Abundance and Productivity of Bacterioplankton in a Eutrophication Gradient of Shimoda Bay

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1 Journal of Oceanography Vol. 49, pp. 657 to Abundance and Productivity of Bacterioplankton in a Eutrophication Gradient of Shimoda Bay TAKESHI NAGANUMA* and HUMITAKE SEKI Institute of Biological Sciences, University of Tsukuba, Tennoudai, Tsukuba 305, Japan (Received 11 September 1992; in revised form 8 May 1993; accepted 20 May 1993) Abundance, specific growth rate, and productivity of bacterioplankton were investigated over a year in Shimoda Bay, Japan, with reference to the eutrophication parameters such as dissolved organic carbon (DOC) concentration. The results from six stations in the bay indicated the formation of a eutrophic gradient ranging from eutrophic (at a river mouth) to mesotrophic (at the bay mouth) levels. Both DOC concentration and UV absorbance were found to have good correlations to bacterioplankton abundance and productivity within the eutrophication range of Shimoda Bay. 1. Introduction Bacteria in aquatic ecosystems have been shown to play crucial roles in geochemical cycles and eutrophication processes through decomposition, remineralization, nutrient uptake and biomass production (e.g., Seki, 1982, 1992; Cole et al., 1988; Rheinheimer, 1991). Free-living bacterioplankton may have lower metabolic activity than epibacteria that are attached to particles, but they are far more abundant in most water bodies (e.g., Pedrós-Alio and Brock, 1983). Thus, bacterioplankton can make considerable contribution to the geochemical cycles and eutrophication processes. To investigate the eutrophication processes, traversal observation is advantageous for the areas where eutrophication gradients are formed. This is because the multi-stational approach can give clear resolution of the gradient in a relatively short period on the assumption that the eutrophication process can be tracked representatively by the traversal observation. Examples of this approach are seen in the studies of eutrophication gradients such as Kiel Fjord to Kiel Bay, Germany (Rheinheimer, 1991) and Shimoda Bay, Japan (Yamaguchi and Seki, 1977), both of which focussed mainly on biomass, i.e., total bacterial number, chlorophyll concentration etc. In another Shimoda Bay study, Seki et al. (1975) investigated the bacterial uptake and turnover rate of dissolved organic materials with reference to the eutrophication gradient. However, few studies have been done on the bacterial population dynamics (total number, growth rate, production, etc.) regarding eutrophication gradients. Shimoda Bay, about 120 km southwest of Tokyo, is a small bay with an approximately 3- km NNE-SSW axis. The River Inohzawa, the major input of the eutrophication materials (Maeda et al., 1973), flows in at the western end of the inner bay. On the other hand, the outer part of the bay is influenced by less-polluted Kuroshio counter current (Seki et al., 1975). These topographical and hydrological conditions make Shimoda Bay an excellent field for studying the *Present address: DeepStar Program, Japan Marine Science and Technology Center, 2-15 Natsushimacho, Yokosuka 237, Japan.

2 658 T. Naganuma and H. Seki eutrophication gradient and its biological influences. In this communication, we report the bacterioplankton population dynamics over the eutrophication gradient of Shimoda Bay, with special reference to the dissolved organic carbon (DOC) concentration and UV absorbance. 2. Materials and Methods Surface seawater samples were collected at six stations in Shimoda Bay (Fig. 1) monthly from April 1987 through April 1988, except from November 1987 through January Water temperature was measured in situ. Salinity was calculated from temperature and conductivity measured with a Toho-Dentan EST-3 salinometer (Toho-Dentan Co., Ltd., Tokyo). Concentration of dissolved oxygen (DO) was determined by the Winkler method (Strickland and Parsons, 1972). For total bacterial counts, portions of the water samples were fixed on the boat with formalin (sample: formalin, 20:1 v/v). The surface seawater samples were filtered through Whatman GF/F filters. Concentrations of inorganic nitrogen (as the sum of NH 4+, NO 2 and NO 3 ) and inorganic phosphate (as PO 4 3 ) in the filtrate, as well as chlorophyll a on the filters, were determined according to Strickland and Parsons (1972). Concentration of dissolved organic carbon (DOC) was measured by the wet oxidation method using persulfate (Menzel and Vaccaro, 1964; Strickland and Parsons, 1972) with an infra-red photometer, UNOR (Maihak, Hamburg). As a DOC-related parameter, absorbance at 280 nm (A 280 ) was also measured with a quartz cell having a 10-cm light path (Krom and Fig. 1. Locations of the sampling stations in Shimoda Bay and the Shimoda Marine Research Center (SMRC).

