Large-Scale Autotrophic Culture ofEuglena gracilis*

zyxwvutsrqp zyxw 297 J. PROTOZOOL. 14(2), 297-299 (1967). Large-Scale Autotrophic Culture of Euglena gracilis” HARVARD LYMAN and H. W. SIEGELMAN Microbiology Division, Medical Research Center and Biology Department, Brookhaven National Laboratory, Upton, New York 11973 z zy SYNOPSIS. Large quantities of autotrophic Euglena gracilis can be obtained in a short time using simple and inexpensive equipment. Cultures grown in flasks on a rotary shaker are tramferred to a polyethylene drum containing 180 liters of medium. The drum, which is illuminated with both internal and external fluorescent lamps, contains a coil of vinyl tubing carrying tap water for temperature control and 2 vinyl tubes for air and CO,. Sterilization of the drum or the medium was unnecessary, but can be accomplished by gassing ithe drum with ethylene oxide or overnight exposure to germicidal lamps and by dispensing the medium into the drum through a large Millipore apparatus. Yields of 289-330 g of fresh weight material have been obtained after one week’s growth. Shorter growth periods yielded 134-160 g of fresh weight material. Ten g of purified chloroplasts have been obtained from 160 g of cells grown in this manner. E of 8 F96T12 “Cool-White” fluorescent lamps ppovides the external source (Fig. 1). One to 8 F48T12 “Warm White” lamps, inserted into large glass tubes closed at the end within the culture are put thru the perforations in the cover of the drum. The diameter of the glass tubes is 47 mm. A large loop of vinyl tubing 11 mm ID X 17 mm OD is placed within the drum for the circulation of cold tap water for cooling when mare than 2 lamps are in the drum. The temperature is mahtained at 26 C k 2 C. Medium: The constituents of the medium are shown in Table 1. Concentrated solutions of the ingredients are made and added to demineralized tap water or distilled water in the drum to give the desired final concentrations. The usual volume in the drum is 180 liters. Demineralized tap water is prepared with 2 columns (15 an diameter X 50 cm high bed volumes) of anion and cation exchange resin, Bio-Rex RG 1-X8 20-50 mesh and Bio-Rex RG SOW-XS, respectively. No attempt is made to sterilize the drum, medium, or the water. Cell counts were made with a Coulter Electronic Cell Counter, Model B. Total chlorophyll was determined by Arnon’s method(2). Harvesting of cultures: Cultures were harvested in a Sorvall RC-2B refrigerated centrifuge using the Sorvall continuous-flow apparatus with an 8-channel distributor at 15,OOO rpm with a flow rate of 1700 ml/min. zyxwvut zyxwvut zyxwvuts zyxwvuts uglena gracilis excels for studies of chloroplast synthesis and replication( 13), photosynthesis(S,11,12,15), phototaxis(6,16), and the effects of drugs, antibiotics, and carcinogens( 1,8,9). Autotrophically grown cells are preferred for photosynthetic studies and chloroplast isolation since the entire photosynthetic apparatus is present in much higher concentration than in light-grown, heterotrophic cells( 10,14). The chloroplasts of the autotrophic cells are much larger and easier to isolate as intact organelles (7). Bach(3) has described a method for the mass culture of heterotrophic cells, and Bergeron( 4 ) has designed an apparatus for the continuous culture of autotrophic cells. Both culture methods are suitable for growing autotrophic Euglena gracilis if only small amounts of material are needed. The present paper describes a procedure for growing autotrophic Euglena with yields of 300 g fresh weight. Large amounts of Euglena may be grown in a short time by a simple method using inexpensive equipment. The method is also employed for culturing several blue-green and red algae. N zyxwvutsrqponm MATERIALS .4ND METHODS Znoczilzim : Five liters of a sterile autotrophic culture of Euglena gracilis variety bacilluris, strain Z, were grown in five 2-liter Erlenmeyer flasks on a rotary shaker with 400-500 foot-candles of fluoescent illumination. The cells were grown to the end of the logarithmic growth phase in the medium shown in Table 1 and aerated with 5% CO? in air. A dense inoculum (1.5-2 X 10’ cells/ml) reduces the possibility of contamination in the large culture vessel described below. Large-scale culture: The 5 liters of inoculum are transferred to a 50-gal polyethylene drum containing 175 liters of the medium shown in Table 1. The polyethylene drum is covered with a tightfitting polyethylene lid which is perforated to allow the insertion of fluorescent lamps and vinyl tubing for aeration and cooling (Fig. 1). Aeration and stirring are accomplished by passing air scrubbed by a “Koby Junior” disposable air purifier and flow equalizer (Koby Corp., Melrose, Mass.) in vinyl tubing (5 mm I D X 7 mm OD) thru 2 cylindrical gas diffuser stones (Fisher Scientific Co.) at the bottom of the drum. C02 from a cylinder of 99.5% CO? is introduced in a separate vinyl tube at 100 ml/min through one gas diffuser