[7] Isolation and culture of marine nitrogen-fixing unicellular cyanobacteria Synechococcus

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intervals using heavy inocula. Representative strains of these cyanobacteria are available from the culture collection of the Bigelow Laboratory for Marine Science (Boothbay Harbor, ME 04575). The complete collection is housed at the Woods Hole Oceanographic Institution (Woods Hole, MA 02543). Acknowledgments The authors' work is supported by National Science Foundation Grants BSR-8607386 and DCB-8608698. Woods Hole Oceanographic Institution Contribution No. 6438.

[7] Isolation a n d C u l t u r e of M a r i n e N i t r o g e n - F i x i n g U n i c e l l u l a r C y a n o b a c t e r i a Synechococcus By AKIRn MITSUI and SHULI CAO Introduction Nitrogen fixation by cyanobacteria contributed significantly to the nitrogen budget in the marine environment. Trichodesmium species (Oscillatoria spp. or LPP group) have been well known as marine N2-fixing cyanobacteria. 1 More recently, however, unicellular aerobic N2-fixing cyanobacteria of the genus Synechococcus have been found and isolated from the marine environment.2-4 Since these strains can carry out photosynthetic 02 evolution and oxygen-labile N2 fixation in the same cell, they are good material for both ecological and biochemical studies. This chapter describes methods of sample collection, enrichment, and isolation. Culture methods for one of the fast-growing aerobic N2-fixing marine Synechococcus species are also described. ~G. E. Fogg, in "Environmental Role of Nitrogen-Fixing Blue-Green Algae and Asymbiotic Bacteria" (U. Gramhall, ed.), p. 11. Swedish National Research Council, Stockholm, 1978. 2 A. Mitsui, Proc. Int. Ocean Dev. Conf., 5th 1, 29 (1978). 3 A. Mitsui, E. J. Phlips, S. Kumazawa, K. J. Reddy, S. Ramchandran, T. Matsunaga, L. Haynes, and H. Ikemoto, Ann. N.Y. Acad. Sci. 413, 514 (1983). 4 A. Mitsui, S. Kumazawa, E. J. Phlips, K. J. Reddy, T. Matsunaga, K. Gill, B. R. Renuka, T. Kusumi, G. Reyes-Vasquez, K. Miyazawa, L. Haynes, E. Duerr, C. B. Le6n, D. Rosner, H. Ikemoto, R. Sesco, and E. Moffat, in "Biotechnology and Bioprocess Engineering" (T. K. Ghose, ed.), p. 119. United India Press, New Delhi, 1985.

METHODS IN ENZYMOLOGY, VOL. 167

Copyright © 1988by Academic Press, Inc. All rights of reproduction in any form reserved.

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Sample Collection Nitrogen-fixing cyanobacteria can be encountered in a variety of marine environments. Samples should be placed into sterilized plastic bags. Samples taken from the water column can be obtained with a discrete depth sampler, a number of which are available. 5 Sediment samples can be obtained using a grab sampler or similar apparatus. Finally, since some cyanobacteria are known to grow epiphytically, samples of macroalgae and seagrasses can be taken along with scrapings from rocks and other stationary objects. Enrichment In the laboratory or aboard the sampling vessel each sample is subdivided into eight subsamples, and each subsample is inoculated into sterilized test tubes or 50-ml flasks containing different enriched, combinednitrogen-free artificial seawater media. The eight different nitrogen-free media represent a range o f p H and salinity regimes (e.g., pH values of 6.5, 7.5, 8.5, and 9.5 at 30%° salinity, and salinities of 9, 18, 27, and 36%0 at an initial pH of 8.0). The basic enriched culture medium is composed of the following in 1 liter of distilled water: MgSO4" 7HzO, 5 g; KC1, 600 mg; CaC12"2H20, 370 mg; tris(hydroxymethyl)aminomethane, 50 mg; KH2PO4, 50 mg; disodium EDTA, 30 mg; FeSO4.7H20, 3.89 mg; vitamin B~2, 10/zg; and micronutrient solution, l0 ml. Micronutrient solution is composed of the following in I liter of distilled water: H3BO3, 3.426 g; MnC12.4H20, 432 mg; Na2MoO3"2H20, 130 mg; ZnC12, 32 mg; COC12.4H20, 1.2 mg; and CuSO4, 0.3 mg. NaCI is added in varying amounts to obtain the desired salinities. These subsamples are placed on illuminated shelves at 100 /zE/m2/sec at approximately 25-30 ° for 2-6 weeks. When growth is visually evident the subsamples are transferred to fresh, sterile, nitrogen-free media of the same pH and salinity. This process is repeated for at least 3 cycles before initiating the isolation procedures so that culture parameters become stabilized and any nitrogencontaining contaminants from the samples are minimized in the culture, thus enriching the nitrogen-fixing cyanobacteria in the subsample. Isolation Subsequent to the enrichment process the subsamples are examined microscopically, and those subsamples containing unicellular forms are 50. T. Lind, "Handbook of Common Methods in Limnology," 2nd Ed. Kendal/Hunt, Dubuque, Iowa, 1985,