3 Bacterioplankton in a Eutrophication Gradient of Shimoda Bay 659 Sholkovitz, 1977) using a double-beam spectrophotometer, UV-200 (Shimadzu Co., Kyoto). As an attempt to characterize the DOC composition, the ratio of A 280 /DOC was compared. Total numbers of bacterioplankton (N t ) were counted by the combination of membrane filtration and epifluorescence microscopy (Hobbie et al., 1977; Zimmermann et al., 1978). Specific growth rates (µ) of bacterioplankton were determined by the simulated in situ method with chemostats (Naganuma and Seki, 1985). Sea water that had been collected at the respective stations the day before the chemostat operation was used as the in situ seawater media for chemostat culture. The media were GF/F-filtered and autoclaved. The chemostats were operated at the in situ temperatures (within ±1.5 C). In this sense, it was taken as the simulated in situ specific growth rate that was actually measured. Biomass productivity of bacterioplankton (µgc/l/day) was calculated by multiplying N t and µ (Moriarty, 1990), assuming a cell volume of 0.1 µm 3 (by microscopy, data not shown), a specific gravity of 1.1 and a carbon content of 22% of the wet weight (Bratbak and Dundas, 1984; Simon and Azam, 1989). Correlation between physico-chemical and bacterial parameters was analyzed by the least square method. The data collected at Station 1 (at a river mouth) in July 1987 were excluded from the correlation analyses in Figs. 4 and 5 because temporal environmental perturbation due to heavy rainfall and intense river flow was observed there. This fact was evidenced by the very low salinity (2.0 ) at Station 1 on July 21, Results The eutrophication parameters such as concentrations of inorganic nitrogen, inorganic phosphate, DOC (and A 280 ) and chlorophyll a characterized Shimoda Bay as a eutrophic-mesotrophic system (e.g., Seki, 1986). In the bay, distinct formation of a eutrophication gradient, ranging from eutrophic (Station 1) to mesotrophic (Station 6), was observed (Fig. 2; each of the Station 1 values was taken as 25). The peak values of the gradient were mostly seen at Station 1. Some parameters, for example chlorophyll a concentration on the annual average (Table 1), were the highest at Station 2. The DOC concentration (mgc/l) and A 280 were shown to correlate significantly (p < 0.01) within the observed range (Fig. 3). The overall correlation was expressed as: Fig. 2. Schematic characterization of the eutrophication gradient in Shimoda Bay. Relative units of each parameter are based on the annual average values in Table 1.

4 660 T. Naganuma and H. Seki [A 280 ] = 0.015[DOC] (r = 0.700, n = 60). Despite the overall correlation of high significance, there seemed to be a tendency that the data from Station 1 (Station 4, 5 and 6) deviated above (below) the line of correlation (Fig. 3). This tendency was represented by the ratio of A 280 /DOC (mgc/l), which was ranging from (Station 1) to (Station 4, 5 and 6) on the annual average (Table 1). The overall average of the A 280 /DOC ratio was ± Table 1. Annual averages of physico-chemical, primary production-related, and microbiological parameters in surface water of Shimoda Bay. Data were collected from April 1987 through April Station Physico-chemical Temperature ( C) 18.7± ± ± ± ± ±4.4 Salinity ( ) 19.6± ± ± ± ± ±1.6 DO (mgo 2 /l) 7.52± ± ± ± ± ±0.53 Inorg. N (µg-at/l) 25.8± ± ± ± ± ±5.3 Inorg. P (µg-at /l) 1.2± ± ± ± ± ±0.3 DOC (mgc/l) 6.7± ± ± ± ± ±1.8 A 280 (1000 ) 156±52 113±51 116±47 79±33 78±35 69±37 A 280 /DOC (1000 ) 24±13 20±9 19±3 15±5 16±5 16±5 Primary production-related Chl. a (µg/l) 2.18± ± ± ± ± ±0.75 Microbiological Bac. No. ( 10 8 /l) 11.5± ± ± ± ± ±1.7 Bac. Growth (/h) 0.21± ± ± ± ± ±0.09 Bac. Productivity ( µgc/l/day) 140±99 119±85 77±56 68±48 41±36 36±36 Fig. 3. Relationship between dissolved organic carbon (DOC) concentration and UV absorbance at 280 nm of the Shimoda Bay surface water. Each data point represents a given station for a given month.

