Note. Upward Movement of Chromatium sp. in the H2S-Layer of Lake Kaiike Causing a Bloom at its Upper Boundary

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1 Jpn. J. Limnol., 56, 3, , 1995 Note Upward Movement of Chromatium sp. in the H2S-Layer of Lake Kaiike Causing a Bloom at its Upper Boundary Michiro MATSUYAMA Abstract Two kinds of large-celled bacteria, Chromatium sp. and Macromonas sp., bloom at an upper boundary of the H2S-containing layer of Lake Kaiike throughout all seasons. An isolated strain of Chromatium sp. was incubated in an inorganic medium under different levels of photosynthetically available radiation (PAR). In the absence of light the bacterial cells moved upward in a glass tube filled with the medium. Above a PAR level of lƒêmole m-2 s-1, the bacterium was able to grow. Vertical measurement of PAR in the lake on 24 July 1994 showed that PAR was 10 ƒêmole m-2 s-1 at 4.5 m depth, where the two bacterial species began to appear, and that it was reduced to a level below 0.1 pole M-2 s-1 at their peaks (5.0 m depth). More than 75% of the Chromatium population existed in a light-limiting state. Individual Chromatium cells appeared to move upward in the H2S-containing layer and to meet the light requirement at its upper boundary. Key words: Chromatium sp., bloom, light-limiting, upward movement, Lake Kaiike Two kinds of large-celled bacteria, Chromatium sp. and Macromonas sp., bloom at an upper boundary of the H2S-containing layer of meromictic Lake Kaiike on Kamikoshiki Island throughout all seasons (bacterial plate). The maximum number of Chromatium sp. cells in the lake was on the order of 10 5 `10 6 cells ml-1 (MATSUYAMA, 1987), while Macromonas sp. was usually one order of magnitude more abundant than Chromatium sp. Little information has been presented on the Macromonas bloom. The bacterial plate was regularly found just below the algal compensation point, where surface light was reduced below a 10% level (MATSUYAMA, 1985). This study was conducted to answer the question of what factors are responsible for the bloom of Chromatium sp. at such a light-limiting layer. Chromatium sp., isolated from Lake Kaiike and maintained in the inorganic medium of PFENNIG (1965), was used in this study. The bacterial cells from the exponential growth phase was inoculated into a newly prepared PFENNIG'S medium. A series of glass tubes (17.7 cm tall, 2.1 cm i.d.) were filled with the inoculated medium and tightly stoppered. The tubes were then allowed to stand upright in a water bath at 25 Ž laterally illuminated with an incandescent lamp. Black nylon nets of different mesh sizes were placed before the tubes to adjust the light intensity. Light was provided in a 12-h light and dark cycle. Light intensity was presented as photosynthetically available radiation (PAR) and measured by using a quantum sensor with a flat response between 400 `700 nm (LI-Cor Underwater Radiation Sensor, Type SA, LI-Cor Inc. USA). At 1, 3, 6, 8, 10, 13 and 15 days incubation, the glass tubes at different PAR levels were carefully opened so as not to disturbe the bacterial profiles. One ml of sample was taken with a pipette from a surface layer to measure the cell number with a Thoma hemocytometer. This procedure was repeated to collect the bacterial cells floating at 3, 6, 9, 12 and 15 cm depths. The bacterial culture in the continuous dark, which was attained by wrapping

2 206 MATSUYAMA Fig.1. Vertical profiles of Chromatium sp. (left) and H2S concentration (right) in glass tubes filled with inorganic medium of PFENNIG (1965). The bacterium was left in continuous dark. Numbers indicate incubation time (days). Initial bacterial number was 2 ~10 7 cells ml-1 Fig 2. Vertical profiles of Chromatium sp. in glass tubes exposed to different levels of photosynthetically available radiation (PAR) provided in a 12-h light and dark cycle. A: 0.6, B: 1.0, C: 2.1, D: 5.9ƒÊmole m-2 s-1). Numbers indicate incubation time (days). Initial bacterial number was 5 ~10 6 cells ml-1.