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singled out for further isolation procedures. This is initiated with a serial dilution process using the same enriched medium as above. The serial dilution is followed by plating on sterile agar media (enrichment media above in 2% agar), Streaking is also employed to help isolate monoclonal colonies. After a period of growth on illuminated shelves, apparently pure colonies are picked from the surface of the agar with sterile implements and reinoculated into sterile, combined-nitrogen-free liquid medium as described above. This process is repeated until observation using phasecontrast light microscopy confirms a pure culture, which is then maintained in transfers of fresh media. Axenic Culture and Maintenance

Scanning electron microscopic observation serves not only to confirm that a single strain has been isolated but also to detect the presence or absence of associated bacteria or fungi in the culture. However, microscopic observation is often insufficient for determining with absolute certainty whether the culture is axenic or not. Purity can be confirmed by using TYG (tryptone-yeast extract-glucose) agar and broth medium, 6 Burk's Azotobacter medium, 7 2% sucrose-enriched cyanobacterial medium, and nutrient broth at varying pH values. If a contaminant is found, antibacterial or antifungal agents are utilized as part of the isolation procedure prior to physiological or biochemical studies. Cycloserine in concentrations of l, 2, 3, 4, and 5 mg/ml and/or cycloheximide at 10, 15, 20, 25, 30, and 35 mg/liter are added to the culture media in this purification process. Classification and taxonomy of unicellular cyanobacteria has been described by Rippka et al. 8 and elsewhere in this volume. 9 Growth Batch Culture

Batch cultures of Synechococcus are grown in water-jacketed glass cylinders (8.5 cm in diameter and 75 cm in height). The top part of the culture cylinder is fitted with a ground glass cap. Media and inoculant are added from the top by disconnecting the ground glass cap under sterile conditions. Aeration and sampling of the culture materials are carried out 6 T. Vaara, M. Vaara, and S. Niemela, Appl. Environ. Microbiol. 38, 1011 (1979). 7 j. Oppenheim and L. Marcus, J. Bacteriol. 101, 286 (1970). 8 R. Rippka, J. Deruelles, J. B. Waterbury, H. Herdman, and R. Y. Stanier, J. Gen. Microbiol. U l , 1 (1979). 9 R. Rippka, this volume [2].