5 Bacterioplankton in a Eutrophication Gradient of Shimoda Bay 661 The bacterioplankton parameters such as abundance and productivity were correlative to the eutrophication gradient in Shimoda Bay (Table 1). On the other hand, specific growth rates (/h), which ranged from 0.14 (generation time of 5.0 hrs, Station 5) to 0.22 (3.2 hrs, Station 2) on the annual average (Table 1), were rather at a steady level among the stations. The correlations of the bacterioplankton abundance (N t ; 10 8 cells/l) and productivity (P; µgc/l/day) to the DOC concentration (mgc/l) and A 280 are shown in Fig. 4. These correlations (n = 59), excluding the values of the heavy rain time of July 1987 (arrowed in Fig. 4), were statistically significant and expressed as: [N t ] = 0.85[DOC] (p < 0.01), [N t ] = 45.4[A 280 ] (p < 0.01), [P] = 11.1[DOC] (p < 0.05), [P] = 641[A 280 ] (p < 0.01). Taking the annual averages for each station, the correlations became still clearer (Fig. 5). All the correlations were highly significant (n = 6, p < 0.005) and expressed as: Fig. 4. Relationships of dissolved organic carbon (DOC) concentration and UV absorbance at 280 nm to bacterioplankton abundance and productivity of the Shimoda Bay surface water. Each data point represents a given station for a given month. Arrowed data points were excluded from the correlation analyses.

6 662 T. Naganuma and H. Seki Fig. 5. Relationships, on the annual average, of dissolved organic carbon (DOC) concentration and absorbance at 280 nm to bacterioplankton abundance and productivity of the Shimoda Bay surface water. Annual average values of the stations (1 to 6) are from Table 1. [N t ] = 2.60[DOC] 7.49, [N t ] = 82.4[A 280 ] 1.63, [P] = 39.1[DOC] 134, [P] = 1218[A 280 ] Compared with the DOC concentration, A 280 always yielded slightly higher correlation coefficient values, though the differences were small. 4. Discussion The eutrophication gradient of Shimoda Bay was reported before (e.g., Yamaguchi and Seki, 1977). Data from the six stations in this study indicated that the eutrophication gradient (in terms of physico-chemical parameters) was still or again formed in Considering the hydrology, topography, and geography of the bay, the eutrophication gradient of Shimoda Bay can be thought to be formed stably for years and to be an ideal area for investigating the physicochemical eutrophication gradient and its biological influences. The DOC concentration was determined by the wet oxidation method in this study. Other methods such as the high-temperature catalytic oxidation method and the ultraviolet photo-