3 Bloom of Chrmnatium in Lake Kaiike207 the tubes with aluminum foil, was opened at 1, 2, 3, 4, 7 and 9 days incubation, and was offered not only for bacterial counting but also for measurement of H2S concentration using a spectrophotometric method of CLINE (1969). Figure 1 shows the change in the bacterial profile cultured in continuous dark together with the HzS concentration. Immediately after inoculation, many bacterial cells moved upward in the glass tubes to exhibit a sharply defined cloudy accumulation in the surface layer. The cellular buoyant density of Chromatium sp. was extremely high due to the deposition of sulfur globules inside the cells (MATSUYAMA, 1991). The buoyancy of the bacterium is due to their own swimming activity. Bacterial upward movement seemed neither phototactic nor H2S-tactic, because H2S concentration was almost unchanged during the incubation. This suggests that it was geotactic, although further studies are necessary to confirm this. Figure 2 shows the change in the bacterial profile in the glass tubes exposed to different PAR levels. H2S was present in all glass tubes throughout the incubation period except for the last two (13 and 15 days incubation) exposed to the highest PAR level (5.9ƒÊmole m-2 S-1). Most bacterial cells were floating in the medium dur ing incubation and forming a purplish red suspension. At a minimum PAR level (Fig. 2-A), many bacterial cells concentrated in the surface layer, although any increase in cell numbers was not observed during incubation. Above a PAR level of 1 umole m-2 s-1, the bacterium was able to grow and the cells spread the tubes. When HzS was depleted bacterium sank to form a red sediment. evenly in (Fig. 2-D), the Figure 3 shows the vertical profiles of Chromatium sp. and Macromonas sp. in Lake Kaiike on 24 July 1994, together with environmental factors such as salinity. Apparatus and methods for sampling of lake water and chemical analyses were the same as described in MA- TSUYAMA and SHIROUZU (1978). Dissolved O2 disappeared at 5.0 m depth, and H2S began to appear at the same depth. Two bacterial species appeared at 4.5 m depth and reached their peaks immediately below. PAR penetrated well into the upper layer, reaching 4.5 m in depth (mean extinction coefficient : 0.71 m-1). However, PAR reaching 5.0 m depth was reduced to 0.024% of the surface (mean extinction coefficient:11.7 m-1). Apparently, this decrease in PAR was due to shading by dense populations of two bacterial species. PAR reaching the sea level at a latitude of Fig.3. Vertical profiles of Chromatium sp. (A) and Macromonas sp. (B), together with photosynthetically available radiation (PAR) in Lake Kaiike on 24 July Vertical profiles of some environmental factors are shown in the left column.

4 208 MATSUYAMA deeper layer of Lake Kaiike, few showed any microscopic difference from those from an upper part of the bacterial plate; sulfur-deficient cells were rarely found; any dividing cells, which frequency could be used as a very simple diagnostic parameter for growth (MATSUYAMA, 1993) were also found. It is suggested that Chromatium cells forming the bacterial plate in Lake Kaiike might be equal in photosynthetic use of light, and that even the bacterial cells found in the deeper layer would move upward in the H2S-containig layer and meet their light requirement at its upper boundary. Individual cells of Chromatium sp. would be expected to diminish the photosynthetic activity (MATSUYAMA, 1993). FOLT et al. (1989) studied the phototrophic bacterial community occupied by Thiocapsa rosea at 6 `8 m depth of monomictic Zaca Lake (California), and found that the bacterial activity was reduced to a minimum level for 11 months (mean doubling time:>1 month) due to extremely low light intensity (PAR reaching the top of the bacterial plate was<1,umole m-2 s-1 compared with nearly 1,000,umole M-2 s-1 at 0.2 m depth). It is suggested that phototrophic bacterial populations blooming in natural waters, or at least Chromatium sp. in Lake Kaiike, may exist in a viable yet nongrowing state, that is to say, the bacteria may barely maintain cell integrity in spite of the large biomass. 30 N is maximum in June (150ƒÊmole m-2 s-1 when averaged for a day time) (CAMPBELL and AARUP, 1989). If PAR of 150ƒÊmole m-2 s-1 was assumed to fall on the lake surface (31 51 N), and incident PAR was assumed to penetrate the water layer in the same manner shown in Fig. 3, PAR reaching depths of 4.5 m and 5.0 m could be calculated as 6.1 and 0.02ƒÊmole m-2 S-1, respectively. Apparently, these rates would be overestimated, because very clear (cloud-free) atmospheric conditions and no losses of PAR at the lake surface were assumed. However, it is safe to say that PAR reaching the bacterial peak (5.0 m depth) was too little to sustain photosynthetic growth (Fig. 1). Bacterial growth seems to be restricted to an upper region of the bacterial plate, that is, the upper boundary of the H2S-containing layer above 5.0 m depth. Integration of the bacterial numbers during 4.5 m to 10 m depth showed that more than 75% of the Chromatium population (at and below the bacterial peak) was incapable of photosynthetic growth. Could these cells be thought to be dying as suggested by GUERRERO et al. (1985)? They studied a bloom of Chromatium spp. in Lake Ciso (Northeast Spain) and divided the bacterial plate into 3 parts: a top region of maximum specific activity (1.75 m) receiving PAR of 60 ƒê mole m-2 s-1 at noon; a peak region of maximum abundance (2.0 m) (<1ƒÊmole m-2 s-1) and a bottom region of inactive cells. They suggested that the cells did not mix within the layers, and that once the cells were trapped in the deeper, darker zone, they were unable to migrate upward. If Chromatium cells found in the water below 5.0 m in Lake Kaiike were assumed to be dying and to sink at a rate of 0.8 cm h-1 (MA- TSUYAMA, 1991), the bacterial cells at its peak would require one month to reach 10 m depth. When an isolated strain of Chromatium sp. was left in continuous dark, sulfur globules rapidly disappeared from the cells; within the first 5 days of incubation, half the cells lost the globules (MATSUYAMA, 1988). However, the bacterium maintained their initial numbers in the prolonged dark culture for 30 days. Among Chromatium cells collected from the