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through a port in the bottom of the cylinder fitted with a three-way valve. Air is vented through a port at the top of the ground glass cap with a sterile air filter. Illumination is provided by two 30-W fluorescent tubes (arranged parallel to the cylinder) for each culture cylinder. A handmade light reflector (aluminum foil) is added to the light fixture. In a routine experiment, 4% CO2-enriched air is used for aeration, which is passed through a 50% H3PO4 solution to remove ammonia, if any, from the air and then through a sterilized cotton air filter. In addition to the bubbling through the cylinder, mixing of the culture is provided by a magnetic stirrer located at the bottom. The basic culture medium is the same as previously described except 18 g NaCI and 2.5 g NaHCO3 are added to 1 liter of culture medium. During the pH adjustment of the culture medium to 7.6, the medium is bubbled with 4% CO2-enriched air. After autoclaving the medium, precipitates formed during the autoclaving are dissolved by bubbling the medium with CO2 gas through a sterilized cotton air filter. The cleared medium is transferred to the sterilized culture cylinder and then bubbled with 4% CO2-enriched air (about 200 ml/min) for pH equilibration before inoculation. The CO2 concentration of the CO2-enriched air is slightly adjusted in order to maintain the pH of the culture medium at 7.6. Seed cyanobacterial cultures are grown in l-liter flasks at pH 7.6 at room temperature (25-27 °) and a light intensity of approximately 150/zE/ m2/sec. When the cultures reach midexponential growth phase, a 50-ml aliquot is inoculated into each glass cylinder containing 3.3 liters of culture medium. After the inoculation of the seed culture, the culture is bubbled with CO2-enriched air as described above (flow rate of about 200 ml/min) and stirred by magnetic stirrer as mentioned above. The light intensity at the surface of the culture cylinders is 150 tzE/m2/sec. The temperature and pH are maintained at 30 - 1° and 7.6 - 0.2, respectively, throughout the culture period. As mentioned above, the pH of the culture is controlled by slight adjustment of the COz concentration of the CO2-enriched air. At optimum culture conditions (34°, pH 7.6, 4% CO2 in air), Synechococcus sp. strain Miami BG 43511 can be grown at a minimum doubling time of 14 hr in N:-fixing conditions, when the temperature is strictly maintained. Since there is a sharp decline of growth above 34 ° in this strain, however, a culture temperature of 30° is recommended for ordinary use. In ammonia or nitrate culture (final concentration, 10 mM) a minimum doubling time of 9-10 hr can be obtained in this strain. Biomass yield in N2-fixing optimum culture conditions at 8 days batch culture is 1.0 g dry weight per liter culture.

[7]

ISOLATION AND CULTURE OF N2-FIXING Synechococcus

-l/-,

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FIG. 1. Changes in turbidity of an aerobic N2-fixing culture of marine Synechococcus sp. strain Miami BG 43511 during and after various dark treatments. A, B, C, and D were treated with 0, 8, 16, and 32 hr dark, respectively, and E was treated with 16 hr dark-16 hr light-16 hr dark. Numbers indicate dark treatment periods (hr). All cultures contained approximately 106 cells/ml (0.075 absorbance units at 600 nm) at the beginning of dark treatments (at 92 hr culture time). See text for other experimental conditions. Relative

changes in absorbance are shown in a log scale.

Synchronous Culture Under the growth conditions described above, synchronization of aerobic N2-fixing Synechococcus sp. strain Miami BG 43511 can be induced by interruption during their early exponential growth phase (-106 cells/ ml) with dark periods. ]°-n2 During the dark period(s), aeration is stopped, but mixing by magnetic stirrer is continued. Measurement of synchronous 10 C. Le6n, S. Kumazawa, and A. Mitsui, Curt. Microbiol. 13, 149 (1985). " A. Mitsui, S. Kumazawa, A. Takahashi, H. Ikemoto, S. Cao, and T. Arai, Nature (London) 323, 720 (1985). 12 A. Mitsui, S. Cao, A. Takahashi, and T. Arai, Physiol. Plant. 69, 1 (1987).

1 10

ISOLATION, IDENTIFICATION, AND CULTURING

[7]

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Fro. 2. Relationship among total cell number, doubling cell percentage, and carbohydrate content in a continuously illuminated batch culture of aerobic N2-fixing marine Synechococcus sp. strain Miami BG 43511. See the legend to Fig. l and text for experimental conditions.

growth can be monitored roughly by hourly measuring the change in absorbance of the culture at 600 nm. Figure 1 shows the changes of absorbance after treatment with various dark periods. A stepwise increase is observed in well-synchronized cultures. More exact measurements, however, should be made of the doubling cells, which appear only during the segregated periods of the cell division cycle, and total cells should also be monitored. For cell counts, a 1-ml sample is taken from the culture cylinder at periodic time intervals and fixed with 50 ~l of 10% Lugol's solution. Cell numbers are measured using a Petroff-Hauser bacterial cell-counting chamber. For each cell count, the total number of cells and the number of doubling (dividing) cells are counted. Doubling cells are counted as one cell. Changes in the carbohydrate content in the culture also provide a good index of growth synchrony under nitrogen-fixing conditions ~L~2(see Figs.