7 Bacterioplankton in a Eutrophication Gradient of Shimoda Bay 663 oxidation method were reported to yield higher DOC values (Gershey et al., 1979; Sugimura and Suzuki, 1988; Suzuki et al., 1992). For example, the DOC concentration by the wet oxidation method was about 78 85% of that determined by the high-temperature oxidation method (Sharp, 1973; Gershey et al., 1979). However, the high-temperature oxidation and UV photo-oxidation methods require relatively expensive instruments, while the wet oxidation method is the simplest and least expensive. Thus, the wet oxidation method is still useful for the work where absolute values are not a prime consideration. In addition, because organic matter in surface water was not resistant to wet oxidation (Sharp, 1973), the wet oxidation method may not be discounted, at least for surface waters. Associated with the DOC determination, UV absorbance (A 280 ) was measured for certain organic compounds that have UV absorption characteristic. Krom and Sholkovitz (1977) reported a good correlation between A 280 values and DOC concentrations (by dry-combustion) for estuarine waters. This was supported by our data from a eutrophic coastal water (Fig. 3). Also it is suggested that the DOC-A 280 correlation is retained over the eutrophication gradient from eutrophic to mesotrophic range. Therefore, UV photometry can be a convenient alternative to conventional DOC measurements, and A 280 could be useful for work (especially field work) where absolute values, again, are not a prime consideration. The difference in the A 280 /DOC ratio among the stations is likely to be respondent to the difference in the DOC composition. If the DOC composition was all the same, A 280 /DOC ratio was expected to be the same. Thus, the difference in A 280 /DOC ratio can be indicative of the difference in local DOC composition. For example, DOC from Station 1 (A 280 /DOC ratio of 0.024) might be mostly reverine, which DOC was reported conservative (Mantoura and Woodward, 1983). In contrast, the DOC of Station 4, 5 and 6, whose A 280 /DOC ratios were as low as , were likely to be influenced by the Kuroshio counter current. The A 280 /DOC ratios from Station 2 and 3 were in-between ( ), which suggests 1) mixing of the riverine and marine DOC and/or 2) autochthonous DOC production (excretion by phytoplankton). The latter can be supported by the high chlorophyll a concentrations observed at these stations (Table 1). The abundance and productivity of bacterioplankton were shown to be well-correlated to the DOC concentration and A 280 (Figs. 4 and 5). The correlations indicate that it is the DOC level that governs the bacterioplankton abundance and productivity, at least within the range of the eutrophication gradient in Shimoda Bay. This look is in good agreement with the general view that bacterial biomass and production in aquatic environments are mainly supported by DOC (Seki, 1982, 1992). Compared with the DOC, A 280 always yielded slightly higher correlation coefficient values, which suggests that A 280 might be more indicative of the DOC readiness for bacterial utilization. While the overall analysis of bacterioplankton-doc (-A 280 ) relationship resulted in the highly significant correlations, the A 280 /DOC ratio was suggested to link to the chemical composition of what is called DOC. Thus, the linkage would be an attractive subject to be studied hereafter, including chemical analysis and laboratory experiment as well as statistical analysis. In particular, recent development of the chemical analysis will facilitate assessing the amount and effect of the individual DOC fractions. In contrast with the variation of bacterioplankton abundance and productivity correlative to DOC and A 280, the specific growth rate was at almost the same level throughout the stations (Table 1). Supposedly, this was mainly because the bacterioplankton population had adapted to the local DOC to grow at a certain rate. Another possible reason is that temperature, which was at the same level throughout the stations, had equally or more profound influence on the