5 Bloom of Chromatium in Lake Kaiike209 References CAMPBELL, J. W. and T. AARUP (1989): Photosynthetically available radiation at high latitudes. Limnol. Oceanogr., 34: CLINE, J. D. (1969): Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol. Oce anogr., 14: FOLT, C. L., M. J. WEVERS, M. P. YODER-WILLIAMS and R. P. HOWMILLER (1989): Field study comparing growth and viability of a population of phototrophic bacteria. Appl. Environ. Microbiol., GUERRERO, R., E. MONTESINOS, C. PEDROS-ALIO, I. ESTEVE, J. MAS, H. VAN GEMERDEN, P. A. G. HOF- MAN and J. F. BAKKER (1985): Phototrophic sulfur bacteria in two Spanish lakes: Vertical distribution and limiting factors. Limnol. Oceanogr., 30: MATSUYAMA, M. (1978): Stratified condition of Lake Kaiike, a small coastal lake on Kamikoshiki Island, Kagoshima Prefecture. Jpn. J. Limnol., 39: MATSUYAMA, M. (1985): Unusual organic matter production in Lake Kaiike, Kamikoshiki Island, Kagoshima Prefecture. Seibutsukagaku, 37: Iwanamishoten, Tokyo. (in Japanese). MATSUYAMA, M. (1987): A large phototrophic bacterium densely populating the O2-H2S interface of Lake Kaiike on Kamikoshiki Island, Southwest Japan. Acta Academiae Aboensis, 47: MATSUYAMA, M. (1988): Low growth rate of a largecelled phototrophic bacterium as a factor to form the dense population at the dissolved O2 -H2S interface of Lake Kaiike. Jpn. J. Limnol, 49: MATSUYAMA, M. (1991): Buoyant density of Chromatium sp.: Its effect on the blooming at an upper boundary of the H2S layer in Lake Kaiike. Jpn. J. Limnol., 52: MATSUYAMA, M. (1993): Frequency of dividing cells of Chromatium sp. blooming in Lake Kaiike as an estimate of growth rate. Jpn. J. Limnol., MATSUYAMA, M. and E. SHIROUZU (1978): Importance of photosynthetic sulfur bacteria, Chromatium sp. as an organic matter producer in Lake Kaiike. Jpn. J. Limnol., 39: PFENNIG, N. (1965): Anreicherungskulturen fur rote and grune Schwefelbakterien. Zb. Bakt., 1. Abt. Orig. Suppl., 1: , Received:21 December 1994 Accepted:5 April 1995