[7]

ISOLATION AND CULTURE OF N2-FIXING I~"11~L~hIIJFIl~

Synechococcus

111

Light

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FIG. 3. Relationship among total cell number, doubling cell percentage, and carbohydrate content in a synchronous culture of aerobic N2-fixing marine Synechococcus sp. strain Miami BG 43511. From Mitsui et alJ 2

2 and 3). Carbohydrate content of the culture is measured by a colorimetric method. 13 To obtain well-synchronized growth in culture, insertion of proper dark and light periods is essential. 12,14 Under the culture conditions described above, a regime of 16 hr dark-16 hr light-16 hr dark yields good synchrony of Synechococcus sp. strain Miami BG 43511.12,14 Longer dark periods reduce the degree of synchrony, and shorter dark periods induce irregular cell divisions. 12 Figures 2 and 3 represent the nonsynchronous (batch) culture without pretreatment and the well-synchronized culture with dark-light-dark pretreatment, respectively. After the 16 hr dark-16 hr light-16 hr dark period, cells grow synchronously during the subse13 M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith, Anal. Chem. 28, 350 (1956). 14 T. Arai and A. Mitsui, Prog. Photosyn. Res. 2, 649 (1987).

FIG. 4. Phase-contrast photomicrographs of the aerobic Nz-flxing marine unicellular cyanobacterium Synechococcus sp. strain Miami BG 43511 during synchronous growth. A, B, and C show cells during the photosynthetic elongation period, dividing period, and small daughter cell period, respectively, under the aerobic diazotrophic synchronous growth conditions. Bar, 10/zm.

[8]

SYMBIOTIC ASSOCIATIONS

1 13

quent 80 hr for 3-4 cell division cycles under continuous illumination (Fig. 3). As shown in Fig. 3, the distribution of doubling cell percentage is very narrow in the first cell division cycle in a well-synchronized culture. Doubling cells disappear from the culture from 8 through 18 hr until the second cell division cycle starts. In the second, third, and fourth cell division cycles, the peak doubling cell percentages decrease and the distribution of doubling cell percentage becomes wider. However, the time of occurrence of doubling cell percentage peaks during synchronous growth indicates that cell division cycles occur at approximately 20-hr intervals under the given culture conditions. The changes of carbohydrate content during well-synchronized growth also exhibit approximately 20hr intervals for 3-4 cell division cycles during the continuous illumination period (Fig. 3). Figure 4 represents phase-contrast photomicrographs of the culture during typical synchronous growth of Synechococcus sp. strain Miami BG 43511. At the start of synchronous growth of this strain, most of the cells were single and small (3.0 × 2.5/zm). During the first 5-hr period, most of the single cells became larger (3.5 × 7.0/zm) and formed septa. At 6 and 7 hr, most cells split into two daughter cells of small size (3.0 × 3.5/zm). These changes in cell size were repeated with each cycle. The cell length of Synechococcus sp. strain Miami BG 43522 is slightly shorter.

[8] S y m b i o t i c Associations By J. C. MEEKS Introduction

Certain filamentous nitrogen-fixing cyanobacteria establish symbiotic associations with ascomycete fungi (to form lichens), with a marine diatom, and with phylogenetically diverse plants that include bryophytes, a fern, cycads (a class of gymnosperm), and an angiospermJ ,2 Compared to free-living cultures, the cyanobacteria in association with a photosynthetic eukaryotic partner have a lower growth rate, a diminished capacity to assimilate ammonium and photosynthetically reduce CO2, and a higher frequency of heterocysts. Thus, symbiotically associated cyanobacteria J. W. Millbank, in "The Biology of Nitrogen Fixation" (A. Quispel, ed.), p. 238. Elsevier, New York, 1974. z G. A. Peters, R. E. Toia, Jr., H. E. Calvert, and B. H. Marsh, Plant Soil 90, 17 (1986).

METHODS IN ENZYMOLOGY, VOL. 167

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