stone at the bobtom of the drum. Air and CO, are metered with needle valves and flow rates are monitored with rotameters. Illumination: Illumination is both internal and external. A bank * Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission. RESULTS Typical growth curves and chlorophyll per cell during growth in the polyethylene drum are shown (Fig. 2 ) . In culture 1, 7 internal lamps were used as indicated. In culture 2, 8 internal lamps were used, with 2 turned on simultaneously at 139 hr and one turned off 17 hr before TABLE Component 1. Growth medium. Concentration (g/liter except where specified) EDTA (disodium salt) KH,PO, MgSO, * 7H20 CaCO, (NH,), SO, Vitamin B, Vitamin B,, (stock s o h tion of 4 mg/100 nil) Metale mix (Hutner’s #49) p H 3.5 Metals mix (Hutner’s #49) g/500 nil stock Component solution 0.50 0.30 0.50 0.06 1.0 0.6 m g 0.125 nil Fe( NH1)?(SO,),-GH,O MnSO, H,O ZnSO, * 7H20 CuSO, * 5H,O (NH,), Mn,O, * 4H,O CoSO, * 7H20 Na,VO, * 1 6 H 2 0 0.130 m g HaBOa - 70 31 22 4 0.72 2.4 0.46 0.57 298 zyxwvuts zyxw zyxwvutsrqp LARGE-SCALE CULTURES OF Euglena Fig. 3 shows a culture grown for only 94 hr and with only 4 internal lamps. The yield in this instance was 134 g. Altho the yield per liter is quite low compared with heterotrophically grown cells, the yield in terms of chloroplast constituents is high. As an example, a harvest of a single large culture yielded 10 g of purified chloroplasts from 160 g of cells. Harvest of 4 cultures can easily provide in excess of 1 kg fresh weight of Euglena. Studies of cellular constituents may require large amounts of cells, and it is possible using the methods described to obtain the necessary material with only a small expenditure of time. Contamination of the cultures has not proved to be a problem altho the medium and the polyethylene drum were not sterilized. The low pH of the medium and the absence of an organic carbon source are probably responsible. The culture apparatus may be sterilized if desired by minor modifications. The drum can be sterilized by gassing with COLD WATER zyxwvutsrqp zyxwvutsrqp Fig. 1. Diagram of the large-scale autotrophic culture apparatus. The bank of external lamps is shown behind the polyethylene drum. One internal lamp is shown in place. The gas-diffuser stones from the air and CO? lines are shown at the bottom of the drum. harvest. The yield from culture 1 was 289 g of fresh weight material and the yield of culture 2 was 330 g. I I 20 I 40 fn I I I 40 60 3 I I 80 100 HOURS I 120 (a) I 140 160 I 80 1 I00 HOURS zyxwvutsrq e 20 I 60 I I 20 I 40 I 60 I 80 I 100 HOURS Fig. 3. Growth of a 96-hr autotrophic culture ( a ) and chlorophyll content of the cells (6). Culture conditions are as described in the text and symbols as in Fig. 2. ethylene oxide or by placing 2 or 3 15-watt germicidal lamps inside overnight. The medium may be dispensed into the drum thru a large sterile Millipore apparatus. 0 E 0.5 I 20 I 40 I 60 I I 80 100 HOURS I I 120 140 I (b) 160 Fig. 2. Growth of autotrophic cultures ( a ) and chlorophyll contents of the cells ( 6 ) . Culture conditions are as described in the text. t, one internal lamp o n ; .1, one internal lamp off. zyxwvu The excellent technical assistance of R. Love, M. Hogan, R. Wright and S. Dunwoody is gratefully acknowledged. REFERENCES H., Bensky, B., Frank, 0. & Zahalsky, A. 1964. Protozoan assays for vitamins and cytotoxicity of carcinogens 1. Aaronson, S., Baker, zyxwvutsrq zyxwvu zyxwv THETESTOF Rosalina jloridana and pharmacological agents, in Developments in Industrial Microbiology, Am. Inst. Biol. Sci., Washington, D.C., 6, 48-58. 2. Arnon, D. 1949. Copper enzymes in isolated chloroplasts. Polyphenol-oxidase in Beta vulgaris. Plant Physiol. 21, 1-15, 3. Bach, M. K. 1960. Mass culture of Euglena gracilis. J . Proto2001. 299 9. Hutner, S. H., Provasoli, L. & Baker, H. 1961. Development of microbiological assays for biochemical, oceanographic, and clinical use. Microchem. J . (Symp. Series) 1, 95-113. 10. Lyman, H. Unpublished observations. 11. Olson, J. M. & Smillie, R. M. 1963. Light-driven cytochrome reactions in Anacystis and Euglena, in Photosynthetic Mechanisms of Green Plants, Nat. Acad. Su.-Nat. Res. Counc., Washington, D.C., 56-66. 12. Perini, F. 1963. The photosynthetic and respiratory systems in Euglena gracilis, in Photosynthetic Mechanisms of Green Plants, Nat. Acad. Sci.-Nat. Res. Counc., Washington, D.C., 291-301. 13. Schiff, J. A. & Epstein, H . T . 1965 The continuity of the chloroplast in Euglena, in Locke, M., Reproduction: Molecular, Subcellular and Cellular, Academic Press, New York, 131-89. 14. Smillie, R. M. Personal communication. 15. Smillie, R. M., Evans, W. R. & Lyman, H. 1963. Metabolic events during the formation of ‘a photosynthetic cell from a nonphotosynthetic cell. Brookhaven S y m p . Biol. 16, 89-108. 16. Wolken, S. S. & Shin, E. 1958. Photomotion in Euglena gracilis. I. Photokinesis. Photokinesis. 11. Phototaxis. J . Protozool. 