This study was supported by the grant from the National Institute of Fisheries Science, Korea (grant number: R2018043).

Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.ecss.2018.02.013.

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