Fisheries Science 62(1), 40-43 (1996)
Effects of Bacterial Coexistence on the Growth of a Marine Diatom Chaetoceros gracilis
Suminto*1 and Kazutsugu Hirayama*2 *1Graduate School of Marine Science and Engineering , Nagasaki University, Bunkyo, Nagasaki 852, Japan *2Faculty of Fisheries , Nagasaki University, Bunkyo, Nagasaki 852, Japan (Received May 12, 1995)
The promotive and suppressive effects of coexistence of bacterial strains on the growth of a diatom Chaetoceros gracilis were investigated under laboratory conditions. Tested bacterial strains were isolat ed from the diatom mass culture tanks at a kuruma prawn farm. To evaluate the effect of each bacterial strain, the growth pattern was compared between cultures with and without bacterial medium addition. In diatom growth with the coexistence of Flavobacterium sp. in the ASP6 medium, the diatom cell yield during a 16-day culture period and specific growth rate were high compared to axenic culture as a control, whereas the duration of lag time and the variation among daily cell densities from four repli cate cultures were as small as those in the control. These facts indicate that this bacterium greatly pro motes diatom growth. However, four tested strains had no apparent effect and seven strains had a sup pressive effect. Key words: Diatom Chaetoceros gracilis, mixed culture, biocontrol, growth promoting effect, coexistent marine bacteria
We reported1) that in a culture tank for mass production ing to Simidu's scheme.6) All bacterial strains were propa
of food diatoms, the actively growing population of bacter gated in modified ZoBell 2216E culture medium (10% sea ia or diatom exhibits a mutually suppressive effect on the water solution of ZoBell 2216E without agar). Following growth of another population. Mitsutani et al.2) have culture, 0.5 ml samples were collected from each strain documented the lysis of Skeletonema costatum by and stored at -80•Ž in sterilized plastic straws till ex Cytophaga sp. isolated from the coastal water of the perimentation. Glycerol (10% by volume) was added to Ariake Sea. On the other hand, Riquelm et al.3) found that each sample. the growth of a marine diatom Asterionella glacialis was supported by the coexistence of the Flavobacterium strain Cultivation of C. gracilis and was strongly promoted by the addition of the Pseudo To prepare an axenic culture of diatom C. gracilis, cells monas strain isolated from a bloom in Maizuru Bay . were picked up from a colony on the agar plate and Fukami et al.4) have also experimented on the promotive washed with sterilized seawater several times . The diatom effect of the addition of bacterial isolates from deep sea was cultured axenically and stocked in the ASP6 medium7) water on the growth of Chaetoceros ceratosporum. These at 23•Ž and 80ƒÊE/m2/s (white fluorescent bulbs) with a studies suggest that artificial control of bacterial popula 14L:IOD light cycle. No bacterial contamination was ascer tion may be an effective technique for the stable mass cul tained using STP medium8) and confirmed by an epifluores ture of diatom. cence microscope (Olympus) after staining with 4•Œ , 6•Œdi We therefore investigated the promotive and suppres amidino-2 phenylindole (DAPI) 9) sive effects of the coexistence of the bacterial strains, isolated from diatom mass culture tanks, on the growth of Mixed Culture a marine diatom Chaetoceros gracilis. Before each experiment, the algal cells of C . gracilis taken from the stock culture were precultured again in the Materials and Methods ASP6 medium. Bacterial strains from frozen stock were also precultured in the above mentioned modified ZoBell Isolation and Preparation of Bacterial Strains 2216E culture medium. The algal and bacterial cells were The water samples from the diatom mass culture tanks1) cultured for 7 days and 15-24 hours , respectively, till they were diluted with sterilized seawater and spread onto reached the late exponential growth phase . Both C. gracili ZoBell 2216E agar plates,5) followed by incubation for 2 s and one of the tested bacterial strains were inoculated days at 25•Ž. From twenty colonies which appeared, simultaneously into four replicates of 100m/Ehrenmeyer twelve bacterial strains were selected. These were abbreviat flasks containing 60m/ASP6 medium . Initial densities of ed as Flavobacterium DN-10, coryneforms DY-8, Altero the algal cells and the bacteria cells were adjusted to about monas DN-4 and DN-1, Micrococcus DN-11 and DY-7, 1•~104 and 1•~105 cells/ml , respectively after the count us Moraxella DN-18 and DY-12, Vibrio DM-6 and DN-5, ing a haemacytometer. These flasks were incubated fo r 28 d and Bacillus DM-9 and DY-13, after identification accord- ays under the same conditions as described above . Effect of Bacteria on a Marine Diatom 41