8 664 T. Naganuma and H. Seki bacterioplankton growth than the variation of DOC concentration. In conclusion, it is indicated that the bacterioplankton abundance and productivity are controlled by DOC concentration and that the controlling manner is consistent over a eutrophication gradient, if the gradient is within the same eutrophication range. Previous work (Naganuma, 1989) demonstrated the same relationship for mesotrophic fresh waters. Thus, it should be further studied to determine if this relationship between DOC concentration and bacterioplankton dynamics still holds also for waters in other eutrophication ranges. Acknowledgements This work, Contribution No. 557 from Shimoda Marine Research Center, the University of Tsukuba (SMRC/UT), would have been impossible without the help of H. Ueda, Y. Tsuchiya and T. Sato, SMRC/UT. The authors are much obliged to Professors H. Watanabe, H. Suzuki and Y. Yokohama, SMRC/UT, for their kind arrangement for the use of a laboratory and instruments. Thanks are also extended to T. Sato for his technical assistance and to D. A. Coury, Marine Science Institute, University of California at Santa Barbara, for reading the manuscript. References Bratbak, G. and I. Dundas (1984): Bacterial dry matter content and biomass estimations. Appl. Environ. Microbiol., 48, Cole, J., J. S. Findlay and M. L. Pace (1988): Bacterial production in fresh and salt-water ecosystems: a cross-system overview. Mar. Ecol. Prog. Ser., 43, Gershey, R. M., M. D. Mackinnon, P. J. le B. Williams and R. M. Moore (1979): Comparison of three oxidation methods used for the analysis of the dissolved organic carbon in seawater. Mar. Chem., 7, Hobbie, J. E., R. J. Daley and S. Jasper (1977): Use of Nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol., 33, Krom, M. D. and E. R. Sholkovitz (1977): Nature and reactions of dissolved organic matter in the interstitial waters of marine sediments. Geochim. Cosmochim. Acta, 41, Maeda, O., M. Zamma and S. Ichimura (1973): Photosynthetic response of estuarine phytoplankton to salinity variations in their habitat. La mer, 11, Mantoura, R. F. C. and E. M. S. Woodward (1983): Conservative behaviour of riverine dissolved organic carbon in the Severn Estuary: chemical and geochemical implications. Geochim. Cosmochim. Acta, 47, Menzel, D. W. and R. F. Vaccaro (1964): The measurement of dissolved organic and particulate carbon in seawater. Limnol. Oceanogr., 9, Moriarty, D. J. W. (1990): Techniques for estimating bacterial growth rates and production of biomass in aquatic environments. p In Methods in Microbiology, Vol. 22, Techniques in Microbial Ecology, ed. by R. Grigorova and J. R. Norris, Academic Press, San Diego. Naganuma, T. (1989): Population dynamics of bacterioplankton communities in mesotrophic aquatic ecosystems. Ph. D. diss., University of Tsukuba, 166 pp. Naganuma, T. and H. Seki (1985): Population growth rate of the bacterioplankton community in a bog, Matsumiike, Japan. Arch. Hydrobiol., 104, Pedrós-Alio, C. and T. D. Brock (1983): The importance of attachment to particles for planktonic bacteria. Arch. Hydrobiol., 98, Rheinheimer, G. (1991): Aquatic Microbiology. 4th ed. John Wiley & Sons, New York, 363 pp. Seki, H. (1982): Organic Materials in Aquatic Ecosystems. CRC Press Inc., Boca Raton, Florida, 201 pp. Seki, H. (1986): Thresholds in eutrophication of natural waters. Environ. Monitor. Assess., 7, Seki, H. (1992): Biogeochemical cycles and microorganisms in the marine ecosystems. Umi no Kenkyu, 1(2), 1 8 (in Japanese with English abstract). Seki, H., Y. Yamaguchi and S. Ichimura (1975): Turnover rate of dissolved organic materials in a coastal region of Japan at summer stagnation period of Arch. Hydrobiol., 75, Sharp, J. H. (1973): Total organic carbon in seawater comparison of measurements using persulfate oxidation and high temperature combustion. Mar. Chem., 1,

9 Bacterioplankton in a Eutrophication Gradient of Shimoda Bay 665 Simon, M. and F. Azam (1989): Protein content and protein synthesis rates of planktonic marine bacteria. Mar. Ecol. Prog. Ser., 51, Strickland, J. D. H. and T. R. Parsons (1972): A Practical Handbook of Seawater Analysis. 2nd ed. Bulletin 167, Fisheries Research Board of Canada, Ottawa, 310 pp. Sugimura, Y. and Y. Suzuki (1988): A high-temperature catalytic oxidation method for the determination of nonvolatile dissolved organic carbon in seawater by direct injection of a liquid sample. Mar. Chem., 24, Suzuki, Y., E. Tanoue and H. Ito (1992): A high-temperature catalytic oxidation method for the determination of dissolved organic carbon in seawater: analysis and improvement. Deep-Sea Res., 39, Yamaguchi, Y. and H. Seki (1977): Microbial biomass in the eutrophic Bay of Shimoda as compared by estimations of several biomass parameters. J. Oceanogr. Soc. Japan, 33, Zimmermann, R., R. Iturriaga and J. Becker-Birck (1978): Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl. Environ. Microbiol., 36,