5 , 39-46. zyxwvutsrqpo zyxwvuts zyxwvutsr zyxwvutsrqp zyxwvuts 7 , 50-2. 4. Bergeron, J. 1963. Studies of the localization, physicochemical properties, and action of phycocyanin in Anacystis nidulans, in Photosynthetic Mechanisms of Green Plants, Nat. Acad. Sci.-Nat. Res. Counc., Washington, D.C., 527-37. 5 . Brody, S. S. & Brody, M . 1963. Aggregated chlorophyll in vivo, in Photosynthetic Mechanisms of Green Plants, Nat. Acad. Sci.-Nat. Res. Counc., Washington, D.C., 455-78. 6. Diehn, B. & Tollin, G. 1966. Phototaxis in Euglena. 11. Physical factors determining the rate of phototactic response. Photochem. Photobiol. 5 , 523-32. 7. Eisenstadt, J. M. & Brawerman, G . 1964. The protein-synthesizing systems from the cytoplasm and the chloroplasts of Euglena gracdis. J . Mol. Biol. 10, 392-402. 8. Hutner, S. H. 1961. Plant animals as experimental tools for growth studies. Bull. Torrey Rot. Club 88, 339-49. J. PROTO’ZZOOL. 14(2), 299-307 (1967). The Test Structure and Composition of the Foraminifer RosaZina fZoridana* ROBERT W. ANGELL Committee on Paleozoology, University of Chicago, Chicago, Illinois 60637 SYNOPSIS. The composition of the test of Rosalina floridana (Cushman) was examined histochemically, and its structure was studied with the electron microscope by means of thin sections and carbon replicas. The test is composed of a thick organic lining overlain by one or more calcite layers bounded above and below by thin membranes. The membranes are fused to organic pore processes composed of coarse fibers that penetrate the calcite layers. The lining, consisting of coarse fibers matted into a laminated sheet, is considered a strengthening element of the test. The membranes covering each calcite layer are composed of fine, beaded fibrils which in aggregate have a striated pattern; they are thought to be the crystal-nucleating agent during calcification and to form a protective covering for the previously deposited calcite layers. The pore processes, which are devoid of an internal entrance for cytoplasm, are considered to be points of attachment for the membranes; they tie the organic test components into a unified whole. The calcite layers and the chambers lack this unity, being separated from each other and from the preceding chambers by membranes so that there are no calcite-to-calcite boundaries between them. An organic, sievelike structure of undetermined function has been found in the foramina of chambers near the prolocular region of the test. Histochemical methods show that the lining contains proteins, polysaccharides, and unidentified substances ; the membranes and the pore processes stain as a pratein-polysaccharide complex free of other substances. URING the last forty years the tests of fossil forami- the mineralogical composition of calcareous tests representing 131 genera of Foraminiferida taken from unindurated marine cores. Rosalina candeiana (d’orbigny) and R . optima (Cushman) , presumably closely related to R . jloridana, were found to have calcitic tests and a Mg content of 10-15 mol per 100 g. They concluded from their study that the mineral type (calcite or aragonite) and the Mg content of the test is a genetically controlled character not influenced by environmental conditions. Wood( 13) , in a re-evaluation of the wall structure of numerous foraminiferal families, found Discorbis vesicularis Lamark, D . concinna (Brady), D. globularis (d’orbigny), D . orbiclaris (Tarquem) , and D . tricamerata (Heron-Allen & Earland) of the family Discorbidiae, to which R . floridana is now assigned, to have perforate radial walls (composed of crystals with their C axes perpendicular to the surface of the chamber) rather than microgranular ones. The work of Wood, Smout, Reiss, and others has culminated in a revision of foraminiferal taxonomy by Loeblich and Tappan(9) in which the laminated or nonlaminated structure of walls and septa is considered to be of importance. The D nifera have been intensively studied because of their usefulness to geologists engaged in stratigraphic studies. A large body of knowledge has accumulated about the test structure of many species, but slight attention has been given to the tests of living foraminifera in which both the mineral and the organic components of the test may be studied without the complications that arise in the interpretation of fossilized material. This paper, part of a study of the process of chamber formation and calcification of the test of Rosalina jloridana (Cushman), considers both the organic and mineral parts of the test with emphasis on the organic structure shown by electron microscopy. Todd and Blackmon ( 2 ) determined by x-ray diffraction *Based on a portion of a thesis submitted to the Interdivisional Committee on Paleozoolopy, The University of Chicago, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. The electron microscope facilities of Whitman Laboratory, Department of Zoology, University of Chicago, were used for this ;search.