Twelve bacterial strains were totally examined in three ex tures. However, the algal growth with Bacillus DM-9 was perimental sets. In order to separate the effect of bacterial remarkably retarded and the variations of daily cell densi addition from that of bacterial culture medium on diatom ties during the whole culture period, especially in the later growth, two controls were employed. C. gracilis was cul period, were large. tured axenically in 60 ml of ASP6 medium with and To evaluate the effect of the coexisting bacteria on the without the addition of 0.1 ml of the modified ZoBell growth of C. gracilis, four indices expressing the growth 2216E culture medium. Triplicates were prepared for each patterns of algal cells were employed. The first index com control in every experimental set. The water samples were pares the diatom cell yield to that of the control with taken daily with a 1 m/ sterilized pipette and then the cell modified ZoBell 2216E. Daily differences between the two numbers of diatom were counted on the haemacytometer means of logarithmic diatom cell densities of four repli under a differential interference microscope (Olympus). cates in the mixed culture (Log. M) and of nine replicates Daily cell densities of two controls were statistically ana in the control culture (Log. C) were integrated for the lyzed in every experimental set using unpaired Student's t whole culture period of 16 days. For every bacterial strain test and among the three experimental sets using ANOVA employed for the experiment, the patterns of the daily Fisher's PLSD.10) differences (Log. M-Log. C) are shown in Fig. 2. Plus and minus values mean the promotive and suppressive effects Results of bacterial coexistence on the growth of diatom, respec tively. Therefore, the integration of the daily differences Algal growth coexisting with Flavobacterium DN-10 or during the culture period (16 days) was used as an index Bacillus DM-9 is shown in Fig. 1, together with that in the which expresses the total effect of bacterial coexistence on two controls with and without addition of modified ZoBell the yield of diatom cells. 2216E culture medium. The densities of the two controls The second index is the highest specific growth rate (k). after 16 days were both 1.5 x 106 cells/ml. Among the dai Specific growth rates were calculated from cell densities at ly densities of the two controls except on the first day, no four serial culture days, except for Flavobacterium DN-10, significant difference was observed (p>.05). The controls for which three serial points were used, because the diatom showed an identical growth pattern of the diatom having reached the stationary phase on the fourth day. The extremely low variation among cell densities. The growth highest value was then selected as k. The higher rates than patterns in different experimental sets can thus be com in the control mean more accelerated cell division in the ex pared in a figure to assess the effect of the bacterial coexis ponential growth phase. tence on algal growth. The cultures ran for 28 days, but in The third index is the duration of lag time. The linear the present report, only data to the 16th day are employed, regression line during the exponential growth phase is ex because after the 16th day the diatom continued growth in pressed as Y=Ak+B, where Y, A and B represent the almost the same trend. In the mixed culture with Flavobac logarithmic cell density, time (day) and a constant, respec terium DN-10, C. gracilis showed faster growth for three tively. The duration of lag time is then calculated as A days in the exponential phase than in the axenic control cul- when Y=1•~10•Ž cells/ml (inoculation density). The lon
ger lag time means the higher suppression of diatom growth in the early period of culture. The fourth index is the largest standard deviation of cell density. From daily standard deviations of diatom cell den sities in four replicated diatom cultures, the largest value was used as an index which indicates the stability of the dia tom culture. The standard deviations were also calculated for controls from the data of nine replicated cultures. The values of the four indices from algal cultures with bacterial additions and controls are shown in Table 1. Data from both controls showed identical values for 3 indices. Of the twelve bacterial strains tested, cultures mixed with Flavobacterium DN-10 and Moraxella DY-12 showed higher values (6.8 and 2, respectively) of the integrated dai ly differences of logarithmic algal cell density (Fig. 2 and Table 1). On the contrary, the values were low when the bacterial strains of Bacillus DM-9, Moraxella DN-18, Vibrio DN-5, coryneforms DY-8, and Bacillus DY-13 were added (Table 1). Table 1 also shows the ratios of the specific growth rate and the largest standard deviation against those in the con Fig. 1. Growth curves of C. gracilis in the axenic culture (control) with trol with the addition of modified ZoBell 2216E, respec and without the addition of modified ZoBell 2216E and cultures mix tively. The growth of diatom cells in the mixed cultures ed with either Flavobacterium DN-10 or Bacillus DM-9. with seven bacterial strains showed higher specific growth Plotted points and bars indicate means and standard deviations of rates than in the control. The specific growth rate in the nine replicate flasks for two controls and of four replicate flasks for mixed cultures, respectively. Standard deviations for both controls mixed cultures with Flavobacterium DN-10 and Moraxella have small values (less than 0.07). DY-12 were 2.8 and 1.6 times greater than that for the con- 42 Suminto and Hirayama
Fig. 2. Daily differences in logarithms of algal cell density between mixed culture (Log. M) and control (Log . C). A, mixed culture with Flavobacterium DN-10 and coryneforms DY-8; B, with Alteromonas DM-4 and DN-1; C , with Micrococcus DN-11 and DY-17; D, with Moraxella DY-12 and DN-18; E, with Vibrio DM-6 and DN-5; F, with Bacillus DM-9 and DY-13 . trol, respectively. However, the mixed cultures with Moraxella DN-18, Vibrio DN-5, and Bacillus DM-9 Discussion showed lower specific growth rates than for the control. The largest standard deviations for the cultures mixed with Two types of controls showed extremely stable and iden Moraxella DN-18 and Bacillus DM-9 were 5.2 and 12 times tical growth patterns (Fig. 1 and Table 1). This indicates larger than that in the control (Table 1), respectively. The that the supply of a small amount of modified ZoBell corrected value of the duration of lag time was obtained 2216E had neither promotive nor suppressive effects on the by subtracting the lag time value of the control from that growth of C. gracilis. The different patterns of diatom in the mixed culture (Table 1). The corrected values of the growth observed between the culture with the bacterial ad index in the algal culture mixed with coryneform DY-8, dition and the controls were induced only by the bacteria Micrococcus DY-17, Moraxella DN-18, Vibrio DM-6, addition, because the amount of bacterial culture medium Bacillus DM-9 and DY-13 had relatively larger values than incidentally supplied into the ASP6 medium at the inocula 2 days. tion of bacterial population was less than that added into the control culture medium. Effect of Bacteria on a Marine Diatom 43
Table 1. The index values expressing growth pattern of C. gracilis in mixed cultures and controls
*1 Ratio of specific growth rate in mixed culture (kM) to that in control (kC). *2Corrected lag time by subtracting the control value from that in mixed culture . *3Ratio of the largest standard deviation (S .D.) in mixed culture to that in control. Marks of a, b, and c represent the results by unpaired Student's t-test; a, significant difference at p <.01 (control
The growth of C. gracilis was characterized by using 2) A. Mitsutani, K. Takesue, M. Kirita, and Y. Ishida: Lysis of four indices (Table 1). A comparison of the index values Skeletonema costatum by Cytophaga sp. isolated from the coastal with those of controls shows the effects of bacterial coexis water of the Ariake Sea. Nippon Suisan Gakkaishi, 58, 2159-2169 tence on the algal growth as follows. The bacteria strain (1992). 3) C. E. Riquelm, K. Fukami, and Y. Ishida: Effects of bacteria on the Flavobacterium DN-10 strongly promoted diatom growth. growth of a marine diatom, Asterionella glacialis. Bull. Japan Soc. The bacterial strains Moraxella DY-12, Alteromonas DM Microbiol. Ecol., 3, 29-34 (1988). -4 and DN-1 and Micrococcus DN-11 had no apparent 4) K. Fukami, T. Nishijima, and Y. Hata: Availability of deep sea effect on diatom growth. Algal growth is suppressed by the water and effects of bacteria isolated from deep seawater on the addition of coryneforms DY-8, Moraxella DN-18, Bacillus mass culture of food microalga Chaetoceros ceratosporum. Nippon DM-9 and DY-13, Vibrio of DN-5 and DM-6, and Suisan Gakkaishi, 58, 931-936 (1991). 5) C. H. Oppenheimer and C. E. ZoBell: The growth and viability of Micrococcus DY-17. sixty-three species of marine bacteria as influenced by hydrostatic pressure. J. mar. Res., 11, 10-18 (1952). Acknowledgments This study was partly supported by a Grant-in-Aid 6) U. Simidu: The taxonomy of marine bacteria, in "Marine Microbi for Scientific Research from the Ministry of Education, Science and Cul ology" (ed. by N. Taga), Tokyo Univ. Press, Tokyo, 1974, pp. 45 ture of Japan (No. 04304020). Authors wish to thank Dr. A. Hagiwara -65. (Faculty of Fisheries, Nagasaki Univ.), Dr. N. Munuswamy (Department 7) L. Provasoli, J. J. A. McLaughlin, and M. R. Droop: The develop of Zoology, Univ. of Madras), and Dr. T. A. Mousseau (Department of ment of artificial media for marine algae. Arch. Microbial., 25, Biological Sciences, Univ. of South Carolina) for their critical reading of 279-308 (1957). this manuscript. We also thank Mr. T. Kotani for technical assistance in 8) L. Provasoli, K. Shiraishi, and J. R. Lance: Nutritional idiosyncra the data tabulations. sies of Anemia and Tigriopus in monoxenic culture. Ann. N. Y. Acad. Sci., 77, 250-261 (1959). References 9) K. G. Porter and Y. S. Feig: The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr., 25, 943-948 1) Suminto and K. Hirayama: Relation between diatom growth and (1980). 10) J. H. Zar: Biostatistical Analysis, 2nd ed., Prentice-Hall Interna bacterial population in semi mass culture tanks of diatom. Bull. tional, Inc., Englewood Cliffs, New Jersey, 1984, pp. 1-260. Fac. Fish., Nagasaki Univ., 74/75, 37-41 (1993).